Analytical Profiles

of
Drug Substances
Volume 15
 EDITORIAL BOARD

Abdullah A. Al-Badr Lee T. Grady

Steven A. Benezra Eugene Inman
Gerald S. Brenner Joseph Mollica
Glenn A. Brewer, Jr. James W. Munson
Nicholas DeAngelis Milton D. Yudis
John E. Fairbrother John Zarembo

Academic Press Rapid Manuscript Reproduction

 Analytical Profiles
of
Drug Substances
Volume 15

Edited h y

Klaus Florey
The Sqiiil)t> Institute lor Medical Research
New Birinswick, New Jersey

Contribu ti rig Editors

Abdullah A. Al-Badr Glenn A. Brewer, Jr.
Gerald S. Brenner Nicholas J. DeAngelis
Joseph A. Mollica

ACADEMIC PRESS, INC.

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Analytical profiles o f drug substances.

Compiled under the auspices o f the Pharmaceutical

Analysis and Control Section, Academy of Pharmaceutical
Sciences.
Includes bibliographical references and indexes.
1. Drugs-Analysis. 2. Chemistry, Pharmaceutical-
Collected works. I. Florey, Klaus. II. Brewer, Glenn A.
I l l . Academy o f Pharmaceutical Sciences. Pharmaceutical
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 CONTENTS

Affiliutiotis of Editors uiitl Contrihutors uii

Preface ix

Amiloride Hydrochloride 1
David J . Mazzo

Aminoglutethimide 35
Hassan Y. Aboul-Enein

Caffeine 71
Mohnmmad Uppal Zubair, Mahmoud M . A . Hassan,
and Ibrahim A . Al-Meshal

Cocaine Hydrochloride 151

Farid J . Muhtadi and Abdullah A . Al-Badr

Ephedrine Hydrochloride 233

Syed Laik Ali

E stradiol 283
Eugene G . Salole

Guanabenz Acetate 319

Charles M . Shearer

Iodamide 337
Davide Pitrb

V
vi CONTENTS

Lithium Carbonate 367

Henry C . Stober

Maprotiline Hydrochloride 393

Soonhee K . Suh and James B . Smith

Penicillin G, Potassium 427

Joel Kirschbaum

Piroxicam 509
Mladen MihaliC, Hrvoje Hofmun, Josip Kuftinec,
Branku Krile, Vesna Capler, Franjo KujfeZ, and
Nikola BlaieviC

Ranitidine 533
Marijan Hohnjec, Josip Kuftinec, Miuenka Malncir,
Milivoj Skreblin, Franjo KujfeZ, Antun Nagl, and
Nikola BlaZeviC

Strychnine 563
Farid J , Muhtadi and Mohumed S. Hifnawy

Vidarabine 647
Wen-Hai Hong, Tsun Chang, and Robert E. Duly

Zomepirac Sodium 673

Mladen ZiniC, Josip Kuftinec, Hrvoje Hofman,
Franjo KajfeZ, and Zlatko MeiC

PROFILE SUPPLEMENTS

Chloramphenicol 70 1
Abdullah A. Al-Badr and Humeida A. El-Obeid

Lidocaine and Lidocaine Hydrochloride 761

Michael F. Powell

Sodium Nitroprusside 78 1
Auke Bult, Oscar R. Leeuwenkamp, and
Wouter P. van Bennekom

Cumulative Index 793

Erratum to Volume 14 796
 AFFILIATIONS OF
EDITORS AND CONTRIBUTORS

Hussun Y. Ahoul-Enein, King Faisal Hospital, Riyadh, Saudi Arabia

Abdulluh A . Al-Badr, King Saud University, Riyadh, Saudi Arabia
Syed Laik Ali, Zentrallaboratorium Deutscher Apotheker, Eschliorn,
Federal Republic of Germany
Ibrcihim A . Al-Meshul, King Saud University, Riyadh, Saudi Araliia
S . A. Renezru, Wellcome Research Laboratories, Research Triangle
Park, North Carolina
Nikolu Bluieuic', Chromos-Aroma, mirisi, Zagreb, Yugoslavia
G. A . Rrenner, Merck Sharp and Dohnie Research Laboratories, West
Point, Pennsylvania
G. A . Brewer, The Squibb Institute for Medical Research, New Bruns-
wick, New Jersey
Auke Bult, Rijksuniversiteit Utrecht, Utrecht, The Netherlands
Vesnu capler, Podravka Institute, Zagreb, Yugoslavia
Tsun Chung, Warner-Lambert, Morris Plains, New Jersey
Robert E . Duly, Warner-Lambert, Morris Plains, New Jersey
N . J. DeAngelis, Wyeth Laboratories, Philadelphia, Pennsylvania
Humeicla A. El-Obeid, King Saud University, Riyadh, Saudi Arabia
J. E . Fairbrother, E. R. Squibb & Sons, Moreton, England
Klaus Florey, The Squibb Institute for Medical Research, New Bruns-
wick, New Jersey
L. T. Grudy, The United States Pharmacopeia, Rockville, Maryland
Muhmoud M . A . Hussun, King Saud University, Riyadh, Saudi Arabia
Mohumed S. Hvnuwy, King Saud University, Riyadh, Saudi Arabia
Wruoje Hofmun, Podravka Institute, Zagreb, Yugoslavia
Murijun Hohnjec, Podravka Institute, Zagreb, Yugoslavia

vii
viii AFFILIATIONS

Wen-Hai Hong, Warner-Lambert, Morris Plains, New Jersey

E. L. Znman, Lilly Research Laboratories, Indianapolis, Indiana
Frunjo Kujfea, Podravka Institute, Zagreb, Yugoslavia
Joel Kirschbaum, The Squibb Institute for Medical Research, New
Brunswick, New Jersey
Brunku Krile, Podravka Institute, Zagreb, Yugoslavia
Josip Kuftinec, Podravka Institute, Zagreb, Yugoslavia
Oscar R. Leeuwenkamp, Rijksuniversiteit Utrecht, Utrecht, The Nether-
lands
Miljenka Malnar, Podravka Institute, Zagreb, Yugoslavia
David J . Muzzo, Merck Sharp & Dohme, West Point, Pennsylvania
Zlatko MeiC, Institute Ruder Boscovii., Zagreb, Yugoslavia
Mluden Mihalid, Podravka Institute, Zagreb, Yugoslavia
J . A. Mollica, CIBA-GEIGY Corporation, Summit, New Jersey
Farid J . Muhtadi, King Saud University, Riyadh, Saudi Arabia
J . W . Munson, T h e Upjohn Company, Kalamazoo, Michigan
Antun Nagl, University of Zagreb, Zagreb, Yugoslavia
Davide Pitrb, Bracco Industria Chimica, Milan, Italy
Michael F. Powell, Syntex Research, Palo Alto, California
Eugene G. Salole, University of Strathclyde, Glasgow, Scotland
Charles M . Shearer, Wyeth Laboratories, Philadelphia, Pennsylvania
Milivoj Skreblin, Podravka Institute, Zagreb, Yugoslavia
James B. Smith, CIBA-GEIGY Corporation, Suffern, New York
Henry C. Stober, CIBA-GEIGY Corporation, Suffern, New York
Soonhee K. Suh, CIBA-GEIGY Corporation, Suffern, New York
Wouter P. van Bennekom, Rijksuniversiteit Utrecht, Utrecht, The Neth-
erlands
M . D . Yudis, Schering-Plough, Kenilworth, New Jersey
J. E. Zarembo, Revlon Health Care, Tuckahoe, New York
Mladen ZiniC, Podravka Institute, Zagreb, Yugoslavia
Mohammad Uppal Zubair, King Saud University, Riyadh, Saudi Arabia
 PREFACE

Although the official compendia define a drug substance as to identity,

purity, strength, and quality, they normally d o not provide other physical
or chemical data, nor do they list methods of synthesis or pathways of
physical or biological degradation and metabolism. Such information is
scattered through the scientific literature and the files of pharniaceiitical
laboratories.
I perceived a need to supplement the official cornpendial standards of
drug substances with a comprehensive review of such information, and
fifteen years ago the first volume of Anulyticul Profiles of Drug Sub-
stances was published under the auspices of the Pharmaceutical Analy-
sis and Control Section of the APhA Academy of Pharmaceutical Sci-
ences. That we were able to publish one volume per year is a tribute to
the diligence of the editors to solicit articles and even more so to the
enthusiastic response of our authors, an international group associated
with pharmaceutical firms, academic institutions, and cornpendial au-
thorities. I would like to express my sincere gratitude to them for making
this venture possible.
Over the years, we have had queries concerning our publication pol-
icy. Our goal is to cover all drug substances of medical value and, there-
fore, we have welcomed any articles of interest to an individual contrib-
utor. We also have endeavored to solicit profiles of the most useful and
used medicines, but many in this category still need to be profiled.

Kla~isFlorey

ix
This Page Intentionally Left Blank
 AMILORIDE HYDROCHLORIDE

David J. Mazzo

1. Introduction
1.1 Therapeutic Category
1.2 History
2. Description
2.1 Chemical Name, Formula, Molecular Weight
2.2 Definition
2.3 Appearance, Color, Odor
3. Synthesis
4. Physical Properties
4.1 Infrared Spectrum
4.2 Nuclear Magnetic Resonance Spectrum
4.2.1 Proton NMR
4.2.2 Carbon-13 NMR
4.3 U1 traviolet Spectrum
4.4 Mass Spectrum
4.5 Optical Rotation
4.6 Thermoanalytical Behavior
4.6.1 Melting Point
4.6.2 Differential Thermal Analysis Behavior
4.6.3 Thermogravimetric Analysis Behavior
4.7 Solubility
4.8 Crystal Properties
4.9 Hygroscopicity
4.10 Dissociation Constant
5. Methods of Analysis
ANALYTICAL PROFILES O F DHUG SUBSTANCES Copyright 8 1986
VOLUME 15 by the American Pharmaceutical Association
1 A11 rights of reproduction in any form reserved.
2 DAVID J. MAZZO

5.1 Identification Tests

5.1.1 Ultraviolet Spectrophotometry
5.1.2 Infrared Spectroscopy
5.1.3 Elemental Analysis
5.2 Spectrophotometric Analysis
5.2.1 Direct Ultraviolet Spectrophotometry
5.2.2 Ultraviolet Spectrophotometry via Flaw
Injection Analysis
5.2.3 Liquid-Liquid Extraction with Ultraviolet
Spectrophotometry or Spectrofluorimetry
5.3 Chromatographic Analysis
5.3.1 Thin-layer Chromatography
5.3.2 High Performance Liquid Chromatography
5.4 Non-Aqueous Titration
6. Stability - Degradation
6.1 Solid State Stability
6.2 Solution Stability
7. Biopharmaceutics and Metabolism
7.1 Absorption and Bioavailability
7.2 Metabolism
7.3 Pharmacokinetics
8. Determination in Biological Matrices
9. Determination in Pharmaceuticals
9.1 Dissolution Testing
9.2 Assay, Dosage Uniformity and Stability Testing

10. References
AMILORIDE HYDROCHLORIDE 3

1. Introduction
1.1 Therapeutic Category (1)
Amiloride hydrochloride dihydrate, hereafter
referred to as amiloride hydrochloride, is a potas-
sium-conserving diuretic with relatively weak
natriuretic and antihypertensive activity. It is
not an aldosterone antagonist and, therefore, is
effective in the absence of aldosterone. Amilo-
ride hydrochloride is indicated as adjunctive
treatment with thiazide diuretics or other kaliu-
retic-diuretic agents in congestive heart failure
or hypertension to aid in the restoration of nor-
mal serum potassium levels and/or to prevent the
development of hypokalemia. Amiloride hydro-
chloride is available for oral dosing as tablets,
is usually well tolerated, and except for hyperka-
lemia, has had significant adverse effects
reported infrequently (1).
1.2 History
Amiloride hydrochloride, a substituted (pyrazine-
carbonyl)guanidine, was first synthesized in the
Merck, Sharp and Dohme Research Laboratories ( 2 ) .
The first non-patent literature reference to ami.10-
ride appeared in 1966 ( 3 ) and the first structure-
activity relationship study was published in 1967
(4). Amiloride hydrochloride has gained steadily
increasing popularity as a therapeutic drug as
well as a pharmacological tool and its actions and
effects have k e n the subject of at least three
international synposia (5,6,7). A search of
Chemical Abstracts from 1966 to 1985 produced 490
bibliographic citations for works dealing with
amiloride hydrochloride.
2. Description
2.1 Chemical N a m e , Formula, Molecular Weight
The current accepted Chemical Abstracts name for
amiloride hydrochloride (MK-870) is 3,5-diamino-
-
N-(diaminomethylene)-6-~hloropyrazinecarbox-
amide monohydrochloride dihydrate. The CAS
registry no. is 17440-83-4.
4 DAVID J. MAZZO

Other names which have been used for amiloride

hydrochloride include 3,5-diamino-y-(amino-
iminomethyl)-6-chlorpyrazinecarboxamide monohydro-
chloride dihydrate, N-amidino-3,5-diamino-6-
chloropyrazinecarbox%nide monohydrochloride dihy-
drate, N-amidino-3,5-diamino-6-chloropyrazin-
amide Gnohydrochloride dihydrate, 1-( 3,5-diamino-
6-chloropyrazinecarboxy1)guanidine monohydro-
chloride dihydrate, 1-(3,5-diamino-6-chlorpyra-
zinoy1)guanidine monohydrochloride dihydrate, as
well as the monohydrochloride dihydrated salts of
guananprazine, amiprddin and amipramizide

0 NH2
II I
CI C N=CN H2
.HCI 02H20
H2N NH2

2.2 Definition
Amiloride Hydrochloride, unless specifically
stated otherwise, is defined as the crystalline,
monohydrochloride dihydrated salt form of the
conpound. Its tradename is M I W R @ . Amiloride,
when referred to, indicates the free base.
2.3 Appearance, Color, Odor
Amiloride hydrochloride is a yellow to greenish
yellow crystalline powder which is odorless or
practically odorless.
3. Synthesis
Amiloride hydrochloride, essentially a substituted
guanidine, has been prepared through a series of
synthetic steps beginning with methyl cyanoacetate and
urea (4,8-14). The synthetic route is presented in
AMILORIDE HYDROCHLORIDE 5

Figure 1. The starting materials (I and 11) are

reacted in sodium isopropoxide and subsequently nitro-
sated. The product (111) is reduced to the amino
compound (IV), treated with glyoxal to form a pteridine
intermediate (V) and hydrolyzed in base which upon
acidification gives 3-aminopyrazinoic acid (VI). This
substituted pyrazinoic acid is then esterified with
methanol (VII) in the presence of sulfuric acid, chlori-
nated (VIII) and converted to the 5-amlnO ester with
ammonia in DMSO. The product (IX) is then reacted with
guanidine to form amiloride which upon reaction with
hydrochloric acid in water forms amiloride monohydro-
chloride dihydrate (X).
4. Physical Properties
4.1 Infrared Spectrum
The infrared spectrum of amiloride hydrochloride
taken in a KBr pellet is Shawn in Figure 2 (15).
A Digilab Model FTS-15C fourier transform infrared
spectrophotometer was used to acquire the spec-
trum. Frequency assignments for some of the
characteristic bands are listed in Table I.
Table I
Infrared Spectral Assignments
for Amiloiide Hydrochioride
Frequency (cm-l) Assignment
3250-3500 N-H stretch (NH )
3150 N-H stretch (NHf
1680 C=O stretch
1640 H2 deformation mode
1600 H2 deformation mode
1240 N-(C&) Stretch
770 C-H out-of-plane mode
The infrared spectrum of amiloride hydrochloride
taken in a mineral oil mull is sham in Figure 3
(15)-
 W a J
4 + a
*d
!4
i;
I 0
41 P
0
44
-0-0
X t $1 I a
a
a
h
.c
5
I tfiu
-+
t 0
U
+ aJ
u
1
0
f
z
Id- +
rB
z--0-2
I" u
+
220"
3" u
0--0--0
6
85.0 I I I I I I 1 I 1 I I I I 1 I

42.:

\
C J I I I I I I I I I 1 1 1 1 I

3500 3300 3100 2900 2700 2500 2300 2100 1900 1700 1500 1300 1100 900 700
I 500
1

Figure 2. I n f r a r e d spectrum of a m i l o r i d e h y d r o c h l o r i d e t a k e n i n a KBr p e l l e t .

 9c I I 1 I I I I I I I 1 1 I I

a
0
c
0
.-
c
e

E
g!
2
I-
s
4!
d
Frequency (cm-’ 1
Figure 3 . I n f r a r e d spectrum of a m i l o r i d e h y d r o c h l o r i d e t a k e n i n a m i n e r a l o i l mull.
AMILORIDE HYDROCHLORIDE

4.2 Nuclear Magnetic Resonance Spectrum (16)

4.2.1 Proton NMR Spectrum
The proton magnetic resonance spectrum of
amiloride hydrochloride was obtained using
a JEOL(USA) C-60HL spectrometer. The
spectrum was acquired from a 1.0 M drug
solution in fully deuterated dimethylsul-
foxide (d6-DMSO). The spectrum is shown
in Figure 4 and the spectral assignments
are listed in Table I1 (17).
Table I1
Proton NMR Spectral Assignments
for Amiloride Hydrochloride
Relative No.
Integral (mm) Protons Assignments
3.8 52 4.4(b) H20
7.4
48.5 4.1 Aromatic -NH2
7.7 NH2
9.8

57(c) 4.9
NH2
11.1
N -H
Notes:

(a) All protons are bound to nitrogen or oxygen

(H20), therefore chemical shift will be strongly
dependent upon sample concentration, temperature
and/or solution pH.

(b) This si.gnal includes a small contribution due to

water originally present in the d6-DMS0.

(C) The total integral resulting from these signals is

reported since the appearance of the spectrum
suggests intermediate kinetic exchange between the
two types of sites.
 (CH314-S

1 1 I I I I I 1 1 I

8.0 7.0 6.0 5.0 4.0 30 2.0 1.o 0

Figure 4 . Proton NMR spectrum of amiloride hydrochloride.

AMILORIDE HYDROCHLORIDE 11

4.2.2 $3 NMR Spectrum

The CI3 NMR spectrum shown in Figure 5
was obtained on a Varian Associates CFT-20
spectrometer using a 1.0 M amiloride
hydroytjloride solution in d6-DMS0 (17).
The C spectral assignments are listed
in Table 111.

Table I11
Cl3 NMR Spectral Assignments for Amiloride Hydrochloride

0 Assignments

109.3 c2
119.9 ‘6
154.2 c3
155.3 C9
155.9 c5
165.3 c7

0 NH2
II I
CI
9
HCI 2H20
H2N NH2

Both the proton and Cl3 NMR spectra are

consistent with the amiloride hydrochloride
structure.
 c9

6-DMSO
(CH 3)4Si

I I I I 1

200 150 100 50 0

F i g u r e 5. CI3 NMR spectrum of a m i l o r i d e h y d r o c h l o r i d e .

AMILORIDE HYDROCHLORIDE 13

4.3 Ultraviolet Spectrum

The ultraviolet absorption spectrum of amiloride
hydrochloride in 0.01 N aqueous hydrochloric acid
is characterized by maxima at approximately 212 mm,
285 nm and 362 MI.
The absorbance in absorban?$ units of a 1% drug
solution in a 1 cm cell (A1 ) is 642,
555 and 617 for the wavelenggs of 212 nm, 285 nm
and 362 nm, respectively. The W spectrum shown
in Figure 6 was obtained using a Cary Model 18
ultraviolet spectrophotomter (18).
4.4 Mass Spectrum
Figure 7 is a depiction of the low resolution mass
spectrum of amiloride hydrochloride obtained using
a LKB nrodel 9000 mass spectrometer in the electron
impact mode with an ionization energy of 70 eV and
a probe temperature sufficiently high to produce
sanple vaporization (19). The spectrum is domi-
nated by the molecular ions of the free base (m/e
= 229, 231). Masses of m/e = 43, 187, 189, 171,
173 and 212, 214 characterize the guanidino substi-
tuent. Loss of the entire guanidino group leads
to m/e = 144, 146 and m/e = 86.
The remaining less abundant ions are most likely
due to losses of CH from the pyrazine ring,
i?
although the loss o CO cannot be totally excluded
on the basis of low resolution spectra. Figure 8
schematically presents the fragmentation of amilo-
ride hydrochloride.
4 - 5 Optical Rotation
Amiloride hydrochloride is not optically active.
 14 DAVID J. MAZZO

8
c
0
e
s
n
a

I I I I
200 250 300 350 L

Wavelength (nm)
Figure 6. U l t r a v i o l e t spectrum of a s o l u t i o n of a m i l o r i d e
h y d r o c h l o r i d e i n 0.1N HC1.

229

loo] 43

40L L
60

20

0
37

40
Y’

Figure 7.
53

60 80
86

09
101

100
116

120
144

143

140
I

160
171

180

Low r e s o l u t i o n mass spectrum of a m i l o r i d e

200

h y d r o c h l o r i d e t a k e n i n t h e e l e c t r o n impact
220
240

mode.
 R I t u
X I I
z z z
\ /
-0
I
X U
z *
Q
I Q “ E
o=y E
0;;
a 0
+ R1
16 DAVID J. MAZZO

4.6 Thermoanalytical Behavior

4.6.1 Melting Point
The melting point (with decomposition) of
anhydrous amiloride hydrochloride is
293.5OC. The dihydrate melts with
decomposition at approximately 288OC (20).
4.6.2 Differential Thermal Analysis
Behavior ( 1 7 )

The differential thermal analysis (DTA)

curve of amiloride hydrochloride is charac-
terized by a water loss endotherm with a
peak teprature of ca. 136OC. An endo-
therm-exotherm combination is observed at
ca. 300OC. Actual peak temperatures are
298OC and 304OC for the endotherm and exo-
them respectively. This thermal event
corresponds to a melting plus deconposition
4.6.3 Thermogravimetric Analysis
Behavior (17)
Thermogravimetric analysis of amiloride
hydrochloride indicates no weight loss
- -
until 90OC. From 90°C to -17OoC,
there is an approximately 12% weight loss
due to dehydration of the dihydrate. No
further weight loss occurs until
-
sublimation/deconposition begins at 280OC.
4.7 Solubility
The solubility of amiloride hydrochloride in a
variety of solvents at room temperature (- 25OC)
is presented in Table IV ( 1 8 ) . Note that these
solubilities are stated in terms of the current
U.S.P. definitions (21).
AMILORIDE HYDROCHLORIDE 17

Table IV
Solubility of Ami1ori.de Hydrochloride at Room Temperature

Solvent Solubi1ity
Acetone Practically insoluble
Chloroform Practically insoluble
Diethylether Practically insoluble
Dimethylsulfoxide Freely soluble
Ethanol Very slightly soluble
Ethylacetate Practically insoluble
Isopropanol Slightly soluble
Methanol Sparingly soluble
Water Slightly soluble

The solubility of amiloride hydrochloride in water

is typical of an organic base with limited aqueous
solubility and increases with a decrease in pH
(Table V) ( 1 8 ) .
Table V
Aqueous Solubility of Amiloride Hydrochloride
As a Function of pH

PH Solubility (mg/mL)
4.8 5.2
7.6 5.1
9.4 0.5
10.0 0.3

4.8 Crystal Properties

Amiloride hydrochloride dihydrate exists as a
crystalline powder. Variations encountered in the
X-ray powder diffraction pattern suggest the
existence of at least two polymorphic forms ( 2 2 ) .
Polymrphism of amiloride hydrochloride has not
been detected by other physical and/or chemical
measurement techniques. Figures 9 and 10 show the
X-ray powder diffraction patterns for the two
18 DAVID J. MAZZO

XI02
5.00

4.50

4.00

3.50

3.00

2.50

2.00

1.50

1 .OO

0.50

0.0 5.0 10.6 15.0 20.0 25.0 30.0 35.0 40.0

Figure 9. X-ray powder d i f f r a c t i o n p a t t e r n of polymorph A

of a m i l o r i d e h y d r o c h l o r i d e .

XI02
3.50

3.00

2.50

2.00

1.50

1 .OO

0.50

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0

F i g u r e 10. X-ray powder d i f f r a c t i o n p a t t e r n of polymorph B
of a m i l o r i d e h y d r o c h l o r i d e .
AMILORIDE HYDROCHLORIDE 19

polymorphs (A and B) seen to date. The patterns

were obtained using a Phillips Electronics model
APD3720 powder diffractometer using copper Ka
radiation.
Tables VI and VII list the interplanar distances
and the relative intensities of the major lines in
the X-ray powder diffraction patterns of
polymorphs A and B, respectively.
Table VI
X-Ray Powder Diffraction of
Amiloride Hydrochloride Polymorph A
Peak Angle (28) I/Imx ( % )

7.6725 29.03
8.9150 22.55
10.2425 15.06
12.8400 2.14
15.3800 30.02
15.6100 14.71
17.1975 16.89
17.8950 7.76
19.3425 4.41
20* 0200 4.22
20.5925 6.08
23.1600 20.85
24.9000 5.86
25.6375 15.42
26.0275 23.43
26.4375 23.87
27.0325 5.86
28.0650 100.0
29.1425 7.51
29.8300 14.02
30.4700 11.11
31.0600 10.22
32.4275 9.08
33.3500 5.64
34.8350 20.44
35.4775 12.04
36.4200 4.61
37.9675 5.01
20 DAVID J. MAZZO

Table V I I
X-Ray Pawder Diffraction of
Ami lor ide Hydrochloride P o l p r p h B

Peak Angle (28) I/Imax ( % )

6.7350 6.49
7.5025 31.95
8 -8375 14.36
9.4075 100.00
10.2150 7.13
12.5850 5.57
13.6050 5.57
15.0775 59.32
15.3400 14.83
16.1350 73.47
17.1500 10.43
18.6225 11.67
18.9125 17.84
19.7450 25.31
20.4850 5.87
22 * 1000 7.47
22.7050 29.20
23.1300 13.43
24.2150 14.83
25.4975 23.47
26.2200 63.21
26.5000 65.20
27.1625 14.36
28.0425 44.17
28.4525 42.53
29.7150 40.14
30.4400 14.36
31.0225 12.10
31.7100 7.13
32.4225 34.09
i 33.2050 19.45
34.5275 37.81
35.2825 12.98
36.2325 7.47
36.4600 3.95
37.7200 6.17
38.7850 10.84
AMILORIDE HYDROCHLORIDE 21

4.9 Hygroscopicity
The monohydrochloride salt of amiloride forms a
stable dihydrate and amiloride hydrochloride,
unless stated otherwise, is supplied as such.
Amiloride monohydrochloride dihydrate may be
converted to the anhydrous form of the sa t by
drying at 100°C at pressures 6.6 x lo-’ atm
for 3 hours (23). No other stable hydrates of
amiloride hydrochloride have been reported (24).
4.10 Dissociation Constant
The dissociation constant of amiloride
hydrochloride as derived from aqueous titration
(18) indicates that amiloride is a moderately
strong organic base with a pKa of approximately
8.7 at 25OC (amidino nitrogen).
The pKa of amiloride hydrochloride and, in fact,
m n y pyrazinylguanidine derivatives has also &en
determined using gas-phase proton affinities,
enthalpies of solution and semi-empirical
calculations (25, 26). These theoretically
derived pKa values agree well with the
experimentally determined value of pKa = 8.7.
5. Methods Of Analysis
5.1 Identification Tests
5.1.1 Ultraviolet Spectrophotometry
Ultraviolet spectrophotometry is used to
identify amiloride hydrochloride. A solu-
tion in 0.1 N HC1 scanned from 200 nm to
400 nm qualitatively exhibits the same
absorbance characteristics at identical
wavelengths as does a similarly prepared
and concomitantly measured solution of an
amiloride hydrochloride standard. Quantita-
tively, equimlar sample and standard solu-
(Amx)
-
tions will exhibit ahorbances at 360 nm
which differ by no more than 3%.
22 DAVID J. MAZZO

5.1.2 Infrared Spectroscopy

Infrared spectroscopy may also be used to
identify amiloride hydrochloride. The
infrared absorption spectrum prepared as a
potassium bromide disk or a mineral oil
dispersion compares qualitatively (with
maxima only at the same frequencies) to the
spectrum of a similarly prepared amiloride
hydrochloride standard.
5.1.3 Elemental Analysis
Elemental analysis has been employed to
identify amiloride hydrochloride. The
results of a weight percent determination
of carbon, nitrogen, hydrogen and chloride
are compared to the respective theoretical
values of 28.85%, 32.45%, 4.34% and
23.47%. Amiloride hydrochloride will
respond positively to the test for chloride
described in the United States Pharmacopeia
(27).
5.2 Spectrophotometric Analysis
5.2.1 Direct Ultraviolet Spectrophotometry
Amiloride hydrochloride exhibits a W
absorption band near 360 nm attributed to
the substituted pyrazine ring system. This
absorption is the basis for the quantita-
tive determination of the drug. Assay of
the conpound is based on a conparison of
the net absorbance at 360 nm of a sample in
0.1 N HC1 with a standard in 0.1 N HC1 of
known concentration. The net absorbance is
calculated by subtracting the drug free
matrix contribution to absorbance at the
wavelength of determination from the absor-
bance of the drug solution at the same wave-
length.
AMILORIDE HYDROCHLORIDE 23

5.2.2 Ultraviolet Spectrophotometry Via Flow

Injection Analysis
Ultraviolet absorbance at 360 nm is also
used as the detection mode for Amiloride
hydrochloride determinations by flow injec-
tion analysis (FIA) (28). In the case of
FIA assay, amiloride hydrochloride sample
and standard solutions are periodically
injected into a flowing stream. The result-
ing changes in W absorbance at 360 nm of
the stream are measured relative to the
drug free stream. Calculation of the con-
centration of amiloride is made in an iden-
tical fashion as direct W spectrophoto-
metry.
5.2.3 Liquid-Liquid Extraction with Ultra-
violet Spectrophotometry or Spectro-
fluorimetry
Amiloride hydrochloride may be determined
in the presence of its degradation products
by a liquid-liquid extraction technique
followed by quantitation by ultraviolet
spectrophotometry or spectrofluorimetry
(18). An alkaline aqueous solution of
amiloride hydrochloride is extracted with
tributyl phosphate. Amiloride is parti-
tioned into the organic layer while its
degradates remain in the aqueous phase.
Amiloride is then determined by ultraviolet
spectrophotometry at 360 nm or fluorimetry
with an excitation wavelength of ca. 360 nm
and an emission wavelength of ca. 420 nm.
-
Sensitivity is 0.2 ppm.
5.3 Chromatographic Analysis
5.3.1 Thin Layer Chromatography
Normal-phase thin layer chromatography on
silica gel using one of two developing
solvent systems has been employed for amil-
oride hydrochloride. In the first system,
a developing solvent of 10% n-propanol in
chloroform is used to develop a spot result-
ing from 5 pL of a 1% aqueous solution of
24 DAVID J. MAZZO

system is approximately zero (R -

the drug. Rf for the analyte in this
0).
Detection is by W absorbance at 254 nm or
360 nm (most sensitive). A sensitivity of
0.01% has been reported. In the second TLC
system, 4 parts 3 N aqueous a m n i u m hydro-
xide are mixed with 30 parts of tetrahydro-
furan to form the developing solvent (23).
A 1 pL spot of a 0.1% aqueous solution of
the drug is developed and detected by W
absorbance at 254 nm or 360 nm. Rf for
the analyte is approximately 0.7.
Sensitivity under these conditions is in
the range of 0.1% to 0.5% of the analyte
concentration.
5.3.2 High Performance Liquid Chromatography
Reversed-phase HPLC is routinely used to
determine amiloride hydrochloride (29).
The method employs an ES Industries C-2(300
mm x 4.6 mm i.d., 10 pm particle size) HPLC
column operated at ambient room temperature
(- 25OC). A mobile phase consisting of 85%
aqueous 0.01M sodium hexane sulfonate (pH =
3.0) in acetonitrile is used to elute amilo-
ride. Flow rate is 2.0 mL/min and detec-
tion is by W absorbance at 280 nm. Under
-
these conditions, amiloride elutes in less
than 8 minutes (k' 2). Typically 20 pL
injections of an approximately 200 pg/mL
drug solution are made.
5.4 Non-Aqueous Titration
Amiloride hydrochloride can be determined by non-
aqueous titration with perchloric acid (23). The
assay involves dissolution of an appropriate
amount (- 450 mg) of amiloride hydrochloride in
100 mL of glacial acetic acid to which is then
added 10 mL of mercuric acetate, 15 mL of dioxane
and an appropriate amount of crystal violet indica-
tor. The well mixed solution is titrated with
0.1N perchloric acid to a blue endpoint. Assay
results must be corrected for a blank titration.
Each equivalent of perchloric acid is equivalent
to 26.61 mg of amiloride hydrochloride.
AMILORIDE HYDROCHLORIDE 25

6. Stability - Degradation
6.1 Solid State Stability
Amiloride hydrochloride is a stable compound at
room temperature in the solid state, i.e. no signi-
ficant degradation has been observed in samples
exposed to no excessive humidity for seven years.
Some darkening of amiloride hydrochloride powder
has been observed upon exposure to intense ultra-
violet light (24 times the strength of direct sun-
light) but no degradation products were detected
by thin layer chromatography, even after 7 days of
exposure (18). Elevated temperatures as high as
100°C for 1 week in the absence of excessive
humidity do not produce degradation. Only pro-
longed (> 1 week) exposure to very high humidity
(> 90% RH) and temperatures greater than 6OoC will
produce significant degradation in the solid state.
6.2 Solution Stability

Aqueous solutions of amiloride hydrochloride are

stable at usual ambient temperatures. A study of
the stability of amiloride hydrochloride in
aqueous solution at elevated temperatures at
various pH levels resulted in the identification
of three degradation products (Figure 11). The
relative amounts of the three degradates vary with
pH. At pH < 1, compound I dominates, at pH -5
both I1 and I11 are present and in alkaline solu-
tion (pH > 1 3 ) , compound 111 predominates. All
three potential degradation products are acidic in
comparison to the parent compound. The degrada-
tion of amiloride hydrochloride in alkaline solu-
tion (pH > 13) follows first-order degradation
kinetics (18). The chemical transformation of
arniloride hydrochloride in solution formulation
with other pharmaceutical agents and excipients
has been shown to be more conplex than that
observed in simple aqueous solution (30).
26 DAVID J. MAZZO

’ 0 0
II ,NH2

NH2
2

It

L“‘x;I&c’xlI
pH> I3
OH-

H2N
0
II
c-o-

NH2
HeN
0
II
C-OH
NH2

m
F i g u r e 11. S o l u t i o n d e g r a d a t i o n p r o d u c t s of
.
a m i l o ri d e h y d r o c h l o r i d e
AMILORIDE HYDROCHLORIDE 27

7. Biopharmaceutics and Metabolism

7.1 Absorption and Bioavailability
It has been shown, using radiolabelled (CI4)
amiloride hydrochloride, that approximately 50% of
an oral dose administered to man is absorbed from
the gastrointestinal tract ( 3 1 , 3 2 ) . The remainder
of the dose is unabsorbed and can be found in the
feces (31-34). Human bioavailability studies
comparing 5 mg tablets of amiloride hydrochloride
to an aqueous reference solution showed that the
pharmaceutical dosage form was fully bioequivalent
( 3 3 ) . Absorption of amiloride hydrochloride is
reduced when administered with food ( 3 3 , 2 5 ) .
7.2 Metabolism
Amiloride hydrochloride which has been absorbed in
man is excreted without metabolism in the urine
( 3 1 , 3 3 , 3 4 , 3 6 ) . No metabolic products have been
detected and/or identified. Since the drug is not
metabolized it probably can be administered to
patients with hepatic dysfunction, providing that
renal excretory function is normal ( 3 4 ) . Amilo-
ride hydrochloride is cleared from the body by
tubular transport (5,7,31-34,36) and, in combina-
tion with hydrochlorothiazide, does not alter the
normal renal potassium excretion or the rate of
urinary variable excretion ( 3 7 ) .
7.3 Pharmacokinetics
Amiloride hydrochloride is rapidly absorbed from
the gastrointestinal tract (31,32,38-40). Onset
of the physiological effect of the drug in man is
usually noted within two hours ( 3 4 , 3 6 ) with peak
serum levels being achieved in between 3 and 4
hours (31,321. Typical pharmacokinetic half-life
values (T/2) range between 6 and 10 hours
( 31,32,36!. Considerable prolongation of the half-
life has been noted in patients with chronic renal
failure ( 3 3 , 3 4 , 3 6 , 3 9 ) . Effects of the drug gen-
erally subside within 24 hours resulting in
urinary levels of less than 0.5% of the adminis-
tered dose ( 3 1 , 3 6 ) . Urinary amiloride concentra-
tions range from 4 pg/mL to 30 pg/mL, during
diuresis ( 3 1 , 3 2 ) with peak renal clearance of
28 DAVID J. MAZZO

amiloride ranging from 400 mL/min to 600 mL/min

(33). A dose-response relationship has been
observed in man (33,34,36). Progressively increas-
ing effects of single doses have been noted from 1
mg to 40 mg with a plateau being reached above 40
mg -
8. Determination in Biological Matrices
Several techniques have been used to determine amilor-
ide in biological fluids. Among them, liquid scintilla-
tion counting for radiolabelled compound, liquid-liquid
extraction with measurement of ultraviolet absorbance
or fluorescence, thin-layer chromatography with W or
fluorescence detection and high performance liquid
chromatography (HPLC)with fluorescence detection have
been the most widely used.
Liquid scintillation counting of C14 labelled
ami1ori.de in urine, serum, plasma, tissue and feces has
been performed routinely (31,32,41) usually with an
internal standard used for quantitation of the analyte.
Liquid-liquid extraction of amiloride from serum,
plasma and/or urine has been performed with a 7%
saturated aqueous sodium carbonate solution in ethyl
acetate (1 part drug containing fluid to 27 parts
extracting solution). The analyte is back-extracted
from the ethyl acetate phase into 0.1 N HC1 and then
determined by spectrofluorimetry (Aexcite = 365 nm,
'emit. = 420 nm) (40). In matrices containing
minimal interferents, ultraviolet absorbance at 360 nm
may also be used.
Normal-phase thin-layer chromatography (TLC)on silica
has also been employed for the determination of
amiloride from biological fluids (31,41,42). General-
ly, an extraction technique similar to that described
above is employed for drug isolation prior to TLC. A
common developing solvent consists of butanol-acetic
acid-water (100:27:73) (41). Other extraction tech-
niques and developing solvents have been reported
(42). Detection of TLC spots is accomplished by
fluorescence with long wavelength W excitation and
emission at ca. 425 nm.
AMILORIDE HYDROCHLORIDE 29

Finally, reversed-phase high performance liquid chroma-

tography with fluorescence detection has become popular
for the determination of amiloride in biological
fluids. Two HPLC systems have been reported (43,44)
with large numbers of samples being processed by one of
these systems ( 4 4 ) . A C18 HPLC column (300 mm x 3.9
mm i-d., 10 pm particle size) is employed with a mobile
-
phase of 60:40 [0.1M NaP04(pH = 4.0)l:CH OH flawing
at 1.0 mL/min. Column temperature is 35OC and detec-
tion is by 'fluorescence with an excitation wavelength
of 368 nm and an emission wavelength of 417 nm. Under
-
these conditions, amiloride elutes in 6 minutes.
Quantitation is performed using an internal standard,
3,5-diamino-N-(aminoiminomethyl)-6-fluoropyrazinecarbox-
amide.
9. Determination in Pharmaceuticals
9.1 Dissolution Testing
The determination of amiloride hydrochloride in
samples resulting from dissolution testing of tab-
lets containing the drug can be accomplished by
ultraviolet absorbance at 360 nm. Either direct
W spectrophotometry or W spectrophotometry vis-
a-vis flow injection analysis may be used (45).
9.2 Assay, Dosage Uniformity and Stability Testing
High performance liquid chromatography is the
technique of choice for the determination of
amiloride hydrochloride in tablets for release
and/or stability purposes although ultraviolet
spectrophotometry may be used for the determina-
tion of dosage uniformity. Reversed-phase HPLC
with W detection at 360 nm has been performed
using a c18 HPLC colum (300 mm x 3.9 mn i.d.,
10 pm particle size) with a mobile phase of 25%
methanol in 0.05 M phosphate buffer (pH = 3.0).
Flow rate was 1.0 mL/min (30). Recently, a fast-
HPLC method was developed (46) which employs a
c18 mini-colurm (50 mm x 4.6 mm i.d., 5 pm
particle size). Using a mobile phase of 20%
methanol in 0.02M phosphate buffer pH = 2 at a
30 DAVID J. MAZZO

flow rate of 4.0 mL/min amiloride is eluted in

-
approximately 0.5 minutes (k' 1). Detection is
again by W absorbance at 360 nm. This method,
because of its speed, has also been used routinely
for dosage uniformity samples.

Acknowledgement
Special thanks go to Mrs. Elizabeth Moyer for typing
the manuscript. Thanks are also in order to Mr. James Ryan
and Dr. James McCauley for obtaining the IR spectra and X-
ray diffraction patterns, respectively. The assistance of
Ms. Florence Berg for literature searching and Mr. William
Shearin for background information retrieval is also
acknowledged. Finally, sincere gratitude is expressed to
Dr. Gerald Brenner for his review of the manuscript.
AMILORIDE HYDROCHLORIDE 31

10. References
Physician's Desk Reference, 36th edition,
Charles E. Baker, Jr., Publisher, Medical
Economics Company, Inc., Oradell, NJ, U.S.A.,
1982, w. 1268.

Cragoe, E. J., Jr., Department of Medicinal

Chemistry, Merck, Sharp and Dohme Research
Laboratories, West Point, PA, U.S.A., internal
communication.
Glitzer, M.S.; Steelman, S. L.; Nature (London)
212 (1966), 191.
Cragoe, E. J., Jr.,; Woltersdorf, O.W., Jr.;
Bicking, J. B.; Kwong, S. F.; Jones, J. H.; J.
Med. Chem. 10 (1967), 66-75.
Amiloride and Epithelial Sodium Transport, A.
W. Cuthbert, G. M. Fanelli, Jr. and A. Scriabine,
Editors, Urban and Schwarzenberg, Inc., Baltimore,
MD, U.S.A., 1979.

International Symposium: Diuresis,

Kaliuresis, and Hypertension, J. E. Salvioli,
Chairman, Biomedical Information Corporation
Publications, Inc., New York, NY, U.S.A., 1981.
Hyams, D. E., in Arrhythmias and Myocardial
Infarction: The Role of Potassium, C. W o o d
and W. Somerville, Editors, Academic Press,
London, England, U.K., 1981, pp. 65-73.
Cragoe, E. J., Jr., Belgium Patent 639,386 (to
Merck & Corrpany, Rahway, NJ, U.S.A., 1964).
Cragoe, E. J., Jr., United States of America
Patent 3,313,813 (to Merck & Campany, Rahway, NJ,
U.S.A.), 1967.
(10) Cragoe, E. J., Jr., Canada Patents 765823 and
7644963 (to Merck & Co., Rahway, W , U.S.A.), 1967.
(11) Cragoe, E. J., Jr., England Patent 1066855 (to
Merck & Co., Rahway, W, U.S.A.), 1967.
32 DAVID J. MAZZO

(12) Cragoe, E. J., Jr., France Patent 1563612 (to

Merck & Co., Rahway, NJ, U.S.A.), 1969.
(13) Cragoe, E. J., Jr., Federal Republic of Germany
Patents 1470053.9 and 1795438.0 (to Merck & Co.,
Rahway, NJ, U.S.A.), 1963.
(14) Cragoe, E. J., Jr., Japan Patents 498389, 502839
and 508194 (to Merck & Co., Rahway, NJ, U.S.A. ),
1963.

(15) Ryan, J. A., Department of Pharmaceutical Research

and Development, Merck, Sharp and Dohme Research
Laboratories, West Point, PA, U.S.A., personal
communication.
(16) Smith, R. L.; COchran, D. W.; Gund, P.; Cragoe,
E. J., Jr.; J. Am. Chem. SOC. 101 (1979), 191.
(17) Hitchings, W. S. and O'Brien, M. J., Department of
Control and Manufacturing Data, Merck, Sharp and
D0hn-e Research Laboratories, West Point, PA,
U.S.A., personal communication.
(18) Rogers, D. H e , Department of Pharmaceutical
Research and Development, Merck, Sharp and Dohme
Research Laboratories, West Point, PA, U.S.A.,
internal communication.
(19) Smith, J. L., Department of Analytical Natural
Product Chemistry, Merck, Sharp and Dohme
Research Laboratories, Rahway, NJ, U.S.A.,
personal communication.
(20) The Merck Index, 10th Edition, Martha
Windholz, Editor, Merck and Company, Rahway, NJ,
U.S.A, 1983, pg. 403.
(21) The United State Pharmacopeia, 21st Revision,
Mack Publishing Company, Easton, PA, U.S.A., 1984,
pg. 7.

(22) McCauley, J. A., Department of Analytical

Research, Merck, Sharp and Dohme Research
Laboratories, Rahway, NJ, U.S.A., personal
communication.
AMILORIDE HYDROCHLORIDE 33

The United States Pharmacopeia, 21st Revision,

Mack Publishing Company, Easton, PA, U.S.A., 1984,
pg. 35.

Giuliano, J., Department of Chemical Data, Merck

Sharp and Dohme Research Laboratories, Rahway, NJ,
U.S.A., personal comnunication.
Aue, D. H.; Webb, H. M.; Bowers, M. T.; Liotta,
C. L.; Alexander, C. J.; Hopkins, H. P., Jr.; J.
Am. Chm. SOC., 98(1976), 854-856.

Bock, M. G.; Schlegel, H. B.; Smith, G. M.; J.

Org. Chem. 46(9) (1981), 1925-1927.
The United States Pharmacopeia, 21st Revision,
Mack Publishing Company,
- . Easton, PA, U.S.A., 1984,
pg. 1185.

Bigley, F. P., Department of Pharmaceutical

Research and Development, Merck, Sharp and Dohme
Research Laboratories, West Point, PA, U.S.A.,
personal camrmnication.
Martin, M., Department of Chemical Manufacturing,
Quality Control, Merck, Sharp and D o h , Ltd.,
Ballydine, Kilsheelan, Clonmel, County Tipperary,
Ireland, personal communication.
Roman, R., Department of Pharmaceutical Research
and Development, Merck, Sharp and Dohme Research
Laboratories, West Point, PA, U.S.A., internal
communication.
Weiss, P.; Hersey, R. M.; Dujovne, C. A.;
Bianchine, J. R.; Clin. Pharmacol. Ther. lO(3)
(1969), 401-406.
Smith, A. J.; Smith, R. N.; Brit. J. Pharmacol.
48(4) (1973), 646-649.
Davies, R. O., in International SyIposium:
Diuresis, Kaliuresis and Hypertension, J. E.
Salvioli, Chairmn, Biomedical Information
Corporation Publications, Inc., New York, NY,
U.S.A., 1981, pp. 43-69.
34 DAVID J. MAZZO

(34) Vidt, D. G.; Pharmacotherapy l(3) (1981), 179-187.

(35) Hyams, D. E., in Arrhythmias and Myocardial
Infarction: The Role of Potassium, C. Wood
and W. Somerville, Editors, Academic Press,
london, England, U.K., 1981, pg. 68.
(36) Laragh, J. H.; Curr. Ther. Res. 32(2) (1982), 173-
188.

(37) Leary, W. P.; Reyes, A. J.; Curr. Ther. Res. 32(3)

( 1982), 432-438.

(38) Baba, W. I.,; Lant, A. F.; Smith, A. J.;

Townshend, M. M.; Wilson, G. M.; Clin. Pharmacol.
Ther. 9L (1968), 318-327.
(39) George, C. F.; Brit. J. Pharmacol 9 (1980), 94-95.
(40) Lant, A. F.; Smith. A. J.; Wilson, G. M.; Clin.
Pharmacol. Ther., 10 (1969), 50-63.
(41) Baer, J. E.; Jones, C. B.; Spitzer, S. A.; RUSSO,
H. F.; J. Pharmacol. Exp. Ther. 157(2) (1967), 472-
485.
(42) Reuter, K.; Knauf, H.; Mutschler, E.; J.
Chromatogr. 233(1982), 432-436.
(43) Yip, M. S.; COates, P. E.; Thressen, J. J.; J.
Chromatogr. 307 (1984), 343-350.
(44) Vincek, W. C.; Hessey, G. A., 111; Constanzer, M.
L.; Bayne M. W.; Pharm. Res. 3 (1985), 143-145.
(45) Konieczny, J. M.; Department of Pharmaceutical
Research and Development, Merck, Sharp and Dohme
Research Laboratories, West Point, PA, U.S.A.,
personal cmnication.
(46) Mazzo, D. J.; Department of Pharmaceutical
Research and Development, Merck Sharp and Dohme
Research Laboratories, West Point, PA, U.S.A.
Chemical Abstracts was searched for amiloride
hydrochloride citations from 1966 to 1985.
 AMINOGLUTETHIMIDE

HASSAN Y ABOUL-ENE I N

1 DESCRIPTION

1.1 Introduction

1.2 Nomencl a t u r e

1.2.1 Chemical Names

1.2.2 Generic Names

1.2.3 Trade Names

1.3 Formulae

1.3.1 Empirical

1.3.2 Structural

1.3.3 Chemical A b s t r a c t R e g i s t r y Number

1.4 M o l e c u l a r Weight

1.5 Elemental Composition

1.6 Appearance, Color, Odor and Taste

1.7 p~ (0.1% S o l u t i o n )

ANALYTICAL PROFILES OF DRUG SUBSTANCES Copyright 0 I986

VOLUME 15 by the American Pharmaceutical Association
35 All rights of reproduction in any form reserved.
36 HASSAN Y. ABOUL-ENEIN

2 PHYSICAL PROPERTIES

2.1 M e l t i n g Range

2.2 Solubility

2.3 D i p o l e Moment

2.4 O p t i c a l A c t i v i t y and Absolute C o n f i g u r a t i o n

2.5 Spectral Properties

2.5.1 U1t r a v i o l e t Spectrum

2.5.2 I n f r a r e d Spectrum

2.5.3 Nuclear Magnetic Resonance

2.5.3.1 PMR
2.5.3.2 1% NMR

2.5.4. Mass Spectrum

3 SYNTHESIS

4 METABOLISM AND PHARMACOKINETICS

5 METHODS OF ANALYSIS

5.1 T i t r i m e t r i c Non-Aqueous A n a l y s i s
5.2 Spectrophotometic A n a l y s i s

5.3 Chromatographic A n a l y s i s

5.3.1 Paper Chromatography

5.3.2 Thi n-Layer Chromatography (TLC)

5.3.3 Gas-Liquid Chromatography (GLC)

5.3.4 High-Performance L i q u i d (HPLC)

6 REFERENCES

7 ACKNOWLEDGEMENT
AMINOGLUTETHIMIDE 37

ANALYTICAL PROFILE
1. Description

1.1 Introduction:

Aminoglutethimide was i n i t i a l l y developed as an

a n t i c o n v u l s a n t i n t h e 1950's b u t was l a t e r withdrawn

from c l i n i c a l use a f t e r r e p o r t s of adrenal i n s u f -

f i c i e n c y (1). It was subsequently shown t o suppress

adrenal s t e r o i d synthesis by i n h i b i t i n g t h e demolase

enzyme system which i s responsible f o r t h e conver-

sion o f cholesterol t o a5 -pregnenolone (2). It

has been used i n t h e treatment o f a d r e n o c o r t i c a l

tumours and Cushing's syndrome ( 3 ) .

Aminoglutethimide i n h i b i t s s t e r o i d aromatase enzyme

i n v o l v e d i n t h e b i o s y n t h e s i s o f estrogens ( 4 ) , e.g.

conversion o f androstenedione t o esterone. It i s

c u r r e n t l y used as an e f f e c t i v e agent f o r t h e t r e a t -

ment o f advanced b r e a s t cancer i n post-menopausal

women (5,6,7).

1.2 Nomenclature:

1.2.1 Chemical Names :

CY, - (p-Aminophenyl) -a- Ethylglutarimide

2 - (p-Aminophenyl) - 2 - Ethy g l u t a r i m i d e

3 - (4-Aminopheny1)- 3 - Ethy - 2,6 -

p i p e r i d i nedi one
38 HASSAN Y. ABOUL-ENEIN

3 - Ethyl - 3 - ( p . aminophenyl) - 2,6 -

d i oxopi p e r i d i ne

1.2.2 Generic Names :

Aminoglutethimide, Ciba 16038

1.2.3 Trade Names:

E l ipten, Cytadren, Orimeten

1.3 Formula

1.3.1 Empirical : C13H16N202

1.3.2 Structural :

CH2CH
3

1.3.3 Chemical Abstract R e g i s t r y Number: 125-84-8

1.4 Molecular Weight: 232.27

1.5 Elemental Composition: C 67.22%; H 6.94%; N 12.06%;

0 13.78%
1.6 Appearance, Color, Odor and Taste: a white t o

creamy w h i t e c r y s t a l 1i n e powder, odor1 ess and

possessing a b i t t e r t a s t e (8,9,10).

1.7 pH (0.1% s o l u t i o n ) : 6.2-7.3

I n t r o d u c e 200.0 mg 2 0.2 mg of aminoglutethimide

i n t o a 200 m l v o l u m e t r i c f l a s k , d i s s o l v e i n 10.0 m l

o f methanol and add approx. 180 m l o f C o p f r e e water

AMINOGLUTETHIMIDE 39

a t 50'C. A f t e r shaking w e l l , cool down t o room tem

p e r a t u r e and f i l l up t o t h e mark w i t h C02-free water

a t room temperature, then determine t h e pH poten-

t i o m e t r i c a l l y (8).

2. P h y s i c a l P r o p e r t i e s

2.1 M e l t i n g Range:

The f o l l o w i n g m e l t i n g range has been r e p o r t e d f o r

aminoglutethimide:

M.p, 'C Reference

151 9
149-150 ( f r o m MeOH o r EtOAc) 10
152-154 11
The h y d r o c h l o r i d e s a l t [CAS 31075-85-11 has a

m e l t i n g range of 223-225' (9).

2.2 Solubility:

V i r t u a l l y i n s o l u b l e i n water, f r e e l y s o l u b l e i n most

o r g a n i c s o l v e n t s e.g. methanol, methylene c h l o r i d e

and chloroform. Poorly s o l u b l e i n e t h y l a c e t a t e , 0.1N

HC1 and absolute ethanol, r e a d i l y s o l u b l e i n acetone

and 100% a c e t i c a c i d ( 8 , 9 ) .

2.3 D i p o l e Moment:

Lee and Kuml e r ( 12) determi ned t h e d i p o l e moment and

s t r u c t u r e o f t h e imide group i n f i v e and s i x membered

c y c l i c imides. Aminoglutethimide has a d i p o l e moment

o f 2.83 )I i n D u n i t determined a t 30' i n dioxane.

40 HASSAN Y . ABOUL-ENEIN

2.4 O p t i c a l A c t i v i t y and Absolute C o n f i g u r a t i o n s :

Finch --
e t a1 (13) resolved t h e o p t i c a l enantiomers of

aminoglutethimide -
v i a r e c r y s t a l l i z a t i o n o f the t a r -

t a r a t e s a l t from methanol. The d e x t r o r o t a t o r y a n t i -

pode (+) aminoglutethimide has a m.p. 114-115' and

[,ID25 = t 163.1 (MeOH).

The 1evorotatorY antipode ( - ) aminoglutethimide( 11)

has a m.p. 114-115' and [a]D25 = -163.6' (MeOH).

The absolute c o n f i g u r a t i o n o f (t) - isomer was d e t e r -

mined t o have R-configuration w h i l e ( - ) - isomer has

S - c o n f i g u r a t i o n around t h e assymmetric carbon as

shown i n Figure 1. It i s o f i n t e r e s t t o mention

t h a t t h e (+)-isomer ( I ) had t h e most o f t h e s t e r o i d

s y n t h e s i s i n h i b i t i o n a c t i v i t y (2-3 more p o t e n t than

t h e racemate), w h i l e t h e ( - ) isomer had very l i t t l e

a c t i v i t y a t dose l e v e l s 1 0 - f o l d h i g h e r .

2.5 Spectral P r o p e r t i e s

2.5.1 U l t r a v i o l e t Spectrum

The u l t r a v i o l e t spectrum o f aminogl u t e t h i m i d e

i n n e u t r a l methanol i s shown i n Fig. 2. It

e x h i b i t s a maximum a t about 242 nm and a

shoulder a t approximately 282 nm. The maxima

a t 242 and 282 nm do n o t change o r s h i f t i n

a c i d i c (0.1N H2SO4) o r b a s i c (0.1N NaOH) media

as shown i n Figures 3 and 4. The u l t r a v i o l e t

AMINOGLUTETHIMIDE 41

": 2

H H
II I
(-) Aminoglutethlmlde (t) Amlnoglutethlmlde

Fig. 1 - The absolute c o n f i g u r a t i o n o f ( - ) - S and (t) -R-

hinogl u t e t h i m i d e

o ~ , l , , l , l , l , , , , , , , , , ~
210 220 240 260 280 300 320 340 360 380 400

Fig. 2 - U l t r a v i o l e t spectrum o f aminoglutethimide i n n e u t r a l

methanol.
42 HASSAN Y. ABOUL-ENEIN

0.8

0.7 -
0.6 -
0.5 -
0.4 -

0
210 220 240 260 280 300 320 340 360 380 400
F i g 4. - U l t r a v i o l e t spectrum o f aminoglutethimide i n
0.1N NaOH.
0.8

0.7 -
0.6 -
0.5 -
0.4 -
0.3

0.1

0 0
210 220 240 260 280 300 320 340 360 380 400

Fig. 3 - Ultraviolet spectrum o f aminoglutethimide i n

0.1NH2S04.
AMINOCLUTETHIMIDE 43

spectra are recorded on a V a r i a n AG UV-VIS

spectrophotometer - Model DMS-90. These data

are i n agreement w i t h p r e v i o u s l y pub1 ished

i n f o r m a t i o n (14).

2.5.2 I n f r a r e d Spectrum

The i n f r a r e d o f aminoglutethimide i n Nujol

m u l l i s presented i n F i g u r e 5 and i s recorded

on Perkin-Elmer spectrophotometer model 5808.

The frequencies and t h e i r s t r u c t u r a l assign-

ments are as f o l l o w s :

Frequency (cm-1) Assignment

3 asym NH2 s t r e t c h i n g v i b r a t i o n
3480
3470 t
3 180 3 sym-NH-imide group

3080 CH aromatic s t r e t c h

C=O imide carbonyl s t r e t c h

1690 o f glutarirnide r i n g

C=C aromatic f o r phenyl

1520 skeletal vibrations

830 & 700 CH-out-of-plane bending

vibration characteristic for

p-substi t u t i o n

Other c h a r a c t e r i s t i c f i n g e r p r i n t bands are

1260, 1200 cm-1. These data a r e i n agreement

w i t h p r e v i o u s l y reported r e p o r t s (8,14).
 MCRONS
2.5 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10 * 12 14 16 1820 25 30 4050
100 100

80-

60-

40-

20-

0 )O
I I 1 I I I I
4Mx) 3;oo 3ooo 2500 2doo 1800 lsbo 14bo 12bo lo00 800 600 400 200
W
A- (an-')

Fig. 5 - I n f r a r e d spectrum o f aminoglutethimide i n Nujol mull.

AMINOGLUTETHIMIDE 45

2.5.3 Nuclear Magnetic Resonance Spectra

2.5.3.1 PMR Spectrum

The 60MHz PMR spectrum o f aminoglu-

t e t h i m i d e i n DMSO-d6 i s shown i n

F i g u r e 6. The spectrum was recorded

on a Varian T60-A NMR spectrometer

u s i n g TMS as t h e i n t e r n a l standard.

The f o l l o w i n g s t r u c t u r a l assignments

have been e l i c i t e d from F i g u r e 6:

Chemical S h i f t (ppm) Ass ig nment s

0.77 t CH3-CH2

1.80 q CH3-CH2

2.27 m CHz-CHz-CH o f t h e g l u t a r i m i d e

r i n g system

6.53 d J=8.5 Hz aromatic H3 8 H5

6.95 d J=8.5 Hz aromatic H2 8 H6

AB system c h a r a c t e r i s t i c o f

p heny 1 p -5 ubs t it ut ion

5.0 s NH2 exchangeable w i t h D20

9.33 s NH exchangeable w i t h D20

s-singlet, d=doublet, m-multiplet, q=quartet t=triplet

Defay and D o r l e t (15) r e p o r t e d t h e

assignments o f NMR s p e c t r a o f some

glutarimide derivatives including

 I ' " " ' ' " " ' ' l " " ' l ' " ' ~ " I " " ~ " ' ' I '
I ' 1 I I ' I I

i !
i

I .
.O
. . . . I
70
1
. . . . i
.o
. . . .
I
I
I0
. .
. . . . i
4 0
. . . I . i . . . . i
3 0 LO
. I . . . i . . . ,
1 0
!
4 '
Fig. 6 - PMR spectrum o f aminoglutethimide in DMSO - d 6 using TMS as the internal standard.
AMINOGLUTETHIMIDE 47

aminoglutethimide and i s i n agreement

w i t h t h e assigned peaks.

2.5.3.2 13C-NMR Spectrum

The 13C-NMR spectrum o f aminogl u t e t h i m i d e

has been determined on a V a r i a n FT80

spectrometer a t ambient temperature.

The sample was d i s s o l v e d i n DMSO-d6 i n a

t u b e w i t h l O m m diameter. Spectral width:

5000 Hz, a c q u i s i t i o n t i m e : 1.638

seconds, p u l s e w i d t h 6 y second and num-

b e r o f d a t a p o i n t s 16384. The n o i s e

decoupled and t h e complete o f f - r e s o n a n c e

s p e c t r a a r e shown i n F i g u r e s 7 and 8,

respectively. The s p e c t r a l assignments

a r e 1 is t e d be1 ow:
6 7

Chemical Shi f t Assignment

( i n ppm r e l a t i v e t o TMS)

176.23 s C=O a t C5

172.92 s C=O a t C 1

147.46 s c11

126.66 d c9 c13
48 HASSAN Y. ABOUL-ENEIN

11
Fig. 7 - 13C-NMR off-resonance spectrum o f aminogl utethimide
I n DMSO-d6.

Fig. 8 - 13C-MNR off-resonance spectrum o f aminoglutethimde

i n DMs0-d~.
AMINOGLUTETHIMIDE 49

126.11 s c8

114.04 d c10 & c12

49.27 s c4

32.35 t c3

29.17 t C2

26.04 t c6

8.86 q c7

s=singlet, d=doublet, t=triplet, q=quartet

2.5.4 Mass Spectrum

The mass spectrum o f a m i n o g l u t e t h i m i d e

o b t a i n e d by chemical i o n i z a t i o n ( C I ) on a

s o l i d probe u s i n g CH4 as i o n i z i n g gas i s shown

i n F i g u r e 9. The ( M t 1 ) peak i s a t m/z 233

(100%) which corresponds t o i t s m o l e c u l a r

weight, a small peak ( M t 29) i s a l s o p r e s e n t

a t m/z 261 (5%). The mass spectrum o f ami-

nogl u t e t h i m i d e o b t a i n e d by he1 ium charge

exchange (HCE) w i t h d i r e c t i n l e t probe i s

shown i n F i g u r e 10.

The s p e c t r a were recorded on a F i n n i g a n 3200

GC/MS connected t o an Incos 2300 d a t a system.

The spectrum ( F i g u r e 10) shows a m o l e c u l a r

i o n peak (Mt) as m/z 232 and a base peak a t m/z

50 HASSAN Y. ABOUL-ENEIN

132. The most prominent fragments a r e l i s t e d

i n Table 1 .

TABLE 1. Most Prominent Fragments o f Aminoglutethimide

Mass m/z Relative Intensity, % Fragment

232 31.2 C13H16N2Ot2s Mt

203 45.2

175 34.4 ClOHllN20+

160 14

147 11.4 ClOH13i

7+
AMINOGLUTETHIMIDE 51

2 I 707504
loo
354

Sample: Aminogknhethimide
conds.: cH4 CI sdid Probe

50

26 1
205
73 94. ,182, 149. 175187 .I 219 1
2$o 'T3 . .304, , ,

MI2 100 150 200 250 300 3 I

Fig. 9 - Chemical i o n i z a t i o n (CI) mass spectrum o f

aminoglutethimde u s i n g CH4 as i o n i z i n g gas.
100 1 1 16992
117

Sample: Aminogkrthethimide
Conds.: Helium Charge Exchange

50

117
175
232

144 190

187
L _"217 . 26 1
MI2 100 D

Fig. 10 - Helium charge exchange (HCE) mass spectrum Of

aminoglutethimide by d i r e c t i n l e t probe.
52 HASSAN Y. ABOUL-ENEIN
+
132 100 C10H12

117 37.6

115 30.5 c9Hf7

+
+

Other peaks appear a t m/z 131 and 130.

Rucker and Bohn discussed t h e f r a g m e n t a t i o n process

o f g l u t e t h i m i d e and aminoglutethimide u s i n g t h e

deutrium l a b e l e d analogs o f these drugs t o c o n f i r m

the s t r u c t u r e o f t h e fragments (16).

3. Synthesis

Aminoglutethimide i s synthesized as shown i n scheme 1

(17). N i t r a t i o n of 2-phenyl b u t y r o n i t r i l e g i v e s t h e

p-nitro derivative. Conjugate Michael a d d i t i o n o f t h e

carbani on of p-ni trophenyl b u t y r o n i t r i 1e t o methyl acry-

1a t e g i v e s methyl 4-cyano-4 ( p - n i t r o p h e n y l ) hexanoate. The

AMINOGLUTETHIMIDE 53

l a t t e r intermediate i s c y c l i z e d i n t h e presence o f a c e t i c

a c i d and 90% s u l f u r i c a c i d [ s i m i l a r t o t h e s y n t h e s i s o f

glutethimide ( 1 8 ) l t o give 2-ethyl-2(p-nitrophenyl) glu-

tarimide. C a t a l y t i c r e d u c t i o n of t h e n i t r o group a f f o r d s

-
(t) ami nogl u t e t h i m i d e ( 17).

4. Metabol ism

A f t e r an o r a l dose o f 250 o r 500 mg o f aminoglutethimide

i n man, t h e drug i s absorbed and excreted unchanged t o a

c o n s i d e r a b l e e x t e n t i n t h e u r i n e a f t e r 48 hours (19),
along w i t h t h e N-acetyl aminoglutethimide which i s con-

s i d e r e d t h e major m e t a b o l i t e (20). It has been suggested

t h a t t h i s m e t a b o l i t e may c o n t r i b u t e t o t h e o v e r a l l phar-

macological a c t i v i t y o f t h e parent drug (20). However,

t h e N-acetyl m e t a b o l i t e has been shown t o be l e s s than

h a l f as e f f e c t i v e i n reducing g l u c o c o r t i c o i d p r o d u c t i o n

as t h e p a r e n t drug (21). Also, i t lacked t h e a n t i f e r -

t i l i t y a c t i v i t y o f aminoglutethimide (22). Sheets and

V i c k e r y ( 2 3 ) showed t h a t t h e a b i l i t y o f aminoglutethimide

t o i n h i b i t c h o l e s t e r o l conversion t o pregnenolone was

l o s t upon a c e t y l a t i o n o f arylamine n i t r o g e n . The authors

e x p l a i n e d t h i s f a c t due t o t h e f a i l u r e o f N-

acetylami nogl u t e t h i m i d e t o b i n d t o cytochrome P45Oscc.

Aminogl u t e t h i m i d e i s among those drugs t h a t a r e p o l y -

m o r p h i c a l l y a c e t y l a t e d i n humans (24). Thus, i t s e f f e c t ,

as w e l l as some s i d e e f f e c t s , may be r e l a t e d t o t h e acety-

54 HASSAN Y. ABOUL-ENEIN

l a t o r phenotype o f a p a r t i c u l a r p a t i e n t t r e a t e d w i t h t h i s

drug (25). The serum h a l f l i f e was found t o be about 7

hours f o r aminoglutethimide (25). The pharmacokinetics,

b i o a v a i l a b i l i t y and b i n d i n g t o blood c o n s t i t u e n t s was

s t u d i e d by Thompson --
e t a1 (26).

Other minor m e t a b o l i t e s o f t h e drug have been i d e n t i f i e d

such as N-formylaminoglutethimide and n i t r o g l u t e t h i m i d e

(27). N-Hydroxyaminoglutethimide has been r e c e n t l y

described as an auto-induced-metabol i t e t h a t appears i n

t h e u r i n e on c h r o n i c dosing w i t h aminoglutethimide (28).

The metabolism o f aminoglutethimide was s t u d i e d i n t h e r a t

by use o f t h e 14C-labeled compound (29). Following oral

doses o f 5 and 50 mg/kg, t h e drug was almost completely

e l i m i n a t e d w i t h i n 48 h r i n t o u r i n e and feces, mostly i n

t h e form of metabolites. I n b i l e duct-cannulated r a t s ,

b i l i a r y e x c r e t i o n of r a d i o a c t i v i t y amounted t o about 52%

w i t h i n 24 h r o f an o r a l l y administered 50 mg/kg dose, w i t h

t h e remainder o f t h e dose being e l i m i n a t e d i n t o urine.

The major u r i n a r y metabolites r e s u l t e d from a c e t y l a t i o n o f

t h e a n i l i n e moiety, h y d r o x y l a t i o n o f t h e g l u t a r i m i d e r i n g

a t p o s i t i o n s 3 and 4, and o x i d a t i v e e l i m i n a t i o n o f t h e

e t h y l sidechain. The p o l a r m e t a b o l i t e s a r e accounted f o r

by aromatic h y d r o x y l a t i o n w i t h subsequent s u l f a t e con-

j u g a t i o n and by a g l u t a r i m i d e ring-opened compound. In

AMINOGLUTETHIMIDE 55

a d d i t i o n , a gamma-butyrolactone d e r i v a t i v e was a l s o iden-

t i f i e d ( F i g u r e 11).

Murray --
e t a1 (30) reported t h a t t h e h a l f - l i f e o f aminoglu-

t e t h im i de admi n i s t e r e d t o p a t i e n t s w i t h mest a s t a t ic breast

carcinoma was 13.3 -

t 2.65 hours f e l l s i g n i f i c a n t l y t o 7.3

-t 2.14 hours a f t e r 6 t o 32 weeks o f therapy. Recently,

f o u r new m e t a b o l i t e s o f am n o g l u t e t h i m i d e have been iden-

t i f i e d i n t h e u r i n e o f pat ents being t r e a t e d c h r o n i c a l l y

w i t h t h e drug (31). These were products of h y d r o x y l a t i o n

o f t h e g l u t a r i m i d e r i n g system, namely 3-( 4-aminophenyl)

-3-ethyl-5-hydroxypiperidine-2, 6-dione and i t s a c e t y l a t e d

analog, 3-( 4-aminophenyl) -3-( l - h y d r o x y e t h y l ) p i p e r i d i n e - 2 ,

6-di one and 3-( 4-ami nophenyl)3-( 2-carboxami do-ethyl ) t e t r a

hydrofuran-2-one, a l a c t o n e formed by rearrangement of

3-(4-ami nopheny1)-3-( 2-hydroxyethyl) p i p e r i d i ne

-2,6-dione. These metabolites were minor c o n s t i t u e n t s

compared w i t h aminoglutethimide and t h e major metabo-

lites. There were marked species d i f f e r e n c e s between

r a t and human inasmuch as almost a l l t h e u r i n a r y meta-

b o l i t e s o f t h e r a t were N-acetylated whereas most of

t h e human m e t a b o l i t e s were not (31). However, 5-hydro-

x y l a t i o n occurs i n both species, t h e -

c i s isomer being

formed e x c l u s i v e l y . A l l i s o l a t e d m e t a b o l i t e s o f amino-

g l u t e t h i m i d e a r e b i o l o g i c a l l y i n a c t i v e as compared t o
56 HASSAN Y. ABOUL-ENEIN

I CH,=CH- COOCH,
Basic catalyst

Cyclization

SCHEME I SYNTHESIS OF AMINOGLUTETHIMIDE

oso;
- -
O
&NH;

H
N
I
0 O
&NH,
N
I
H
0

( !)AminOglutc?himida
oc

’
r c Qco
-N%

cp
X=OH,Y=NH,
OR X=NH,,Y=OH

H,C, OH

’1 ‘

*
&NHcocH3- NHCOCHg-* Ho+NHcocH3

0 c&
o Hy o
0
H
I

“3

NHCOCHS

O N
NIP N H C O C H 3
OJ 3H I
H

FIG.11 PROPOSED METABOLIC PATHWAYS OF AMINOGLUTETHIMIOE IN THE RAT

AMINOGLUTETHIMIDE 57

t h e p a r e n t drug (31,32), The s t r u c t u r e of t h e i d e n t i f i e d

human u r i n a r y m e t a b o l i t e s o f a m i n o g l u t e t h i m i d e a r e shown

i n Table 2.

Table 2.. Identified Human Urlnary Metaboiltes of Aminoglutethlmide

Compound R,
Amlnoglutethimide NH2 H H
Acet yiaminogiutethimide NHCOCH, H H
p-Nitroglutethimlde NO2 H H
Formylaminoglutethimide NHCHO H H
Hydrox yiaminoglutethlmide NHOH H H
p- Amino-5-hydrox yglutethimide NH2 OH H
p-Acetylamino-5-hydroxygiutethlmide NHCOCH, OH H
Lactone from p-amlno-2'-hydroxygiutethimlde Structure shown above
p-Amino-1'-hydroxyglutethimide NH2 H OH

5, METHODS OF ANALYSIS
5.1 T i t r i m e t r i c Non-aqueous

Aminoglutethimide has been assayed i n bu k and phar-

maceutical f o r m u l a t i o n s by non-aqueous t t r a t i o n

u s i n g 0.1 N p e r c h l o r i c a c i d i n g l a c i a l a c e t i c a c i d

(8). The end p o i n t is determi ned p o t e n t iomet r i c a l l y

u s i n g a g l a s s e l e c t r o d e and a calomel e l e c t r o d e as a

reference electrode with a saturated solution o f

potassium c h l o r i d e i n methanol as t h e b r i d g e f l u i d .

Agarwal and Blake(33) determined a m i n o g l u t e t h i m i d e ,

58 HASSAN Y. ABOUL-ENEIN

g l u t e t h i m i d e and bemegride by d i s s o l v i n g t h e sample

i n ( e l m i l l i e q u i v a l e n t o f t h e powdered drug o r

t a b l e t s ) i n dimethylformamide (40 m l ) using 0.1N

sodium methoxide i n benzene-methanol as a t i t r a t i n g

agent. The end-poi n t i s determi ned poten-

t i o m e t r i c a l l y ( w i t h calomel and p l a t i n u m e l e c t r o d e s ) ,

o r v i s u a l l y w i t h a z o v i o l e t as i n d i c a t o r . The coef-

f i c i e n t of v a r i a t i o n i s N 0.7%.

5.2 Spectrophotometric Analysis

B u l t and Klasen(34) determined am1 nogl u t e t h i m i d e

among o t h e r drugs c o l o r i m e t r i c a l l y through i t s reac-

t i o n w i t h cobaltous amine reagent ( P a r r i r e a c t i o n ) .

The v i o l e t c o l o r produced c o u l d be measured a t 555

and 530 nm, r e s p e c t i v e l y . The general procedure i s

described as f o l l o w s :

About 1 mg o f t h e drug i s d i s s o l v e d i n 1.6 m l

ethanol. A f t e r a d d i t i o n o f 0.2 m l cobaltous s a l t

s o l u t i o n (cobaltous) n i t r a t e 6 H20: 120 mg/20 m l

methanol o r cobaltous acetate 4 H20:100 mg/20 m l

methanol) and 0.2 m l cyclohexylamine s o l u t i o n (6 9/20

m l methanol) t h e produced c o l o u r i s compared w i t h

t h a t o f t h e blank (reagents).

Another method used t o assay aminogl u t e t h i m i d e

AMINOGLUTETHIMIDE 59

colorimeterically i n b ological f u i d (urine) i s

based on t h e f o r m a t i o n o f c o l o r e d Sch if f ' s

base w i t h p-dimethylaminobengaldehyde ( E h r l i c h ' s

reagent). The y e l l o w c o l o r formed was measured a t 440

nm. A l i n e a r r e l a t i o n s h i p between c o l o r i n t e n s i t y and

c o n c e n t r a t i o n o f t h e drug was o b t a i n e d f o r t h e range

o f 1 - 1 5 ~g/ml (19). A m o d i f i e d method was a p p l i e d t o

determine aminoglutethimide serum l e v e l s u s i n g

E r l i c h ' s reagent (30). The a u t h o r s r e p o r t e d t h a t

a l t h o u g h several substances c o u l d p o t e n t i a l l y i n t e r -

f e r e w i t h t h e blood assay o f a m i n o g l u t e t h i m i d e , none

was a c t u a l l y found t o do so t o any s i g n i f i c a n t degree.

5.3 Chromatography

5.3.1 Paper Chromatography

Douglas and N i c h o l l s (19) d e s c r i b e d a method

f o r i d e n t i f i c a t i o n and s e p a r a t i o n o f aminoglu-

t e t h i m i d e i n u r i n e o f man by paper chroma-

tography. The chromatograms r u n on Whatman No.

1 paper by ascending t e c h n i q u e u s i n g s o l v e n t

systems shown i n Table 3.

Table 3. Solvents Systems used f o r I d e n t i f i c a t i o n o f

Ami nogl u t e t h i m i d e by Paper Chromatography

Sol v e n t System Rf Local iz i n g Agent

a) n-Bu0H:HOAc :H20 0.73 a) N,N-dimethyl ami n o c i nnaml dehyde

12: 3:5 v/v spray (permanent r e d c o l o r )
(35)
60 HASSAN Y. ABOUL-ENEIN

b) CHC13: MeOH 0.93 b) UV l i g h t

1:l v/v

c) CCl4: HOAc: H20 0.34 c) E r l i ch ' s reagent (ye1 1ow

1: 2: 1 v/v (color)

d) Aqueous NaCl 0.74 d) N i t r o u s acid-naphthylethy-

10% w/v l e n e diamine(35)

e) NaOCl - K I - s t a r c h
( b l ue b l a c k c o l o r ) (36)

Dav es and Nicho l s ( 3 8 ) r e p o r t e d a study o f t h e

chromatographic behavior o f n i n e g l u t a r i m i d e s

i n c l u d i n g aminoglutethimide using Whatman No. 1 paper

impregnated w i t h l i q u i d p a r a f f i n (4% i n hexane),

o l i v e o i l (20% i n acetone) o r t r i b u t y r i n (10% i n

acetone). The most u s e f u l solvent system used were:

a) toluene: a c e t i c a c i d : water 10:5:4

b) carbon t e t r a c h l o r i d e : a c e t i c a c i d : water 1:2:1

c) 10% w/v aqueous sodium c h l o r de

d) 0.066M sodium phosphate (pH7 3)

The compounds were detected by hypochl o r i t e

reagent o r a1 k a l ine hydroxyl ami ne reagent.

5.3.2 Thin Layer Chromatography (TLC)

Several procedures had been p u b l i s h e d f o r detec-

t i o n o f aminoglutethimide by t l c which i s sum-

marized i n Table 4.
 Table 4. Thin Layer Chromatography o f A m i nogl utethimide

Sol vent System Visual iz i ng Agent Comnents/Reference

CHCl :C6H6 Hydroxyl ami ne reagent Alumina 50 t h i c k p l a t e ac i v t e d

1:l a t 150° f o r v ? hour was used t377

C H CH3:CH$OCH3 Ehrl ich ' s reagent used i n determ na i o n o f aminoglutethi-

9&:20 mide i n serum 130j

i s 0 PrOH:CHC13:25%NH40H D i c h l o r o f 1uoroscei n
9:9:2 or
CHCl :CH3COCH3 Mercuric n i t r a t e

CHC13 :CH3C002CH5 UV l i g h t a t 254 nm used i n i d n t ' f i c a t i n o f amino l u t e t h i -

mide i n buyk {Rf:0.47 and t o d e f e c t t h e
35 :15 by-product m-aminoglutethimide (Rf:0.35)
MNSi 1 625 HR/UV p l a t e (Macherey-Nagel
& Co. was 8 used. (8)

CH3COOC2Hg:CH30H: UV l i g h t a t 254 nm a m l i e d t o i d e n t i f y aminoqlutethimide i n

or t a b l e t s (Rf:O 6 ) - S i l i c a " g e l 60F 254
100%CH3COOH by d i a z o t i zat ion and p l a t e used. (8)
spraying w i t h napthyl
17 :3:O. 1 e t h y l enedi ami ne d i hy-
chloride.
62 HASSAN Y. ABOUL-ENEIN

5.3.3 Gas-Liquid Chromatography (GLC)

Adams and Roger(39) r e c e n t l y p u b l i s h e d a rapid,

s e n s i t i v e and s e l e c t i v e GLC assay f o r aminoglu-

tethimide i n biological fluids. The method i s

s u i t a b l e f o r t h e study o f t h e pharmacokinetics

o f t h e drug and i t s N-acetyl m e t a b o l i t e (which

gave an assymmetric peak shape on t h i s system).

The c o n d i t i o n s are as f o l l o w s :

Column: glass column (1.5in x 0.4mm I.D.)

packed w i t h 2% CDMS

(cyclohexanedimethanol succi n a t e ) on

Chromosorb W80-100 mesh ( a c i d washed &

dichloromethylsalisilane t r e a t e d ) .

W80-100 mesh ( a c i d washed & d i c h -

1oromethyl s a l is i lane t r e a t e d ) .

Column temperature : 240'

D e t e c t o r temperature: 350'
C a r r i e r gas : Nitrogen

F1ow r a t e : 100ml/min

Detector : Nitrogen s e l e c t i v e

D u t t reported a gas chromatographic method f o r

t h e i d e n t i f i c a t i o n o f 116 common drugs by

t h e i r m u l t i p l e peaks o f t h e parent and t h e i r

AMINOGLUTETHIMIDE 63

t r i m e t h y l s i l y l y l d e r i v a t v e s i n c l u d i n g ami-

n o g l u t e t h i m i d e (40). The study was done u s i n g

3% OV-17 Gas-Chrom Q 100-120 mesh g l a s s column

(2m x 3mm I.D.) and flame i o n i z a t i o n d e t e c t o r

(FID). The c a r r i e r gas was n i t r o g e n a t a f l o w

r a t e o f 30 ml/min and t h e hydrogen and a i r

f l o w r a t e s were 30 and 450ml/min, respec-

tively. The i n j e c t i o n p o r t and d e t e c t o r tem-

p e r a t u r e s were 300' and t h e oven temperature

was programmed from 120" t o 270' a t lO"/min.

o r a t an isothermal temperature o f 300'.

5.3.4 High-Performance L i q u i d Chromatography (HPLC)

Several HPLC procedures have been p u b l i s h e d f o r

t h e d e t e r m i n a t i o n o f aminoglutethimide and i t s

m a j o r m e t o b o l i t e (N-acetylaminoglutethimide) i n

biological fluids. Table 5 summarises some o f t h e

HPLC systems used f o r t h e a n a l y s i s o f t h e drug.

 Table 5 HPLC Systems Aminoglutethimide

Column Mobile Phase Detection Comnent/Reference

Spherisorb ODS column MeCN: 0.01 M phosphate b u f f e r UV a t 234 nm Applied f o r simple

(30cm X 4mm I.D., pH 6.8 (22:68), f l o w r a t e 1.5 determination o f
r
5 g particle size m l /mi n ami nogl utethimide
& i t s m e t a b o l i t e (41)

Octadecyl (C18) s i l i c a MeCN: t e r t . butylamnonium UV a t 254 nm Applicable f o r assay

reversed phase uncapped phosphate:H20 (100:3: l o o ) , o f t h e drug i n
column (10cm X 0.8cm adjusted t o pH 6.320.2 w i t h plasma (42)
I.D. w i t h 10 m par- orthophosphori c a c i d , f 1ow
t i c l e size).Y r a t e 2 ml/min.

ODS H y p e r s i l (10 cm 11%MeCN i n lOOmM ammonium Applicable f o r a n a l y s i s

long, 3 m p a r t i c l e s i z e phosphate b u f f e r , pH 3.5 o f t h e drug and i t s meta
Y also - 8-20% MeCN i n 15mM b o l i t e s i n plasma. Re-
acetate b u f f e r pH 4.5 - or t e n t i o n times were ap-
lOmM phosphate b u f f e r , pH 6.0, prox.: 4.5 min amino-
f l o w r a t e 2 ml/rnin. E l u t i o n o f glutethimide, 14 m i n . N
n i t r o g l utethimide r e q u i r e d a formylmetabolite, 17 min
step wise increase i n MeCN conc. N-acetylmetabol it e , 28
from 11 t o 23% a f t e r 16 min. m i n. n i t r o g l u t e t h i m i de
(43)
 Table 5 HPLC Systems Aminoglutethimide
Column Mobile Phase Detection Comment/Reference

Nucleosil - 10 (C18) MeCN:HzO:Et3N (25:75:0.05) UV at 254 mm Applied as stabi 1 i ty

stainless steel column flow rate 2 ml/min i ndi cati ng assay (8)
(20 cm x 4.8 mm I.D.)
Nucleosil - C18 (!Iyn MeCN: MeOH:H20 (5:20:75) UV at 235 mn Appl ied for simultaneous
particle size) determination of the
drug and its major meta-
bolite in human plasma
( 44 9451
Retention times f o r
aminoglutethimide =
8.9 min
N-acetylmetabol i te =
12.1 min
Reversed phase C &rate bu fer pH 3-4:
I UV at 254 mn Suitable for pharmaco-
Magnusphere C18 MeOH (500: 280), flow rate kinetic study of amino-
(7pm particle size 1.2 ml/min gl utethimide and its
15 cm x 4.6 cm 1.0.) N-acetyl metabol i te in
stainless steel column biological f1 uids
(plasma, urine saliva),
(39)
66 HASSAN Y. ABOUL-ENEIN

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-
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10 Merck Index, 10th E d i t i o n , Merck & Co. Inc., Rahaway,
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11 A l d r i c h Catalog Handbook o f Fine Chemicals, A l d r i c h

Chemical Co., Milwaukee, Wisconsin, U.S.A., p. 61,
1984-1985.
12 C.M. Lee and W.D. Kumler, -----
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4586 (1961).
AMINOGLUTETHIMIDE 67

13 N. Finch, R. Dziemian, J. Cohen and B.G. Steinetz,

-
Experi e n t i a, 31, 1002 ( 1975).

14 C.M. Lee e t a l , -----

J. Amer. Chem. SOC. 83, 4586 (1961).

15 --
N. Defay and C. D o r l e t , J. Pharm. Belg., -
27 335 (1972).
16 G. Rucker and G. Bohn, Archive -
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(1969).
17 K. Hoffman and E. Urech, U.S. P a t i e n t 2, 848, 455
(1958).
18 E. Tagmann, E. Surry and K. Hoffmann, --
Helv Chim.
--
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19 J.S. --
Douglas and P. J. N i c h o l l s , J. Pharm, Pharmacol.
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17, 115 S (1965).
20 J.S. Douglas and P. 3. N i c h o l l s , 3. Pharm. Pharmacol.
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24, (Suppl .) , 150 P (1970).
21 R.C. Coombes, M. Jarman, S. Harland, W.A. R a t c l i f e ,
T.J. Powles, G.N. Taylor, M. O'Hare, E. Nice, A.B.
F o s t e r and A.M. N e v i l l e , J. Endocrinol, 87, 31P (1980).

22 R. Paul, R.P.
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---
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23 5.3. Sheets and L.E. Vickery, Naunyn-Schmied. Arch.

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Pharmacol , 321, 70 (1982).

24(a) R.C. Coombes, A.B. Foster, S.J. Harland, M. Jarman

--
and E.C. Nice, Br. J. 7 Cancer, 46,-
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24(b) A.M. Adam, H.J. Rogers, S.A. Amiel and R.D. Rubens,
---
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495 (1984).
25 J.S. Schanche, P.E. Lonning, P.M. Ureland and S.
K r i n n s l and, Therapeutic Moni t o r i ng , -
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26 T.A. Thompson, J.D. Vermeulen, W.E. Wagner,

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27 M.H. Baker, A.B. Foster, S.J. Harland and M. Jarman,

-- Pharmacol , 74, 243 P (1981).
B r i t . J,
68 HASSAN Y. ABOUL-ENEIN

28 M. Jarman, A,B. Foster, P.E. Gross, L.J. Griggs,

--
1. Howe and R.C. Coombes, Biomed Mass Spectrum,
620( 1983).

29 H. Egger, F. B a r t l e t t , W. I t t e r l y , R. Rodebaugh, C.
C. Shimanskas, Drug Metab. Dispos., 10, 405 (1982).

F.T. Murray, s. Santner, E. Samojlik and R. J.

30
-- .
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31 A.B. Foster, L.J. Griggs, I.Howe, N. Jarman,

C.S. Leung, 0. Manson, and M.G. Rowlands Drug. Metab.
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32 A.B. Foster, M. Jarman, C-S. Leung, M.G. Rowlands and
G.N. Taylor, J. Med. Chem. 26, 50 (1983)
33 S.P. Agarwal and M.I. Blake, ----
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1668 (1965).
34 A. B u l t and H.B. Klasen, Pharmaceutisch Weekblad,
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109, 389 (1974).
35 J.W. Bridges and R. T. Williams, J. Pharm,
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Pharmacol , 15,565 (1963).

36 J.V. Jackson and M.S. Moss , "Chromatographic and

E l e c t r o h o r e t i c Techniques" e d i t o r I.Smith, Vol. I,
p 406, London, Heinemann.

37 D. Davies and P.J. -

Nicholls, J. Chromatogr. 17 416
(1965).
38 H.J. Uhlmann, Pharm. Z t Ver A otheker Zt
---
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39 A.H. Adam and H.J. Rogers, -
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(1984).
40 M.C. -
Outt , J. Chromatogr. 248, 115 (1982).
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-
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42 B.A. Robinson and F.N. C l i n . Chem.,
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AMINOGIXTETHIMIDE 69

44
.
P.E. Lonning, J.S. Schance, S. Kvinnsland and P.M.
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45 6. Menge and J.P. Dubois, J. Chromatogr., 310 431

(1984).

A c k n o w l e d g e m e n t s

The Author wishes t o thank Dr. K. Scheibli o f

Ciba-Geigy Limited, Basle, Switzerland, for donatf ng

a sample of Aminogl utethimide, batch 9614586, used i n

t h i s profile.
This Page Intentionally Left Blank
 CAFFEINE

l c l b h a d Uppat Zubair
khmoud M.A. Hassan, and
Ibrahim A . AZ-Meshat

1. Description
1.1 Nomenclature
1.1.1 Chemical Names
1.1.2 Generic Names
1.2 Formulae
1.2.1 Empirical
1.2.2 S t r u c t u r a l
1.2.3 S t r u c t u r a l Confirmation by Degradation
1.2.4 CAS Registry Number
1.2.5 Wiswesser Line Notation
1.3 Molecular Weight
1 . 4 Elemental Composition
1.5 Appearance, Colour, Odor and Taste
2. Physical P r o p e r t i e s
2.1 Melting Range

ANALYTICAL PROFILES OF DRUG SUBSTANCES Copyright Q 1986

VOLUME 15 by the American Pharmaceutical Association
71 All rights of reproduction in any form reserved.
72 MOHAMMAD UPPAL ZUBAIR ET AL.

2.2 Sublimation
2.3 Density
2.4 Solubility
2.5 Crystal Structure
2.6 Spectral Properties
2.6.1 Ultraviolet Spectrum
2.6.2 Infrared Spectrum
2.6.3 Nuclear Magnetic Resonance Spectra
2.6.3.1 Proton Spectrum
2.6.3.2 l 3 C - m Spectra
2.6.4 Mass Spectrum
3. Isolation of Caffeine
3.1 From Tea Leaves
3.2 From Green Coffee
3.3 Process for the Extraction of Caffeine from Coffee
beans
3.4 Isolation of Natural Caffeine from the decaffeination
process.
3.5 Isolation of Caffeine from Various Sources
4. Synthesis
4.1 Route 1
4.2 Route 2
4.3 Route 3
4.4 Route 4
4.5 Route 5
4.6 Route 6
5. Biosynthesis
6. Pharmacokinetics and Metabolism
7. Methods of Analysis
7.1 Elementa1 Analysis
7.2 Identification Tests
7.3 Titrimetric
7.4 Spectrophotometric
7.4.1 Colorimetric
7.4.2 Ultraviolet
7.4.3 Infrared
7.4.4 Nuclear Magnetic Resonance
7.4.5 Mass Spectrometric
7.4.6 Photo-nephlometric
7.5 Coulometric
7.6 Polarographic
7.7 Ring Oven (Micro)
7.8 Radioactive Isotopes
7.9 Radiochemical
7.10 Chromatographic Methods
7.10.1 Paper Chromatography
7.10.2 Column Chromatography
7.10.3 Thin-Layer Chrmakxagraphy
CAFFEINE 73

7.10.4 Gas Liquid Chromatography

7.10.5 High Performance Liquid Chromatography.

8. References

9. Acknowledgements
74 MOHAMMAD UPPAL ZUBAIR ET AL.

1. Description

1.1 Nomenclature

1.1.1 Chemical Names

a) lH-Purine-2,6-dione 3,7-dihydro-1 3,7-

trimethyl.

b) 1,3,7-Trimethylxanthine.

c ) Methylt heobromine .
d) 1,3,7-Trimethyl-2,6-dioxo-l,2,3,6-tetrahydro-
purine.

e) 3,7-Dihydro-l,3 ,T-trimethyl-lH-purine-2,6-
dione.

f) 1,3,7-Trimethyl-4,6-dioxopurine.

1.1.2 Generic Names

Anhydrous caffeine; Caffeine; Coffeniwn; Coffeine;

Coffeina; Cuavanine; Methyltheobromine; No-Doz;
Thiene.

1.2 Formulae

1.2.1 Empirical

C8H10N402
1.2.2 Structural
CAFFEINE 75

Discovered by Robiquet i n coffee i n 1821, while

searching f o r quinine which he believed t o be
present. I n 1827, Oudry found an a l k a l o i d i n
tea and c a l l e d it t h i e n e . I n 1838, J o b s t and
Mulder proved t h e i d e n t i c a l c h a r a c t e r of t h e two
p r i n c i p l e s (1).

1.2.3 S t r u c t u r a l Confirmation by Degradation ( 2,3)

Fischer has reported t h a t c a f f e i n e on o x i d a t i o n

gave dimethylalloxane and methyl urea, i n d i c a t i n g
s i m i l a r i t y with t h e skeleton s t r u c t u r e of u r i c
acid.

With t h e above information it becomes evident

t h a t : t h e t h i r d methyl group i s e i t h e r a t
p o s i t i o n 7 o r 9 and t h e remaining oxygen atom i s
e i t h e r at p o s i t i o n 6 o r 8.
P o s i t i o n of t h e methyl group; Fischer a l s o
i s o l a t e d another oxidation product which on
hydrolysis, gave N-methylglycine, carbondioxide
and ammonia. Thus, t h i s t h i r d oxidation product
must be N-methylhydantoin.

+ NH3 + C02
co - C02H
N-methylhydantoin

Therefore, it follows t h a t c a f f e i n e contains two

r i n g s t r u c t u r e s t h a t of dimethylalloxane and t h a t
o f methylhydantoin. The following two skeleton
76 MOHAMMAD UPPAL ZUBAIR E T A L .

s t r u c t u r e s were proposed:

F i n a l l y Fischer i s o l a t e d f o u r t h oxidation product,

N , N ' -dimethyloxamid@ CH3NHCOCONHCK3. Exami-
nation of ( I ) and (11) showed t h a t only ( I ),
could give r i s e t o t h e formation o f t h e oxamide.
Therefore ( I ) must be t h e skeleton of c a f f e i n e .

P o s i t i o n of oxygen atoms: t h e following two

s t r u c t u r e s were p o s s i b l e with regard t o t h e
p o s i t i o n of oxygen atoms.

0
a3 y3
I

1%
0 I
">
N/

CH3
I11 IV

By analogy, it would appear t h a t t h e more l i k e l y

s t r u c t u r e o f I11 would be t h a t of u r i c a c i d .
CAFFEINE 77

F i s c h e r , f u r t h e r confirmed t h e c a f f e i n e s t r u c t u r e
with t h e following degradation s t u d i e s :

c12 CH30H
Caffeine > - * Chlorocaffeine
NaoH 'l Met hoxy c a f f c i n e
dilute IlCl,
boil
Oxycaf feine+CII Cl
3
1.2.4 CAS Registry Number

a ) Anhydrous c a f f e i n e [ 58-08-21
b ) Monohydrat e [ 5743-12-4 I
1.2.5 Wiswesser Line Notation

T 56 BN DN FN
VNVJ B F H

1 . 3 Molecular Weight

Anhydrous C8HloN4O2 = 194.19

Monohydrate C8HloN402.H20 = 212.21

1 . 4 Elemental Composition
C, 49.48% ; H , 5.13% ; N , 28.85% ; 0, 16.48%.
1 . 5 Appearance, Color, Odor and Taste
White powder o r white, g l i s t e n i n g n e e d l e s , u s u a l l y
matted t o g e t h e r . It i s o d o r l e s s and has a b i t t e r
taste.
2. Physical P r o p e r t i e s
2 . 1 Melting range
Between 235' and 237.5'. determined a f t e r drying at 80'
f o r 4 hours.
Hexagonal prisms by sublimation, m.p. 238? (4)
2.2 Sublimation

Caffeine sublimes a t 178? Fast sublimation i s obtained

a t 160-165' under 1 mm p r e s s u r e a t 5 mm d i s t a n c e .
78 MOHAMMAD UPPAL ZUBAIR ET AL.

2.3 Density

d i 8 1.23 ( 5 ) .

2.4 S o l u b i l i t y (1)

1 gm of anhydrous c a f f e i n e dissolves i n about 50 ml

water, 6 ml water at 8 0 ° , 75 m l alcohol, about 25 m l
alcohol a t 6 0 ° , about 6 m l chloroform, 600 ml e t h e r ,
50 m l acetone, 100 ml benzene and 22 m l b o i l i n g benzene.
Freely soluble i n pyrrole; i n tetrahydrofuran contain-
ing, 4% water, a l s o soluble i n e t h y l a c e t a t e and
s l i g h t l y i n petroleum e t h e r .

Being a weak base, c a f f e i n e does not form s t a b l e salts,

and even i t s salts of strong a c i d s , such as t h e hydro-
chloride o r hydrobromide, are r e a d i l y hydrolysed by
water. The s o l u b i l i t y of c a f f e i n e i n water i s
increased by t h e presence o f organic a c i d s o r t h e i r
a l k a l i salts, e.g., benzoates, s a l i c y l a t e s , cinnamates,
o r c i t r a t e s and this i s t h e reason f o r t h e use of
s e v e r a l such preparations.

2.5 Crystal S t r u c t u r e

Sutor ( 6 ) has determined t h e c r y s t a l s t r u c t u r e of

c a f f e i n e , 1,3,7-trimethyl-2,6-dihydroxy purine (Fig. 1).
Crystallographically, it is nearly isomorphous with
theophylline (Fig.. 2) (7), and a comparison of bond
lengths i n t h e two has yielded information concerning
t h e e f f e c t s of a s u b s t i t u e n t i n t h e imidazole r i n g on
t h e purine ring. The c r y s t a l s of c a f f e i n e are mono-
c l i n i c , space group P21/a, w i t h a = 14.8 ? 0.01,
b = 16.7 ? 0.1, C = 3.97 ? 0.03 A'; 8 = 97.0 ? 0.5'.

Systematic absences are hoc with h = 2n + 1, OK0 w i t h

K = 2n + 1; space group ~ 2 1 / a' q h . Its f i n a l Fourier
projection i s shown i n Fig. 3. Table 1 shows t h e
f r a c t i o n a l coordinates r e f e r r e d t o t h e monoclinic
c r y s t a l axes. The c r y s t a l s t r u c t u r e w a s solved by an
application of t h e isomorphus-replacement method and
a consideration of t h e possible hydrogen bond system
i n t h e c r y s t a l . Bond lengths and bond angles within
t h e molecule are shown i n Fig. 4, Fig. 5 and Table 2.
CAFFEINE 79

Fig. 2: Crystal structure of theophylline.

10

15

Fig. 1: Caffeine, showing the numbering

system used.
80 MOHAMMAD UPPAL ZUBAIR ET AL.

Fig. 3: The f i n a l UKO Fourier projection of

c a f f e i n e , contours are arbitrary but
equal i n t e r v a l s ; ZUO contour broken.
CAFFEINE 81

Table 1. The F r a c t i o n a l C o o r d i n a t e s Referred t o t h e

Monoclinic C r y s t a l Axes.
X Y Z X* Y"

c2
0.2h.6 0.2225 0.9002 0.2414 0.2225
c4 0.1003 0.2533 0.1295 0.1019 0.2541
0.0835 0.1769 0.1944 0.0841 0 1759
c5
'6 0.1456 0.1151 0.1155 0.1463 0.1143
'8 0.4798 0.2481 0.3638 0.4801 0.2480

clo 0.2879 0.0841 0.8790 0.2891 0.0832

c12
0.1960 0.3641 0.9209 0 * 1959 0.3638
1' 4 0.4533 0 3950 0.4584 0.4536 0.3947
N1
0.2193 0.1418 0.9735 0.2196 0.1415
0.1796 0.2764 0.9848 0.1801 0.2769
N3
N 0.ooog 0.1749 0.3376 0.0020 0.1749
7
0.0407 0.3008 0.2440 0.0403 0.3008
N9
O11
0.3057 0.2397 0.7614 0.3063 0.2400
0.1374 0.0404 0.1616 0.1363 0.0404
' 3
1
0.0166 0.4790 0.2705 0.0184 0.4705
' 5
1
H1
0.413 0.239 0.474 - -
H2
0.487 0.438 0 599 - -
H3 0.435 0.437 0.278 - -
H4 0.395 0.363 0.510 - -
H5
0.263 0.362 0.857 - -
H6 0.228 0.396 0.105 - -
H7
0.142 0 * 377 0.783 - -
H8 0.348 0,100 0.772 - -
H9
0.300 0.033 0.022 - -
H1O 0.257 0.060 0.676 - -
x* and y* represent t h e weighted x and y coordinates from t h e
hKO and hKL projections.
82 MOHAMMAD UPPAL ZUBAIR ET AL.

Fig. 4: Bond angles and C-H bond lengths in the

caffeine molecule.

10

13 14

Fig. 5: Bond lengths in the caffeine molecules.

CAFFEINE 83

Table 2. Bond Lengths and t h e i r Standard Deviation

Bond Standard
Bond
length ( A ) deviation ( A )

C2-N1 1.42 0.014

C -N 1.35 0.016
2 3
C4-N3 1.42 0.021

c4-c5 1.32 0.014

'5-'6 1.44 0.015

C6-N1 1.36 0.021

c4 -N9 1.31 0.019

'8"g 1.34 0.019

C8-N7 1.32 0.012

C -N
5 7 1.41 0.021

C1O-N1 1.48 0.016

C12-N3 1.50 0.013

C14-N7 1.47 0.018

c2-011 1.19 0.023

'6"13 1.26 0.013

A comparison is made of t h e intramolecular d i s t a n c e s

with t h o s e i n o t h e r purines and a pyrimidine,Table 3,
and i n d i c a t e s t h a t s t e r i c hindrance must be allowed
for, i n a t h e o r e t i c a l c a l c u l a t i o n of bond l e n g t h i n
r e l a t i v e l y complicated molecule of t h i s t y p e .
Evidence f o r and a g a i n s t t h e e x i s t e n c e o f a s h o r t
hydrogen bond between water molecules i s given.

C r y s t a l s t r u c t u r e of 2 : l molecular complexes of c a f f e i n e
w i t h hexaaquamagnesium (11) bromide and hexaaquamanga-
nese (11) T r i i o d i d e i o d i d e c r y s t a l s t r u c t u r e s of
a4 MOHAMMAD UPPAL ZUBAIR ET AL.

Table 3. S i g n i f i c a n t l y D i f f e r e n t Bond Lengths i n a Comparison

of C a f f e i n e w i t h Theophylline, Adenine Hydrochloride
and U r a c i l .

Caffeine with theophylline

Si g n i f ic a n t Actural
Bond
d i f f e r e n c e (A') d i f f e r e n c e (A')

0.04 0.05
c4-c5
C -N 0.05 0.07
5 7
Caffeine with u r a c i l

S i g n if i ca n t Actual
Bond
difference (Ao) d i f f e r e n c e (A')

0.06 0.08
c4 -N3
0.05 0.09
c4-c5
Caffeine w i t h adenine h y d r o c h l o r i d e

Bond Significant Actual

d i f f e r e n c e (Ao) d i f f e r e n c e (A')

C2"1 0.05 0.05

C -N 0.05 0.05
2 3

C4-N3 0.06 0.06

c4-c5 0.05 0.05

C4"g 0.05 0.05

( C ~ H ~ O N ~ O ~ ) ~ M)@r2
~ - ( Oand
H~ (C~H~ON~O~)~M~(OH,)~I.I
have been determined by X-ray d i f f r a c t i o n methods (83.

The c r y s t a l S t r u c t u r e s O f (C8H10N402)2Mg(OH2)6Br2 ( I )
and (C8HloN402)2Mn( OH2 161. I 3 (I1) have been determined
by X-ray d i f f r a c t i o n methods; c r y s t a l s o f I are tri-
c l i n i c , s p a c e group P1, w i t h Z = 1, i n a u n i t c e l l of
dimension: a = 9 . 6 2 0 ( 7 ) , b = 1 0 . 7 7 9 ( 8 ) , c = 7.645(6) Ao,
a = 1 0 7 . 0 3 ( 7 ) , 8 = 1 0 8 . 8 8 ( 7 ) , y 72.71(8)O; c r y s t a l s o f
CAFFEINE 85

II are monoclinic, s p a c e group P2 / n , w i t h Z = 4 , i n a

u n i t c e l l of dimensions a = 1 2 . 4 0 k ( 8 ) , b = 2 9 . 6 5 2 ( 1 2 ) ,
c = 9.419(6) Ao, B = 108.39(7)O. The s t r u c t u r e s of I
3nd I1 have been s o l v e d from d i f f r a c t o m e t e r d a t a by
P a t t e r s o n and F o u r i e r methods and r e f i n e d by f u l l m a t r i x
l e a s t - s q u a r e s t o R = 0.046 f o r I and 0.090 f o r 11. Both
compounds c o n t a i n o c t a h e d r a l hexaaquametal (11) c a t i o n s ,
uncoordinated c a f f e i n e molecules and bromide a n i o n s i n
I and t r i i o d i d e and i o d i d e a n i o n s i n 11, h e l d t o g e t h e r
by a network o f hydrogen bonds. The t r i i o d i d e a n i o n s
are unsymmetrical [ 1 ( 1 ) - 1 ( 2 ) = 2.89, 1 ( 2 ) - 1 ( 3 ) = 2.95A0,
I ( 1 ) - I ( 2 ) - I ( 3) = 1 7 8 O ] , a r r a n g e d i n l i n e a r systems
w i t h a weak ' h e a d - t o - t a i l ' i n t e r a c t i o n , t h e d i s t a n c e
being o f 3.62 A O .

2. 6 Spectral Properties
2.6.1 U l t r a v i o l e t Spectrum

The W spectrum of c a f f e i n e i n methanol (Fig. 6 )

was scanned from 190 t o 440 nm, u s i n g DMS 9 0 .
Varian Spectrophotometer. It e x h i b i t e d a Amax
at 270 nm. Other r e p o r t e d W s p e c t r a l d a t a are
shown below:

Solvent nm E l % , 1 cm Ref.
'max -
Methanol 272 - 9
Trichloro- 278 - 10
ethylene

Ethanol 273 5 19 11

0.1N H C 1 272 470 11

0.1N H C 1 27 5 490 12

0.1N NaOH 275 490 12

2.6.2 I n f r a r e d Spectrum

The I R spectrum o f c a f f e i n e as KBr d i s c ( F i g . 7 )

w a s recorded on a P e r k i n E l m e r - 580 B I n f r a r e d
spectrophotometer t o which an I n f r a r e d d a t a
s t a t i o n is a t t a c h e d . The s t r u c t u r a l assignments
m
Q)

Fig.6 UV Spectrum of' CnFFeirle in /Ye!~anbl

 2.5 3.0 4.0 5.0 MICk&’5 6.0 zo 80 9.0 10 f2 14
100

wavenumber lob0
I

2500
.
2000 f800
1

f60d
1

f/oO fOm 8W
1 s50
88 MOHAMMAD UPPAL ZUBAIR ET AL.

have been c o r r e l a t e d with t h e following

frequencies Table 4 ) .
Table 4 . I R C h a r a c t e r i s t i c s of Caffeine.
-1
Frequency cm Assignment

3110, 2950 and CH s t r e t c h

CH3
1700 c=o
1650 C = N
Other c h a r a c t e r i s t i c bands are:
1600, 1550, 1480, 1455, 1430, 1400, 1360, 1285,
1235, 1185, 1020, 970, 860, 760 and 745.

The i n f r a r e d d a t a f o r c a f f e i n e were also reported

and are a s follows:
3100, 2970, 1700, 1660, 1550, 1480, 1360, 1240,
1020, 980, 750, and 610 cm-1 ( 9 ) .

P r i n c i p a l peaks are 1658, 1695 and 745 cm-I (11).

Major peaks 747, 1454, 1480, 1548, 1658 and
1698 cm-1 ( 1 2 ) . The region from 19-22 p i s
important f o r i d e n t i f i c a t i o n of c a f f e i n e (13).

2.6.3 Nuclear Magnetic Resonance Spectra

2.6.3.1 Proton Spectrum

The PMR spectrum of caffeine i n CDCl3

(Fig. 8 ) was recorded on a Varian ~ 6 0 - A ,
60 MHz NMR spectrometer using TMS
( t e t r a m e t h y l s i l a n e ) a s an i n t e r n a l
reference. The following s t r u c t u r a l
assignments have been made (Table 5 ) .

Table 5. PMR C h a r a c t e r i s t i c of C a f f e i n e
Croup Chemical S h i f t ( 6 ) ppm
1-N-CH3 3.53 s
3-N -CH 3.33 s
7-N-CH3 3.98 s
8-H 7.54 s
~~

s = singlet
c

A - _ . -
I 1 I I
I . . . . l . . . . I . . . I
3.0 2.0 1.0
 11
27,87 CH3

I 53

The structure o f c a f f e i n e f o r carbon c h e m i c a l s h i f t s .

CAFFEINE 91

O t h e r r e p o r t e d d a t a are 3 . 4 , 3 . 6 , 4.0 and

7.6 ppm (7,14). Stamm(15) has i n v e s t i -
&ed t h e a s s o c i a t i o n o f c a f f e i n e with
sodium benzoate and o t h e r compounds and
r e p o r t e d a l a r g e chemical s h i f t of t h e
NMR s i g n a l s o f t h e t h r e e methyl Kroups.
Also metal p o r p h y r i n / c a f f e i n e complexes
have been i n v e s t i g a t e d ( 1 6 ) .

2.6.3.2 13C-NMR Spectra

The n a t u r a l abundance C-13 NMFf n o i s e

decoupled and s i n g l e frequency o f f -
resonance decoupled (SFORD) s p e c t r a
( F i g s . 9 and 1 0 ) were o b t a i n e d at 20 M l z
on a Varian FT-80A, 80 MHz F o u r i e r
t r a n s f o r m NMR s p e c t r o m e t e r u s i n g a broad
band 1 0 mm probe. The sample w a s run a t
c o n c e n t r a t i o n C a 1-2 M i n d e u t r a t e d
chloroform w i t h t e t r a m e t h y l s i l a n e as an
i n t e r n a l r e f e r e n c e s t a n d a r d . The chemi-
c a l s h i f t s were measured at 5 KHz
s p e c t r a l width. The carbon chemical
s h i f t s are a s s i g n e d on t h e basis o f t h e
t h e o r y o f chemical s h i f t and SFORD
s p l i t t i n g p a t t e r n and i s shown i n Table 6.

Table 6. Carbon Chemical S h i f t s o f

Caffeine .
Carbon Multipli-
Chemical S h i f t ( 6 ) ppm
No.

c-2 151.69 S

C-4 148.73 S

c-5 107.55 S

C-6 155.35 S

C-8 141.53 d
C-10-CH3 29.70 9
C-11-CH3 27.87 q
C-12-CH3 33.54 9

s = s i n g l e t ; d = doublet; q = quartet.
1000
8d 0
EbO

Fly.9 '3C -NMR Norse -decoupled Spectrum oP Cp.Lf'eine in CU c/jr

 h
93
94 MOHAMMAD UPPAL ZUBAIR ET AL.

2.6.4 Mass Spectrum

The mass spectrum of c a f f e i n e obtained by e l e c t -
ron impact i o n i z a t i o n ( E I ) i s shown i n Fig. 11.
It was recorded on Finigan-Mat 1020 GC/Mass
spectrometer. The spectrum w a s scanned from 40
upto 440 a.m.a. Electron energy was 70 e V . The
mass s p e c t r a l data are shown i n Table 7.

Table 7 . The Most Prominent Fragments of

Caffeine.

m/e Relative i n t e n s i t y Fragment

I
194 loo Base peak (M+)

109 66
C5H7N3
82 37 -
67 54 -
55 80 -
Other mass s p e c t r a l d a t a were a l s o reported
( 9 $171 19411001 67" 661 , 1091661 , 821 391 42[ 281 ,
40[18] and 41[16]. A comparison of mass s p e c t r a l
d a t a o f theobromine, theophylline and c a f f e i n e
w a s reported by S p i t e l l e r and Friedmann (18).

Dunbor and Wilson have published an isotope mass

spectrum method f o r i d e n t i f i c a t i o n of t h e geogra-
p h i c a l o r i g i n of c a f f e i n e (19).

3. I s o l a t i o n of C afPeine
3.1 From Tea Leaves : Finely powdered t e a leaves (100 g )
are e x t r a c t e d with ethanol i n a soxhlet apparatus f o r
3 h r s (20). The c a f f e i n e so e x t r a c t e d i s then adsorbed
on magnesium oxide. It i s t h e n desorbed after treat-
ment with 10% H2SO4 and i s e x t r a c t e d i n t o chloroform
and i s r e c r y s t a l l i s e d .

3.2 From Green Coffee : A procedure f o r t h e e x t r a c t i o n of

c a f f e i n e from green coffee has been reported ( 2 1 ) .
Green c o f f e e beans were steamed f o r 2 t o 3 hours t o
e q u i l i b r a t e t h e water content between 40 and 50%.
100.0

1 55

50.0
1- 1
J
67
1 I

-- -1J4- I --
203 - - - - .222- .
1. .I
- - II
60 100 120 140 160 180 200 220 240

F i g . 11. EX-fiass Spectrum of C a f f o i w .

96 MOHAMMAD UPPAL ZUBAIR ET AL.

Caffeine was then e x t r a c t e d with CHC13 and c a f f e i n e

upto 95% of t h e t h e o r e t i c a l amount was recovered.

3.3 A process f o r t h e e x t r a c t i o n of c a f f e i n e from c o f f e e

beans by leaching w i t h water has been developed ( 2 2 ) .
The highest y i e l d o f 90.4% was obtained when t h e
average c o f f e e p a r t i c l e s i z e w a s 1.4095 mm, t h e water/
coffee r a t i o of 9:1, a t 7 5 O C and t h e e x t r a c t i o n was
c a r r i e d out f o r t h i r t y minutes.

3 . 4 Natural c a f f e i n e is a l s o obtained as a by-product,

from t h e d e c a f f e i n a t i o n process of coffee and t e a .
Some of t h e s e method a r e summarised and presented i n
Table 8.

3.5 Caffeine can a l s o be obtained from various sources.

Table 9 summarises t h e source, b o t a n i c a l c h a r a c t e r i -
s t i c s , occurrance and t h e caffeine/theobromine content
i n each case ( 3 7 ) .
CAFFEINE 97

Table 8. I s o l a t i o n o f C a f f e i n c by Decaf f e i n a t i o n Processes.

Material Solvent -
Ref.

Green c o f f e e F l u o r i n a t e d hydro- 23
beans* carbons

Coffee* and t e a With o i l s such as 24

extracts (also corn o i l , o l i v e
o i l caffeine). o i l , safflower o i l .

Coffee base* Benzyl a l c o h o l 25

I n s t a n t coffee - 26

Black tea* With supercr it 27

carbon dioxide.

Tea waste - 28

Green coffee* Alcohols 29

Caffeine" S u p e r c r i t carbon 30
dioxide with
water .
Coffee and t e a With s u p e r c r i t 31
carbon dioxide.

Roast ed co f f ee* With super c ri t 32

(aqueous carbon dioxide and
extracts of) recovery of aroma.

Raw coffee* Superc rit carbon 33

dioxide.

Coffee* Propane and butane 34

Coffee* Aqueous ( s o l u t i o n s ) 35
Coffee" Aqueous (steam) 36
 Table 9. Plant Sources of Caffeine

Caffeine Information about t h e p l a n t

Source (theobromine) Main region
con%ent ( 4 % ) Botanical c h a r a c t e r i s t i c s Native region
of c u l t i v a t i o n

Seeds 0,l-0.8 Tree, 4-15 m i n height when Mexico, Central and West Africa
(beans) of (0.2-2.7) growing w i l d , but pruned t o South America
Thdobroma 6 m under c u l t i v a t i o n . F r u i t s
cacao furrowed l e a t h e r y pods, shaped
(stercdi- l i k e l a r g e cucumbers and con-
.
aceae ) t a i n i n g 20-25 cream coloured
almond-shaped seeds.

Seeds 0.8-2.4 C. arabica: Shrub o r s m a l l E a s t Africa South America

(beans) of t r e e , 4-6 m i n height when
Coffea arabica, growing wild but pruned t o
C. Ziberica, 2-3 m under c u l t i v a t i o n .
C. excelsa or Fruits ellipsoidal berries
C. robusta ( c h e r r i e s ) , r e d t o . dark
(Rubiaceae) purplish, containing 2 s i l v e r y
skinned seeds (Beans) within
t h e pulp.
Continued Table 9 .

Caffeine
Information about t h e p l a n t
Source (theobromine)
Main region
content ( % ) Botanical c h a r a c t e r i s t i c s Native region
of c u l t i v a t i o n

Seeds 2-6 Large, climbing o r creeping Amazon v a l l e y of S t a t e of

(beans) of p l a n t with smooth stem. Brazil Amazons,
Paul linia F r u i t ovoid, nut-like, about Brazil.
cupana as l a r g e as a grape and
(Sapindaceae) u s u a l l y containing 1 seed,
t h e s i z e o f a hazel-nut, with
white, mealy covering.

Bark of 2.7 Extensive, woody l i a n a . Stem Colombia, Equador, Not c u l t i -

Pau l linia yoco s t o u t , up t o 12 cm i n diameter Peru vated.
(Sapindaceae) a t t h e base, with a milky-
white a s t r i n g e n t sap.

Leaves of 1.1-1.g Tree, 20-30 m i n height when Large a r e a i n v a l l e y s Paraguay and

I l e x paragua- growing w i l d , 4-6 m under of t h e Parana, South of
yensis c u l t i v a t i o n . Leaves Persis- Paraguay and Upper Brazil
( Aqui f o l i a c e a e ) t e n t a l t e r n a t e , f i n e l y toothed Uruguay r i v e r s .
at t h e margin and dark green
i n colour.
Continued Table 9.

Caffeine
Information about t h e p l a n t
Source (theobromine) Kain region
content ( I ) B o t a n i c a l c h a r a c t e r i s t i c s n a t i v e region
of c u l t i v a t i o n

Leaves and 0.1-1.6 Shrub o r s m a l l tree, c l o s e l y E a s t c o a s t of Not c u l t i-

shoots of r e l a t e d t o I. paraguayensis North America vat ed .
IZex vomitoria from V i r g i n i a
( Aqui f o l i a c e a e ) t o Mexico.

Leaves of 3-4 Shrub o r s m a l l t r e e , 9-15 m A s s a m , China, S r i Lanka,

Camellia i n height when growing wild, Japan. I n d i a , China
sinensis about 1.5 m under c u l t i v a t i o n . Jaoan .
(Theaceae) Leaves a l t e r n a t e , e l l i p t i c a l
on s h o r t s t a l k s , l e a t h e r y and
w i t h t o o t h e d margins.

Seeds ( n u t s ) 1.5-3.5 C. acwninuta: Slender t r e e , Southern Nigeria Xest Africa

of Cola n i t i a k 6-9 m t a l l , t r u n k commonly
and C. acwninata branching near t h e base. Bark
(Stercdiaceae). rough and corky, grey i n colour.
Most o f t h e caf- F o l i a g e s p a r s e , confined t o t h e
feine i n kolas t i p s o f branches. F r u i t c o n s i s t s
not derived from of up t o f i v e f o l l i c l e s borne at
t h e k o l a nut but r i g h t angles t o t h e s t a l k or s l i -
added i n t h e g h t l y downwards. F o l l i c l e s , rus-
form o f s y n t h e t i c s e t , rough t o t h e touch. Each f o l -
caffeine. l i c l e c o n t a i n s up t o 14 pink o r r e d
seeds, covered with white s k i n .
Cotyledons 3-6.
 Continued Table 9.

Caffeine
Information about t h e p l a n t
Source (theobromine)
content ($) Botanical c h a r a c t e r i s t i c s Native r e g i o n Main r e g i o n
of c u l t i v a t i o n

I n Africa, c. nitida: More r o b u s t t r e e , S i e r r a Leone, West A f r i c a ,

t h e seeds 9-12 m h e i g h t , t r u n k Ivory coast, West I n d i e s
a r e used unbranched for at least 1 m. Ghana South America
inainly as Bark smooth w i t h f i n e l o n g i -
a msstica- t u d i n a l cracks. Foliage
tory , but dense, n o t c o n f i n e d t o t h e
+ t h e y . Kay t i p s o f t h e branches. F r u i t
0, &so be used c o n s i s t s o f up t o f i v e
f o r prepara- f o l l i c l e s , u s u a lly bent
t i o n of a upwards. F o l l i c l e s g r e e n ,
drink (Cola). smooth t o t h e t o u c h . Seeds
up t o 1 0 i n number, w i t h 2
cotyledons and of t h e shape
and s i z e of horse
chestnuts .
102 MOHAMMAD UPPAL ZUBAIR ET AL.

4 . Synthesis

4.1 Route 1

Uric acid with methyliodide gave 1,3,7-trimethyluric

acid, which was converted into 8-chloroderivative and
its subsequent dehalogenation afforded caffeine
(38-40).

0
H
N

0 L% H

Uric acid
N
H
)co
CH31
NaOH >

C'H3
1,3,7-Trimethyluric acid
CAFFEINE 103

I + . 2 Route 2

C a f f e i n e h a s a l s o been s y n t h e s i s e d from uric a c i d v i a

a n o t h e r r o u t e (41-42).
0 0
~ ~ ; > ~ ~ ~ ) ; H ; c H o

>L2-
0

-~2~~~
H H N
H H
0 0
HCOM12 NHCHo

0 hYICOFHCH0 0 MCONHCHO
H H
0 0

I
cH3 Caffeine
104 MOHAMMAD UPPAL ZUBAIR ET AL.

4 . 3 Route 3

A d i f f e r e n t r o u t e f o r t h e p r e p a r a t i o n of c a f f e i n e from
u r i c a c i d has been r e p o r t e d ( 4 3 ) . Uric a c i d is f i r s t
c o n v e r t e d i n t o 8-methylxanthine by t h e a c t i o n o f
a c e t i c anhydride (44-46), a p r o c e s s which i n v o l v e s t h e
i n t e r m e d i a t e formation o f a d i a c e t y l d e r i v a t i v e . The
secondary amino groups are t h e n methylated i n an
a l k a l i n e s o l u t i o n t o g i v e 1,3,7,8-tetramethylxanthine.
F i n a l l y , t h e methyl group o r i g i n a l l y i n t r o d u c e d i n t h e
.
8 - p o s i t i o n i s e l i m i n a t e d ( 47 )

H I

I
0

-co*

I
CH3 Caffeine
CAFFEINE 105

4 . 4 Route 4
A l k y l a t i o n o f x a n t h i n e d e r i v a t i v e s has a l s o been
c a r r i e d o u t f o r t h e s y n t h e s i s of c a f f e i n e . M e t h y l a t i o n
o f theobromine g i v e s c a f f e i n e (48-50), w h i l e 1-
methylxanthine h a s a l s o been c o n v e r t e d t o c a f f e i n e v i a
t h e o p h y l l i n e (48,49,51,52). 1,7-Dimethylanalogue
( p a r a x a n t h i n e ) on a l k y l a t i o n a f f o r d s c a f f e i n e ( 5 3 ) ,
w h i l e x a n t h i n e on t r e a t m e n t w i t h a n e x c e s s o f methyla-
t i n g agent g i v e s r i s e t o a ' c a f f e i n e d e r i v a t i v e
(41,49,54,55).
4.5 Route 5

C a f f e i n e i s u s u a l l y commercially s y n t h e s i z e d by T r a u b ' s
methods; ( A and B, g i v e n below: ) ( 56)
(A)

CH3

k-.--.@
NaN1i2
/N H - C H 3
HOC
o= c
'NH-CH~
+
CN'
%I$

- 0
1
NH2

H C ~ ~
0
~ Z n / i%
H
0
2
a3

S ~

i i . HCOOH, A
0

1h3
CH31/E t OH
CH a >
N Caffeine
106 MOHAMMAD UPPAL ZUBAIR ET AL.

(B) In this method 4-amino-5-formamido-l,3-dimethyl-

uracil is cyclised and methylated in one step in
sodium ethoxide containing methyliodide to
afford initially theophylline and subsequently
caffeine (57).

0 0 CHz

I
cH3 (33

4.6 Route 6
It involves ring closure of 5-ethoxycarbonyl-1-methyl-
4-( N-methyl) ureidoimidazole, and subsequent methyla-
tion in alkaline medium gives caffeine (58).

(33
Caffeine
CAFFEINE 107

5. B i o s y n t h e s i s
It h a s been r e p o r t e d ( 5 9 - 6 2 ) t h a t b i o s y n t h e s i s o f p u r i n e
r i n g system probably proceeds i n n e a r l y t h e same manner
i n a l l organisms as shown i n Scheme I . I n i t i a l l y , t h i s
pathway was proposed f o r micro-organisms and animals b u t
r e c e n t evidence s u g g e s t s t h a t it may a l s o hold good f o r
h i g h e r p l a n t s . I n o s i n e monophosphate i s t h e f i r s t com-
pound in t h i s b i o s y n t h e t i c pathway t o c o n t a i n a complete
p u r i n e r i n g system (59) and it h a s a c e n t r a l p o s i t i o n i n
t h e purine m e t a b o l i s m . It can be converted t o x a n t h o s i n e
monophosphate i n an " I + dependent r e a c t i o n as shown
below:

OH

RIBOSE R I BOSE-P

I n o s i n e monophosphate Xanthosine monophosphate

Xanthine, which o r i g i n a t e s from x a n t h o s i n e monophosphate

by e l i m i n a t i o n o f phosphate and r i b o s e , i s t h e s t a r t i n g
material f o r formation o f c a f f e i n e and o t h e r p u r i n e s . I n
t h e p l a n t Coffea arabica, t h e x a n t h i n e is c o n v e r t e d , t o
theobromine and t o c a f f e i n e , e i t h e r v i a N3-methylxanthine
108 MOHAMMAD UPPAL ZUBAIR ET AL.

p-o'it=? OH

5-Phosphoribosyl
OH dH

5-Phosphoribosyl-
OH P-Ribose

Glycinamide
-1-- pyropkosphate 1- amine ribonucleotide

- -
H H
H272
,Cao
o=c,
,!j

NH
H2r"'$
NH=C,
NH
-;
.

ZHN
1
:
)I
I
P-Ribose I
P-Ribose P-Ribose

a-N-Formylglycinamide- N-Formylglycinamide 5-Aminoimidazo-

ribonucleotide ribonucleotide le-ribonucleotide

H:'x:)
P I
+

I
P-Ribose P-Ribose P-Ribose
5-Aminoimidazole- 5-Aminoimidazole-4- 5-Aminoimidazole-4
4-carboxylic acid N-succinocarboxamide -carboxamide
ribonucleotide ribonucleotide ribonucleotide
OH

P-Ribose

5-Formamidoimidazole-4- Inosine-5-monophosphate
carboxamide-ribonucleotide

Scheme I. Biosynthesis of inosine monophosphate.

CAFFEINE 109

o r N 7-methylxanthine Scheme 11. The methyl groups

o r i g i n a t e from methionine ( 6 3 - 6 5 ) .

0 CHJ CH
3 3
H N -Methylxanthine CH3 R I

0
H I I
Xanthine \ The0bromin e
CH3
Caffeine

N 7 -Methylxanthine
Scheme 11. Formation of theobromine and c a f f i n e from xanthine.

6 . Pharmacokinetics and Metabolism

The absorption of c a f f e i n e from t h e g a s t r o i n t e s t i n a l t r a c t
i s r a p i d b u t i r r e g u l a r (67,68). It i s d i s t r i b u t e d i n
various t i s s u e s of t h e body i n approximate proportion t o
t h e i r water content ( 6 3 ) . ‘The absorption o f c a f f e i n e is
pH-related, an i n c r e a s e i n pH increases i t s absorption
( 6 7 ) . Within t h e t i s s u e , c a f f e i n e i s r a p d i l y broken down
(69), involving metabolite r e a c t i o n s , such as N-demethyla-
t i o n and oxidation, and r i n g cleavage ( 6 7 ) . The drug
metabolising enzymes of t h e l i v e r a r e stimulated following
t h e ingestion o f l a r g e amount o f c a f f e i n e ( 6 7 ) .

The degree o f c a f f e i n e degradation and degradation products

excreted i n t h e u r i n e o f d i f f e r e n t species seems t o vary
 4;
As pa rt ic acid

C 1 Fragments
\ 0

N/
\ Fragments

H1-CHp-C 0 0 H
Glycine

Amide N of g lut amin e

Fig. 12: Origin of various atoms in the biosynthesis of caffeine.

CAFFEINE 111

considerably ( 7 0 ) . Blood l e v e l c o n c e n t r a t i o n i n four

a n i m a l s p e c i e s , followinr: o r a l a d m i n i s t r a t i o n o f 25 mg/kF:
[1-C1’I] c a f f e i n e , have been reported by Burger ( T O ) ,
(Table 1 0 ) . The plasma h a l f - l i f e f o r humans i s between
4 t o 10 hours ( 6 7 ) .
14
Table 1 0 . Plasma Concentration o f [1-C ] Caffeine i n
D i f f e r e n t A n i m a l Species.

Rat Hamster Rabbit Rhesus monkey

Half-life ( h )
Radioactivity 5.4 3.5 11.0 19.0
Caffeine 2.8 3.1 3.7 2.8

Absorption h a l f -
time ( h )
Radioac t i v i t y 0.1 0.5 0.7 0.9
Caffeine 0.1 0.1 0.7 -
Peak plasma con-
c e n t r a t i o n of 18 18 22 13
c a f f e i n e (pg/ml).

Caffeine metabolism i n t h e rat l i v e r s l i c e s and p o s t n a t a l

developing rats has been s t u d i e d (71). Rao e t al, (1973)
have proposed a b i o t r a n s f o r m a t i o n r o u t e f o r c a f f e i n e i n
rats (72) (Scheme 111).
112 MOHAMMAD UPPAL ZUBAIR ET AL.

Caffeine >--
Hydroxylationl
1,3,7-Trimethyldihydrouric acid

I.
( unchanged, 9%) Reduct'on (11.4%)

dehydrogenation
N-demet hylat ions N-demethylations

1,3,7-Trimet hyluric acid

I
(trace) >

I
J. 3-Methyluric acid (trace)
Theobromine ( 5.1%)

Paraxanthine (8.a%)
Oxidat ion
Theophylline (1.2%)

3,6,8-Trimethylallantoin (1.3%)
I
I
I Oxidat ion
I
I
V
1,6,8-Trimethylallantoic acid
I
hydrolysis

I
J.
Glyoxylic acid
Met hylur ea
1,3-Dirnethylurea

Scheme 111. Biotransformation of caffeine in rat.

CAFFEINE 113

An i n t e r e s t i n g sulphur containinE metabolite o f c a f f e i n e

was i s o l a t e d from t h e u r i n e of mouse, r a t , r a b b i t and
horse ( 7 3 ) , and i s shown i n Scheme I V .

ru 0

0 n
Caffeine U

H3Nx-3
II
0 CH S-CHij
2-11

0’

I
“i
N/

I1 I11
Scheme I V . The sulphur metabolite of c a f f e i n e .

I n case o f humans, u s u a l l y 45% o f a dose is excreted i n

u r i n e i n 48 hours as 1-methylxanthine (67) and 1-methyluric
a c i d (67,74). Other breakdown products excreted i n t h e
u r i n e include, t h e o p h y l l i n e , lY7-dimethylxanthine,
7-methylxanthine and 1,3-dimethyluric a c i d , along with
some unchanged c a f f e i n e ( 6 7 ) ,

Further s t u d i e s on human metabolism of (1-methyl-14C) and

(2-14C)caffeine have been reported (75,76). Radiolabelled
c a f f e i n e was administered ( 7 5 ) o r a l l y a t 5 mg/kg t o a d u l t ,
male volunteers. Blood, s a l i v a , expired C02, u r i n e , and
feces were analysed f o r t o t a l r a d i o l a b e l l e d equivalents of
114 MOHAMMAD UPPAL ZUBAIR ET AL.

c a f f e i n e and i t s metabolites. High-performance l i q u i d

chromatography (HPLC) was t h e main technique used f o r t h e
separation of c a f f e i n e and i t s metabolites with quantita-
t i o n by l i q u i d - s c i n t i l l a t i o n counting. The h a l f - l i f e of
c a f f e i n e i n both serum and s a l i v a was approximately 3 h r s . ,
with t h e amount of c a f f e i n e i n s a l i v a samples almost 65 t o
85% of t h a t found i n serum samples. The main metabolites
found i n serum and s a l i v a were t h e dimethylxanthines,
paraxanthine, theophyllinc and theobromine ( 7 7 ) . It has
been reported (76) t h a t demethylation of t h e 3-methyl
groups seems t o be t h e most important pathway i n man, and
t h e plasma concentration of t h e dimethylxanthines depend
on t h e i r urinary excretions a s w e l l as t h e i r own metabolism
(76). Effect of c a f f e i n e on alcohol metabolism(78), basal
metabolism (75) and i t s e f f e c t on exercise performance
have a l s o been s t u d i e d (79).
Caffeine metabolism i n sheep h a s a l s o been studied (80-82).
7. Methods o f Analysis
7 . 1 Elemental Analysis

The elemental composition of c a f f e i n e i s : (83)

Element % Theoretical
C 49.48 %
H 5.19 %
N 28.85

0 16.48 %
7.2 I d e n t i f i c a t i o n T e s t s

1. Addition of t a n n i c a c i d s o l u t i o n t o c a f f e i n e
s o l u t i o n gives a p r e c i p i t a t e , which dissolves on
f u r t h e r addition of t h e reagent ( 8 4 ) .
2. Addition of iodine s o l u t i o n and hydrochloric a c i d
t o c a f f e i n e s o l u t i o n gives a brown p r e c i p i t a t e ,
which n e u t r a l i s e s on addition of sodium hydro-
c h l o r i d e (84).

3 . Reaction of c a f f e i n e w i t h potassium c h l o r a t e i n
hydrochloric a c i d , and subsequent exposure t o
ammonia, gives purple colour, which disappears on
addition of a s o l u t i o n of a fixed a l k a l i (85).
CAFFEINE 115

4 . Addition of gold c h l o r i d e s o l u t i o n t o c a f f e i n e
s o l u t i o n a f f o r d s small rods ( 8 6 ) .

5 . Addition of mercuric c h l o r i d e s o l u t i o n t o c a f f e i n e
s o l u t i o n gives long needles ( 8 6 ) .

6. Comparison of t h e i n f r a r e d spectrum of t h e c a f f e i n e
sample with t h a t of a reference s t a n d a r d i s a l s o
employed ( 8 5 ) .

7.3 T i t r i m e t r i c
Several methods have been reported f o r t h e analyses of
c a f f e i n e by acid-base t i t r a t i o n procedures , e i t h e r by
using d i f f e r e n t i n d i c a t o r s (87-100) o r by potentio-
metric methods (101-108), f o r t h e end-point d e t e c t i o n .

An iodometric t i t r a t i o n method (88) w a s developed f o r

t h e determination of c a f f e i n e . It i s mixed with H2SO4
(1:l) and I B r s o l u t i o n , and t h e mixture i s d i l u t e d t o
100 m l . After f i l t r a t i o n , t o a p o r t i o n of t h e
f i l t r a t e i s added K I s o l u t i o n , and t h e equivalent
amount of iodine l i b e r a t e d i s t i t r a t e d with Na2S204,
using s t a r c h s o l u t i o n as i n d i c a t o r . A blank is a l s o
c a r r i e d out a t t h e same t i m e .

Collado e t a l . , have reported a method (87) f o r t h e

determination o f c a f f e i n e , phenazone, phenacetin and
phenobarbitone, i n analgesic t a b l e t s . For t h e estima-
t i o n of c a f f e i n e , phenazone is p r e c i p i t a t e d with
p i c r i c a c i d and f i l t e r e d o f f , and c a f f e i n e i s
determined i o d i m e t r i c a l l y i n a c i d s o l u t i o n .

Caffeine can be determined i o d i m e t r i c a l l y from ice-

cream and cocoa admixtures (109,110). 40 g of t h e
sample is heated with 1 0 m l o f water, and 1 ml of 15%
aqueous NaOH, on a b o i l i n g water bath f o r 30 minutes.
The mixture i s cooled and 3 m l of 30% Pb(N03)2 i s
added. It i s d i l u t e d t o 100 m l with water and i s
f i l t e r e d . 1 0 M l o f t h i s sample i s used t o determine
.
c a f f e i n e t i t r imet ri c a l l y

The U.S. Pharmacopoeia1 method (108) c o n s i s t s i n

dissolving about 400 m g of f i n e l y powdered c a f f e i n e
i n 40 m l of a c e t i c anhydride. After mixing with 80 m l
of benzene it i s t i t r a t e d with 0.1N p e r c h l o r i c a c i d ,
determining t h e end-point potentiometrically.
116 MOHAMMAD UPPAL ZUBAIR ET AL.

Caffeine has been assayed f o r another non-aqueous

t i t r a t i o n procedure ( 9 3 ) . It is dissolved i n w a r m
benzene and a f t e r cooling, couple o f drops of a s u i t -
able i n d i c a t o r , such as Sudan I V o r Nile b l u e A , are
added before t i t r a t i o n with 0.1N HCL04 i n g l a c i a l
a c e t i c acid.

Caffeine i n cacao s h e l l s has a l s o been determined (100)

by non-aqueous t i t r i m e t r i c method. About 2 g of t h e
ground and de-fatted sample i s mixed with magnesium
oxide and water. The mixture i s heated on a water
bath f o r 30 minutes and then it i s e x t r a c t e d i n t o
chloroform i n a soxhlet apparatus. The chloroform
e x t r a c t is d r i e d at lO5'C f o r 25 minutes and t h e
r e s i d u e is first t i t r a t e d f o r t o t a l theobromine and
c a f f e i n e , and t h e n f o r theobromine only.

7.4 Spectrophotometric
7.4.1 Colorimetric
A sample containing 100 mg i s dissolved i n 100 m l
o f water and i s f i l t e r e d (111). A n a l i q u o t o f
5 m l i s d i l u t e d t o 25 m l and 1 ml of d i l u t e
hydrochloric a c i d , and 1 m l of 10% molybdophos-
phoric a c i d are added t o it. The mixture after
heating on a water b a t h i s c h i l l e d i n i c e and
centrifuged. The p r e c i p i t a t e is washed and
dissolved i n 25 ml o f acetone and t h e e x t i n c t i o n
is measured a t 440 mu. The r e s u l t s with
compounded tablets show recoveries o f c a f f e i n e ,
100.2% o f t h e o r e t i c a l amount.

C o r t e ' s method (112,113) c o n s i s t s i n l i b e r a t i n g

c a f f e i n e w i t h concentrated H2SO4, followed by
e x t r a c t i o n w i t h CHC13. It i s then estimated by
a modified c o l o r i m e t r i c method involving
formation o f i t s periodide ( 1 1 4 ) .

I n another method (115) c a f f e i n e (110 t o 400 pg)

i s dissolved i n a f r e s h l y prepared s o l u t i o n of
0.5 m l of acetylacetone and 5 m l o f 2N NaOH.
T h i s mixture i s heated at 80°C, and a f t e r cooling,
a s o l u t i o n o f p-dimethylaminobenzaldehyde and
20 m l o f conc. H C 1 are added. The mixture i s
f u r t h e r heated at 8OoC, and after cooling, 10 m l
o f water i s added and e x t i n c t i o n of t h e r e s u l t i n g
blue s o l u t i o n i s measured a t 615 mu.
 117
CAFFEINE

E f f o r t s t o optimise conditions (116) have

r e s u l t e d i n a n improved colorimetric method (117)
f o r t h e estimation of c a f f e i n e .

A small amount of c a f f e i n e is dissolved i n a

mixture of 3% aqueous a c e t i c a c i d s o l u t i o n , and
10% aqueous pyridine s o l u t i o n . The mixture i s
then made up t o 18 m l with water and 2 ml of
N a O C l s o l u t i o n i s added. Add 2 m l of 0.1N
Na2S203, followed by 3 m l of 1 N N a O H , s o l u t i o n
and d i l u t e t o 50 ml with water. The e x t i n c t i o n ,
i n 4 cm c e l l i s measured a t 460 mp.

Iodine has a l s o been employed as a c o l o r i m e t r i c

reagent f o r t h e determination of c a f f e i n e (118).
3N i o d i n e (1 ml) s o l u t i o n i s mixed with 1%
s o l u t i o n of c a f f e i n e ( 5 m l ) followed by 50%
H2SO4 ( 0 . 5 m l ) and i s set a s i d e f o r 1 0 minutes.
The p r e c i p i t a t e i s f i l t e r e d and washed before
dissolving it i n acetone. Its e x t i n c t i o n i s
measured a t 525 mp.

A colorimetric method f o r t h e determination of

c a f f e i n e i n pharmaceutical preparations has been
developed (119-121). The t e s t s o l u t i o n i s
t r e a t e d with an equal volume of 10N NaOH a t 12OoC
t o convert c a f f e i n e i n t o c a f f e i d i n e . It i s t h e n
coupled with d i a z o t i s e d s u l p h a n i l i c a c i d and t h e
e x t i n c t i o n is measured a t 451 mp. Other drugs
present i n usual c a f f e i n e pharmaceutical prepara-
t i o n s do not i n t e r f e r e .

Caffeine present i n beverages can a l s o be

estimated c o l o r i m e t r i c a l l y (121-122). It i s
e x t r a c t e d from t h e beverage by t h e known (123)
method and is t r e a t e d with 296 s o l u t i o n o f
malonic a c i d i n a c e t i c anhydride. The mixture
i s heated a t 90°C, and a f t e r cooling it i s made
up t o 25 m l with methanol. The e x t i n c t i o n of t h e
r e s u l t i n g greenish-yellow s o l u t i o n i s measured
a t 430 nm.

7. 4.2 U l t r a v i o l e t

Spectrophotometric methods f o r t h e estimation of

c a f f e i n e i n pharmaceutical preparations and i t s
mixtures with o t h e r drugs and compounds have been
developed (124-129). These i n v a r i a b l y involve
118 MOHAMMAD UPPAL ZUBAIR ET AL.

preliminary separation of c a f f e i n e , before i t s

spectrophotometric estimation.

An i n d i r e c t spectrophotometric method f o r t h e
determination of c a f f e i n e i n quaternary mixtures
such as NaOBz, p-EtoC6H)+NHAc, aminopyrine and
c a f f e i n e has been reported (1.29). The percentage
of these components can be estimated by a
compact c a l c u l a t i o n scheme a f t e r determination of
e x t i n c t i o n c o e f f i c i e n t s a t t h e given wavelengths.

Several u l t r a v i o l e t spectrophotometric methods

have been developed f o r t h e estimation of
c a f f e i n e i n coffee and decoffeinated coffee
( 130-136), t e a ( 137-141) and beverages (142-146).

Caffeine can a l s o be determined i n b i o l o g i c a l

f l u i d s spectrophotometrically (147). O.1N-NaOH
i s used t o e x t r a c t c a f f e i n e , and a mixture of
s o l i d N a C l and Na2SOb is added t o enhance t h e
e x t r a c t i o n o f c a f f e i n e i n t o ether-chloroform.
The c a f f e i n e i s then t r a n s f e r r e d t o an aqueous
a c i d i c s o l u t i o n , t h e s o l u t i o n i n t h e reference
c e l l i s made strongly a c i d , and t h e contents of
t h e sample c e l l are adjusted t o pH 1.3.

7.4.3 Infrared
I n f r a r e d spectrophotometric method has been used
(148,149) f o r t h e a n a l y s i s of caffeine. A pro-
cedure f o r t h e determination of c a f f e i n e i n
pharmaceutical preparation containing aminopyrine
and phenacetin has been reported (148), and t h e
region 650 t o 400 cm-l o f t h e spectrum, i n KBr
d i s c , w a s used f o r r a p i d and simultaneous
determination of t h e t h r e e components.

7.4.4 Nuclear Magnetic Resonance

A simultaneous q u a n t i t a t i v e NMR method f o r t h e
a n a l y s i s of a s p i r i n , phenacetin and c a f f e i n e i n
pharmaceutical preparations has been reported
(150). The method involves comparing t h e
i n t e g r a l s of t h e CH2-signal ( a t 6 ppm) of
piperonaldehyde, used as an i n t e r n a l standard,
w i t h t h o s e of a s p i r i n methyl s i n g l e t (2.3 ppm) ,
CAFFEINE 119

phenacetin, e t h y l t r i p l e t ( a t 1 . 3 ppm) and

c a f f e i n e methyl s i n g l e t ( a t 3.4 ppm). The
average percent recoveries and standard deviations
were 95.61 f 0.37, 96.34 k 0.47 and 101.26 -+ 1.46
f o r a s p i r i n , phenacet i n and c a f f e i n e respectively.

MMR-shift technique i n forensic chemistry f o r

t h e analyses of mixed samples of c a f f e i n e has
been reported (151). NMR s t u d i e s o f metal
porphyrin c a f f e i n e complexes (151) and associa-
t i o n o f c a f f e i n e with sodium benzoate have a l s o
been reported (152,153).

7.4.5 Mass Spectrometric

A new and simple a n a l y t i c a l method using d i r e c t -
i n l e t chemical i o n i s a t ion mass spectrometry, has
been reported (154) f o r simultaneous determina-
t i o n of c a f f e i n e and o t h e r components of a n a n t i -
cold drug. These components were detected as
quasimolecular i o n s , and were q u a n t i t a t e d by
using an accumulation program.
2
I n another method (155) B E = constant, l i n k e d
scan has been used as a t o o l f o r q u a n t i f i c a t i o n s
with reversed-geometry mass spectrometers. D-
l a b e l l e d analogs may be used as i n t e r n a l
standards, t h u s providing very simple clean-up
procedures. The method i s applicable t o c a f f e i n e
present i n beverages.

Caffeine w a s analysed together with o t h e r drugs

l i k e a c e t y l s a l i c y l i c a c i d and phenacetin by
mass spectrometry (156).

7.4.6 Photo-nephlometric
Caffeine can be estimated i n t e a by using photo-
nephlometer (157). . A c a l i b r a t i o n curve i s
obtained by mixing 2 ml o f 0.01M sodium tungsto-
phosphate, 4 m l of 2.5M HNO3 and 0.5 t o 2.5 m l
of 0.001M c a f f e i n e s o l u t i o n . It i s t h e n d i l u t e d .
t o 50 m l and examined i n a photo-nephlometer.
Other compounds present i n tea do not cause any
i n t e r f e r e n c e . With some modifications, t h i s
120 MOHAMMAD UPPAL ZUBAIR ET AL.

procedure can be used f o r t h e determination of

c a f f e i n e i n presence of various d r u g s .

7.5 Coulometric
Kalinowska has used coulometric technique f o r t h e
estimation of c a f f e i n e (158-162). About 0.05 of
c a f f e i n e i s dissolved i n hot water, and then mixed
w i t h 6 m l o f 30% KOH. The mixture i s f u r t h e r heated
i n a b o i l i n g water b a t h , a f t e r cooling and d i l u t i o n
w i t h water, it i s n e u t r a l i s e d with hydrochloric a c i d .
About 1 m l o f t h i s s o l u t i o n i s used f o r t h e determina-
t i o n o f c a f f e i n e with coulometrically generated
c h l o r i n e , with a c u r r e n t o f 5 mA. The end-point i s
determined by t h e dead-stop end method. The c o e f f i -
c i e n t o f v a r i a t i o n w a s about 0.5%.

7.6 Polarographic

Caffeine, although not reducible a t dropping-mercury

e l e c t rode can be determined polarographically a f t e r
i t s oxidation w i t h bromine ( 1 6 3 ) . The wave h e i g h t s
of t h e r e s u l t i n g s u b s t i t u t e d parabanic a c i d s are then
evaluated. This method i s q u i t e s e n s i t i v e and t h e
results obtained are within +- 3%. B a r b i t u r a t e s ,
a c e t y l s a l i c y l i c a c i d , ephedrine, codeine, papaverine
and d i g i t o x i n do not i n t e r f e r e ; however, phenazone,
phenacetin and amidopyrine should be removed b e f o r e
t h e reaction.

In another method (164), t h e c a f f e i n e i s analysed by

anodic d i f f e r e n t i a l phase voltametry a t a glassy C
e l e c t r o d e , t h e a e t e c t i o n l i m i t i s 0.5 ppm at pH 1.2.
Oxidation products o f c a f f e i n e are i d e n t i f i e d by
cathodic d i f f e r e n t i a l pulse polarography.

7.7 Ring-Oven (Micro)

A r e l a t i v e l y s p e c i f i c colour r e a c t ion between chloro-

form e x t r a c t o f c a f f e i n e , a l k a l i n e acetylacetone
s o l u t i o n and a c i d p-dimethyl aminobenzaldehyde solu-
t i o n , has been used for t h e micro determination o f
air-borne p a r t i c l e s of c a f f e i n e . It i s possible t o
determine 0.5 pg of c a f f e i n e by t h i s method with a
mean e r r o r of -+ 3% (165).
Table 11. Parameters Used f o r Paper Chromatography of Caffeine

No. support Developing Solvent Detection Ref.

1. S & S. 2043 Consisted of s a l i c y l i c a c i d Chromatogram immersed i n 1% 169

Paper impregnated ( 0 . 3 g) dissolved i n AgNO3 d r i e d and immersed
with ethanolic butanol ( 30 m l ) ; t h e solu- i n 0.5% aqueous K 2 C r 2 0 p .
s a l i c y l i c acid t i o n being t r e a t e d dropwise
( 0 . 3 g i n 30 ml) with H20, t o t h e f i r s t
and dried. turbidity.

2. Whatman DE20 Develop with 0.2N-aq. W light 170

Anion-exchange ammonia f o r 105 minutes.
paper.

3. Whatman No. 4 Mobile phase w a s a xylene- -- 171

paper tetralin-n-amyl alcohol
mixture and t h e s t a t i o n a r y
phase w a s water a c i d i f i e d
t o pH 3.2.
122 MOHAMMAD UPPAL ZUBAIR ET AL.

7.8 Radioactive Isotopes

Caffeine can be estimated (166) by means of isotope-
d i l u t i o n a n a l y s i s with [1-14C] c a f f e i n e . It i s pre-
pared by methylation o f theobromine with [ C14]methyl-
iodide. About 1 0 mg o f t h e p u r i f i e d sample is
subjected t o combustion i n Baker's apparatus, using
t h e Van Slyke-Folch reagent. The l i b e r a t e d C02 i s
absorbed i n 0.25N-NaOH and 1.88% BaC12 s o l u t i o n is
added. The p r e c i p i t a t e d Ba2CO3 i s f i l t e r e d o f f and i t s
s p e c i f i c r a d i o a c t i v i t y i s measured with a Geiger-Muller
tube. 30-80 Mg o f c a f f e i n e , even i n presence of
a c e t y l s a l i c y l i c a c i d , theobromine, aminophenazone o r
phenacetin give s a t i s f a c t o r y r e s u l t s .

Noakes has reported (167) another radiocarbon measure-

ment method f o r t h e estimation of natural-product
p u r i t y i n case of c a f f e i n e .

7.9 Radiochemical
Caffeine was a l s o analysed by using r a d i o a c t i v e t r a c e r
techniques (168). It i s p r e c i p i t a t e d from t h e s o l u t i o n
of t h e sample i n hydrochloric a c i d with P3*-labelled
phosphomolybdic a c i d , and after f i l t r a t i o n , t h e t r a c e r
I-phosphomolybdate i s dissolved i n acetone and
c o l l e c t e d i n a sample g l a s s holder. The a c t i v i t y of
each p r e c i p i t a t e i s determined and t h e amount calcu-
l a t e d from a standard curve. The method i s simple and
gives reproducible results with s m a l l amount of
samples.

7.10 Chromatographic Methods

7.10.1 Paper Chromatoaraphy

Some paper chromatographic systems used f o r t h e

determination o f ' c a f f e i n e have been summarised
i n Table 11.

7.10.2 Column Chromatography

Various column chromatographic systems used

(175-182) f o r t h e determination o f c a f f e i n e
are presented i n Table 12.

I n another method (182) column chromatographic

technique has been employed i n i t i a l l y f o r t h e
Table 12. Summary of Conditions Used f o r t h e Column Chromatography of Caffeine

~ ~~ ~~ ~~ -~ ~~

support Eluent Detect ion Sample Ref.

I. Celite 545 and 4 N -

&SO4 covered by a
l a y e r of a mixture
of c e l i t e 545 and
N aq. NaHCO3.

11. Basic aluminuim Mixture o f - Pharmaceuticals 173

oxide C H C 1 3 and and c o f f e e
diethyl
ether.

111. Alumina (previous- ( i ) CHC13 ( i ) Extinction o f t h e e l u e n t Coffee and i t s 174

l y a c t i v a t e d at i n C H C l measured a t mixtures
800°C f o r 6 h r s . ) ( i i ) H20 257, 27? and 297 m u .
i n a 100 m l ( i i ) For determination i n
burett e . aqueous s o l u t i o n t h e
e x t i n c t i o n i s measured
a t 250, 273, and 296 mu.
IV. ( i ) Celite and ( i ) Etyyl 276 mu Beverage and 175
( i i )Column of ether tablets.
c e l i t e contain- ( i i )CHC13
i n g 4N-H2S04.
 Continued Table 1 2 .

support Eluent Detection Sample Ref.

V. Mgo-Celite 545 H2° 272 my Caffeine-containing 176

(l:l,w/w) and caffeine-free
coffee

VI. Celite 545 CHC13 276.5 my Coffee and decaffei- 177

nated c o f f e e

VII. Dowex 50W-X2 40% aqueous methanol 273 my Pharmaceuticals 178

VIII. Multiple column Elute phenacetin with -- Pharmaceutical combi- 179

I- containing seg- ether-CHClg (8:l) n a t i o n of a s p i r i n ,
N
l h ment s , 50 m l . Dismantle phenacetin, and
( a ) C e l i t e 545 t h e column e l u t e caffeine
( 3 g) and 1% a s p i r i n from segment
tartaric acid ( c ) and determine
soh. caffeine .
(b) Celite 545
( 3 g) and 12%
H2SO4 s o l n .
( 3 ml),
( c ) C e l i t e 545
( 2 g) and 8.4%
NaHCO3 s o l n .
( 2 ml) and
(a) Celite 545
( 3 g) and 22.1%
K3P04 soln. ( 3 m l ).
Continued Table 12.

support Eluent Detection Sample Ref.

IX. Polyamide (0.5 g) A f t e r passing percu- 272 nm Tea 180

late, t h e caffeine
i s washed w i t h H20,
2 x 3 x 10 ml.

X. S i l i c a gel (parti- H20-methanol (19:l) 254 nm S o f t d r i n k s and food 181

c l e s i z e 0.04 to
0.063 mm) i n
column
(30 cm X 1 cm)
126 MOHAMMAD UPPAL ZUBAIR ET AL.

i s o l a t i o n of c a f f e i n e from a sample of
decaffeinated c o f f e e , followed by i t s t i t r a t i o n
w i t h 0.01M H C l O 4 using methyl v i o l e t as
indicator.

7.10.3 Thin-Layer Chromatography

A summary of some of t h e TLC systems i n v e s t i -

gated f o r t h e a n a l y s i s of c a f f e i n e a r e given
i n Table 13. Combined TLC -
colorimetric/spec-
trophotometric methods f o r t h e determination of
c a f f e i n e i n pharmaceutical preparations and
beverages, have been reported (183,184). The
method i s based on t h e spectrophotometric
estimation of t h e q u a n t i t a t i v e l y e l u t e d c a f f e i n e
from TLC p l a t e s a f t e r separation from accompany-
ing substances. TLC and high-performance TLC
(1985) followed by densitometry has a l s o been
employed f o r t h e determination of c a f f e i n e i n
drugs ( 186,187 ) , beverages (185,188,189) and
c o l a seeds (189). The s o l u t i o n from the
aspirin-phenacetin-caffeine t a b l e t s i s chromato-
graphed on Whatman chemically bonded K C 1 8
reversed-phase p l a t e s containing fluorescent
phosphor and using 1:l MeOH-O.5M N a C l system
(186). Caffeine w a s then determined by using a
densitometer.

7 .lo.4 Gas Liquid Chromatography

Gas l i q u i d chromatographic methods have been
employed f o r t h e estimation of c a f f e i n e , and
v a r i a b l e parameters used f o r some o f these a r e
summarised i n Table 1 4 .

7.10.5 High Performance Liquid Chromatography

High pressure l i q u i d chromatography HPLC method

has wide a p p l i c a t i o n f o r t h e estimation o f
c a f f e i n e i n host o f d i f f e r e n t samples. A summary
o f v a r i a b l e parameters i n a few cases is given
i n Table 15.
Table 13. Summary of Conditions Used f o r t h e TLC o f Caffeine

support Mobile phase Detection Sample Ref.

Siluf ol W 254 nm Drugs 190

K i e s e l g e l 60F254 Spect rophotometri- Caffeine 191

( l a y e r t h i c k n e s s 0.2 mm) ca l l y
or
C e l l d o s e Fpj4 ( l a y e r -
t h i c k n e s s 0.1 mm)

K i e s e l g e l GF CHC13-cyclohexane- W 254 nm Coffee 192

2 54
a c e t i c a c i d (8:2:1)

Silica gel G Spray w i t h 2% HgC12 193

s o l u t i o n containing
1 0 mg of methyl r e d p e r
100 m l . or
Sprayed w i t h 2% HgC12
s o l u t i o n d r i e d and
sprayed w i t h K I s o l u t i o n .

K i e s e l g e l 60 Chloroform-acetone (9:l) Chromatograms evalu- Blood 194

a t e d by remission den-
s i t o m e t r y at 273 nm
w i t h a double beam
Continued Table 13

support Mobile phase Detect ion Sample Ref.

~

Kieselgel F Cyclohexane-acet one Determined by t h e i r Drugs 195

254
(4:5) quenching e f f e c t on
t h e background
fluorescence.

Silica gel G -- Drugs 196

(powders)
 Table 1 4 . Summary of Conditions Employed f o r t h e GLC o f Caffeine

Flow (ml/min)
Column support Mesh Length Temp. C a r r i e r gas Sample Ref.

31, Chromosorb W - - 210oc Argon 197

OV-17

Tenax 0.3 m 25OoC Flavor a n a l y s i s 198

GC o r and beverages
5% OV17 Chromosorb 1.0 m 2 0 0 ~ ~
WHP
c
SE-30 Chromosorb 80-100 2.0 m 180Oc N2(30 ml/min) (flame Drug 199
G ionisation detector).

Varaport 3% Dexsil - 2.0m 280Oc o r N2(16 ml/min) (flame Drug 200

30 300 ( 310°C ionisation detector).
i f sample
contains
papave-
rine)

Chromo- 3.5% s i l i - 1.5 190°C N2(55 ml/min) H-flame Drug b i o l o g i c a l 201

sorb W cone SE-30 glass i o n i s a t ion material
 m
0
N
I
u
0
0
a3
rl
I
0
(u
rl
I I
0
0
rl
L-
d
I
i3
bp
m
130
 Table 15. Summary o f HPLC Conditions f o r t h e Determination o f Caffeine

Column Retent i o n
Mobile phase Flow (ml/min) time (m,in) Detect i o n Sample Ref.

Partisif 10 Aqueous s o l u t i o n 1.1 d m i n - U.V. Tea and c o f f e e 204

SCX of 15 mM potas- (280 nm)
sium c i t r a t e , pH
3.0 and 10%
(v/v) methanol
lJ Bondapak MeOH+Wat er+HOAc U.V. Cocoa and 205
~ C18, 1 0 pm ( 20+79+1) chocolate
W, particle products
size

Bondapak MeOH-O.lM, pH 2a h i n 10 U.V. B 1ack t e a 206

C18 7:0, citrate- (254 nm) infusion
phosphate b u f f e r
(20 :80)

Corasil I Hept ane+ethanol 300 p . s . i . U.V. Purine alka- 207

or (10:l). (270 nm) loids
C o r a s i l I1

Cl8 ( R a d i a l Wat er+MeOH+HOAc 3.0 ml/min U.V. Animal d i e t s 208

Pak A ) c a r t - (74 :25: 1) (280 nm)
ridge
 Continued Table 15.

Retention
Column Mobile phase Flow (ml/min) time (min) Detect ion Sample Ref.

Bondapak 0.025 M NaH2P04 2 mUmin 0.48 U.V. Drugs 209

C18 i n MeOH+wat e r (254 and
(2:3) t o PH 7 280 nm)
using NaOH

Spherosil Iso-octane+diiso- 1d/min Aprox . - Drugs 210

XOA 600 and propyloxide+MeOH+ 4 min
+, XOA 800 t r ie thylamine+
to
0 water (34.93+49.51+
14.58+0.20+0.78)

Silica gel Methylene chloride: 50ml h-l - U.V. Caffeine 211

(M&N Nucleo- ethanol : water (280 nm)
sil 50-5.5 (936:47:17)
IJM)
PhenylfCora- Acetonitrile-water; - - - Drugs 212
s i l and 1,4 dioxan-wat er ;
C o r a s i l I1 methanol-water;
1-4-dioxan-ac e t o-
n i t r i l e - w a t e r and
various mixtures of
chloroform with
methanol
 Continued Table l>.

Colunln Mobile phase Retention

Flow ( d / m i n ) time (min) Detection Sample Ref.

Zipax SCX O . O M HNO

3
4 d/min U.V. Coffee 213
(254 nm)
-- 5% G l a c i a l a c e t i c - - u-Y. Beverages 214
acid

Bond e l u t , Chloroform - - - Beverages 215

C 1 8 bonded
~ silica
0
0
1.1 Bondapak Methanol-vat er 1.0 d / m i n - U.V. Plasma 216
c18 (30:70) (273 nm)
Bondapak MeCN-Water (8:92) - - - Beverages 217
c18
Spherisorb- - U.V. Decaffeinated , 218
ODS w i t h (272 nm) i n s t an t coffee
gradiant and o t h e r
elution beverages

10-pM P a r t i - MeCN-H20 ( 3 0 : 7 0 ) - U.V. Appetite/supp- 219

s i l ODS-2 (254 nm) r e s s a n t formu-
lations
 Continued Table 15.

Retention
Column Mobile phase Flow (ml/min) time (min) Detection Sample Ref.

YWGCH200x 50% s o h . of MeOH 1.0 ml/min U.V. Drugs 220

5mm i n 0.002M K2HPO4 (254 nm)
a d j u s t e d t o pH 8.0
by H3P04.

Hypersil 5% Isopropanol - U.V. - 221

+ dichloromethane (280 nm)
x Ion 25% aqueous e t h a n o l 15ml/hour U.V. - 222
exchange (254 nm)
r e s i n , Dowex-
AG 50w-x8( H+)

Nucleosil- MeCN-H20-HOAc 223

5 C18 ( 13:87 :1)

Finepak MeOH-NH4OH ( 99 :1 224

gel-110 or and
Finepak MeCN-NH40H ( 99 :1)
SIL-CIS
 Continued Table 15.

Column Mobile phase Flow ( d / m i n ) Retention

time (min) Detection Sample Ref.

Bondapak 0.01M sod. 3 mUmin - U.V. Umbil i c a 1 225

C18/corasil acetate buffer, (254 nm) cord plasma
and pH 5.0-MeOH-
LI Bondapak t et r ahydro f u r an
C18 (95:h:l)

c
Radial-Pak Acetonitrile i n - Biological 226
C 1 8 reversed 0.1 mol/L potassium fluids
phase column phosphate b u f f e r , ( neonates )
pH 4.0 (9.5/90.5
by vol. ) .
Part i s i l Containing H20, ace- - - Various drugs 227
50DS-3RAC o r t o n i t r i l e H3PO4 and sample s
5C8RAC NaOH with o r without including
( Radial-PAK LI hexylamine caffeine
Bondapak C 1 8
cartridge,
HS/5C18 o r
HS/3C18 o r
partisil
1O-ODS- 3
Continued Table 15.

Column Mobile phase Flow (ml/min) Retention

time (min) Detection Sample Ref.

Shodex Aqueous 70% - 270 nm Horse u r i n e 228

D-814 methanol at 4OoC. and by
(50 cm X d i f feren-
8 mm) t i a l ref-
r a c t ometr y

Radial- 0.M-phosphat e 254 nm Caffeine and 229

PAK C18 b u f f e r (pH 4) - t heophylline
acetonitrile r eonat e s
(181:19).
Micro packed - - U.V. Caffeine , 230
fused-silica, a s p i r i n and
used 1 0 and phenacet i n
5 cm columns
were used.

p Bondapak 1 0 m M-Na a c e t a t e 3 254 nm Metabolites 2 31

C 1 8 (30 cm buffer o f pH 5 i n Umbilical
x 3.9 cm) methanol-t e t ra- cord plasma
with Bonda- hydrofuran o f bovine
pak C18/ (95:h:l).
Corasil
guard column
CAFFEINE 137

8. References

1. Remingtons Pharmaceutical S c i e n c e s , Managing E d i t o r ,

J . E . Hoover, 1 5 t h Ed. Mack P u b l i s h i n g Co., Easton,
Penn. 18042, U.S.A. (1975) p . 1068.

2. Organic Chemistry, I . L . F i n a r , Longman Group L t d . , 5 t h ed.

Val. 2 ,(19751, P. 805.
3. E . F i s c h e r , Annalen., 215, 257 ( 1 8 8 2 ) .

4. The United S t a t e s Pharmacopoeia X I X , Mack P u b l i s h i n g

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Madison, J . Kozarek, and P. C e c i l l i a , J . Ass. Off
Analyst Chem., 2, 1258 (1976).

214. D. Smyly, B. Woodward and E. Conrad; J . Assoc. Off.

Anal. Chem. , 59, 1 4 (1976).

215. s. Reid, J. Good and S Thomas, J . Agri. and Food Chem.

-
30(4), 775 ( 1 9 8 2 ) .
216. S. 0 ' Connell and F. Zurzola; J . Pharm. S c i e n . , 7 3 ( 7 ),
1009 (1984).

217. J. Blauch, and S. Tarka; J. Food S c i . , 4 8 ( 3 ) , 745 (1983).

218. C. Trugo, R. Macrac and J. Dick; J . S c i . Food A g r i . ;

34(3), 300 (1983).
219. H. Tan and C. S a l v a d o r , J . Chromatogr. , g(1,)
111
(1983).
220. Y . S h i and N. L i ; Nanjing Yaoxueyuon Xuebao; 2 0 ( 3 ) , 23
(1982) , CA 99:93845m.

221. R . Smith; J . Food Chem., 7, 4 1 (1981).

222. H. Tong-Jung, L. Chi-Chow and C . Mei-Yun; J . Chinese

Chem. SOC. 25, 153 (1978).

223. M. Nishizawa, T. Chonan, I . S e k i j o and T. S u g i i ,

Hokkaidoritsu E i s e i Kenkyushoho , 32, 7 ( 1 9 8 2 ) .
150 MOHAMMAD UPPAL ZUBAIR E T A L .

224. S. Kikuchi and T. Ohata; Iwate-ken Eisei Kenkyusho

Nenpo; 25, 1 3 (1982).

225. R . Hartley, J . Cookman and I. Smith, J . Chromatogr.

-
306, i g i (1984).
226. Ching Nan Ou and Vicki Frowley, Clin. Chem., =(11),
1934 (1983).
227. I.S. Lurie and S.M. Carr; J. Liq. Chromatogr.; 6(9),
1617 (1983).
228. I. Matsue, Y. Matsumoto, T. Matsumoto and Momose;
Yakugaku Zasshi, =( 71, 800 (1983).
229. C.N. Ou and V.L. Frawley; C l i n . Chem., =(11), 1934
(1983).
230. T. Takeuchi, D. Ishii and A. Nakanishi; J. Chromatogr.,
285(10, 97 (1984).

231. R . Hartley, J . R . Cookman and I.J. Smith; J.

Chromatogr , . s,191 (1984).
9. Acknowledgements

The authors would l i k e t o thank M r . Mohammad Saleem Mian

of t h e Pharm. Chem. Dept., f o r h i s a s s i s t a n c e i n t h e
l i t e r a t u r e review and Mr. Khalid N.K. Lodhi of t h e
Central Laboratory of t h e College of Pharmacy, King Saud
University f o r h i s t e c h n i c a l a s s i s t a n c e .
 COCAINE HYDROCHLORIDE

FAR10 J. MUHTAUZ and ABDUllAH A. Al-BAUR

1. Description
1.1 Nomenclature
1.2 Formulae
1.3 Molecular Weight
1.4 Elemental Composition
1.5 Appearance, Color, Oder and Taste
2. Physical Properties
2.1 Melting Range
2.2 Solubility
2.3 Optical Rotation
2.4 X-rays Diffraction
2.5 Spectral Properties
3. Isolation of Cocaine
4. Synthesis of Cocaine
5. Biosynthesis of Cocaine
6. Metabolism of Cocaine
7. Pharmacokinetics
8. Drug Stability

ANALYTICAL PROFILES OF DRUG SUBSTANCES Copyright 8 1986

VOLUME 15 by the American Pharmaceutical Association
151 All rights of reproduction in any form reserved.
152 FARID J. MUHTADI AND ABDULLAH A. AL-BADR

9. Methods of Analysis
9.1 Identification Tests
9.2 Microcrystal Tests
9.3 Titrimetric Methods
9.4 Spectrophotometric Methods
9.5 Counter-Current Extraction
9.6 Chromatographic Methods
9.7 Radio-immunoassay
References
COCAINE HYDROCHLORIDE 153

1. Description

1.1 Nomenclature

1.1.1 Chemical Names

- [ 1R - (exotexo ) 1-3- (Benzoyloxy) -8-methyl-8-

azabicyclo [ 3.2.11 octane-2-carboxylic acid
methyl ester.

- ( - ) 3-(Benzoyloxy)-8-methyl-8-azabicyclo
[ 3.2.11 octane-2-carboxylic acid methyl ester
[l(R), 2(R), 3(s)1.
- 3 &Hydroxy-1 a H, 5 a H-tropane-2%-carboxylic
acid methyl ester benzoate.

- 8-Azabicyclo [3.2.1] octane-2-carboxylic acid

3-(benzoyloxy)-8-methyl-methyl ester hydro-
chloride [lR-(exo-exo)l.

- 2 f3-Carbomethoxy-3~-benzoxytropane.
- Methyl-3 B-hydroxy-1 a H, 5 a-tropane-2 6-
carboxylate, benzoate (ester) hydrochloride.

- (lR, 2R , 3 S , 5S)-3-Benzoyloxy-2-methoxy car-

bony1 tropanium chloride.

- (lR, 2R , 3s , 5S)-2-Methoxycarbonyl-tropane-3-
yl-benzoate .
- 2(R) -Carbomethoxy-3( S) - ( - )-benzoxy-1 ( R ) -
tropane.

1.1.2 Generic Names

Cocaine; 1-Cocaine; %-Cocaine; Benzoylmethyl-

ecgonine; Methylbenzoylecgonine; Ecgonine methyl
ester benzoate.

Cocaine hydrochloride; Cocaine muriate; N6uro-

caine hydrochloride.
154 FARID J. MUHTADI AND ABDULLAH A. AL-BADR

1.2 Formulae

1.2.1 Empirical

C17H21N04 for cocaine.

C H NO C1 for cocaine hydrochloride.

17 22 4
1.2.2 Structural

/CH3

The structure of cocaine was confirmed by the

total synthesis of cocaine which was achieved
by several authors (1-3).

1.2.3 CAS Registry Numbers

[50-36-2] for cocaine.

153-21-41 for cocaine hydrochloride.

1.2.k Wiswesser Line Notations

T 56 A ANTJ A - F V O l
GOVR - & GH LV (Cocaine hydrochloride) (4).
1.2.5 Stereochemistry

The ecgonine moiety of cocaine possesses four

COCAINE HYDROCHLORIDE 155

d i s s i m i l a r c h i r a l c e n t r e s a t C 1 , C2, C 3 and
c5 and so t h e r e a r e e i g h t p a i r s of enantio-
mers p o s s i b l e .

H
Since, however, only t h e c i s f u s i o n of t h e
n i t r o g e n bridge i s p o s s i b l e i n p r a c t i c e , C 1
and C5 t h e r e f o r e have only one c o n f i g u r a t i o n
( t h e c i s form), and so t h e r e are only f o u r
p a i r s of enantiomers a c t u a l l y p o s s i b l e , t h r e e
p a i r s of which have been prepared s y n t h e t i c a l -
l y (5).
The r e l a t i v e c o n f i g u r a t i o n s of cocaine and Y-
cocaine have been e x c l u s i v e l y determined by
chemical methods (6-11).

J
N

%
COOCH3
0
If
CH O-C-%H
65 H 5
Cocaine Y -Coc a i n e

The a b s o l u t e c o n f i g u r a t i o n of (-)-cocaine w a s
e s t a b l i s h e d by t h e following methods.
a ) C o r r e l a t i o n w i t h L(+)-glutamic a c i d through
ecgoninic a c i d ( 1 2 ) .
N-CH3
(-1 Cocaine
L
( - ) Ecgonine

COOH

(-IEcgoninic a c i d
156 FARID J. MUHTADI A N D ABDULLAH A. AL-BADR

L(+) glutamic acid

H H

C02H

COOH

CONH2 OH- CN CN

(-1 ecgoninic acid

b ) X-ray crystallographic study of 1-cocaine

hydrochloride (13).
c) The stereoselective synthesis of dl-cocaine
(3).
From the above data the absolute configuration
of (-)-cocaine is presented in Fig. 0.
COCAINE HYDROCHLORIDE 157

Fig.0 Perspective view of the molecule of cocaine ( 1 3 ) .

158 FARID J. MUHTADI AND ABDULLAH A. AL-BADR

1.3 Molecular Weight

303.35 (cocaine)
339.81 (cocaine hydrochloride )

1.4 Elemental Composition

C , 67.30%; H , 6.98%; N, 4.62%; 0 , 21.10%.

(cocaine)
C , 60.08%; H , 6.53%; N, 4.12%; 0 , 18.83%; C 1 ,
10.43%. (cocaine hydrochloride)

1.5 Appearance, Color, Odor and Taste

Colorless c r y s t a l s o r a white c r y s t a l l i n e powder,

odorless, s l i g h t l y v o l a t i l e . It has a b i t t e r t a s t e ,
numbs tongue and l i p s . (Cocaine).

Colorless granule c r y s t a l s , or a white c r y s t a l l i n e

powder, odorless and hygroscopic. It has a s l i g h t l y
b i t t e r t a s t e , numbs tongue and l i p s . (Cocaine
hydrochloride).

1.6 Dissociation Constant

pKa a t 15' = 5.59(cocaine); pKa 8.4 (cocaine H C 1 )

1.7 Loss on Drying

When d r i e d t o constant weight a t 80' l o s e s not more

than 0.5% of i t s weight. ( 1 4 ) .

2. Physical Properties

2.1 Melting Range

lg70
About 195'
(14
(15 ] cocaine hydrochloride
2.2 Solubility

One gram dissolves i n 600 m l water, 270 m l water a t

80°, 6.5 ml alcohol, 0.7 m l chloroform, 3.5 m l e t h e r ,
12 ml o l i v e o i l , a l s o soluble i n acetone, e t h y l ace-
t a t e and carbondisulfide (Cocaine).
COCAINE HYDROCHLORIDE 159

One gram d i s s o l v e s i n 0.4 m l w a t e r , 3.2 a l c o h o l ,

2 m l hot a l c o h o l , 12.5 m l chloroform, a l s o s o l u b l e
i n g l y c e r o l and acetone. (Cocaine hydrochloride)

2.3 O p t i c a l Rotation
Cocaine
[a]D20 - 16' (C=4 i n C H C l ) ; [a]D18 -35'(50% alcohol).
3
[aID - 79' t o -81° (0.6 g i n 2.5 m l of M hydro-
c h l o r i c and s u f f i c i e n t water
t o produce 25 m l ) ( 1 4 ).
Cocaine hydrochloride
[aID - 72' (C=2 aqueous s o l u t i o n pH 4.5)
[alD - 70' t o -73O (2.5% w/v s o l u t i o n ) (14 )

2.4 X-ray D i f f r a c t i o n

The c r y s t a l and molecular s t r u c t u r e of 1-cocaine

w a s determined by X-ray d i f f r a c t i o n . Babe and
Barnes ( 1 3 ) have achieved t h i s .
Three dimentional study of t h e hydrochloride and t h e
hydrobromide s a l t s revealed t h e c r y s t a l s t o be ortho-
rombic with space group P21 21 21; f o r t h e hydroch-
l o r i d e , a = 7.633, b = 10.300, c = 21.459 A'; for t h e
hydrobromide, a = 7.68, b = 10.68, c = 21.65 R o . The
study a l s o revealed t h a t t h e stereochemical configura-
t i o n of cocaine molecule agrees with t h a t deduced
from chemical evidence. The p i p e r i d i n e r i n g of t h e
tropane nucleus has t h e c h a i r form, with C 3 d i s p l a c e d
l e s s , and N displaced more than u s u a l from t h e plane
of t h e r i n g . The benzoxy s i d e chain on C 3 i s equato-
r i a l and t h e carbomethoxy s i d e chain on C2 i s a x i a l .
Intramolecular bond l e n g t h s , bond angles and t o r s i o n
angles of cocaine hydrochloride a r e t a b u l a t e d i n
t a b l e s 1, 2 and 3 r e s p e c t i v e l y .
160

1.494
1.382
1.406
1.380
1.370
1.360
1.384
COCAINE HYDROCHLORIDE 161

Table 2. Bond Angles ( ” ) of Cocaine Hydrochloride

C1-N-C 103.6
5
C1 - N - C17 112.4
C5 - N - C17 112.5
c3 - 01- c8 117.4
118.1
‘15- ‘4- ‘16
N - C1- c2
109.1
N - C1- c7
101.1
c2 - cl- c7 112.5
c1 - c2- c3 109.1
162 FARID J. MUHTADI A N D ABDULLAH A. AL-BADR

c1 - c2- ‘15
109.3 Cg - 50- 51 120.2

‘3 - ‘2- ‘15 114.7 5 0 - cll-5 2 119.6

o1 - c3- c2 114.5 ‘11- ‘12- ‘13 121.1
o1 - c3- c4 108.3 ‘12- ‘13- ‘14 120.7
c2 - c3- c4 112.8 ‘9 - ‘14- ‘13 118.6
c3 - c4- c5 110.4
‘3 - ‘15- ‘4 121.4
N - C5- C4 108 ‘3 - ‘15- ‘2 122.3
N - C5- c6 102.3 ‘4 - ‘15- ‘2 115 9

Table 3. Torsion Angles ( ” ) of Cocaine

Hydrochloride (16).

C c2 72
5 -N-C1-
C5-N- - c7
c1 -47
C17- N - C1 - C2 -166
C17- N - - c7 75
C1 - N - C5 - C4 -72
C1-N- ‘5 - ‘6 46
C17- N - C5 - C4 166
C17- N - c5 - c6 -75
c8 - 01- c3 - c2 77
c8 - 01- cg - c4 -157
c3 - 01- c8 - O2 1
c3 - ol- C8 - c9 179
‘16- ‘4- ‘15- ‘3 8
c16’ 04- C l 5 - c2 -178
N -
C1- C2 - C3 -59
N - Cl- ‘2 - ‘15 67
c7 - cl- c2 - c3 52
c7 - cl- c2 - ‘15 178
N - C1- C7 - 28
‘6
c2 - cl- c7 - c6 -88
c1 - c2- c3 - o1 172
c1 - c2- c3 - c4 47
Cl5- c2- c3 - o1 49
COCAINE HYDROCHLORIDE 163

2.5 Spectral Properties

2.5.1 U l t r a v i o l e t Spectrum

The UV spectrum of cocaine hydrochloride i n

methanol (Fig. 1) was scanned from 190 t o 400
nm using DMS 90 Varian Spectrometer. It exhi-
b i t e d t h e following UV d a t a (Table 4 ) .
Table 4. W C h a r a c t e r i s t i c s of Cocaine Hydrochloride

hmax. nm - E A(%%, 1 em)

202 4375 128.75

2 30 7136 210.0
272 433.3 12.75
282 390.8 11.50

Other reported UV s p e c t r a l d a t a f o r cocaine i n

ethanol hmax. 230, 274, 2 8 1 mu (17) , f o r cocaine
hydrochloride i n water h m a x . 233 and 274 mu ( 1 7 ) .

2.5.2 I n f r a r e d Spectrum

The I R spectrum of cocaine hydrochloride as

KBr-disc was recorded on a Perkin E l m e r 580 B
I n f r a r e d spectrophotometer t o which an i n f r a -
red d a t a s t a t i o n i s attached (Fig. 2 ) .
The s t r u c t u r a l assignments have been c o r r e l a -
t e d with t h e following frequencies (Table 5 ).

Table 5 . I R C h a r a c t e r i s t i c s of Cocaine Hydrochloride

-1
Frequency em A s s i gnment
3020 CH ( s t r e t c h )

2780-2500 H3C - N+H

0
I1
1730 C-OCH3 (ester)
0
II
1715 C-0- (ester)
1600 C=C (aromatic )
1155,1028 C-0-C (ether )
730 monosubstituted aroma-
tics
 I

190 140 wavelength 340 440

 !
T------
:
5
E
c
d
C
a
c
t
c
Y
c
ci
:
*9
3
165
166 FARID J. MUHTADI A N D ABDULLAH A. AL-BADR

The I R exhibited t h e following other c h a r a c t e r i s t i c

bands :-

1488, 1455, 1430, 1395, 1375, 1365, 1315, 1300, 1268,

1250, 1232, 1210, 1168, 1135, 1108, 1072, 1010, 980,
950, 855, 830, 815, 795, 755, 682 cm-l,
Other I R data f o r both cocaine and cocaine hydro-
chloride have been a l s o reported ( 4,17).

2.5.3 Nuclear Magnetic Resonance Spectra

2.5.3.1 Proton Spectra (PMR)

The PMR s p e c t r a of cocaine hydrochlo-

r i d e i n D20 and cocaine i n CDC13 and
i n TFA ( T r i f l u o r o a c e t i c a c i d ) were
recorded on a Varian T ~ O A ,60 MHZ
NMR Spectrometer using TMS (Tetra-
methylsilane) as an i n t e r n a l refer-
ence. These a r e shown i n Fig. 3 and
Fig. 4 respectively. The following
s t r u c t u r a l assignments have been made
(Table 6 ).

PMR w a s used as a t o o l t o confirm the

configuration of both cocaine and
pseudococaine (18 ) and provided
evidence of t h e p r e f e r r e d confirmation
(19).
 I I I I
C 1 I I I I 1 * 1 1 1 1 1 1 1 1 1 1 1 . l l l l l l l l I I I I I I 1 , I , I , , , 1

8.0 ZO 6.0 5.0 PPM(6J tO 3.0 2.0 1.0

FIG. 3. THE PMR OF COCAINE HYDROCHLORIDE IN D20

 m
-J
u
9
E
w
f
c
V
0
u
u.
0
a
z
Y
X
-
I-
c
v
=r
2
LL
168
I !-

FIG. 4(B) THE PflR OF COCAINE IN TFA

170 FARID J. MUHTADI AND ABDULLAH A. AL-BADR

Table 6. PMR C h a r a c t e r i s t i c s of Cocaine

and Cocaine Hydrochloride.

Chemical Shift (Ppm)

Group Cocaine Cocaine
hydrochloride CDC13 TFA

Aromatics
13, 17 HS
1 4 , 15, 16 HS
3 H
1 s 5 HS
0-CH3
N-CH3

s = s i n g l e t , d = doublet, m = m u l t i p l e t , q = q u a r t e t
bs = broad s i n g l e t , s i = s i x t e t .

Other PMR data f o r cocaine ( 3,18,20 ) and cocaine hydro-

chloride ( 21) have a l s o been reported.

2.5.3.2 13C-NMR

The 13C-NMR noise decoupled and off

resonance s p e c t r a are presented i n
Fig. 5 and Fig. 6 respectively. Both
were recorded over 4000 Hz range i n D20
and i n Dioxan on a Varian FT 80 A-80
MHz spectrometer, using LO mm. sample
tube and dioxan a s a reference stan-
dard a t 22O.
The carbon chemical s h i f t s a r e assign-
ed on t h e bases of t h e a d d i t i v i t y
p r i n c i p a l s and o f f resonance s p l i t -
t i n g p a t t e r n (Table 7 ).
t ' I I 1 ' I ' I ' I ' I ' I ' I I I I l ' l ' l ' l ' l ' l I l ' l ~ l ' l ' I

FIG. 5 . THE I3C-NMR NOISE DECOUPLED SPECTRUM OF COCAINE HYDROCHLORIDE

 I

l ~ I ~ I ~ l ~ l ~ ~ ~ l ~ l ~ ~ ~ ~ ~ l

FIG. 6. THE I3C-NHR OFF RESONANCE SPECTRUM OF COCAINE HYDROCHLORIDE

COCAINE HYDROCHLORIDE 173

Table 7. Carbon Chemical S h i f t s of Cocaine H C 1

Carbon no. Chemical S h i f t s Carbon no. Chemical S h i f t s

[ PPm 1 [ PPm 1
~~

C
9

5 2

‘13’ ‘17

‘15
‘14’ ‘16
c3

c5

s = s i n g l e t , d = doublet, t = t r i p l e t , q = quartet

Other l3C-NMR d a t a f o r cocaine ( 2 0 ~ 2 2) have a l s o been

reported.
174 FARID J. MUHTADI AND ABDULLAH A. AL-BADR

2.5.4 Mass Spectrum

The mass spectrum of cocaine hydrochloride i s

presented i n Fig. 7 . This was obtained by
e l e c t r o n impact i o n i z a t i o n on a Varian MAT 1020
by d i r e c t i n l e t probe a t 270'~. The e l e c t r o n
energy w a s 70 eV. The spectrum scanned t o
mass 310 mu.
The spectrum (Fig. 7 ) shows a molecular ion
peak # a t m/e 303,with a r e l a t i v e i n t e n s i t y
of 15.60%. The base peak i s 82 with a rela-
t i v e i n t e n s i t y 100%.
The most prominent fragments t h e i r r e l a t i v e
i n t e n s i t i e s and some proposed ion fragments
are given i n t a b l e 8.

Table 8. Mass Fragments of Cocaine H C 1

Elk Relative i n t e n s i t y % -
Ions
30 3 15.60 M+ (cocaine)
272 6.75 -
198 11.37
183 9.26 -
182 84.84 183-H
122 9.85 -
105 29.97
97 10.03

96 24.06

94 35.19 96-2H

83 35 83
 DATI: KSU a1396

1n.e

1
176 FARID J. MUHTADI AND ABDULLAH A. AL-BADR

m/e Relative i n t e n s i t y % -
Ions

82 100.00 e C H 3

81 10.63 82-H
77 36.66 -
68 6.39
55 8.29
51 14.54
42 32.43 [ CH2--N=CH2 1+
41 9.45 42-H

Other reported mass s p e c t r a of cocaine has been

a l s o reported ( 23,24 ).
COCAINE HYDROCHLORIDE 177

3. Isolation of Cocaine
Cocaine occurs in the leaves of ErythroqZon coca
(Bolivian coca leaves), ErythroqZon truxiZZense (Peru-
vian and Javanese coca leaves) and other species of
ErythroqZon family Erythroxyzaceae ( 25,26).
Javanese leaves are usually the richest in total
alkaloids, of which, the chief alkaloid is cinnamyl-
cocaine, while the South American leaves contain less
total alkaloids but higher percentage of cocaine ( 2 7 , 2 8 ) .
Java coca leaves are used to isolate cocaine commer-
cially. The method depends upon isolating the total
alkaloids including cocaine, hydrolyzing these alkaloids
either to (-)-ecgonine or to ecgonine methyl ester, ( - ) -
cocaine is then synthesized by methylation and benzoyla-
tion or by simple benzoylation (Fig. 8).
The Procedure

Powdered coca leaves are moistened with sodium

carbonate solution and percolated till exhaustion with
benzene.
- The benzene extract is shaken with dilute sulfuric acid.
The collected acid extract is rendered alkaline with
an excess of sodium carbonate. The resulting precipita-
ted total alkaloids are extracted with ether.
- The ether extract is dried with anhydrous sodium sulfate, ~

filtered and the ether is distilled off.

- The residue consisting of the total crude alkaloids is
dissolved in methyl alcohol and the resulting solution
is heated with sulfuric acid or with alcoholic hydrogen
chloride (this treatment splits off any acids from ecgo-
nine and simultaneously esterfies the carboxyl group
to carboxymethyl group).
- After dilution, with water, the organic acids which have
been liberated are removed with chloroform. The aqueous
solution is then concentrated, neutralized and cooled
with ice, whereupon methylecgonine sulfate crystallizes
out and collected.
- This is now benzoylated by heating with benzoyl chloride
or benzoic anhydride at about 150°C. Upon adding water
and sodium hydroxide, cocaine is precipitated and extrac-
ted with ether.
- The ether extract is concentrated to crystallization, the
crystallized cocaine is collected and recrystallized from
178 FARID J. MUHTADI AND ABDULLAH A. AL-BADR

Fig. 8. The Commercial Preparation of Cocaine (27).

,CHI
COOCH,

COCAINE RELATED
ALKALOIDS H

/
COMPLETE
HYDROLrSlS

,CHI
J CHaOH HCI
PARTIAL
HYDROLYSIS

H
METHYLATION
AND
BENZOYLATION
/ BENZoYLAT1oN
J
H
COCAINE HYDROCHLORIDE 179

a mixture of acetone and benzene to give colorless

prisms (Fig. 8 ) .
Cocaine hydrochloride
This is prepared by adding cocaine to an alcoholic
solution of hydrochloric acid and the resulting salt is
purified by subsequent recrystallization.
4. Synthesis of Cocaine
4.1 Partial Synthesis
Cocaine can be synthesized by methylation and benzoy-
lation of (-) ecgonine. Thus upon heating a mixture
of (-)-ecgonine, benzoic anhydride and methyl iodide
at looo, (-)-cocaine is resulted (29).
(-)-Ecgonine is esterified with methanol to yield
(-)-ecgonine methyl ester and this upon simple
benzoylation with benzoyl chloride gives (-)-cocaine
(30-32).
4.2 Total Synthesis
Since cocaine is an ester alkaloid consists of
ecgonine methyl ester and benzoic acid, schemes for
the total synthesis of both are required.
4.2.1 Total Synthesis of Ecgonine
Two schemes for the total synthesis of ecgo-
nine are known.
Scheme I: Willstatter's total synthesis of
ecgonine (1) .

Suberone (cycloheptanone) [ 11 is reduced to

suberol which is treated with hydrogen iodide
to give suberyl iodide [ 2 ] . This is treated
with potassium hydroxide in ethanol to give
Cycloheptene [ 3 ] . Cycloheptene is brominated
to give 1,2-dibromocycloheptane [4] which is
treated with dimethylamine to yield dimethy-
laminocyclohept-2-ene [5]. The latter is
converted to cyclohepta-193-diene[6] by ex-
haustive methylation. [6] is brominated at
1,4-positions to give 1,4-dibromocyclohept-
2-ene [7]. Elimination of two moles of the
180 FARID J. MUHTADI AND ABDULLAH A. AL-BADR

SCHEME I; Willstatter s t o t a l s y n t h e s i s of cocaine

-Q exhaust
methyln.
[5l N(CH3)2
(Me2NH QBr

[41
Br

Br

-quinoline
150°C
COCAINE HYDROCHLORIDE 181
182 FARID J. MUHTADI AND ABDULLAH A. AL-BADR

hydrogen bromide of [7] i s e f f e c t e d by quin-

oline t o give cycloheptatriene [8].
Substance [8] i s t r e a t e d with hydrogen brom-
i d e t o give bromocyclohepta-3,5-diene [9]
which i s reacted with dimethylamine t o give
dimethyl aminocyclohepta-2,4-diene [ l o ] . The
l a t t e r is t r e a t e d with sodium i n ethanol f o l -
lowed by bromination t o give 1,2-dibromo-5-
dimethylamino-cycloheptane [ l l ] .
warmed i n e t h e r when intramolecular alkyla-
This i s

t i o n occurs t o give 2-bromotropane methobro-

mide [12]. Hydrogen bromide i s eliminated
from [12] by t h e a c t i o n of a l k a l i t o y i e l d
t r o p i d i n e methobromide [13]. This i s t r a n s -
formed t o t r o p i d i n e methochloride [14] by t h e
action of potassium iodide followed by t h e
a c t i o n of s i l v e r chloride. Substance [ 141
is pyrolized t o give tropidine [15].
Hydrogen bromide i s added t o an acetic a c i d
s o l u t i o n of t r o p i d i n e [15] t o y i e l d 3-bromo-
tropane [16] which i s hydrolysed with 10%
s u l f u r i c a c i d a t 200-210' t o give pseudo-
t r o p i n e [17]. $-tropine [17] i s oxidized
with chromium t r i o x i d e t o give tropinone [18].
This ketone upon treatment w i t h sodium,
(Kolbe-Schmitt type of reaction) gives t h e
intermediate [19] which i n the presence of
carbon dioxide gives sodium tropinone carbo-
x y l a t e [20]. Upon reduction of [20] followed
by acid treatment y i e l d s (+)-ecgonine [21].
(+)-Ecgonine [21] i s resolved with ( + ) - t a r t -
a r i c a c i d t o furnish (-) -ecgonine.
Scheme 11: Total synthesis by adaptation
of Robinson's tropinone synthesis (2).
Succindialdehyde [ 11 i s condensed with methy-
lamine [2] t o give biscarbinolamine [3]. This
i n t u r n condensed with acetondicarboxylic
acid monomethyl e s t e r [4] t o give t h e conden-
s a t e [5]. The l a t t e r i s decarboxylated t o
y i e l d ecgoninone methylester [6]. This 8-
k e t o e s t e r i s reduced with sodium amalgam t o
give ecgonine methyl ester [ 7 ] , which i s
hydrolyzed t o (2) -ecgonine [8]. [8] i s
resolved with ( + ) - t a r t a r i c t o render (-) -
ecgonine.
COCAINE HYDROCHLORIDE 183

SCHEME

(+ CHO
I I : Total
H
\NCH3
H
/
-(7"'
synthesis by adaption of Robinson's method

CH-OH
\
CH-OH
t
CH2COOCH3
\c=o
I
C H2-COOH
[I1 [21 C 31 [41

COOCH3 COOCH3

COOH
[61 151

COOH

( - 1-ecgonine (-)-cocaine
184 FARID J. MUHTADI A N D ABDULLAH A. AL-BADR

Synthesis of Cocaine

[-I -Ecgonine i s e s t e r i f i e d first with methanol and

hydrochloric a c i d , then with benzoyl c h l o r i d e t o
y i e l d (-) -cocaine.

An a l t e r n a t e s y n t h e s i s of tropinone has been reported (33).

Tropinone can be converted i n t o ecgonine by Willstatter’ s
method (Scheme I ) .

Diyne d i e s t e r [l] i s condensed with methylamine [2] t o

give t h e p y r r o l i d i n e d i e s t e r [3]. C a t a l y t i c hydrogena-
t i o n gives [4] which on Dickmann c y c l i z a t i o n , hydrolysis
and decarboxylation y i e l d s tropinone [S].

0
COCAINE HYDROCHLORIDE 185

4.2.2 Total Synthesis of Benzoic Acid

Benzoic acid is known acid and occurs in sever-
al sources in nature.
It can be prepared by heating gum benzoin when
benzoic acid sublimes (34). It can also be
prepared from hippuric acid which on boiling
with mineral acids is hydrolysed to glycine
and benzoic acid (34).
It is prepared exclusively from toluene as
follows:-
Toluene is converted into benzotrichloride by
treatment with chlorine and this is hydrolysed
by lime water to calcium benzoate, from which
benzoic acid is precipitated by the addition
of hydrochloric acid and purified by recrystal-
lization from water. Benzoic acid is prepared
in large quantities by catalytic oxidation of
toluene (34).
Benzoyl chloride required to prepare cocaine
is prepared by warming benzoic acid with phos-
phorus pentachloride or preferably thionyl
chloride or by the action of chlorine on benz-
aldehyde (34).
C6H5. COOH + S0Cl2 = C6H5. COCl + HC1 + SO2
C6H5. CHO + C12 = C6H5. COCl + HC1.

4.3 Stereoselective Synthesis of dl-Cocaine

Stereoselective synthesis of dl-cocaine was described
(3).
Mesylate olefin [I] is brominated to the bromo-ester
[Z] which is converted into the nitroester [3]. This
is condensed with acrolein [4] in the presence of meth-
anol containing sodium methoxide to give the dimethyl-
acetal [S]. The nitrogroup of [5] is reduced with zinc
and aqueous ammonium chloride solution to produce the
hydroxylamine acetal [6] which upon acidification
generates the hydroxylamine aldehyde [7]. [7] is
cyclized to the nitron [8] which is converted into
the cycloadduct [9].
The cycloadduct [9] is methylated with methyliodide
in methylene chloride affords methiodide [9] which is
treated with activated zinc in 50% aqueous acetic acid
at 7Ooc in order to effect the scission of the nitrogen-
oxygen bond to provide ecgonine methylester, which is
then benzoylated to afford dl-cocaine [lo].
186 FARID J. MUHTADI AND ABDULLAH A. AL-BADR

Stereoselective Synthesis of dl-Cocaine

c; C02Me
NQcH3 H

N-CH3
COOCH3

OCOC6Hg

H
[lo1
COCAINE HYDROCHLORIDE 187

4.4 Synthesis of Radioactive Cocaines

"-methyl -14C] cocaine i s prepared by N-methylat ion

Of norcocaine with [ ~ n e t h y l - ~ ~iodide
C] (35).

[Ester methyl-14C] cocaine i s prepared by methylation

of benzoylecgonine with diazomethane-l4C ( 3 5 ) .

H
[ B e n z ~ y l - a - ~ ~ cocaine
C] i s prepared by benzoylation
of e cgon i n e met hy 1e s t e r with benz oy l c h l o r ide -a- l 4 C
( 351.

H
M e t h y l - t r i t i a t e d d e r i v a t i v e s of (-)-cocaine and (+) -
$-cocaine were prepared ( 3 6 ) .
188 FARID J. MUHTADI AND ABDULLAH A. ALBADR

5. Biosynthesis of Cocaine

It has been assumed t h a t t h e t r o p i n e and ecgonine

moieties of hyoscyamine and cocaine r e s p e c t i v e l y a r i s e d
from t h e aminoacid o r n i t h i n e (37,38). The aromatic carb-
oxylic a c i d s of both a l k a l o i d s are b u i l t up from pheny-
l a l a n i n e which i s formed i n p l a n t s from shikimic a c i d
( 38 ,39 ).
Using r a d i o a c t i v e t r a c e r technique, it w a s found t h a t t h e
administration of [ 3' -14C3 phenylalanine t o Erythroxy Zrm
novogranatense yielded r a d i o a c t i v e cocaine i n which a l l
t h e a c t i v i t y resided i n t h e carboxyl group of t h e benzoic
acid moiety of cocaine (40 ) .
Feeding sodium [ L-14C 1 a c e t a t e and [methyl-14C J methio-
nine i n t o t h e same species, r e s u l t e d i n t h e i s o l a t i o n of
l a b e l l e d cocaine ( 4 1 ) .
It w a s a l s o found t h a t feeding sodium [l-C] a c e t a t e t o
E. coca p l a n t s , radioactive cocaine was i s o l a t e d i n which
about 60% of t h e a c t i v i t y were l o c a t e d i n t h e e s t e r methyl
group, 30% i n t h e carboxyl group of t h e benzoic a c i d moie-
t y and 8.7% i n t h e ecgonine residue (42).
Leete ( 43 ) has f e d t h e following radioactive precursor
t o Erythoxy Zon coca p l a n t s : -
DL-[ 5-14C1 o r n i t h i n e hydrochloride , DL-[2,3-13C ,5-14C1
ornithine hydrochloride , DL-[ 2-14CI o r n i t h i n e hydrochlo-
r i d e , [ 2-13C , 1 4 C I-N-methyl-Al-pyrrolinium a c e t a t e , [ 2-14CI
-N-methyl-Al-pyrrolinium chloride , sodium [ l - k ] a c e t a t e
and [ carboxyl-14C 1 n i c o t i n i c acid. Radioactive cocaine
r e s u l t e d from each o f t h e above precursors with v a r i a b l e
l e v e l of r a d i o a c t i v i t y .
Cocaine containing a s i g n i f i c a n t l e v e l of r a d i o a c t i v i t y
was obtained by p a i n t i n g t h e leaves of t h e Erythroxylon
coca with an aqueous s o l u t i o n of DL-[5-14C] o r n i t h i n e
hydrochloride ( 43 ). A systematic degradation of t h i s
cocaine indicated a l l t h e a c t i v i t y was l o c a t e d a t t h e
brigehead carbons ( C 1 and C 5 ) of i t s tropane moiety and
equally divided between t h e s e p o s i t i o n s (43).

Leete t h e r e f o r e , proposed t h e following biosynthetic

pathway of cocaine (43).
Ornithine [11 i s incorporated i n t o cocaine via B-N-
methylornithine [ 2 ] which i s formed by N-methylation of
[I]. [ 2 ] w a s i s o l a t e d i n radioactive form a f t e r feeding
[ 5-14C 1 or [ 5-3HI o r n i t h i n e t o Atropa belladonna (44).
Decarboxylation of [ 2 1 y i e l d s N-methylputrescine [ 3 1 , an
e s t a b l i s h e d precursor of t h e tropane nucleus of hyoscya-
mine and scopolamine (45-47). Oxidation of [31 affords
k-methylaminobutanal [4]. This i s cyclized t o give
COCAINE HYDROCHLORIDE 189
Leete’s Scheme: Biosynthesis of Cocaine
COOH

COOH COOCH3
/

CH3

[81 I COOCH3

-kH
COOCH3
/

0- 0
II
190 FARID J. MUHTADI AND ABDULLAH A. AL-BADR

N-methyl-Al-pyrrolinium salt [ 51 which is condensed with

acetoacetate [61 to afford hygrine-1'-carboxylic acid [ T I .
171 is dehydrogenated and esterified to give dehydro-
hygrine-1'-carboxymethylester [ 81. The latter is cyclized
to yield ecgoninone methylester [ 91. Stereospecific redu-
ction of [ g ] affords ecgonine methylester [ l o ] .
Ecgonine methylester [lo] is finally esterified with benz-
oic acid [Ill to give cocaine [121.

Benzoic acid is formed from the aminoacid phenylala-

nine ( 39 ) which is formed in plants from shikimic acid

shikimic
acid

*CH *COOH
1 2
CH-NH*
I
COOH
COCAINE HYDROCHLORIDE 191

6. Metabolism of Cocaine

Cocaine i s w e l l absorbed from a l l s i t e s of a p p l i c a t i o n s

including mucous membranes and t h e g a s t r o i n t e s t i n a l muco-
sa (48-49). Absorption i s enhanced i n t h e presence of an
inflammation (48,49).
After absorption, cocaine i s degraded by plasma e s t e r a s e s
( 5 0 ) and i n some animals by hepatic enzymes ( 5 1 ) t o a
number of metabolites (52-63). Some cocaine i s excreted
unchanged i n t h e u r i n e (51,52).
The major metabolites of cocaine i n man and animals a r e
benzoylecgonine, ecgonine, ecgonine methylester and nor-
cocaine (52-57). Minor metabolit,es. i n man a r e ecgonine
e t h y l e s t e r , cocaethylene, m-hydroxycocaine and ecgonidine
methylester ( 5 6 , 5 7 ) , t h e s e metabolites have been i d e n t i -
f i e d i n multiple i n t o x i c a t i o n and overdoses of cocaine
( 5 7 ) . Other metabolites which can be detected i n animals
are benzoylnorecgonine and norecgonine ( 58-60).
The metabolism of cocaine i n man and animals i s presented
i n Fig. 9, and t h e s t r u c t u r e s of cocaine and it.s metabo-
l i t e s a r e shown i n Fig. LO ( 5 2 ) .

7. Pharmacokinetics

The pharmacokinetics of cocaine have been reported by

s e v e r a l authors. Peak serum l e v e l s occur i n 3-5 minutes
following intravenous administration; i n 20-60 minutes
following i n t r a n a s a l administration and i n 60-90 minutes
after oral administration (64).
Plasma l e v e l s of cocaine 30 and 45 minutes post-administra-
t i o n of 1 . 5 mg/kg of a t o p i c a l i n t r a n a s a l 10% cocaine
s o l u t i o n , were 331 and 320 n g / d r e s p e c t i v e l y ( 6 5 ) .
Following i n t r a n a s a l a p p l i c a t i o n of 1-5 mg/kg of cocaine
t o 1 3 s u r g i c a l p a t i e n t s , plasma l e v e l s reached peak con-
c e n t r a t i o n s of 120-474 ng/ml a t 15-60 minutes, and then
decreased overthe next 3-5 hours (66).
Plasma l e v e l of cocaine a f t e r smoking one c i g a r e t t e (75 mg
of cocaine) i n 3 minutes w a s 251 ng/ml with a range of
91-462 ng/ml (67).
The average plasma l e v e l of cocaine a f t e r smoking 3 cigar-
e t t e s for 1 5 minutes was 478 ng/ml with a range of 226-
684 ng/ml (67). The h a l f - l i f e of cocaine i n t h e plasma
a f t e r o r a l or n a s a l administration i s approximately one
hour (68). The mean h a l f - l i f e of cocaine f o r intravenous
i n j e c t i o n s i n four human s u b j e c t s was 41.4+-8.2 minutes
and t h e range was 19 t o 64 minutes (69).
192 FARID J. MUHTADI AND ABDULLAH A. AL-BADR

Ecgonine (mn, r u t , dogl

(man, r u t , dogl

Ec gonine ethy les t er

(man)

m-Hydroxycocaine
(man)

Benzoylnorecgonine Norcocaine
(rat, dog) (man, r u t , dog, monkey)

Norecgonine
(dog)

Fig. 9. The Metabolism of Cocaine (Cocaine Metabolites).

 COCAINE HYDROCHLORIDE 193

P
3~~woJJ
N Fig.10. The Structures of
Cocaine Metabolites.

n
COCAINE

BENZOVLECCONINE ECGONINEi3HYLESTER

BEWZOVLNORECGO)(INE ECGONINE UETHVLESTEA

/"I
N

8=Tmo+Q H OH

m-HVOROXYCOCAlNE

ECQONIMNE METHYLESTER

H ECGONINE NORECGONINE
194 FARID J. MUHTADI A N D ABDULIAH A. AL-BADR

8. Drug S t a b i l i t y

Cocaine hydrochloride i n t h e dry state, s t o r e d i n a well

closed container a t room temperature, showed no decompo-
s i t i o n a f t e r f i v e years when examined by d i f f e r e n t physi-
c a l and chemical d a t a including spectrophotometrical evid-
ence (70).
Solution of cocaine hydrochloride i s l i a b l e t o develope
fungus growths and should contain a preservative. Stored
i n a i r t i g h t containers, protected from l i g h t ( 4 9 ) .
Aqueous s o l u t i o n s containing cocaine hydrochloride 5% and
phenol 0.5% remained clear and c o l o r l e s s f o r a year a t
0' t o 4', room temperature and 37'. A f a l l i n pH from
4.6 t o 3.9; 2.7 and 2 . 1 respectively, suggesting a chemi-
c a l change, such s o l u t i o n should therefore be s t o r e d i n a
cool place (49).
Factors a f f e c t i n g t h e s t a b i l i t y of cocaine i n s o l u t i o n s
were studied. No hydrolysis of cocaine was observed a t
pH values below 4.0, but hydrolysis w a s r a p i d when pH w a s
g r e a t e r than 5.5. Decreasing i o n i c s t r e n g t h a l s o increa-
sed t h e rate of hydrolysis. With proper pH conditions,
solutions of cocaine were s t a b l e a t temperatures up t o 24'
f o r up t o 45 days (71).
Brompton's c o c k t a i l containing morphine, cocaine and alco-
hol has a l i m i t e d s h e l f l i f e of about 3 weeks. This cock-
t a i l should be s t o r e d i n well-closed l i g h t - r e s i s t a n t con-
tainers (72).
COCAINE HYDROCHLORIDE 195

9. Methods of Analysis

9.1 I d e n t i f i c a t i o n Tests

The following i d e n t i f i c a t i o n t e s t s are mentioned

under cocaine i n t h e B r i t i s h Pharmacopoeia ( 1 4 ) .
- The l i g h t absorption i n t h e range 230 t o 3501x11 of a
2-cm l a y e r of 0.001% w/v s o l u t i o n i n 0 . 0 1 M hydro-
c h l o r i c a c i d e x h i b i t s a w e l l defined maximum only a t
about 233 nm; with absorbance about 0.86.
- 0 . 1 g of cocaine i s heated w i t h 1 ml of s u l f u r i c
a c i d f o r f i v e minutes a t looo, upon c o o l i n g and
c a u t i o u s l y mixing with 2 ml of water; t h e aromatic
odor of methyl benzoate i s p e r c e p t i b l e , and when t h e
s o l u t i o n i s cooled and allowed t o s t a n d f o r some
hours, c r y s t a l s of benzoic a c i d s e p a r a t e .
- 50 mg of cocaine i s d i s s o l v e d i n 1.65 m l of 0.1 M
hydrochloric a c i d , 8.5 ml of a 5% w/v s o l u t i o n of
alum and 5 m l of potassium permanganate s o l u t i o n
are added t o t h e cocaine s o l u t i o n w i t h s t i r i n g f o r
s e v e r a l seconds; c h a r a c t e r i s t i c r e c t a n g u l a r v i o l e t
p l a t e s are formed.
- A s a t u r a t e d s o l u t i o n i s a l k a l i n e t o phenolphthalein
solution.
The followings a r e i d e n t i f i c a t i o n t e s t s mentioned
under cocaine hydrochloride (14 ) .
- To 0.5 m l of a 2% w/v s o l u t i o n , 0.5 m l of water and
0 . 1 m l of a 3% w/v s o l u t i o n of chromium t r i o x i d e a r e
added; a yellow p r e c i p i t a t e i s formed which r e d i s -
s o l v e s on shaking. Upon a d d i t i o n of t h e same reagent
or hydrochloric a c i d , t h e p r e c i p i t a t e reappears.
- 0 . 1 g of cocaine hydrochloride i s heated with 1 m l
of s u l f u r i c a c i d f o r f i v e minutes on a water b a t h ,
2 m l of water a r e added c a r e f u l l y , methyl benzoate
recognisable by i t s odor i s produced. On c o o l i n g t h e
s o l u t i o n , c r y s t a l s a r e deposited.
- 50 mg a r e d i s s o l v e d i n 1 . 5 ml of water, 8.5 ml of a
5% w/v s o l u t i o n of alum and 5 m l of a 1% w/v s o l u t i o n
of potassium permanganate a r e added and t h e r e s u l t -
i n g mixture i s shaked f o r a few seconds; a c r y s t a l -
l i n e p r e c i p i t a t e i s slowly formed, which when exami-
ned under a microscope, can be seen t o c o n s i s t of
c h a r a c t e r i s t i c rectangular v i o l e t c r y s t a l s .
- S p e c i f i c o p t i c a l r o t a t i o n of a 2.5% w/v s o l u t i o n ,
-TO0 t o -73', c a l c u l a t e d w i t h r e f e r e n c e t o t h e un-
d r i e d substance.
- Yields t h e r e a c t i o n s c h a r a c t e r i s t i c of a l k a l o i d s and
t h e r e a c t i o n s c h a r a c t e r i s t i c of c h l o r i d e s mentioned
i n (14).
196 FARID J. MUHTADI AND ABDULLAH A. AL-BADR

The following i d e n t i f i c a t i o n t e s t s a r e mentioned

under cocaine i n t h e United S t a t e s Pharmacopeia (73).
- The u l t r a v i o l e t absorption spectrum of a 1 i n 75,000
s o l u t i o n i n d i l u t e hydrochloric a c i d (1:120 ) e x h i b i t s
maxima and m i n i m a a t t h e same wavelengths a s t h a t
of a similar s o l u t i o n of USP Cocaine Hydrochloride
RS, concomitantly measured, and t h e r e s p e c t i v e molar
a b s o r p t i v i t i e s , c a l c u l a t e d on t h e d r i e d b a s i s , a t t h e
wavelength of maximum absorbance a t about 233 nm do
not d i f f e r by more than 3.0%.
- It meets t h e requirements under i d e n t i f i c a t i o n -
Organic Nitrogenous Bases (181) , USP Cocaine Hydro-
chloride RS.
- 100 g a r e dissolved i n a mixture of 0.4 m l of d i l u t e
hydrochloric a c i d (1i n 1 2 ) and water t o make 5 m l ,
upon adding 5 drops of chromium t r i o x i d e solution
(1:20); a yellow p r e c i p i t a t e i s formed, and it quick-
l y r e d i s s o l v e s when t h e mixture i s shaken. Upon
addition of 1 m l hydrochloric acid; a permanent
orange-colored c r y s t a l l i n e p r e c i p i t a t e i s formed.
- 1 0 mg a r e dissolved i n 1 m l d i l u t e hydrochloric a c i d
(1:600) and evaporated on a steam bath j u s t t o dry-
ness. The residue i s dissolved i n 2 drops of water,
1 m l of potassium p e m n g a n a t e s o l u t i o n (1i n 300)
i s added; a v i o l e t c r y s t a l l i n e p r e c i p i t a t e i s formed,
and it appears brownish v i o l e t when c o l l e c t e d on a
f i l t e r paper, and shows c h a r a c t e r i s t i c violet-red
c r y s t a l l i n e aggregates under t h e low power of a
microscope, similar t o those obtained from USP Coca-
ine Hydrochloride RS.
- 1 0 mg of t h e s a l t i s dissolved i n water (1:20), s i l -
ver n i t r a t e TS i s added dropwise t o t h e s o l u t i o n , a
white p r e c i p i t a t e i s Tormed which i s i n s o l u b l e i n
n i t r i c acid.

9.2 Microcrystal Tests

30 mg of cocaine hydrochloride dissolved i n 25 m l of

water. The following microcrystal t e s t s were perfor-
med and microscopically examined.
- Mercuric chloride s o l u t i o n gives with cocaine after
1 5 minutes, c l u s t e r c r y s t a l s which are shown i n
Fig. 11 (74).
- M a r m ' s reagent gives with cocaine feathery needles
which formed a f t e r 1 5 minutes, these are presented
i n Fig. 12 (74).
- A concentrated s o l u t i o n of cocaine hydrochloride
(30 mg i n 12.5 ml water) gives with potassium per-
manganate s o l u t i o n (1%), sharp jagged irregular blades
COCAINE HYDROCHLORIDE 197

@@

FIG. 13 : MIcXCCFWTAKS OF COCAINE WIlM pd. PBWJGUU'IE.

198 FARID J. MUHTADI AND ABDULLAH A. AL-BADR

FIG. 14 : YICIMCRYSTALS CW CXXAINE WI?H LJNl ICDIDE.

COCAINE HYDROCHLORIDE 199

a f t e r 7-10 minutes as i n Fig. 1 3 ( 7 4 ) .

- Lead i o d i d e reagent y i e l d s w i t h cocaine immediately,
dentated i r r e g u l a r r o s e t t e s as presented i n F i g . 14
(17,74).
- Gold c h l o r i d e reagent y i e l d s with cocaine immediately,
comb-shaped r o s e t t e s as shown i n Fig. 15 (17,74).

The a n a l y t i c a l methods of determination of cocaine

and i t s m e t a b o l i t e s i n b i o l o g i c a l materials , have
been reviewed by s e v e r a l a u t h o r s (52, 75-78).

9.3 T i t r i m e t r i c Methods

9.3.1 Non-aqueous T i t r a t i o n

B r i t i s h Pharmacopeia 1980 (14) and U . S .

Pharmacopeia XX ( 7 3 ) described a non-aqueous
t i t r a t i o n f o r cocaine and i t s hydrochloride.
The following a r e t h e procedures described
i n t h e US Pharmacopeia XX:-

Cocaine

Dissolve about 600 mg o f cocaine, p r e v i o u s l y

d r i e d and a c c u r a t e l y weighed, i n 50 m l of
g l a c i a l a c e t i c a c i d , add 1 drop of c r y s t a l
v i o l e t TS, and t i t r a t e with 0.1N p e r c h l o r i c
acid V !: t o a green end-point. Perform a
blank determination, and make any necessary
c o r r e c t i o n . Each m l of 0.1N p e r c h l o r i c a c i d
i s equivalent t o 30.34 mg of C17H21N04.

Cocaine hydrochloride

Dissolve about 500 mg of cocaine hydrochloride,

a c c u r a t e l y weighed, i n a mixture of 40 m l of
g l a c i a l a c e t i c a c i d and 1 0 m l of mercuric
a c e t a t e TS. Add 2 drops of q u i n a l i d i n e r e d
TS, and t i t r a t e with 0.1N p e r c h l o r i c a c i d VS.
Perform a blank determination, and make any
necessary c o r r e c t i o n . Each m l of 0.1N per-
c h l o r i c a c i d i s equivalent t o 33.98 mg o f
C17H21N0h. H C 1 .

9.3.2 Potentiometric T i t r a t i o n

Diamandis and Hadjiioannou (79) published a

p o t e n t l o m e t r i c method f o r t h e determination
200 FARID J. MUHTADI A N D ABDULLAH A. AL-BADR

of cocaine and some common a l k a l o i d s w i t h a

p i c r a t e - s e l e c t i v e e l e c t r o d e . The method i s
based on formation o f t h e i n s o l u b l e p i c r a t e s
i n = 1 M c o n c e n t r a t i o n and were determined by
d i r e c t potentiometry or by potentiometric
t i t r a t i o n w i t h 8 mM- or 40 t o 80 mM sodium
p i c r a t e a t pH 6 , a p i c r a t e - s e l e c t i v e e l e c t r o d e
being used f o r i n d i c a t i o n . End-point s were
determined with use o f Gran p l o t s , f o r which
t h e mean e r r o r w a s ? 2%.

9.4 Spectrophotometric Methods

9.4.1 Infra-red Spectrometry

Moss et a1 ( 8 0 ) had analysed t h e i n f r a - r e d

s p e c t r a of s e v e r a l drugs for s i m i l a r i t i e s by
techniques of numerical taxonomy including
cocaine. Preliminary r e s u l t for t h i s s e t of
drugs i n d i c a t e t h a t an expanded m u l t i - v a r i a t e
approach t o drug c l a s s i f i c a t i o n may be u s e f u l .

Moore (81) have described i . r . d a t a for t h e

i d e n t i f i c a t i o n of cis and t r a n s - cinnamoyl
cocaine i n i l l i c i t cocaine s e i z u r e s .

9.4.2 Ultraviolet

Kolosova ( 8 2 ) r e p o r t e d t h e use o f u l t r a v i o l e t
spectrum for t h e d e t e c t i o n of a l k a l o i d s
( i n c l u d i n g cocaine) and b a r b i t u r a t e s i n
o b j e c t s of l e g a l chemical examination. The
drugs can be d e t e c t e d i n b i o l o g i c a l m a t e r i a l s
by u l t r a v i o l e t absorption i n t h e region
200-400 nm.

-
C i s and t r a n s - cinnamoyl cocaine i n i l l i c i t
s e i z u r e s were i d e n t i f i e d (81). The isomeric
a l k a l o i d s were found i n t h e l e v e l of = 1% in
over h a l f of t h e i l l i c t cocaine samples
i n v e s t i g a t e d . The author have presented t h e
UV d a t a as w e l l as some o t h e r s p e c t r a l d a t a
and g.1.c. c o n d i t i o n s o f a n a l y s i s .

L-cocaine had been determined by c i r c u l a r

dichroism ( 8 3 ) . The samples a r e d i s p e r s e d
i n water o r methanol, and, a f t e r removal of
COCAINE HYDROCHLORIDE 20 1

i n s o l u b l e material, t h e p r i n c i p l e a b s o r p t i o n
bands o f L-cocaine a r e measured a t 275 or
245 nm i n a s p e c t r o p o l a r i m e t e r . The l i m i t o f
d e t e c t i o n i s 1 0 uM ( a s c a l c u l a t e d from t h e
245 nm b a n d ) . I n term o f a n a l y s i s t i m e , t h e
method compares f a v o u r a b l y w i t h o t h e r methods
now i n u s e ; however, any DL-racemate w i l l
remain u n d e t e c t e d . R e s u l t s can b e a c c e p t e d
a s proof o f t h e presence o f L-cocaine i n
samples.

T o f f o l i and Avico ( 8 4 ) have r e p o r t e d chemical

e v a l u a t i o n o f c r u d e c o c a i n e and o f t h e r e si-
due a f t e r i n d u s t r i a l e x t r a c t i o n o f c o c a i n e
t h e r e f r o m . The a u t h o r s have d e s c r i b e d a pro-
cedure f o r t h e d e t e r m i n a t i o n o f c o c a i n e i n
c r u d e c o c a i n e and a n o t h e r procedure f o r t h e
d e t e r m i n a t i o n o f ecgonine and anhydroecgonine
i n t h e residue a f t e r e x t r a c t i o n of cocaine.
The procedure f o r d e t e r m i n a t i o n of c o c a i n e i n
crude c o c a i n e i s as follows:-

D i s s o l v e 0.5 t o 1 g o f sample i n 3% o f H2SO4

(10 m l ) a t 0'. S t i l l a t O o , add 6% potassium
permanganate s o l u t i o n ( 8 m l ) and 10% H2SO4
(1m l a t a t i m e , and a t 5 minutes i n t e r v a l s
u n t i l t h e solution i s decolorised. Set aside
f o r 30 minutes, t h e n add f i n e l y powdered
o x a l i c a c i d u n t i l t h e p r e c i p i t a t e formed has
dissolved. Extract t h e resulting c l e a r
colorless solution t w i c e w i t h e t h e r and
d i s c a r d t h e e x t r a c t s . Mzke t h e s o l u t i o n
a l k a l i n e w i t h aqueous ammonia and e x t r a c t f o u r
times w i t h e t h e r . Dry t h e combined e x t r a c t s ,
e v a p o r a t e and d r y t h e r e s i d u e f o r two hours
a t 60°, t h e n for 1 2 hours over H2SO4 and
weigh. D i s s o l v e t h e r e s i d u e i n e t h a n o l ( 2 0 m l )
i n t h e same c o n t a i n e r , add w a t e r ( 2 0 m l ) and
re-weigh. From t h e weight of s o l u t i o n ,
calculate t h e dilution ( w a t e r ) required) t o
g i v e a d e n s i t y o f 0.926 a t 20°, i . e . , 54%
( v / v ) of e t h a n o l c o n t e n t . Measure t h e o p t i c a l
r o t a t i o n and hence c a l c u l a t e t h e amount o f
cocaine. D i l u t e an a l i q u o t o f t h e s o l u t i o n
1:lOOO w i t h 0.1N H C 1 and measure t h e e x t i n c -
t i o n a t 274 nm; f o r c o c a i n e i n 0.1N H C 1 ,
EiZm = 38.2 2.0. Two maxima are i n f a c t
202 FARID J. MUHTADI A N D ABDULLAH A. AL-BADR

observed ( a t 274 and 233 nm), w i t h molecular

e x c i t a t i o n c o e f f i c i e n t o f 1130 and 13,500,
r e s p e c t i v e l y . The d i r e c t weighing method
t h e most r e l i a b l e of t h e s e methods. Chromato-
graphy and e l e c t r o p h o r e s i s o f t h e sample
b e f o r e and a f t e r o x i d a t i o n w i t h potassium
permanganate shows t h a t o n l y cocaine remains
after t h i s treatment.

Spectrophotometric a s s a y o f c o c a i n e and some

n a r c o t i c s and a l k a l o i d s i n g a l e n i c a l composi-
t i o n s have been r e p o r t e d ( 8 5 ) . To a s s a y
c o c a i n e i n aqueous s o l u t i o n of i t s s a l t , t h e
e x t i n c t i o n o f t h e d i l u t e d sample i s d e t e r -
mined a t t h e wavelength f o r maximum a b s o r p t i o n
(257 t o 286 nm) and compared w i t h t h a t of
p r o g r e s s i v e l y d i l u t e d samples o f s t o c k solu-
t i o n s . The method i s c h i e f l y designed f o r u s e
on aqueous p r e p a r a t i o n s ( e . g . from ampoules).

9.4.3 Fluorescence Analysis

Shih-Chung Tu (86) have r e p o r t e d f l u o r e s c e i n

chloride spot t e s t f o r t h e determination of
c o c a i n e and some o t h e r drugs c o n t a i n i n g amino
and h e t e r o c y c l i c n i t r o g e n . The d e t e r m i n a t i o n
i s based on t h e formation of thodamine dyes.
One mg f l u o r e s c e i n c h l o r i d e and 1 drop o f t h e
drug (0.01-0.1%)were evaporated t o d r y n e s s ,
fused w i t h one drop of ZnC12; t h e melt w a s
d i s s o l v e d i n 2 drops 10% a l c o h o l i c H C 1 solu-
t i o n . The f l u o r e s c e n c e was determined w i t h
u l t r a v i o l e t l i g h t ; t h e c o l o r depends on t h e
molecular s t r u c t u r e . The t e s t (2-4 y ) was
2-5 times more s e n s i t i v e t h a n t h e F e i g e l t e s t -
t u b e method.

9.4.4 Phosphorimetry

Harbaugh e t a1 ( 87) have r e p o r t e d q u a l i t a t i v e

and q u a n t i t a t i v e a n a l y s i s of cocaine. The
phosphorescence emission s p e c t r a , l i f e times,
and r e l a t i v e s i g n a l s (peak e m i s s i o n s ) of
c o c a i n e and some o t h e r drugs were determined
w i t h use o f t h e a p p a r a t u s and procedures pre-
v i o u s l y d e s c r i b e d ( 8 8 ) . For a mdticomponent
m i x t u r e , t h e parameters c i t e d i n d i c a t e which
COCAINE HYDROCHLORIDE 203

o f t h e drugs can be s e p a r a t e d s p e c t r a l l y o r
t e m p o r a r a l l y o r by a combination o f t h e two
t e c h n i q u e s . Some mixtures, however , cannot b e
s e p a r a t e d . Examples are given f o r t h e d e t e r -
mination o f t h e components o f s e v e r a l synthe-
t i c b i n a r y m i x t u r e s o f t h e drugs. I n some
i n s t a n c e s where t h e emission s p e c t r a s e v e r e l y
o v e r l a p . Time-resolved phosphorimetry pro-
v i d e s a u s e f u l means f o r i d e n t i f y i n g t h e s e
drugs even i n some o f t h e i r m i x t u r e s .

E x t e r n a l heavy-atom e f f e c t on d e t e c t i o n l i m i t s
and l i f e times o f phosphoresence i n t i m e -
r e s o l v e d l a s e r - e x c i t e d phosphorimetry have
been r e p o r t e d by B o u t i l i e r and Winefordner
(89). I n t h e instrument d e s c r i b e d , r a d i a t i o n
from a p u l s e d n i t r o g e n o r f l a s h lamp-pumped
dye laser w a s d i r e c t e d v i a a f i l t e r t o t h e
sample c o n t a i n e d i n a q u a r t z c e l l ( 3 0 cm X
2 mm i . d . ) mounted i n an n.m.r. s p i n n e r assem-
b l y . The e m i t t e d r a d i a t i o n was examined i n a
system i n c o r p o r a t i n g a monochromator, photo-
multiplier, gated amplifier, signal generator,
boxcar i n t e g r a t o r , c h a r t r e c o r d e r , o s c i l l o -
scope, s i g n a l o v e r a g e r , t a p e punch and mini-
computer. Phosphoresence l i f e - t i m e and detec-
t i o n l i m i t s were determined a t 77 K f o r solu-
t i o n s o f phenanthrene and o f s e v e r a l drugs
( i n c l u d i n g c o c a i n e ) i n ethanol-HzO (1:9). I n
t h e presence o f A g
'
, d e t e c t i o n l i m i t s were
o f t en improved.

9.4 . 5 Colorimetry

Tomasch and Majer ( 9 0 ) have e s t i m a t e d some

pharmaceutical e s t e r s which a r e used i n
pharmacy. The method o f H i l l ( 9 1 ) w a s modi-
f i e d and adopted f o r t h e d e t e r m i n a t i o n o f t h e
e s t e r s . F i v e m l o f t h e e t h a n o l i c s o l u t i o n of
t h e e s t e r (1mg/ml) i s added t o 1 ml o f 10%
hydroxylamine h y d r o c h l o r i d e i n 80% e t h a n o l ,
2 m l o f 10% sodium hydroxide i n 90% e t h a n o l
and 5 m l p e r o x i d e - f r e e e t h e r ; t h e m i x t u r e i s
shaken w e l l , a f t e r 20 minutes t r e a t e d w i t h
2 ml HC1 d i l u t e d 1 : 3 and 1 ml o f 10% o f f e r r i c
c h l o r i d e (FeC13) i n 0.1N H C 1 , d i l u t e d t o 25 ml,
f i l t e r e d from sodium c h l o r i d e and d e t e c t e d
c o l o r i m e t r i c a l l y a t 320 nm.
204 FARID J. MUHTADI A N D ABDULLAH A. AL-BADR

The u s e o f c o b a l t t h i o c y a n a t e i n t h e c o l o r i -
m e t r i c a n a l y s i s of c o c a i n e and some o t h e r
o r g a n i c b a s i s have been r e p o r t e d ( 9 2 ) . The
a u t h o r s have d i s c u s s e d e a r l i e r unpublished
work by o t h e r a u t h o r s on t h e p r e c i p i t a t i o n and
s e p a r a t i o n of a l k a l o i d s and o r g a n i c b a s e s w i t h
t h i o c y a n a t e s some metals and w i t h c o b a l t i n
p a r t i c u l a r . The p r e c i p i t a t i o n o f such com-
pounds w i t h CO(SCN)z from aqueous medium
r e s u l t s i n two t y p e s o f complexes, v i s , a b l u e
t y p e ( t h e more common) which t h e c o b a l t forms
a complex anion w i t h SCN-, and a r e d , brown o r
v i o l e t t y p e i n which t h e c o b a l t forms a com-
p l e x c a t i o n w i t h o r g a n i c base. Condition for
t h e d e t e r m i n a t i o n of v a r i o u s q u a t e r n a r y
ammonium compounds and of c o c a i n e i n non-
aqueous medium by e x t r a c t i o n w i t h benzene,
chloroform o r dichloromethane a r e d i s c u s s e d ;
a s u i t a b l e r e a g e n t i s prepared by d i s s o l v i n g
CO(SCN)p i n methanolic HSCN ( o b t a i n e d by
c a t i o n exchange) t o form a s o l u t i o n of
H2CO(SCN)2. This i s d i l u t e d w i t h chloroform
o r dichloromethane t o g i v e 0.2N s o l u t i o n ,
which i s added t o a s o l u t i o n o f t h e b a s e i n
t h e same s o l v e n t . The e x t i n c t i o n o f t h e b l u e
s o l u t i o n i s measured a t 625 nm.

Other c o l o r i m e t r i c methods have been r e p o r t e d

( 93-95 1 *

9.4.6 Mass Spectrometry

The mass a n a l y s e d ion-kinetic-energy s p e c t r o -
meter w a s a p p l i e d t o t h e d e t e r m i n a t i o n o f
cocaine i n coca l e a v e s and u r i n e by u s e o f
s i n g l e o r m u l t i p l e - i o n monitoring t e c h n i q u e
(96); s e n s i t i v i t y w a s maximized by u s e o f
s i n g l e - i o n monitoring ( w i t h i s o b u t a n e as rea-
gent g a s ) . The d e t e c t i o n l i m i t f o r c o c a i n e i n
1 pg of coca l e a f b e i n g < 1 ng. Multiple-
r e a c t i o n monitoring provided similar s e n s i -
t i v i t y t o , and was more s e l e c t i v e t h a n s i n g l e -
i o n monitoring. The e f f e c t o f t h e method o f
sample i n t r o d u c t i o n w a s important. For q u a n t i -
t a t i v e analysis, isotope dilution with analyte
l a b e l l e d w i t h s t a b l e i s o t o p e was p r e f e r r e d ;
a l t e r n a t i v e l y , s t andard-addit i o n , e x t e r n a l -
COCAINE HYDROCHLORIDE 205

s t a n d a r d or c a l i b r a t i o n graph methods could be

used. The graph ( b a s e d on s i n g l e - r e a c t i o n
monitoring) on peak a r e a v s amount f o r solu-
t i o n o f c o c a i n e i n methanol w a s r e c t i l i n e a r
f o r 1 t o 1 5 0 ng. S i n g l e r e a c t i o n monitoring
permitted detection of cocaine i n unreacted
samples ( c o n t a i n i n g l a r g e q u a n t i t i e s of many
o t h e r d r u g s ) and o f benzoylecgonine i n u r i n e .

Cooks e t a 1 ( 9 7 ) have reviewed mass analyzed

i o n k i n e t i c energy spectrometry ( M I K E ) and
p r e s e n t e d i t s a p p l i c a t i o n t o t h e d i r e c t analy-
s i s o f cocaine and cinnamoylcocaine i n coca
p l a n t (Erythroxylum c o c a ) t i s s u e s .

Moore (81) have p r e s e n t e d m . s . d a t a for t h e

i d e n t i f i c a t i o n o f cis and t r a n s - cinnamoyl-
c o c a i n e i n i l l i c i t c o c a i n e s e i z u r e s . The
n.m.r. d a t a and some o t h e r s p e c t r a l d a t a have
a l s o been p r e s e n t e d .

9.5 Counter-Current E x t r a c t i o n

The s e p a r a t i o n o f t h e m i x t u r e s o f o r g a n i c s u b s t a n c e s
by a s i m p l i f i e d method o f c o u n t e r - c u r r e n t e x t r a c t i o n
had been r e p o r t e d ( 9 8 ) . The o p t i m a l c o n d i t i o n s were
found f o r t h e s e p a r a t i o n o f two component a l k a l o i d
and o t h e r o r g a n i c compound m i x t u r e s by t h e method
o f c o u n t e r - c u r r e n t e x t r a c t i o n i n f u n n e l s . The depen-
dance o f t h e e x t r a c t i o n w i t h chloroform for q u a n t i t a -
t i v e s e p a r a t i o n o f c o c a i n e , and o t h e r a l k a l o i d s , on
t h e pH v a l u e s o f t h e b u f f e r s o l u t i o n i s p r e s e n t e d .

9.6 Chromatographic Methods

9.6.1 Thin Layer Chromatography

Clarke (17) d e s c r i b e d t h e f o l l o w i n g system:

Glass p l a t e s , 20 X 20 cm, c o a t e d w i t h a slurry
c o n s i s t i n g o f 30 gm o f s i l i c a g e l G i n 60 m l
o f water t o g i v e a l a y e r 0.25 mm t h i c k and
d r i e d a t 110' f o r 1 hour. A sample 1 . 0 p 1 o f
a 1% s o l u t i o n i n 2 N a c e t i c a c i d , t a k e n by a
micro d r o p , i s used. The s o l v e n t system con-
s i s t s of s t r o n g ammonia s o l u t i o n : methanol
(1.5 : 1 0 0 ) . It should be changed a f t e r two
r u n s . Solvent i s allowed t o s t a n d i n t h e t a n k
206 FARID J. MUHTADI AND ABDULLAH A. AL-BADR

f o r one hour. The assending chromatogrem i s

developed i n a t a n k 21 X 21 X 10 cm, t h e end
of t h e t a n k being covered with f i l t e r paper t o
a s s e s t evaporation. T i m e of run 30 minutes.
The l o c a t i o n reagent i s an a c i d i f i e d iodopla-
t i n a t e spray, and t h e R f value is 0.60.

Several solvent systems have been used f o r

t h i n - l a y e r chromatographic s e p a r a t i o n of
cocaine andareshown i n Table ( 9 ) .

Table ( 9 ) Thin-Layer Chromatography of Cocaine

- -
Absorbent and Solvent Visualizing
Rf Ref.
Systems agent
--
S i l i c a g e l F 1500 LS 254 W a t 230 nm a f t e r 0.73 102
a c t i v a t e d a t 110' f o r 2 e x t r a c t i o n i n t o chlo-
hours methanol : aqueous roform
ammonia (100:1.5)
ethanol : chloroform (1:l W a t 230 nm a f t e r 0.31 102
e x t r a c t i o n i n t o chlo-
roform.

S i l i c a g e l 60 F254 0.25 m
a ) Chloroform : methanol Sprayed by 5% H2SO4 0.90 103
.
conc ammonia ( 9 :10 :1) followed by iodoplat i
nat e
b ) Ethyl a c e t a t e : metha- Sprayed by 5% H p O 4 0.87 103
nol : water : conc. followed by iodoplat i
ammonia ( 8 5 :10 :3 :1) nat e .
Activated precoated s i l i c c Acidic i o d o p l a t i n a t e 0.79 105
g e l p l a t e s of 0.25 mm ( AIPA)
t h i c k n e s s (Merck)
E t h y l a c e t a t e : n propanol:
28% ammonium hydroxide
( 40: 30: 3)

Precoated s i l i c a g e l G , Dragendoff t h e n 0.73

26 X 20 cm, 0.25 mm l a y e r iodoplat i n a t e
*ethyl a c e t a t e : methanol
17:2 and 20 m l of 50%
ammonia i n a beaker.
COCAINE HYDROCHLORIDE 207

Continued Table ( 9 )

Absorbent and Solvent Visualizing

Systems agent

Chloroform : methanol
(1:l) and 20 m l o f 50%
ammonia i n a beaker.

The above two system are

placed i n t h e center of a
tank.

Other t h i n l a y e r chromatography systems have

a l s o been r e p o r t e d ( 1 7 , 99-101, 104, 106-109,
111-119).

9.6.2 Paper Chromatography

Clarke (17)d e s c r i b e d t h e f o l l o w i n g system:

Whatman No. 1, s h e e t 1 4 X 6 i n c h , i s b u f f e r e d
by d i p p i n g i n a 5% s o l u t i o n o f sodium dihyd-
rogen c i t r a t e , b l o t t i n g , and d r y i n g a t 25'
f o r one hour. It c a n b e s t o r e d i n d e n i n i t e l y .
A sample o f 2.5 1~.1 o f a 1% s o l u t i o n ; i n 2 N
a c e t i c a c i d i f p o s s i b l e , o t h e r w i s e i n 2N
h y d r o c h l o r i c a c i d , 2 N sodium hydroxide, or
e t h a n o l s o l v e n t , 4.8 g o f c i t r i c a c i d i n a
m i x t u r e o f 130 m l of water and 870 m l o f n-
butanol. ( T h i s s o l v e n t may be used for
s e v e r a l weeks i f w a t e r i s added from t i m e t o
t i m e t o keep t h e s p e c i f i c g r a v i t y a t 0.843 t o
0.844). The chromatogram i s ascending, i n a
t a n k 8 X 11 X 15% i n c h , 4 s h e e t s being run a t
a t i m e . Time o f run, f i v e h o u r s , R f v a l u e
0.38, l o c a t i o n i s done under u l t r a v i o l e t
l i g h t , s t r o n g a b s o r p t i o n and t h e l o c a t i o n
reagent i s iodoplatinate spray; strong
react ion.

Bastos e t a1 ( 1 2 0 ) r e p o r t e d a method f o r
r o u t i n e i d e n t i f i c a t i o n of c o c a i n e m e t a b o l i t e s
i n human u r i n e . The sample ( 1 0 m l ) w a s
a d j u s t e d t o pH 8 t o 9 w i t h NaHC03 and e x t r a c t e d
208 FARID J. MUHTADI AND ABDULLAH A. AL-BADR

chloroform : e t h a n o l ( 3 : 2 ) ( 5 m l ) ; t h e lower
l a y e r ( c o n t a i n i n g , e.g. unmetabolized
c o c a i n e ) was r e j e c t e d . The upper l a y e r w a s
s a t u r a t e d w i t h K2CO3 and c e n t r i f u g e d , and
t h e pH o f t h e e t h a n o l i c phase was a d j u s t e d
t o < 7 with d i l . H C 1 . The e x t r a c t e d meta-
b o l i t e s were converted i n t o t h e i r b u t y l
d e r i v a t i v e s w i t h butanol-H2S04 and t h e solu-
t i o n w a s washed w i t h t o l u e n e . The s o l u t i o n
was t h e n s a t u r a t e d w i t h NaHC03 and t h e
d e r i v a t i v e s were e x t r a c t e d w i t h cyclohexane
( 1 0 m l ) . The e x t r a c t w a s e v a p o r a t e d , and t h e
r e s i d u e w a s d i s s o l v e d i n chloroform and
a p p l i e d t o a polygram s i l i c a g e l MN s h e e t .
The chromatogram w a s developed w i t h e t h y l
acetate-methanol-water ( 7 : 2 : 1 ) , t h e n f o r a
s h o r t d i s t a n c e i n t h e same d i r e c t i o n w i t h
chloroform-acetone-aqueous ammonia( 5 :94 :1) .
A l t e r n a t i v e l y , t h e s e two s o l v e n t s were used f o r
t w o - d i r e c t i o n a l s e p a r a t i o n . S p o t s were d e t e c -
t e d by s p r a y i n g w i t h i o d o p l a t i n a t e r e a g e n t .
El-Darawy and Mobarak ( 1 2 1 ) s t u d i e d t h e
chromatographic s e p a r a t i o n o f some a l k a l o i d s
i n c l u d i n g c o c a i n e , on carboxymethylcellulose
cation-exchange paper. From 5 t o 8 p1 of
methanolic s o l u t i o n (1mg p e r m l ) o f each
a l k a l o i d were a p p l i e d t o t h e paper and a f t e r
development o f t h e chromatogram, t h e s p o t s
were d e t e c t e d by viewing under 254 nm r a d i a -
t i o n or by s p r a y i n g w i t h Dragendorff r e a g e n t .

9.6.3 Gas Chromatography

Clarke ( 1 7 ) d e s c r i b e d t h e f o l l o w i n g system:
Column: 5% SE-30 on 60-80 mesh chromosorb W
AW. 5 ft X 118 i n c h i n t e r n a l diameter
s t a i n l e s s s t e e l column. Column t e m p e r a t u r e
230°, Carrier g a s : Nitrogen g a s flow:
30.7 m l p e r minute, D e t e c t o r : flame i o n i s a -
t i o n , hydrogen 22 ml p e r minute. R e t e n t i o n
time i s 0.57 r e l a t i v e t o codeine.

S e v e r a l r e p o r t s had been p u b l i s h e d concerning

t h e chromatographic i d e n t i f i c a t i o n and separa-
t i o n of c o c a i n e and i t s m e t a b o l i t e s as shown
i n Table 1 0 .
Table (10) G a s Chromatography of Cocaine

Column C a r r i e r gas Detector Remarks Ref.

U- shaped Nitrogen Flame i o n i s a - - The benzoyl ecgonine ( a cocaine m e t a b o l i t e ) 99

column 25 ml/min. t ion i s t h e d e t e c t e d substance which w a s o b t a i n e d
( 6 f t x 0.25 operated a t from u r i n e .
in. ) 2000. - The m e t a b o l i t e i s s e p a r a t e d by TLC t h e n w a s
s u b j e c t e d t o GLC a f t e r being converted i n t o
2.5% pf SE-30
i t s methyl d e r i v a t i v e s by t r e a t m e n t w i t h 1,l-
on G a s Chrom
dimethoxytrimethylamine at 70' t o 80° f o r
Q (100-120
1 hour.
mesh)
- Down t o 1 g o f t h e m e t a b o l i t e p e r m l of u r i n e
w a s e a s i l y detected.
- Recovery of t h e m e t a b o l i t e a t t h e e x t r a c t i o n
s t a g e w a s = 100% but only 30 t o 40% w a s
recovered by t h e prepared TLC.

3% of ov-1 - Tetracosane w a s used as i n t e r n a l standard. 122

- The cocaine H C 1 c o n t e n t s of 6 samples analysed
ranged from 6 t o 100%.
- Recoveries were 98.7 t o 103%.
- C o e f f i c i e n t of v a r i a t i o n were 0.89 t o 3.16.
- The method w a s adopted as o f f i c i a l f i r s t
a c t ion.
-
 Continued Table (10 ) .
Column Carrier gas Detector Remarks Ref.

2-M g l a s s - Nitrogen - Used f o r t h e i d e n t i f i c a t i o n n a r c o t i c s and

column packei! 30 ml/min-: psychotropics through p y r o l y t i c GC.
with 5% of - Oven tem- - The sample i n aqueous s o l u t i o n (1t o 2 ~1
polyoxyet hy- p e r a t u r e i: containing 50 t o 100 pg o f salt of t h e d r u g ) ,
lene glycol programmed i s placed on t h e f i l t e r e d w i r e and d r i e d i n a
20 M on Chro- t o 230' at stream o f hot a i r (100'). Pyrolysis i s
mosorb W ao min-1. c a r r i e d out a t 610' for 1 0 s , and t h e product
(100-120 mesh) i s t h e n s e p a r a t e d i n t h e column.
t
o
r
- S a l t a n a l y s i s i s p r e f e r r e d (because o f reducec
0 volatility).

4 f t X 0.125in Helium Nitrogen- - Blood + i n t e r n a l standard i s b u f f e r e d a t pH 9

packed with 30 ml/min-l phosphorous and e x t r a c t e d w i t h 1-chlorobutane and t h e
Chromosorb W temperature detector e x t r a c t ( p l u s H C 1 ) i s evaporated a t 60' under
(100-120 mesh) 230'. nitrogen .
coated with 3% - C a l i b r a t i o n graph a r e r e c t i l i n e a r f o r 0 . 2 t o
of ov-1. 1 Ug m l - 1 f o r t h e drugs (cocaine and o t h e r
basic drugs).
Nitrogen
Glass column 1.4 nWmin-1 Flame i o n i s a , - Ethyl morphine i s used a s t h e i n t e r n a l
(20 m X 0.35 tion standard.
mm i . d . 1
wall-coat ed
with SE-30.
 Continued Table ( 1 0 )

Column Carrier g a s Detector Remarks Ref.

-
A Fye 104 Nitrogen Rapid method f o r t h e d e t e r m i n a t i o n of c o c a i n e . 126
Chromatograph temperature
w i t h a column 2200.
(1.5 m X 4 mm)
of Gas-Chrom Q
(100-120 mesh)
s u p p o r t i n g 3%
of ov-17
N
~1
C1
Glass column Helium Flame i o n i s a - - Used f o r t h e d e t e r m i n a t i o n o f t h e drug, h e r o i i 127
( 6 f t x 2 mm) 30 ml/min-' t ion and morphine.
packed w i t h a The column - The powdered sample w a s e x t r a c t e d w i t h chlo-
1:l m i x t u r e o f w a s program- roform : methanol ( 3 : l ) c o n t a i n i n g r e s m e t h r i n
3% o f OV-17 on med ( 2 O min-1 as i n t e r n a l s t a n d a r d .
Varaport 30 f o r 190' t o
(80 t o 1 0 0 nesh 24O0.
and 5% o f SE-3C
on Chromosorb W
(80 t o 1 0 0 mesh
Cont h u e d Table (10 )

~ ~~ ~~ -
Column Carrier gas Detector Remarks Ref.
-
A silanised - Nitrogen Flame i o n i s a - Used f o r t h e chromatographic a n a l y s i s of t h e 128
g l a s s column 25 ml/min-' t ion cocaine o f Erythroxylum coca from t h r e e
(2.4 m X 2 mm) 2200 l o c a t i o n i n Peru.
packed w i t h 6% - Androst-4-ene-3,17-dione w a s used as i n t e r n a l
of OV-1 on standard .
Chromosorb W
AW - DMCS (100
t o 200 mesh).
~~ -
Conventional H e l i u m (205') Nitrogen - - Cocaine and benzoyl ecogonine were q u a n t i t a - 130
OV-1 column phosphorous t e d a f t e r JETUBE e x t r a c t i o n and d e r i v a t i s a -
detector tion.
- The propyl e s t e r of benzoyl ecgnonine i s used
as t h e i n t e r n a l s t a n d a r d .
-
(74 cm x
2 mm) Helium - Used t o determine ecgnonine methyl e s t e r , a 131
column packed ( 20 min-l) major m e t a b o l i t e of cocaine, i n u r i n e , a f t e r
2% of OV-101 on ( temp-pro- o r a l a d m i n i s t r a t i o n of cocaine.
Gas-Chrom-Q AW gramming f r o n - Phencyclidine i s t h e i n t e r n a l standard.
DMCS (100-120 140° t o 240' - The e f f l u e n t i s monitored by 70 e V m . s . a t
mesh) a t 100 min-1) m/e 82 f o r cocaine and i t s e s t e r and a t m / e
200 for t h e i n t e r n a l standard ( p h e n c y c l i d i n e ) I
COCAINE HYDROCHLORIDE 213

Other g a s chromatographic procedures f o r t h e

s e p a r a t i o n and i d e n t i f i c a t i o n have been
p u b l i s h e d (53,56,81,99,129,132-150).

9.6.4 Gas Chromatography-Mass Spectrometry (GC-MS)

Cocaine h a s been determined by GC-MS by

several authors:-

a ) J i n d a l and Vestergaard ( 1 5 1 ) have r e p o r t e d

a method f o r q u a n t i t a t i o n of c o c a i n e and
i t s p r i n c i p a l m e t a b o l i t e , benzoylecgonine
by glc-ms u s i n g s t a b l e i s o t o p e - l a b e l l e d
analogues as i n t e r n a l s t a n d a r d s . E x t r a c t
t h e c o c a i n e from u r i n e a t pH 9 i n t o
chloroform and t h e n i n t o 0.1N H C 1 , a d j u s t
t h e s o l u t i o n t o pH 9 w i t h aqueous ammonia,
r e - e x t r a c t i n t o chloroform, e v a p o r a t e t h e
e x t r a c t t o d r y n e s s a t 40' under n i t r o g e n ,
and d i s s o l v e t h e r e s i d u e i n benzene.
Analyse a p o r t i o n of t h e s o l u t i o n of
glc-ms on a g l a s s column ( 1 . 8 m X 2 mm)
c o n t a i n i n g 1 . 5 % of OV-1 on Gas Chrom 9,
( 1 0 0 t o 200 mesh) and o p e r a t e d a t 205'
w i t h helium a s c a r r i e r g a s ( 2 0 m l m i n - l ) .
Use N-( t r i d e u t e r o m e t h y l ) c o c a i n e as t h e
i n t e r n a l s t a n d a r d , and compare t h e i o n
i n t e n s i t i e s a t m / e 303 and 306. E x t r a c t
benzoylecgonine and t h e i n t e r n a l s t a n d a r d ,
N-( trideuterornethy1)-benzoyecogonine, from
u r i n e a t pH 7 i n t o chloroform : i s o p r o p y l
alcohol ( 4 : 1 ) , evaporate t h e e x t r a c t t o
dryness a t 60° under n i t r o g e n , convert
benzoylecgonine i n t o t h e e t h y l e s t e r w i t h
e t h a n o l i c diazomethane, e v a p o r a t e a t 40'
under n i t r o g e n , and d i s s o l v e t h e r e s i d u e
i n chloroform. E x t r a c t t h e s o l u t i o n w i t h
0.1N - H C 1 , a d j u s t t h e aqueous phase t o
pH 9, and e x t r a c t w i t h benzene, e v a p o r a t e
t h ? e x t r a c t t o dryness a t 40' under
n i t r o g e n , d i s s o l v e t h e r e s i d u e i n benzene,
and submit on a l i q u o t t o glc-rns as f a r
c o c a i n e , but compare i n t e n s i t i e s a t rn/e
317 and 320. About 2 ng m l - I o f c o c a i n e
and 5 ng m l - 1 of benzoylecgonine can b e
determined w i t h a p r e c i s i o n o f about 5%.
2 14 FARID J. MUHTADI A N D ABDULLAH A. AL-BADR

Lawry e t a1 ( 5 7 ) have i d e n t i f i e d two

novel c o c a i n e m e t a b o l i t e s i n b i l e by g a s
chromatography and by g a s chromatography-
mass spectrometry i n a c a s e of a c u t e
i n t r a v e n o u s c o c a i n e overdose. The drug
i s e x t r a c t e d from t h e sample i n t o 1-
c h l o r o b u t a n e and back-extracted i n t o weak
a c i d . The a c i d e x t r a c t i s made a l k a l i n e
and t h e compound i s e x t r a c t i n t o c h l o r o -
form f o r g l c w i t h 3% of OV-1 o r OV-17 as
s t a t i o n a r y phase. For g l c , m s , t h e OV-1
column i s used, w i t h t e m p e r a t u r e programm-
i n g and electron-impact m.s. The com-
pounds i d e n t i f i e d i n c l u d e c o c a i n e , t h e
known m e t a b o l i t e s , methylecgonine, nor-
c o c a i n e , and benzoylecgonine, and a l s o a
hydroxycocaine and methylecgonidine.

Chinn e t a1 (152) have d e s c r i b e d a g a s

chromatography-chemical i o n i s a t i o n mass
spectrometry of c o c a i n e and i t s metabo-
l i t e s i n b i o l o g i c a l f l u i d s . The sample,
e.g. blood, u r i n e , aqueous t i s s u e homoge-
n a t e o r v i t r e o u s humour, i s t r e a t e d w i t h
sodium f l u o r i d e t o i n h i b i t enzymic hydro-
l y s i s o f cocaine. The one p o r t i o n ( p l u s
t r i d e u t e r a t e d c o c a i n e as i n t e r n a l standard)
i s made a l k a l i n e w i t h K2HP04 and e x t r a c t e d
w i t h toluene-heptane-isoamyl a l c o h o l
( 7 : 2 : 1 ) ; t h i s e x t r a c t c o n t a i n s c o c a i n e and
norcocaine. A second p o r t i o n ( p l u s
t r i d e u t e r a t e d benzoylecgonine as i n t e r n a l
s t a n d a r d ) i s s a t u r a t e d w i t h sodium chlo-
r i d e and e x t r a c t e d ( a t pB 7 ) w i t h c h l o r o -
form-isopropyl a l c o h o l (9:l); t h e e x t r a c t
i s evaporated a t 60°, and t h e r e s i d u e i s
t r e a t e d by R method similar t o t h a t o f
J a i n et al(115) t o form t h e propyl e s t e r
of benzoylecgonine which i s p u r i f i e d by
e x t r a c t i o n . The f i n a l e x t r a c t c o n t a i n s
c o c a i n e and t h e ester o f benzoylecgonine.
Each e x t r a c t i s a n a l y s e d by g l c a t 205'
[column 1 . 2 m X 2 mm; 3% of OV-1 on Gas
Chrom Q (100 t o 1 2 0 mesh); methane (=20 m l
min-l) as c a r r i e r g a s and r e a g e n t gas f o r
t h e subsequent 120 e V m . s . 1. The mass
s p e c t r a are monitored a t m / e 304 f o r
COCAINE HYDROCHLORIDE 2 15

c o c a i n e , 307 ( f o r d e u t e r a t e d c o c a i n e ) ;
290 for norcocaine and 332 and 335 for
t h e d e r i v a t i v e s o f benzoylecgonine and
d e u t e r a t e d benzoylecgonine r e s p e c t i v e l y .
Jindal _ et - a1 (153) have a l s o determined
c o c a i n e and i t s b i o l o g i c a l l y a c t i v e meta-
b o l i t e , n o r c o c a i n e , i n human u r i n e . The
sample (1m l ) w a s t r e a t e d with ['H3]-
norcocaine ( 5 6 n g ) and, a f t e r adjustment
t o pH 8.5, w a s e x t r a c t e d w i t h cyclohexane.
The r e s i d u e from e v a p o r a t i o n of t h e
e x t r a c t was t r e a t e d w i t h t r i f l u o r o a c e t i c
anhydride, t h e m i x t u r e was evaporated and
an a l i q u o t o f a s o l u t i o n o f t h e r e s i d u e
i n benzene w a s s u b j e c t e d t o g.c.-m.s. w i t h
u s e o f a g l a s s column (1.8 m X 2 mm)
packed w i t h 1.5% o f OV-1 on Gas Chrom Q
(100 t o 120 mesh) and o p e r a t e d a t 205O.
The mass s p e c t r o m e t e r was o p e r a t e d i n t h e
s e l e c t e d - i o n monitoring mode a t 70 e V ,
and t h e i n t e n s i t i e s o f t h e i o n s a t m/e
303 and 306 and m / e 263 and 266, were used
t o measure c o c a i n e and n o r c o c a i n e ,
respectively.

J i n d a l e t a1 (154) have a l s o p u b l i s h e d a
g a s - l i q u i d chromatographic - mass s p e c t r o -
m e t r i c d e t e r m i n a t i o n o f Lidococaine
( L i g n o c a i n e ) i n an i l l i c i t sample o f
c o c a i n e . The sample, i n chloroform, w a s
i n j e c t e d on t o a g l a s s column ( 1 . 8 m X
2 mm) s i l a n i s e d w i t h 5% of d i c h l o r o d i -
m e t h y l s i l a n e i n t o l u e n e , packed w i t h 3%
o f OV-17 on Gas Chrom Q (100 t o 200 mesh)
and o p e r a t e d a t 200°, w i t h flame i o n i s a -
t i o n d e t e c t i o n . Two peaks were r e s o l v e d ,
one w a s due t o c o c a i n e and t h e o t h e r t o
l i g n o c a i n e a s w a s shown by combined g . c . -
70 e V m . s . ( t h e molecular i n n w a s a t m / e
234 and t h e b a s e peak w a s a t m/e 8 6 ) .

Clark ( 1 5 5 ) has d e s c r i b e d a mass-

s p e c t r a l q u a n t i t a t i o n method for t h e
a n a l y s i s of cocaine hydrochloride i n
powders. A s o l u t i o n c o n t a i n i n g 4 mg each
o f cocaine h y d r o c h l o r i d e and [ 2Hs]-cocaine
216 FARID J. MUHTADI A N D ABDULLAH A. AL-BADR

i n 25 m l of methanol i s a n a l y s e d by
g.1.c.-m.s. on Finnigan model 9500 and
model 3300 i n s t r u m e n t s l i n k e d by a j e t
s e p a r a t o r . A 0.2 1-11p o r t i o n o f s o l u t i o n
i s i n j e c t e d ( v i a a p o r t a t 240') i n t o
a g l a s s column (120 cm X 2 mm) packed w i t h
3% of OV-1 on Gas Chrom Q (100 t o 120
mesh) and o p e r a t e d a t 190°, w i t h helium
as c a r r i e r g a s ( 4 0 ml m i n - l ) . The spec-
trometer i s operated i n r e p e t i t i o n scan
mode ( e v e r y 2s) from m / e 75 t o 310.

Lewin e t a1 (156) r e p o r t e d a combined

g.c.-m.s. o f c o c a i n e and i t s t h r e e i s o -
mers ( f o u r i s o m e r s ) and of t h e correspond-
i n g ecgonine methyl e s t e r s , w i t h u s e o f a
column (1.8 m X 2 mm) o f 2% of OV-17
and chemical i o n i s a t i o n , w i t h i s o b u t a n e
o r NH3 as r e a g e n t as, i s s t u d i e d ; f r a g -
mentation p a t t e r s are d i s c u s s e d i n d e t a i l .

Other g.c.-m.s. methods have a l s o been

p u b l i s h e d (62, 81, 157-159).

9.6.4 High-pressure Liquid Chromatography (HPLC)

S e v e r a l HPLC systems for t h e i d e n t i f i c a t i o n

and a n a l y s i s of c o c a i n e have been r e p o r t e d
in the literature:-

Olieman e t a1 ( 1 6 0 ) r e p o r t e d a method f o r
a n a l y s i s o f c o c a i n e , pseudococaine,
a l l o c o c a i n e and allopseudococaine by ion-
p a i r reverse-phase high-performance
l i q u i d chromatography. The compounds can
be s e p a r a t e d by l i q u i d chromatography on a
column o f o c t a d e c y l s i l y l - s i l i c a w i t h t h e
a d d i t i o n t o t h e e l u e n t of n-heptanesulfo-
n a t e and i d e n t i f i e d by peak area measure-
ments at d i f f e r e n t u l t r a v i o l e t wavelength.

- Masoud and Krupski (161) have d e v i s e d an

HPLC method for t h e a n a l y s i s of c o c a i n e i n
human plasma a f t e r being i s used as a n
a n a e s t h e t i c for n a s a l s u r g e r y . Blood i s
c o l l e c t e d i n a t u b e c o n t a i n i n g sodium
f l u o r i d e and D-glucose c i t r a t e phosphate
COCAINE HYDROCHLORIDE 217

s o l u t i o n (pH 5 . 7 ) , and t h e plasma ( w i t h

amethocaine H C 1 added as i n t e r n a l
s t a n d a r d i f d e s i r e d ) i s made a l k a l i n e w i t h
sodium c a r b o n a t e s o l u t i o n and e x t r a c t e d
w i t h e t h e r . The drugs are back-extracted
i n t o acetic acid, then t h e acid layer i s
made a l k a l i n e and t h e drugs are e x t r a c t e d
i n t o hexane. T h i s e x t r a c t i s evaporated
under n i t r o g e n a t 40' and t h e r e s i d u e i s
d i s s o l v e d i n t h e mobile phase [methanol :
0.05 phosphate b u f f e r (pH 6 . 6 ) (3:1)] f o r
h . p . 1 . c . a t 40' on a column (25 cm X
2.6 mm) o f Perkin-Elmer ODs-HC SIL-X-1
f i t t e d w i t h a brownless RP-18MPLC guard
column's e l u t i o n i s a t 0.8 m l min-l and
d e t e c t i o n i s at 232 nm. R e t e n t i o n times
are 5.2 min f o r c o c a i n e and about 8 min.
f o r amethocaine.

Poochikian and Cradock ( 1 6 2 ) have d e t e r -

mined c o c a i n e i n t h e p r e s e n c e o f i t s
h y d r o l y s i s p r o d u c t . The drugs were
s e p a r a t e d from each o t h e r and from t h e
i n t e r n a l s t a n d a r d ( 4 - c h l o r o p y r i d i n e ) by
h . p . 1 . c . on a column (30 cm X 4.6 mm i . d . )
of p Bondapak C 1 8 ( 1 0 pm) o p e r a t e d a t
ambient t e m p e r a t u r e w i t h 1 5 mm phosphate
b u f f e r (pH ? , ) - a c e t o n i t r i l e ( 3 : l ) as t h e
mobile phase (0.8 ml min-1) and d e t e c t i o n
a t 235 nm. The d e t e c t i o n l i m i t s were 3 ng
f o r cocaine.

Lewin - e t_a 1 (156) have i d e n t i f i e d and

q u a n t i t a t e d isomeric c o c a i n e s by HPLC.
Cocaine i s s a t i s f a c t o r i l y s e p a r a t e d from
i t s t h r e e isomer by h . p . 1 . c . on a column
o f p a r t i s i l - 1 0 PXS o p e r a t e d w i t h i s o -
propyl alcohol-hept ane-diethylamine
(25:75:0.1) as mobile phase at a flow
r a t e i n c r e a s i n g ( d u r i n g 1 2 m i n u t e s ) from
0.48 t o 4 m l min-1; t h e e l u t e i s monitored
a t 230 nm. C a l i b r a t i o n graphs are based
on peak a r e a s r e l a t i v e t o t h o s e of N N-
dibenzylbenzamide ( t h e i n t e r n a l s t a n d a r d ) .

Evans and Morarity ( 1 6 3 ) have r e p o r t e d t h e

a n a l y s i s of cocaine and i t s m e t a b o l i t e s
218 FARID J. MUHTADI AND ABDULLAH A. AL-BADR

HPLC. The plasma or t i s s u e homogenate

i s mixed w i t h aqueous i n t e r n a l s t a n d a r d
( L i g n o c a i n e ) and s o l i d sodium f l u o r i d e
( t o i n h i b i t enzymic h y d r o l y s i s o f c o c a i n e
and n o r c o c a i n e ) , t h e pH i s a d j u s t e d t o 9
( c a r b o n a t e b u f f e r s o l u t i o n ) and t h e mix-
t u r e i s e x t r a c t e d w i t h chloroform-iso-
p r o p y l a l c o h o l (3:2). The e x t r a c t i s
evaporated under n i t r o g e n a t 40' and a
s o l u t i o n o f t h e r e s i d u e i n Ij20 i s sub-
m i t t e d t o h.p.1.c. on a column ( 3 0 cm X
4 mm) o f p Bondapak C 1 8 , w i t h water -
a c e t o n i t r i l e - methanol ( 8 : l : l ) c o n t a i n i n g
1% o f a c e t i c a c i d and 0.3M i n EDTA as
mobile phase ( 2 ml min-1) and d e t e c t i o n
at 235 nm. R e t e n t i o n t i m e f o r cocaine
9.7 , benzoylecgonine 2 . 9 , Lignocaine 4 . 2
and norcocaine 11.1 min.

F l e t c h e r and Hancock (164) have r e p o r t e d

p o t e n t i a l e r r o r s i n benzoylecgonine and
c o c a i n e a n a l y s i s . Cocaine H C 1 s o l u t i o n
( 5 0 pg m l - l ) were a d j u s t e d t o pH v a l u e s
between 2 and 9.4 and were analysed
immediately, or a f t e r being set a s i d e f o r
u p t o 6.75 hours by h.p.1.c. on a column
( 1 0 c m X 4.6 mm) of H y p e r s i l 5-ODs
( 5 pm) w i t h aqueous 55% methanol a d j u s t e d
t o pH 3.8 w i t h H3PO4 as mobile phase
( 2 m l min-l) . Benzoylecgonine ( r e t e n t i o n
t i m e 1 . 4 m i n u t e s ) and c o c a i n e ( r e t e n t i o n
t i m e 3.4 m i n u t e s ) were d e t e c t e d a t 232 nm.

Noggle e t a1 (165) have p u b l i s h e d a

l i q u i d chromatographic procedure f o r
i d e n t i f i c a t i o n o f cis and t r a n s - cinna-
moylcocaine i n i l l i c i t cocaine. A metha-
n o l i c s o l u t i o n o f t h e sample w a s a n a l y s e d
by h.p.1.c. w i t h u s e o f a s t a i n l e s s s t e e l
column ( 3 0 cm X 4 mm) o f 1.1 Bandapak-C18,
w i t h phosphate b u f f e r (pH 3)-methanol
(2:l) as mobile phase ( 2 ml min-1) and
w i t h two u l t r a v i o l e t d e t e c t o r s , a t 254 and
280 nm, r e s p e c t i v e l y , i n series. The
r e l e v a n t f r a c t i o n s were c o l l e c t e d , made
a l k a l i n e w i t h aqueous ammonia and
e x t r a c t e d w i t h dichloromethane. Residues
o b t a i n e d on e v a p o r a t i o n were d i s s o l v e d
COCAINE HYDROCHLORIDE 219

separatdyin methanol for the analysis by

ultraviolet spectrophotometry and by mass
spectrometry.

Development of a standardized analysis

strategy for basic drugs using ion-pair
extraction and high-performance liquid
chromatography, philosophy and selection
of extraction technique have recently been
reported (166).
Others HPLC methods have also been
reported (167,168).

9.7 Radio-immunoassay
Mule' --
et a1 (169) reported the evaluation of the
radio-immunoassay for benzoylecgonine (a cocaine
metabolite) in human urine. The 251-radio-immuno-
assay (RIA) for benzoylecgonine in urine was
evaluated by comparison with gas liquid chromato-
graph and thin-layer chromatography and the enzyme-
multiplied immunoassay technique. By radio-immuno-
assay, a statistically significant concentration,
2 pgllitre, was observed for urinary benzoylecgonine.
The coefficient of variation for the radio-immuno-
*
assay was 2.58 0.38% interassay and 2.20 f. 0.14%
interassay. There was cross-reactivity with cocaine
(more reactive than benzoylecgonine and other
members of the tropane family of alkaloids. There
was agreement between results by radio-immunoassay
and gas liquid chromatography in 95.5% of the
samples, between radio-immunoassay and thin-layer
chromatography in 87.0% and between radio-immuno-
assay and enzyme-multiplied immunoassay technique
in 84.5%. The percentage of true false-positive
was 3.5% for the radio-immunoassay in comparison
to gas-liquid chromatography, 8.8% in comparison to
thin-layer chromatography and 9.1% in comparison to
enzyme-multiplied immunoassay technique. True
false-negative were insignificant (0. to 1 . 0 % ) .
Gas liquid chromatography and radio-immunoassay
results correlated highly ( 4 = 0.908). Gas-liquid
chromatography, therefore, was the best comparison
method for the evaluation study. Radio-immunoassay
for benzoylecgonine is sensitive, reproducible and
reliable for the detection of cocaine in urine.
220 FARID J. MUHTADI A N D ABDULLAH A. AL-BADR

Budd (170) r e p o r t e d a c o c a i n e radio-immunoassay-

s t r u c t u r e v e r s u s r e a c t i v i t y . He t e s t e d s e v e r a l
a l k a l o i d s f o r benzoylecgonine antibody-binding
a c t i v i t y i n t h e Roche r . i . a k i t s . Benzoylecgonine
has t h e optimum antibody-binding a c t i v i t y ; change
of any o f t h e s u b s t i t u e n t s ( e x c e p t e s t e r i f i c a t i o n
of t h e carboxy-group) reduced t h e b i n d i n g , coca-
e t h y l e n e w a s t h e only drug t h a t i n t e r f e r r e d w i t h
t h e Roche a s s a y a t t h e r a p e u t i c o r overdose l e v e l s ,
but it was seldom encountered under t h e s e conditions.
Thus t h e k i t was c o n s i d e r e d t o be s u i t a b l e f o r
a s s a y i n g cocaine and i t s m e t a b o l i t e s .

Baum-gartner - et - a1 (171) p u b l i s h e d a method f o r

r a d i o - i m u n o a s s a y of c o c a i n e i n h a i r . The drug was
detected i n h a i r o f 13 p a t i e n t s from a drug-abuse
c l i n i c who acknowledged having used c o c a i n e i n
v a r y i n g amounts d u r i n g t h e l a s t s i x months. A
c o r r e l a t i o n was observed between t h e amount of
c o c a i n e used and t h e q u a n t i t y t r a p p e d i n t h e
i n t e r i o r o f h a i r grown d u r i n g t h e s i x month p e r i o d .
I n c o n t r a s t t o h a i r a n a l y s i s , u r i n a l y s i s by t h i n -
l a y e r chromatography w a s n e g a t i v e i n a l l c a s e s .
I n d i c a t i n g t h a t , c o c a i n e h a s not been used by t h e
p a t i e n t s w i t h i n 48-72 hours b e f o r e t h e u r i n e
c o l l e c t i o n . Hair a n a l y s i s t h u s appears t o be f a r
superior t o urinalysis for establishing h i s t o r i e s
of drug use.

Other immunoassay methods have a l s o been r e p o r t e d

(110, 172-174).

ACKNOWLEDGEMENT

The a u t h o r s would l i k e t o thank Mr. Uday C . S h a n a and

Tanvir A . B u t t , b o t h o f College of Pharmacy, King Saud h i -
v e r s i t y €or t h e i r v a l u a b l e s e c r e t a r i a l a s s i s t a n c e i n t y p i n g
o f t h i s manuscript,
COCAINE HYDROCHLORIDE 22 1

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COCAINE HYDROCHLORIDE 227

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COCAINE HYDROCHLORIDE 229

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230 FARID J. MUHTADI A N D ABDULLAH A. AL-BADR

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COCAINE HYDROCHLORIDE 23 1

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This Page Intentionally Left Blank
 EPHEDRINE HYDROCHLORIDE

SYED LAIK ALI

1. History
2. Nomenclature
3. Description
3.1. Name, Formula, Molecular Weight
3.2. Appearance, Colour, Odour
4. S ynthesis
5. Physical Properties
5.1. Solubility
5.2. Loss on Drying
5.3. Melting Point
5.4. Specific Optical Rotation
5.5. Sulphates
5.6. Sulphated Ash
5.7. pH Value
5.8. Dissociation Constant
5.9. Ultraviolet Spectrum
5.10. Infrared Spectrum
5.11. Nuclear Magnetic Resonance Spectrum
5.12. Mass Spectrum
6. Stereochemistry
7. Colour and Identification Reactions
8. Stability and Degradation
9. Methods of Analysis
9.1. Ti trimetry
9.2. Visible and UV Spectrophotometry
9.3. Fluorimetry
9.4. Chromatographic Methods
Thin Layer Chromatography , Paper Chroma-
tography, Gas-Liquid Chromatography, High
Performance Liquid Chromatography
9.5. NMR Assay
9.6. Radioimmunoassay
9.7. Isotachophoresis
10. Drug Metabolism and Pharmacokinetics
11. Acknowledqement
References
ANALYTICAL PROFILES O F DRUG SUBSTANCES Copyright 0 1986
VOLUME 15 by the American Pharmaceutical Association
233 All rights of reproduction in any form reserved.
234 SYED LAIK ALI

Ephedrine Hydrochloride

Ephedrine and ephedrine hydrochloride have been

mostly used synonymously in this monography. Ephe-
drine is quite often cited in literature but it is
presumed to be valid also for ephedrine hydrochlo-
ride. Aqueous or acidic solutions of ephedrine hy-
drochloride are often converted into ephedrine
before other chemical or chromatographic opera-
tions and measurements. Casually ephedrine sulfate
has been also included in this monograph. Physical
properties, spectra etc. are given for ephedrine
hydrochloride.

1. History
Ephedra is a Phanerogame-Gymnosperme from the
family of Gnetaceen. There are 30 different
types of this plant-species known which grow
in Asia, mediteranian countries and America.
Specially ephedra vulgaris, ephedra equise-
tina and ephedra sinica contain ephedrin with
its other isomers. A certain ephedra species
has been used in ancient Chinese medicine
since ages. Already in 5 0 0 0 B.C. an ephedra-
plant was widely used in China under the name
Ma-Huang. Ma-Huang has been mentioned as me-
dicine of moderate therapeutic range in the
first Chinese pharmacopoe, published under
the government of Shen Lung in 1 7 6 0 B.C. A
detailed description of the plant and its
pharmacological action is given in the
Chinese pharmacopoe of modern times in 1 5 9 6 .
According to this pharmacopoe Ma-Huang was
applied as Diaphoreticum, Antipyreticum and
Sedativum for the treatment of coughs and
colds as well as a stimulant for blood-cir-
culation ( 1 ) . These therapeutic effects were
mostly confirmed later for ephedrin which was
discovered and isolated in pure form by Nagai
(2) in 1 8 8 7 as a main alkaloid of ephedra
from ephedra sinica.
EPHEDRINE HYDROCHLORIDE 235

2. Nomenclature
Ephedrine hydrochloride is ( I R , 2 . S ) - ( - ) - / 3 -
hydroxy-N d-dimethyl-phenethylammonium chlo-
ride. The formula is illustrated at the next
Page

3. Description
3.1. Name, Formula, Molecular weight
Ephedrine Hydrochloride
CloH15NO HC1 201.70

3.2. Appearance, Colour, Odour

Colourless crystals or white, crystalline,
orthorohombic needless or powder, odourless,
with a bitter taste.

4. Synthesis
Ephedrin was synthesised for the first time
by Spzth ( 3 ) as illustrated in Fig. 1. Ra-
cemic ephedrin was then further split up into
its components. Addition of methylamin to
I-phenylpropylenoxyd leads also to the syn-
thesis of ephedrin (4). Ephedrine could fur-
ther be synthesised through amination of
A-Brompropiophenon with methylamine or ben-
zylmethylamine. A-Brompropiophenon was syn-
thesised through bromination of propiophenon
and propiophenon itself was prepared with
Friedel-Crafts reaction using benzene and
propoyl chloride (5). The resulting product
methylaminoketon or benzylmethylaminoketone
was then hydrogenated in presence of palla-
dium as catalyst and a racemic mixture of
ephedrin was obtained (Fig. 2). This was
further reacted with sodium dibenzoyl tar-
236 SYED LAIK ALI

Ephedrine Hydrochloride

0 CHOH-
I
CH - C H 3 CI -

STRUCTURAL FORMULA

FIG, 1

CH3 y 3
I

FIG, 2
EPHEDRINE HYDROCHLORIDE 237

trate to convert ephedrine into its optical

isomers. Dibenzoyl tartrate of (-)ephedrine
was then treated with hydrochloric acid to
give (-)ephedrine hydrochloride in 60 %
yield. This process was used on an industrial
scale by C-H-Boehringer Company in Germany
during second world war (5). Later on this
method was further modified by using di-
benzoyl(+)-tartaric acid or mandelic acid for
the resolution of racemic o( -methylamino-
propiophenone into its optical isomers in
90 % yield. The resulting product was then
catalytically hydrogenated to (-)ephedrine
(6). A biochemical process for the production
of optical active (-)ephedrine-isomer is
applied by Knoll Company in Germany. L-phenyl
acetyl carbinol is obtained through an acyl
condensations with benzaldehyde during the
fermentation process of a Melasse-solution.
This compound is then reacted with methyl-
amine and simultaneously reduced to (-)ephe-
drine in presence of active aluminium or a
platinum catalyst (Fig. 3 ) . Ephedrine is then
converted into ephedrine hydrochloride which
is recrystallised from water into optically
pure isomer ( 7 , 8 ) . Upon heating ephedrine
hydrochloride is decomposed into phenyl ethyl
ketone and methyl amine.

5. Physical properties

5.1. Solubility
It is freely soluble in 4 parts of water, and
in 17 parts of alcohol ( 9 6 % ) , very slightly
soluble in chloroform and practically inso-
luble in ether (9).

5.2. Loss on Drying

Not more than 0.5 %, determined with 1.00 g

by drying to constant weight in an oven at
100" - 105°C (9).
 1. H2N -CH3(Amination)
2. HZ (Reduction)
3 Q
HO - C - H HO - C- H
I H ~ C - H N - C I- H
c =o
I I
CH3 H3

FIG, 3
EPHEDRINE HYDROCHLORIDE 239

5.3. Melting point

217O - 22OOC (9)

5.4. Specific optical Rotation

-33.5O to -35.5O, determined by dissolving
5.00 g in sufficient water to produce 50.0
ml, diluting 1 0 ml to 20 ml with water and
calculated with reference to the dried sub-
stance (9).

5.5. Sulphates
1 0 0 ppm with limit test for sulphates (9).

5.6. Sulphated Ash

Not more than 0.1 %, determined with 1 .O g
(9).

5.7. pH-Value
The pH of a 0.5 % aqueous solution is 5.9.

5.8. Dissociation constant

The Pk, of ephedrine hydrochloride in water
at 2 O o C is reported to be 9.68 ( 1 1 ) . This
value is attributed to the ammonium cation of
the molecule.

5.9. Ultraviolet Spectrum

The ultraviolet spectra of ephedrine hydro-
chloride were taken with a Perkin-Elmer UV
spetrophotometer 5 7 1 at a concentration of

238
240 SYED LAIK ALI

0 . 5 mg/ml in methanol, 0.1N HC1 and 0.1N

NaOH. In all three solvents the spectra show
absorption bands at almost identical wave
lenghts of 2 5 0 , 2 5 6 and 2 6 2 nm.
The molecular extinction coefficients and
A 1 % of ephedrine hydrochloride are reported
Icm to be ( 1 2 )
Methano 1 0.1N HC1 0.1N N a O H
Absorption 2 5 0 nm 2 5 0 nm 2 5 1 nm
Maximum 2 5 6 nm 2 5 6 nm 2 5 7 nm
2 6 2 nm 2 6 2 nm 2 6 3 nm
A1 %' 8.4 7.4 7.8
1 cm 10.8 9.4 9.7
8.2 7.2 7.4
170 150 160
€ 220 190 195
165 145 150

The UV spectra are shown in Fig. 4.

5.10. Infrared Spectrum

The infrared spectrum of ephedrine hydro-
chloride is given in Fig. 5 . The spectrum
was obtained with a Perkin-Elmer 1 4 2 0 Ratio
Recording Infrared Spectrophotometer from a
KBr pellet. The structural assignments may
be correlated with the following band fre-
quencies:

Frequency ( cm-11 Assignments

3330 Stretching vibra-
tions of OH
2700-2840 Amine halide salt
stretching bands
2480 NH2+ stretching
1 4 5 0 and 1 4 9 0 Characteristic
vibrations of the
aromatic ring
7 5 0 and 6 9 8 C-H out of plane
deformation, mono-
substitued benzene
EPHEDRINE HYDROCHLORIDE 24 1

- Methanol
- - 0,lN - HCI
---- 0,l N - Na OH

FIG, 4
'UV SPECTRUM OF EPHEDRINE HYDROCHLORIDE
I N DIFFERENT SOLVENTS
 MlKR ONS
3 4 5 6 7 8 91012U1

I
coo0 3000
. I .
2OOO 1800 1600 UOO 1200 loo0 800 625 1

WAVE NUMBER CM O1

FIG, 5
IR SPECTRUM OF EPHEDRINE HYDROCHLORIDE, O R PELLET, PERKIN-ELMER
1420 S PECTROPHOTOMETER
EPHEDRINE HYDROCHLORIDE 243

5.11. Nuclear Magnetic Resonance Spectrum

The nuclear magnetic resonance spectrum of
ephedrine hydrochloride was obtained with a
Varian T-60 NMR spectrometer in D20. The
following spectral assignments are made for
the spectrum reproduced in Fig. 6.
Chemical shift Assiqnment
1.20 doublet CH3 at CH
2.90 singlet CHS at NH2
3.60 multiplet CH at NH2
5.28 doublet CH at OH
7.53 singlet aromatic protons
OH and NH2 deuterated.

5.1 2. Mass spectrum

In the highest mass regions molecular ion
peak ist not observed. Prominent ion-peaks
observed are m/e, 31, 59, 78 and 108. The
mass spectrum of ephedrine hydrochloride is
given in Fig. 7.
Instrument: Varian Mat 44
Sample temperature: (direct inlet)
5OoC - 25OOC in 5 min
Source temperature: 25OOC
Electron energy: 80eV
Some ions of this spectrum can be correlated
to the structure as following:
NH CH3
M-C6H5CHOHHCl = 59; M-CHOHCHCH3HC1 = 78
M-CHCH3HC1 = 108
NHCH3
 a:
W
I-
W
r
0
a:
I-
u
W
a
cn
0
Lo
I-
z
a
c(
cc
a
>
1 W
n
%
U
a:
0
-1
I
u
0
a:
n
>
I
W
z
U
a:
n
W
I
II
W
LL
0
r
3
a:
I-
u
W
a
v)
cc
z
I
EPHEDRINE HYDROCHLORIDE 245

FIG, 7
MASS SPECTRUM OF EPHEDRINE HYDROCHLORIDE
246 SYED LAIK ALI

6. Stereochemistry
Ephedrine has two asymmetric centres; there
exist four steroisomers and two inactive
forms 5 ephedrin and 2 pseudoephedrin. The
erythro-configuration of asymmetric centres
in (-)ephedrine was confirmed by Freudenberg
(13) through the synthesis from D(-Imandelic
acid and L(+)-alanin. According to the Cahn-
Ingold-Prelog nomenclature the absolute con-
figuration of asymmetric carbon atoms in
(-)ephedrine could be defined as 1R,2S. Hyne
has found with nmr measurements the dieder
angle of 90' between OH and NHCH3 in ephe-
drine base (14). Gauche-Confirmation A and B
are strongly favoured in free ephedrine base
and its salts (15). Ephedrine hydrochloride
dissolved in D20 lies 90% in A and B forms
and 10% in trans-form C (Fig. 8). Testa (16)
found good agreement of CD and ORD results
with nmr values.

7. Colour and Identification Reactions

To 10 mg ephedrine hydrochloride in 0.1 ml
water when 0.2 ml of copper sulphate solution
(12.5%) and 1 ml of strong sodium hydroxide
solution (10 N) are added, a violet colour is
produced. When this solution is shaken with
2 ml ether, the ether layer turns purple and
the aqueous layer remains blue (17). 50 mg of
the substance dissolved in 1 ml water is mi-
xed with 4 ml 0.1N NaOH, shaken with 3 ml
CCl4 for ten seconds and then allowed to
stand for 2 minutes. The organic layer is
separated and treated with few copper tur-
nings. A turbidity appears rapidly which
turns in few minutes into a copious pre-
cipiate (18, 19). L- and D,L-ephedrines could
be distinguished through their crystalline
modification. An aqueous ephedrine salt
solution is acidified with 3 drops of 15%
 I
u" Ln
I I,
0
w
n
CI
CK
0
0 0 - I
I
- 0
w o
- C K
L L ~
>-
I
w
m
I
i
i
I 0
Xi 0
I I
x
248 SYED LAIK ALI

H2SO4, 2 ml potassium iodobismutate solution

(prepared by mixing 2 parts of 7 % basic bis-
muth nitrate in nitric acid, 1 part potassium
iodide and making upto volume of 1 0 ml with
water) are added and left to stand overnight.
L-ephedrine is identified under microscope
with needle-like crystals, whereas D,L-ephe-
drine is visible as dark-red prisims (20).
Ephedrine hydrochloride gives in weak alka-
line solutions with ninhydrine a violett co-
lour (21 1 .

8. Stability and Degradation

Decomposition of (-)-ephedrine was less than
1 % after the prolonged passage of air through
cold (2OOC) or refluxing neutral or basic
aqueous solutions (0.2% W / V ephedrine hydro-
chloride in phosphate buffer pH 7 . 4 or in 1%
sodium hydroxide). On exposure to heat ephe-
drine hydrochloride is decomposed in phenyl
ethyl ketone and methyl amine. GLC-MS pro-
vided almost the sole means of identifying
some of the breakdown products of ephedrine
due to their similar GLC properties and la-
bility on tlc. Significant losses occurred
during the extraction of small quantities of
ephedrine from aqueous media using either
regular or analytical grades of diethyl
ether. The losses were, at least in part,
caused by reaction of the ehedrine with
aldehydic impurities in the ether. The
addition of n-butanol to the ethereal extract
before evaporation reduced the "breakdown"
that occurred if the extracts were allowed to
boil dry in the water bath. For this reason
the routine use of aldehyde-free n-butanol is
of value.
Different substituted oxazolidines were iden-
tified through GLC-MS as break-down products.
Consistent low levels of ephedrine "break-
down" were achieved by prior washing of the
EPHEDRINE HYDROCHLORIDE 249

ether with 1 0 % sodium metabisulphate solution

followed by IN hydrochloric acid and finally
with sodium hydroxide,5N. Negligible decom-
position was observed on refluxing ephedrine
( 0 . 5 % ) in ether sturated with aqueous 20%
NaOH for 8h or ephedrine (8%) in ethanolic
sodium hydroxide for 3h. Ephedrine base sto-
red in ether ( 1 0 0 mg/ml) at room temperature
in light for several weeks decomposed to give
oxazolidines. This is in contrast to the
small amount of decomposition that occurred
upon ultraviolet irradiation of aqueous solu-
tion of ephedrine. Solutions of ephedrine
base (3% W / V ) in ether or benzene were exten-
sively degraded by ultraviolet light over 18h.
The major decomposition products yielding
peaks upon glc examination after extraction
of ephedrine solutions with ether and concen-
tration of these extracts arise from addition
and condensation of ephedrine with acetalde-
hyde, propionaldehyde and formaldehyde impu-
rities in the solvent. Oxidation of (-)-ephe-
drine base with nickel peroxide, active sil-
ver carbonate and with active manganese dio-
xide gave benzaldehyde, a mixture of oxazo-
lidines and 2-methyl-amino-I-phenyl-I-propa-
none as oxidation products. The degradation
products were identified through glc and
glc-ms analysis ( 2 2 ) .

9. Methods of Analysis
9.1. Titrimetry
The assays of halogen salts of organic bases
can be carried out either by a two-phase-ti-
tration or through non-aqueous titration of
the substance. The two-phase titration app-
lies ethanol 9 6 % and chloroform as solvent,
phenolphthalein as an indicator and 0.1N
sodium hydroxide as the titrant. The deter-
250 SYED LAIK ALI

mination of ephedrine hydrochloride in this

medium is rather uncertain and gives about
3 % lower results ( 2 3 ) . Ephedrine hydrochlo-.
ride is determined in german pharmacopoeia
(DAB 7 ) through dissolving it in water, aci-
difying with 3 N nitric acid, additon of ex-
cess of O . l N silver nitrate solution and
back-titration of silver nitrate with 0 . 1 N
ammonium thiocyanate using ferric ammonium
sulphate as an indicator ( 2 4 ) . In non-aqueous
medium either ephedrine hydrochloride is
dissolved in glacial acetic acid, mercuric
acetate and then few drops of crystal violet
indicator solution are added. The solution is
then subsequently titrated with 0 . 1 N perchlo-
ric acid to an emerald-green end-point (USP
XXI). Another method of non-aqueous titration
is to dissolve the substance in warm mercuric
acetate solution, to add acetone and then to
titrate it against 0 . 1 N perchloric acid using
saturated solution of methyl orange in ace-
tone as an indicator until a red colour is
obtained (26, 27). An indirect non-aqueous
titrimetric method was devised for the deter-
mination of hydrochlorides of nitrogen-bases.
The chloride-ion interference was prevented
without the use of mercuric acetate reagent.
The method depends on the treatment of a so-
lution of the hydrochloride of the organic
base with an excess of standard perchloric
acid solution in acetic acid and the hydrogen
chloride displaced is removed by boiling. The
excess of perchloric acid is determined by
titration against the basic titrant sodium
acetate in g.lacia1 acetic acid using either
potentiometric or visual end-point detection
with a crystal violet indicator. Potentio-
metric titrations showed that the point of
the maximum inflection in the titration curve
coincided with the appearance of the violet
colour of the indicator. The mean percent
recovery of 99.66 % obtained indicates that
the proposed method is equivalent in accuracy
and precision to the non-aqueous titrimetric
EPHEDRINE HYDROCHLORIDE 25 1

method most commonly used by official compen-

dia (28). Sanchez determined ephedrine by its
reaction with alkaline iodine solution at
5OoC to form iodoform and titration of the
excess of iodine in acid solution. Appli-
cation of this method to standard solutions
of ephedrine hydrochloride has given reco-
veries within 2 3% of the theoretical values.
However, this method could not be applied to
the determination of ephedrine HC1 in cough
mixtures due to interferences from other con-
stituents (29). Horak and Gasperik used a
method for the determination of ephedrine
based on the liberation of methylamine by
alkaline hydrolysis. Good results were ob-
tained for the determination of ephedrine HC1
in pharmaceutical injections and tablets (30).
9.2. Visible and UV-Spectrophotometry
UV spectrophotometric determination of ben-
zaldehyde or substituted benzaldehydes formed
by periodate oxidation of ephedrine and other
drugs with vicinal hydroxyl and amine func-
tions has provided simple and sensitive assay
methods for them. Ephedrine hydrochloride in
dilute HC1 solution exhibits a molar absopti-
vity of 190 at its about 258 nm maximum. Oxi-
dation of it to benzaldehyde affords an €-va-
lue of about 14400 at its maximum at about
241 nm in hydrocarbon solvents, about a
75-fold again in sensitivity (31). Periodate
oxidations are among the most elegant reac-
tions used in organic chemistry, because they
are often quantitative within minutes at room
temperature in aqueous media. Malaprade in-
troduced periodic acid as a reagent for the
oxidation of 1,2-glycols in 1928 (32). Ni-
colet and Shinn first reported the use of
periodate for oxidation of ethanolamine deri-
vatives. They found that ethanolamines with
primary or secondary amine functions were
rapidly and quantitatively cleaved to al-
252 SYED LAIK ALI

dehydes and ammonia or a primary amine (33).

Wickstrom studied the rate of periodate oxi-
dation of ephedrine as a function of pH, ti-
trating excess of oxidant iodometrically. He
found that periodate consumption was too slow
to measure at pH 3.0, very slow at pH 6.0 and
stoichiometric within 10 min at pH 7.5 or hi-
gher ( 3 4 ) . Oxidation potential of periodate-
iodate couple is -1.6V in acid solution and
about 0.7 V in alkaline solution (35). Wick-
strom found that the reaction products of
periodate oxidation of ephedrine are benzal-
dehyde, acetaldehyde and methylamine. The
determination of ephedrine via colorimetry of
acetaldehyde distilled from the reaction mix-
ture was suggested ( 3 4 ) . Chafetz studied
among others the periodate oxidation of ephe-
drine (36). It was quantitatively oxidised to
benzaldehyde in 1 0 min in a bicarbonate me-
dium. Extraction of benzaldehyde in n-hexane
and its spectrophotometric determination was
recommended. Spectrophotometry of the benzal-
dehyde produced by periodate oxidation will
not distinguish between compounds such as
ephedrine and phenylpropanolamine. It will
further not discriminate steroisomers such as
ephedrine (erythro-configuration) and pseudo-
ephedrine (threo-configuration) ( 3 1 ) .
Wallace conducted a reaction for the deter-
mination of ephedrine in biological samples
at the temperature of boiling hexane, about
69OC, with a reaction time of 3 0 min. The
hexane layer was separated, washed with di-
lute acid and benzaldehyde determined spec-
trophotometrically either as such or after
conversion to its semicarbazone (37, 38).
Chafetz ( 3 1 ) further showed that the carbonyl
compounds analogs of ephedrine are oxidised
to benzoic acid by periodate and do not
interfere in the assay of ephedrine. It is
further demonstrated that N-acetylephedrine
does not react with periodate under the assay
conditions.
EPHEDRINE HYDROCHLORIDE 253

An application of orthogonal functions to the

UV spectrophotometric determination of ephe-
drine hydrochloride in tablets is reported
(39). The method is applicable for the deter-
mination of a single substance in the pre-
sence of irrelevant absorption from exci-
pients such as lactose, starch, sucrose, ge-
latin, talc, stearic acid and magnesium
stearate. The choice of polynomial, number of
points, wavelength range and intervals are
illustrated. Glenn's method of orthogonal
functions proved to be powerful in discoun-
ting irrelevant absorption contribution ( 4 0 ) .
A colorimetric method for the quantitative
determination of ephedrine hydrochloride in
presence of chlorpheniramine maleate and
guaiacolsulfonate potassium in a cough syrup
containing colouring agents is described
( 4 1 ) . Ephedrine hydrochloride is assayed
using bromothymol blue as a dye in which
interference from chlorpheniramine maleate is
taken into consideration. The extinction of
the chloroform extract is measured at 4 2 0 nm
against the reagent blank. A collaborative
study for the on-column periodate reaction
method for analysis of ephedrine in solid
dosage forms is reported. Ephedrine is se-
parated from water-soluble impurities and
strong acids by elution from a weakly basic
celite column, and further cleaned up by re-
tention on a weakly acidic column while the
weak acids, weak bases and organic-soluble
neutrals are eluted. Ephedrine is eluted from
the column after neutralisation with N H 3 and
is converted to benzaldehyde via on-column
periodate reaction and determined spectro-
photometrically ( 4 2 , 43). An UV spectropho-
tometric determination of ephedrine hydro-
chloride in an antiasthma capsule preparation
containing aminophylline and amobarbital is
reported ( 4 4 ) . On a prepared column contai-
ning alginic acid ephedrine HC1 is retained
from an ethanolic solution; aminophylline and
amobarbital pass through the column. Subse-
254 SYED LAIK ALI

quently ephedrine HC1 is eluted with 0.1N HC1

and determined spectrophotometrically at 2 5 7
nm. A rapid second and fourth derivative UV
spectrophotometric assay procedure is des-
cribed for the determination of ephedrine or
pseudoephedrine in pharmaceutical formula-
tions. The method has been applied success-
fully to Ephedrine Elixir BP, Ephedrine HC1
tablets BP, Ephedrine Nasal Drops, Paedria-
tric Belladonna and Ephedrine mixture, tab-
lets, capsules containing aminophylline and
amobarbital and coloured syrups containing
tripoldine hydrochloride and codeine phos-
phate. A simple extraction procedure avoids
interference from colouring agents in certain
formulations. Specificity, accuracy and pre-
cision of the method has been assessed. The
second derivative absorption spectrum of
ephedrine HC1 shows enhanced resolution of
the fine structure. Discrimination against
broad spectral bands in favour of narrow
bands is one of the advantages in derivative
spectroscopy. The second derivative assay of
Ephedrine Elixir BP eliminates the interfe-
rence of the broad absorption bands of ex-
cipients ( 4 5 ) . Colouring agents or other
coformulated drugs are removed from an ephe-
drine formulation by a simple solvent extrac-
tion procedure before the derivative spectro-
scopy. The recovery of ephedrine by the ex-
traction method was 99.2 % ( 4 5 ) . Ephedrine
hydrochloride has been determined directly or
after separation on cellulose thin layer pla-
tes photometrically using p-dianisidine in
aqueous solutions at pH 7. The yellow colour
has been measured at 4 2 4 nm. The method per-
mits the determination of ephedrine in the
range of 5 0 - 2 0 0 pg with a relative standard
deviation of 2 4 % (46). Spectrophotometric
determination of ephedrine is done at 629 nm
after reacting it with periodic acid 3-methyl-
benzthiazolin-2-on-hydrazon (47). Organic
bases like ephedrine are reacted with
EPHEDRINE HYDROCHLORIDE 255

bromthymol blue, subsequently extracted with

chloroform or methylene chloride and the ex-
tinction determined at 4 2 0 nm. The dyestuff
itself does not dissolve in organic phase. An
alkaline reagent like tetraethyl ammonium
hydroxide solution gives a blue coloured
organic phase which could be determined at
6 2 0 nm ( ( 4 8 , 4 9 ) . The coloured reaction pro-
duct of bromcresol green with ephedrine can
either be extracted at pH 5-6.2 with chloro-
form and the extinction is measured at 4 2 0 nm
or the organic phase is further treated with
an alkaline reagent and the extinction of the
coloured aqueous solution is determined at
6 2 0 nm ( 5 0 ) . Secondary alkyl amines react
with carbon disulphide to give dithiocarba-
mates which form with cupric salts coloured
chelates. Ephedrine hydrochloride has been
determined spectrophotometrically at 4 3 7
after extracting its coloured chelate with
chloroform ( 5 1 , 52, 5 3 ) .

9.3. Fluorimetry
A fluorimetric method has been developed for
the determination of ephedrine hydrochloride
as its dansyl derivative in ephedrine ta-
blets. After dansylation the dansyl deriva-
tive is separated on a thin layer plate and
determined directly densitometrically at
5 2 6 nm ( 5 4 ) . In another method the dansyl
derivative of ephedrine hydrochloride is
eluted from the plate with ethanol and deter-
mined at its fluorescence maximum of 5 0 5 nm
( 5 5 ) . Dansyl derivative of ephedrine has its
excitation maximum at 3 5 4 nm and emission
maximum at 4 7 6 nm ( 5 6 ) .
256 SYED LAIK ALI

9.4. Chromatographic Methods

Thin layer chromatoqraphy
Beckett and Choulis have separated ephedrine
from other compounds such as adrenalin, nor-
adrenalin etc., on cellulose, silicagel G and
aluminium oxide plates using n-butanol + gla-
cial acetic acid and water (40 + 1 0 + 50) or
with water saturated with n-butanol as mobile
phases. On cellulose tlc plates ephedrine and
its salts give two spots. The use of alkaline
mobile phases leads to only one spot (57).
Choulis has used the mobile phases n-butanol-
acetic acid-water (4+1+5) or phenol-0.1N HC1
(85+15) with cellulose tlc plates for separa-
tion of ephedrine (58). Waldi recommended the
converting of ephedrine to its corresponding
triacetylderivative before applying it on si-
licagel G plates. Having decreased its hy-
drophilic character it was possible to deve-
lop the chromatogram with chloroform - metha-
n o l (9+1) as mobile phase. The Rf-value of
ephedrine was found to be 0.51 ( 5 9 ) . Ephe-
drine could be detected by spraying tlc pla-
tes with a 0.2 % ninhydrin solution in etha-
nol or n-butanol and heating the chromatogram
to 13O-14O0C ( 6 0 ) . The best modification with
ninhydrin reagent is to use 0.3 % solution in
n-butanol + 3 m l acetic acid ( 6 1 , 6 2 ) . A si-
milar reaction can be carried out with Folin's
reagent which re- sults in the formation of
light rose-coloured spot for ephedrine ( 6 1 ) .
A spectraldensitometric method is described
for the determination of ephedrine in ephedra
herb and ephedrine extracts. After t l c sepa-
ration on silicagel G plates with the mobile
phase n-butanol-glacial acetic acid-water
(4+1+5) the chromoplate was dried, subse-
quently immersed in a 0.2 % ninhydrine so-
lution in ethanol and finally dried at llO°C.
EPHEDRINE HYDROCHLORIDE 257

The violet spots of ephedrine at Rf-value 0.3

were evaluated densitometrically at a measu-
ring wavelength of 540 nm and a reference wa-
velength of 680 nm ( 6 3 ) .
Ephedrine hydrochloride in tablets along with
other components has also been determined
densitometrically after its conversion to the
dansyl derivative and chromatographic separa-
tion on at 105'C activated silicagel 60
(Merck) tlc plates using benzene + ethanol +
glacial acetic acid (90 + 10 + 1 ) as a mobile
phase. Spectrodensometric measurement was
carried on with a 526 nm filter. This proce-
dure was on account of its great sensitivity
and good reproducibility very useful in con-
tent uniformity determinations of ephedrine
H C 1 in tablets and capsules (54). A tlc sepa-
ration of ephedrine after its conversion to
the corresponding dansyl derivative was car-
ried on silica gel tlc plate with toluene +
methanol + acetone (9+1+1) as mobile phase.
The Rf-value for ephedrine was found to be
0.50. The spots were evaluated using spec-
trofluorimeter equipped with a tlc scanning
attachment; excitation wavelength was 360 nm
and the emission wavelength was set between
500-510 nm using a U V filter. The improved
selectivity and sensitivity have permitted an
analysis in 10-100-fold excesses of other
drugs. Detection limits are reported in the
range of 1-10 ng or better. The reproduci-
bility of the method is limited by the
derivatization step, but a relative standard
deviation of less than 2 % could be obtained
(56). Lang has applied cellulose tlc plates
and mobile phases of n-butanol-glacial acetic
acid-water (4+1+5) or n-propanol+benzene+gla-
cia1 acetic acid + water (40+30+10+1) for the
separation of ephedrine HC1 in pharmaceuti-
cals. The ephedrine spot was localised,
scratched from the tlc plate, extracted with
the solution of diazotized p-dianisidine
258 SYED LAIK ALI

reagent, the solution was filtered and then

measured in a spectrophotometer at 424 nm
against corresponding blank reagent (46).
Waldi has separated ephedrine from alkaloids
of various other groups on a silica gel tlc
plate with the mobile phase chloroform-die-
thyl-amine, (19+1), Rf:0.29 (64). Simulta-
neous detection of ephedrine along with a
wide variety of commonly abused drugs in a
urine screening program using tlc techniques
has been reported. A detailed extraction pro-
cedure for the urine samples is given. Gelman
precoated silica gel glass microfilter sheets
with a layer thickness of 250 pm were used.
Different solvent systems and detection rea-
gents were used. The specific colour reac-
tions obtained there may not be achieved on
glass plates coated with silica gel. Solvent
systems ethyl acetate-cyclohexane-methanol-
ammonia (70+15+10+5)and ethyl acetate-cyclo-
hexane-ammonia (50+40+0.1) were found sui-
table for the separation of ephedrine. Micro-
gram amounts of ephedrine could be detected
with the detection reagents 0.5 % ninhydrin
in n-butanol, mercury(II)sulfate, iodoplati-
nate and iodine-potassium iodide solutions
(65). NBD-C1 derivative of ephedrine was
separated along with the corresponding deri-
vatives of other drugs on silica gel G plate
with solvent systems diethyl ether-benzene
( l + l ) , ethyl acetate-cyclohexane ( 2 + 3 ) and
ethyl acetate-cyclohexane ( 3 + 2 ) with Rf-va-
lues 0.42, 0.18 and 0 . 3 3 respectively. The
spots showed yellow fluorescence in UV light
254 nm (66).
Separation and determination of ephedrine
EPHEDRINE HYDROCHLORIDE 259

along with other doping agents has been ob-

tained through overpressurised TLC and HPTLC
silica gel 60 F254 plates using an eluent
n-butanol-chloroform-methyl ethyl ketone-
water-acetic acid ( 2 5 + 1 7 + 8 + 4 + 6 ) with an exter-
nal membrane pressure of 1 . 0 mPa. In compa-
rison with the classical TLC, the resolution
was improved, the development time was shor-
ter and detection limit was lower. The quan-
titative evaluation was carried out with a
scanner at 210 nm ( 6 7 ) .
Ephedrine hydrochloride has been determined
densitometrically at 270 nm after its separa-
tion on silica gel 60 HPTLC plates with the
mobile phase ethyl acetate-glacial acetic
acid-water ( 2 7 + 6 + 4 ) and nitration with ni-
trous gases ( 1 0 0 % nitric acid). After chro-
matography the HPTLC plate is heated in a
drying chamber for 1 5 min at 16OoC and then
exposed while still hot to nitrous gases for
1 0 min. The relative standard deviation for
ephedrine hydrochloride was found to be
1.75 8 ( 6 8 ) .

Paper Chromatography
By using papers impregnated with alkaline
buffers, such as boric acid-NaOH, pH 1 0 , and
ether saturated with water as mobile phase it
is possible to separate (+)ephedrine (Rf:
0 . 7 0 ) from (+)pseudoephedrine (Rf: 0 . 3 8 )
( 6 0 ) . Ephedrine and pseudoephedrine can be
estimated after elution in the form of copper
dithiocarbaminate complexes ( 6 0 ) . Dittrich
quantitates ephedrine in paper chromatography
by using the iodine fixation properties of
the ephedrine followed by iodometric determi-
nation after elution with KI solution ( 6 9 ) .
Other solvent systems for the paper chromato-
graphic separation of ephedrine are n-buta-
nol-acetic acid-water ( 4 + 1 + 5 ) , Rf: 0 . 7 3 ( 6 1 )
260 SYED LAIK ALI

isobutanol+formic acid+water (100+12+10), Rf:

0.50 (70), n-butanol saturated with water,
Rf: 0.64 (71), cyclohexane-diethylamine (9+1),
Rf: 0.47 (72).

Gas Liquid Chromatography

The separation of optical amines by GLC can
be achieved by using either an optically ac-
tive stationary phase after making deriva-
tives with a suitable optically inactive rea-
gent or an optically active reagent such as
N-trifluoroacetyl-L-prolylchloride (TPC) to
form diastereoisomers followed by chromato-
graphy on an optically inactive stationary
phase. TPC has been used for the resolution
of numerous asymmetric amines, including
ephedrine ( 7 3 , 74, 75). TPC-derivative of
ephedrine was prepared through addition of
0.1 M TPC solution in chloroform to the amine
in chloroformic solution. After 10-15 min the
solution was injected into a gas chromato-
graph with FID detector on a 3 % SE 30 packed
column on Chromosorb G (AW, DMCS treated,
100-120 mesh), at 17OOC oven temperature
isotherm and 22OoC injection block tempera-
ture using nitrogen (25 ml/min) as carrier
gas. A quantitative determination of the
enantionmetric percentages of ( - ) ephedrine,
( + ) and ( - 1 norephedrine-TPC derivatives was
possible. The method was found to be suitable
for biological studies (76). Ephedrine was
determined by treating the sample with sodium
periodate to form benzaldehyde and gaschro-
matographing a solvent extract of this mix-
ture. The oxidation step was included because
ephedrine did not give a well-resolved peak
(77).
A rapid GLC method for the determination of
ephedrine hydrochloride in suspension for-
mulation along with other components such as
EPHEDRINE HYDROCHLORIDE 26 1

theophylline and phenobarbital with A-naph-

thylamine as an internal standard is descri-
bed. The analysis was performed on a 3 % OV
1 7 on Gaschrom Q 1 0 0 - 2 0 0 mesh packed column
using a flame ionisation detector. The in-
jection port, column and detector tempera-
tures for the assay of ephedrine hydrochlo-
ride were maintained at 200, 150 and 2 0 0 ' C
respectively. For the assay of ephedrine
hydrochloride the sample was diluted with
water, the pH was adjusted to pH 1 1 with 2 0 8
NaOH and the solution was extracted twice
with chloroform. The chloroform solution was
injected directly into gas chromatograph.
Several additional substances such as flavou-
ring or colouring agents were extracted by
chloroform along with ephedrine, but they did
not interfere with ephedrine assay. The mean-
recovery in the assay of synthetic mixtures
for ephedrine hydrochloride was 99.2 2 0.6 %
and for commercial suspension 95.8 0.8 %
( 7 8 ) . A method for the assay of ephedrine
hydrochloride is recommended by the joint
committee of the pharmaceutical society and
the society for analytical chemistry of Great
Britain ( 7 9 ) . The assay procedure is a modi-
fication of the method proposed by Beckett
and Wilkinson ( 8 0 ) . Ephedrine hydrochloride
tablets, elixir, syrup and nasal drops have
been analysed. An internal standard phen-
dimetrazine bitartrate was added to the
sample prior to its extraction. The aqueous
solution is made alkaline with 20 % NaOH and
the liberated ephedrine base is extracted
with diethylether, the ether extract dried
with anhydrous sodium sulphate, evaporated
and made up to volume with ether. This so-
lution is injected into gaschromatograph with
a FID. The packing used in the 1 m, 4 mm i.d.
glass column was 8 0 - 1 0 0 mesh, acid washed,
silanised Chromosorb G impregnated with 2 %
carbowax 6000 and 5 % of potassium hydroxide.
The oven, injector and detector temperatures
262 SYED LAIK ALI

maintained were 150, 200, 2OO0C, respectively

with nitrogen as carrier gas (35 ml/min). The
retention times for phendimetrazine bitartra-
te base and ephedrine were found to be 3 and
5 min respectively. The collaborative study
carried on found this procedure adequate for
the determination of ephedrine in certain
pharmaceutical preparations. An examination
by mass spectrometry and IR analysis of the
ephedrine peak confirmed that ephedrine base
was eluted intact. Some tailing of the ephe-
drine peak was noted by some collaborators.
In general coefficients of variation within
laboratories were not greater than 3 % (80).
An electron-capture GLC procedure for deter-
mination of plasma ephedrine concentrations
is described (81). The procedure is capable
of determining 2 ng/ml of ephedrine. Pentane
extraction of the drug and the internal stan-
dard 3,4-dimethoxyamphetamine and formation
of the N-pentafluorobenzyl derivatives were
followed by GLC determination. The analysis
was performed on a 0.9 m x 2 mm i.d. glass
column filled with 3 % OV 225 on Chromosorb
W, AW-DMCS, 100-120 mesh. The injection port,
oven and detector temperatures were 250, 235
and 325OC respectively. An electron-capture
63 Ni detector was operated with a standing
current of 3.0 n amp. Argon-methan (95:5) as
a carrier gas was maintained at 93 ml/min.
The retention times of N-pentafluorobenzoyl
derivatives of ephedrine and internal stan-
dard were reported to be 1.40 and 3.82 min
respectively. Different GLC stationary phases
such as OV-7, OV-17 were tried but were not
suitable for quantitation. The drug and in-
ternal standard were poorly resolved with the
former and broad peaks were obtained with the
later. A OV-25 column gave sharp peaks for
ephedrine and internal standard derivatives,
but the norephedrine, the major metabolite of
ephedrine, could not be separated. Formation
of N-trifluoroacetyl, N-pentafluoropropionyl,
EPHEDRINE HYDROCHLORIDE 263

N-heptafluorobutryl and N-pentafluorbenzoyl

derivatives and their glc-mass spectrometric
identification are discussed together with
comparative electron-capture sensitivities of
these derivatives with Nickel-63 detector.
The detection of the N-pentafluorobenzoyl
derivative of ephedrine is at least 100-fold
greater in sensitivity than detection of the
N-trifluoroacetyl derivative (81). Hepta-
fluorobutyryl ephedrine derivatives following
benzene extraction of alkaline serum were
used for electron-capture analysis of blood
levels at therapeutic dosages (82). The pro-
cedures reported earlier were found insuffi-
cently sensitive for clinical use (83). The
determination of ephedrine plasma levels were
performed through GLC with FID using a 8 %
carbowax 20 M + 2 % KOH on Chromosorb W co-
lumn. Ephedrine was extracted from plasma
with diethylether after alkalising the sample
with NH40H (84). GLC with FID determination
of ephedrine was performed on a 2.0 % Carbo-
wax 20 M and 5 % KOH on Chromosorb G (100-120
mesh), AW, DMCS column at oven temperature of
100°C (22). Gas chromatography has been fur-
ther applied for the specific quantitative
determination of ephedrine and its metabolite
norephedrine in urine (85, 86). A 1.83 m,
4 mm i.d. 3 % OV 1 on Gaschrom Q, 100-200
mesh column at 14OOC oven, 140' injection
block and 23OOC detector temperatures was
used to monitor urinary excretion of ephe-
drine in man through gas chromatography with
FID (87). Derivatives of ephedrine with
trialkylsilyl groups attached to the hydroxyl
function of the molecule were synthesised and
tested gas chromatographically on a 2 m 3 %
OV 17 on chromosorb G, AW, DMCS glass column
at oven temperatures between 175-185OC (88).
N-TFA-L-alanine and N-TFA-L-alanyl chloride
were used for the preparation of high vola-
tile diastereomeric derivatives of ( - ) and
(+)-ephedrine which were separated along with
264 SYED LAIK ALI

the derivatives of other chiral amino alco-

hols and amines on a 30 m glass capillary
column OV-17 or SE 30 at a column temperature
of 2OO0C with FID (89). The separation of the
N,O-pentafluoropropionyl derivatives of the
enantiomers of ephedrine and of some analo-
gues has been carried out using chirasil-val
(90). N,O-Bis-heptafluorobutyryl derivatives
of ephedrine and other analogues were separa-
ted on a 18-m glass capillary column coated
with XE-60-L-valine-(R)- d-phenylethylamide
(91). Excellent separations of diastereomeric
derivatives of several amino alcohols of the
ephedrine type have been obtained on a column
with a chiral stationary phase after N-acy-
lation with L- A-chloroisovaeryl chloride and
o-trimethylsilylation (92). KGnig and Benecke
reported the resolution of a number of amino
alcohols including the N-demethylated analo-
gues of ephedrine and norephedrine on a GLC
chiral modified OV-225 phase (93).

High Performance Liquid Chromatoqraphy

The separation and quantitation of ephedrine
is carried out by means of HPLC after its
conversion into 4-nitrobenzamide with 4-ni-
trobenzoyl chloride. A procedure for the
preparation of derivatives is given. The
determination was carried out on a 20 cm
spherisorb 5 pm column using a solvent mix-
ture of isooctane methylene chloride + etha-
nol + water (400+87+8+5)as a mobile phase
with 1.8 ml/min flow-rate and the ephedrine
derivative was detected at 337 nm. The de-
tection limit is given as about 5 ng per
20 pl injected pure derivative with a re-
lative standard deviation of less than -f 6 %.
The method could also be applied for the
analysis of plasma samples (94). The quan-
titative determination of combination of
antihistamine and decongestant drugs inclu-
EPHEDRINE HYDROCHLORIDE 265

ding phenylepherine, dl-ephedrine, 1-ephe-

drine, chlorpheniramine etc. contained in
solid and liquid dosage forms are described.
All active ingredients except the ephedrine
optical isomers were separated from other
ingredient with ion-paired HPLC. Elixirs,
syrups, tablets and timed-release capsules or
tablets were analysed. The chromatographic
separation was done on a 4 mm i.d. x 30 cm
p-Bondapak phenyl column with a mobile phase
water-methanol-glacial acetic acid (55+44+1
V / V ) containing enough heptanesulfonic acid
sodium salt to yield a 0.005 M solution. The
flow rate was 2.0 ml/min and the detection
wavelength 254 nm. The column used is capable
of resolving almost all of the compounds
except the stereoisomers 1-ephedrine and
d-ephedrine. The reproducibility of the
method was excellent with a coefficient of
variation of 0.9 % (95). Simultaneous de-
termination of ephedrine sulphate, hydro-
xyzine hydrochloride and theophylline in
tablets by HPLC involved a 1 0 m Bondapak c18
6"
column with acetonitrile-aque us ammonium
carbonate solution (50+50) at pH 7.0 as the
eluent and UV detection at 254 nm. 0.1 %
aqueous ammonium carbonate buffer was pre-
pared and adjusted to pH 7.0 with acetic acid
(96). The chiral forms of ephedrine were
analysed as the corresponding oxazolidines
formed by reaction between the propanolamine
and 2-naphthaldehyde. HPLC separation was
carried out on a column of 25 cm x 4.6 mm
i.d., 5 pm aminopropyl-bonded silica gel
modified with (R)-N-(3,5dinitrobenzoyl)phe-
nylglycine using a mobile phase n-hexane-
isopropanol (99.5+0.5) with a flow rate
1.0 ml/min at 254 nm ( 9 7 ) . Following their
conversion to dithiocarbamate ligands and
subsequently to nickel complexes, the separa-
tion and quantitation of enantiomeric mix-
tures of ephedrine and pseudoephedrine were
accomplished by liquid chromatography with
266 SYED LAIK ALI

ternary solvent mixtures. Formation of nickel

complexes prior to chromatography and on-co-
lumn formation using nickel(I1)ions in the
mobile phase has been studied ( 9 8 ) . Ephedrine
and pseudoephedrine in formulated products
were determined on a 1 0 pn alkylphenylp-Bon-
dapak column with acetonitrite-water-monoba-
sic sodium phosphate ( 1 % acetonitrile in
0.05 M monobasic sodium phosphate aqueous
solution) as the eluent and UV detection at
2 1 0 nm ( 9 9 ) . A HPLC method is described in
which ephedrine hydrochloride is measured
after its oxidation to benzaldehyde through
periodate simultaneously with theophylline
and phenobarbital in tablets with butabar-
bital as the internal standard. A pH of 7.8
was selected for rapid oxidation of ephedrine
and a detection wavelength of 2 4 1 nm was
chosen which is near to the maximum for
benzaldehyde and barbiturates and to the
minimum for theophylline. Chromatographic
column was a reversed phase C18 phase bonded
on silica and the mobile phase consisted of
acetonitrile ( 2 4 0 ml) mixed with 0.01 M phos-
phate buffer, pH 7.8 ( 7 6 0 ml). Benzaldehyde
obtained from ephedrine had a retention time
of 1 1 . 7 min. The chromatogram showed no in-
terference from the excipients and other oxi-
dation products. Procedures are provided for
the assay of conventional and sustained-ac-
tion tablet formulations ( 1 0 0 ) . In another
HPLC method the simultaneous assay of ephe-
drine hydrochloride, theophylline and pheno-
barbital in tablets is reported. 2 5 cm x 4.5
mm i.d., 1 0 pm Partisil ODS I1 column and a
methanol-0.007 M monobasic potassium phos-
phate ( 3 7 + 6 3 , pH 2 . 3 ) as mobile phase was
used at detection wavelength 2 5 4 nm. A me-
thanolic extract of the powdered sample
containing salicylamide as the internal
standard was injected into the chromatograph
( 1 0 1 ) . In the determination of ephedrine
using reversed-phase ion-pair liquid chroma-
tography, a chromatographically pure sample
EPHEDRINE HYDROCHLORIDE 267

was observed to give three peaks under cer-

tain mobile phase condition. The peak due to
ephedrine was found to vary from symmetrical
to almost completely resolved split peaks in
mobile phases containing only P I C B7 (heptane
sulfonic acid). When sodium sulphate was in-
cluded in the mobile phase peak-splitting was
more pronounced. A proposal, that peak split-
ting was the result of the composite inter-
play of two discrete chromatographic mecha-
nism, was investigated. The results of ana-
lysis by GC/MS confirmed that each peak was
due to ephedrine, however, only one of the
three split peaks was found to contain ion-
pairs. It is postulated that peak splitting
is a physical phenomenon on reversed-phase
column and the separation of these drugs by
ion-pair HPLC is based on a mixed rather than
a single mechanism ( 1 0 2 ) . Derivatization of
ephedrine with dansyl chloride and a sensi-
tive, specific HPLC method for its determi-
nation in complex pharmaceutical dosage forms
is reported. A 25 cm x 2 . 8 m i.d. column
filled with Merck silica ge! SI 1 0 0 , 1 0 pm
and diisopropyl ether saturated with conc.
Ammonia-isopropanol ( 9 9 + 1 ) were used for
separation. The detection was carried out
simultaneously with a fluorescence detector,
3 5 4 nm excitation, 4 7 6 nm emission, and a
fixed-wavelength 2 5 4 nm UV detector (56).
Determination of ephedrine sulfate in cough-
cold mixtures along with various other anal-
gesic and antihistamine compounds was per-
formed on a Corasil C 1 8 column with the mo-
bile phase acetonitrile-water ( 6 0 : 4 0 ) with
1 % ammonium acetate and the pH adjusted to
7.40 (103).

HPLC retention characteristics of ephedrine

have been measured along with 8 4 other basic
drugs of forensic interest. Chromatography
was carried on using 250 x 5 mm i.d. column
packed with Spherisorb S5W at 2 5 4 nm. The
268 SYED LAIK AL1

eluent consisted of methanol-aqueous ammonium

nitrate buffer (9+1). The buffer was prepared
by adding 94 ml ammonia (35 % ) and 21.5 ml
nitric acid (70 % ) to 884 ml water and then
adjusting the pH to 10.1 with ammonia. The
flow-rate was 2 ml/min. Because of the alka-
line nature of the eluent, a short column,
dry packed with silica (40 pm) was included
between the pump and injector to minimise
dissolution of the analytical packing mate-
rial (104).
Resolution of the enantiomers of ephedrine
and other related compounds through a simple
HPLC method is described. A 150 mm x 4.6 mm
column was packed with Ultrasphere O D s , 5 pm
particle size and the mobile phase was prepa-
red by mixing 400 ml acetonitrile with 600 ml
water containing 1.4 g of monobasic ammonium
phosphate. The flow-rate was 1.0 ml/min and
the column eluent was monitored at 254 nm.
Ephedrine or ephedrine hydrochloride is deri-
vatised with the chiral reagent 2,3,4,6-tetra-
0-acetyl-P-D-glucopyranosyl isothiocyanate
(GITC) and the separation of the resulting
diastereomeric thioureas is performed by
reversed phase HPLC. The derivatisation
method is extremely simple, the chiral rea-
gent is commercially available, chemically
and stereochemically stable. The resolution
of ephedrine, pseudoephedrine and norephe-
drine is considerably better (105). R-4-Me-
thylbenzyl isothiocyanate, a commercially
available chiral compound, was evaluated as a
chiral derivatizing agent for the separation
of among others ephedrine's enantiomers
through HPLC on a 150 x 4.6 mm Ultrasphere
ODS 5 pm column with a water-acetonitrile
(50:50) mobile phase at 1.0 ml/min flow-rate
(106).
The enantiomers of ephedrine were resolved as
their cyclic oxazolidine derivatives which
EPHEDRINE HYDROCHLORIDE 269

were produced by the condensation of the

amino alcohol with 2-naphthaldehyde. The
enantiomeric resolution of ephedrine was
performed on an ionically bonded chiral
stationary phase. The column was a stainless
steel Regis-packed pirkle type I-A (250 x 4.6
mm) with an d-aminopropyl packing of 5 pm
spherical particles modified with (R)-N-(3,5-
dinitrobenzoy1)phenyl-glycine. The mobile
phase consisted of hexane-isopropanol
(99.5+0.5), the flow-rate was 1.0 ml/min and
the detection wavelength was set at 254 nm
(107).
Ephedrine is converted to metal (copper and
nickel) dithiocarbamate complexes by means of
a pre-column derivatisation method. Chro-
matography is done on a Lichrosorb RP 18
column with mixtures of acetate buffer (pH
5.8) and organic solvents like methanol,
acetonitrile or ethanol as mobile phases. The
complexes were detected amperometrically
(applied potential + 0.7 V VS SCE) using a
thin-layer electrolytic cell fitted with
glassy carbon working and auxiliary elec-
trodes. The spectrophotometric detector was
set at 325 nm for nickel chelates and at 270
nm for copper chelates. The procedure is
described to have a great sensitivity (about
-1 2
10 M) and good selectivity for the more
substituted amino drugs (108).

9.5. NMR-Assay
A rapid NMR method is described for the
determination of ephedrine hydrochloride in
tablets. The NMR spectrum of ephedrine hydro-
chloride in D20 displays chemical shifts for
methyl proton doublet at 1.16 ppm, N-methyl
proton singlet at 2.83 ppm and aromatic pro-
tons singlet at 7.43 ppm. The doublet at
1.16 ppm was chosen for quantitative work.
Acetamide was used as an internal standard on
270 SYED LAIK ALI

the basis of its methyl three proton singlet

at 2 ppm, which is sufficiently separated
from the 1.16 ppm doublet of ephedrine hy-
drochloride and the solvent signal at
4.75 ppm to allow satisfactory determination.
The results for synthetic mixtures and tab-
lets are comparable to those obtained by
other methods. The relative standard devia-
tions were 0.5 and 1.2 % for the pure drug
and tablets respectively. In addition the NMR
method furnishes a specific means of identi-
fication of ephedrine (109).

9.6. Radioimmunoassay
Stereospecific radioimmunoassay for l-ephe-
drine and d-ephedrine in human plasma after
administration of a single 50 mg oral dose of
dl-ephedrine hydrochloride has been reported
(110). Separate RIAS developed for d-ephe-
drine and 1-ephedrine were used to measure
the concentrations of the enantiomers of
ephedrine in the blood of two volunteers
dosed with racemic ephedrine. The RIAS were
validated by comparing the sum of the con-
centrations of the enantiomers with total
ephedrine concentrations determined by a
nonstereoselective GLC-ECD method. Fig. 9
shows graphically plasma concentrations of d-
and 1-ephedrine (110). Budd has presented a
comparison of GC and EMIT (enzyme multiplied
immunoassay technique) methods for the ana-
lysis of ephedrine and other related drugs.
For GC analysis an Apiezon-KOH column was
used ( 1 1 1 ) .

9.7. Isotachophoresis
The utility of isotachophoresis has been
examined for the simultaneous determination
of all the six ephedrine alkaloids in ephedra
EPHEDRINE HYDROCHLORIDE 271

HOURS

FIG, 9
D-EPHEDRINE (4) AND L-EPHEDRINE ( A ) CONCENTRATIONS
I N THE PLASMA OF A HEALTHY HUMAN VOLUNTEER FOLLO-
WING A S I N G L E 50 MG ORAL DOSE OF DL-EPHEDRINE
HYDROCHLORIDE (110)
272 SYED LAIK ALI

herb extracts. Ephedra herb was extracted

with 50 % ethanol. All analysis were carried
out on a Shimadzu model Ip-2A Isotachopho-
retic analyzer equipped with a Shimadzu po-
tential gradient detector with a 4 0 mm PTFE
capillary ( 1 . 0 mm i.d.1 as the pre-column and
a 1 5 0 mm FEP capillary (0.5 mm i.d.) connec-
ted in series. The leading electrolyte was
prepared by adding p-alanine or histidine to
0.005 M barium hydroxide to adjust the pH.
0.1 % Triton X-100 was added to improve se-
paration by enhancement of viscosity and
reduction of electrodenosmosis. 0.01 M Amme-
diol was used as the terminating electrolyte.
The analysis was first proceeded at 2 0 0 pA
for 1 2 min and was then continued at 1 0 0 PA.
Calibration curves for the six ephedrine
alkaloids were constructed in the range of
0.2 - 2.0 mg/ml. The ephedra extract (20.0
mg) was dissolved in water ( 1 . 0 ml) and an
aliquot ( 0 . 0 1 0 ml) was injected directly
(112).

10. Drug Metabolism and Pharmacokinetics

The uses of ephedrine indicate that the drug
has sympathomimetic actions grossly resem-
bling those of epinephrine. It is readily
absorbed from the upper gastrointestinal
tract. Good effects have also been achieved
from the lower tract also through its em-
ployment in a retention enema. The gastro-
intestinal absorbability and the long du-
ration of action has been shown to be asso-
ciated with the attachment of a methyl group
on the alpha carbon. Ephedrine is not oxi-
dized by enzymatic action, but conjugation
occurs in some species ( 1 1 3 ) . Ephedrine is
readily and completely absorbed from the GI
tract, peak plasma concentrations being
achieved about an hour after oral admini-
stration. It is resistant to metabolism by
EPHEDRINE HYDROCHLORIDE 273

monoamine oxidase and is largely excreted

unchanged in the urine, with some deaminated
and N-demethylated metabolites. Half-life
lies between 3-6 h. Elimination is enhanced
and half-life is accordingly shorter in acid
urine ( 1 1 4 ) . The relative metabolism and
urinary excretion of the ephedrines are
dependent upon the urinary pH and in certain
circumstances the volume also (115). Intra-
subject variation was observed in both the
lag time and the rate constant for absorption
in case of (-)ephedrine. Ephedrines were
readily absorbed within 3 hours of admini-
stration. Mean overall elimination t 1/2
value for unchanged (-)ephedrine was 3 . 0 3 h.
It appears that ephedrine and norephedrine,
when present in the body as metabolites, are
eliminated faster than when they are admini-
stered per se. A possible explanation for
this phenomenon is that the kinetics of eli-
mination of the ephedrines is dose-dependent;
elimination is faster at low body drug levels
than at high levels. The kinetics of absorp-
tion, metabolism and excretion of (-)-ephe-
drine has been elucidated using analog com-
puter analysis of urinary excretion data from
three male subjects under constant acidic
urine control ( 1 1 6 ) . In a feasibility study
25 mg single oral doses of ephedrine sulfate
in the form of a commercial syrup and two
commercial capsules were administered in
crossover fashion to three non-smoking sub-
jects. Urinary pH was not controlled, but
adequate urinary flow rates were maintained
by regulated water intake. There were no
significant differences in average amounts of
ephedrine excreted during any of the 1 1 samp-
ling intervals, in average peak excretion
rates, nor in average times of occurrence of
the peak excretion rate. The average elimi-
nation half-life of ephedrine was 5.99 h and
average urinary pH 6.30. The urinary excre-
tion data was adequately described by the
two-compartment open model with first-order
 HOURS

F I G , 10
EPHEDRINE CONCENTRATIONS I N THE PLASMA OF A HUMAN VOLUNTEER
(67.6 KG) FOLLOWING A S I N G L E 24 MG DOSE OF EPHEDRINE HYDRO-
CHLORIDE I N A COMBINATION TABLET (81)
EPHEDRINE HYDROCHLORIDE 275

absorption and lag time. The baseline concen-

tration of ephedrine in plasma 1 5 hours after
the night dose was 20 ng/ml. Following ad-
ministration of one tablet containing 1 5 mg
ephedrine sulphate the concentration rose to
9 5 ng/ml after 4 h, falling to 6 5 ng/ml at
6 h (84). Ephedrine is readily absorbed after
oral or percutaneous administration; gastro-
intestinal absorption is increased by anta-
cids but decreased by kaolin. After an oral
dose of 22 mg of ephedrine hydrochloride
plasma concentrations of 40-140 ng/ml are
obtained and after an oral dose of 3 3 mg peak
plasma concentrations of 6 5 - 120 ng/ml are
attained during therapy: effective broncho-
dilator plasma concentrations are in the
range of 35 - 80 ng/ml and plasma half-life
lies between 3 - 1 1 hours. Metabolic reac-
tions are N-demethylation and oxidative de-
amination followed by conjugation. Upto about
9 5 % of a dose may be excreted in the urine
in 24 hours, 55 - 7 5 % as unchanged drug, 8 -
20 % as the N-demethylated metabolite and
4 - 1 3 % as deaminated metabolites such as
benzoic acid, hippuric acid and l-phenyl-
propane-lI2-diol. The rate of urinary excre-
tion of ephedrine is pH-dependent and is
increased in acidic urine ( 1 1 7 ) . A combi-
nation tablet containing ephedrine hydrochlo-
ride (24 mg) theophylline (130 mg) and pheno-
barbital (8 mg) was administrated and ali-
quots of plasmas collected showed peak plasma
concentration of over 100 ng/ml. Plasma con-
centrations over 24 h in the subject fit a
one-compartment model from which the elimi-
nation half-life was calculated as 4.8 h.
Fig. 1 0 graphically illustrates this pheno-
.
menon (81 )

11. Acknowledgement
Mrs. Monica Hediger has put a lot of efforts
276 SYED LAIK ALI

and taken great pains in typing this manus-

cript. Dr. Wolfgang Stcber has kindly perfor-
med the mass spectrometric measurements. The
author is greatful to them for their assis-
tance.
EPHEDRINE HYDROCHLORIDE 277

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This Page Intentionally Left Blank
 ESTRADIOL

E u g e n e G. Salole

1. Foreword

2. De s c r i p t i o n

3. Synthesis

4. Physical Properties
4.1 I n f r a r e d Spectrum
4.2 N u c l e a r M a g n e t i c Resonance S p e c t r a
4.3 U l t r a v i o l e t Spectrum
4.4 Mass S p e c t r u m
4.5 D i p o l e Moment, O p t i c a l R o t a t i o n a n d pKa
4.6 S o l u b i l i t y , C o m p l e x a t i o n and
Distribution Ratios
4.7 Crystal Properties

5. Methods o f A n a l y s i s
5.1 E x t r a c t i o n and P u r i f i c a t i o n
5.2 A b s o r p t i o n and F l u o r e s c e n c e
Spectrophotometry
5.3 Chromatography
5.4 P r o t e i n - b i n d i n g Assays
5.5 Bioassay

6. S t a b i 1i t y

7. Metabolism, Pharmacokinetics and Bio-

availability

8. D e t e r m i n a t i o n i n Body F l u i d s

9. Determination i n Pharmaceuticals

Acknowledgements
References
ANALYTICAL PROFILES OF DRUG SUBSTANCES Copyright 0 1986
VOLUME 15 hy the American Pharmaceutical Association
283 All rights of reproduction in any lorn1 reserved.
284 EUGENE G. SALOLE

1. Foreword

E s t r a d i o l i s t h e most p o t e n t e s t r o g e n i c hormone,
s e c r e t e d i n n o r m a l p r e - m e n o p a u s a l women m a i n l y b y
the ovaries. The l a s t o f t h e m a j o r n a t u r a l h o r m o n e s
t o b e i s o l a t e d (12mg w e r e e x t r a c t e d f r o m 4 t o n o f
p o r c i n e o v a r i e s i n 1935, a l t h o u q h i t h a d p r e v i o u s l y
been s y n t h e s i z e d f r o m e s t r o n e ) , i t s m a i n c l i n i c a l
u s e i s i n p o s t - c l i m a c t e r i c replacement therapy, f o r
w h i c h i t i s a d m i n i s t e r e d ( a l o n e and i n c o m b i n a t i o n
w i t h o t h e r hormones) by t h e o r a l , t r a n s d e r m a l ,
s u b c u t a n e o u s and i n t r a v a g i n a l r o u t e s i n a v a r i e t y o f
d o s a g e f o r m s 1 92.
The i n t e n s e i n t e r e s t i n e s t r o g e n s s i n c e t h e t u r n
o f the c e n t u r y has generated a vast, and s t i l l
r a p i d l y e x p a n d i n g , l i t e r a t u r e ; t h i s modest p r o f i l e
t h e r e f o r e a i m s p r i n c i p a l l y t o c o l l a t e some o f t h e
p h y s i c o c h e m i c a l d a t a on e s t r a d i o l w h i c h w o u l d be o f
i n t e r e s t t o pharmaceutical s c i e n t i s t s . Reviews o f
t h e b i o c h e m i s t r y , m o l e c u l a r p h a r m a c o l o q y and o t h e r
b i o l o g i c a l a s p e c t s o f e s t r a d i o l may b e f o u n d i n t h e
b o o k s e d i t e d by Chaudhury3 and M a k i d ; i t s c h e m i s t r y
i s c o v e r e d b y F i e s e r and F i e s e r 5 and i n B e i l s t e i n ' s
Handbook6.

2. Description

Estra-1,3,5(lO)-triene-3,17~-diol; 17p-estradiol;
E2; obsolete terms include dihydrofollicular
hormone, dihydrofolliculin, d i h y d r o t h e e l i n and
dihydroxyestrin ('17a-estradiol' i s a misnomer i n
the literature prior to ca. 1952); Chemical
A b s t r a c t s S e r v i c e r e g i s t r y number 5 0 - 2 8 - 2 .

'1 EH24'2
M 272.3E7
-r
HO (see Sect. 4.7)

A white, odorless, tasteless, c r y s t a l l i n e powder.

ESTRADIOL 285
3. Synthesis

The t o t a l s y n t h e s i s o f e s t r a d i o l f r o m a c y c l i c
p r e c u r s o r s h a s b e e n a c c o m p l i s h e d 6 , 7, b u t i t i s
u s u a l l y p r e p a r e d by t h e sodium b o r o h y d r i d e r e d u c t i o n
o f e s t r o n e , i t s e l f o b t a i n e d by t h e p y r o l y s i s o r
reductive aromatization o f androstenedione8.
Syntheses o f i s o t o p i c a l l y - l a b e l l e d e s t r a d i o l h a v e
been described9.

4. Physical Properties

4.1 I n f r a r e d Spectrum
The I R spectrum o f estradiol hemihydrate is
i l l u s t r a t e d i n F i g . 1; d e t a i l e d a s s i g n m e n t s h a v e
b e e n p r o p o s e d by Smakula e t a l l 0 .
The s p e c t r u m i n F i g . 1 is i d e n t i c a l t o t h a t
r e p o r t e d by N e u d e r t and R o p k e l l and Hayden e t a l l 2 . ,
and t o one s u p p l i e d by t h e MRC S t e r o i d R e f e r e n c e
C o l l e c t i o n , London, b u t q u i t e d i f f e r e n t f r o m t h e
spectra in the Sadtler and Sammul et al.,
c ~ m p i l a t i o n s l ~w, h i c h a l s o d i f f e r f r o m e a c h o t h e r
( s e e S e c t . 4.7).

4.2 N u c l e a r M a g n e t i c Resonance S p e c t r a
I H - and I3C-NMR spectra o f e s t r a d i o l hemihydrate i n
a c e t o n e - d 6 w e r e o b t a i n e d a t 2 5 0 . 1 3 a n d 62.9MHz,
respectively. Assignments f o r the p r o t o n spectrum
(Fig. 2 ) are:

b/PPm
7.09 ( I H , d, J = 8.3Hz, H - I )
6.59 ( I H , ad,-J = 8 . 3 , 2.8Hz, H - 2 )
6.52 ( I H , d, J-= 2.8Hz, H - 4 )
4.10 (3H, 6r.s, 2xOH & l / 2 H 2 0 )
3.67 ( I H , dd, - J = 8.8, 8.1Hz, H - 1 7 a )
2.76 (2H,
2.28 ( I H , K)
2.20-1.10 (12H,-m)-
0.77 (3H, 2, M e - 1 8 ) .

The following assignments for the I3C-NMR

spectrum (Fiq. 3) concur w i t h the l i t e r a t u r e 1 4 :

d/ppm d/PP

155.89 (5,C-3) 44.04 (5, C-13)

138.38 -
(5, C-5) 40.03 - C-8)
(7,
100-

I I I I I I 1 I I I I J
4000 cm-1 3000 2000 1800 1400 1000 600
Fig. 1 I R spectrum o f e s t r a d i o l hemih d r a t e : Pye-Unicam SP3-200 spectrometer, KC1 disc
T
( p o l y s t y r e n e marker a t 1602cm- ).
 .
Fig. 2 'H-NMR spectrum o f e s t r a d i o l hemihydrate: Bruker WM250 spectrometer a t 250.13MHz,
acetone-d s o l u t i o n , TMS i n t e r n a l standard.
6
 -%-

Fig. 3 I3C-NMR spectrum o f e s t r a d i o l hemihydrate: Bruker WM250 spectrometer a t 62.9MHz,

acetone-d s o l u t i o n , TMS i n t e r n a l standard.
6
E STRADIOL 289
132.09 (s, C-IO) 37.76 (t, C-12)
126.94 (7,C - 1 ) 31.02 (T, C-16)
115.96 (3,C-4) 30.30 (T, C-6)
113.58 (Ti, C-2) 28.13 (5, C-7)
81.86 (3,C-17) 27.26 ( t , C-11)
50.96 (3,C-14) 23.80 ( f , C-15)
44.90 - C-9)
(7, 11.63 - C-18).
(4,

4.3 U l t r a v i o l e t Spectrum
I n 2Xv/v m e t h a n o l , e s t r a d i o l h e m i h y d r a t e e x h i b i t s
U V m a x i m a a t 221nm ( A l # l c m 2 8 9 ) a n d 280nm ( A I X l c m
751, w i t h a s h o u l d e r a t 287nm ( F i g . 4 ) . Absorbance
values quoted f o r e s t r a d i o l i n o t h e r media are:

max/nm A 1X 1 c m

0.1M h y d r o c h l o r i c a c i d q 5 2 78 76
80Xv/v methanol,
alkalinifiedll 285.6 69
0.1M s o d i u m h y d r o x i d e A 5 238 34 1
296 102
6M ammonia16 240 309
297 98.

The a b s o r p t i o n s p e c t r u m o f e s t r a d i o l u n d e r g o e s
b a t h o c h r o m i c s h i f t s w i t h r i s e i n pH ( i s o s b e s t i c
point at 285.6nmll); spectra i n concentrated
s u l f u r i c a c i d a r e i l l u s t r a t e d i n R e f . 17.

4.4 Mass S p e c t r u m
T h e f o l l o w i n g a s s i g n m e n t s o f a h i q h - r e s o l u t i o n mass
spectrum, o b t a i n e d u s i n g an A E I (Kratos) MS9
spectrometer ( e l e c t r o n impact i o n i s a t i o n , d i r e c t
i n s e r t i o n ) agree t o s i x s i g n i f i c a n t f i g u r e s w i t h t h e
e x p e r i m e n t a l mass d a t a f o r e s t r a d i o l h e m i h y d r a t e :

-
m/z Rel.Int.

2 72 100
213 46
186 14
185 13
172 27
160 43
159 29
158 13
146 28
145 18
133 23.
 nm
Fig. 4 UV spectrum o f estradiol hemihydrate: Cecil CE505 spectrophotometer, 0.33mg d1-I
in Z.OXv/v methanol.
ESTRADIOL 29 1

A simulated low-resolution p l o t o f the spectrum i s

i l l u s t r a t e d i n F i g . 5. The c h e m i c a l i o n i z a t i o n mass
s p e c t r u m has been d e s c r i b e d 1 8 .

4.5 D i p o l e Moment, O p t i c a l R o t a t i o n a n d pKa

T h e d i p o l e moment o f a n h y d r o u s e s t r a d i o l i n d i o x a n e
was m e a s u r e d a t 2.330 ( 7 . 7 7 2 ~ 1 0 - j ~C m ) l l .

S p e c i f i c o p t i c a l r o t a t i o n values reported are:

[ a l ~( 2 2 - 2 4 ' C , dioxane) +76'11

(IE'C, ethanol) +78"19.

Values a t d i f f e r e n t w a v e l e n g t h s and t e m p e r a t u r e s ,
and i n o t h e r s o l v e n t s , a r e q u o t e d i n B e i l s t e i n 6 .

Spectrophotometrically determined apparent pKa

v a l u e s f o r t h e p h e n o l i c OH a r e :

10.12 2 0.025 SD20

10.30 ;t0.10 M S E ~ ~
10.71 2 0.02 S D I 6 .

4.6 S o l u b i l i t y , Complexation and D i s t r i b u t i o n

Ratios
T h e s o l u b i l i t i e s o f e s t r a d i o l i n some a q u e o u s a n d
o r g a n i c media a r e l i s t e d i n Tables 1-3. These
v a l u e s , p a r t i c u l a r l y f o r aqueous s o l u t i o n s , s h o u l d
be accepted c a u t i o u s l y , because the apparent
s o l u b i l i t y o f e s t r a d i o l depends upon t h e d e t e r m i n i n q
procedure. For i n s t a n c e , t h e e q u i l i b r i u m
c o n c e n t r a t i o n s a c h i e v e d b y u n s a t u r a t e d s o l u t i o n s may
d i f f e r f r o m t h o s e a t t a i n e d by i n i t i a l l y s u p e r -
saturated solutions26, and whereas s h a k i n q an
aqueous s u s p e n s i o n for 48h r e s u l t e d i n s o l u b i l i t y o f
0.319mg d l - I a t 25'C, u l t r a - s o n i c a t i o n f o r 0.5h
i n s t e a d p r o d u c e d a c o n c e n t r a t i o n o f 0.613mg d l - I
2 8 ; t h e f i l t e r s u s e d t o c l a r i f y s u p e r n a t a n t s may
adsorb e s t r a d i o l t o a degree dependent on i t s
c o n c e n t r a t i o n and t h e i r c o m p o s i t i o n 2 9 .

A l t h o u g h t h e 25mg d l - I c o n c e n t r a t i o n r a p i d l y
achieved by a 1 : 9 s o l i d c o - p r e c i p i t a t e o f e s t r a d i o l
i n p o l y v i n y l p y r r o l i d o n e 4 0 0 0 0 was a t t r i b u t e d t o t h e
s t e r o i d b e i n g in m o l e c u l a r d i s p e r s i o n 2 5 , t h e a q u e o u s
solubility o f estradiol may be modified by
a s s o c i a t i o n w i t h a v a r i e t y o f compounds. The a m i n o
acids tyrosine (Table I ) , a r g i n i n e and l y s i n e
t9
C D I
W N
T
E
N
S
I
T
Y

Fig. 5 Simulated (low-resolution) mass s p e c t r u m o f e s t r a d i o l h e m i h y d r a t e .

TABLE 1

S o l u b i l i t y (mg d1-I) o f E s t r a d i o l i n Aqueous S o l v e n t s

Temperature/OC

20 25 35 37 42.5 50 Ref.

Water 0.399 22
0.17 0.30 0.56 0.77 0.97 23
0.02 M Sodium c h l o r i d e 0.38 24
0.20 M Sodium c h l o r i d e 0.34
0.40 M Sodium c h l o r i d e 0.28
Phosphate b u f f e r ,
pH7.2, 0.05 I 0.38f0.046
pH7.2, 0.10 I 0.36+0.016
pH7.2, 0.20 I 0.34-0.024
0.005 M L - t y r o s i n e i n
phosphate b u f f e r ,
pH7.2, 0.10 I 0.6620.049
Phosphate b u f f e r ,
pH7.4, 0.15 I 0.512 25
0.02 M Sodium
deoxycholate i n
phosphate b u f f e r ,
pH 7.4, 0.15 I 3.92
TABLE 2
Solubility (mg d1-I) o f Estradiol in Organic Solvents26

Temperature/OC
~ ~

0 15 25 40

Acetone 2594.1 4291.2 7068.8 8914.8 13715.2

Benzene - 21.7 46.7 80.1 198.3
Chloroform 140.5 251.2 411.4 642.7 762.0
Cyclohexane - 0.5 1.3 2.2 7.6
Dichloromethane 45.1 108.4 192.6 267.1 -
Dioxane - 7075.2 12085.6 19868.4 30968.0
Ethanol 1541.8 2387.2 3134.4 3727.4 4890.7
95% v/v Ethanol 1060.4 1606.9 2908.8 4186.3 4805.3
Diethyl ether 533.2 700.8 754.5 836.7
Hexane < 0.5 < 0.5 < 0.5 0.9 -
Methanol 862.1 1811.3 2548.8 3525.6 5342.4
Tetrahydrofuran 25447.2 28006.4 29222.4 33788.8 43313.6
Toluene 4.3 14.8 33.1 51.5 95.0
TABLE 3
27
Solubility (mg g-') o f Estradiol in Organic Solvents at 2 2 O C

1 -Decanol 28
Dimethyl sulfoxide > 500
Ethyl oleate 16
Ethylene glycol 16
Ethylene glycol:
polyethylene glycol 4 0 0 , 1:l W/W 78
Glycerol 1.5
Polyethylene glycol 400 105"
Polysorbate 80 (Tween 80) 36
Propylene glycol 75
80% w/w Propylene glycol 2.8
60% w/w Propylene glycol 0.52
40% w/w Propylene glycol 0.10
20% w/w Propylene glycol 0.023
Propylene glycol:
glycerol, I :I w/w 25

2
* 167x10 rngdl-A at 35OCZ2
296 EUGENE G. SALOLE
p r o m o t e s o l u b i l i t y 2 4 , a s do p r ~ g e s t e r o n ea~n d~ human
serum a l b u m i n 3 0 ( w h i c h admixed w i t h p r o p y l e n e g l y c o l
has been used t o p r e p a r e i n t r a v e n o u s i n j e c t i o n s o f
100mg d l - 1 3 1 ) . P o l y s o r b a t e 2 0 ( a t 20'C) a n d s o d i u m
d o d e c y l s u l f a t e ( a t 40'C) m a x i m a l l y s o l u b i l i z e d
e s t r a d i o l a t 1 3 a n d 25mmol p e r m o l s u r f a c t a n t ,
r e s p e c t i v e l y 3 2 ; w i t h egg l e c i t h i n v e s i c l e s t h e r a t i o
was f o u n d t o c h a n g e f r o m 0 . 0 1 7 6 t o 0 . 0 4 2 2 m m o l p e r
mol l i p i d depending on their method of
preparationz8. p - c y c l o d e x t r i n was f o u n d t o c o m p l e x
w i t h e s t r a d i o l i n aqueous s o l u t i o n ; the white
amorphous powder isolated by interfacial
co-precipitation exhibited a rapid rate o f
d i s s o l u t i o n , w i t h a s o l u b i l i t y a t 25'C o f 12mg d l - 1
33. I n c o n t r a s t , b o t h u r e a and d i q i t o n i n f o r m o n l y
s p a r i n g l y s o l u b l e complexes w i t h e s t r a d i o l : columnar
c r y s t a l s ( o f orthorhombic symmetry) o f t h e 1:l
complex p r e c i p i t a t e f r o m a s o l u t i o n i n b e n z e n e o f
e s t r a d i o l and urea i n 1 : l O mole r a t i o 3 4 , whereas
n e e d l e s o f t h e d i g i t o n i d e c o m p l e x ( m . p s 2 6 5 " C ) may
b e o b t a i n e d by m i x i n g s o l u t i o n s o f t h e s t e r o i d w i t h
1- 4 7 i W / v d i g i t o n i n i n 8 0 X v / v e t h a n o l 3 5 . Apparently
t h e u r e a complex i s n o t o f t h e c l a t h r a t e t y p e 3 4 , and
l i k e the d i g i t o n i d e i s r e a d i l y cleaved (by warming
i n w a t e r and d r y p y r i d i n e , r e s p e c t i v e l y ) .
Estradiol i s s u f f i c i e n t l y s o l u b l e i n peanut and
sesame o i l s f o r them t o b e u s e d as v e h i c l e s f o r
intramuscular injections36.

D i s t r i b u t i o n r a t i o s o f e s t r a d i o l (and i t s e s t e r s )
i n s e v e r a l dozen systems have been c o m p i l e d by
E n g e 1 1 7 ; a s e l e c t i o n o f v a l u e s a t room t e m p e r a t u r e
a r e l i s t e d i n T a b l e 4. Lundberg23 has determined
t h e thermodynamic parameters a s s o c i a t e d w i t h t h e
p a r t i t i o n i n g o f e s t r a d i o l b e t w e e n o c t a n o l and w a t e r .

TABLE 4

Distribution Ratios o f Estradiol17

So 1v e n t s y s t e m L
Benzene/Wat e r cc
Benzene/l.54M H y d r o c h l o r i c a c i d #:
Benzene/O.lOM Sodium h y d r o x i d e 0.23
Benzene/l.OM Sodium h y d r o x i d e 0.04
Benzene:petroleum e t h e r , l:l/Water 24
50Sv/v M e t h a n o l / C a r b o n t e t r a c h l o r i d e 2.10
Water/Carbon t e t r a c h l o r i d e 0.08
 ESTRADIOL 297
D i e t h y l ether/Water 55
D i e t h y l ether/l.6M Hydrochloric a c i d 50
D i e t h y l ether/O.lOM Sodium h y d r o x i d e 2.0
D i e t h y l e t h e r / l . O M Sodium h y d r o x i d e 0.7
E t h y l acetate/Water 28
Hexane/Water 1.07
Petroleum e t h e r (35-6O0)/Water 0.79

4.7 Crystal Properties

E s t r a d i o l e x h i b i t s a v a r i e t y o f s o l i d - s t a t e phases
and t r a n s f o r m a t i o n s , w h i c h h a s l e d t o a d e q r e e o f
c o n f u s i o n i n t h e somewhat f r a g m e n t e d l i t e r a t u r e ,
e.g. d i f f e r e n t I R s p e c t r a i n r e f e r e n c e w o r k s ( S e c t .
4.1). This section attempts t o put the c r y s t a l
properties o f estradiol into perspective by
p r e s e n t i n g a r e s u m e o f p u b l i s h e d w o r k , some o f w h i c h
i s reinterpreted i n the l i g h t o f a close comparison
o f d a t a a n d some u n p u b l i s h e d r e s u l t s .
The m o s t n o t e w o r t h y p r o p e r t y o f e s t r a d i o l i s i t s
t e n d e n c y t o a d o p t t h e h e m i h y d r a t e d form, i n which
phase i t c r y s t a l l i s e s f r o m n o t o n l y p a r t i a l l y
aqueous s o l u t i o n s b u t a l s o f r o m e t h 1 a c e t a t e l o ,
chloroform12, absolute e t h a n o l 2 5 9 r7 and o t h e r
a p p a r e n t l y anhydrous solvents38. In ignorance o f
' t h i s c h a r a c t e r i s t i c some c r y s t a l l o g r a p h i c d a t a h a s
b e e n e r r o n e o u s l y a s c r i b e d , e.g. t h e X-ray powder
d i f f r a c t i o n d a t a o f P a r s o n s a n d B e h e r 3 9 ( T a b l e 51,
c i t e d i n t h e J o i n t Committee o n Powder D i f f r a c t i o n
Standards File, properly refers to estradiol
hemihydrate, similarly the 'anhydrous' single
c r y s t a l a n a l y s i s r e p o r t e d b y N o r t o n e t a140., and
t h e ' m o n o h y d r a t e ' d a t a c i t e d i n S t r u c t u r e ReportsLL1
are erroneous.

TABLE 5

X-ray Powder D i f f r a c t i o n D a t a f o r E s t r a d i o l
H e m i h ~ d r a t e ~ ~

d/i
- 1/10 -
d/i 1/10

7.50 4 2.90 2
6.71 9 2.80 4
6.03 4 2.65 1
5.64 10" 2.56 2
5.00 4 2.49 4
4.78 9 2.40 2
4.63 4 2.32 1
298 EUGENE G . SALOLE
4.32 2.27
4.08 2.21
3.92 2.14
3.72 2.08
3.35 2.00
3.24 1.93
3.13 1.86
3.02 1.79

* These d a t a were o b t a i n e d by v i s u a l a n a l y s i s o f
Debye-Scherrer photographs; t h e d i f f r a c t o m e t e r c u r v e
p r e s e n t e d b y R e s e t e r i t s e t a l Z 5 . ,o i n d i c a t e s a
d o u b l e t ( s t r o n g p e a k s a t 5.64 a n d 5.69A).

The crystallo raphic parameters o f estradiol

h e m i h y d r a t e a r e 37 :

a = 12.055 2 0.003
-
5 = 19.280 f 0.003
c 6.632 f
-
7 = 4 I,
= 1.21q cm-3
cm-3
I
I~ 1.209
orthorhombic, space group P21212. -
The w a t e r m o l e c u l e s a r e l o c a t e d on t h e l a t t i c e
b i n a r y axis, i n a s s o c i a t i o n with t h e D-rings o f
s t e r o i d a l molecules packed 'head-to-tail', and
p a r t i c i p a t e i n the hydrogen b o n d i n g which s u p p o r t s
the lattice. The d i f f e r e n t i a l t h e r m a l a n a l y s i s
(DTA) c u r v e o f e s t r a d i o l h e m i h y d r a t e e x h i b i t s
e n d o t h e r m i c p e a k s b e g i n n i n q a t 112 a n d 174'C p r i o r
t o t h e m e l t i n g e n d o t h e r m a t 179°C ( F i g . 6a)42;
s i m i l a r c u r v e s have been o b t a i n e d u s i n q d i f f e r e n t i a l
s c a n n i n g c a l o r i m e t r y (DSC)25,38,43. Simultaneous
e f f l u e n t g a s a n a l y s i s o f DSC s p e c i m e n s i n d i c a t e d
t h a t t h e t w o p r e - m e l t i n q endotherms were a s s o c i a t e d
with solvent 1 0 ~ ~ 3 8 ~ 4 3 T h. e r m o g r a v i m e t r i c a n a l y s i s
was i n c o n c l u s i v e ( d u e t o t h e s m a l l m a s s c h a n g e s
i n v o l v e d ) , b u t K a r l F i s c h e r t i t r a t i o n o f a sample
w h i c h had b e e n m a i n t a i n e d f o r I h a t 148'C ( i . e .
a b o v e t h e t e m p e r a t u r e o f t h e f i r s t DTA p e a k , w h i c h
was a b o l i s h e d ) i n d i c a t e d 1.8Xw/w r e s i d u a l m o i s t u r e
(cf. 3.5#W/w initially). Thermomicroscopic
examination o f hemihydrated c r y s t a l s had suggested
t h a t some s t r u c t u r a l r e a r r a n q e m e n t o c c u r r e d p r i o r t o
melting30; the pre-melting endotherm-exotherm
d o u b l e t on DTA ( F i g . 6 b ) a n d DSC25,43 curves
o b t a i n e d a t l o w h e a t i n g r a t e s would s u p p o r t t h i s
 I

l-
a

I 1 1 1 1 1 1 L
80 120 160 2 O O 0 C 160 190 O C

Fig. 6. DTA curve of estradiol hemihydrate at ( a ) loo C. min.-l and ( b ) 2 O C. min.-l heating
rate: Stanton Redcroft 671B analyser, alumina reference, 8 mg. samples, open cups,
static ambient atmosphere.
300 EUGENE G . SALOLE
view. I t appears t h e n t h a t e s t r a d i o l hemihydrate
d e s o l v a t e s i n t w o s t a g e s , a t a b o u t 1 1 2 a n d 174'C,
t h e complete l o s s o f l a t t i c e water r e s u l t i n g i n
s i m u l t a n e o u s t r a n s f o r m a t i o n t o an a n h y d r o u s p h a s e .
Smakula e t a l l 0 . , f i r s t reported t h e apparent
polymorphism o f e s t r a d i o l ; on t h e b a s i s o f X-ray
powder diffraction and I R spectroscopy they
i d e n t i f i e d four c r y s t a l l i n e m o d i f i c a t i o n s (Forms A
t o 0) a n d a n a m o r p h o u s , g l a s s y p h a s e , a l l o f w h i c h
t r a n s f o r m e d when s u b j e c t e d t o h e a t a n d / o r g r i n d i n g ,
a s s u m m a r i s e d i n t h e f o l l o w i n g scheme:

Form B b
-. Form C 4,- Form D
(m.p. 178-9'C) 0
\
\ /

11
\ I /
\
\ heating -
L r// grinding ---
Amorphous f o r m - - -b Form A

Independently, Kuhnert-Brandstatter44 concluded

(from the thermomicroscopic examination o f melts,
l a r q e l y ) t h a t e s t r a d i o l was d i m o r p h i c : Form I 1 (m.p,
169°C) b e i n g t h e u n s t a b l e monotrope o f Form I (m.p,
178"C), t h e two m o d i f i c a t i o n s d i f f e r i n g i n h a b i t ,
b i r e f r i n g e n c e and I R s p e c t r u m 4 5 . Close s c r u t i n y o f
t h e r e p o r t s shows t h a t Form A o f S m a k u l a e t a l . , i s
i n f a c t e s t r a d i o l h e m i h y d r a t e and Forms C and D
correspond t o Kuhnert-Brandstatter and c o l l e a g u e s '
F o r m s I a n d 11. It appears then t h a t anhydrous
e s t r a d i o l i s d i m o r p h i c and t h a t i n t h e p r o c e s s o f
d e s o l v a t i o n t h e hemihydrate transforms t o anhydrous
F o r m I, t h e a p p r o x i m a t e i n t e r p l a n a r - s p a c e s a n d
corresponding r e l a t i v e i n t e n s i t i e s (extrapolated
f r o m t h e X - r a y powder d i f f r a c t o m e t e r c u r v e p r e s e n t e d
b y R e s e t a r i t s e t al25., f o r a dehydrated sample) o f
which are:

7.63 3 4.23 3
6.44 5 4.06 3
5.77 10 4.01 3
5.55 4 3.88 1
5.26 3 3.71 1
4.86 4 3.32 2.
4.62 2

As n o t e d a b o v e , g r i n d i n g transformed t h e anhydrous
E STRADIOL 30 1
m o d i f i c a t i o n s o f e s t r a d i o l t o the hemihydrate, which
i s i t s e l f a f f e c t e d by comminution. Grinding
c r y s t a l l i n e e s t r a d i o l hemihydrate r e s u l t e d i n no
change i n I R spectrum, b u t t h e D T A c u r v e e x h i b i t e d
o n l y a s i n g l e , e n l a r g e d , p r e - m e l t i n g peak a t about
lZO'C42; s i m i l a r changes i n DSC c u r v e s were r e p o r t e d
f o r samples which had been m i l l e d ( t h e s e a l s o
exhibited diffuse X-ray powder diffraction
p a t t e r n s ) 4 3 o r o b t a i n e d by r a p i d p r e c i p i t a t i o n f r o m
ethanolZ5. These changes suggest t h a t comminution
can s t r u c t u r a l l y deform t h e c r y s t a l l i n e h e m i h y d r a t e
t o t h e e x t e n t t h a t d e h y d r a t i o n and s i m u l t a n e o u s
t r a n s f o r m a t i o n t o a n h y d r o u s Form I a r e f a c i l i t a t e d ,
o c c u r r i n g a t a t e m p e r a t u r e a b o u t 60'C l o w e r t h a n
usual. ( I t has been noted that commercial
' m i c r o n i z e d ' e s t r a d i o l a p p a r e n t l y v a r i e s i n degree
o f c r y s t a l l i n i t y , some b a t c h e s , e v e n f r o m t h e s a m e
s u p p l i e r , e x h i b i t i n g 'deformed' DTA c u r v e s . )
E s t r a d i o l a l s o forms solvates with organic
solvents: a hemisolvate w i t h methanol38 and a
m o n o s o l v a t e with ethanolz5, both o f which desolvate
( a t 1 5 5 a n d 1 1 9 " C , r e s p e c t i v e l y ) t o F o r m 138. The
c r y s t a l l o g r a p h i c parameters o f the monosolvate w i t h
p r o p a n o l are46:

-a = 12.215i
-
-c = 26
4 *. 62 75 1 !
z- = 4 space group -
P212121.

The p r e s e n c e o f p r o p a n o l i n t h e l a t t i c e was f o u n d t o
only very s l i g h t l y perturb the conformation o f
estradiol molecules46, which may explain the
a p p a r e n t f a c i l i t y w i t h w h i c h some o r g a n i c s o l v e n t s
s u b s t i t u t e f o r water o f c r y s t a l l i s a t i o n .
Estradiol crystals precipitate i n a variety o f
h a b i t s (Table 6); i n view o f i t s tendency t o
incorporate solvent o f c r y s t a l l i s a t i o n , these very
p r o b a b l y r e p r e s e n t s o l v a t e d forms, t h e hemihydrate
i n particular.

TABLE 6

E s t r a d i o l C r v s t a l Habits26947

Habit Crystallisation solvent

Amorphous Acetone, benzene,

dioxane, ether.
302 EUGENE G. SALOLE
Bladed Dioxane, e t h a n o l ,
aq.ethano1, e t h e r ,
tetrahydrofuran.
Platy Chloroform,
dichloromethane,
hexanol, isopropanol,

P r isma t i c
methanol .
Benzene, c h l o r o b e n z e n e ,
methanol.

The o u t s t a n d i n g s o l i d - s t a t e c h a r a c t e r i s t i c o f
estradiol i s the tenacity with which i t adopts the
h e m i h y d r a t e form. I n t h e experience o f t h i s author
( a n d other^^^,^^) c o m m e r c i a l s a m p l e s a r e i n v a r i a b l y
composed o f t h i s p h a s e a n d , a s d e s c r i b e d a b o v e , i t
has been demonstrated t h a t water i s s e l e c t i v e l y
incorporated i n t o the l a t t i c e when estradiol
crystallises from solution, that anhydrous
c r y s t a l l i n e and amorphous phases t r a n s f o r m t o t h e
h e m i h y d r a t e ( i t was i n t e r e s t i n q t o n o t e t h a t t h e D T A
c u r v e o f s h a v i n g s from t h e s u r f a c e o f a p r o p r i e t a r y
i m p l a n t p r e p a r e d b y f u s i o n was t y p i c a l o f t h e
h e m i h y d r a t e ) and t h a t d e h y d r a t i o n i s d i f f i c u l t ,
u s u a l l y o c c u r r i n q c o m p l e t e l y o n l y a t temperatures
close t o melting. C l e a r l y t h e presence o f water
molecules i s a crucial requirement for the
crystallographic stability of estradiol under
normal conditions; on t h i s b a s i s i t has been
s u g g e s t e d t h a t w a t e r may e v e n p l a y a s i g n i f i c a n t
part i n the hormone's interactions with i t s
receptors48. However, p r o p e r c o g n i s a n c e o f t h i s
c h a r a c t e r i s t i c has n o t been taken by t h e major
p h a r m a c e u t i c a l c o m p e n d i a , a l t h o u g h t h e NF X I V 4 9
s t a t e d t h a t e s t r a d i o l i s ' h y g r o s c o p i c ' and t h e USP
X X I 5 0 i m p o s e s a 3.5% l i m i t o n w e i q h t l o s s o n d r y i n q
a t 105'C ( a t w h i c h t e m p e r a t u r e t h e r e may n o t b e a n y
d i s c e r n a b l e l o s s 3 8 o r s u b s e q u e n t a l t e r a t i o n i n DTA
curve). The e v i d e n c e s u m m a r i s e d h e r e p r e s e n t s a
s u b s t a n t i a l case f o r t h e m o l e c u l a r formula and
r e l a t i v e m o l e c u l a r mass o f e s t r a d i o l t o b e p r o p e r l y
acknowledged as :

5. Methods o f A n a l y s i s

5.1 E x t r a c t i o n and P u r i f i c a t i o n
In biological fluids estradiol exists i n the 'free'
ESTRADIOL 303
f o r m and c o n j u g a t e d as s u l f a t e s and glucuronides.
B e i n g an e s t r o g e n o f r e l a t i v e l y l o w p o l a r i t y ,
e s t r a d i o l can be q u a n t i t a t i v e l y e x t r a c t e d w i t h
diethyl ether or benzene:petroleum ether, the
o r g a n i c p h a s e washed w i t h p H 1 0 . 5 c a r b o n a t e b u f f e r t o
remove non-steroidal acidic impurities, then
back-extracted i n t o 0.1M sodium h y d r o x i d e and
re-extracted into organic solvent after
a c i d i f i c a t i o n w i t h s u l f u r i c acid. I t s highly polar
c o n j u g a t e s may b e e x t r a c t e d w i t h e t h y l a c e t a t e
( a f t e r s e p a r a t i o n o f t h e u n c o n j u g a t e d f r a c t i o n ) and
hydrolysed by e i t h e r r e f l u x i n g w i t h h y d r o c h l o r i c
acid or incubation with sulfatases or
p - g l u c u r o n i d a s e enzymes4.
S e v e r a l techniques have been used t o p u r i f y
e s t r a d i o l e x t r a c t s , i n c l u d i n g p r e c i p i t a t i o n as a
complex with u r e a or d i g i t o n i n ( t h e l a t t e r i s
selective for the - e p i m e r ; c o m p l e x e s may b e c l e a v e d
I?
w i t h warm w a t e r a d p y r i d i n e , r e s p e c t i v e l y , a n d t h e
s t e r o i d e x t r a c t e d ) , c o u n t e r - c u r r e n t d i s t r i b u t i o n and
v a r i o u s c h r o m a t o g r a p h i c methods.

5.2 A b s o r p t i o n and F l u o r e s c e n c e
Spectrophotometry
S e v e r a l methods f o r t h e c o l o r i m e t r i c a n a l y s i s o f
e s t r a d i o l have b e e n d e v e l o p e d on t h e b a s i s o f t h e
Kober r e a c t i o n , where a sample i s h e a t e d w i t h a
phenol (e.g. p-naphthol) and sulfuric acid
()60Xw/v), t h e y e l l o w s o l u t i o n d i l u t e d t o an a c i d
c o n c e n t r a t i o n o f 30-50%W/v and r e - h e a t e d t o q i v e a
p i n k s o l u t i o n which absorbs maximally a t around
530nm. I n t h e more s e n s i t i v e and a c c u r a t e I t t r i c h
modification, the re-heating a f t e r d i l u t i o n i s
o m i t t e d a n d t h e c o l o u r e d complex i s e x t r a c t e d i n t o
2Xw/v p-nitrophenol i n chloroform, giving a
solution with yellow-green fluorescence. The
n a t u r a l f l u o r e s c e n c e o f e s t r a d i o l is e n h a n c e d i n
c o n c e n t r a t e d s u l f u r i c and o t h e r m i n e r a l a c i d s q 7 .
B a r t o s and Pesez51 c o m p r e h e n s i v e l y d i s c u s s t h e
c o l o r i m e t r i c and f l u o r i m e t r i c methods f o r e s t r a d i o l .

5.3 Chromatography
The e n t i r e r a n q e o f c h r o m a t o g r a p h i c t e c h n i q u e s has
been a p p l i e d t o t h e a n a l y s i s o f e s t r a d i o l ; paper
c h r o m a t o g r a p h i c s e p a r a t i o n s have been d i s c u s s e d a t
l e n g t h by Bush52 and E n q e l l 7 , and Stah153 l i s t s a
number o f t h i n - l a y e r s y s t e m s , e.g.

adsorbent - s i l i c a q e l G, 250pm l a y e r ,
304 EUGENE G. SALOLE
a c t i v a t e d a t 150'C f o r 3h;
s o l v e n t system - (a) ch1oroform:ethyl acetate,
80:20, ( b ) acetone:
,
d i c h l o r ome t h a n e 20: 8 0 ;
location - 1OO%W/w a n t i m o n y t r i c h l o r i d e
i n g l a c i a l a c e t i c a c i d spray,
d e v e l o p e d a t 95'C f o r 5 m i n t o
b r i g h t r e d s p o t s a t ( a ) Rf0.32,
( b ) Rf0.56.

The d e v e l o p m e n t o f m o d e r n c h r o m a t o g r a p h i c m e t h o d s
f o r e s t r a d i o l c o n t i n u e s apace. The p o w e r f u l
c o m b i n a t i o n o f g a s o r l i q u i d c h r o m a t o q r a p h y and mass
s p e c t r o m e t r y i s u s e d t o d e t e c t and a s s a y e s t r a d i o l
i n b i o l o g i c a l f l u i d s 5 4 , and s e v e r a l h i g h - p e r f o r m a n c e
l i q u i d chromatographic (HPLC) systems for the
steroid and i t s metabolites have been
d e v e l o p e d 5 5 , 56.

5.4 P r o t e i n - b i n d i n g Assays
A v a r i e t y o f t h e s e h i g h l y s e n s i t i v e and ( w i t h c a r e )
s p e c i f i c methods have been developed f o r e s t r a d i o l .
S e v e r a l r a d i o i m m u n o a ~ s a y s 5 ~a r e a v a i l a b l e a n d
d e v e l o p m e n t s i n n o n - i s o t o p i c t e ~ h n i q u e s ~e.g.
~ ,
c h e m i l u m i n e s c e n c e i m m u n o a ~ s a y s ~h~a ,v e made t h e m
v i a b l e a l t e r n a t i v e s f o r r o u t i n e analyses.

5.5 Bioassay
Initially, e s t r a d i o l i n b i o l o g i c a l f l u i d s was
assayed by b i o l o g i c a l m e t h o d s based o n changes i n
the vaginal epithelium or uterine mass of
oophorectomized rodents60. Although often hiqhly
s e n s i t i v e and s p e c i f i c (even i n t h e presence o f
i m p u r i t i e s ) , t h e precautions necessary f o r p r e c i s i o n
make t h e m e x p e n s i v e a n d l a b o r i o u s a n d t h e y a r e
therefore now consigned to validating the
p h y s i c o c h e m i c a l methods which have superseded.
Modern c y t o c h e m i c a l bioassays61, though s e n s i t i v e
and e l e g a n t , have a l s o n o t been a d o p t e d f o r r o u t i n e
use.

6. Stability

E s t r a d i o l c a n w i t h s t a n d b o i l i n g i n d i l u t e a c i d s and
a l k a l i , and s o l i d m a t e r i a l r e m a i n s c h e m i c a l l y s t a b l e
for at least five years under temperate
conditions6*. However, i t has been r e p o r t e d t h a t
e s t r a d i o l a p p l i e d t o s i l i c a g e l TLC p l a t e s a n d
exposed to the atmosphere for Ih underwent
ESTRADIOL 305
s u b s t a n t i a l decomposition, b u t n o t p r i m a r i l y due t o
o x i d a t ion63.

7. M e t a b o l i s m , P h a r m a c o k i n e t i c s and
Bioavailability

E s t r a d i o l is b i o s y n t h e s i s e d i n n o r m a l p r e - m e n o p a u s a l
women p r i m a r i l y b y c o m p o n e n t s o f t h e o v a r i e s ( i . e .
f o l l i c l e s , c o r p u s l u t e u m and s t r o m a ) , f o l l o w i n q t h e
classical route for s t e r o i d s from acetate through
cholesterol, pregnenolone and a n d r o s t e n e d i o n e
(Fig. 7). S e c r e t i o n v a r i e s w i t h t h e phase o f t h e
m e n s t r u a l c y c l e , i s e p i s o d i c and t o a n y c t e r o h e m e r a l
r h y t h m , so p l a s m a l e v e l s f l u c t u a t e r a p i d l y ; t y p i c a l
s e c r e t i o n r a t e s d u r i n g t h e f o l l i c u l a r , m i d c y c l e and
l u t e a l p h a s e s a r e 8 0 , 4 0 0 a n d 200pq d - 1 , p r o v i d i n q
t o t a l e s t r a d i o l plasma l e v e l s o f about 6 , 30 and
15ng d l - l , r e ~ p e c t i v e l y ~ ~O.n l y 1-37; o f ' f r e e '
( i . e . unconjugated) hormone c i r c u l a t e s unbound t o
protein, about 40% b e i n g bound t o sex hormone
bindin g l o b u l i n a n d 6 0 % t o a l b u m i n (K=6.4x108 and
1.8x1O2 M - I , r e s p e c t i ~ e l y ) ~ ~ T h. e e x t r a g l a n d u l a r
( p r i n c i p a l l y i n the l i v e r , adipose t i s s u e and s k i n )
a r o m a t i s a t i o n o f androgens i s v i r t u a l l y t h e s o l e
s o u r c e o f e s t r a d i o l i n p o s t - m e n o p a u s a l women a n d
a c c o u n t s f o r a b o u t 70% o f t h e s t e r o i d i n males64.
The p r o d u c t i o n o f e s t r a d i o l b y t h e f e t o p l a c e n t a l
u n i t d u r i n g pregnancy h a s been r e v i e w e d 4 .
A m u l t i p l i c i t y o f transformations are involved i n
the catabolism o f estradiol, estrone (the subject o f
a recent Analytical Profile66) beinq a principal
metabolite (Fig. 8). I n humans, e s t r a d i o l is
e x c r e t e d m a i n l y i n u r i n e as g l u c u r o n i d e and s u l f a t e
conjugate^^^, i n w h i c h f o r m s i t a l s o u n d e r g o e s
e n t e r o h e p a t i c r e c i r c ~ l a t i o n s~o ~ t h a t a l t h o u g h
2 3 - 6 8 % o f a d o s e may b e r e c o v e r e d f r o m b i l e a s
conjugates (mostly sulfoqlucuronides), these are
normally hydrolysed by mucosal enzymes and
i n t e s t i n a l f l o r a ( m a i n l y i n t h e l o w e r i l e u m and
u p p e r c o l o n ) and t h e f r e e s t e r o i d m o s t l y r e a b s o r b e d ,
l e a v i n g o n l y 10-30% t o be e x c r e t e d i n faeces ( c f .
50-80% as conjugates i n urine). Due t o
enterohepatic c y c l i n g the complete elimination o f
e s t r a d i o l i s delayed for 3-6d; the metabolic
c l e a r a n c e r a t e i n women i s t y p i c a l l y 1 3 0 0 1 d - l 64.
The d e c l i n e i n b l o o d l e v e l s a f t e r i n t r a v e n o u s
i n f u s i o n i n d i c a t e d t h a t e l i m i n a t i o n was b i p h a s i c ,
w i t h a h a l f - l i f e o f 20 and 70min68; however t h e
r e s u l t s a f t e r b o l u s i n j e c t i o n showed t h a t t h e c u r v e
306 EUGENE G. SALOLE

7%

A4 pathway A5 pathway
I40

y 3 1 p r egnenolone

C=O

no ~.
17a-hydroxypregnenole

17a- hydroxyprogesterooe
no&
dehydroeplandrosterone

oestradiol

Fig. 7 Ovarian b i o s y n t h e s i s o f e s t r a d i o l
64
.
cH30no
& '
2-methox yoestrone

2-hydroxyoestrone

4 -hydroryot?slfone im
Ga-hydroxyOBJtm

Fig. 8 Metabolism o f estradiol 6 4 .

308 EUGENE G. SALOLE
was b e s t d e s c r i b e d b y t h e sum o f t h r e e e x p o n e n t i a l s ,
t h e i n i t i a l v o l u m e o f d i s t r i b u t i o n b e i n g 1 0 . 9 ( * 1.1
S E M I 169.
To e l i c i t a c l i n i c a l r e s p o n s e i n r e p l a c e m e n t
therapy ( a s s o c i a t e d w i t h plasma l e v e l s i n t h e
p r e - m e n o p a u s a l f o l l i c u l a r phase range, c f . I n g d l - 1
post-menopausally64) , e s t r a d i o l administered o r a l l y
must be f i n e l y - d i v i d e d , i.e. w i t h an a v e r a g e
p a r t i c l e s i z e o f o n l y a f e w pm70. However, a l t h o u q h
capsules, aqueous suspensions and t a b l e t s o f
m i c r o n i z e d m a t e r i a l ma be absorbed as r a p i d l y as
ethanolic solutions71-~3, the bioavailability o f
e s t r a d i o l by t h e o r a l r o u t e i s v a r i a b l e ( w i t h plasma
c o n c e n t r a t i o n s p e a k i n g a t 0.5-8h), incomplete (due
t o e x t e n s i v e i n t e s t i n a l and h e p a t i c m e t a b o l i s m ) a n d
affected by diet and medication74. Oral
a d m i n i s t r a t i o n a l s o r e s u l t s i n a s u b s t a n t i a l and
sustained increase i n c i r c u l a t i n g l e v e l s o f estrone,
an u n d e s i r a b l e f e a t u r e because o f t h e apparent
association with endometrial carcinoma75.
Notwithstanding these disadvantages, t h e o r a l r o u t e
remains c l i n i c a l l y useful.
The i n t r a n a s a l i n s t i l l a t i o n o f a n e s t r a d i o l
suspension i n s a l i n e r e s u l t e d i n r a p i d a b s o r p t i o n ,
b u t t h e r i s e i n p l a s m a l e v e l s was s h o r t - l i v e d a n d
accompanied by an i n c r e a s e i n c i r c u l a t i n g e s t r o n e ,
suggesting local catabolism76. Similarly,
a b s o r p t i o n f r o m t a b l e t s p l a c e d s u b l i n g u a l l y was
rapid, a 0.5mg dose producing serum levels
e q u i v a l e n t t o 2mg o r a l l y , b u t a g a i n e s t r o n e l e v e l s
were r a i s e d , p e r h a p s due t o c a t a b o l i s m w i t h i n the
r e ticuloendoth e l i a l ~ y s t e m ~ ~ - ~ ~ .
E s t r a d i o l i s w e l l absorbed from s o l i d p e l l e t s
p r e p a r e d by f u s i o n and i m p l a n t e d s u b c u t a n e o u s l y i n t o
t h e abdominal w a l l or buttocks, and has been
administered i n this form since the late
1 9 3 0 ' ~ ~ ~ , * P~l a.s m a l e v e l s c a n i n c r e a s e b y 5 0 %
w i t h i n an h o u r o f i m p l a n t a t i o n E 2 and r e m a i n i n t h e
pre-menopausal r a n g e f o r s e v e r a l months83,84 w i t h o u t
large increases i n c i r c u l a t i n g estrone occurring
concomitantly, since first-pass metabolism i s
avoided. However, a l t h o u g h e s t r a d i o l i m p l a n t s a r e
w e l l t o l e r a t e d ( c f . progesterone, implants o f which
a r e e x p e l l e d o r form s t e r i l e abscesses i n a b o u t 20%
o f p a t i e n t s ) and can remain c l i n i c a l l y e f f e c t i v e f o r
over three years85, inter-patient variation in
a b s o r p t i o n i s s u b s t a n t i a l E 3 and t h e occasionally
f a i l t o p r o d u c e a s u s t a i n e d response52,84
due t o e n c a p s u l a t i o n i n f i b r o u s tissue8°,8 I, perhaps
since
ESTRADIOL 309

sometimes i m p l a n t s e x c i s e d a f t e r s e v e r a l months
appear i n p r i s t i n e c o n d i t i o n 8 z ) .
The v a g i n a l r o u t e o f a d m i n i s t r a t i o n h a s b e e n
a d v o c a t e d as t h e most a p p r o p r i a t e f o r replacement
therapy because, apart from beneficial local
e f f e c t s , s y s t e m i c a b s o r p t i o n occurs within minutes
and, s i n c e t h e p o r t a l c i r c u l a t i o n i s bypassed,
l e v e l s o f estrone a r e r e l a t i v e l y low. However,
d e s p i t e e a r l y work on m i c e showing t h a t e s t r a d i o l
was an order of magnitude more potent when
a d m i n i s t e r e d i n t r a v a g i n a l l y i n aqueous g l y c e r i n t h a n
i n o l i v e o i 1 8 6 , d e l i v e r y by t h i s r o u t e has y e t t o be
optimised: n e v e r t h e l e s s a v a r i e t y o f f o r m u l a t i o n s
have been used, i n c l u d i n g s a l i n e s ~ s p e n s i o n s ~ ~ , ~ 7 ,
creams88 polysiloxane rings89, hydrophilic
pessariesbO and even o r a l t a b l e t s 9 ' .
E s t r a d i o l is r e a d i l y a b s o r b e d t h r o u q h t h e s k i n ;
some p a r a m e t e r s m e a s u r e d i n v i t r o a r e 9 z ; 9 3 :

permeability constant - 106k,/cm h-'

h y d r a t e d whole s k i n ' 3888
hydrated dermis 55080
h y d r a t e d s t r a t u m corneum 300
distribution ratio -
s t r a t u m corneum/water 46.

Despite the mediocre partition coefficient,

e s t r a d i o l p e n e t r a t e s t h e s t r a t u m corneum w i t h i n
minutes but is only slowly, variably and
i n c o m p l e t e l y absorbed i n t o t h e systemic c i r c u l a t i o n
a f t e r t o p i c a l a p p l i ~ a t i o n ~ 4 , ~T ~h i. s is b e c a u s e t h e
percutaneous absorption o f e s t r a d i o l i s h i g h l y
dependent on t h e v e h i c l e z 7 (perhaps also the s i t e
a n d mode o f a p p l i c a t i o n ) a n d i t i s t o s o m e d e g r e e
c a t a b o l i s e d by t h e s k i n 9 6 . Nevertheless, although
maximum p l a s m a l e v e l s a r e a c h i e v e d o n l y a f t e r
several hours, they are sustained (perhaps f o r days)
due t o t h e ' r e s e r v o i r e f f e c t ' o f t h e s t r a t u m corneum
and l o c a l r e t e n t i o n i n u n d e r l y i n g dermal t i s s u e ,
with t h e a d d i t i o n a l advantage o f c i r c u l a t i n q e s t r o n e
b e i n g maintained a t low, pre-menopausal l e v e l s .
Replacement t h e r a p y has been s u c c e s s f u l l y a c h i e v e d
by the topical application o f estradiol in
hydro-aldoholic e1s94,95 and transdermal
therapeutic system^^^,^^.
8. D e t e r m i n a t i o n i n Body F l u i d s

The e n t i r e a n a l y t i c a l a r m a m e n t a r i u m is a p p l i e d t o
310 EUGENE G. SALOLE
t h e a n a l y s i s o f e s t r a d i o l i n body f l u i d s and
t i s s u e s , and more s p e c i f i c m e t h o d s a r e c o n t i n u a l l y
being developed. C o l o r i m e t r i c and f l u o r i m e t r i c
methods based o n t h e K o b e r r e a c t i o n r e m a i n u s e f u l
f o r r o u t i n e assay o f t h e r e l a t i v e l y l a r g e amounts o f
e s t r a d i o l i n u r i n e 9 9 , b u t f o r b l o o d and o t h e r f l u i d s
more s p e c i f i c and s e n s i t i v e t e c h n i q u e s a r e a p p l i e d ,
n o t a b l y radioimmunoassay, w h i c h has been a p p l i e d t o
p l a s m a l o o , s a l i v a 1 Ol a n d f a e c e s l o 2 , a n d HPLC55956.
A l t h o u g h gas c h r o m a t o g r a p h y - m a s s s p e c t r o m e t r y h a s
b e e n u s e d t o a s s a y b o d y f l u i d s , e.g. semenqo3, b e i n g
the definitive i t i s usually reserved
f o r s p e c i a l circumstances. The r e v i e w b y B u s h 1 0 5 i s
recommended a s a n i n t r o d u c t i o n t o t h e c o m p l e x a n d
delicate business o f e s t r o g e n e x t r a c t i o n and
analysis.

9. Determination i n Pharmaceuticals

P h y s i c o c h e m i c a l methods a r e u s u a l l y chosen f o r t h e
a n a l y s i s o f e s t r a d i o l i n medicines. Thin-layer
chromatography remains useful for routine
i d e n t i f i c a t i o n , as i n s t a b i l i t y t e s t i n q o f s o l i d
dosage forms62. Creams h a v e b e e n a s s a y e d b y q a s
c h r o m a t o g r a p h y l o 6 and t h e h i g h l y f l u o r e s c e n t d a n s y l
d e r i v a t i v e o f e s t r a d i o l has been used t o a n a l y s e
s o l i d a n d p a r e n t e r a l f o r m u l a t i o n s d i r e c t l y l 0 7 and
a f t e r HPLC s e p a r a t i o n l o f l . HPLC w i t h a n o v e l
light-scatterin
.d e t e c t o r may f i n d a p p l i c a t i o n t o
p h a r m a c e u t i c a l s ?09

Acknowledgements

The a u t h o r t h a n k s D r . P e t e r B l a d o n , Department o f
P u r e and A p p l i e d C h e m i s t r y , f o r t h e NMR and mass
s p e c t r a a n d D r . P e t e r G. W a t e r m a n , D e p a r t m e n t o f
Pharmacy, f o r a s s i s t a n c e w i t h t h e i r i n t e r p r e t a t i o n .
The a u t h o r i s p a r t i c u l a r l y g r a t e f u l t o Mrs. Maureen
R e i d a n d Mrs. E l i z a b e t h C a r r u t h e r s , D e p a r t m e n t o f
Pharmacy, for photographing the figures and
painstakingly typing the manuscript, respectively.

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 GUANABENZ ACETATE

Charles M. Shearer

1. Description
1.1 Name, Formula, Molecular Weight
1 . 2 Appearance, Color, Odor
2 . Physical Properties
2.1 Infrared Spectrum
2 . 2 Nuclear Magnetic Resonance Spectrum
2 . 3 Ultraviolet Spectra
2 . 4 Mass Spectrum
2 . 5 Melting Range
2 . 6 Differential Scanning Calorimetry
2.7 Solubility
2 . 8 Crystal Properties
2 . 9 Dissociation Constants
2 . 1 0 Electrochemical Properties
3 . Synthesis
4 . Stability and Degradation
5 . Metabolism and Pharmacokinetics
5.1 Metabolism
5 . 2 Pharmacokinetics
6. Identity
7 . Methods of Analysis
7.1 Elemental Analysis
7 . 2 Phase Solubility Analysis
7.3 Direct Spectrophotometric Analysis
7 . 4 Titrimetric Analysis
7.5 Chromatographic Analysis
8. References

ANALYTICAL PROFILES OF DRUG SUBSTANCES Copyright 0 1986

VOLUME 15 by the American Pharmaceutical Association
319 All rights of reproduction in any form reserved.
320 CHARLES M. SHEARER

1. Description
1.1 Name, Formula, Molecular Weight
The name used by Chemical Abstracts for guanabenz
is hydrazinecarboximidamide, 2-[(2,6-dichlorophenyl)meth-
ylene]. It is also called (2,6-dichlorobenzylidene)amino-
guanidine and N-(2,6-dichlorobenzylidene)-N'aminohydraz~ne
( 1 ) . The drug code number (1) is NSC-68982 and the Chemi-
cal Abstracts Registry number is 5051-62-7 for guanabenz
and 23256-50-0 for guanabenz acetate. This compound can
exist (2) as the E-isomer (CAS-60329-04-6) or the Z-
isomer (CAS:60329-05-7). Guanabenz acetate is the E-
isomer.

c1OH 12N402C 2 Mol. Wt. = 291.14

1.2 Appearance, Color and Odor

Guanabenz acetate is'a white to off-white
practically odorless crystalline powder.

2. Physical Properties
2.1 Infrared Spectrum
An infrared absorption spectrum of a potassium
bromide dispersion of guanabenz acetate (Wyeth Reference
Lot C-12258) is presented as Figure 1. The spectral band
assignments are listed in Table I.

Table I

IR Spectral Band Assignments

Wave number (cm-l) Vibration Mode

3400 NH2 stretch
3000 to 2600 protonated nitrogen stretch
1685 C=N stretch
1600 NH' and C=C stretch
1600 and 1395 COO- stretch
780 three adjacent ring
hydrogen deformations
 WAVELENGTH (MICRONS1
2.5 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10 12 16 20 25 30 35 4045
100

80

Y
V
5 60
E
I
z
vl
2 40
+
7
s

20

0
4000 3500 3000 2500 1800 1600 1400 1200 1000 600 4w 200
FREOUENCY (CM-lI

F i g u r e 1 - I n f r a r e d Spectrum of Guanabenz Acetate,

(Wyeth Reference S t a n d a r d , L o t C-12258) KBr p e l l e t
322 CHARLES M . SHEARER

The UV absorptivities found for guanabenz

acetate (Wyeth Reference Standard, Lot C-12258) are given
in Table 111.

Table I11

Ultraviolet Spectra Characteristics

Solvent Max (nm) Absorptivity

0.1N HC1 270 44.5
0.1N NaOH 295 43.4
absolute ethanol 312 45.2

2.4 Mass Spectrum

The mass spectrum of guanabenz acetate (Wyeth
Reference Standard, Lot C-12258) was obtained (5) by
direct injection of the sample into a MS-25 spectrometer.
The ionizing beam energy was 70 eV. Figure 5 is a bar
graph of the mass spectrum with the molecular ion at m/e
230. Identification of the pertinent masses is presented
in Table IV.

Table IV

Mass Spectrum Fragmentation Pattern

m/e
- Species
230 M+
195 M+ - C1
153 M+ - C1 - CN2H2
123 M+ - C1 - CNqHq

2.5 Melting Range

Wyeth Reference Standard, Lot C-12258 of guana-
benz acetate melts at 188 - 190°C (dec) by USP Class 1
( 6 ) . The Merck Index ( 1 ) reports a value of 192.5OC
(dec).
GUANABENZ ACETATE 323

2.2 Nuclear Magnetic Resonance Spectrum

The nuclear magnetic resonance spectrum sample
(Wyeth Reference Standard, Lot C-12258) was prepared in
deuterated dimethylsulfoxide containing tetramethylsilane
as internal reference ( 3 ) . The spectrum was obtained on
a 300 MHz Varian XL-300 spectrometer and is presented as
Figure 2 . The spectral assignments are listed in Table
11.

Table I1

NMR Spectral Assignments

Ass igned Chemical Number of

Proton Shift (ppm) Type Protons
02CCH3 1.88 Singlet 3
Aromatic 7 . 2 5 to 7 . 6 0 Multiplt-t 3
Methine 8.26 Singlet 1
exchangeable 6 . 0 0 to 9 . 5 Broad
OH

2.3 Ultraviolet Spectra

The ultraviolet spectra of the neutral guanabenz
species (in 0.1N NaOH) and the protonated species (in
0.1N HC1) are presented in Figure 3 .
In nonaqueous solvents such as methanol or abso-
lute ethanol (see Figure 4 ) guanabenz forms another taut-
omer and the spectrum is considerably changed ( 4 ) . This
tautomer is the more conjugated species with the
following structure.

Cf H
I I

It was demonstrated that the ultraviolet spectra

of an N-methylated guanabenz, which could not undergo
such a tautomeric shift, had a maximum in either water or
methanol near that shown by guanabenz in water.
I a 1 I I 1
10 9 8 7 6 5 4 3 2 1 0
PPm

Figure 2 - NMR Spectrum of Guanabenz Acetate

(Wyeth Reference Standard, Lot C-12258) in
deuterated dimethylsulfoxide
 0.7 -

0.6 -

0.5 -
W

Figure 3 - U l t r a v i o l e t S p e c t r a of Guanabenz A c e t a t e (Wyeth

Reference Standard, Lot C-12258) i n (A) 0 . 1 N
NaOH and ( B ) 0 . 1 N H C 1
0.8

0.71

0.6

0.5 -
8
z
a
2 0.4-
0
VI
M
a
0.3 -

0.2 -

0.1 -

0.0; 1 1 I I I I , I , I , I , , , , , , ,
Figure 5 - Mass Spectrum of Guanabenz Acetate
(Wyeth Reference Standard, Lot C-12258)
GUANABENZ ACETATE 327

2.6 Differential Scanning Calorimetry

The DSC thermogram of guanabenz acetate (Wyeth
Reference Standard, Lot C-12258) is shown in Figure 6.
The thermogram was obtained (7) at a heating rate of
10°C/min using a Perkin-Elmer DSC-2. The thermogram
shows no endotherm or exotherm other than that associated
with the decomposition melt.
2.7 Solubilit
d i n g approximate solubilities at room
temperature have been reported.
Solvent Solubility (mg/mL) Reference
Water 11 8
Alcohol 50 8
Propylene Glycol 100 8
Chloroform 0.6 9
Ethyl Acetate 1 9

2.8 Crystal Properties

The X-ray powder diffraction pattern of guanabenz
acetate (Wyeth Reference Standard, Lot C-12258), obtained
(7) with a Phillips diffractometer using CuKq radiation
is presented as Figure 7. The calculated "d" spacings
are given in Table V.

Table V

X-Rav Powder Diffraction Pattern

d
- 1/10 d
- 1/10
-
15.0 9 41.8 8
18.5 38 42.7 26
25.6 L 43.3 5
27.7 2 b5.3 15
29.1 4 46.9 57
31.0 11 47.7 1[ I
31.3 14 51.2 10
33.1 1 I10 53.5 7
34.2 30 55.7 3
35.8 90 56.3 3
40.2 81; 58.3 10
58.6 10
 0
n
z
W
I
0
15

I I I 1 1 I 1 I I I

40 80 120 oc
160 200 240

Figure 6 - D i f f e r e n t i a l Scanning C a l o r i m e t r i c Thermogram of

Guanabenz Acetate (Wyeth Reference S t a n d a r d , Lot C-12258)
 I

,
ze
Figure 7 - X-Ray D i f f r a c t i o n P a t t e r n of Guanabenz Acetate
(Wyeth Reference S t a n d a r d , L o t C-12258)
330 CHARLES M. SHEARER

The single crystal X-ray crystallographic struc-

ture determination of guanabenz acetate indicates that
guanabenz (base) and acetic acid are hydrogen bonded and
that the entire salt lies substantially within a single
plane (10). On the basis of bond lengths and angles the
bonding for guanabenz acetate in the crystal may be rep-
resented as given below.

. .

The crystal system is orthorhombic, space group

of 2Pbca-D15 (NO. 61). The lattice constants are A =
2R
17.326(3) .f, b=13.436(2) 6: and c=10.999(2) 6: with eight
molecules per unit cell.
2.9 Dissociation Constants
The ionization constants for guanabenz acetate
were determined by potentiometric titration [method of
Parke and Davis (1111 in 40% ethanol/water to be 5.8
(acetate) and 8.1 (guanidinium). Spectrophotometric
determination in water for the guanidinium ion also gave
8.1 (12).

2.10 Electrochemical Properties

Guanabenz acetate at a concentration of 0.1
mg/mL is reduced with a half-wave potential of about
-0.9V vs SCE in 0.1M acetic acid and about -1.1V vs SCE
in 0.1M NaH2P04. The half-wave potentials and the limiting
diffusion currents are concentration dependent in these
solvents (12).
GUANABENZ ACETATE 33 1

3. Synthesis
Guanabenz can be synthesized (13) as the acetate
by the condensation reaction of 2,6-dichlorobenzalde-
hyde with aminoguanidine in the presence of acetic
acid.

4. Stability and Degradation

Guanabenz acetate can decompose by
- hydrolysis
- to
the starting materials in its synthesis, aminoguanidine
and 2,6-dichlorobenzaldehyde. It can also hydrolyze
forming 2,6-dichlorobenzaldehyde semicarbazone. These
reactions occur very slowly even under reflux in strong
acid or base (2).

It has been shown by spectral evidence that

guanabenz acetate exists in the more stable E-config-
uration (2). The Z-isomer is formed from guanabenz by
irradiation in solution with ultraviolet light (2,14).
The reaction is reversible in the presence of acid (14)
and an equilibrium between the two isomers is formed (2).

5. Metabolism and Pharmacokinetics

5.1 Metabolism
In man the major metabolite of guanabenz is
(E)-p-hydroxyguanabenz and its glucuronide. Other
identified metabolites are Z-guanabenz, (Z)-p-hydroxy-
guanabenz, 2,6-dichlorobenzyl alcohol glucuronide and
guanabenz glucuronide. These metabolites and those
reported below for studies in the Rhesus monkey were
characterized by comparison of their Rf's in thin-
layer chromatography to that of authentic sample
(15).

The following metabolites have been identified

(16) following administration of guanabenz to Rhesus
monkeys: Z-guanabenz, 2,6-dichlorobenzyl alcohol, 2,6-
dichlorobenzaldehyde, p-hydroxyguanabenz and its
glucuronide conjugate and 2,6-dichlorobenzaldehyde azine.

After oral or i.v. administration of

guanabenz acetate to rats, thin-layer chromatographic
analysis of urine and bile revealed the metabolites:
Z-guanabenz, 2,6-dichlorobenzyl alcohol, p-hydroxy-
guanabenz, 3-hydroxyguanabenz, and sulfates and/or
glucuronides of these (17).
332 CHARLES M. SHEARER

5.2 Pharmacokinetics
Meacham et al. (15) reported on the disposition
of 14C guanabenz in hypertensive patients. Maximum con-
centrations of guanabenz were reached at 2 to 5 hours
after dosing. The major route of elimination was into
urine (80%), but guanabenz itself accounted for less than
1% of the total. Kinetic parameters for guanabenz were
estimated by fitting the plasma and urine data to a 2-
compartment model.

A study similar to the one described above, but

using Rhesus monkeys was also described by Meacham et al.
(18). The results of this study, which were similar to
those obtained in human patients, indicates that the
Rhesus monkey may serve as a satisfactory model for man
in disposition studies of guanabenz.

The pharmacokinetics of guanabenz in rats were

reported by De Marchi (19) and by Yokozama (20,211.

6. Identity
Infrared spectroscopy can be used directly upon the
drug substance for its identification.

Thin-layer chromatography, ultraviolet and nuclear

magnetic resonance spectrophotometry will readily dis-
tinguish guanabenz from the 2-isomer (2).

7. Methods of Analysis
7 . 1 Elemental Analysis
The elemental analysis of guanabenz acetate
(Wyeth Reference Standard, Lot C-12258) is presented
below.
Element % Calculated % Reported (22)
C 41.26 41.00
H 4.15 4.05
N 19.24 19.34
c1 24.35 24.50
7.2 Phase Solubility Analysis
Phase solubility analysis (23) on guanabenz
acetate (Wyeth Reference Standard, Lot C-12258) using 12%
methanol in acetone as the solvent gave a purity of 100
+
- 0.4%.
GUANABENZ ACETATE 333

7.3 Direct Spectrophotometric Analysis

A stability-indicating assay by ultraviolet
spectrophotometry has been reported (2). It will assay
intact guanabenz in the presence of its degradation pro-
ducts.

7.4 Titrimetric Analysis

Guanabenz acetate can be titrated with perch-
loric acid in acetic acid after dissolving the sample
in glacial acetic acid (24,251.

7.5 Chromatographic Analysis

7.51 Thin-Layer Chromatography
The following systems using silica gel
plates have been reported:

Solvent Rf Reference
-
chloroform/methanol (10:l) 0.7 14
chloroform/methanol/
ammonium hydroxide
(48.5:50:1.5) 0.71 18
ethyl acetate/methanol/
water/ammonium hydroxide
(68:26 :4:2 ) 15
benzene/acetone/acetic
acid (70:10:20) 15
chloroform/methanol/
ammonium hydroxide
(60:40:1) 25

7.52 Gas Chromatography

Knowles et al. (26) describe a sensitive
gas chromatographic assay for guanabenz in urine and
plasma. The method involves the acid hydrolysis of
guanabenz to 2,6-dichlorobenzaldehyde which is chromato-
graphed on a 2 mm i.d. x 3 m column packed with 1%
neopentylglycol succinate on 60/80 mesh Chromosorb G
AW-DMCS at 200' and detected by an electron capture
detector.
334 CHARLES M . SHEARER

7.53 High Performance Liquid Chromatography

Guanabenz can be separated (2,271 from its
degradation products by an eluant consisting of 430 mL
methanol/570 mL distilled water13 mL phosphoric acid on a
octadecyl reverse-phase column.

7.6 Radioligand Binding Analysis

Fluck et al. (28) describe a radioligand assay
specifically for guanabenz. It is based upon its dif-
ferential binding of guanabenz and its metabolite to
cerebral4 2 receptors. It can be used in analysis of
guanabenz in plasma and urine. It will distinguish
guanabenz from the 2-isomer.

8. References
1. Merck Index, 10th ed., Merck and Co., Rahway,
N.J., (1983) page 656.

2. C.M. Shearer and N.J. DeAngelis, J. Pharm. Sci.,

-
68, 1010 (1979).

3. B. Hofmann, Wyeth Laboratories, Inc., personal

communication.

4. R. Rees, Wyeth Laboratories, Inc., personal

communication.

5. C.A. Hetzel, Wyeth Laboratories, Inc., personal

communication.

6. J.T. Stoklosa, Wyeth Laboratories, Inc., personal

communication.

7. L. Sivieri, Wyeth Laboratories, Inc., personal

communication.

8. J.M. Bissinger, Wyeth Laboratories, Inc.,

personal communication.

9. D.N. Gutekunst, Wyeth Laboratories, Inc.,

personal communication.

10. R. McCaully, Wyeth Laboratories, Inc., personal

communication.

11. T.V. Parke and W.W. Davis, Anal. Chem., -

26, 642
(1954).
GUANABENZ ACETATE 335

12. C.M. Shearer, Wyeth Laboratories, Inc.,

unpub 1ished work.

13. W.F. Bruce and T. Baum, Ger. Offen. 1802394

14. T. Tsujikawa, E. Mizuta and M. Hayashi, -
J.
Pharm. SOC. (Japan), 96, 125 (1976).

15. R.H. Meacham, M. Emmett, A.A. Kyriakopoalos,

S.T. Chiang, H.W. Ruelius, B.R. Walker, R.G.
Narins and M. Goldberg, Clin. Pharmacol. Ther.,
-
27, 44 (1980).

16. R.H. Meacham, C. Kick, H. Ruelius and

S. Sisenwine, Fed. Proc. Fed. Am. SOC. Exp. Biol.,
39, 850 (1980).
-
17. A. Mizamoto, Y. Soda, J. Mori, N. Yokozama, H.
Nobuharu, K. Horisaka, F. Shichino and H. Tatsumi,
Iyakuhin Kenkyu, -13, 1214 (1982). C.A.
98:83214v (1983).

18. R.H. Meacham, S.T. Chiang, C.J. Kick, S . F .

Sisenwine, W.J. Jusko and H.W. Ruelius, Drug
Metab. Disp., -9, 509 (1981).

19. G. DeMarchi. P. Gomarasca, E. Marmo and

113, 225
C. Scolastico, Boll. Chim. Farm., -
(1974).

20. N. Yokozama, K. Horisaka, Y. Soda, I. Mori,

H. Sakamoto, K. Ohata, A. Shimada, F. Shichino,
K. Murrai and H. Tatsumi, Iyakuhin Kenkyu, -
13,
1190 (1982). -C.A. 98:83212t (1983).

21. N. Yokozama, K. Horisaka, Y. Soda, I. Mori,

A. Shimada, F. Shichino, K. Murai and H. Tatsumi,
Iyakuhin Kenkyu, - 13, 1207 (1982). C.A.
98:83213u ( 1983).

22. M. Politowski, Wyeth Laboratories, Inc., per-

sonal communication.

23. E. DiEgidio, Wyeth Laboratories, Inc., personal

communication.
336 CHARLES M. SHEARER

24. P.E. Leander, Wyeth Laboratories, Inc.,

personal communication.

25. United States Pharmacopeia/National Formulary

XXI, Supp. 1, page 1.725, U.S. Pharmacopeia,
Rockville, Md. (1984).

26. J . Knowles, G.R. White, C.J. Kick, T.B. Spangler

and H.W. Ruelius, J . Pharm. Sci., 71,710 (1982)

27. C. Snodgrass-Pilla, Wyeth Laboratories, Inc.,

personal communication.

28. E . R . Fluck, C . A . Homon, J . A . Knowles and H.W.

Ruelius, Drug. Dev. Res., - 3, 91 (1983).
 IODAMIDE
Davide Pitre

1. Description
1.1 Nomenclature
1.1.1 Chemical Names
1.1.2 Generic Names
1.1.3 Trade Names
1.2 Formula, Molecular weight
1.3 Appearance, Color, Odor

2. Physical Properties
2.1 Spectra
2.1.1 Infrared Spectrum
2.1.2 Nuclear Magnetic Resonance Spectra

2.1.2.1 'H-NMR
13
2.1.2.2 C-NMR
2.1.3 Mass Spectrum
2.1.4 Ultraviolet Spectrum
2.2 Physical Properties of Solid State
2.2.1 Differential Thermal Analysis
2.2.2 X-Ray Powder Diffraction
2.2.3 Melting Range
2.2.4 Eutectic Temperature
2.3 Solution Data
2.3.1 Solubility
2.3.2 PKa
2.3.3 Partition Coefficients
2.3.4 Index of Refraction
2.3.5 Specific Gravity
2.3.6 Osmotic Properties

3. Manufacturing Procedures
3.1 Synthesis

4. Stability
ANALYTICAL PROFILES OF DRUG SUBSTAKCES Copyright 0 1986
VOLUME 15 by the Anirrirari Pharmaceutical Association
337 All rights of reproduction in any furin reserved.
338 DAVIDE PITRE

5. Analysis of impurities
5.1 Free aromatic amine
5.2 Free Iodine and free Halides
5.3 TLC of free amino compounds

6. Metabolism and Pharmacokinetics

6.1 Metabolism
6.2 Pharmacokinetics
6.3 Protein Binding
6.4 Acute toxicity

7. Polarography
7.1 Elemental composition
7.2 Identification tests
7.3 Official methods
7.3.1 Organically bound iodine
7.3.2 Titrimetry
7.4 Chromatography
7.4.1 Paper chromatography
7.4.2 Thin layer chromatography
7.4.3 High pressure Liquid Chromatography

8. Determination of Iodamide in Body fluids and tissue

IODAMIDE 339

1. Description

1.1 Nomenclature

1.1.1 Chemical Names

Benzoic acid, 3-( acetylamino)-5-[( acetylamino) me-

thyl] -2,4,6-triiodo -
n-Toluic acid,&, 5-diacetamido-2,4,6-triiodo
CAS Reg. N. (240-5857

1.1.2 Generic Names

Iodamide (BAN, DCF, USAN)

Iodamidum (NFN)
Ametriadinic acid
Deriv.
Iodamide Meglumine (USAN)

1.1.3 Trade Names

Angiomiron (Schering AGBerlin)

Contraxin (Takeda - Osaka)
Isteropac (Bracco-Milano)
Opacist (Bracco-Milano)
Uromiro ( Bracco-Milano)
Uromiron (Schering A.G.-Berlin)
Urombrine (Dagra-Dienmarc
- Trade names of 1-deoxy-1-(methylamino)-D-glucitol
salt
Renovue (Squibb)
Jodomiron (Erco-Vedback)
340 DAVIDE PITRE

1.2 Formula, Molecular weight

COOH

CH COHN CH NHCOCH3
3 2
1
C H I N 0 Mol.wt. 627.93
12 11 3 2 4

1.3 Appearance, Color, odor

White, fine, crystalline powder, odorless and with

a very bitter taste (2).

2. Physical Properties

2.1 Spectra

2.1.1 Infrared Spectrum

The infrared spectrum in potassium bromide pellet

was reported by E. Felder et a1 ( 3 ) . The spectral band assi-
gnments are reported in Table I.
IODAMIDE 34 1

Table n. 1

Values of i n f r a r e d a b s o r p t i o n
~

-r
Freq ency
(cm 1
Type of
vibration
Assignment

3380
~ N H -CONH groups
3200

2980+2500 4 C H and <OH a s s -CH -CH and -COOH groups

2' 3

1728 qc = 0 -COOH group

1660
qc = 0 -CONH "Amide I band" groups
1635

1525 $NH + (CN -CONH "Arnide I1 band"groups

1425 &OH + 'fC-0 -COOH group

1460 6CH -CH group

2

1370 bCH -CH group

3

1296
VCN + SNH -CONH "Amide I11 band'lgroups
1280

1210
60H + ((2-0 -COOH group
1193

985

758

738

695

685

650
342 DAVIDE PITRE

2.1.2 Nuclear Magnetic Resonance Spectra

2.1.2.1 'H-NMR
1
The H-NMR spectrum of Iodamide, recorded in DMSO
solution with a EM 390 Varian spectrometer at 90 MHz, is re-
ported in Fig.1 (4). The spectral assignment is shown in
Table 4.

Table N.' 2
1
H-NMR data

Multiplicity n. Protons Assignment

10.00 S 1, exch. +NHCO-

7.93 t 1, exch. 4- CHgHCO

4.67 d 2 $-CH2

3.0+4.0 v.1. N1 -COOH

2.01 S 3 &NHCOCH3

0.90 S 3 B-CH2-NHCOCH
-3
 caiu;r
LOCI PO5 L p p m SPECTRUM AMPL boo SWEEP TIME 5 rrun NUCLEUS ,
' SAMWE
(yr&h OPERATOR 9
LOCKPOWER amx5 mG
FILTER o.05 sec SWEEP WIDTH -do ppn ZERO REF '(WF OAT€ 5!Jz!%
DECOUPLE 415

DECOUPLING P O w E R L m G RF POWER 6.41 WG END OF SWEEP^.^^ SAMPLE TEMP ,/ o SOLVFN~ )M% SPECTRUM No=

Fig. 1 - 'H-NMR Spxtrun o f lodanide i n DMSO - d6

344 DAVIDE PITRE

2.1.2.2 13C-NMR

The 13C-NMR spectrum, recorded in DMSO solution

with a XL-100 Varian spectrometer at 25.2 MHz, is shown in
Fig.2. The assignments are presented in Table 3 ( 4 ) .

p b l e n. 3
C-NMR data
-
Line n. Intensity Assignment

1 84 169.6 -COOH

2 100 168.8
-+ ~CH~NHCO
$NHCO
3 86 167.5

4 27 149.0 C -C and C aromatil

1 3 5
5 77 143.7
6 104 143.6 carbon

7 39 107.5 C -C and C aromatir

2 4 6
8 35 97.0
9 47 95.8 carbon

10 32 56.1 $-g2
11 69 22.8 &CH2NHCOCH3

12 74 22.0 $-NHCOCH,

2.1.3 Mass Spectrum

The main fragmentation patterns observed in EI/MS

were reported by E. Felder et a1 ( 3 ) . In order to better cha-
racterize the molecular ion which in the EI spectrum is very
scarcely abundant (0.001%), the mass spectrum of iodamide was
recorded using a Fast Atom Bombardment (VG Micromass 70-70E,
thioglycerol mixture). The normalized spectrum is shown in
Fig. 3 from which it appears that the pseudomolecular ion fi
+H
+
at m/z = 629 represents the base peak.
C
.-
.
?
m
m m
Ln
VI
N
N
m
E
-
m
t I I
00 88 68 48 28
21
IODAMIDE 347

2.1.4 Ultraviolet Spectrum

The ultraviolet absorption spectra of Iodamide in

methanol, 0.1N NaOH and 0.1N HC1 were reported by E. Felder
et a1 ( 3 ) . The relevant spectrophotometric data are reported
in Table 4.
Table n. 4

2.2 Physical Properties of Solid State

2.2.1 Differential Thermal Analysis

Differential thermal analysis was carried out in a

Perkin-Elmer model DSC-1 calorimeter(heating rate of 4O/min).
In the temperature range from 40°-350° only one endothermic
transition was seen at 284O. A thermogravimetric analysis
carried out under nitrogen with RH Cahn electrobalance and a
Stanton-Redcroft temperature programmer showed decomposition
with loss of iodine at 280° ( 3 ) .

2.2.2 X-Ray Powder Diffraction

The X-ray powder diffraction pattern of Iodamide

was determined with a Philips Powder Diffractometer PW 1710
using nichel-filtered copper radiation with the following in-
strumental conditions: tube LFF, Cu, 40 KV, 40 mA; slits lo-
0.1 mm-lo; detector PW 1171 proportional counter plus dis-
3
criminator; scale 2x10 cps; time constant 1"; scanning speed
0.004°/sec; paper speed 1 crn/lo 28; specimen holder Niskanen
plus internal standard.
348 DAVIDE PITRE

Interplanar distances (d(8)=1.54051/2d Sin 4 and relative

intensities are reported in Table 5.

Table n. 5
X-Ray Powder Diffraction Pattern of Iodamide

N d (A) I/I,xlOO

1 10.52 70 31 2.92 11
2 8.91 2 32 2.91 10
3 7.11 6 33 2.86 12
4 6.32 5 34 2.81 6
5 6.04 4 35 2.75 3
6 5.75 2 36 2.69 4
7 5.70 3 37 2.65 2
8 5.43 2 38 2.63 11
9 5.26 100 39 2.59 4
10 5.14 8 40 2.56 4
11 5.02 12 41 2.54 10
12 4.64 6 42 2.48 3
13 4.21 11 43 2.43 3
14 4.14 2 44 2.40 3
15 4.07 3 45 2.38 3
16 4.03 4 46 2.35 3
17 3.86 14 47 2.31 4
18 3.81 6 48 2.29 3
19 3.77 4 49 2.28 3
20 3.65 5 50 2.20 3
21 3.50 12 51 2.13 3
22 3.43 6 52 2.11 7
22 3.43 6 53 2.01 5
23 3.31 8 54 1.96 4
24 3.22 3 55 1.93 3
25 3.19 9 56 1.75 5
26 3.12 4 57 1.72 2
27 3.07 4 58 1.71 2
28 3.03 4 59 1.67 2
29 2.98 2 60 1.61 2
30 2.96 3 PLUS OTHER LINES :2
IODAMIDE 349

2.2.3 Melting Range

Iodamide shows in thermal microscopy a melting ran-

ge between 200° and 270°, with decomposition (3). M.p. 255-7O
(5).

2.2.4 Eutectic Temperature

The following melting points of Iodamide, that have

the advantage of being very sharp, have been reported (3)

Mixture M.p.

N-Phenylacetamide 110.10

p-Ethoxyacetamide 131.8O

Salophen 186.7O

1-Cyanoguanidine 183. Oo

N-Phenylbenzamide 158.8O
350 DAVIDE PITRE

2.3 Solution Data

2.3.1 Solubility

Iodamide is sparingly soluble in methanol, slightly

soluble in water and in ethanol and practically insoluble in
ether and in chloroform (2).
Water solubilities, expressed in g/100 ml, are 0.30 at 20°,
0.32 at 40° and 0.39 at 60°.
Water solubilities, as a function of pH, are reported in
Table n. 6 (3).

Table n. 6
Solubility in water as a function of pH at 20°

PH Solubility
-
+0.05) g/100 ml

1.0 0.03
2.0 0.14
3.0 1.16
3.6 5.41
3.8 11.1
4.0 18.7
4.5 52.9
5.0 73.?

2.3.2 -
PKa

The apparent ionization p y t a n t , determined in

methylcellosolve/water (W/W) was pK = 4.15; extrapolation
MCS
25
to aqeuous solution gave a pK = 1.88 (7).
*2O
Other Authors (6) reported a pKa = 3.7, determined by
titration.
IODAMIDE 351

2.3.3 -
Partition Coefficients

The n-octanol/buffer partition coefficients at pH

between 1 and 8 are listed in Table 7 (3).

Table n. 7
n-octanol/aqueous phase partition coefficients

Aqueous phase Part. Coeff.

0.1 M Citrate buffer pH 1 2.71

0.1 M Citrate buffer pH 2 1.02

0.1 M Citrate buffer pH 3 0.12

0.1 M Citrate buffer pH 4 0.02

0.1 M Citrate buffer pH 5 40.01

0.1 M Phosphate buffer pH 6 QO.01

0.1 M Phosphate buffer pH 7 60.01

0.1 M Phosphate buffer pH 8 so. 01

-3
A partition coefficient of 0.9 x 10 in chloroform/water at
pH 3.3 was reported (6).

2.3.4 Index of Refraction

The refraction index (n2') of the aqueous solutions

of the meglumine and sodium salts of Iodamide is given in
Table 8.
352 DAVIDE PITRE

2.3.5 Specific Gravity

The specific gravity (420

0
) of water solutions of
the meglumine and sodium salts of Iodamide is reported in
Table 8.

2.3.6 Osmotic Properties

Osmolalities of aqueous solutions of the sodium and

meglumine salts of Iodamide are reported in Table 8 (4).
IODAMIDE 353

Table n. 8
Osmotic properties
Sodium salt of Iodamide (M.W. = 650.0)

20 20
Iodamide-Na n Osmol/kg
mg I/ml d20 D
g/100 m l

5.00 29.3 1.0282 1.3410 0.1464

10.00 58.6 1.0595 1.3493 0.2869
20.00 117.2 1.1217 1.3658 0.5668
30.01 175.8 1.1850 1.3822 0.8740
40.00 234.3 1.2467 1.3989 1.202
50.01 293.0 1.3092 1.4152 1.591
60.00 351.5 1.3707 1.4314 2.025
70.01 410.1 1.4347 1.4488 2.527
a0 .oo 468.6 1.4979 1.4645 3.092

Meglumine salt of Iodamide (M.W. = 823.2)

20 20
Iodamide-MGA n Osmol/kg
mg I/ml d20 D
g/100 m l

10.09 46.6 1.0499 1.3491 0.2234

20.17 93.1 1.1052 1.3655 0.4346
30.24 139.6 1.1593 1.3819 0.6532
40.33 186.2 1.2133 1.3989 0.8955
50.42 232.8 1.2671 1.4158 1.165
60.48 279.2 1.3202 1.4325 1.490
70.57 325.8 1.3749 1.4490 1. a84
80.63 372.3 1.4297 1.4652 2.370
354 DAVIDE PITRE

3. Manufacturing Procedures

3.1 Synthesis

The starting step in the synthesis of Iodamide (1) (1)

is the introduction of an acylaminomethyl group in position 5
of a benzoic acid molecule, either substituted or not in po-
sition 2 and/or 4 with a chlorine atom, followed by the in-
troduction of an amino group in position 3 by means of nitra-
tion and reduction.
The compound thus obtained is subsequently iodinated and ace-
tylated. If the starting product is benzoic acid ( x = H, X =
1
H) the synthesis has no industrial value because the reaction
leads mostly to an undesired compound with the nitro group in
position 4. The synthesis was therefore studied using 2-chlo-
ro benzoic acid (X = C1, X = H), 4 chlorobenzoic acid (X =
1
H, X = C1) and 2,4-dichlorobenzoic acid (X = X = C1) as a
1
starting products in which the introduction of the chain is
easier. Nowdays the industrial synthesis is performed start-
ing from 2-chlorobenzoic acid using the scheme through 3-ace-
tylaminomethyl-6-chlorobenzoic acid (IVb). This product is
nitrated to (Vb), reduced to (VI) and iodinated to 3-acetyl-
aminomethyl-5-amino-2,4,6-triiodobenzoic acid (VII) which is
finally acetylated to Iodamide.
 P
!
r
z
U z
rN “ 50
I N r
urn
r
U
=N
r
5
r
L
N

1:”
B
rN
rN U
U
X
4
r u
I1 11 X
4
z 4 4 v
I1 11
x
4.4
u r
.x 4
x
356 DAVIDE PITRE

4. Stability

Iodamide, stored in the solide state at room tempe-

rature (150-25OC) and at 40°C/75%/RH for 12 months, is physi-
cally and chemically stable.
Injectable solutions of Iodamide Sodium salt or Me-
glumine salt stabilized with sodium edetate, have not shown
any evidence of chemical change after storage for 60 months
at room temperature (15O-25OC).

5. Analysis of impurities

5.1 Free aromatic amine

For the determination of free aromatic amine an of-

ficial method (2), based on the Bratton-Marshall colorimetric
reaction, was reported. The procedure has been automated (9)
for bulk product and injectable solutions.

5.2 Free Iodine and free Halides

The method reported by Jap.P. IX for detection of

free iodine consists in the extraction with chloroform of a
suspension of Iodamide obtained by acidification of an
aqueous solution of the sodium salt. Chloroform must be co-
lorless. Free halides are tested with silver nitrate after
acidification with nitric acid of an aqueous solution of the
ammonium salt of Iodamide and filtration.

5.3 TLC of free amino compounds

The following de-acetylation products may be con-

sidered as by-products of Iodamide ( A ) :
3-amino-5-aminomethyl-2,4,6-triiodobenzoic acid (B)
3-amino-5-acetylaminomethyl-2,4,6-triiodobenzoic acid (C)
3-acetamido-5-aminomethyl-2,4,6-triiodobenzoic acid (D)
The Rf values of these compounds in the solvent system n-
amylalcoho1:acetic acid:water 4:1:5, using Silica-gel 60 F
254
pre-coated plates (Merck), are summarized in Table 9 (10).
IODAMIDE 357

Table 9
Rf of de-acetylation products of Iodamide

Compound Rf

0.14
0.08
0.3
0.03

6. Metabolism and Pharmacokinetics

6.1 Metabolism

Chromatographic studies on urine of normal subjects

showed that Iodamide was excreted unchanged in the first 4
hours after administration, as unchanged compound together
with amounts of an unidentified metabolite from 4 to 12 hours
and mostly as the metabolite after 12 hours. However, the to-
tal amount of excreted metabolite is quite small (less than
1.5 per cent of the injected dose) (13).

6.2 Pharmacokinetics

Urinary excretion in rabbits after i.v. adminis-

tration was found to be (14):
Sodium salt of Iodamide: 84.9% after 195 min.
Meglumine salt of Iodamide: 79.6 after 180 min.
Plasma concentrations in rabbits, after i.v. administration
of the meglumine salt of Iodamide, were the following

Minutes 7 10 15 20 27

% dose -
42.3+11.3 -
24.8+9.1 -
18.1+2.0 -
11.9+1.9 -
3.56+1.37

The same Authors report a 97% excretion in 24 hours after

i.v. administration to rats and give the content in blood,
liver, kidney, spleen and thyroid after 30 min., 1 hour, 2
and 24 hours.
358 DAVIDE PITRE

Urinary e x c r e t i o n i n mouse is 0.6% a f t e r 4 hours when

Iodarnide is a d m i n i s t e r e d o r a l l y (300 mg/kg).
As r e g a r d s humans, urinary excretion after i.v. adminis-
t r a t i o n , is o f 84.7% a f t e r 4 hours and 94.2% a f t e r 72 h o u r s
while f e c a l e x c r e t i o n is 0.5%.
Pharmacokinetic parameters f o r i . v . a d m i n i s t r a t i o n and f o r
i n f u s i o n a r e s i m i l a r and w e have T 1 j o f about 3 min f o r t h e
2, I
d i s t r i b u t i o n h a l f - l i f e and T 1 o f about 69 minutes f o r t h e
disposition h a l f - l i f e (16).
P
2~

6.3 P r o t e i n Binding

Plasma p r o t e i n binding o f Iodamide i n 2 p a t i e n t s

v a r i e d between 4 and 13 p e r c e n t (17). Other a u t h o r s r e p o r t
t h a t t h e b i n d i n g t o serum p r o t e i n is n e g l i g i b l e (13).

6.4 Acute t o x i c i t y

...........................................
Animal i.v. i.p.
----------.--------------_.----------------. .
Mause 9 (16) 11 (18)

Rat 11.4 (16) 17.5 (16)

Rabbit 13.2 (16) 15.5 (16)
Dog dose of 1 5 ml/kg of a 50% solu-
t i o n do n o t cause l e t h a l i t y
.........................................
The a c u t e t o x i c i t y o f t h e sodium s a l t of Iodamide i n mouse
was found t o be 17.3 g/kg ( 1 9 ) .
H.J. Herms r e p o r t s a DL = 16.5 g/kg a f t e r i . v . adrninis-
50
t r a t i o n o f Iodamide ( c a l c u l a t e d as a c i d ) t o r a t s ( 1 9 ) and on
i n t r a c e r e b r a l t o x i c i t y o f 0.2 g/kg i n mice a f t e r a d m i n i s t r a t -
i o n of t h e meglumine s a l t .
IODAMIDE 359

7. Polarography

Iodamide exhibits a wave at 0.6 volt in 0.1M phos-

phate buffer at pH 9.5 (10).

7.1 Elemental composition

Element % Calc. % Found

C 22.95 22.93
H 1.77 1.78
I 60.63 60.75
N 4.46 4.42
0 10.19 10.12

7.2 Identificatipn Tests

Iodamide may be identified by the following methods

(2) (13):

a) dissolve 0.01 g in 5 ml of hydrochloric acid and heat in

a water bath for 5 minutes. The solution must respond to
the qualitative test for aromatic primary amines.

b) Heat strongly: violet vapours of iodine are evolved

c) Infrared spectrum recorded on 3 m g , previously dried at

-1 -P
105O for 4 hours, by the PO assium bromi e disk meth d
exhibits maxima at 3 85 cm , 1369 cm , 1269 cm ,
-P
1210 cm-l and 1191 cm
-? .
d) The UV spectrum of a solution, containing about 10
mg/ml, &
a buffer at pH = 9 must show a maximum at 238
nm and E of about 526.
lcm
360 DAVIDE PITRE

7.3 Official methods

7.3.1 Organically bound iodine

The reductive dehalogenation method with zinc-

sodium hydroxide is used to free organically bound iodine.
The iodide thus formed is determined by titration in acidic
solution with standardized silver nitrate in presence of
tetrabromophenolphthalein ethyl ester until the color of the
precipitate turns from yellow to ireen (2).
The determination can be carried out in a simpler way through
a reduction with sodium borohydride (10) according to the me-
thod first proposed by Egli (11).

7.3.2 Titrimetry

Iodamide can be titrated directly with 0.1N NaOH,

after dissolving it in CH OH and adding the same volume of
H 0, using phenol red (0.02% of sodium salt) as an indicator,
2
or potentiometrically using a glass electrode (10).

7.4 Chromatography

7.4.1 Paper chromatography

Ascending paper chromatography was performed using

Whatman N1 filter paper and the following systems:
a) n-C H OH : CH COOH : H 0 = 3:1::2
4 9 3 2
b) CH CHOHCH3 : NH40H 25% = 8:2
3

The product was visualized as a blue spot on a white back-

ground (Rf 0.67 (a), 0.63 (b)/ after spraying with soluble
stark, exposing to UV light (254 nm) and again spraying with
2N HC1.
IODAMIDE 36 1

7.4.2 Thin layer chromatography.

Thin layer chromatographic methods for identifica-

tion and separation of Iodamide and related compounds are
summarized in the following Table 10.

Table 10
Thin layer systems of separation of Iodamide (12)

Solvent Rf Rf
a b
system

I 0.43 0.59
I1 0.50 0.55
I11 0.34 0.47
IV 0.70 0.74
V 0.51 0.59
VI 0.44 0.74
VII 0.24 0.33
VIII 0.28 0.50

a) Silica gel 60 F (Merck) pre-coated TLC plates

254
b) Cellulose F (Merck) pre-coated TLC plates

- Solvent system:

I = n-C4H9OH : CH3COOH : H2°

- 3:l:l

I1 = iso-C H OH : CH CHOHCH : NH40H 25%

49 3 3
- 2:5:3

I11 = iso-C H OH : CH CHOHCH3 : NH40H 25%

49 3
- 5:2:2

IV = CH COOC H : CH3COOH
3 2 5
: H20 - 3:2:2

V = n-C H OH : NH40H 25%

3 7
- 7:3

VI = CHCl ' CH COOH :

3' 3 H2°
- 5:5:1

VII = CHCl ' CH OH : NH40H 25%

3' 3
- 6:3:1
VIII = n-C H OH : CH3COOH :
49 H2°
- 4:1:5
362 DAVIDE PITRE

- Detection system

a) 0.5% aqueous starch solution with subsequent exposure of

the plates to UV light (254 nm): formation of brown
spots;

b) 1:l aqueous solution of 10% cerium sulfate and 5% sodium

arsenite: white spots on a yellow background.

7.4.3 High pressure Liquid Chromatography

A HPLC method for quantitative determination of

Iodamide and for separation of possible impurities was deve-
loped (10).

Instrumental conditions:

- Apparatus: Hewlett-Packard, mod. 1084B

- Column: 25 cm x 4 mm Hibar(R), packed with Lichrosorb

RP-18 (5 um)
/
- Injection: 10 ul of a 01 mg/ml aqueous solution (PH 7.5)
/
- Eluant A: 0.076 M phosphate buffer, pH 7.5, in water

- Eluant B: 1:l (v/v) methano1:phosphate buffer pH 7.5)

- Flow: 1.2 ml/min

- Gradient prophile : minutes % eluant B

0 15
5 15
15 40
16 40
17 15
22 stop
IODAMIDE 363

- Column temperature: 4OoC

- Detection : UV, 240 nm

: @ 7.2 min
- tR

8. Determination of Iodamide in Body fluids and tissue

The following methods are used for this determi-

nation:

a) Total iodine determination, after decomposition with

perrnanganate in hot acid or alkaline medium (16)(23) ac-
cording to the methods of White and Rolf (22);

b) Total iodine (13) (14) determination with a PBI Techni-

con Autoanalyzer (23);

c) Spectrophotometric determination in diluted urine of dog

(6);

d) X-Ray Fluorescence analysis after irradiation with 241Am

(15);
131
e) Total radioactivity of I material determined in a
gamma counter (17).
364 DAVIDE PITRE

References

1) E. Felder, D. PitrB, L. Fumagalli, Helv. Chim. Acta, 48,

259 (1965)

2) Jap. P., IX (1976)

3) E. Felder, D. Pitr&, M. Grandi, I1 Farmaco Sc. ed., 32,

755 (1977)

4 ) M. Grandi, Bracco SPA, Personal communication

5) Index Merck

6) A.J.M. Engelen, J.F. Rodrigues de Miranda, E. J. Ariens,

Invest. Rad. g, 210 (1973)

7) E. Felder, D. Pitrb, M. Grandi, I1 Farmaco Sc. ed., 28,

485 (1973)

8) M. Svoboda, I. Fiala, Rontgenbl., 19, 273 (1966)

9) E. Felder, D. PitrB, M. Grandi, J. Pharm. Sc., 64, 684

(1975)

10) U. Tiepolo, Bracco SPA, Personal communication

11) R. Egli, Z. Anal. Chem., 247, 39 (1969)

12) M. Brocchetta, Bracco SPA, Personale communication

13) L.T. Di Fazio and coll., J. Chem. Pharm. 35 (1978)

14) D. PitrB, M. Fonberg-Broczek, Rad. Med., 3, 729 (1970)

15) V . E . Zajcik, I.S. Arnosov, E.I. Frolova, V.I. Jakoder,

Radiol. diagn. , 24, 429 (1983)

16) F. Bonati, G.F. Rosati, M.G. Poletto, Arzneim. Forschr.,

-
15, 222 (1965)
IODAMIDE 365

17) A.C. Bollerup, B. Hesse, E. Sciven, Europ. J. Clin.

Pharmacol., 2, 63 (1975)

18) F. Bonati, G.F. Rosati, Riv. Rad., 12, 66 (1972)

19) H.J. Herms, Rontgenbl., 21, 3 (1958

20) F. Bonati, G.F. Rosati, V. Zanichelli, Radiol. Med.,

-
52, 577 (1966)

21) H.L. White, D. Rolf, Proc. SOC. Exp. Biol. Med., 2,1
(1940)

22 H.L. White, D. Rolf, Proc. SOC. Exp. Biol. Med., 45,

433 (1940)

23) M. Riley, N. Grochman, "A fully automated method for the

determination of serum protein-bound iodine", Technicon
Symposium, N.Y., 1964
This Page Intentionally Left Blank
 LITHIUM CARBONATE
Henry C. Stober

1. Description
1.1 Name, Formula, Molecular Weight
1.2 Appearance, Color, Odor
1.3 Drug Properties

2. Physical Properties
2.1 Infra-red Spectroscopy
2.2 Raman spectroscopy
2.3 Atomic Emission and Absorption Spectroscopy
2.4 Melting Point
2.5 Thermogravimetric Analysis
2.6 Dissociation Constant
2.7 Conductivity
2.8 Microscopy
2.9 Index of Refraction
2.10 Density
2 . 1 1 Crystal Structure
2.12 X-Ray Powder Diffraction
2.13 Polymorphism
2.14 Solubility
2.15 Dissolution

3. Preparation of Lithium Carbonate

4. Stability
4.1 Solution
4.2 Solid State (Light, Thermal, Humidity)

5. Methods of Analysis
5.1 Identification Test for Lithium
5.2 Identification Test for Carbonate
5.3 Microchemical Test for Lithium
5.4 Microchemical Test for Carbonate
5.5 Volumetric Analysis
5.6 Atomic Emission and Absorption Spectroscopy
5.7 Other Spectrometric Techniques
5.8 Conductivity
5.9 Ion Selective Electrodes
5.10 Ion Chromatography

6. Medicinal History

7. Pharmacology
ANALYTICAL PROFII.ES OF DRUG SUBSTANCES Copyriglit Q 1986
VOLUME 15 hy the American Pharmaceutical Association
367 All rights o!' reproduction i i i any form reserved.
368 HENRY C.STOBER

LITHIUM CARBONATE

1. Description

1.1 Name, Formula, Molecular Weight

Chemical Name
Lithium Carbonate

Nomenclature
The following nomenclature is used in Chemical
Abstracts: Carbonic Acid, Dilithium Salt
[ 554-13-21

Trademarks
The following trademarks are listed in the Merck
Index (1): Camcolit; Candamide; Carbolith;
Ceglution; Eskalith; Hypnorex; Lithane; Lithobid;
Lithonate; Lithotabs; Plenur; Priadel; Quilonum
retard.

Molecular Formula and Weight (1)

Li2CO3 73.89
C: 16.25% Li: 18.78% 0: 6 4 . 9 6 %

1.2 Appearance, Color, Odor

White, granular, odorless, light alkaline powder
(1,2) *
1.3 Drug Properties
Lithium Carbonate, by virtue of the therapeutic
properties of lithium, is used for the treatment
of manic depressive psychoses. The drug is
listed in the United States Pharmacopea (21, the
British Pharmacopoeia (3)) and the Modern Drug En-
cyclopedia (4), as well as Remington's Pharmaceu-
tical Sciences (5).

2. Physical Properties

2.1 Infra-red Spectroscopy

The infra-red spectrum of lithium carbonate
dispersed in KBr is shown in Figure 1 (6).
Literature sources have demonstrated that the
infra-red spectrum of lithium carbonate is
consistent with its known crystal structure ( 7 ,
8). The infra-red spectrum of lithium carbonate
 n
d
I
E
U
UI
n W
0
0
03
0
0
0
rl
0
0
hl
rl
0
0
-3
4
0
3
\D
rl
0
0
c3
rl
0
0
0
hl
0
0
Ln
N
3
3
3
m
3
3
n
m
370 HENRY C.STOBER

has also been obtained as a film on sodium

chloride plate and as Vaseline and flurolub
suspensions ( 7 , 9 ) . A summary of the character-
istic infra-red bands for lithium carbonate is
presented in Table I.

TABLE I

Characteristic Infra-red Bands of Lithium Carbonate (7)

Frequency (cm-l) Wavelength (p) Relative Intensity

2558 3.91 W
2494 4.01 W
1842 5.43 W
1806 5.54 W
1495 6.69 vs
1437 6.96 vs
1088 9.19 M
866 11.55 S
846 11.82 W
741 13.50 W
7 12 14.04 vw

The infra-red absorption band at 1088 cm-l can be

used to uniquely quantitate lithium carbonate in
the presence of other alkali carbonates ( 1 0 ) .
The observed frequencies related to the isotopic
species 6LizC03 and 'Li~C03 have been reported by
Tarte ( 1 1 ) .

2.2 Raman Spectroscopy

The Raman spectra of crystalline and molten
lithium carbonate have been reported by Brooker
and co-workers ( 1 2 , 1 3 ) . Major bands are ob-
served at 1091 and 1459 cm-l.

2.3 Atomic Emission and Absorption Spectroscopy

Lithium carbonate can be made to exhibit the
characteristic emission spectrum of lithium.
Three typical analytical emission lines are
obtained for lithium containing aqueous solu-
tions. These are summarized along with their
relative intensities in Table I1 ( 1 4 ) .
LITHIUM CARBONATE 371

The line at 670.8 nm is particularly intense and

imparts a deep red color to an oxidizing flame.

TABLE I1

Lithium Analytical Emission Lines

Wavelength (nm) Relative Intensit9

670.8 1
323.3 235
610.4 3600

+'relative amount of lithium required for 1%

response

Typical sources of excitation include

air-acetylene and nitrous oxide-acetylene flames.
More recently electrothermal and argon plasma
excitation techniques have become available.
Because of its greater sensitivity the line at
670.8 nm is most often used for the analysis of
lithium by flame emission spectroscopy, atomic
absorption spectroscopy and plasma spectros-
copy (14, 15).

2.4 Melting Point

The melting behavior of lithium carbonate has
been evaluated by DTA using both heating and
cooling programs. Lithium carbonate has been
reported by various sources to melt in the
temperature range of 714O to 733OC (16-25) depend-
ing on the atmosphere employed (ie. C02 or air)
and the degree of dissociation of Li2CO3 to Liz0
and C02 that occurs as the melting point is
approached (16, 18, 20, 21).

Typical thermal properties reported in the

literature for lithium carbonate are summarized
in Table 111.
372 HENRY C. STOBER

TABLE I11

Thermal Properties of Lithium Carbonate

Melting Point (23) 993OK; 72OOC

Heat Capacity (23) 23.2 cal/mole /deg
Specific Heat (24) 0.315 cal/g
Heat of Fusion (24) 10.7 kcal/mole

2.5 Thermogravimetric Analysis

Data obtained for a typical lot of lithium
carbonate (H2O < 0.5%)- by thermogravimetric
analysis is presented in Table IV. Under the
conditions employed the compound is essentially
weight stable up to 200OC with only water loss.
Above 200OC a gradual continuing weight loss is
observed. This behavior is consistant with that
reported by Machaladze and co-workers (21).

TABLE IV

Lithium Carbonate: Thermogravimetric Behavior (26)

TGA (N2 atmosphere) Perkin-Elmer TGS-1

Scan rate 10°C/minute

RT to 90°C 0.11% weight loss

900 to 200oc 0.08% weight loss
ZOOo to 45OOC 0.63% weight loss
above 45OOC continuing weight loss

2.6 Dissociation Constant

The pKa's for the first and second ionization
steps of the conjugate acid of the carbonate ion
are reported in the literature to be 6.38 and
10.25 respectively (27).
LITHIUM CARBONATE 373

2.7 Conductivity
The relationship between the equivalent conduc-
tance (4) and the concentration of lithium
carbonate is typical of a strong electrolyte. A
plot of& versus JC yields straight line for
concentrations less than 0.01N. The equivalent
conductance at infinite dilution (4')for lithium
carbonate was determined to be 110.2 R/cm2/Eq
at approximately 25OC, from this plot (6).

2.8 Microscopy
USP Lithium Carbonate is a microcrystalline solid
that is birefringent under crossed polars. The
solid has been observed to exist as polycrystal-
line aggregates ranging from approximately 10 to
85 micrometers in diameter (6).

2.9 Index of Refraction (ND25)

The refractive indices of Lithium Carbonate have
been reported as 1.428, 1.567 and 1.572 (25).
Lithium Carbonate is biaxial and is optically
negative.

2.10 Density
The density reported for Lithium Carbonate is
2.11 g/cc 125):

2.11 Crystal Structure

The crystal structure of Lithium Carbonate is
monoclinic w'th unit cel dimensions of a = 8.39
k,
A, b = 5.00 c = 6.21 , and f3 = 114.5+ (28).
The crystal lattice belongs to the space group
Cg - C 2/C and there are four lithium carbonate
mofecules per unit cell (29, 7).

2.12 X-Ray Powder Diffraction

Major lines present in the x-ray powder diffrac-
tion pattern of USP grade lithium carbonate are
presented in Table V. Strong lines are observed
at 31.6, 21.4, and 30.6 degrees 28 for copper Ka
radiation. These values are in good agreement
with the literature values of 28 = 31.4, 21.0 and
30.3 [ASTM Data (30)] and 28 = 31.8, 21.3, and
30.6 [JCPDS Data (3111.
374 HENRY C. STOBER

T y p i c a l i n s t r u m e n t a l and experimental c o n d i t i o n s
used t o o b t a i n t h e x-ray powder d i f f r a c t i o n
p a t t e r n of l i t h i u m carbonate d e p i c t e d i n F i g u r e 2
(32) a r e p r e s e n t e d below.

I n s t r u m e n t a l Conditions

Spectrometer: Diano 8535 D i f f r a c t o m e t e r

Generator: 30 KV, 13 mA
Tube T a r g e t : cu 0

Radiation: Cu, N i F i l t e r e d , Ka = 1.542 A

Optics : lo Beam S l i t , MR S o l l e r S l i t , 0.1'
D e t e c t o r S l i t , 3 O Take-Off Angle
Goniometer: Scan Rate: 2 degrees 29/minute
Detection: SPG-10 D e t e c t o r
Rate meter 2500 cps f u l l s c a l e
P u l s e Height S e l e c t i o n , EL = 0.2V

Sample
-
Sample was ground, s i e v e p t E r o u g h a
10
Preparation: No. 100 US Standard Sieve, and back-
packed i n t o an aluminum sample h o l d e r .

X-Ray Powder D i f f r a c t i o n P a t t e r n o f
Lithium Carbonate (USP)

MAJOR LINES

28 Degrees * dJ;J, 1/10 Hdc

21.4 4.15 84
23.5 3.79 18
29.5 3.03 24
30.6 2.92 81
31.6 2.83 100
34.1 2.62 31
36.1 2.49 19
36.9 2.43 40
39.7 2.27 19
48.8 1.87 15

f 28 degrees read t o n e a r e s t 0 . 1 degrees 28

nh
*k Interplaner Distance (1): d = 2 Sin 8
 Figure 2: X-Ray Powder Diffraction Pattern of Lithium Carbonate

.
h
u
m
2 .
u
H

$
C

-74
I
u
cd
4
d
b

s
ry

* .
7 U
1

15
I

19 23 27 31 35 39 43 47 Degrees 28
376 HENRY C . STOBER

+d** RelativeIntensity in percent based on strongest

signal. Under the experimental conditions employed
the relative intensities are subject to change due to
variations in sample handling and particle size and
are only included as a guide for identifying strong
lines.

The Hanawalt indices for lithium carbonate are 2.8lX,

4.168 and 2.928 (33).

2.13 Polymorphism
Recent literature indicates that up to its fusion
temperature lithium carbonate exists as only one
crystal form (18). An earlier report in the
literature indicated the presence of a
polymorphic transition at 166OC (17). The
possibility of lithium carbonate existing as a
stable c1 and a metastable f3 phase, under atmo-
spheric conditions, also has been proposed (34).
It should be noted, however, that in the
crystalographic sources consulted (30, 31, 33)
only one form of lithium carbonate is listed,
implying that other crystalline forms are
uncommon.

Impurities present in lithium carbonate may

account for some of the thermal effects noted.
In this respect, Reisman reported an anomalous
transition at -410OC and an additional heat
effect at 35OOC (18). The later was suspected to
be due to the presence of Li20. Impurities such
as Li20, Na2C03 and K2CO3 are known to form
eutectics with lithium carbonate and may account
for some of the anamolies observed. The ternary
eutectic with Na2C03 and K2CO3 melts at 397O (19)

2.14 Solubility
The following equilibrium solubility data was
obtained either experimentally at 37OC (6) or
from the literature as indicated in Table VI.
LITHIUM CARBONATE 377

TABLE VI

Solubility of Lithium Carbonate in Common Solvents

Solvent Solubility Source

(g/lOO ml)

Water, O°C 1.5 (25)

Water, 37OC 1.0 experimental
Water, 100°C 0.7 (25)
0.1N NaOH, 37OC 1.1 experimental
1.ON NaOH, 37OC 1.7 experimental
0.05M tris-buffer, 37OC 1.1 experimental
Ethanol Insoluble (25)
Acetone Insoluble (25)

Lithium carbonate decomposes in strong mineral

acids to yield carbonic acid, carbon dioxide and
the conjugate salt. The solubility of lithium
carbonate in water has been extensively studied
as a function of temperature and has been ob-
served to have a negative temperature coefficient
of solubility; the solubility decreasing signifi-
cantly with increasing temperature (35, 36). The
heat of solution of lithium carbonate in water at
25OC has been reported as -14,800 20.021 kJ mol-'
(37) *
When considering the solubility of lithium
carbonate in aqueous solutions, it should also be
noted that lithium forms insoluble salts with
several common anions including phosphate,
fluoride and the carboxylate anion of the C14
- C ~ Bfatty acids (25).

The solubility product of lithium carbonate in

water at 25OC is 1.7 x (38) when the concen-
tration of the lithium and carbonate ions are
expressed in moles/liter.

2.15 Dissolution
The intrinsic dissolution rate of lithium carbon-
ate in aqueous solutions was reported by Wall and
co-workers (39) using the rotating disc method.
They found linear dissolution rate profiles for
378 HENRY C. STOBER

lithium carbonate in water, simulated gastric

fluid and tris buffer. Dissolution studies in
simulated intestinal fluid containing phosphate
were complicated by the precipitation of trilith-
ium phosphate onto the disc.

Dissolution rate determinations for various

experimental and commercial lithium carbonate
preparations have been reported in the literature
(40 - 4 4 ) . Ritschel and Parab ( 4 3 ) evaluated
seven (six conventional and one sustained re-
lease) lithium carbonate commercial prepara-
tions. For the conventional preparations they
found a good correlation between the ENSLIN
number and t ( 5 min.), t (10 min.) and MRT (mean
residence time). The ENSLIN number (amount of
water in mL absorbed by 1 g of powdered sub-
stance) is a measure of the hydrophilicity, or
wetting, of the formulation.

The wetting of pharmaceutical powders, including

lithium carbonate, has also been evaluated by
Lerk and co-workers ( 4 5 ) using contact angle
measurements. The contact angle 8 obtained for
lithium carbonate by these workers was SO0 indi-
cating hydrophilicity.

3. Preparation of Lithium Carbonate

Lithium carbonate is primarily prepared from the

mineral spodumene, LiAlSizOs ( 4 6 ) . Other mineral
sources of lithium include petalite (LiAlSi4010),
amblygonite (LiAl[F,OH]PO4) and lepidolite
(K2Li3A14Si702[0H,F]3). Spodumene is the most commer-
cially important o f the lithium ores because of its
relative abundance and its relatively high lithium
content (3.757,).

In the manufacturing process employed by the Lithium

Corporation ( 4 7 ) spodumene crude ore, which also
contains such components as mica, quartz and feldspar,
is crushed to a fine sand. The crushed spodumene is
separated from the other components by flotation. The
“purified” spodumene is subjected to intense heat
(approximately llOO°C) and milled to a fine powder to
increase its surface area and reactivity. The powdery
LITHIUM CARBONATE 379

spodumene is treated with strong sulfuric acid at

25OoC to produce lithium sulfate (LiZSO4). The
lithium sulfate produced is separated from the residual
insoluble components of the ore by aqueous dissolution.
The resulting lithium sulfate solution is reacted with
sodium carbonate to produce lithium carbonate, which
remains in solution. Impurities yielding insoluble
carbonates are precipitated in this step. The lithium
carbonate solution is further purified by pH adjustment
and filtration and concentrated by evaporation. Lith-
ium carbonate is precipitated from the concentrated
solution by further treatment with sodium carbonate.
Pharmaceutical grade material is also further processed
to meet compendia1 and special requirements such as
particle size and bulk density. The flow chart depicted
in Scheme I summarizes the process just described.

Other processes reported to utilize spodumene involve

treatment with limestone to produce lithium hydroxide
( 4 6 ) and direct recovery of lithium carbonate by
digestion with aqueous sodium carbonate at 200°C ( 4 8 ) .
The production of lithium carbonate from lithium
hydroxide has been accomplished using carbonization
(COz) ( 4 9 ) and treatment with urea (50).

A procedure for purifying lithium carbonate by suspen-

sion in boiling water is described in Volume I of
"Inorganic Synthesis" ( 5 1 ) . The procedure is based on
the fact that lithium carbonate is less soluble in hot
water than in cold water, in contrast to the salts
that are present as impurities. A zone melting
procedure for producing single crystals of lithium
carbonate has also been described ( 5 2 ) .

4. Stability

4.1 Solution
The predominant stability problem for lithium
carbonate in aqueous solutions occurs in acid
solutions, in which it decomposes to yield the
lithium salt of the acid, bicarbonate, carbonic
acid and ultimately, carbon dioxide.

Solutions of lithium carbonate are also incompat-

ible with a variety of cations and anions.
380 HENRY C . STOBER

SCHEME I
Preparation of Lithium Carbonate from Spodumene

I Mined
Spodumene
Crushina
Milling
I

'
Fine
Spodumene

I Ore Ore

1
Powdery
Spodumene

1
-
I
Sintering
(1lOO"C)

Size
Reduction

(250OC)

Solution Solution

I
Lithium
Carbonate
Filtration
Evaporation
Sodium
Carbonate

Solid
LITHIUM CARBONATE 38 I

Cations such as calcium and barium, whose carbon-

ate salts are much less soluble than lithium
carbonate, and anions such as phosphate, that
form less soluble lithium salts, should be
excluded from lithium carbonate preparations.

4.2 Solid State

Light
-
Pharmaceutical grade lithium carbonate was
subjected for one week to visible light, whose
intensity was 600 foot candles (6). No decompo-
sition as noted by changes in physical appearance
(color, texture), weight and titrimetric assay
for carbonate was observed.

Therma1
Samples of lithium carbonate were stored in open
weighing dishes at 25OC and 105OC, respectively,
for one week (6). A small increase in the
titrimetric assay for carbonate from 99.4% to
99.6% was observed for the sample stored at
105OC. No change in the assay was noted for the
sample stored at 25OC. No measureable changes in
weight or physical appearance were observed for
either sample. This stable behavior for lithium
carbonate in the solid state is confirmed by the
literature (16, 17, 21). Accordingly, the lowest
temperature at which lithium carbonate has been
reported to begin to dissociate to lithium oxide
and carbon dioxide is 2OOOC (21). Other workers
have reported that dissociation occurs only near
the melting temperature (15, 16).

Humidity
Samples of lithium carbonate stored for one week
at 25OC/85% RH, 35OC/lO% RH and 35OC/85% RH were
essentially weight stable (6). This is consis-
tent with the literature (8, l o ) , which indicates
that lithium carbonate is not very hygroscopic.

5. Methods of Analysis

5.1 Identification Test for Lithium (2)

When lithium carbonate is moistened with hydro-
chloric acid, it imparts an intense crimson color
to a non-luminous flame.
382 HENRY C. STOBER

5.2 Identification Test for Carbonate (2)

Lithium carbonate effervesces upon the addition
of an acid, yielding a colorless gas, which when
passed into a solution of calcium hydroxide,
immediately causes a white precipitate to form.

5.3 Microchemical Test for Lithium

The microscopic identification of lithium is
practical oniy in materials in which lithium is
present in relatively high concentrations.
Identification has been achieved by preparation
of the tri-lithium phosphate salt, which forms
star-like clumps (53), the pyroantimonate salt,
which gives hexagons (53) and an orange-brown
aurate salt (54). In the absence o f other alkali
metals lithium reacts with zinc uranylacetate to
yield regularly developed octahedra (55).

5.4 Microchemical Test for Carbonate

Absorption of carbonate, evolved upon acidifica-
tion of a lithium carbonate preparation, by a
hanging drop of lead acetate, gives rise to
acicular crystals occurring singly or in irregu-
lar aggregates. Both bicarbonate and carbonate
give a positive response under these conditions.
When added directly, thallus acetate does not
precipitate with bicarbonate, but forms colorless,
long, slender needles with carbonate (56).

5.5 Volumetric Analysis

Lithium carbonate is analyzed in the USP ( 2 ) , BP
( 3 ) and ACS Reagent Chemicals (57) by titration
of the carbonate anion. Excess strong acid is
added to a solution of lithium carbonate and the
residual acid remaining after neutralization is
back-titrated with sodium hydroxide. Differences
exist between these methods with respect to the
indicators used and the emphasis placed on
expulsion of carbon dioxide after acidification.
Alternatively, potentiometric end point detection
can be accomplished using glass and calomel
electrodes.
LITHIUM CARBONATE 383

5.6 Atomic Emission and Absorption Spectroscopy

Solutions of lithium carbonate are frequently
analyzed for lithium using emission techniques
such as flame photometry, plasma spectroscopy and
by atomic absorption. The methods typically
employ analysis at a wavelength of 670.8 nm,
which is the most sensitive specific lithium line
(2, 1 4 ) . These techniques are particularly
effective for the analysis of lithium in dosage
forms and biological fluids (2, 40, 4 4 , 58). A
detailed discussion of the analysis of lithium in
biological fluids and tissues by flame photometry
and atomic absorption is presented in the text
"Lithium Research and Therapy" (59).

5.7 Other Spectrometric Techniques

Lithium carbonate has been determined
spectrophotometrically in tablets using the color
formed upon complexation of lithium with a
"crowned" dinitrophenylazophenol ether (60). The
purple color produced is stable and the absor-
bance of the solution measured at 560 nm is
linear over the concentration range from 25 to
250 ppb. Field desorption mass spectrometry
(FD-MS) in conjunction with a multichannel
analyzer has proven to be a useful tool for trace
analysis of lithium (61). The method also allows
for the determiation of the isotopic distribution
of the lithium salt analyzed.

5.8 Conductivity
Although nonspecific, conductimetric methods have
been used for the analysis of strong electrolytes
(62). The methods tend to be easily automated.
The technique is comparatively simple and appli-
cable to the dissolution rate determination of
dosage forms providing that the other conducting
components of the formulation are not present in
significant amounts. It has been used success-
fully in the authors laboratory for the determi-
nation of the dissolution rate of lithium
carbonate sustained release formulations in water
(63). Precautions were required against absorp-
tion of atmospheric Cop. This was accomplished
by performing the dissolution rate studies with
the vessels under a steady stream of nitrogen.
384 HENRY C. STOBER

5.9 Ion Selective Electrodes

Several workers have explored the usefulness of
ion selective electrodes for the analysis of
solutions of lithium (64, 65, 66). The elec-
trodes are limited by comparatively poor Li+/Na+
selectivity. This deficiency is especially
apparent in biological fluids where comparatively
high levels of Na+ are encountered. Using
elecrodes based on PVC membranes containing ETH
1810 as a neutral carrier, Metzger and co-workers
(64) obtained recoveries of lithium in serum to
within +_lo% over the clinically relevant concen-
tration range.

5.10 Ion Chromatography

Aqueous solutions of lithium carbonate can be
readily analyzed for lithium by ion
chromatography. A polystyrene-divinyl benzene
sulfuric acid cation exchanger in the hydrogen
form (Waters, IC-PAC-C) and 2 mM nitric acid
eluent is employed (67). Under these conditions
lithium at the ppm level is readily separated
from sodium and potassium at comparable concen-
trations. The carbonate anion can also be
determined using a polymethacrylate anion ex-
changer in the quaternary ammonium ion form
(Waters, IC-PAC-A).

6. Medicinal History (68, 69)

Lithium, the lightest of the alkali metals, was

discovered in 1817 by Arfwedson. Its salts were later
found in the spa waters of Germany and England, where
it was believed that it was therapeutic in the treat-
ment o f rheumatoid arthritis and gout. Its use-
fulness in the treatment of gout was attributed to the
high solubility of lithium urate. The use of lithium
as a uricosuric prompted J. F. Cade in Australia to
give animals a lithium salt to decrease the nephrotox-
icity of uric acid. He noted that the lithium salt
produced a calming effect in the animals and proceeded
to use lithium salts clinically as a sedative. During
the 1950's and 1960's the clinical use of lithium
LITHIUM CARBONATE 385

salts was intensely investigated in Europe, where it

became accepted as an effective and safe treatment for
manic depressive illness.

Lithium salts were not accepted in the United States

until 1970. This was due in part to concerns about
the safety of lithium salts after several deaths were
reported (1949-50) among patients using large amounts
of lithium chloride as a salt substitute. Although
several lithium salts such as the chloride and bromide
have been used, lithium carbonate is preferred because
of its relative stability (it is less hygroscopic than
the halogen salts) and it is less irritating to the
gastrointenstinal tract.

7. Pharmacology (69, 70)

Lithium carbonate is usually administered as 300 mg

tablets or capsules. Each 300 mg of the carbonate
contains 8.12 mEq of lithium. The usual daily dose of
lithium carbonate for prophylactic therapy is 600 mg
to 1200 mg.

Lithium is readily absorbed after oral administration

in the gastrointestinal tract, peaking in the plasma
within 1 to 3 hours and tends to distribute evenly
throughout the total body water space. Lithium is not
bound to plasma proteins. There is some lag in
penetration into the cerebrospinal fluid, but there is
no absolute barrier to its entry into the brain.
Equilibration of lithium between the blood and the
brain is almost complete within 24 hours.

The "metabolism" of the lithium ion is almost entirely

via the kidneys. About half of a single dose of lith-
ium is excreted in 24 hours. An important feature of
the renal excretion of lithium is that its rate is not
typically increased by the administration of most
diuretics. Increased administration of sodium appears
to have minimal effect on the normal excretion of
lithium, while depressed in-vivo levels of sodium fac-
ilitate lithium retention. These physiological fac-
tors have important implications for the management of
lithium intoxication. The therapeutic plasma range
(0.5 - 1.5 mEq/L) and the toxic plasma levels (>1.6
mEq/L) are very close and monitoring is performed at
386 HENRY C. STOBER

the onset of therapy. The mechanism of action of

lithium ion in manic depression is not clearly under-
stood. Lithium interferes with the action of the
catecholamines in the brain. This supports the
popular hypothesis that in mania catecholamines may be
functionally overactive in the brain. The role played
by lithium may also be related to competition with
sodium ions in body sites, such as electrolyte balance
across cell membranes, including those of the neurons.

Acknowledgement
The typing assistance o f Mrs. Frances Ghiazza in the
preparation of this manuscript is appreciated.
LITHIUM CARBONATE 387

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Pretsch and W. Simon, Anal. Chem. Acta., 131, 117
(1985).

67. Waters L. S. Series Ion/Liquid Chromatographs,

Operators Manual, Chapter 3, Millipore Corp., Waters
Chromatography Div., Milford, MA (1985).

68. Lithium Research and Therapy, F . N. Johnson, ed.,

Chapters 1 and 2. Academic Press, New York, NY
(1975).

69. Handbook o f Psychopharmacology, L. L. Iversen, S. D.

Iversen and S. H. Snyder, ed., Chapter 6 , Plenem
Press, New York, NY (1978).

70. Lithium Research and Therapy, F . N. Johnson, ed.,

Chapters 16, 17, and 18, Academic Press, New York,
NY (1975).
This Page Intentionally Left Blank
 MAPROTILINE HYDROCHLORIDE

Soonhee K. Suh and James B. Smith

1. Description

1.1 Name, Formula, Molecular Weight

1.2 Appearance

2. Physical and Chemical Properties

2.1 Infrared Absorption Spectrum

2.2 Nuclear Magnetic Resonance Spectrum
2.3 Ultraviolet Absorption Spectrum
2.4 Mass Spectrum
2.5 Polymorphism
2.6 Melting Range
2.7 Differential Scanning Calorimetry
2.8 X-Ray Diffraction
2.9 Thermogravimetric Analysis
2.10 Dissociation Constant
2.11 Solubility
2.12 Partition Coefficient

3. Synthesis

4. Stability - Degradation

5. Drug Metabolism and Pharmacokinetics

5.1 Metabolism
5.2 Methodology
5.3 Pharmacokinetics
ANALYTICAL PROFILES OF DRUG SUBSTANCES Copyright 0 1986
VOLUME 15 by the American Pharmaceutlcal Association
393 All rights of reproduction in any form reserved.
394 SOONHEE K. SUH AND JAMES B. SMITH

6. Toxicity

7. Methods of Analysis

7.1 Identification
7.2 Elemental Analysis
7.3 Nonaqueous Titration
7.4 Phase Solubility Analysis
7.5 Thin-Layer Chromatography
7.6 Gas Chromatography
7.7 High Pressure Liquid Chromatography
7.8 Gas Chromatography -
Mass Spectrometry
7.9 Double Radio-Isotope Derivative Method
7.10 Colorimetric Assay

References
MAPROTILINE HYDROCHLORIDE 395

1. Description

1.1 Name, Formula, Molecular Weight

Maprotiline hydrochloride is 1-(3-methylarnino-
propyl)-dibenzo[b,e]bicyclo[2.2.2.]octadiene
hydrochloride.

CH2 CH 2 CH2NHCH 3 *HC1

I

C20H23N0HC1 Molecular Weight 313.87

1.2 Appearance
Maprotiline hydrochloride occurs as a white to
off-white, odorless, fine, crystalline powder.

2. Physical and Chemical Properties

2.1 Infrared Absorption Spectrum

The infrared absorption spectrum obtained from a
mineral oil dispersion of maprotiline hydro-
chloride on a Perkin Elmer Model 281B IR
spectrophotometer is shown in Figure 1. The
spectral assignments are listed in Table I.

Table I
-1
Wavenumber, cm Assignment

2720-2780 N-CH3

2455 -NH2 -
1596 Aromatic stretch

746-755 o-substituted benzene

ring
Percent
transmission
1O(

80

60

40

20

4000 3500
I
W ’ 2500
3000 , 2000
I 1800
I 1600
I 1400
1 1200
I 1000
I 800
I 600
I
MAPROTILINE HYDROCHLORIDE 397

2.2 Nuclear Magnetic Resonance Spectrum (NMR)

The IH-NMR spectrum of maprotiline hydrochloride
is shown in Figure 2. The spectrum was deter-
mined on a Varian CFT-20 NMR instrument at ambi-
ent temperature. The sample was dissolved in
deuterated dimethylsulfoxide containing
tetramethylsilane as an internal standard. The
spectral assignments are shown in Table 11.

Table I1

No. of
Chemical
~.
Shift Multiplicity Protons Assignment
(PPm)
1.65 m g, h
2.45 m e, f

2.66 S d

3.19 t C

4.24 t b

7.0 - 7.4 m aromatic H

9.7 s (broad) a

e f c a d a
H~-CH~-CH~-NH-CH~OHC~

* H H

2.3 Ultraviolet Absorption Spectrum

The ultraviolet absorption spectrum of maproti-
line hydrochloride in methanol (0.1 mg/ml) exhib-
its two maxima at 265 and 272 with A(l%,lcm)
values of 40.0 and 49.7, respectively. A typical
spectrum obtained from HP 8450A W/Visible
spectrophotometer is shown in Figure 3 .
398 SOONHEE K. SUH AND JAMES B. SMITH

I I I I I I I I I I
Id.0 910 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0
PPm

Figure 2
NMR Spectrum of Maprotiline Hydrochloride
MAPROTILINE HYDROCHLORIDE 399

Absorbance

0.70

0.60

0.50

0.40

0.30

0.20

0.10

1
%
I240 260 280 300 320 340 350
Wavelength (nm)

Figure 3
Ultraviolet Absorption Spectrum of Maprotiline Hydrochloride
400 SOONHEE K. SUH AND JAMES B. SMITH

2.4 Mass Spectrum

Figure 4 shows the mass spectrum of maprotiline
hydrochloride obtained at 70 ev on a Kratos MS25
spectrometer using solid probe insertion. An
interpretation of the fragmentation ions is given
in Table 111.

Table I11

m/z Species

277 M+
247 M - NHCH3
218

203

CH=CH~
19 1

178

70

44
MAPROTILINE HYDROCHLORIDE
401

Relative
Intensity

1 Ot

9c

80

70

60

50

40

30

20.

10-

0- r'

MassKharge
Figure 4
Mass Spectrum of Maprotiline Hydrochloride
402 SOONHEE K. SUH AND JAMES B. SMITH

2.5 Polymorphism
Maprotiline hydrochloride is polymorphic and
known to exhibit several crystal forms (1, 2, 3 ) .
A typical production batch is a mixture with
varying ratios of crystal form A and B. The two
crystal forms show no difference in solubility
characteristics at room and physiological temper-
atures. In the solid state, they show differ-
ences in IR, DSC and x-ray powder diffraction
patterns. Crystal form A is thermodynamically
stable under 104OC, while the B form is
metastable. Under conditions such as storage
and tablet compression, no transformation
of form B to form A, or the reverse, has been
observed. However, a transformation of form B
to form A has been observed upon melting.

2.6 Melting Range

MaDrotiline hvdrochloride melts between
-
237 246OC with partial decomposition.

2.7 Differential Scanning Calorimetry (DSC)

The existence of polymorphism has also been shown
by differential scanning calorimetry. A typical
batch of maprotiline hydrochloride frequently
exhibits two melt endotherms indicating the pres-
ence of two crystal forms. The thenogram shown
in Figure 5 was produced on a Perkin Elmer DSC-2,
scanning 10°C per minute. Crystal forms A and B
show extrapolated melting points at 242OC and
239OC, respectively.

2.8 X-Ray Diffraction

The x-ray powder diffraction pattern obtained
for a typical batch of maprotiline hydrochloride,
as shown in Figure 6, is relatively weak with
the strongest reflection occurring at approxi-
mately 16.4 k 0.2 degrees 28. The characteristic
reflection patterns attributable to crystal form
A and B , although very weak signals, appear at
3.85 and 3.60 angstrom, respectively. They are
the basis for quantitative determination of a
mixture by x-ray diffraction (4).

2.9 Thermogravimetric Analysis

The TGA of maprotiline hydrochloride typically
exhibits a mean weight l o s s of about 0.2% between
MAPROTILINE HYDROCHLORIDE 403

0 ; I I I 1 I I I I I
310 340 370 400 430 460 490 520 550 580
TemperatureOK
Figure 5
DSC Scan of Maprotiline Hydrochloride

I I 1 I I 1 I I
5 10 15 20 25 30 35 40
Degrees Two Theta

Figure 6
X-ray Powder Diffraction Pattern of Maprotiline Hydrochloride
404 SOONHEE K. SUH AND JAMES B. SMITH

room temperature and 175OC. Above 175OC a r a p i d

weight l o s s due t o decomposition and/or
s u b l i m i n a t i o n i s observed.

2.10 D i s s o c i a t i o n Constant
The pKa v a l u e determined i n water a t 25OC i s 10.5
*0.2-(5). Also, v a l u e s of 9.0 k 0 . 1 and 9.4 k
0.1 determined i n 80% e t h y l e n e g l y c o l monomethyl
e t h e r i n water have been r e p o r t e d (1, 6 ) .

2.11 S o l u b i l i t y
Maprotiline hydrochloride i s f r e e l y soluble i n
methanol and chloroform, s l i g h t l y s o l u b l e i n wa-
t e r , and p r a c t i c a l l y i n s o l u b l e i n i s o o c t a n e .
S o l u b i l i t i e s i n some commonly used o r g a n i c sol-
v e n t s and aqueous s o l u t i o n s a r e shown i n Table I V .

Table I V

Solubility Refer-
(mg/ml) ence
Chlorof orm 25 -- 100 7
Chloroform 30 142 9
Methano 1 25 -- 100 7
Dimethylformamide 25 -- 30 7
Acetone 25 -- 0.1 7
Ether 25 -- 0.02 7
Water 25 5.6 10.3 8
Water 30 5.7 11.1 9
Water 37 5.6 13.2 8
0.1N H C 1 25 1.0 3.1 8
0.1N H C 1 37 1.0 4.4 8
Gastric Fluid 37 1.1 3.7 1
(simulated)

I n t e s t i n a l Fluid 35 7.5 5.3 1

(simulated)

Phosphate B u f f e r RT 7.4 1.3 1

Phosphate B u f f e r RT 11.0 0.29 10-4 1

MAPROTILINE HYDROCHLORIDE 405

2.12 Partition Coefficient

The partition coefficient data shown in Table V
were obtained at 25OC as the ratio of the concen-
tration found in the organic phase to that of in
the aqueous phase (5, 9 ) .

Table V

Organic Aqueous @ Partition

Phase Phase Coefficient

Chlorof o rm Water -- 0.30

Chloroform 0.1N HC1 -- 4.7
Hexane Water -- 0
n-Octanol 0.1 Hydrochloric 1.1 15.0
Acid
n-Octanol 0.1M Glycine 3.0 3.51
Buffer + HC1
n-Octanol 1/15M Phosphate 5.2 1.25
Buffer
n-Octanol 1/15M Phosphate 7.4 27.3
Buffer
n-Octanol 0.1M Glycine 9.0 708
buffer + NaOH

3. Synthesis
Maprotiline hydrochloride is synthesized by the fol-
lowing route shown below (10,ll):

CH=CHCHO CH=CHCH0

CH=CHCH=NCH3 CH~CHZCH~NHCH~*HC~
I I
406 SOONHEE K. SUH AND JAMES B. SMITH

4. -
Stability Degradation
Maprotiline hydrochloride is a very stable compound
and resists degradation even in very harsh environ-
ments (9, 12). When maprotiline hydrochloride was
refluxed for 94 hours in 0.1N HC1 and 0.1N NaOH, no
degradation product was observed in the sample re-
fluxed in acid, while one trace impurity was detected
in the sample refluxed in base. An aqueous solution
(0.5%) stored at 100°C for one week showed no
decomposition.

Maprotiline hydrochloride is stable after storage for

5 years at room temperature, one year at 5OoC, 12
weeks at 40°C/70% relative humidity and 600 f.c. light.

5. Drug Metabolism and Pharmacokinetics

5.1 Metabolism
The lipophilic properties of maprotiline produced
by the tetracyclic-propyl portion of the molecule
account for the in vivo distribution behavior of
this antidepressant. In rodents, following
intravenous administration, maprotiline is
quickly distributed into organs, with the highest
concentrations found in the lungs, adrenal and
thyroid glands, and heart. The lowest concen-
trations are found in the blood. High tissue
levels of maprotiline as compared to blood appar-
ently also occur in man, as indicated in a post-
mortem study of a suicide from an overdose of
maprotiline ( 1 3 ) . Following intravenous admin-
istration of 14C-labeled maprotiline in man, 57%
and 30% of the dose, respectively, were found in
urine and feces after 21 days. Maprotiline is
excreted in urine predominantly (90%) as
metabolites, 75% of which are glucuronides.
Principal metabolic transformations involve
N-demethylation, deamination, aromatic as well as
aliphatic hydroxylations and aromatic methoxy
derivative formation ( 1 3 ) .

5.2 Methodology
The method most often used in the determination
of the pharmacokinetic parameters of maprotiline
has been a double radio-isotope derivative tech-
nique coupled with thin layer chromatography for
specificity ( 4 8 , 14). A similar double
radio-isotope method, with a gas chromatographic
MAPROTILINE HYDROCHLORIDE 407

determination for specificity, has been described

for the tricyclic nortriptyline (15).

Using an internal standard and a nitrogen detec-

tor, maprotiline in plasma has been determined by
gas chromatography as the free base (33). When
compared to the double radio-isotopic technique,
this procedure was found to be comparable in the
range of 20 to 150 ng/ml (16). Nitrogen detec-
tion by gas chromatography after reaction with
acetic anhydride was sensitive to 50 ng/ml for
the determination of maprotiline and its
N-desmethyl metabolite in blood (30).

Sensitivity to 10 ng of maprotiline in biological

samples was achieved using gas chromatography and
electron capture detection. Maprotiline was
chromatographed as the heptafluorobutyramide af-
ter reaction with heptafluorobutyric anhydride
(17, 34). Using the same anhydride with either
N-desmethylclomipramine or isotope labeled mapro-
tiline as the internal standard, maprotiline can
be determined with the combination of gas chrom-
atography and chemical ionization mass spectro-
metry in biological fluids at concentrations of
0.5 to 150 ng/ml (46).

Reversed phase high perfomance liquid chromato-

graphy on a Nucleosil C-18 column with 35% ace-
tonitrile, 65% 0.05 M phosphate buffer (pH 2.7) .
has also been utilized for the determination of
maprotiline in human plasma. Ultraviolet absorb-
ance at 214 run provided sensitivity to 2 ng/ml of
plasma after isolation of maprotiline as the free
base (41).
5.3 Pharmacokinetics
The biological half life of maprotiline following
a single intravenous dose of 50 mg to 6 healthy
volunteers was found to be 43 hours (13). The
half life of 40 hours reported in a similar study
using normals and a 75 mg intravenous dose, is in
excellent agreement (14).

Availability of an oral dose of 50 mg of maproti-

line compared to an equal intravenous dose was
comparable as determined by AUC measurements
(13). Peak levels of maprotiline appear in blood
or plasma relatively slowly following oral admin-
408 SOONHEE K. SUH AND JAMES B. SMITH

istration. Maximum maprotiline levels have been

found to occur within about 8-24 hours post ad-
ministration (13, 18, 19).

The relatively large apparent volume of distribu-

tion (13, 14, 19) indicates maprotiline is highly
tissue-bound, as the aforementioned post-mortem
study indicated (13).

When normal subjects received simultaneously a 50

mg maprotiline tablet and 50 mg of tridueterated
maprotiline hydrochloride in an aqueous solution,
blood levels for the isotope labeled and unla-
beled drug were essentially superimposable. Peak
blood level averaged about 50 ng/ml (19).
In five depressive patients receiving seven day
incremental doses of 25 mg, 50 mg, and 75 mg,
plasma levels of maprotiline were clearly dose
related. At the end 7, 14, and 21 days, maproti-
line plasma levels were respectively, 18-46
ng/ml, 64-103 ng/ml and 96-155 ng/ml. Although
therapeutic activity was observed at all plasma
levels (18-155 ng/ml), the incidence of side ef-
fects appeared to be related to the 75 mg twice
daily dosage regimen (20). Proportional blood
levels have also been reported under steady state
conditions (about 7 days) for maprotiline doses
of 50 mg, 100 mg, and 150 mg (13).

Several reviews and summaries of the pharmacolo-

gy, pharmacokinetics, toxicity, and efficacy of
maprotiline are available (21, 22).

6. Toxicity

The standard intravenous LD50 established

for maprotiline hydrochloride in male rats is 27.3
mg/kg. Maprotiline hydrochloride is therefore
classified as moderately toxic when administered
intravenously to rats (23).

7. Methods of Analysis

7.1 Identification
Maprotiline hydrochloride is best identified by:
1)- infrared absorption; 2) ultraviolet absorption;
3 ) a test for chloride.
MAPROTILINE HYDROCHLORIDE 409

7.2 Elemental Analysis

The elemental composition determined for a typi-
cal sample of maprotiline hydrochloride on a
Perkin Elmer Model 240 CHN Analyzer is given below.

Element Theory, % Found, %

Carbon 76.53 76.71

Hydrogen 7.71 7.78
Nitrogen 4.46 4.44

7.3 Nonaqueous Titration

Maprotiline hydrochloride may be titrated with
acetous perchloric acid in glacial acetic acid
containing mercuric acetate. The titration can
be carried out potentiometrically using a glass
electrode and a calomel electrode containing gla-
cial acetic acid saturated with lithium chloride.

7.4 Phase Solubility Analysis

Phase solubility analysis of maprotiline hydro-
chloride has been carried out using the following
systems:

Solvent: n-propanol
Approximate Solubility: 1 6 . 4 mg/g
Temperature: 25OC

7.5 Thin-layer Chromatography

A number of thin-layer chromatographic systems
have been developed for the identification and
the determination of the drug and compounds re-
lated to the drug.

System I
Adsorbent: Silica Gel G or GF254, 2501.1
thicknes s

Mobile Phase: sec-Butyl Alcohol/Ethyl Acetate12N

Ammonium Hydroxide ( 1 2 0 : 6 0 : 2 0 )
saturated with ammonia vapor

Detection: a ) After exposing the plate under

hydrogen chloride vapor for 30
minutes, irradiate it with high
intensity W light for 5 minutes.
Observe under longwave W source.
b) Iodine vapor
410 SOONHEE K. SUH AND JAMES B. SMITH

System I1
Adsorbent : Silica Gel G or GF254 2 5 0 ~
thickness

Mobile Phase: sec-Butanol/Ethyl Acetate/5N

Ammonium Hydroxide ( 1 4 : 4 : 5 )
Detection: After exposing the plate under
hydrogen chloride vapor for 30
minutes, irradiate with high
intensity W light for 5 minutes.
Observe under longwave W source.
System I11
Adsorbent : Silica Gel GF254, 250p thickness

Mobile Phase: n-ButanolIAcetic Acid/Water

(70:10:20)

Detection: a) Iodine vapor

b) After exposing the plate to
chlorine vapor for 1-2 minutes,
spray with sodium hypochlorite
(2%), followed by a second spray
of a mixture of 10 ml of KI (1
in loo), 10 ml of soluble starch
( 3 in 100) and 3 ml of methanol.
c) W 254nm
System IV
Adsorbent: Silica Gel GF254, 250p thickness

Mobile Phase: Chloroform/Methanol (95:5) satu-

rated with ammonia gas

Detection: Same as System I11

System V
Adsorbent : Silica Gel HF, 250p thickness

Mobile Phase: n-Butanol/acetic acidlwater

(70:10:20)

Detection: Spray the plate with sodium hypo-

chlorite (2%) in 0.125N sodium
hydroxide solution. Dry the plate
at 4OoC for 1 hour. Spray the
plate with a mixture (1:l) of KI
(0.83%) and o-tolidine (0.2% in
2% acetic acid).
MAPROTILINE HYDROCHLORIDE 411

System VI
Adso rbent : Silica Gel HF, 250p thickness

Mobile Phase: BenzeneIDiethylamine (9:l)

Detection: Same as System V

System VII
Adsorbent : Silica Gel HF, 250p thickness

Mobile Phase: Chloroform/Methanol/Acetic Acid

(7:2:1)

Detection: Same as System V

System VIII

Ad so rbent : Silica Gel GF2s4, 250p thickness

Mobile Phase: Methanol/Ethylacetate/l,Z-

Dichloroethane/water (3:3:3:1)

Detection: Same as the detection method b ) in

System 111.

7.6 Gas Chromatography

System I: The following system has been used

to determine the drug substance
for routine toxicological analysis
(24).
Column: SE-30 coated fused silica capil-
lary, 3.5 to 15m x 0.25 mm ID

Temperature: Column programmed from 100°C to

295OC at 5OC/min. Injector & de-
tector temperatures not reported

Carrier: Helium, linear velocity p = 45

cm/second at 100°C

Detection: Flame Ionization

System I1 The following system has been used

f o r the determination of the drug
substance and other basic drugs in
liver (25).
412 SOONHEE K. SUH AND JAMES B. SMITH

Column : AR-glass capillaries coated with

0.3% (W/V) SE-52, 15 m x 0.26 mm ID

Temperature: Injector at 25OoC, detector at

3OO0C, column temperature
programmed by S°C/min. from 190
to 22OOC followed by 15OC/min. to
28OOC and then by 8OC/min. to the
final temperature of 3OO0C, at
which it was held for 5 min.

Carrier: Helium, linear velocity 35-40

cm/sec.

Detection: Nitrogen Phosphorus Detector

System I11 The following system has been used
for the identification of the drug
and its silylated derivatives
along with a number of other drug
substances and their silylated
derivatives (26).

Column: 3% OV-17 on Gas Chrom Q (100-120

mesh) Glass, 2 m x 3 mm ID

Temperature: Injection port and detector at

3OO0C, column at 3OO0C for iso-
thermal run or 120 to 27OOC at
10°C/min., hold at 27OOC for 16
min. for programmed run.

Carrier: Nitrogen, 30 mllmin.

Detection: Flame Ionization

System IV: The following system has been em-

ployed for the determination of
the drug substance in plasma,
derivatizing it with acetic
anhydride (27).

Column : 5% OV-17 on Gas Chrom Q (100-120

mesh), 1.83 m x 2 mm ID glass

Temperature: Injector and detector at 3OO0C and

column at 255OC

Carrier: Helium, 30 ml/min.

MAPROTILINE HYDROCHLORIDE 413

Detection: Alkali Flame Ionization

System V: The following system has been used
to determine the drug substance
and a number of other neutral and
basic drugs in blood ( 2 8 ) .

Column : a) 3% OV-101 on Chromosorb

(100-120 mesh), 2 m x 0.6 cm ID
glass.
b) 3% OV-17 on Chromosorb
(100-120 mesh) 2 m x 0.6 cm ID
glass.

Temperature: Injector and detector at 3OO0C and

-
column from 170 290°C programmed
at a rate of 4OC/min.

Carrier: Helium, 30 ml/min.

Detection: Nitrogen Phosphorous Detector

System VI The following system has been used
for the assay of the drug substance
in serum (29).

Column: 1.4% Carbowax 20M plus 1.4% KOH on

Gas Chrom Q (60-80 mesh), 1.8 m x
2 mm ID glass

Temperature: Injection port and detector at

25OoC and column at 210°C

Carrier: Nitrogen, 30 ml/min.

Detection: Flame Ionization

System VII The following system has been em-
ployed for the assay o f the drug
substance in biological fluids
derivatizing it with acetic
anhydride (30).

Column: 3% HI-EFF-8BP on Chromosorb W HP

(100-120 mesh), 0.30 m x 3 mm ID.

Temperature: Injector and detector at 3OO0C and

column at 245OC
Carrier: Nitrogen, 40 ml/min.
414 SOONHEE K. SUH AND JAMES B. SMITH

Detector: Nitrogen S e l e c t i v e Flame I o n i z a t i o n

System VIII The following system h a s been em-

ployed f o r t h e d e t e r m i n a t i o n of
t h e drug s u b s t a n c e i n plasma,
d e r i v a t i z i n g it w i t h h e p t a f l u o r o -
b u t y r i c anhydride ( 3 1 ) .

Column a ) 3% SP-2250 (OV-17) on

Supelcoport 80-100 mesh, 2 m x 4
mm I D
b) Open t u b u l a r column coatded
w i t h SE-30, 25 q x 0.37 mm I D

Temperature : I n j e c t o r and d e t e c t o r a t 3OO0C and

column a t 26OoC

Carrier: a) Helium, 50 ml/min.

b) H e l i u m , 1 . 5 ml/min.

Detection: Nitrogen S e l e c t i v e D e t e c t o r

System I X The f o l l o w i n g system has been used

f o r t h e d e t e r m i n a t i o n o f t h e drug
s u b s t a n c e and o t h e r drugs i n se-
rum, d e r i v a t i z i n g it w i t h
t r i f l u o r o a c e t i c anhydride (32).

Column: 3% OV-17 on Gas Chrom Q (100-120

mesh), 138 cm x 2 mm I D g l a s s

Temperature: I n j e c t o r a t 23OoC, d e t e c t o r a t
27OOC and column from 220 - 275OC
programmed a t 8OC/min.

Carrier: N i t r o g e n , 35 ml/min.

Detection: Nitrogen Phosphorous D e t e c t o r

System X The f o l l o w i n g system h a s been used

t o determine t h e drug s u b s t a n c e i n
plasma (33).

Column 3% OV-17 on gas chrom Q (100-120

mesh), 2 m x 2 mm I D g l a s s

Temperature : I n j e c t o r and d e t e c t o r at 3OOOC and

column a t 265OC
MAPROTILINE HYDROCHLORIDE 415

Carrier: Not r e p o r t e d

Detection: N i t r o g e n Phosphorous D e t e c t o r

System X I : The f o l l o w i n g system has been used

f o r t h e a s s a y of t h e drug sub-
stance i n biological f l u i d s
d e r i v a t i z i n g with heptafluorobu-
t y r i c anhydride (34).

Column: 3% JXR ( m e t h y l s i l i c o n e ) on Gas

Chrom Q , 4 ' x 3 mm I D

Temperature: I n j e c t i o n p o r t a t 25OoC, column a t

23OoC, d e t e c t o r a t 28OOC

Carrier: N i t r o g e n , 40 ml/min.

Detection: 6 3 N i E l e c t r o n Capture
System XII: The f o l l o w i n g system has been used
t o determine t h e drug s u b s t a n c e
and i t s t r i f l u o r o a c e t y l a t e d d e r i v -
a t i v e i n b i o l o g i c a l sample (35).

Column: 2% D e x s i l 300 on Chromosorb W ,

AW-DMCS (80-100 mesh), 2 m g l a s s
column (column I D n o t r e p o r t e d )

Temperature: I n j e c t o r a t 3 1 O o C , column a t 28OOC

and d e t e c t o r t e m p e r a t u r e n o t
reported
Carrier: N i t r o g e n , 30 ml/min.

Detection: Flame I o n i z a t i o n
System X I 1 1 The f o l l o w i n g system has been used
t o determine t h e drug s u b s t a n c e i n
plasma and u r i n e a s h e p t a f l u o r o b u -
t y r i c d e r i v a t i v e (36).

Column: a ) 0.75% OV-17 o r

b) 0.5% XE-60 and 0.25% DC
LSX-3-0295 on s i l a n i z e d Chromosorb
G (80-100 mesh), 320 cm x 1.8 mm
I D g l a s s column
Temperature: I n j e c t o r a t 255OC, d e t e c t o r a t
21OOC and column a t 245OC
416 SOONHEE K. SUH AND JAMES B. SMITH

Carrier : Nitrogen a t 10 - 15 ml/min.

Detect i o n : 3H E l e c t r o n Capture
System XIV: The following system h a s been used
t o determine t h e drug s u b s t a n c e i n
blood a s h e p t a f l u o r o b u t y r i c d e r i v -
a t i v e (17).

Column: 1.5% 08-225 on Supelcoport packed

i n a s i l a n i z e d g l a s s column, 1.8 m
x2mmID

Temperature : I n j e c t o r a t 250 - 26OoC, column a t

200 - 23OOC and d e t e c t o r a t
320 - 330OC.

Carri.er: Nitrogen

Detection: 63Ni Electron capture

7.7 High P r e s s u r e Liquid Chromatography
System I The following system h a s been em-
ployed € o r t h e a n a l y s i s of t h e p u r e
drug s u b s t a n c e and dosage forms
(37).

Column: ZorbaxB CN (DuPont) , 4.6 mm I D x

250 mm

Mobile Phase: Methanol/O.O8M sodium a c e t a t e

(95:5) w i t h t h e a p p a r e n t pH ad-
justed t o 7.5 ,.,
8.0

Flow Rate: 2 mljmin.

Detection: U l t r a v i o l e t a b s o r p t i o n a t 272 nm
System I1 The f o l l o w i n g system h a s been em-
ployed f o r t h e a n a l y s i s of t h e
pure drug s u b s t a n c e and dosage
forms ( 3 7 ) .

Column: Zorbaxb CN (DuPont), 4.6 mm I D x

250 mm

Mobile Phase: Tetrahydrofuran/O.O25M sodium ace-

t a t e (9O:lO) w i t h t h e apparent pH
adjusted t o 7.4 8.0 -
MAPROTILINE HYDROCHLORIDE 417

Flow Rate: 1 ml/min.

Temperature: 4OoC

Detection: Ultraviolet absorption at 272 nm

System I11 The following system has been em-
ployed for the analysis of the
drug substance and a number of
other basic drugs (38).

Column: Spherisorb 5 Silica, 4.9 mm ID x

250 mm

Mobile Phase: a) Methanol/Hexane (85:15) with

0.02 v/v% (1.85 mM) Perchloric Acid
b) Same as the above except 0.05
v/v% (4.63 mM) Perchloric Acid
c) Same as the above except 0.1
v/v% (9.25 mM) Perchloric Acid

Flow Rate: 2.0 ml/min.

Detection: Ultraviolet Absorption at 215 nm

System IV The following system has been em-
ployed for the analysis of the
drug substance and a number of
tricyclic antidepressants (39).

Column: Hypersil 5 I.rm ODs, 4.6 mm ID x 70

or 100 mm
Mobile Phase: a) Acetonitrile/lO mM Sodium
Dihydrogen Phosphate (50:50) at pH
2 with 80 mM of sodium lauryl
sulfate
b) Same a s the above except the
addition of 5 mM of tetrabutylam-
monium bromide

Flow Rate: 2.0 mllmin.

Detection: Ultraviolet Absorption at 254 nm

System V The following system has been em-
ployed for the determination of
the drug substance from biological
fluids (40).
418 SOONHEE K. SUH AND JAMES B. SMITH

Column: VBondapak CIS, 3.9 mm ID x 300 mm

Mobile Phase: Acetonitrile/O.l M Potassium Phos-

phate, pH 2.5 (30:70)

Flow Rate: 2 ml/min.

Detection: Ultraviolet Absorption at 205 nm

System VI The following system has been used

for the assay of the drug sub-
stance in human plasma (41).

Column: Nucleosil c18

Mobile Phase: Acetonitrile/O.O5M Phosphate Buff-

er, pH 2 . 7 (35:65)

Flow Rate: Not indicated

Detection: Ultraviolet Absorption at 214 nm

System VII The following system has been used

for the qualitative identification
of the drug substance and other
basic drugs (42).

Column: Three types o f columns have been

used.
a) 3-7 pm silica
b) mercaptopropyl bonded-phase
silica
c) aliphatic strong cation
(n-propylsulphonic acid) bonded-
phase silica, 5 mm ID x 250 m m

Mobile Phase : Methanol/ZM Ammonium Hydroxide/lM

Ammonium Nitrate (27:2:1)

Flow Rate: 1 ml/min.

Detection: Ultraviolet Absorption at 254 nm

System VIII The following system has been used

for the assay of biological sam-
ples by derivatizing maprotiline
with p-nitroazobenzenecarbonyl
chloride ( 4 3 ) .
MAPROTILINE HYDROCHLORIDE 419

Column: Lichrosorb SI 100 (5p), 3 mm ID x

100 mm and 3 mm ID x 300 mm con-
nected by two-way switching valve

Mobile Phase: 1.1% t-Butanol, 23% Dichlorometh-

ane, 0.05% Morpholine, 0.05% water
and make to 100% with Hexane

Flow Rate: Not indicated

Detection: Ultraviolet Absorption at 337 nm

System IX The following system has been used
for the analysis of the drug sub-
stance and a number of tricyclic
antidepressants (44)

Column: Partisil 5 pm, 3.0 mm ID x 150 mm

Mobile Phase: Dichloromethane/Methanol/Acetate

Buffer pH 3.2 (90+10+0.15)

Flow Rate: 1.0 ml/min.

Detection: Ultraviolet Absorption at 254 nm

7.8 Gas Chromatography - Mass Spectrometry (GC-MS)
System I The following system has been used
for the determination of the drug
substance and other neutral and
basic drugs in blood (28).

Column: 3% 33-30 on Chromosorb (100-120

mesh), 2 m x 0.6 cm ID glass

Temperature: Injector at 270OC and column from

220 -
270OC programmed at a rate
of 10°C./min.

Carrier: Helium, 30 ml/min.

MS EI and
CI Source: 70 eV
CI Reagent
Gas : Methane
Detection: EI selected ion-monitoring at
m/e = 59 and 191 CI selected
ion-monitoring at m/e = 278
420 SOONHEE K. SUH AND JAMES B. SMITH

System I1 The following system has been used

for the determination of the drug
substance and its major metabolite
derivatized with trifluoroacetic
anhydride using stable isotope-
labeled analog as internal standard
(45).
Column: 1% OV-17 on Gas Chrom Q (100 - 120
mesh), 1.8 m x 2 mm ID glass

Temperature: Injector at 24OoC, column at 255OC,

separator at 25OoC, and ion source
at 275OC

Carrier: Helium, 20 ml/min.

MS EI Source: 70 eV
Detection: EI selected ion-monitoring at
m/e = 345

System 111 The following system has been used

for the determination of the drug
substance in biological fluids,
derivatizing it with heptafluoro-
butyric anhydride. Isotope labeled
analogs of the drug substance were
used as internal standard (46).

Column: 1.5% Poly 5-179 on Chromosorb W AW

DMCS (80-100 mesh), 1 m x 2 mm ID
glass

Temperature: Injector at 250 - 26OoC, column at

240 - 25OoC, GC-MS interface at
245 - 27OoC and ion source at
245 - 265OC

Carrier: Not reported

MS CI Source: 40 - 100 eV

CI Reagent
Gas : Methane
Detection: CI selected ion monitoring at
m/e = 474
MAPROTILINE HYDROCHLORIDE 42 1

System I V The f o l l o w i n g system has been used

f o r t h e d e t e r m i n a t i o n o f t h e drug
s u b s t a n c e and o t h e r t r i c y c l i c an-
t i d e p r e s s a n t s i n blood plasma,
using deuterated i n t e r n a l stan-
d a r d s (47)

Column: 3% OV-17 on chromosorb HPW-DMCS,

(80-100 mesh), 40 cm x 2 mm I D
glass

Temperature: I n j e c t o r a t 3OO0C, column a t

24OoC, s e p a r a t o r a t 35OOC and i o n
s o u r c e a t 24OOC

Carrier: H e l i u m , 15 m l / m i n .

MS C I Source: 70 eV

C I Reagent
Gas :
. Methanol Vapor

Detection: C I s e l e c t e d ion-monitoring a t
m/e = 378/286 +
[ (M+1) Ludiomil/ (M+l) d e u t e r a t e d
Ludiomil]

System V The f o l l o w i n g system has been used

f o r t h e d e t e r m i n a t i o n of t h e drug
s u b s t a n c e and i t s t r i f l u o r o a c e t y -
lated derivative i n biological
sample (24).

Column: 1%OV-17, 2 m g l a s s ( s o l i d s u p p o r t
and column I D n o t r e p o r t e d )

Temperature: Column a t 15O-32O0C, programmed a t

a r a t e of 15OC/min. Temperature
f o r i n j e c t o r and i o n s o u r c e n o t
reported.

Carrier: Not r e p o r t e d

MS E I Source: 70 eV

Detection: E I s e l e c t e d ion-monitoring a t
m / e = 345
422 SOONHEE K. SUH AND JAMES B. SMITH

7.9 Double Radio-Isotope Derivative Method

The double radio-isotope derivative technique for
the assay of the drug substance in biological
material has been employed by Riess ( 4 8 ) . A bio-
logical sample spiked with a definite amount of
14C-labeled maprotiline standard was extracted
with heptane containing 1% amyl alcohol. The
extracted sample ( 14C-standard plus unknown) was
derivatized with 3H-acetic anhydride. After the
addition of an amount of unlabeled acetyl deriva-
tive of maprotiline as carrier, the sample was
purified by two-dimensional thin-layer
chromatography. The purified sample was analyzed
for both 3H activity and 14C activity by liquid
scintillation counting with the discriminator-
ratio method. Appropriately prepared standard
samples allow calculation of the overall individ-
ual yield of the 14C-internal standard in each
sample. B means of the 14C-yield found, the
41
recovered H activity value can be corrected to
the equivalent of a theoretical 100% yield. The
resultant 3H activity value can be converted to
the concentration units by the 100% 3H activity
value resulting from an appropriate number of
tests samples which have been treated in exactly
the same way as the unknown and contain a defi-
nite, known amount of trial compound.

7.10 Colorimetric Assay

Maprotiline hydrochloride can be assayed by the
dithiocarbamic acid copper complex method. Ma-
protiline, a secondary amine, undergoes the fol-
lowing reaction with carbon disulfide and cupric
ion in an alkaline medium.

0 0 KOH
0 0 cu
2

The absorbance of the colored complex is measured

at 437 run. The method is specific to the secon-
dary aliphatic amine.
MAPROTILINE HYDROCHLORIDE 423

Acknowledgement

The authors express appreciation to Frances Ghiazza

and Richard Brown for their help in the preparation of
this manuscript.

References

1. Stahl, P., CIBA-GEIGY, Ltd., Personal Communication.

2. Marti, E. and Stahl, P . , CIBA-GEIGY, Ltd., Personal

Communication.

3. Kuhnert-Brandstaetter, M., Wurian, I., and Geiler, M.,

Sci. Pharm. , 50(3), 208-16 (1982).

4. Mfiller, R., CIBA-GEIGY, Ltd., Personal Communication.

5. Jakel, K., Moser, P., and Stahl, P., CIBA-GEIGY, Ltd.,

Personal Communication.

6. Smith, J., Hagman, H., and Mollica, J., CIBA-GEIGY,

Personal Communication

7. Steiner, H., CIBA-GEIGY, Ltd., Personal Communication.

8. Stahl, H . , CIBA-GEIGY, Ltd., Personal Communication.

9. Mollica, J., Pucket, J., and Rhem, C., CIBA-GEIGY,

Personal Communication.

10. Beck, D., Bernasconi, R., Schenker, K., and Wilhelm,

M. (CIBA-GEIGY A.-G.) Ger. Offen. 2, 207, 111 (C1. C
0 7 C ) , 07 Sep., 1972, Swiss Appl. 257671, 23 Feb. 1971.

11. Wilhelm, M., and Schmidt, P . , Helv. Chim Aceta 52,

1385-95 (1969)

12. Cohen, J., CIBA-GEIGY, Personal Communication.

13. Riess, W., Dubey, L., Fcnfgeld, E. W., Imhof, P.,

Hfirzeler, H., Matussek, N., Rajagopalan, T. G.,
Raschdorf, F., and Schmid, K., J. Int. Med. Res., 3,
Supplement (2)16 (1975).
424 SOONHEE K. SUH AND JAMES B. SMITH

14. Maguire, K. P., Norman, T. R., Burrows, G. D.,

Scoggins, B. A., Eur. J. Clin. Pharmacol. g,
249 - 254 (1980).

15. Maguire, K. P., Burrows, G. D., Coghlan, J. P . ,

-
Scoggins, B. A., Clin Chem., 22/6, 761 764 (1971).

16. Witts, D. J., Proceedings of an International

-
Symposium Verdala, Malta, November, 1976, Edited
by Jukes, A., Published by CIBA Laboratorie, Horsham,
England, 169 -
173 (1977).

17. Alkaxay, D., Khemani, L., Volk, J. Analytical Letters,

-
15(B18), 1493 - 1503 (1982).

18. Jones, R. B., Luscombe, D . K., Proceedings of an

Intefnational Symposium, Verdala, Malta, November, 1976,
Edited by Jukes, A., Published by CIBA Laboratories,
Horsham, England, 135 -
143, (1977).

19. Alkalay, D., Wagner, W. E., Carlsen, S., Khemani, L.,

Volk, J., Bartlett, M. F., LeSher, A., Clin. Pharmacol.
Ther., 11, 5, 693 -
703, (1980).

20. Mathur, G. N., Jones, R. B., Luscombe, D. K., Proceed-

ings of an International Symposium, Verdala, Malta,
November, 1976, Edited by A. Jukes, Published by CIBA
Laboratories, Horsham, England, 157 - 168 (1977).

21. Wells, B. G., Gelenberg, A. J., Pharmacotherapy, (11,

2, 121 - 139 1981).

22. Stimmel, G. L , Drug Intelligence and Clinical Pharma-

CY, 14,585 - 590 (1980).
23. Huber, K., CIBA-GEIGY, Personal Communication.

24. Anderson, W. H., and Stafford, D. T., J. High Resolut.

6, 247-54 (1983).
Chromatogr. Chromatogr. Commun. -

25. Eklund, A., Jonsson, J., and Schuberth, J., J. Anal.

Toxicol. 7 , 24-28 (1983).

26. Dutt, M. C., J. Chromatogr.; 3,

115-24 (1982).

27. Charette, C., McGilveray, I. J., and Midha, K. K., J.

Chromatogr., 224, 128-132 (1981).
L
MAPROTILINE HYDROCHLORIDE 425

28. Cailleux, S., Turcant, A., Premel-Cabic, A., and

Allain, P., J. Chromatogr. Sci., 2 , 163-176 (1981).

29. KErkkainen, S . , and SeppPlE, E., J. Chromatogr., 221,

319-326 (1980).

30. Sioufi, A., and Richard, A . , J. Chromatogr. 221,

393398 (1980).

31. Rovei, V., Sanjuan, M., and Hrdina, P. D., J.

Chromatogr., 182, 349-357 (1980).

32. Conner, J.,. Johnson, G., and Solomon, H., J.

Chromatogr. 143, 415421, (1977).

33. Witts, D., and Turner, P., Br. J. Clin. Pharmacol., 4,

249-52 (1977).

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Chromatogr. 114, 167-173 (1975).

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33, 6571 (1974).
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36. Borga, O., and Garle, M., J. Chromatogr., 68, 77-88

(1972).

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Communication.

38. Flanagan, R., Storey, G., and Bhamra, R., J.

Chromatogr., 247, 15-37 (1982).

39. Hung, C. T., and Taylor, R. B., J. Chromatogr., 240,

6173 (1982).

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175-7 (1983).

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43. Hesse, C., proceedings of the 9th Materials Research

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1, 41724 (1979).
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426 SOONHEE K. SUH AND JAMES B. SMITH

45. Jindal, S. P., Lutz, T., and Vestergaard, P . , J.

Pharm. Sei. 69(6), 6847 (1980).

46. Alkalay, D., Carlsen, S., Khemani, L., and Bartlett,

M. F., Biomed. Mass Spectrom. 6(10), 4358 (1979)
47. Lapin, A., Eur. J. Mass Spectrom. Biochem., Med.
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48. Riess, W., Anal. Chim Acta -

68, 363-76 (1974).
 PENICILLIN G , POTASSIUM

(POTASSIUM BENZYLPENICILLIN)

JOEL KIRSCHBAUM

1. Introduction

1.1 History
1.2 Names, Formula and Molecular Weight
1.3 Appearance, Color, Odor and Precautions
1.4 Biosynthesis, Synthesis, and Commercial
Production
1.5 Reactions, Stability, Degradation and
Polymerization
1.6 Mode of Antibacterial Action
1.7 Non-Microbiological Effects
1.8 Pharmacokinetics and Metabolism

2. Physical Properties

2.01 Physical Properties of Crystalline

Benzylpenicillin, Potassium
2.02 X-Ray Powder Diffraction
2.03 Mass Spectrometry
2.04 Infrared Spectrometry
2.05 Thermal Analysis
2.06 Microscopy and Particle Size
2.07 Surface Area
2.08 Cohesion (Stickiness)
2.09 Hydration
2.10 Polymorphism

3. Spectrometry in Solution

3.1 Nuclear Magnetic Resonance Spectrometry (NMR)

3.2 Ultraviolet Spectrometry
3.3 Optical Rotatory Dispersion and Circular
Dichroism Spectrometry
ANALYTICAL PROFILES OF DRUG SUBSTANCES Copyright 0 1986
VOLUME 15 by the American Pharmaceutical Association
427 A11 rights of reproduction in any Corm reserved.
428 JOEL KIRSCHBAUM

4. Bulk Solution Properties

4.1 Solubilities in Aqueous and

Nonaqueous Solvents
4.2 Partition Coefficients
4.3 Ionization
4.4 Molecular Aggregation and Critical
Micelle Concentration

5. Methods of Analysis

5.1 Compositional Analysis

5.2 Titration
5.3 Colorimetric and Spectrophotometric
Methods
5.4 Chromatographic Methods of Analysis
5.5 Electrochemical Methods of Analysis
5.6 Biologically-Based Assays
5.7 Isotopic Assays
5.8 Contamination Methods
5.9 Comparison of Methods

6. Possible Future Analytical Problems

7. Acknowledgements

8. References
PENICILLIN G, POTASSIUM 429

1. Introduction

1.1 History

In 1928, Alexander Fleming at St. Mary's

Hospital in London noticed the partial lysis of
colonies of staphylococci on a plate that had been
contaminated by PeniciZZium notation. Previous
observations of the antagonism of the growth of
bacteria by fungi of the genus PeniciZZiwn had been
made between 1870 and 1895, however Fleming further
found that this fungus, when cultured, gave a
"mould broth filtrate" that was relatively non-
toxic, possessed selective antibacterial activity
and could be used as a local antiseptic. He
believed that "the trouble of making it seemed not
worthwhile". For the next few years, little new
information on the properties and purification of
this substance was obtained. As the second world
war approached, sulfa drugs were the only known
useful way to treat infections chemically.
Exploratory research by E.B. Chain, Howard Florey,
N.G. Heatley and E.P. Abraham, all at Oxford
University, yielded small quantities of impure
penicillin.
Because of the bombings of Britain, Florey and
Heatley went to the United States to obtain aid,
after a close escape in Lisbon from being arrested
for swimming topless. Work in the United States
with deep fermentation (rather than surface growth)
and corn steep liquor as a growth promoter, with
the efforts of personnel at Squibb, Pfizer, Merck
and several other organizations, resulted in the
eventual production of sufficient antibiotic for
both battle casualties, and, subsequently,
civilians. The Germans concentrated on sulfa
drugs, although there was sufficient fermentation
capacity, considering beer production.
Penicillin G was first crystallized at Squibb
(as a sodium salt) by MacPhillamy, Wintersteiner
and Alicino, where the existance of sulfur in the
molecule was first noted by Alicino due to its
characteristic odor. The B-lactam structure was
supported by R.B. Woodward, especially since many
other possible reactive groups could be eliminated
by such evidence as potentiometric titrations that
indicated the absence of a basic functionality.
430 JOEL KIRSCHBAUM

The structure of benzylpenicillin was established

by Dorothy Hodgkin and Barbara Lois in May, 1945, '
using three-dimensional x-ray crystallography.
Thus, it was shown that penicillin contained a
previously unknown ring system. The B-lactam ring
is the source of antibacterial activity.
Fleming, Chain and Florey shared the Nobel
prize in 1945. Fleming received popular honors for
the discovery of penicillin. For example, in the
fishing village of Gijon, Spain, the aggrandized
photograph of Sir Alexander was paraded about at
the yearly fiesta with the caption, "To the Holy
Virgin we pray: for us, many Sardines; for the
wizard who gave us penicillin, Glory".

1.2 Names, Formula and Molecular Weight

Penicillin G Potassium is the United States

adopted name (10). The preferred chemical names
are (1) 4-Thia-l-azabicyclo[3.2.Olheptane-2-
carboxylic acid,-3,3-dimethyl-7-0~0-6-[(pheny-
lacetyl)amino]-,monopotassium salt , [ 2S-(2a, Sa,
68)]-; and (2) Monopotassium (2S,5R,6R)-3,3-
dimethyl-7-oxo-6-(2-phenylacetamido)-4-thia-l-
azabicyclo[3.2.0]heptane-2-carboxylate. The
chemical abstracts systematic number for potassium
penicillin G is CAS-113-98-4; CAS-61-33-6 is for
penicillin G itself. Below is the structure,
C H KN 0 S, with molecular weight of 372.48
dAftA&s f l f ) . Other names include benzyl-
penicillin potassium,

0l6 H H PU-9
H I I H i I / :
aC-C-N-C, c 5
H17 15 14
22 23

potassium benzylpencillinate, benzylpenicillinic

acid potassium salt, Cilloral, Cosmopen, Cristapen,
Crystapen, Eskacillin, Forpen, Hipercilina,
Hydsorb, Hylenta, Liquapen, M-Cillin, Megacillin
tablets, Monopen, Notaral, Penisem, Pentid, Scotil,
SK-Penicillin G, Sugracillin and Tabilin. Benzyl-
PENICILLIN G, POTASSIUM 431

penicillin has been formulated in tablets (12),

ointments (13,14), lotions (15) and intravenous
solution (16). It has been added to drinking water
(17) and sutures (181, and conjugated to lysine to
minimize immune reactions (19).

1.3 Appearance, Color, Odor and Precautions

Potassium benzylpenicillin is a white, free-

flowing crystalline powder with a faint pungent
odor characteristic of penicillins. The compound
itself should not be mixed with acids, alcohol,
bases, ephredrine, glycerol, iodine and iodides,
naphthalene oils, oxidizing agents, resorcinol,
salts of heavy metals, vitamin B 1 and zinc oxide
(cf. Section on Stability).
The significant adverse reaction to benzyl-
penicillin is the induction of an allergic reaction
as servere as anaphylactic shock leading to death.
Numerous studies (20) have shown that high and low
molecular weight compounds were responsible (21);
including penicillin itself, penicilloyl
derivatives (such as penicilloyl-lysine and
penicilloyl-albumin), oligomers and degradation
products (such as penicilloic acid, benzyl-
penicilloate, monopenicilloyl amides, D-
penicillamine and D-penicillamine-L-
cysteine) (22, 23) which can elicit an immediate
reaction in patients allergic to penicillin. As
expected, different commerical preparations of
benzylpenicillin vary in their ability to evoke
allergic reactions (24).

1.4 Biosynthesis, Synthesis, and Commercial

Production

1.41 Biosynthesis

Beta-lactam compounds are formed in

nature as secondary metabolites of both
eukaryotic and prokaryotic organisms. The
penicillins, cephalosporins and Ja-methoxy-
cephalosporins are products of biosynthetic
pathways with many identical steps (26). The
reaction of an activated form of
L-a-aminoadipic acid (11, Fig. 1 ) with
L-cysteine to form a common precursor
 Activated Form of 8-(L-a-Aminoadipy1)-L-Cysteine
L-ct-Aminoadipic Acid L-u-Aminoadipic Acid Y = OH (Activated Form: Y = SR or OR)
I II 111

+(L-o-Aminoadipy1)-L-Cysteinyl-D-Valine lsopenicillin N Penicillin G

IV V

F i g u r e 1. B i o s y n t h e s i s of P e n i c i l l i n G.
PENICILLIN G, POTASSIUM 433

6-(L-a-aminoadipyl)-L-cysteinyl-D-valine (IV,
27). Cyclization proceeds via an enzyme,
cyclase, dependent on ferrous ion and
ascorbate oxidation to yield isopenicillin N
(28). Isopenicillin N is converted to
penicillin N through the action of an
epimerase which converts the L- to a D- form,
leading to both penicillins and cephalosporins
(29), Fig. 3. Interconversions between
penicillins are possible using enzymes (30) in
the reactions shown below: 6-APA is
6-aminopenicillanic acid,
35
[ SlPenicillin V + 6 5 Y A ~ P 4 V y+ [35S] 6-APA
Penicillin G + [ Sl6-A A d [ SIPenicillin G +
6-APA
35
[ SIPenicillin V+ a Penicillin V+[ 35S]
Penicillin G Penicillin G

A chemical synthesis similar to the

biosynthetic sequence involving the carbon-
hydrogen bond-breaking steps of IV at the
cysteinyl-6- and valinyl-&positions has been
devised (31).

1.42 Synthesis

The first successful synthesis of

penicillin (25) is shown in Figure 2 and, when
modified, can be used for related penicillins.

1.43 Commercial Production

Originally, penicillin G was so rare that

it was repurified from the urine of patients.
Now, with large-scale production (32), the
cost is 2 0 ~(U.S.) a day based on a four
tablet-a-day regimen and a 100% profit margin.
Penicillin G is also used to synthesize other
B-lactam antibiotics. Radioayhive penicillin
G has been produced from L[1- C]-valine by
mycelia of PeniciZZiwn chrysogenwn immobilized
in an alginate gel (35).
-
Phenoxylacetyl
chloride
HCI
NaOH

carbodiimide

Na+
C02H C02H
Penicillin Salt

Figure 2 . Synthesis of a Penicillin..

PENICILLIN G , POTASSIUM 435

1.5 Reactions, Stability, Degradation and

Polymerization

1.51 Reactions,

Potassium penicillin G can be converted

to the sodium salt using cation exchange resin
(36,37) and to other salts (38,39).

Antimicrobial activity is lost by hydro-

lysis of the 8-lactam ring, which is catalyzed
by various 8-lqctamases induced or naturally
occurring i n V Z V O (40-42); cf. stability
section V . i . for studies of hydrolysis i n
VitrO. Other reactions of the 8-lactam group
include aminolysis (43,44) and amino group
conjugation (45,46). Benzylpenicillin has
been converted to penicillanic acids (47) and
penicillamine $48,491 enzymatically, to
5,5-dimethyl4 -thiazoline-4-carboxylic acid
(50) and, via an oxygen bridge at C-6, to a
dimer (51).
Removal of the acyl side chain, either
chemically (52,53) or enzymatically (54-56)
leaves an active nucleus, 6-aminopenicillanic
acid, that can be reacted with other groups to
create various semi-synthetic penicillins.
The side chain can undergo various reactions
(57-59).
The carboxylic acid moiety may readily be
converted to esters (60,61). The 8-lactam can
be reduced (62) to an amino alcohol and the
sulfur oxidized (63-65). Penicillin sulfoxide
can be converted to cephalosporins (66) as
well as the protected ester (67). To
investigate monocyclic 8-lactams, penicillin
was desulfurized with Raney nickel (68).
Benzyl-penicillin reacts with metals like
mercury (11) acetate to form the azetidinone
(69) via 1,5-bond scission (70), cf. the
stability section above for less lucid
metallic effects.
A penicillin antigen can be formed by the
conversion of benzylpencillin to D-benzyl-
436 JOEL KIRSCHBAUM

penicillenic acid followed by u-ipling with

8-alanine (71). Other artificial antigens
were created via coupling with lysine (72),
polyacrylamide (73) and diaminocarboxymethyl-
Ievan (74).

Noncovalent bonding of benzylpenicillin

was studied in microorganisms (75), to serum
albumin by NMR (76) with the binding sites
localized (77), to serum (78) and to tissues
(79) as well as bacterial proteins (cf.
section on mode of action).

1.52 Stability and Degradation

The kinetics and stability of penicillin G

have been studied extensively. Even the effect of
high concentrations of potassium benzylpenicillin
was investigated, since it was found that micellar
penicillin G is 2.5 times as stable as the non-
micellar solution under the conditions of constant
pH and ionic strength ( 8 0 ) .

Effect of pH

The stability of a commercial preparation of

potassium penicillin G in water, 0.1 M NaOH and 0.1
E HC1 was studied using ultraviolet sgectrophoto-
metry (81).
Solvent Wave- Time (hr)
and Concen- length
tration (nm) -
0 -
1 3-5 -2 -4
Water 262 Molar 164 163 163 164 164
2.7 x 10-3M 256 Abs. 251 250 250 251 250

0.1 HCA4 273 Molar 1435 1406 1385 1392 1343

1.4 x 10 M Abs.

0.1 M NaOF 263* Molar 160 160 162 164 178

2.9x 10- M 257 Abs. 223 223 222 223 234
251, 218 217 215 214 219
246 211 209 205 203 200
* Shoulder, Instrument: Perkin-Elmer Model 320
PENICILLIN G, POTASSIUM 437

From these data, and especially from the

studies discussed below, benzylpenicillin is shown
to be most stable at neutral pH. Low temperature
also enhances stability. The activation energies
were reported to be 17.6, 21.0 and 22.7 kcal/mole
at pH 1.2, 4 . 5 4 and 9.57, respectively.

Several pathways have recently been proposed

for the degradation of benzylpenicillin (I) at low
pH. Evidence for scheme 1 is based on kinetics and
the existance of penicillamine as demonstrated by
HPLC and differential pulse polarography ( 8 2 ) . An
alternative route to benzylpenillic acid involves
intermediate I1 depicted in scheme 2 ( 8 3 ) .
Evidence is based on (a), the increased stability
of benzylpenicillins by the incorporation of
electron-withdrawing suhstituents in the a-position
of the amide side-chain and (b), that benzyl-
penicillinic acid in ethanol yields benzylpenillic
acid in 25% yield. Scheme 3, showing the
conversion of benzylpenicilloic acid to benzyl-
penillic acid via a carbinolamine intermediate
(IV), is based on HPLC data ( 8 4 ) showing such a
degradation at ,pH 2.5 of benzylpenicilloic acid to
benzylpenillic acid. Another alternative scheme
(851, not shown here, lacks such a pathway for the
formation of benzylpenillic acid from benzyl-
penicilloic acid, although the kinetics are
supported by NMR.
438 JOEL KIRSCHBAUM

Bsnzylpenlclllln Bsnzylpm;;~ Acld

P " y ' )HOpC

=\,y

Bsnzylpinamaldlc Acld
H COpH Ph JYZCOzH
Banzylpenllllc Acld

BenzylpenlcIIIolc Acid

Penlclllarnlna + Benzylpenllloildehyde
COzH
+cop
Bsnzylpsnlllolc Acld

8.nzylpenllllo Acld 111

Scheme 2
H

ti+
:0: 0enzylpenkillolc Acld
ti' 'H
0~nzylp.nklllln
J
cozn
0mqlpenolllc Acld H IV

Scheme 3
Figure 3. Possible Pathways for the
Degradation of Benzylpenicillin
PENICILLIN G , POTASSIUM 439

Below are tabulated other selected references

describing the stability of benzylpenicillin under
various conditions.

Stability Condition Comments Reference

Bulk (Pure) P e n i c i l l i n
pH, ionic strength, temp. Study side-chains 86
Acid Study mechanism 87
Acid Study kinetics 88
PH Kinetics 89,90
Ionic strength, temp. Effect ionic 91
strength
Acids, bases, temp. Determine best 92
stability
Buffers Effect phosphate 93
buffers
Intravenous Solutions
Dextrose ti Sucrose, Used HPLC 94
Inactivated
In 10 Solutions Hydroxylamine assay 95
In 7 Solutions Retained activity 96
In 4 Solutions Student experiments 97
In 3 Solutions Study effect time 98,99
and temperature
In 2 Solutions 6-8 hr stability 100
In dextrose + 10 drugs Predict stability 101
In minibags Effect temp. 102
In dextrose Effect pH 103
Hyperalimentation Effect temp. 104-107
and nutrients
Y-Injection Study 108-110
incompatibilities
Unit Doses Effect time 111

Other Fomulations
Sensitivity discs Effect temperature 112
Topical Metals degrade 113
Suppositories Effect excipients 114
440 JOEL KIRSCHBAUM

Temperature
Heat Study kinetics solids 115
Heat Effect water 116
Heat Predict stability 117
Freezing Effect dextrose, 118
NaCl with time
Freezing Effect mode freezing 119
Freezing Effect various thaws 120
Freez ing Study phase 121
transitions
Chemicals (also see Intravenous Solutions)

Alcoho1s Little effect 122

Amikacin Little effect 123
Ascorbic Acid Little effect 124
Broth High productivity = 125
low stability
Broth Production depends on 126
syn. rate and
pencillin stability
Magnesium Sulfate Depends on pH 127
Metals Penicillin + 128
penicilloic acid
Metals In culture medium 129
Preservatives Crystallize 130
Silica Surface acidity 131
causes degradation
Sucrose 1:l Molar complex 132
Sucrose Study kinetics 133
pH 6-10
Surfactants Stability depends on 134
ionic type
Surfactants Micelles-penicillin 135
3 1pplex
Tobramycin [ 8-methyl- HI [ Clbenzylpenicillin 136
Hydroxyl.amineprevents loss
Tromethamine Aminolysis 137
Water Causes degradation 138
PENICILLIN G , POTASSIUM 44 1

Radiation
Solid State Decomposition & 139,140
dimerization
uv Bacterial inhibited 141
none

Th deute ium isotope effect with pH (pH 4-10)

was studied (142) and demonstrated a water-catalyzed
rearrangement of penicillin G to benzylpenicillenic
acid (scheme 1). A microcomputer was used to help
predict the stability of penicillin formulations
(143). De rada on products of double-labeled
3 f.i
[B-methyl- HI [ C]benzylpenicillin were separated
(144) using a cation-exchange column (ef. thin-layer
and high-performance liquid chromatography section
for additional references) after storage under
various conditions.
Of particular importance in the treatment of
infection with penicillin G is the complex interplay
possible with certain other drugs (145).

1.53 Polymerization

Polymers of such 8-lactam antibiotics as benzyl-

penicillin elicit a passive cutaneous anaphylactic
reaction in sensitized animals. Various methods have
been used to clarify the antigenic determinants
relative to the allergic reactions evoked by the
administration of penicillin G containing polymers
and 8-lactam reaction products with proteins.
Dextran gels Sephadex G-10 and G-25
(size-exclusion chromatography) was used to separate
various reaction products of penicillin G ( 1 4 6 ) .
Aged, 25% solutions of penicillin G were
chromatographed on G2000SW and G-25 columns (147).
Polymer contents increased with duration of storage.
These major fractjons were found after 14 days
storage in the dark, following chromatography on
Sephadex G-25. Two fractions consisted of equimolar
phenylacetyl and thiazolidine moities and had C:N:S
ratios similar to that of penicillin G. These
polymers appeared to be trimers and decamers. The
third fraction was mainly N-formylpenicillamine,
benzylpenilloic acid, benzy penicilloic acid and
1
benzylpenillic acid, using H NMR and IR spectra, and
thin-layer chromatography (148). These appear to be
442 JOEL KIRSCHBAUM

products resulting from the opening of the 8-lactam

ring, as was previously suggested in a prior study of
penicillin polymers (149) using gel exclusion
separations and I R , W and NMR techniques.

1.6 Mode of Antibacterial Action

Penicillin enabled the cure of many common,

often serious infections such as meningococcal
meningitis, pneumococcal pneumonia, group A
streptococcal pharyngitis as well as most
staphlococcal and many skin infections. Prior to the
advent of penicillin, bacterial endocarditis was
almost uniformly fatal, with less than a 1%rate of
spontaneous healing; however, treatment with
penicillin resulted in cures. Unfortunately,
resistance to penicillin G emerged rapidly. However,
synthetic modifications to the 8-lactam structure
preserved many of the therapeutic gains and, in some
compounds, produced new advantages.
The targets of 8-lactam antibiotics are the
enzymes involved in synthesizing the cell wall
surrounding most bacteria. The cell wall of gram-
positive bacteria consists of 5 0 to 100 molecular
layers of peptidoglycan, which is a long linear
polysaccharide chain of alternating N-acetyl-
glucosamine and N-acetylmuramic acid extending in one
direction, and cross-linked by short peptides in a
second dimension. These peptidoglycan strands have a
dipeptide terminus, acyl-D-alanyl-D-alanine, that may
assume a similar conformation to penicillin (150).
The peptide bond cleaved during transpeptidation
(which leads to incorporation and insolubilization
into the peptidoglycan cell wall) would be in the
same position as the highly reactive CO-NH bond in
the 8-Iactam ring of penicillin. Normally, the
transpeptidase is thought to react with its R-D-
alanyl-D-alanine substrate to form an acyl-enzyme
intermediate as the terminal D-alanine is
simultaneously eliminated (151). Cross-links are
formed as a free amino group from an adjacent glycan
strand reacts with an amino acceptor to yield the
transpeptidase product and concurrently regenerate
the enzyme. Penicillin inhibits this reaction,
presumably by acting as a structural analog of the
substrate, via reaction of the 8-lactam with the
active site, to form an inactive penicilloyl-enzyme
PENICILLIN G, POTASSIUM 443

(Fig. 4 ) . Inhibition of peptide cross-linking

results in the production of linear, soluble,
peptide-substituted glycan chains that are not linked
to the insoluble cell wall, but instead are excreted
as peptideglycan. This often results in the lytic
death of the microorganism. (Except when beta-
lactamases enable cell wall synthesis to proceed and
thus account for penicillin resistance). Evidence
for the peptidoglycan hypothesis is 1) the existance
of proteins that covalently bind penicillins ( 1 5 2 ) ,
2) the reversal by hydroxylamine of penicillin
binding to membranes to give a penicilloylhydroxamate
with the concomitant restoration of cell wall
biosynthetic activity, and 3 ) the binding of both a
penicilloyl moiety and an acyl moiety (derived from
substrate) to the same amine acid residue at the
active site of sensitive enzymes in stoichiometric
quar.tities ( 1 5 3 , 1 5 4 ) . Additional evidence is from
the evolutionary relationship between 8-lactamases
and penicillin-sensitive enzymes of cell wall
biosynthesis, as shown by sequence homology and
similar secondary structure. When 4 8 strains of the
soil bacterium Rhizobium japonicwn were screened for
their responses to several widely used antibiotics,
25% were found to be resistant to penicillin G,
tetracycline, neomycin, chloramphenicol and
streptomycin. The occurence of multiple drug
resistance in a soil bacterium that is not a
vertebrate pathogen suggested that the therapeutic
use of antibiotics may not be required to develop
multiple drug resistance ( 1 5 5 ) . Resistance appears
to depend ( 1 5 6 ) on 8-lactamase activity and
penicillin-binding protein alterations. Evidence
against this theory is the lack of analogy to the
dipeptide of portions of both rings of penicillin and
the slight activity of some penicillins and
cephalosporins with structural fyalogies to acy1-D-
alanyi-D-alanine. Solid state N-NMR studies using
L- [ E- 5N]lysine and benzylpenicillin indicated that
disruption of the mechanism for control of pepti-
doglycan synthesis probably is a contributing factor
in the death of bacteria ( 1 5 7 ) .

1.7 Non-Microbiological Effects

In addition to the antimicrobial activity of

potassium benzylpenicillin, in rabbits penicillin
\
CT
I
N
C
6I
w
0=0 I
:X
Z X' I
"-0
I
+ w
z0 c
rd
.rl
IZ c
0
I
a .d
\
a
0 .... 0
rz
I
CT I
U
PENICILLIN G, POTASSIUM 445

administered intramuscularly increased cytochrome C

oxidase activity and decreased the concentrations of
free SH and -S-S- (158). Red blood cells in healthy
children showed higher potassium concentrations after
the addition of therapeutic concentrations of
benzylpenicillin (159). Sodium excretion in urine
was significantly higher (160). A diet containing 4%
potassium penicillin G resulted in the inhibition of
collagen biosynthesis and cross-linking (161).
Suppression of intestinal bacteria by benzyl-
penicillin resulted in changes in the concentrations
of amino acids in tissues (162). Lipid metabolism in
microorganisms was also affected by penicillin G
(163). Symptoms of Lyme arthritis (spirochete
infection-caused) are relieved by penicillin (164).

1.8 Pharmacokinetics and Metabolism

In man, 15-30% of the total oral dose is

absorbed, due, to a great extent, to the low
stability of penicillin G in stomach acid. Lower
percentages are absorbed if benzylpenicillin is
administered during a meal (165-167). Disappearance
of drug from the gut lumen depends on the intrinsic
absorption rate and the non-enzymatic rate of
degradation (168). Approximately 60% of the
antibiotic is bound to serum proteins. Peak serum
concentrations after a 500 mg oral dose (fasting) are
1.5-2.7 pg/mL of total drug and 0.6-1 pg/mL of free
drug. This concentration can inhibit streptococci,
staphylococci and pneumococci. Infusion of 500
mg/kg/hour gave 16 pg/mL total drug and 5.6 pg/mL
free drug (165).
For potassium penicillin G , the half-life is 5
min. (Orally active penicillins typically give values
of 180 and 1160 min.), with the reaction following
pseudo-first order kinetics (169). The effect on
bacteria of this rapid decline in concentrations was
studied (170).
Distribution of injected potassium benzyl-
penicillin depends on the dose (171). Lower
concentrations are found in cerebrospinal fluid than
in serum (172), due to the blood brain barrier. High
doses of penicillin provide a minimum inhibitory
concentration. Bone shows no detectable penicillin
even when serum concentrations are as high as 9 pg/d
446 JOEL KIRSCHBAUM,

(173). Alveolar macrophages also show restricted

entry (174). Penicillin is principally excreted in
the urine (175), usually in unmetabolized form
(10-30%). Biliary excretion also occurs (176,177)
but is almost negligible. Biotransformation results
mainly in inactive penicilloic acids. Pharmaco-
kinetics were also studied in turkeys (178), horses
(179), swine (180), and rabbits (181). A synthetic,
oxygen carrying, resuscitation fluid (perfluorocarbon
emulsion Fluosol DA 20X) showed no significant
difference in pharmacokinetic parameters (182).

2. Physical Properties

2.01 Single Crystal X-Ray Diffraction

The original three-dimensional structure was

obtained by Dorothy Crowfoot Hodgkin and coworkers
(1831, which served to establish the structure of the
B-lactam ring and the location of the sulfur atom.
However, due to the large fluctuations in the
background intensity of electron density and the
abnormal interatomic distances, additional
refinements were required to define the benzene ring
(184). Fig. 5 depicts the structure obtained using
MO-:~ radiation measuring 1875 reflections using 1'
min 0-20 scans (185). The bond distances and
angles are tabulated in Table 1. Crystal data a e as
follows: P2 2 2 U/A' = 1,770.9, D /g cm-5 =
1.359; a = 9.103; (!!;, b =36.342 (3) andmc = 30.015
( a ) , and 2 = 4.
Of greatest interest is that four of the five
atoms of the thiazolidine ring tend to be co-planar,
the conformation of the thiazolidine ring depends on
the local environment and the interior angle of the S
atom is 94.7". The potassium ion is co-ordinated by
seven oxygen atoms. Biological activity correlates
with the conformation of the thiazolidine ring (186).
The three-dimensional structure was compared to the
spatial characteristics of glycyglycine, a model of
the D-alanyl-D-alanine terminus of the precursors of
bacterial cell-wall peptidoglycan cross-links (187)
and to acyl-D-alanyl-D-alanine (188) itself.
Structures were obtained for the related
6-aminopenicillanic acid (189), benzylpenicillin 1 '-
diethyl carbonate ester (190) and phenoxy- methyl-
anhydropenicillin (191), whose stereochemical
structure was related to other penicillins.
PENICILLIN G, POTASSIUM 447

These data may be used to calculate a

conformational energy map depicting the conformations
possible for the 8-lactam-thiazolidine ring system
with respect to the side-chain (192).

Figure 5 . Three-Dimensional, X-Ray Derived,

Structure of Potassium Penicillin G.
Table 1. Bond distances and angles

Distances (A)

s ( 1)-c (2) 1.84 7 ( 10)

s (1) -c (5) 1.818 (9)
C (2) -C (3) 1.57(1)
c (3) -N (4) 1.46(1)
N(4) -C (5) 1.45(1)
N (4) -C (7) 1.38(1)
C (5 1-C (6) 1.56(2)
C (6) -C (7) 1.52
C (7) -0 (8) 1.21
c (2) -c (9) 1.52
c (2)-C( l o ) 1.53
C (3)-C (11) 1.55
c ( 11)-0 (12) 1.23
C (1 1)-0( 13) 1.25
C (6) -N( 14) 1.43
N( 14) -C (15) 1.34
C ( 15) -0 ( 16) 1.22
C(15)-C(17) 1.51
C (17) -C (18) 1.45
C ( 18)-C (23) 1.33
C (23) -C (22) 1.37
c (22) -c (2 1) 1.28
C(2 1)-C(20) 1.32
C(20) -C( 19) 1.41
448 JOEL KIRSCHBAUM

Angles (") Degrees

c (2)-S (1)-c (5) 95.2(4)

s (1) -c (2) -c (3) 106.2(6)
c (9)-c (2) -c (10) llO(1)
C (2) -C (3)-N( 4) 105.2( 7)
C(2)-C(3)-C (1 1) 113.9 (7)
N(4)-C(3)-C(ll) 111.3 (7)
C (3)-N(4)-C (5) 119.4(7)
C (3) -N(4)-C (7) 125(1)
C (5) -N(4) -C( 7) 94.1(8)
s(1)-c(5)-c( 7) 105.1(6)
S (1) -C (5) -C( 6) 119.7 (6)
N(4) -C (5)-C (6) 88.3(7)
C (5) -C (6)-C( 7) 84.1(7)
C(5)-C(6)-N(14) 117.3(7)
C(7)-C (6)-N( 14) 115(1)
N(4) -C (7)-C (6) 93(1)
N (4)-C (7)-0 (8) 131 (1)
C (6)-C (7)-0 (8) 137(1)
C (3) -C ( 11) -0 ( 12) 117(1)
C(3)-C(11)-0(13) 116(1)
0 (12) -C (11)-0 (13) 127(1)
C (6)-N ( 14)-C ( 15) 121.1(1)
N( 14)-C (15) -C (17) 117(1)
C (17) -C (15) -0( 16) 122(1)
C (14)-C (15)-0 (16) 121(1)
C( 15) -C( 17)-C (18) 118(1)
C (17) -C (18)-C (23) 124(1)
C( 17)-C( 18)-C( 19) 122(1)
C (18)-C (23)-C (22) 128(2)
c (23) -c (22) -c (21) 115(2)
C(20) -C(21)-C(22) 125(2)
c (21) -c (20) -c ( 19) 118(2)
C (20) -C (19)-C (18) 120(2)
C(23)-C (18)-C( 19) 114(1)
2.02 X-Ray Powder Diffraction
To observe x-ray diffraction patterns, a Philips
APD 3720 Automated Powder Diffractometer unit
emitting CrK radiation at 2.2897"A was used (193).
The upper poQtion of Figure 6 shows the powder x-ray
diffraction pattern for potassium penicillin G, the
lower portion a stick plot of the crystalline peaks,
using the same data, where the y-axis (height)
represents intensity and the x-axis represents the
diffraction angle at the peak maximum in degrees 20.
Below are the data for the most intense peaks.
PENICILLIN G, POTASSIUM 449

1.00 - P E N I C I L L I N G POTASSIUM
- DEG 213 [ C R ) ->
0.81 .
0.64.
0.49 .
T
I
0.36.
N
T
0.25 . E
0.16. ;
I
0.09 . T
0.04
0.01
.
. A"r
1.00
0.81 .
0.64.
0.49 .
0.36.
0.25 .

25.0 30.0 35.0 40.0 45.0

9 .OO PENICILLI N G P O T A S S IU M -DEG 20 (CR)->

8.10 ?.
I
N
7.20 T
E
N
6.30 S
I
T
5.40 Y

4.50
I
3.60

2.70

1.80

0.90

0.0 10.0 20.0 36.0 40 .6 56 .0

Figure 6. Powder X-Ray D i f f r a c t i o n P a t t e r n s ; Upper

P o r t i o n s , Conventional P l o t ; Lower P o r t i o n , S t i c k P l o t .
450 JOEL KIRSCHBAUM

Peak Angle D Spacing (A) 111 Max (X)

8.6250 15.2248 100.0
26.1050 5.0692 20.9
28.7600 4.6098 10.08
44.6600 3.0132 9.74
29.9850 4.4255 8.95
36.1800 3.6870 8.63
28.4400 4.6606 5.54
40.2250 3.3294 5.09
29.7400 4.4611 4.81
19.2250 6.8561 4.39

2.03 Mass Spectrometry

Positive and negative mass spectra (Fig. 7) of

potassium benzylpenicillin were obtained (194) from a
thioglycerol matrix using a 8 KeV Xe fast atom beam.
The resulting secondary ions were mass analyzed using
a VG-ZAB-2F mass spectrometer. Fig. 7 shows fast
atom bombardment MS/MS spectra of the potassiated
parent (top) and of the deprotonated molecule
(bottom). The suggested fragmentation is shown
below, which is general for many penicillins and
highly diagnostic.

367'

I C02K
I
I
PENICILLIN G, POTASSIUM 45 1

1 1

2 8

2 2
I

. POTASSIUM PENICILLIN G
- POSITIVE FA8 MASS SPECTRUM
Y l

POTASSIUM PENICILLIN G
NEGATIVE FAB MASS SPECTRUM

Figure 7. Mass Spectroscopy of Potassium Benzylpenicillin

Positive (top) and Negative (bottom) Fast
Atom Bombardment Mass Spectra.
452 JOEL KIRSCHBAUM

+
Decarbgxylation from the (M+K) parent accounts
for the 367 fragment observed in its MS/MS spectrum
(top). The dipotassiated fragment at m/z 270 is
analogous to its protonated counterpart (195) as
shown below. This intense daughter ion is formed by
cleavage of the B-lactam ring with oxygen migrqtion.
A similar relationship exists fgr the 236
dipotassiated fragment with its (160 ) protonated
analog (196). This daughter ion arises from a
reverse 2+2 Diels-Alder cleavage. The remainder is
observed in the negative FAB MS/MS spectrum at 174-.
Decarboxylation from the (M-H)- parent yields
the 327- fragment. Although the 192- daughter ion is
the base peak in the mass spectrum, its intensity is
small in the MS/MS spectrum (bottom). This fragment
was assigned (197) as C H CH2CONHCHCHS, which
originates predominantlg 5rom the deprotonated
molecule of the free acid rather than from the
monopotassium salt.
Using electron-impact mass spectrometry, the ion
at m/z 334 was found to correspond to the free acid
of potassium penicillin G (198). Pyrolysis mass
spectrometry was used to characterize some
penicillins and cephalosporins (199). Fragments were
assigned the following structures: m/e 92, d-CH ;
117, ,&CH2CZN, and 104, 8'-HC=CH2. Benzylpenicihn
was reacted with BF3-methanol to yield a derivative
amenable to gas-liquid chromatography (see section
5.43). The product was characterized by MS.

2.04 Infrared Spectrometry

Fig. 8 shows the infrared spectra of a

commercial preparation of potassium benzylpenicillin
using mineral oil and potassium bromide (200). The
instrument used was a Perkin-Elmer Model 983 ratio
recording (dispersive) infrared spectrometer. Below
are the interpretations of these spectra (200,201).

Absorption (Scm-') Assignment

1772 B-lac tam

1666 hide, I
1486 hide, I1
1606 -
Carboxylate as "2-
PENICILLIN G , POTASSIUM 453

Mineral O i l Mull

in=e.ee T
ie 35'ee 3eee me me me ieee cn-i see

KBr P e l l e t

I
reee 35ie 3iee nie 2eie me ieee cn-i 5ee

F i g u r e 8. I n f r a r e d S p e c t r a of Potassium B e n z y l p e n i c i l l i n .
 JOEL KIRSCHBAUM

The results are similar to those found in a

study of the IR spectra of penicillins (202). The IR
spectra of various derivatives and 6-aminopenicillin
were obtained and correlated with structure (203).

2.05 Thermal Analysis

A sample of a commercial preparation of

potassium penicillin G started melting with
decomposition at 218OC (204) using the USP procedure
for the melting range of class IB compounds (205).
Thermogravimetric analysis (206) of potassium
benzylpenicillin, using a heating rate of 20°C/min,
showed no loss in weight up to 15OoC.

I I I I I I 1
50 100 150 200 250 300 350
TEMPERATURE, OC

Differential thermal analysis of potassium

penicillin G gave an exotherm with decomposition at
235-240°C, using a heating rate of 15'min. This is
in good agreement with differential scanning
calorimetry which also yielded an exotherm with
decomposition at 237O-242"C (206).
Thermograms of penicillin and stearic acid,
alone and mixed together, were used to demonstrate
the formation of an unstable mixture (207). Thermal
conductivity and Prandtyl numbers (208,209) for
water-butanol solutions of benzylpenicillin were
calculated.
PENICILLIN G, POTASSIUM 455

2.06 Microscopy and Particle Size

A commercial preparation of potassium penicillin

G was found to contain rectangularly-shaped crystals
1 to 3 pm wide and 4 to 25 pm long (206). There was
no visual evidence of polymorphism.
Particle size was determined after dispersal in
acetonitrile using a Malvern Model 3600E particle
size analyzer. The table below gives the percentage
of penicillin U S size ranges.

Microns Percent Penicillin

-
564 262 1.6
262 - 160 4.4
160 - 113 6.3
-
113 84 7.5
-
84 65 8.8
-
65 50 9.8
50 - 39 10.4
39 - 30 11.3
30.3- 23.7 11.8
23.7- 18.5 8.8
18.5- 14.5 5.9
14.5- 11.4 5.3
11.4- 9.1 3.4
9.1- 7.2 1.9
7.2- 5.8 1.4
2.07 Surface Area

A s measured by nitrogen gas adsorption (206),

the surfac5 area of one lot of potassium penicillin G
was 0.78 m I g . 2
The surface area was determined to be 0.5 m /g
€or potassium benzylpenicillin (2102. After grinding
in a jet mill it increased to 2.1 m Ig, using gas
permeability with a PSKh-4 instrument.

2.08 Cohesion

Cohesion (stickiness) was measured by

determining the resistance to breakdown of a cylinder
of compressed antibiotic powdes (210). Potassium
benzylpenicillin was 0.52 g/cm before grinding and
11.7 after grinding in a jet mill. The respective
measurement errors are 11%and 20%.
456 JOEL KIRSCHBAUM

2.09 Hydration

The crystals are not solvated with water, based

on the thermogravimetric and differential thermal
analyses previously described.

2.10 Polymorphism

There is weak evidence for polymorphism from

infrared spectrometry and no evidence from x-ray
diffraction studies.

3.0 Spectrometry in Solution

3.1 Nuclear Magnetic Resonance Spectrometry (NMR)

3.11 1H-NMR

Figure 9 is the 270 MHz proton NMR spectrum

(211) of potassium benzylpenicillin in D20
obtained on a J E O L FX-270 NMR spectrometer using
a 5 rnm C/H dual probe at 30". The HDO peak at
4.70 ppm from DSS was used as reference. The
interpretation of the spectrum is given below.

Chemical shift (PPM) Number of Protons Assignment

6 1.47 ( s ) , 1.50 (8) 6 CH3's

6 3.47, 3.56(ABq, J=14 Hz) 2 CH
6 4.22 (s) 1 -
N-&I-COO'
6 5.43 (d, J = 4 Hz) lactam C-H's
6 5.47 (d, J = 4 Hz)
6 7.2 (m) 5 Aromatic

The results are similar to those found in a

study of the identification of penicillins and
cephalosporins (212). The nuclear Overhauser effect
helped elucidate the three-dimensional conformation.
Stereof hemist13 of various derivatives were studied
using H and C NMR spectroscopy (213) and with
lanthanide-induced shifts (214). Metal complexation
depends on pH (215). A study of the 100 MHz proton
magnetic resonance spectra of benzylpenicillin in
various solvents that cleave non-covalent bonds (216)
showed significant shifts, supporting the hypothesis
of self-association previously introduced to explain
concentration dependences of H NMR spectra (217).
PENICILLIN G, POTASSIUM 457

Penicillin G was found to interact with guanosine

using 250 MHz NMR and various solvents (218).
Binding to bovine serum albumin was studied by 100
MHz N M R , and the 1 terature summarized (219).
1
Spin-echo 400 MHz H NMR was used to study the
metabolism (220). The kinetics of spontaneous first
order degradation of benzylpenicillin in aqueous -2 h-l
solution to penicilloic acid gave k = 0.7 x 10
(221). The conversion of penicillofc acid to labile
seconda$y Efoducts followed first kinetics (k2 =
6 x 10- h ) using computer simulation.

3.12 13C-NMR

Figure 10 depicts the I3C-NMR spectrum of

benzylpencillin in D 0 also using a JEOL FX-270
Fourier transform NMl? spectrometer (211). The
reference was p-dioxane at 67.6 ppm from TMS for the
assignments tabulated below (cf. reference 222).

Chemical Shift (PPM) Assignment

175.6, 175.1, 174.5 c = 0's

135.5

130.4, 129.9

128.3
8
74.2 N-?H-COO
I
67.7 N-CH-CH-S

65.5
\b / CH
.
C
H
:
-I

59 .O N -tH-CH-S
I I
43.1 CH2
31.9, 27.6 CH3 ' s
458 JOEL KIRSCHBAUM

7 6

1
5
1:
........................................................

.I 3

Figure 9. 270 MHz H-NMR spectrum of Potassium

Benzylpenicillin in D20. See text for details.
2
. I,,,

I
, L
0

m no kD u(0
Ju
Itp
I
100 eb
1.W
c

*
..MyI

to

Figure 10. 13C-NMR Spectrum of Potassium

Benzylpenicillin. See text for details.
PENICILLIN G , POTASSIUM 459

13C-NMR spectrometry was used to study the

structures of a series of penicillins (223),
penicillins and cephalosporins (224,225) and
penicillin derivatives (226). These papers also
summarize prior NMR studies.

3.13 I5N-NMR

Although 15N-NMR spectrometry produces sharp

resonances, because of it-3 spin of 1/2 its
sensitivity is 1.04 x 10 that of the proton. In
-A
addition, its lower natura abundance (0.37%) results
in a sensitivity 3.8 x 10 that of the proton. To
optimize signal strength, a 2.5 M concentration of
the soluble methyl ester was used. (Other penicillin
and cephalosporin derivatives were also studied.)
Two thousand transients were obtained in 1.8 hours to
give f5S/N ratio of 10. Natural abundance spectra
gave N chemical shifts relative to an ammonium
chloride reference of 134.30 ppm for the lactam and
85.13 ppm for the amide ('fZ6). These results are in
exact agreement with the N-NMR chemical shifts that
were obtained in 1,4-dioxane (224). The potassium
salt o f benzylpenicillin gave shifts of 143.7 and
90.7, respectively, in water, and 142.6 and 86.6 in
dimethylsulfoxide using 1.2 to 2 second repetition
rates over 6 to 16 hours, also using ammonium
chloride.

3.2 Ultraviolet Spectrometry

Figure 11 shows the ultraviolet spectrum of a

commercial preparation of potassi m benzylpenicillin
-Y
at a concentration of 2.738 x 10 M in water,
obtained with the aid of a Perkin-Elmer Model 320
Spectrophotometer (227). At 262 nm the E value is
164 and at 256 nm, E = 251. Using traditional
nomenclature, the E (l%, 1 cm) values are 4.41 and
6.73, respectively. These data are in generally good
agreement with previous results (228).
 1
460 JOEL KIRSCHBAUM

240

2g01
290

300

310
310.
p .

-
.

Potassium Pen G
320 - H20
H20

330 - 00510g/50 ml-Sol A

5 ml Sol A/100 ml-Sol 8
Initial
340 r

I I I I I I I I 1 I

n
-1 -
1 I I I I
200 250 300 350 400
WAVELENGTH, nm
Figure 12. The optical rotatory dispersion (-) and
circular dichroism (---) spectra of potassium benzyl-
pencillin in water at pH 6.5. Reprinted here with
the permission of the authors and Academic Press.
PENICILLIN G, POTASSIUM 46 1

3.3 Optical Rotatory Dispersion and Circular

Dichroism Spectrometry

Figure 12 shows the ORD and CD spectra of

potassium benzylpenicillin in water at pH 6.5 ( 2 2 9 )
as reproduced with the permissions of the author and
publisher. Two maxima are visible, one at 230 nm and
one below 200 nm. ORD is more informative than CD.
The B-lactam functionality produces the 230 nm
positive Cotton effect, because 6-aminopenicillanic
acid exhibits a similar spectrum, and hydrolysis of
the B-lactam bond leads to the loss of this
absorption (cf. Methods of Analyses, Section 5 ) . The
B-lactam (amide) function lacks the ground-state
symmetry of the ketone groups. A further complica-
tion of the spectra was induced by interaction with
the n-electrons of the sulfur atom and lack of
plagarity of the blcyclo ring system. Tge [.0Imax x
10- was 2.92 at 243 nm and [OIma x 10- nm was
+3.14 at 234 nm in 0 . 1 M citrate guffer, pH 6.5
(229,230).

4.0 Bulk Solution Properties

4.1 Solubilities in Aqueous and Nonaqueous Solvents

Solubilities of a commercial preparation of

potassium penicillin G were determined ( 2 3 1 ) at room
temperature in various solvents with about one minute
of mixing.

Solvent Solubility (mg/mL)

Acetonitrile 0.1

Chloroform 0.2

Dimethylsulfoxide >loo
Ethanol 10

Hexanes 0.2

Isopropanol <O. 04
462 JOEL KIRSCHBAUM

Table Continued
Solvent Solubility (mg/mL)

Methanol > 100

Methylene chloride <O .04

Methanol-water ( 1 : l ) >loo
0.9 M NaCl >loo
0.1 M HC1 >loo
Aqueous buffer, pH 2 >loo
Aqueous buffer, pH 4 >loo

Aqueous buffer, pH 7 >loo

Aqueous buffer, pH 10 >loo

0.1 M NaOH >loo

4.2 Partition Coefficients

Apparent partition coefficients P were
determined (232) in octanol-water at v%Pous pH
values at 37' with the help of spectrophotometry at
260 nm, and are summarized below. The intrinsic
partition coefficient for the unionized form, P was
U

E P
-aPP
4.86 0.37
5.07 0.32
5.15 0.23
5.47 0.15

1.70 in octanol. In 2-methylpropanol, the intrinsic

partition coefficient for the ionized form was -0.30,
The salt form effects apparent partition
coefficients.
Using thin-layer chromatography, partition
coefficients were calculated for benzylpenicillin
PENICILLIN G , POTASSIUM 463

distributed in various amounts of acetone in an

aqueous, sodium acetate - veronal buffer, pH 7.4
using siliconized silica plates (233). Benzyl-
penicillin was visualized using an alkaline solution
of KMn04. Hansch ZIT values were also obtained.
However, the silica gel plates may have exerted a
buffering effect on the system.
Instead, n-octanol-impregnated microcrystalline
cellulose tlc plates were used with a developing
solvent of 0.5 M 6-aminohexanoic acid (pH 3.5)
saturated with n-cotanol (234). Benzylpenicillin was
visualized on wet plates using NH3 vapor, and
spraying with 10% acetic acid in acetone followed by
starch-iodine solution. Log P, the partition
coefficient of the organic acid was calculated to be
1.76. This value agrees well with the calculated
log P results of 1.72 obtained using high-
octanol
performance liquid chromatography with an
octadecylsilane column, a mobile phase of 0.035
ammonium chloride-aqueous methanol solution adjusted
to an apparent pH of 7.4, and detection at 254 nm
(235) for benzylpenicillin partitioned between
n-octanol and water.
Serum protein binding was found to correlate to
partition coefficients of the penicillins (236).
Partition coefficients were determined in an organic
phase of chloroform and an aqueous phase containing
tetrab'utylammonium ion and n-dodecylamine to form ion
pairs (237).

4.3 Ionization

Almost all pencillins and cephalosporins are

ionized at physiological pH, making knowledge of the
predominant species important in the design of
suitable 8-lactam antibiptics and their dosage forms.
In addition, binding of C-benzylpenicillin to such
micro organisms as Staphylococcus aureaus is a
function of pH (238). The pK of benzylpenicillin at
25" and in water at a concentration of 0.0099 M was
2.73 2 0.03 and for 0.0093 M was 2.71 f 0.05 using
titration with 0. M HC1 (239). Titration with 0.01 N
iodine of benzylpenicillin in aqueous solution at 60"
464 JOEL KIRSCHBAUM

gave a pK of 2.78 ( 2 4 0 ) . Potentiometric titration

was used to determine that the apparent pK value for
penicillin G, at 20" and ionic strength 0.15, in 20%
methanol was 3.82 and in 30% methanol was 4.10 ( 2 4 1 ) .

4.4 Molecular Aggregation (Self-Association) and

Critical Micelle Concentration (CMC)

Potassium penicillin G is capable of self-

association in aqueous solution via non-covalent bond
formation ( 2 4 2 ) . Light-scattering properties of
aqueous and 0.15 M and 0.5 M potassium chloride
solutions indicated the existance of dimers and
trimers at critical micelle concentrations,
respectively, of 0.32 and 0.30 mollkg. (The a values
of 0.7 imply that most of the monomers constituting
the micelle are not in close association withltheir
countkions.) These results agree well with H nmr
studies ( 2 1 7 ) from 0.01 M to 1 M at 30" that
indicated a CMC of 0.275 and 0.251 M, respectively,
for the aromatic and methylene protons. The
benzylpenicillin ions appear to aggregate in aqueous
solution primarily through hydrophobic interactions
of the benzyl side chains. A CMC of 0.25 M w a s also
found ( 2 4 3 ) using cryoscopic and dye-solubilization
methods. Unpublished studies using an analytical
ultracentrifuge also showed aggregation ( 2 4 4 ) .
Potassium penicillin G also interacts with
lipids, such as phosphatidylcholine and lysophos-
phatidylchloride, as shown by viscosity studies
( 2 4 5 ) . This also indicates that hydrophobic
interactions can exist.

5.1 Methods of Analysis

5.1 Compositional Analysis

5 . 1 1 Elemental Analysis

Elemental analysis ( 2 4 6 ) of a commercial

preparation of potassium penicillin G gave the
following contents, in percent, C, 51.43
( 5 1 . 5 9 ) ; H, 4.62 ( 4 . 6 0 ) ; N, 7.52 ( 7 . 5 2 ) S , 8.44
( 8 . 6 1 ) and K, 10.54 ( 1 0 . 5 0 ) . The value for
sulfur is in excellent agreement with the
previously determined content of 8.47 ( 2 4 7 ) .
PENICILLIN G , POTASSIUM 465

5.12 Water Content

Water content was determined ( 2 4 8 ) by

mixing 5 to 200 mg with 1 to 5 mL of dehydrated
isopropanol and centrifuging. The supernatent
(3 ~ 1 )
-+
was injected into a gas chromatograph.
Potassium benzylpenicillin yie ded a value of
0.11% (detection limit 3 x 10 g, linear range
0.20 to 0 . 2 % ) . In another laboratory, automated
stop-flow Karl Fischer titrimetry ( 2 4 9 ) gave a
value of 0.42% (relative standard deviation
0.034%, n=5). These differences probably
reflect differences between samples. When gas
chromatography ( 2 5 0 ) was used to analyze
residual acetic acid, 0.003% was found (recovery
of 9 9 % ) .

5.2 Titration

The analysis of penicillins and their

formulations using titration methods has been
known and practiced since 1946 ( 2 5 1 ) when
Alicino utilized the unreactivity of intact
penicillin to iodine, while the hydrolysis
product, the penicillinoate, reacts with iodine.

Titriant Comments References

PH pH-Stat alkalimetric method 252

PH Alkalimetric and complexometric 253

Iodometric Measure iodine consumption 25 1

before and after hydrolysis
Iodometric In broth, uses penicillinase 254
Iodometric Automated, broth 255,256
Iodometric Automated, sensitive 25 7
Iodometric Dosage forms 258
Iodometric Na metaperiodate and arsenite 259
treatment
Bg (11) Uses untreated and hydrolysed 260
penicillin
Hg (11) Complexes with penicillamine 261,262
Fe (111) Tablets 263
466 JOEL KIRSCHBAUM

Titriant Comments References

p-Chloromercuri- After penicillinase cleavage 264

-benzoate
3-Bromo-4,4- Brominating agent, comparison 265
dimethyl-2- with N-bromosuccinimide
oxazolidinone
Penicillin Used to determine Ag and Cu (11) 266
using ion specific electrodes

5.3 Colorimetric and Spectrophotometric Methods

5.31 Colorimetric and W Spectrophotometric

Analysis

Potassium benzylpenicillin has been

determined using ultraviolet spectrophotometry
virtually from the first production of purified
antibiotic (267,268). The technique advanced to
the point of using orthogonal polynomials (269)
to determine benzylpenicillin in the presence of
its degradation products.
Penicillin G was also indirectly quantified
by W after reaction with thiols (270).
Penicillins in deuterium oxide or dimethyl-
sulfoxide solution were quantified on the basis
of the IR ahso
-f bance of the 6-lactam band at
about 1760 cm . Accuracy was claimed to be 22%
(201) and stability in solution can be
monitored.
Below is a tabulation of colorimetric
assays €or benzylpenicillin. Note that benzyl-
penicillin has been shown to interfe.rewith the
determination of other substances (271,272).

Method or Reagent Comments Reference

Hydroxylamine Automated, compendia1 273

Hydroxylamine Bulk drug + broths 274
Hydroxylamine In broths 275

Hydroxylamine In broths 276

Hydroxylamine Automated, dissolution 277
Hydroxylamine Automated, tablets + 278
capsules
Hydroxylamine Automated, liquid 279
formulations
PENICILLIN G, POTASSIUM 467

Hydroxylamine Intravenous solutions 280

Chromotropic acid Estimation, 281
identification
Chromotropic acid Characteristic colors 282,283
H2S04-HCH0 for different
penicillins
Copper Sulfate, UV In broths 284
Copper Sulfate, UV Various penicillins 285
Copper Acetate, UV Tablets 286
Arsenomolybdate In broths, automated 287
Mercuric Chloride Penicillin + penicilloic 288
acid
Mercuric Chloride Also analyze degradants 289
Mercuric Chloride Automated, sensitive 290

Mercuric Chloride Stabilized assay, 29 1

sensitive
Mercuric Chloride + Other penicillins 29 2
Starch-Iodine In sensitivity discs 293
Rhodamine 6 And other compounds 294

Azure R Via solvent extraction 295

Methylene Blue Quantify at 6 4 0 nm 296

2-(N-methylamino) t Penicillamine 297
phenyl sulfate +
Cr (VI)
Ferrous Nitrate Penicillin as reagent 298-300

5.32 Optical Rotatory Dispersion and Circular

Dichroism Methods

Changes in optical activity of benzylpenicillin

due to hydrolysis of the B-lactam ring by
penicillinase were measured using a Cary 60 recording
spectropolarimeter with a Cary Model 6 0 0 1 circular
dichroism accessory (301). Since the change in -1
spe ific rotation at 255 nm, [ A a ] 2 5
-!i deglml g
dm , is 5053.9 and the precision 02 e%? instrument
is 0.22, the assay has the potential of being
precise. Actual precision was found to be 0.12, with
pH and temperature control. Accuracy was verified
using microbiological and iodometric assays (302).
The method is limited by the error introduced by
mutarotation of penicilloic acids,
468 JOEL KIRSCHBAUM

5.33 Fluorescence Methods

Although penicillin G is only weakly fluorescent

after treatment with 0.1 M sodium hydroxide (303), it
can be derivatized with 5-dimethylaminonaphthalene-
1-sulphonylhydrazine. This permits the assay of both
the intact penicillin and its biotransformation
product, penicilloic acid (304).

5.4 Chromatographic Methods of Analysis

5.41 High-Performance Liquid Chromatography (HPLC)

This author prefers chromatographic method6

since similar compounds are usually resolved from
each other due to selective interactions of the
analyte with the mobile and stationary phases via
weak, non-covalent, bonds. HPLC is preferred by many
analysts and regulatory agencies because of its
specificity, ease of use and high sample capacity.
Fig. 13 shows a chromatogram of potassium penicillin
G and potassium phenylacetate isolated from a
fermentation broth (305). An octadecylsilane column
(12% loaded or covered) was used with a mobile phase
of 0.1 M phosphate-methanol-acetonitrile (60:40:5)
apparent pH 4.15, flowing at 1 mL/min. into an
ultraviolet detector set to 220. Penicillins V, F,
and K and 6-aminopenicilloic acid do not interfere.
Table 2 , below, summarizes HPLC methods. Partition
coefficients were determined by HPLC (cf. section
4.2).

5.42 Thin Layer Chromatography

Table 3 , below, summarizes thin-layer

chromatographic methods for potassium benzyl-
penicillin arranged in chronological order. Use of
a-acceptors as spray reagents for the detection of
penicillin was described (359).

5.43 Gas-Liquid Chromatography

Table 4, below, summarizes the GLCsystems

developed for bulk benzylpencillin.
PENICILLIN G, POTASSIUM 469

Figure 13. Chromatogram of broth sample with detector

set at 220 nm. A, potassium phenylacetate; B,
potassium penicillin G. See text for further details.
 Table 2. High-Performance Liquid Chromatography of Potassium Benzylpenicillin

Column Mobile Phase Detection Comments Reference

Bulk o r Pure Material

C18 MeOH-0.5% ammonium carbonate (3:7) 254 nm Resolves Pen. V. 306

C18 MeOH-0.05 M KH2P04 (35:65) + 0.1% 254 nm Resolves other 307

tetrabutylammonium bromide, penicillins +
apparent pH 3.35 degradation products
!A
4
C18 H 0-MeOH (3:7) containing 0.02 -
M 220 nm Describes effects of 308
0 2dicyclohexyl-18-crown 6 varying mobile phase.
Resolves other B-lactams

C18 CH30H-H20 + PIC Reagent B-7 254 nm Resolves Pen. V + 309

dibenzylethylenediamine

C18 Acetonitrile-MeOH-0.01 MKH2P04 256 nm Resolves other 310

(19:11: 70) penicillins

C18 MeOH-H,O (2:l) containing 1mM each Postcolumn Sensitive, also 311
Na2H604 and NaH2P04 Rxn resolves penicillioates

C8 .
MeOH-0 01 M NaH2P04 (7 :13) 225 nm Resolves other 312
penicillins
 Table 2. HPLC (Continued)

Column Mobile Phase Detection Comments Reference

C8 MeOH-0.05 M phosphate buffer (53:47) 274 nm Resolves Pen V. & 313,314

apparent pH 3.5-3.3 or 254 nm preservatives

Anion 0.02 M NaN03 in 0.01 M borate, 254 nm Resolves ampicillin & 315
pH 9.15 degradation products

Stability

C18 1% KH2P04-acetonitrile (4:l) pH 4.15 254 nm Resolves degradation 3 16

products

C18 Acetonitrile-phosphate buffer + 254 nm Resolves deg. pro. 317

0.008 tetrabutylammonium Mobile phase optimized
chloride, pH 7.5 (3:7)

C18 Acetonitrile-0.01 M KH2P04 (1:4), 254 nm Resolves deg. pro. & 3 18

pH 4.1 other penicillins
Anion 0.03 M citric acid-0.0067 M sodium 254 nm Resolves deg. pro. 319
phosphate buffer, pH 2.7, 37"

Anion 15.4 mL 0.1 M citric acid + 7 mL RI Study stability in acid 320

0.2 M Na2HP04 diluted to 650 mL,
pE 3.8
 Table 2. HPLC (Continued)

Column Mobile Phase Detection Comments Reference

Anion 0.68 g KH2P04 + 0.1 g NaN03/liter, 254 nm Resolves deg. pro. 321
pH 5.1

Formu Zations
C 18 0.01 M ammonium acetate-acetonitrile 254 nm Penicillin in infusates 322
( 4 : 1)

C18 0.01 M KH2P04-acetonitrile ( 4 : l ) 240 nm Effect of excipients 323

(dextrose and sucrose
adverse)

C18 0.42% KH2P04, 0.6% sodium heptane- 258 nm Controlled-release 324

1-sulfonate, pH 3.17-acetonitrile products
(71.5 : 28.5)
C18 MeOH-H20-acetic acid (40:60:0.5) 232 nm Study kinetics decomp. 325
eyedrops

Biological Matrices

C18 0.01 M H PO acetonitrile 220 nm 0.05 ppm in tissues 326

3 4-
(4:l to 2:3)
 Table 2. HPLC (Continued)

Column Mobile Phase Detection Comments Reference

C18 0.01 M NaH2P04, 0.01 M EDTA- 325 nm Derivatize with 327

acetonitrile (72:28), pH 6.5 (NaOH) imidazole-HgC1
2
reagent, broths

C18 acetonitrile-pH 7 buffer (23:77) 230 nm In body fluids 328

C 18 phosphate buffer (ionic strength Deriva- In body fluids 329

0.1)-methanol (6:4) pH optimum tization
le
4
w C18 Gradient: 90% 0.05 M Na formate, pH 240 nm In urine 220
5-102 formate-acetonile (5:3) to
75:25

Misc. and Related Projects

C8 phosphate buffer, pH 7-MeOH (6:4) 220 nm Pen. G in broth & 330

6-aminopenicillanic
acid

Anion 0.003 M NaHC03/0.0024 M Na2C03 Conduc- Cation analysis & 331

Cation 0.005 M HN03 tivity Pen. G

C 18 50X MeOH in 0.1 M phosphate buffer, 254 nm Sep. procaine, 332

pH 4.8; 0.1 MH3P04 to 40% acid benzathine and Na pen.
in 9 min.
 Table 2. HPLC (Continued)

Column Mobile Phase Detection Comments Reference

Silica Acetonitrile- CC12H2 (1:199) 230 nm Prep. LC of Derivatives 333

220,
C18 15-20% MeOH-0.01 M potassium 254 nm Sep diastereoisomers of 334
phosphate, pH 7 penicilloic acids
(allergens)

C18 Acetonitrile-0.01 M sodium phosphate 325 nm Derivatization with 335

buffer, pH 6.5 (1:4) containing imidazole plus metal
0.01 M EDTA salts

C18 Acetonitrile-0.05 M KH PO (17:83) 254 nm Study epimerization of 336

adjusted to pH 4.0 (43P64) benzylpenicilloic acid
 Table 3. Thin-Layer Chromatography of Potassium Penicillin G

Coating Developing Solvent Visualization Comments Ref.

Silica Org. Phase isoamyl acetate-CH3OH- 10% Aq. FeC13 (20 mL) + For 10 337
HCO H-H20 (65:20:5:10) or 5% aq. potassium penicillins
Acegone-acetic acid (95:5) ferricyanide (10 mL)
+ 20% H2S04 (70 mL)
Cellulose 0.1 M NaCl or 0.3 M citric acid
MW300 saturated with n-butanol

Silica CHCl :isopropanol-H20 (60:40:4) iodine-azide Sep. most 338

3 penicillins

Sephadex 0.25 M phosphate buffer, pH 6 Bioautography Sep. other 339

G-15 containing 0.5 M NaCl antibiotics

Silica Acetone-acetic acid (19:l) or -

UV. then Ref 337 reagent Resolves 340
isoamyl acetate-CH OH-HCO H-H20 or Bratton-Marshall degr. products
3 2
(13:4:2:1) (Org.) reaction

Polyamide H20-HOAc-isopropanol (50:15:8.5) 0.5% Br solution or Detects 341

or H 0-HOAc-isoprOH-MeOH 0.25% Na fluorescein other penicillins
(50: $6 :7.1 :5)
 Table 3. TLC (Continued)

Coating Developing Solvent Visualization Comments Ref.

Silica n-BuOH-H20-EtOH-AcOH (5:2:1.5:1.5) First 2 M NaOH, then Sep. and 342
~-BU~H-H,O-ACOH(4:1: 1) iodine-azide, followed detection of
Acetone-&OH (95:5) by 1% starch penicillins and
85% aq. acetone cephalosporins
and their
degradation
products.

A Cellulose BuOH-MeOH-HOAc-H20 (45:30:9:36) Bioautography Detects as 343

-4
Q, little as
0.001 pg in
feed and food

Silica Acetone-HOAc (95:5) See reagent ref 337 For degradation 344
products

Silica Acetone-CHC13-HOAc (10:9:1) W densitometry Quantify in 345

230 nm syrups and tablets

Silica Acetone-CHC13-HOAc (10:g:l) Ferricyanide (ref 337) also degr. 346

then I2 products

Silica n-BuOH-HOAc-H20 (12:3:5) 1 mL 2% PtC14, 0.1 mL 0.05 pg limit 347

20% KI, 0.1 mL 44: HCl
and 20 mL acetone
 Table 3. TLC (Continued)

Coating Developing Solvent Visualization Comments Ref.

Silica CHC13-MeOH-H 0 (80:20:2.5) or UV, 254 nm Resolves many 348
Citrate buf ger antibiotics

Silica Plates impregnated with 2% NaAc, Bioautography as contaminant 349,

adjusted to pH 7.4 barbital. - 350
Barbital acetate buffer-acetone
(94:6)

Silica BuOAc-AcOH-MeOH-n-BuOH-phosphate 10% Fe C1 -2% hexa- For various 35 1

3
buffer, pH 7.3 (80:4:5:15:20) cyanoferrate- penicillins
HC1 (1:2:6)
Silica BuOH-MeOH-AcOH-H20(37.5:25:7.5:16) Chloroplatinate In chicken muscle 352

Glass
Fiber BuOH-H20-HOAc (4:l:l) or hexane- Sep. various 353
BuOH-H20-EtOH-HOAc (5:10:4:3:3) or penicillins
hexane-acetone-H0Ac (1:9:1)
acetone-CHC1 HOAc (10:9:1)
3-
Si Benzene (caution; replace, if Spray of 50% H SO Seperate and 354
possible, with toluene)-CH OH- followed by 126'C4 identify various
3 or 5% FeCl in 0.5 M
acetone ( 15 :3 :2)
HC1 or 0.13 aq. fast
green (diazotized),
drying, then 0.5 M
NaOH spray
 Table 3. TLC (Contined)

Coating Developing Solvent Visualization Comments pef.

Silica CHC13:MeOH:HOAc (90:8:2) or Bioautography or A classification 355

CHCl :MeOH:H20 (20:4:5) or colorimetry and identification
3 system for 45
Potassium phosphate, pH 3-washed
plates with BuOH-HOAc-H20 (2:l:l) antibiotics is
or H 0:Na citrate:citric acid given.
2
(100:20:5) or MeOH or EtOH or H20

Silica Ethyl acetate W, 254 nm Identify 10 356

penicillins

Silica Acetone-CHC13-HOAc (10:g:l) 10% FeC13-5% K Contamination 357

ferricyanide-20%
H2S04 (2: 1:7)

Silica 0.5 M NaCl or acetonitrile-water I2 vapor 18 penicillins 358

(4:l) or EtOAc-CH30H-HOAc(20:10:1) studied in various
or CHC13-EtOH-HOAc(100:50:7.5) or normal-and reversed-
EtOAc-acetone-H 0 (1:2:1) or phase mobile phases
isoamylacetate-6H3OH-HC02H-H20
(13:4:1:2) (upper layer) or acetone-
HOAc (95:5) or EtOAc-acetone-HOAc-
H 0 (5:2:2:1) or n-BuOH-HOAc-H20
(2 :1 :1) or ~ - B ~ o A ~ - ~ - B ~ o H - H o A ~ -
0.066 M phosphate, pH 6.0 (90:9:25:15)
or n-BuOAc-n-BuOH-HOAc-O.l% EDTA,
diNa in 5% NaH2P04 (10:1:6:2)
 Table 3. TLC (Continued)

Coating Developing Solvent Visualization Comments -

Ref.
Silica Buffers are 2 M NH40Ac adjusted to I2 vapor 18' 358
Silanized pH 5.0 or 6.2 or buffer pH 6.2-MeOH (17:3) penicillins
or buffer pH 6.2-MeOH-acetonitrile (7:1:2) studied in various
or buffer pH 6.2-acetone-EtOH(7:2:1) or normal- and reversed-
buffer pH 6.2-acetone-EtOH(6:3:1) or buffer . phase mobile phases.
pH 6.2-MeOH-EtOH(5:4:1) or buffer pH 5-
acetonitrile (17:3) or buffer pH 5.0-
ethylene glycol monoethyl ether (4:l) or
buffer pH5-acetonitrile-ethylene glycol
monoethylether (8:l:l) or buffer pH 5-
THF(3:l) or buffer pH 5-acetone-ethylene-
glycol monoethyl ether (15:3:2) or buffer
pH 5-EtOH(3:1) or
buffer pH 5-EtOH-ethylene glycol monoethyl
ether (15:3:1) or buffer pH 5-acetone (7:3) or
buffer pH 5-EtOH-ethylene glycol monoethyl
ether (7:1:2) or buffer pH 5-EtOH (13:7) or
buffer pH 5-MeOH-acetonitrile (12:3:5) or
buffer pH 5-MeOH-EtOH (5:4:1) or 0 . 3 M NaCl
in 0.05 M potassium phosphate buffer pH 5.6-
acetone (2:l) or 0.1 M NaC1-acetone (2:l) or
0.05 M potassium phosphate buffer pH 6.0-
acetone (4:1)
 Table 4 . Gas-Liquid Chromatography of Benzylpenicillin

Column Carrier T(Co1umn) Detection Reference

0.4% SE-52 on acid-washed, silanized Ar 240°C Strontium, 800V 360

Gas-Chrom I (100-120 mesh) ;
130 cm x 4 mm, Penicillin converted
to methyl ester by reaction with
diazomethane.

2% OV-17 on Supelcoport He, H2, 275°C Flame Ionization 361

(80-100 mesh); 66Omm x 4 mm, Air
conditioned with HMDS to silylate
reactive sites. Penicillins silylated
within 10 min. with HMDS in pyridine.

3% XE-60 on Gas Chrom Q (80-100 mesh) He 100°C Flame Ionization 362

2 m x 0.32 cm. Pyrolysis unit 2OoC/
millisec to 875°C.
5% FFAF' on Chromosorb W (AW-DMCS, Air, 190°C or Flame Ionization 363
80-100 mesh), 3 m x 0.32 cm. 14O"-23O0C
Penicillin reacted with BF in at 16" min.
methanol. 3

3% OV-1 on Gas-Chrom Q (60-80 mesh), He 50" to 200°C MS 363

2.75 m x 0.63 cm glass. Benzyl- at 16"/min.
penicillin was derivatized with BF
in methanol. 3
PENICILLIN G, POTASSIUM 481

5.44 Paper Chromatography

Details of several methods for resolving

potassium benzylpenicillin are to be found in ref.
338 and 364-366.

5.5 Electrochemical Methods of Analysis

5 . 5 1 Electrophoresis

Electrophoresis was used to separate and detect

benzylpenicillin mixed with other penicillins in
various electrolytes ( 3 6 7 , 3 6 8 ) in animal tissues
( 3 6 9 ) and feedo ( 3 7 0 ) and in food ( 3 7 1 ) .

5.52 Polarography and Related Techniques

Penicillin G was analzyed using potentiometric

( 3 7 2 , 3 7 3 and references therein), polarographic
(374-379 and references therein), voltammetric ( 3 8 0 )
and coulometric ( 3 8 1 ) methods of analysis. For other
electrochemical methods see the following sections on
enzyme electrodes and electrophoresis.

5.53 Enzyme Electrodes and Flow Injection

Penicillin-sensitive electrodes were developed

with high selectivity based, in general, on the
immobilization of a penicillinase on an electrode.
Potassium penicillin G could be quantified in
fermentation broths and capsule formulations from 3.5
to 1100 pg/mL, with a precision of +3%, measuring
changes in pH as the penicillin was converted to
penicilloic acid ( 3 8 2 ) . Changes in pH were measured
and shown t o be linear with increasing concentrations
of penicillins ( 3 8 3 , 3 8 4 ) in buffered solutions using
penicillinase-coated pH electrodes. Care must be
taken to allow equilibration to occur to achieve
reproducibility and linear s nsitivity to
concentrations as low as lo-' M ( 3 8 5 ) .
An enzyme electrode sensitive to sodium and
potassium ions was used to analyze for potassium
benzylpenicillin ( 3 8 6 ) , with generally non-linear
results,
Using a co-cross-linked penicillinase-albumin
layer over a pH-sensitive, field effect transister
gave an enzyme electrode generally insensitive to
482 JOEL KIRSCHBAUM

variations in temperature and ambient pH v riation.

The sensitivity is approximately 2.5 x 10
-8
international units ( 3 8 7 ) .
As many as 150 analyses per hour could be
performed using flow injection ( 3 8 8 ) equipment in
conjunction with penicillinase immobilized on a glass
surface with glutaraldehyde, and pH monitoring.
Penicillin G was determined in pharmaceutical samples
in the range of 0.05-0.50 mM after dilution.

5.6 Biologically-Based Assays

5.61 Microbiological Assays

The original assays for benzylpenicillin were

predominately microbiological ( 3 8 9 , 3 9 0 ) . More ,
recently, microbiological tests were developed to
assay penicillin G in broths ( 3 9 1 ) , bulk material
(392-395), formulations ( 3 9 6 - 3 9 8 ) , and tissues and
body fluids (399-402). There have been many
publications describing assays for residual
penicillin G in animal tissue and milk (403-410).
The rate of microbial growth was investigated in
the presence of various B-lactam antibiotics
( 4 1 1 , 4 1 2 ) , with the resistant organism followin
apparent first order regrowths. Inhibition of 44
c02
release by bacteria was studied ( 4 1 3 ) .
Technical advances resulted in rapid assays
based on light scattering ( 4 1 4 ) and changes in pH
( 4 1 5 , 4 1 6 ) . The factors affecting accuracy were
studied ( 4 1 7 - 4 2 3 ) , as was susceptibility ( 4 2 4 - 4 2 8 ) ,
and the effect of infection ( 4 2 9 ) . Bioassays can be
automated ( 4 3 0 , 4 3 1 ) . Unfortunately, space
limitations preclude a more complete discussion of
this once-essential method.

5.62 Immunoassay

Antibiotic immunoassays ( 4 3 2 ) were applied to

potassium penicillin G to analyze bulk penicillin
( 4 3 3 ) , penicilloyl derivatives ( 4 3 4 , 4 3 5 ) and
penicillins for allergy producing compounds
(436,437).

5.63 Enzyme-Aided Assays (Hydrolysis)

Penicillins in mixtures can be quantified on the
PENICILLIN G, POTASSIUM 483

basis of different rates of hydrolysis at acid pH

with the final measurements performed by UV spectro-
photometry (438). 8-lactam antibiotics, including
potassium benzylpenicillin, could be estimated in
biological fluids on the basis of these antibiotics'
ability to inactivate the R39 DD-carboxypeptidase
(4391, a rather expensive enzyme.
Penicillin acylase was used to distinguish
penicillin G from ampicillin or cloxacil.lin in bovine
urine (440).
The rate of reduction of cytochrome c linked to
an enzyme system containing benzylpenicillin and
B-lactamase from Bacillus cereus was measured at
550 nm to determine activity (441).
Differential UV spectrometry of various
penicillins in the presence of B-lactamases, a
convenient and widely used method, showed that the
changes in absorption were linear with time (442).
Kinetic properties of the 8-lactamases, like the
Michaelis constant and rate constant (V 1, could
max- induced
also be measured. The rate of 8-lactamase
penicillin hydrolysis was also followed by the
starch-iodine reaction (443) as well as by iodometric
titration (444,445), and acidimetric titration (446).
B-lactam-containing antibiotics could be determined
within 20 min. using the "Penzym assay" (447).
Hydroxylamine assays were once used routinely
for B-lactamase studies; cf section 5.31 and
reference (448).

5.7 Isotopic Assay

14C-Labeled potassium penicillin G was added t o

bone cement, pellets molded and the rate of release
into aqueous liquids measured using a scintillation
counter (449). Subsequent, in V ~ V Oexperiments
involved measuring concentrations of antibiotic in
the bone adjacent to the antibiotic-impregnated
cement.
B-Lactam antibioticslyere screened in milk based
on a competition between C-penicillin and beta-
lactam antibiotics for sites on a microbial cell wall
that specifically binds B-lactam (450).
484 JOEL KIRSCHBAUM

5.8 Contamination Methods

Assays were developed to examine benzyl-

penicillin €or contamination, as summarized below.

Contaminant Comment Reference

Ethylene oxide In powders & liquids 45 1

Cephalosporins 1-2 uglg 452

Pyrogens Rabbit test 453

Pyrogens Limulus (LAL) 454

Particulates Microscopy 455

Particulates Scanning EM 456

5.9 Comparison of Methods

Presently, most reliance is placed on HPLC. One

company virtually ceased all microbiological testing
of B-lactam antibiotics, because of their relatively
uncomplicated structures, in favor of chemical
testing. However, in the opinion of the author, an
HPLC specialist, at least one biological use test is
mandatory prior to human use, and safety tests ensure
lack of adverse reactions. Various assays involving
titration, spectrophotometry with Hg (11) solution
and other non-selective methods ( 4 5 7 ) can only give
maximum contents. In the secondary literature may be
found assays based on iodometry, hydroxylamine
colorimetry, sterility, identity, safety, pyro-
genicity, moisture, pH, content, crystallinity, heavy
metals content and residue on ignition ( 4 5 8 , 4 5 9 ) .

6. Possible Future Analytical Problems

The rapid excretion and facile inactivation in vivo

of henzylpenicillin decreases its usefulness as a
therapeutic agent. However, these difficulties can be
overcome.
The rapid renal excretion (tl of 30 min.) can be
obviated by large, frequent or coatinuous intravenous or
intramuscular infusion doses. A less wasteful alternative
involves formulating potassium penicillin G in waxes or
PENICILLIN G, POTASSIUM 485

oil.-like liposomes (460) from which, after injection, the

penicillin is slowly released. [Slightly soluble salts of
penicillin G, like benzathine and procaine are varients of
this approach (461)l. Renal excretion can be blocked 9 0 %
by administering probenecid [(p-(dipropylsulphamoyl)
benzoic acid] which inhibits active tubular secretion.
Concentrations of both drugs need to be monitored.
Inactivation in V ~ V Oof benzylpenicillin is due
principally to the hydrolytic action of B-lactamases
(462). The B-lactam clavulanic acid, found by the
systematic screening of fermentation products of
Streptomyces cZavutigerus actinomyces, possesses little
antibacterial activity but is a potent inhibitor of some
6-lactamases of plasmid and chromosomal origin.
Administration in combination with clavulanic acid lowers
the minimum inhibitory concentrations of many penicillins
and cephalosporins. This is an alternative t o using
synthetic and semi-synthetic @-lactam antimicrobial
agents, like methicillin, moxalactam, cefotaxime and the
monobactams, which are resistant to hydrolysis by
6-lactamases. The amoxacillin-clavulanic acid combination
product is called Augmentin, and the potassium
clavulanate-ticarcillin injectable is Timentin. Some
other B-lactamase inhibitors currently being investigated
( 4 6 3 , 4 6 4 ) are sulbactam, mecillinam, halopenicillanic
acids 6-B-sulfonamidopenicillanic acid sulfones
6-(methoxymethy1ene)penicillanic acid, acetylmethylene
penicillanic acid and 6-aminopenicillanic acid (465-467).
These suicide or "Kamikaze" substrates lead to a "dead1'
6-lactamase (468) via an acyl enzyme intermediate, Fig.
14. The sulbactam-ampicillin combination product is
sultamacillin (469). Such formulations of two ingredients
require monitoring of bulk, formulations, and body tissue
and fluid concentrations similar to those needed for
penicillin itself, at least with respect to pharma-
cokinetic studies.
The recent isolation of biosynthetic genes from
Streptomyces through molecular cloning techniques should
make possible improvements in fermentation yields of
penicillin, and, via gene transfer between organisms,
novel antibiotics which are "hybrid" compounds (470).

7. Acknowledgements

The author gratefully acknowledge the assistance of

the many contributors from the Squibb Institute for
Medical Research cited as "personal communication". In
p’,
0
‘coo-
OH I
sulbactam

Acyl Enzyme,_l,

deacylation
and turnover

0 - 0
coo - /
sop
, -

.eNq-
7
A
Ser
coo -

“waiting room”
p+-] n.L
Ser
coo -
malonic semialdehyde

H &
+

y
sop -
t

coo -

Figure 14. Inhibition of a B-Lactamase by

Sulbactam (Redrawn from Ref. 4 6 8 )
PENICILLIN G, POTASSIUM 487

many instances, the information was especially obtained

for this summary.
Special thanks go to Dr. G. Brewer, Dr. B. Kline and
S . Perlman for their critical reading of this MS, to
Mrs. M. George for several computer-assisted literature
searches, to Robert Bragalini for programming, to
P. Moebus, and L. Moore for obtaining many of the
references, to Ms. Linda J. Boor f o r typing the
manuscript, and to Mr. J. Alcantara for drawing the
figures. General review articles and books are listed
below (Ref. 1-9). The literature was reviewed to
August, 1985.

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J . Bone Joint Surg. 65A, 798 (1981).
450. S.E Charm and R.K. Chi, J . Assoc. Off. Anal. Chem.,
6 5 , 1186 (1982).
451. T. Kiss and H.K. Kovacs, Acta Pharm. Hung., 5 2 , 280
(1982); CA, 98, 78230t (1983).
452, M.A. Abdalla, A.G. Fogg, J.G. Baber and C. Burgess,
AnaZyst, 108, 53 (1983).
453. Z.N. Enikeeva and G.Y. Kivman, Pharm. Chem. J . , 1 5 ,
832 (1982).
PENICILLIN G, POTASSIUM 507

454. T.J. Novitsky, S.S. Ryther, M.J. Case and

S.W. Watson, J . Parent. Sci., TechnoZ., 3 6 , 11
(1982).
455. R.L. Longe, Can. Anaesth. Soc. J . , 2 7 , 62 ( 1 9 8 0 ) ; CA,
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(1982).
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460. G. Gregoriadis, Biochem. SOC. Trans., 2 , 117 ( 1 9 7 4 ) .
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4 6 4 . R.B. Sykes and K. Bush i n The Chemistry and Biology
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465. Anonymous, Med. WorZd News, Sept. 10, 1984, p. 10.
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S . Omura, Nature, 3 1 4 , 642 ( 1 9 8 5 ) .
This Page Intentionally Left Blank
 PIROXICAM

Mladen Mihalib, Hrvoje Hofman, Josip Kuftinec,

Branka Krile, Vesna Caplar, Franjo Kajfe2,
N i k o l a Blaievi6

1. Foreword, History, Therapeutic Category

2. Description
2.1. Name, Formula, Molecular Weight
2.2. Appearance, Color, Odor, Taste
3 . Synthe6 is
4. Physical Properties
4.1. Spectra
4.1.1. Infrared
4.1.2. Nuclear Magnetic Resonance
4.1.3. Ultraviolet
4.1.4. Mass
4.2. Solid Properties
4.2.1. Polymorphism
4.2.2. Crystal Properties
4.3. Solution Properties
4.3.1. Solubility
4.3.2. Acidity /pK,/
4.3.3. Partition Coefficient
4.3.4. Dipole Moment
5. Methods of Analysis
-5.1. Elemental
5.2, Chromatographic Methods
5.2.1. Thin Layer
5.2.2. High Performance Liquid
-
6. Stability Degradation
6.1. Thermal Stability
6.2. Photostability
7. Drug Metabolic Products, Pharrnacokinetics, Bio-
availability
ANALYTICAL PROFILES OF m u e SUBSTANCES Copvnght 0 1986
VOLUME 15 hy the American Pharmaceutical Association
509 All rights of reproduction in any form reFerved
510 MLADEN MIHALIC ET AL.

7.1. Pharmacokinetics
7,2. Metabolism
8, Toxicity
9. Identifieation and Determination in Body Fluids and
Tissues
lo. Identification and Determination in Pharmaceuticals
11. References
PIROXICAM 511

1. Foreword, History, Therapeutic Category

Piroxicam belongs to the class of acidic, non-steroid
anti-inflammatory (NSAI) drugs. The compound is quite ef-
ficient in the treatment of rheumatoid arthritis and other
inflammatory disorders in humans /1,2/. The drug is high-
ly potent, has a long half-life of over 30 hours which
makes it suitable for once daily dosage /2-4/. It does
not have unwanted cardiovascular or central nervous ef-
fects /4/.
Piroxicam was first developed by Pfizer and Co. about
fifteen years ago / 5 / and in seventies it entered into
medicinal praxis.
Piroxicam is used as an effective analgesic and anti-
-inflammatory agent in rheumatoid arthritis, osteoarthri-
tis, ankylosing spondylitis and acute pain in musculoske-
letal disorders and acute gout / 6 / . It has been shown to
be an effective analgesic in fracture, dental, postopera-
tive and postpartum pain.
2. Description
2.1. Name, Formula, Molecular Weight
Piroxicam is 4-hydroxy-2-methyl-N-(2-pyridyl)-2H-1,2-
-benzothiazine-3-carboxamide-l,l-dioxide. There are two
possible tautomeric forms:
H

-
2

‘CH3

C H N O S
15 13 3 4
Mol . wt . 331.36
2.2. Appearance, Color, Odor, Taste
Piroxicam is an odorless, colorless crystalline pov-
der of a bitter taste.
3. Synthesis
The most important synthetic pathways to piroxicam
are outlined in Scheme 1. The first one /5, 7 - 9 / starts
with sodium saccharin which is alkylated at nitrogen using
chloroacetic acid esters /lo/. 3-Acyl-2H-1,2-benzothiazin-
-4(3H)-one-l, 1-dioxide is prepared by the base catalysed
 SCHEME 1.
9
W N N a + ClCH&OOR

02

SNHCH2C OOR
02
d C HzC 0OH A-
2eq.RONa
ROH
02
PIROXICAM 513

r e a r r a n g e m e n t of t h e 5-membered i n t e r m e d i a t e c o m p r i s i n g
a l k o h o l y s i s followed by a Dieckmann r i n g c l o s u r e /11-13/.
N-Methyl d e r i v a t i v e i s r e a d i l y o b t a i n e d by r e a c t i o n w i t h
methyl i o d i d e /11, 14/. The p r o d u c t i s t h e n t r e a t e d w i t h
a p p r o p r i a t e amine i n r e f l u x i n g x y l e n e t o o b t a i n t h e c o r -
r e s p o n d i n g carboxamide.

4. P h y s i c a l P r o p e r t i e s
k h ? % % r e d
Piroxicam e x i s t s i n two d i f f e r e n t i n t e r c o n v e r t i b l e
c r y s t a l polymorphs. T h e i r i n f r a r e d s p e c t r a d i f f e r o n l y
s l i g h t l y i n f i n g e r p r i n t r e g i o n , b u t t t e band o f -NH and
-OH s t r e i c h i n g which l i e s a t 3385 cm- i n n e e d l e form and
3330 cm- i n c u b i c form is s i g n i f i c a n t /l5/. The o t h e r
c h a r a c t e r i s t i c bands /Fig.l./ may be a t t r i b u i e d t o t h e
-
f o l l o w i n g group v i b r a t i o n s : 1635,or 1625 cm- ( s t r e t c h i n g
of t h e amide c a r b o n y l ) , 1525 cm ( s t r e t c h i n g o f t h e se-
cond ami e b a n d ) , 1440 cm- ( 3 a s . - C H Af-C=C- stretching),
1322 cm-'( 3 sym.-CH ), 1155 and 10703'c y - o r 1050-1070
cm ( -SO 2- N - ) , 770 3and 730 o r 740 cm- ( o r t h o - d i s u b s t i -
t u t e d p h e n y l ) . The s p e c t r a a r e t a k e n i n K B r d i s c s a t Pye-
-Unicam SP3-200 i n f r a r e d s p e c t r o p h o t o m e t e r .

4.1.2. Nuclear Magnetic Resonance

P r o t o n magnetic r e s o n a n c e spectrum was r e c o r d e d i n
C D C l s o l u t i o n by Bruker WP8oPS s p e c t r o m e t e r . It i s cha-
r a c t z r i z e d by one s h a r p s i n g l e t a t 2.93 ppm ( 6 s c a l e )
f o r t h e N-methyl group, two broad s i n g l e t s , one a t 9.00
ppm (amide h y d r o g e n ) , and a n o t h e r a t 13.20 ppm ( h y d r o x y
g r o u p ) . Both hydrogens a r e exchangeable by a d d i t i o n o f
D20. A m u l t i p l e t a t 6.93-8.50 ppm b e l o n g s t o a r o m a t i c pro-
t o n s and c o r r e s p o n d s t o f o u r p r o t o n s from t h e benzene,
and f f y r p r o t o n s from t h e p y r i d i n e r i n g , s e e F i g . 2 /15/.
C-Nuclear magnetic spectrum was r e c o r d e d i n C D C l
s o l u t i o n by J e o l FX-loo s p e c t r o m e t e r . It i s r a t h e r com- 3
p l e x and d i f f i c u l t t o a s s i g n p r o p e r l y . The s i g n a l s of t h e
b e n z o t h i a z i n e moiety i n t e r f e r e w i t h t h o s e from t h e p y r i -
d i n e r i n g ( F i g . 3 ) . The v a l u e s f o r chemical s h i f t s a r e
p r e s e n t e d i n Table 1. Note t h a t a s s i . g n a t i o n s of s i g n a l s
t o C-5 and C-6 might be i n v e r t e d /15/.

4.1.3. Ultraviolet
The u l t r a v i o l e t s p e c t r a o f piroxicam i n 0.1 M H C 1 and
methanol r e c o r d e d by SP8-loo U V / V I S Pye Unicam s p e c t r o -
photometer a r e shown i n Fig. 4 /l5/. These s p e c t r a a r e
c h a r a c t e r i z e d by two maxima i n 0.1 M H C 1 : a t 242 nm ( & =
= W o o ) and 339 nm ( € = 2 2 5 0 0 ) , and t h r e e i n methanol: a t
 -
4
I

LOO0 3500 3000 2500 2000 1800 1600 1400 1200 1000 800 -1 600
cm
F i g . 1. Infrared s p e c t r a o f piroxicam i n K B r p e l l e t s , n e e d l e form /A/ and cubic form
/B/. Instrument: Pve-Unicam sP3-200.
PIROXICAM 515

I
I I f I 1 I
14 12 10 8 6 4 2
PPmO
Fig. 2. '€I-Nuclear magnetic resonance spectrum of
piroxicam in CDCl Instrument: Bruker WP
80PS at 80 Mz. 3*
 I I I I I I t I I I 1 1 I I I I

200 180 160 140 120 100 80 60 LO

2o PPm 0
Fig. 3. 13C-Nuclear magnetic resonance spectrum of piroxicam in CDCl
Instrument: Jeol FX-loo at 25.05 MHz. 3.
PIROXICAM 517

1.0.

A
- 0.1M HCI
---- MeOH

0.8
I
I
I
I
I
I
I
I
I
I
0.6 I
I
I
I
I
I
I
I
I
I
I
I

0.4

0.2

Fig. 4. Ultraviolet spectra of piroxicam.

Instrument: Pye-Unicam sPS-loo.
518 MLADEN M I H A L I ~ET AL.

T a b l e 1. f 2 e v a l u e s f o r chemical s h i f t s of piroxicam i n
C-NMR spectrum i n CDCl
3
OH 0

C-atom Chemical s h i f t (ppm) Multiplicity

c- 1 117 7 s
c- 2 159.3 S
c- 3 128.8 6
c- 4 126.6 d
c- 5 132.9 d
C- 6 133-3 d
c- 7 124.2 d
C- 8 135.2 6
c- .9 167 5 6
c-lo 150-5 6
c-11 115.8 d
c-12 13% 0 d
c-13 120.4 d
C-14 147.2 d
c-15 39.2 9

256 nm (~=12700), 290 nm ( f = l o o o o ) and 358 nm (~=14600).

The second maximum i n 0.1 M H C 1 i s s u i t a b l e f o r s p e c t r o -
photometric d e t e r m i n a t i o n of piroxicarn i n p h a r m a c e u t i c a l
dosage forms.

4.1.4. Mass
The f r a g m e n t a t i o n was s t u d i e d by K r a t o s M-25 s p e c t r o -
m e t e r l i n k e d t o d a t a system DS+5oS, and i t i s shown i n
Fig. 5 /15/. A m o l e c u l a r i o n M ~ 3 3 1i s p r e s e n t w i t h 39%
i n t e n s i t y r e l a t i v e to t h e b a s i c i o n m/e=l73. The pathway
of f r a g m e n t a t i o n i s shown i n Scheme 2.

4.2. Solid Properties

4.2.1. Polymorphism
Piroxicam e x i s t s i n two d i f f e r e n t i n t e r c o n v e r t i b l e
c r y s t a l polymorphs w & t h m e l t i n g p o i n t s of 196-198"C /need-
l e form/ and 199-201 C / c u b i c form/. The two forms a r e
PIROXICAM 519

100
73
120
C.....

80
117.

78

60
11

s 21
%
CI

'3i L O 331
c 2 7
Q,
CI

.-c
d

!
20

0
100

Fig. 5. Mass spectrum of piroxicam at 2300 C.

Instrument: Kratos M-25.
 SCHEME 2.

-121 1 OH
1-94

m / e 173
+

I
m/e 76
m / e 117 1m / e 146
CH3]
m / e 104
PIROXICAM 521

distinguished by infrared absorption and, even more, by

X-ray powder diffraction (refer to the corresponding chap-
ters). There are no data about a possible different acti-
vity of the particular form.

4.2.2. Crystal Properties

When allowed to crystallize from an ethanolic solu-
tion by fast cooling, piroxicam precipitates as needles,
while by slow cooling from the same solution precipitates
in cubic form. By crystallization from an aqueous ethanol
or aqueous acetone piroxicam crystallizes with one Bole-
cule of water which disappears at approximately 120 C and
the melting point does not change.
The measurements of a sample obtained by grinding
the crystals of piroxicam at room temperature were taken
by a General Electric XRD-6 spectrogoniometer. The value
of 2 0 , interplanar distances d=nh/2 sin@, and relative
intensities I/Io based on highest intensity of loo, for
cubic and needle form of piroxicam, respectively, are gi-
ven in Tables 2 and 3. The data are obtained with a rate
meter T.C. 0.5, lo 0 0 0 cps. The corresponding spectra of
the X-ray diffraction pattern are presented on Figs. 6
and 7.
The space group of piroxicam (monoclinic, P2 /c) is
determined using Weissenberg photographs taken wigh CuK&
radiation /16/. The cell dimensions were defined from dif-
fractometer measurement~:~a=7.127(2), b=15.136(7), c =
=&3.949(6) 8, 4~97.3 (4) , Z=4, Vdt91.15 8 , Dd.481 mg
m , MoKu//h.=o.7107 , &=0.244 mm- /; final h0.050 for
2289 observed reflexions [ I 7 2 C?‘( I)]. The piroxicam mole-

Table 2. X-Ray characteristics of cubic form of piroxicam

11.74 24 7 5387
12.30 37 7 1967
14.62 87 6 0595
16.25 19 5 4552
16.70 46 5.2041
17.70 loo 4.9091
2i.0 0 21 4.0773
22.56 20 3.9416
26.72 72 3.3367
27.40 99 3.2554
27.92 34 3.1959
34.26 19 2.6176
522 MLADEN MIHALIC ET AL.

l . . . . , . l , . , , , , . , , , , . , l
25 20 15 10 5' e

Fig. 6. X-Ray diffraction pattern of piroxicam cubic form.

Instrument: General Electric XRD-6
spec trogoniom8t er .

20 15 10 5' 0

Fig. 7. X-Ray diffraction pattern of piroxicam needle form.

Instrument: General Electric XRD-6
spectrogoniometer.
PIROXICAM 523

Table 3. X-Ray characteristics of needle form of piroxicam

2 0" I/Io

9.18 23 9.6345
10.60 7 8.3468
15-78 loo 5.6166
18.16 17 4.8855
19.64 15 4.5206
20.20 9 4.3965
21.90 lo 4.0443
22.85 24 3.8923
25.22 34 3 5316
26.90 0 3*3051
30.24 8 2.3539

cule is not far from being planar (-bc plane). The thi-
azine ring exhibits a half-chair conformation. An amide
group is involved in an intramolecular hydrogen bond with
the hydroxy group. It also forms an intermolecular hydro-
gen bond with the oxygen atom bonded with the sulphur
atom, connecting piroxicam molecules in an infinite chain
along axis. The molecular packing is also influenced by
van der Waals interaction.
Piroxicam monohydrate, unlike the piroxicam structure,
exists in a zwitterionic form /17/, the enolic hydrogen
having been transferred to the pyridine nitrogen. Two
intramolecular hydrogen bonds are formed by an internal
rotation of the neutral structure (between enolate oxygen
and hydrogen on amide nitrogen, and between carbonyl oxy-
gen and the hydrogen on pyridine nitrogen). The side chain
and the atoms in the thiazine ring are planar. The crys-
tals are prismatic (space group P1) with following cell
b=12.909( 4) c=10.481( 3) 2 d =
dimensions:
=99.31( 2), ;:$78';1:\: z,
f'=102.64(2j0, V-1 37.9(7)8 , -1
Z=4, taken with CuKa radiation, hd.5410 /lc=21.3 cm
/17/*
4.3. Solution Properties
4.3.1. Solubility
Piroxicam is not soluble in water and cyclohexane,
sparingly soluble in diisopropyl ether and in toluene,
and only slightly more soluble in lower aliphatic alco-
hols methanol, ethanol and isopropanol . It is soluble
in some polar organic solvents such as dimethylformamide
(1 g/lo mil,dimethylsulphoxide ( 1 g/lo n i l ) , chloroform
(1 g/20 ml , and somewhat less soluble in dioxane ( 1 g/
524 MLADEN MIHALIC ET AL.

/40 ml), acetone (1 g / 5 0 ml) and ethyl acetate (1 g/80

m l ) /15/*
4.3.2. Acidity (PK 1
Potentiometrit titration of piroxicam solution in a
mixture of dioxane and water (2:l) gave a pK value of
6.3, effected by the enolic hydroxyl group a? C-4 /4,7,
18/.
4.3.3. Partition Coefficient
Piroxicam has a partition coefficient of 1.8 between
n-octanol and aqueous buffer pH=7.4 /4/.
4.3.4. Dipole Moment
The dipole moment+of giroxicam was determined in di-
oxane solution at 20.0-0.1 C, using a Dipolmeter DM 01
( Wiss.-Techn. Werkstatten, D 812 Weilheim). The value
found was 3.6820.06 D /15/.
5 . Methods of Analysis
5.1. Elemental Analysis
Piroxicam C H N 0 S (331.36) calc.: C 54.37 %
15 13 3 4 H 3.96 %
N 12.68 %
0 19.31 %
s 9.68 %
100.00 %
3 . 2 . Chromatographic Methods
3.2.1. Thin Laser
The identity and purity of piroxicam may be checked
by the use of 0.2 mm thick Fertigplatte Merck F and
suitable solvents or solvent mixtures, which are254
with cor-
responding Rf values of piroxicam listed in Table 4 /15/.
Table 4. TLC characteristic6 of piroxicam
Solvent Rf
chloroform 0.1
acetone-cyclohexane 0.56
dichloromethane-ethanol 20:l 0.58
chloroform-ethanol 1o:l 0.64
acetone 0.68

5.2.2. High Performance Liquid

The chromatogram was made by a Pye-Unicam LC-3-XP
chromatograph with UV-LC-3 detector. The column used was
03-

b-

N-
d

I
3O-
-.
526 MLADEN MIHALICET AL.
Whatman-Partisil PXS l0/25 PAC. The applied mobile phase
was acetonitrile-water-acetic acid 25-75-5 ml, 1.2 ml/
/min .The sample of piroxicam was dissolved in 0.1 N
NaOH in concentration of 1.0 mg/ml and gives one peak on
HPL chromatogram /19,20/, Fig. 8.
6, Stability - Degradation
6-1. Thermal Stability
The sample of pirox'cam was gept in a brown powder-
-glass in the dark at 20bC and 40 C. After two years no
change in color, smell, taste and shape of crystals could
be observed in samples kept at either temperature. TL and
HPL chromatograms did not show degradation products. Con-
tents in piroxicam obtained by analytical determinations
at various times of exposure are shown in Table 5. /15/.
Table 5. Content of piroxicam kept at two temperatures
at various times of exposure
Content of piroxicam inosample %
Time months 2ooc 40 C
0 99.9 100.1
3 loo. 0 99.9
6 loo. 1 99.8
9 98.8 99-6
12 loo. 1 99.7
24 99.8 99.5

6-2. Photostability
Samples of piroxicam 0.5 g were filled into color-
less 50 m l bottles and we:e igradiated for 72 hr with
light of 300-830 nm at 30-0.5 C. At 24 hr intervals, some
of the samples were examined for changes in appearance,
smell and taste. Piroxicam contents were determined by
HPLC /15/. The results are presented in Table 6.
Table 6. Photostability of piroxicam after irradiation
Time hours Content of piroxicam %
0 99.8
24 99.7
48 99.6
72 99-6

7. Drug Metabolic Products, Pharmacokinetics, Bioavaila-

bili ty
Piroxicam's pharmacological and pharmacokinetic pro-
file'has been rationalised in terms of chemical groupings
 I
0
L
t
I f
Q
ZI I" O0
0
F
0
0
I
-
"8:
I
0 0
c '0 .-Q
L
\ / \ /
0
528 MLADEN MIHALICET AL.

of the molecule. The enolic pdrtion has a relatively low

pKa which leads to virtually complete ionizations and
prolongs the activity of the drug. The group is also ne-
cessary for prostaglandin biosynthetase inhibition, the
proposed mode of action of piroxicam. The sulphoxide
group is lipophilic, enhancing absorption and facilitating
the passage of piroxicam across the gut-blood barrier.
The heterocyclic side-chain increases acidity and lipophi-
licity, it slows hydroxylation and therefore prolongs the
half-life. The N-methyl group increases efficacy by a
greater inhibition of PG synthetase than that provided by
a larger chain.
7.1. Pharmacokinetics
Piroxicam is readily ab6orbed after oral or rectal
administration and accumulates after repeated doses to
reach steady-state after about 7 days. The drug is exten-
sively metabolised to apparently inactive metabolites and
has a half-life of about 40 hours in man.
Peak plasma concentrations are attained about 2 hours
after a single oral dose and at about 5.5 hours after rec-
tal administration as suppositories /2U. Due to the ex-
tended plasma half-life of piroxicam, plasma concentrati-
ons remain very stable over the next 24-48 hr. Mean peak
plasma concentrations are roughly related t o dosage bet-
ween lo and loo mg, being 0.85,l,i,g/ml and 13.5,bg/ml after
a single lo or loo mg dose, respectively /22/. At concen-
trations of between 5 and 3o&g/ml, piroxicam is 99.3%
bound to plasma proteins /21,22/. Piroxicam penetrates
into the synovial fluid of patients with rheumatoid arthri-
tis and attains concentrations of about 40% of those in
plasma.
Piroxicam is highly protein-bound and thus might be
expected to displace other protein-bound drugs. Its ab-
sorption and disposition are unaffected by concomitant
administration of aspirin and antacids.
7.2. Metabolism
Available data sumest that piroxicam is extensively
metabolised in man /237-by the routes of 5' -pyridine ring
hydroxylation, glucuronide formation, cyclodehydration
a n d amide hydrolysis leading to decarboxylation, ring con-
traction and N-dealkylation /24/. The metabolic pathways
recognized for piroxicam are shown in Scheme 3. The prin-
cipal metabolite in man is that produced by hydroxylation
of the pyridyl ring (metabolite B ) and exists either free
or conjugated with glucuronic acid. The metabolite is at
least 1000 times less active than piroxicam in inhibiting
PIROXICAM 529

prostaglandin synthetase /25/.

About 10% of a single oral dose of piroxicam is re-
covered unchanged in the urine within the first 8 days
after administration. The elimination of half-life of pi-
roxicam is extended due to a low clearance rate and has
most often been calculated at 38 hours range 31 to 56.7
hours in healthy subjects /21-26/. Half-life appears not
to be related to dose or plasma concentration.
The formation of the cyclodehydrated metabolite ( A )
is a major pathway in the dog, while the rat seems to hy-
droxylate the pyridyl ring in the para-position to form
metabolite B. The latter compound, either free or conjuga-
ted with glucuronic acid, is the major human metabolite,
accounting for up to 60% of a daily dose in urine and fe-
ces. Laboratory animals also deamidate, decarboxylate,
and cause ring contraction, all in varying amounts. These
latter processes seem to be of minor importance in man
/n/.
None of the metabolites of piroxicam have significant
antiinflammatory activity in laboratory animals / 2 8 / . The
lack of activity for the metabolites, together with their
very low concentration in plasma [at or below the limits
of analytical detection), leads to the hypothesis that
piroxicam is intrinsically active, and its metabolites do
not contribute to that activity. Apparently, piroxicam
embodies a unique combination of functional groups which
are all required for its action.
8. Toxicity
Acute toxicity of piroxicam is low: the LD for
orally applied piroxicam is 360 mg/kg in the mo?ge, 270
mg/kg in the rat and over 700 mg/kg in the dog. When ad-
ministered intraperitoneally, the LD values are 360 and
220 mg/kg in the mouse and rat, respzgtively / 6 , 2 9 / .

9. Identifioation and Determination in Body Fluids and

Tissues
Piroxicam in plasma was assayed spectrophotometrical-
ly at 355 nm. The sensitivity limit of-the assay was ap-
proxirnFtely 0.5/Ccg/ml /22/. As well, piroxicam in plasma
was assayed fluorometricakly after hydrolysis with 6N
H SO4 for 20 hours at lo5 C. The released 2-amino-pyridine
wis examined in an Xminco Bowman spectrofluorimeter with
excitation at 310 nm and measurement at 380 nm. A Gilford
Model 410 digital absorbance meter and a Gilford Model
4006 data lister were interfaced with the spectrofluori-
meter for recording of the fluorescence values. The assay
was linear and sensitive down to 0 . 5 /ccg/ml. High concen-
530 MLADEN MIHALICET AL.

t r a t i o n s of s a l i c y l a t e were found n o t t o i n t e r f e r e i n t h e
a s s a y of piroxicam /22/.

l o . I d e n t i f i c a t i o n and D e t e r m i n a t i o n i n P h a r m a c e u t i c a l 6
Piroxicam i n c a p s u l e s was determined s p e c t r o p h o t o -
m e t r i c a l l y by measuring t h e e x t i n c t i o n of t h e s o l u t i o n a t
340 nm, 1 mg/loo m l , a g a i n s t R s u i t a b l e b l a n k , and t h e
c o n t e n t of piroxicam i n 1 c a p s u l e was c a l c u l a t e d /15/.

Acknowledgements. The a u t h o r s a r e t h a n k f u l t o d r . A. Nagl,

L a b o r a t o r y of General and I n o r g a n i c Chemistry, F a c u l t y of
S c i e n c e , U n i v e r s i t y of Zagreb, f o r X-ray d i f f r a c t i o n ~ p e -
c t r a l d a t a , and t 9 d r . Z. Meib, ”Ruder Bogkovib” I n s t i t u -
t e , Zagreb, f o r C n u c l e a r magnetic resonance d a t a .

11. References
1. N.E. P i t t s and R.R. P r o c t o r , i n W.M. O’Brien and E.H.
Wiseman / E d i t o r s / flPiroxicamlf Academic P r e s s , London
1978, P 97-108.
2. C.M. Williamson, Curr. Med. Res. Opin. 8, 622 /l9U3/.
3. E.H. Wiseman, D.C. Hobbs, Am. J. Med. 72, 9 /1982/.
4. E.H. Wiseman, Y.-H. Chang, J.G. Lombardino, Arzneim.-
-Forsch. /Drug Res./ 26, 1300 /1976/.
5. P f i z e r and Co., Ger. Offen. 1,943,265 /1969/, C.A.
2, 1 2 0 6 4 7 ~/1970/.
6. R.N. Brogden, R.C. Heel, T.M. S p e i g h t , G . S . Avery,
Drugs 22, 165 /1981/.
7. J . G . Lombardino, E.H. Wiseman, W.M. McLamore, J. Med.
Chem. 14, 1171 /1971/.
8. J.G. L G b a r d i n o P f i z e r Inc. U.S. Pat. 4,289,879
/1981/, C.A. 96, 20llow /1982/.
9. H, Zinnes, N . T Lindo, J.C. S i r c a r , M.L. Schwartz, J.
Shavel, Jr., G. D i Pasquale, J. Med. Chem. l6, 44
/1973/
l o . H. Zinnes, R.A. Comes, F.R. Z u l e s k i , A.N. Caro, J.
Shavel, J. Org. Chem. 2,2241 /1965/.
11. C.B. S c h a p i r a , I . A . P e r i l l o , S. Lamdan, J. H e t e r o c y c l .
Chem. 17,1 2 8 1 /1980/.
12. H. Zinnes, N.A. Lindo, J. Shavel, Jr. Warmer-Lambert
Co. U.S. P a t . 4,074,048 /1978/, C.A. 88, 190868b
/1976/.
13. I.A. P e r i l l o , C.B. S c h a p i r a , S. Lamdan, J. H e t e r o c y c l .
Chem. 20, 155 /1983/.
14. H.L. Rice, G.R. P e t t i t , J. Amer. Chem. SOC. 76, 302
/I 954/
15. M. Mihalib, H. Hofman, F. KajfeB, J. K u f t i n e c , N. B l a -
i e v i b , M. Z i n i b , Acta Pharm. Jugoslav. 2,13 /1982/.
16. B. Kojib-Prodib, 2. Ruii&-ToroH, Acta C r y s t . B 38,
2948 /1982/.
PIROXICAM 53 1

17. J. Bordner, J.A. Richards, P. Weeks, E.B. Whipple,

Acta Cryst. C 40, 989 /1984/.
18. J.G. Lombardino, E.H. Wiseman, J. Chiaini, J. Ned.
Chem. 2,493 /1973/.
19. T.M. Twomey, S. R. Bartolucci, D.C. Hobbs, J. Chroma-
tog. a, 104 /1980/.
20. K.D. Riedel, H. Laufen, J. Chromatog. 276, 243 /1983/.
21. P. Schiantarelli, D. Acerbi, c). Bovis, Arzneim.-Forsch.
(Drug Res.) 2,92 /1981/.
22. D.C. Hobbs, T.M. Twomey, J. Clin. Pharmacol. 2,270
/1979/-
23. T. Ishisaki, T. Nomura, T. Abe, J. Pharmacokinet. Bio-
pharm. 2, 369 /1979/.
24. T.M. Twomey, D.C. Hobbs, Fed. Proc. Fed. Am. SOC. Exp.
Biol. x, 271 /1978/.
25. E.H. Wiseman, J.A. Boyle, Clinics in Rheumatic Disea-
ses 5, 585 /1980/.
26. J.G. Lombardino, E.H. Wiseman, Med. Res. Rev. 2, 127
/1982/.
27. D . C . Hobbs, T.M. Twomey, Drug. Metab. Dispos. 4, 114
/1981/.
28. JOG. Lombardino, J. Med. Chem. 24, 39 /1981/.
29. E.H. Wiaeman, Royal Society of Medicine International
Cougress and Symposium Series, No. 1, pp. 11-23 /1978/.
This Page Intentionally Left Blank
 RANITIDINE

Marijan Hohnjec, Josip Kuftinec, Miljenka Malnar,

Milivoj gkreblin, Franjo Kajfei, Antun Nagl,
Nikola Blaievid

1. Foreword, History, Therapeutic Category

2, Description
2.1. Nomenclature
2.1.1. Chemical Name
2.1.2. Generic Name
2.1.3. Trade Name
2.2. Formula
2.3. Molecular Weight
2.4. Appearance, Color, Odor
3. Synthesis
4. Physical Properties
4.1. Spectral Properties
4.1.1. Infrared Spectra
4.1.2. Ultraviolet Spectrum
4.1.3. f'jjoton Magnetic Resonance
4.1.4. C-Nuclear Magnetic Resonance
4.1.5. Mass Spectrum
4.2. Solid Properties
4.2.1. Melting Characteristics
4.2.2. X-Ray Diffraction
4.3, Solution Properties
4.3.1. Solubility
4.3.2. Acidity (pKa)
5. Methods of Analysis
5.1. Chromatographic Methods
5.1.1. Thin Layer
5.1.2. High Pressure Liquid
5.2. Spectrophotometric Determination
ANALYTICAL PROFILES OF DRUG SUBSTANCES Copyright Q 1986
VOLUME 15 by the American Pharmaceutical Association
533 All rights of reprodiirtion in any form reserved
534 MARIJAN HOHNJEC ET AL.

6. Stability -
Degradation
7. Drug Metabolism, Pharmacokinetics, Bioavailability
8. Identification and Determination in Body Fluids and
Ti 6 sues
9. Identification and Determination in Pharmaceuticals
lo. References
RANITIDINE 535

1. Foreword, History, Therapeutic Category

Ranitidine is a new histamine H -receptor antagonist
which, unlike cimetidine that contaizs an imidazole ring,
has a furane ring structure /1,2/. This substituted amino-
-alkyl-furane derivative is more potent than cimetidine
in inhibition of gastric acid secretion induced by vari-
ous stimuli and lacks cimetidine's anti-androgenic and
hepatic microsomal enzyme inhibiting effects.
The drug has been used in the treatment of duodenal
and gastric ulceration. In the recommended dosage of 150
mg twice daily, ranitidine is as effective as cimetidine
and has therefore the advantages of less frequent doeing
and fewer side effects. Ranitidine appears to be the drug
of choice in the treatment of the Zollinger-Ellison syn-
drome because of its increased potency and lesser effect
on endocrine function compared to cimetidine. The compo-
und is given orally a6 a tablet (150 m a of ranitidine ba-
se) and as an injection Eolution ( 5 0 mg/5 ml).
The first synthesis of ranitidine was reported in
1973 /3/ followed by pharmacological and clinical studies
in 1979 /1,4/ and 1980 /5-7/. Finally, ranitidine was in-
troduced on to the market in 1981.
2. Description
2.1. Nomenclature
2.1.1. Chemical Name
The Chemical Abstracts name for ranitidine is N-[2-
-1 [ 5-1:(dimethylamino)methyl] -2-furanyl]methyl]thiolethyl]
-N'-methyl-2-nitro-l,l-ethene diamine. The CAS registry
-
No. is 66357-35-5. The other name is 2-[ [ [ 5-(dimethylamino)-
methyl-2-furanyl]-methyl]thio]-ethylamino-2-methylamino-
-1-nitroethene.
2.1.2. Generic Name
Ranitidine
2.1.3. Trade Name
Zantac
2.2. Formula
CHN02
!
0 \
-HCI
NCH2 0 CH2SCH2CH2NH NHCH3
C13H22N,+03Se HC1
CH3
536 MARIJAN HOHNJEC ET AL.

2.3. Molecular Weight

350.869
2.4. Appearance, Color, Odor
Ranitidine is marketed only as the hydrochloride
salt. It is a white to yellowish solid with little or no
odor. A slight sulfur-mercaptan odor may be present.
3. Synthesis
The first synthesis of ranitidine, as shown in Sche-
me I, started with the reaction of 5-dimethylaminomethyl-
-2-furanyl-methanol (I)with 2-mercaptoethylamine by me-
ans of aqueous hydrochloric acid to give 2-[[(5-dimethyl-
aminomethyl-2-furanyl)methyl] thiolethaneamine ( 11) This
intermediate is then condensed with N-methyl-1-methBlthio-
-2-nitro etheneamine (IV)by heating in water at 50 C for
4 hours. Compound IV is obtained in the reaction of 1,l-
-bis(methylthio)-2-nitroethene (111)with methylamine in
refluxing ethanol /3/.
Alternatively, ranitidine ( V ) may be prepared by con-
densation of 5-dimethylaminomethyl-2-furanyl methyl mer-
captan (VI) with aziridine derivative VII /8/ and by the
reaction of amine I1 with 1-nitro-3-methyl ketene imine
(1x1 /9/ - Scheme 11. Other patented methods for raniti-
dint preparation are also shown in Scheme 11. The reaction
of nitromethane with pseudothioureido compound VIII /lo/
and carbodiimide XI /11/ as well as the condensation of
aziridine with nitroethene intermediate X /12/ and mer-
capto dfamino nitroethene XI1 with 5-dimethyl-aminomethyl-
-2-furanylmethanol (I)/3/ leads to ranitidine in modera-
te yields.
4. Physical Properties
4.1. spectral ropert ties
The IR absorption spectrum of the crystal form 1 ra-
nitidine hydrochloride is shown in Figure 1. Figure 2 is
the infrared spectrum of the form 2 ranitidine hydrochlo-
ride. These spectra were recorded with KBr-pelleted Barn-
ples using a Pye Unicam SP-200 Infrared Spectrophotometer
/13/. Our results are in good agreement with the publish-
ed data for the cryeta1 form 2 ranitidine hydrochloride
/14/. In the IR spectrum of the crystal form 1 (Fig. 1)
the well-known bandrs characteristics of a nitro group at-
tached to a saturated carbon atom.!:i stretching vibra-
tion frequencies at 1554 and 1382 cm /15/, are hardly
vi_efble as well as the band6 in the range of 1505-1540
cm characteristic of a nitro group attached to a substi-
 cv
cv 0
0 7
7 z >
=
0
w
=:I
m
o
I w
0
II
I
m
11 0
I
0 / \ '2
m' m
m
' c*) m m
X
0
I
0
f
0
I
0 cv
I
0
cv
I
7 0
I"
I"
7
I" *
I" >
I I"
0
0
cv
I
I"
0
m 0
I I"
0
-2
I/
0 \=
U
c
L
538
0
4000 3500 3000 2500 2om 1600 1200 cm-1 800

Fig. 1. Infrared spectrum of ranitidine hydrochloride form 1 in KBr pellet.

Instrument: Pye-Unicam SP3-200.
0
10
0
r
c,
al
rl
0
o
m d
k
f4
D k4
8 C
.rl
ru
0
0
9 E
0
k
al
0
2
2. .
k
0
d
s
0
D
0 0
5 k
T¶
A
.c
0
z
r
0
8
N
0
ul
N
D
D
n
D
8 N
M
.rl
k
D
D
D
Y
RANITIDINE 54 1

tuted ethenf group /16/. There is, however, a strong band

at 1620 cm- which corresponds to the stretching vibrati-
on of the C=N double bond in an aci-nitro group of nitro-
nic acid /l7(. Markedly 6trong bands appearing at 2640
and 2560 cm- are characteristic of the N+-H bond which
exists in the protonated tertiary arnine group H-N’R in
V hydrochloride. The properties of the IR spectrum Jug-
gest that ranitidine hydrochloride exists mostly in the
tautomeric form denoted by B in Scheme 111. Thic c o n c l u -
sion is in agreement with other authors’ findings for si-
milarly substituted nitroethenes in which nitronic acid
forms are stabilized by conjugation /18/. Other spectro-
scopic data also suggest that ranitidine hydrochloride
exists predominantly in the form represented by formula
B. These conclusions were also confirmed by single crys-
tal X-ray diffraction studies reported by B. Kojit-ProdiE
et a1 /19/. Namely, the bond lengths in the 2-ethyl-2-me-
thylamino-1-nitroethene residue are in agreement with the
structure B.
SCHEME I11

CHN02
II
C0\ &\
RNH HNCH3 RN HNCH3
A B

R=
CH3
‘NCH2 ‘ 0
/
‘k CH2SCH2CH2-
CH3
4.1.2. Ultraviolet Spectrum
The ultraviolet spectrum of ranitidine hydrochlorlge
was recorded in an aqueous solution, concentrations lo
g / l , using a Pye Unicam sP8-loo UV-spectrophotometer, and
is shown in Figure 3 /13/. In the ultraviolet spectrum of
V hydrochloride an expected bathochromic shift of the ab-
sorption maximum, characteristic for nitro group -olefin
542 MARIJAN HOHNJEC ET AL.

0.7

0.6

0.5

g 0.4
c
d
a
L
0

0.i

0.'

nm
Fig. 3. Ultraviolet spectrum of ranitidine hydro-
chloride in an aqueous solution.
Instrument: Pye-Unicam SP8-loo.
RANITIDINE 543

conjugation, is present. The spectrum shows two absorp-

tion maxima, at 228 nm (&=23,485) and at 313 nm ( & =
=16.030). The measurement of the absorption at 313 nm is
very convenient for quantitative determination of raniti-
dine hydrochloride even in the presence of intermediates
in the ranitidine synthesis.
4.1.3. Proton Magnetic Resonance
The proton magnetic resonance spectra of ranitidine
base (Figure 4) and ranitidine hydrochloride (Figure 5 )
were recorded with a WP 80PS instrument by Bruker /13/.
The spectra illustrated were obtained using deuterated
chloroform and deuterated dimethyl sulfoxide solutions
containing approximately 80 mg/ml of the compound with
tetramethylsilane as the internal standard. The curve de-
noted by B in both spectra represents the case when D 0
was added to deuterated organic solvent solutions, The 2
spectrum of ranitidine base shows a singlet at 2.24 ppm
(due to six protons of the N ( C H ) group) and a broad
2
doublet at 3.00 ppm (due to prozons of the CH NH group)
which, upon addition of D 0, contracts into a3singlet.
Spin decoupling at 3-40ppm2
- the position of a multiplet
due to the methylene protons of the NHCH group - causes
a contraction of the upfield triplet at ?.84 ppm to a
singlet. As the coupling constant J ( C H 2 S , C H N ) is 7.5 H e ,
the triplet at 2.84 ppm sensitive to decoupfing influen-
ces must correspond to the two protons of the methylene
group connected with the sulfur atom, which points away
from the ring. Further signals may be assigned as follows:
the singlet at 3.50 ppm ( 2 H ) corresponds to protons of
the C H N group in 5-position of the farane ring. The next
2
downfield singlet at 3.72 ppm ( 2 H ) corresponds to the
protons from the methylene bridge linking the ring to the
S-atom. The pair of doublets at 6.22 ppm ( 1 H ) and 6.26
ppm (1H) aDrresponds to ring protons in 3- and 4-positions,
respectively (J 4=3,75 H e ) . The nearest singlet potiiti-
the nitroethene group CHNO .
oned at 6.56 ppJ'[lH) corresponds to the vinyl proton of
In the spectrum of ranitidi-
ne hydrochloride (Figure 53 the signal for this proton is
situated between signals for the furane ring protons.
This proton is in both cases displaced by a deuteron upon
addition of D 0, which indicates a tautomeric prototro-
pic shift in $he nitroethene terminal. This observation
has been confirmed by A. Sega, et al. /20/ in their inve-
stigation of the H/D exchange in 2,2-disubstituted nitro-
ethenes. Finally, the broad singlet centered at 10.33 ppm
(1H) cannot be definitely assigned to either the proton
NH, or that of the nitronic acid group =N-OH. The contrac-
 L *
B

I I I I I

11 10 9 8 7 6 5 L 3 2 1 PPm 0
Fig. 4. Proton magnetic resonance spectrum of ranitidine base in CDCl
Instrument: Bruker WP8oPS 3.
 I I I I I l l I I I I I 1 I I I I 1 1 I I I
10 9 8 7 6 5 4 3 2 1 0
PPm
Fig. 5. Proton magnetic resonance spectrum of ranitidine hydrochloride in DMSQ-d
Instrument: Bruker w P ~ o P s . 6'
546 MARIJAN HOHNJEC ETAL.

t i n g e f f e c t of D 2 0 a d d i t i o n upon t h e C H NH- m u l t i p l e t con-

f i r m s t h a t t h e n e i g h b o u r i n g N-atom b i n d 2 a p r o t o n . The i n -
f l u e n c e of D 0 d o e s n o t extend t o t h e CH N a m u l t i p l e t
2 2
which c o n f i r m s t h e a b s e n c e of a p r o t o n from N-atom. One
s h o u l d e x p e c t , t h e r e f o r e , t h a t r a n i t i d i n e i s more l i k e l y
t o b e r e p r e s e n t e d by formula B (Scheme 111) t h a n by f o r -
m u l a A.

4.1.4. 13C1Nucl e a r MaRnet i c Resonance

The &'C-NMR s p e c t r a o f r a n i t i d i n e h y d r o c h l o r i d e were
r e c o r d e d w i t h a J e o l FX-loo s p e c t r o m e t e r a t 25.05 MHz. The
samples were d i s s o l v e d i n DMSo-d6 and s p e c t r a were r e c o r -
d e d i n broad-band, o f f - r e s o n a n c e and NOE modes /l3/. The
broad-band and o f f - r e s o n a n c e s p e c t r a were shown i n F i g . 6 .
I n b o t h modes i t was p o s s i b l e t o a s s i g n a l l carbon atoms.
The d e t e r m i n a t i o n s of C-H c o u p l i n g c o n s t a n t s were d i f f i c u l t
f o r groups N ( C H ) and NHCH , because t h e r e s p e c t i v e s i p

ponding t o t h e s o l v e n t DMSO-d6 .
n a l s were s i t u a ? e g w i t h i n tAe r a n g e of t h e s e p t e t c o m e s -
T h e r e f o r e , t h e y were
determined u s i n g NOE measurements i n CD OD a t 20.1 MHz.
3
T a b l e I. I3C-NMR spectrum of r a n i t i d i n e h y d r o c h l o r i d e i n
CD OD a t 20.1 MHz; c h e m i c a l s h i f t s ( d ) and C-H
coapling c o n s t a n t s (J
C-H
C-atom c3 (ppm) JC,H ( Hz)

158.0 doublet 6.3

C-5 f u r a n e 155. o doublet 12.6
C-2 f u r a n e 145. o doublet 12.6
C-4 f u r a n e 116.6 quartet 128.1 and 5.0
C-3 f u r a n e 110.2 quartet 123.9 and 5.0
CH=N02H
N-CH,
99.3
53.8
42.8
doublet
triplet
quartet
189.2
148 3
126.3
.
NHCH 42.3 quartet 139.1
-CH 3- 31.7 triplet 150.1
-sc6 - 28.9 triplet 142.2
-CH,S- 28.6 triplet 139 1

4.1.5. Mass Spectrum

The mass spectrum was r e c o r d e d w i t h a n MS-25 mass
s p e c t r o m e t e r w i t h d a t a s y s t e m DS 50s from K r a t o s , Manche-
s t e r . Samples f o r mass s p e c t r a were d i r e c t l y i n t r o d u c e d
i n t o t h e i o n source. The e l e c t r o n impact i o n i s a t i o n was
a p p l i e d a t 70 eV ( 4 7 3 K s o u r c e t e m p e r a t u r e ) and s p e c t r a
were r e c o r d e d a t a s c a n n i n g speed of 3 sec/scan. The spe-
 I I 1 1 I I I I 8

16 0 1LO 120 100 80 60 LO 0

*O PPm
Fig. 6. 13C-NMR broad-band ( A ) and off-resonance ( B ) spectra of ranitidine hydrochloride
in DMs0-d~. Instrument: Jeol FX-loo at 25.05 MHz.
548 MARIJAN HOHNJEC ET AL.

NHCH?
58 224
138 176

loo I
297
269
224
1
I
.dl
281

100 7
8
1

42
cI
z
L
$50-
.-
I
d

8 169
1
II, 1
0
40 60 80 100 120 140 160 180 200
m/e

Fig. 7. Mass spectrum and fragmentation of ranitidine.

Instrument: Kratos MS-25.
RANITIDINE 549

Table 11. X-Ray d i f f r a c t i o n d a t a of r a n i t i d i n e hydrochlo-

r i d e form 1
InterDlanar Relative
8 ("I distance intensity **
d* ( 8 ) I/I-
7-02 6.31 22
7.21 6.14 34
7.59 5.84 39
7.76 5.71 79
8.43 5.26 loo
8.66 5.12 34
lo. 36 4.29 21
10.81 4.11 87
11.16 3.98 64
11 97 3.72 24
11.34 3.61 76
12.12 3.40 59
13- 07 3. 17 73
14.24 3-13 45
14.49 3.08 42
14.18 2.04 21

*d = n h / 2 s i n e
**Based on t h e h i g h e s t i n t e n s i t y a d j u s t e d t o 1.00.
550 MARIJAN HOHNJEC ET AL.

L I 1 . 1 r l ' ' . " ' ' ' . . " . .

25 20 15 10 5' 8

Fig. 8. X-Ray diffraction pattern of ranitidine hydro-

chloride form 1. Instrument: General Electric
XRD-6 spectrogoniometer.
RANITIDINE 551

Table 111. X-Ray diffraction data of ranitidine hydro-

chloride form 2
In t e rplana r Relative
distance intensity**
Q ( O )
d* (8) I/In-
4.2 l o . 47 20
793 6.08 13
7.7 5.75 21
8-3 5.34 37
9.1 4.87 12
9.6 4.62 11
lo. 2 4.36 loo
10.5 4.23 15
11.4 3.88 14
11.8 3.77 58
12.1 3.67 23
12.4 3.60 12
12.9 3.44 15
13.8 3.23 21
14.4 3. l o 19
14.8 3.02 7
16.0 2-79 20
18.2 2.46 9
it d = n h / 2 sin@
**Based on the highest intensity adjusted to 1.00.
Fig. 8. X-Ray diffraction pattern of ranitidine hydrochloride form 2.
Instrument: General Electric XRD-6 spectrogoniometer.
RANITIDINE 553

ctra were evaluated using the attached data system /l3/.

The mass spectrum of ranitidine and its main fragments
are shown in Figure 7. The molecular ion on the ranitidine
spectrum could not be detected. The abundance of the ion
m/e 297, which differs by 17 mass units from the proposed
molecular ion, suggests the presence of a hydroxyl group
in the structure of ranitidine. Loss of OH from the mole-
cular ion was confirmed by exact mass measurements of the
m/e 297 by high resolution mass spectrometry. This frag-
mentation is in accordance with our proposal that the
structure of ranitidine is described by formula B (Sche-
me 111). According to Martin, et al. /21/ the ion m/e 297
was generated by loss a molecule of water from the proto-
nated molecular ion. It should be noted that this mass
spectrum was obtained by the chemical ionization mode,
while we used an electron impact technique.
4.2. Solid Properties
4.2.1. Melting Characteristics
The ranitidine base is difficult to crystallize, but
its hydrochloride can be conveniently crystallized, par-
ticularly from isopropanol. The melting range of ranitidi-
ne hydrochloride depends on the polymorphic form in which
this compound is crystallized. When ethylacetate is added
to an ethanolic solution of ranitidine hydrochloride the
crystalAine form 1 is obtained gith the melting range of
135-136 C /ref. 4: m.p. 133-134 C/. The form 2 is cryatal-
lized from isopropanol-HC1 solutionowith the melting range
of 143-144OC /ref. 14: m.p. 141-142 C/.
4.2.2. X-Ray Diffraction
The X-ray diffraction patterns were determined with
a Mod. XRD-6 spectrogoniometer from General Electric,
Schenectady. The spectra were taken with a monochromatic
radiation which was obtained from the CuKw line (15.42
nm) excited at 35 kV and 20 mA /13/. The X-ray powder dif-
fraction spectra for both forms of ranitidine hydrochlori-
de are given in Figures 8 and 9. Tables I1 and I11 list
the interplanar distances, the diffraction angle and the
relative peak intensities.
4.3. Solution Properties
-4.3.1. Solubility
The solubility of ranitidine hydrochloride in various
solvents at room temperature is summarized in Table IV.
4.3.2. Acidity ( P K
For the detergination of the pKa value the spectro-
554 MARIJAN HOHNJEC ET AL.

T a b l e IV. S o l u b i l i t i e s o f r a n i t i d i n e h y d r o c h l o r i d e

Solvent Solubility

acetic acid freely soluble

water very f r e e l y soluble
methanol soluble
ethanol sparingly soluble
ethylacetate very s l i g h t l y soluble
isopropanol v e r y s l i g h t l y sOlublt?
dioxane insoluble
chloroform insoluble

p h o t y n e t r i c method was used /22/. The o b t a i n e d v a l u e wa8

2.19-0.04 /13/.

5. Methods of A n a l y s i s
5.1. Chromatographic Methods
7.1.1. Thin Layer
The p u r i t y of r a n i t i d i n e h y d r o c h l o r i d e can b e quick-
l y a s s e s s e d by TLC o v e r s i l i c a g e l . Table V shows i t s R f -
- v a l u e s w i t h s e v e r a l s o l v e n t systems /13/. S p o t s were

Table V, R f - v a l u e s

S o l v e n t s and Rf-values

A B C D E F
0.50 0.44 0.36 0.64 0.73 0.39
A= EtOAc/MeOH/Et NH (3:3:1), B= CHC13/i-PrOH/Et NH ( 4 : 3 :
:2 ) , C= dioxane/$eOH/DMF ( 6 :3 :2 1, D= MeCN/MeOH/$% NH OH
( 5 : 2 : 1 ) , E= EtOAc/MeOH/25% NH40H ( 1:5:1), F= EtOAc/i-hOH/
/25% NH40H (4:3: 1).

l o c a t e d e i t h e r under a n UV lamp, o r by s t a i n i n g t h r o u g h
exposure t o i o d i n e vapors.

samples of a 20 mg.cm ’
Suggested p r o c e d u r e f o r t h e i d e n t i f i c a t i o n of r a n i -
t i d i n e by t h i n - l a y e r :romatography: f i v e , 1 5 and 30 /u1
m e t h a n o l i c s o l u t i o n , and t h e same
volume of a m e t h a n o l i c s o l u t i o n o f e q u a l c o n c e n t r a t i o n s
of t h e s t a n d a r d s u b s t a n c e a r e a p p l i e d t o a s i l i c a g e l
p l a t e and chromatograms developed w i t h e t h y l a c e t a t e h e -
t h a n o l / d i e t h y l a m i n e (3:3:1). The s p o t s must have t h e same
f l u o r e s c e n c e i n t e n s i t y under UV-radiation, and t h e same
shade of s t r a i n subsequent t o exposure t o i o d i n e vapor.
All R v a l u e s must be c l o s e t o 0.5.
f
RANITIDINE 555

5.1.2. High Pressure Liquid

The HPLC analyses were performed with a HPLC appara-
tus, LC-3-XP with an UV-LC detector. The HPL chromatogram
is shown in Figure lo /13/. The chromatograms were run
through a column filled with Li-Chromosorb RP-8 (5,Um)
using a mixture of acetonitrile, methanol, w ter, and
concentrated ammonia (250, 20, 6 and 0 . 0 5 cm3, respecti-
vely) having a pH-value of 7.4. The elution was carried
out under 13.78 to 17.22 MPa (20.000- 5.000 p.s.i,) pres-
sure, maintaining a flow rate of 1 cm$.min-l. The efflu-
ent was monitored optically at 217 nm.

2.2. Spectrophotometric Determination

curately weighed into a lo
ved by swirling with 50 c
de up to the mark Ten cm'
' zy
Ten mg of "unknown" ranit'dine hydrochloride is ac-
volumetric flask, dissol-
water and the solution ma-
of the resulting solution is
diluted to loo cm3 in another volumetric flask, and the
absorbance of the final dilution is measured at 313 nm
against water. The same procedure is carried out with a
standard sample of known ranitidine concentration /U/.
Calculation: AUMsP
C H C1N 0 S in unknown, % = A loo
13 23 4 3 s u
A = absorbances, M = masses [ g), u = unknown, s = standard,
P = % of C13H,JC1N 0 S in the standard.
43
6. Stability - Degradation
The stability of ranitidine hydrochloride in tablets
was t % sted in two ways: one series of samples was kept
at 40 C and 50-6096 relative humidity f o r five days, and
the other at 6ooC and loo% relative humidity during the
same period. According to the spectrophotometric deter-
mination, the degradation amounted to -5% (under the
first conditions) and 11.5%, respectively.
The thin-layer chromatographic analysis on silica gel
shows five different degradation products in both cases.

7. Drug Metabolism, PharmacQkinetics, Bioavailability

Metabolic studies of "C-ranitidine in rat and dog
showed that ranitidine was mainly metabolised by oxidati-
on to give N-oxide X I I I , S-oxide XV, and desmethyl rani-
tidine X I V . The relative amount of each metabolite for-
med was found to vary with the species /23/. Thin-layer
chromatographic analyses of the urine collected from vo-
lunteers given oral and intravenous doses of ranitidine
556 MARIJAN HOHNJEC ET AL.

Fig. lo. HPL chromatogram of ranitidine hydrochloride.

Instrument: Pye-Unicam LC-3-XP.
RANITIDINE 557

CHN02
II
2SC H2CH2 NHCNHCH3
t Xlll
0

H
XIV

CHN02
H II
3c)NCH2 CH2SCH2CH2NHCNHCH3
i
0
xv
showed that ranitidine was the major component present.
Compound XI11 was the major metabolite, and small quanti-
ties of compound XIV and XV were also detected / 2 4 / . Rani-
tidine is a potent inhibitor of gastric secretion after
oral administration. It is four to seven times more potent
than cimetidine. In doses of 20, 40 and 80 mg, ranitidine
reduces hydrogen output by 29%, 50%, and 70%, and gastric
secretion volume by 2196, 37%, and 47%. With the same doses
it also reduce6 pepsin activity by 8%, 50% and 49%. Serum
concentration of 0.08 ,ug/ml of ranitidine reduces gastric
acid output by 50% /25,26/. Following oral and parenteral
administration ranitidine blood concentration curve has a
pronounced secondary peak. After oral administration ig
+humans, the first peak in the plasma occurs within 1.1-
- 0 . 4 h, and the second peak within 3:o h. These peak pla-
sma concentrations are not influenced by food /27/. Fol-
lowing intravenous administration there is a biexponential
decline in the plasma levels from 576556 ng/ml after 4
min to 1022 ng/ml after 8 h. In healthy subjects the cere-
558 MARIJAN HOHNJEC ET AL.

brospinal fluid concentration of ranitidine is one twen-

tieth, to one-thirtieth of that in the plasma sampled at
the same time. The distribution half-life of ranitidine
+
is 6.1-0.9 min., elimination half-life being 1.9-0.1 h,
the volume of distributfon is 96-115+7 1, and systemic
plasma clearance is 709-62 ml/min. The reported bioavaila-
bility after a single dose was between about 40 and 88%,
but mostly around 50% /28,29,30/. In the research of the
effects on hepatic drug metabolism, it has been found
that ranitidine does not enhibit the microsomal drug oxi-
dative function /31/. Ranitidine is 15% protein bound.
Half of the oral dose of ranitidine is readily absorbed,
and half of the+absorbed amount is found unchanged in the
urine. Only 1.3-0.3% of the intravenous dose and 2.6toe2%
of the oral dose is converted into the desmethyl metabo-
lite.
8. Identification and Determination in Body Fluids and
Tissues
Ranitidine may be determined in the serum, plasma,
and urine by high pressure liquid chromatographic analy-
sis /32,33,24/. Martin, et al. /21/ used the on-line high
-
performance liquid chromatography mass spectrometry for
the identification and structure determination of raniti-
dine and its metabolites in the urine. The separation of
ranitidine and its metabolites is usually carried out by
extraction of the biological medium with methylene chlo-
ride from an aqueous alkaline solution ( 2 M NaOH or 5M
KOH), followed by mixing, addition of an internal standard
and centrifugation /33/. The addition of 10% isopropanol
to methylene chloride increased the recovery of ranitidine
in this extraction procedure /32/. The organic gayer was
then evaporated to dryness under nitrogen at 45 C. When
treateg in this way, the dry residue was stable for 7 days
at -20 C. Before chromatography, the residue was dissol-
ved in a methanol-dibasic ammonium phosphate mixture /33/.
Ranitidine was also assayed in the urine by HPLC procedu-
re using direct injection and no internal standard /24/.

XVI
RANITIDINE 559

During usual HPLC analyses metiamide /32/ and N-methyl-

-N' -[ 3-[ ( 3-dimethylaminomethyl)phenoxy] propyl] -2-ni tro-
-1,l-ethenediamine hydrochloride XVI /33/ were used as
internal standards. The columns used for HPLC were the re-
verse phase p Bondpak c-18 (Waters Associates), Spheri-
sorb ODs (Phase Separations, Clwyd, Great Britain) and
Spherisorb S5 CN. An example of the conditions of analy-
sis is shown in Table VI /32/.
Table VI. Conditions of HPLC analysis
Parameter Assay conditions required
Mobile phase 92/8 mixture Reagent A/Reagent B
Column U, Bondpak c-18
Temperature ambient temperature ( 20-25OC)
Pressure 1000 - 3 0 0 0 psi
Absorbance full scale 0.005
Flow rate 2 ml/min
Wavelength 228 nm
Internal standard met iamid e
Chromatography time 8 min
A - ranitidine, B - metiamide
9. Identification and Determination in Pharmaceuticals
For the determination of ranitidine hydrochloride con-
tent in a tablet dosage form we recommend the following
procedure /13/; Crush 20 tablets in a mortar, Quantitati-
vely transfer the mass of powder equiv lent to lo mg of
ranitidine hydrochloride into R 250 cm3 volumetric flask.
Add loo cm3 of water and sheke the resulting suspension
automatically for 20 minutes. Make up to volume, mix well
and centrif ge 20 ml aliquot at 2000 G for 5 minute
Pipet lo cm' of the clear supernatant into a loo cm3*vo-
lumetric flask and make up to volume. Measure the absor-
bance of the solution at 313 nm against water and compare
it to that of an appropriate standard solution. Calcula-
tion :
C H C1N 0 S contents, mg per tablet =
13 23 4 3
AU*MS
ut .
ut -
the average mas6 of one tablet
For other symbols see chapter 5.
Acknowledgements. The authors would like to thank Dr B.
RugEi6 and Z. Boii6evi6 of the Institute "Ruder Bo5kovib1I,
Zagreb, for the pK determination, and to Dr M. Milun of
"CHROMOS", Researca and Development, Zagreb, for NMR
560 MARIJAN HOHNJEC E T A L .

measurements .
l o . References
-I. N.R, Peden, J.H.B. Saunders, K.G. Wormsley, Lancet 1,
690 /1979/*
2. W. Domschke, G. Lux, S. Domschke, G a s t r o e n t e r o l o g y

3*
-
79, 1-26? /1980/*
B. J. P r i c e , J, W. C l i t h e r o w , J. W. Bradshaw ( A l l e n and
Hanburys Ltd.) , Ger. Offen. 2,734,070; C O A O 88,
190580b /197a/.
4. H. J. Ruoff, U. Gladziwa, K.F. Sweng, G a s t r o e n t e r o l o g y
76, 1230 /1979/*
m

5. R. S t a b l e s , M.J. Daly, Agents A c t i o n s lo, 191 /1980/.

6. K. K e t t , F. Aadland, A. B e r s t e d , Scan. J. G a s t r o e n t e -
r o l . 2,249 /i980/.
7. E.P. Woodings, G.T. Dixon, C. H a r r i s o n , P. Carey,
D.A. Richard?, Gut 21, 187 /1980/.
8. R. Toso, V. s u n j i 6 ( C R C ) , Ger. Offen. 3,118,813 /1982/;
C.A. 96, 181127~/1982/.
xg.
lo.
9. Lek, Pa$, Appl. 2608/82.
Glaxo, Eur. Pat. 55,626 /1982/; C.A. 97, 1 9 8 0 9 4 ~/1982/.

12.
11.

13.
Evers, Neth. Pat. 8303-4354 /1984/.
Glaxo, Eur. Pat. 59082 /1982/.
M. Hohnjec, S. Rendit, T. Alebit-Kolbah, F. K a j f e i ,
N. B l a i e v i t , J. K u f t i n e c , Acta Pharm. J u g o s l . 2,131
11981~
14. D.L. Crookes ( G l a x o ) , Ger. Offen. 3,139,134; C.A. 97,
61014g /1982/.
15 L.J. Begiamy "The I n f r a r e d S p e c t r a of Complex Molecu-
lestt, 2 Ed., Methuen and Co., London 1957, p 333.
16. R. Gompper, H. S c h a e f e r , Chem. Ber. loo, 591 /1967/.
17 H. Feuer, "The Chemistry of t h e N i t r o and N i t r o s o Gro-

18 . ups1!, John Wiley, N e w York 1969, p 383.

W.L.F. Armarego, J.T. Batterham, K. S c h o f i e l d , R.S.
Theobald, J. Chem: SOC. ( C ) , 1969, 1433.
19 B. Koji6-Prodi6, 2. RuiiE-Toro5, R. Toso, Acta C r y s t .
B 2, 1837 /1982/*
20. A , Sega, R. TOSO, V. g u n j i 6 , L. K l a s i n c , A. S a b l j i 6 ,
D. S r z i 6 , Gazz. Chim. I t a l . 111,217 /1981/.
21 .- L.E. Martin, J, Oxford, R. J.N. Tanner, Xenobiotica
11, 831 /1981/.
22. N,N. Ferguson i n "Advances i n A n a l y t i c a l Chemistry
and I n s t r u m e n t a t i o n 1 t Ed. C.N. R e i l l e y , Vol. 4, I n t e r -
s c i e n c e Publ., N e w York 1965, p. 411.
23. J.A. B e l l , F.A.A. Dallas, W.N. J e n n e r , L.E. Martin,
Biochem. Sac. Trans. 8 , 93 /1980/.
24. P.F. Carey, L.E. Martin, P.E. Owen, J. Chromatog. 225,
161 /1981/.
RANITIDINE 56 I

25. P.A, L e b e r t , S,M. 'Mac Leod, W.A. Mahon, S. J. S o l d i n ,

H.M. Vandenberghe, C l i n . Pharmacol. Ther. 2, 539
/1981/.
26. D.C. B r a t e r , C l i n . Pharmacol. Ther. 32, 484 /1982/.
27. A.M. Van Hecken, T.B. Tjandramaga, A. M u l l i e , R. Ver-
b e s s e l t , P.J. De Schepper, B r . J. C l i n . Pharmacol,
14, 195 /1982/.
28. T J . McNeil, G.W. Mihaly, A. Anderson, A.W. M a r s h a l l ,
R.A. Smallwood, W . J . Louis, B r . J. C l i n . Pharmac.
12, 411 /19a1/.
29. T N . Brogden, A.A. Carmine, R.C. Heel, T.M. S p e i g h t ,
G.S. Avery, Drugs &, 267 /1982/.
3 0 . D.A. Henry, G.KStchingman, M.S.S. Langman, B r i t . Med.
J. 281, 775 /1980/*
31. S. Rendi;, F. K a j f e i , H.H. Ruf, Drug Metabolism and
D i s p o s i t i o n 2,137 /1983/.
32. H. M. Vandenberghe, S. M. Mac Leod, W. A. Mahon, P.A.
L e b e r t , S.J. S o l d i n , T h e r a p e u t i c Drug Monitoring 2,
379
33. G.W.
/ww.
Mihaly, O.H. Drummer, A. Marshall, R.A, Smallwood,
W.J. Louis, J. Pharm. S c i . 69,1155 /1980/.
This Page Intentionally Left Blank
 STRYCHNINE

Farid J. Muhtadi and Mohamed S. Hifnawy

Introduction

1. Description

1.1 Nomenclature
1.2 Formulae
1.3 Molecular Weight
1.4 Elemental Composition
1.5 Appearance, Color, Odor and Taste

2. Physical Properties

2.1 Melting Range

2.2 Solubility
2.3 Optical Rotation
2.4 Crystal Structure
2.5 Spectral Properties

3. Isolation

4. Total Synthesis and Degradation

5. Biosynthesis

6. Metabolism and Toxicity

ANALYTICAL PROFILES OF DRUG SUBSTANCES Copyright 0 1986
VOLUME 15 by the American Pharmaceutical Association
563 All rights of reproduction in any form reserved.
564 FARID J. MUHTADI AND MOHAMED S. HIFNAWY

7. Methods of Analysis

7.1 Identification Tests

7.2 Microcrystal Formation
7.3 Titrimetric Methods
7.4 Compleximetric Determinations
7.5 Spectrophotometric Methods
7.6 Chromatographic Methods
7.7 Radiometric Determinations
7.8 Polarographic Determination.

Acknowledgements
References.
STRYCHNINE 565

Introduction

Strychnine is an indole alkaloid occurs in numerous

strychnos plants ofthe family Loganiaceae. The most impor-
tant of which are the seeds of strychnos nux-uomica L. and
the beans of strychnos ignatii Berg.
These species contain up to 5.3% of the total alkaloids of
which approximately one-half is strychnine.
Strychnine was first discovered in 1817 by the French Pharma-
cists, Pierre Pelletier and Joseph Caventou, and these workers
were also responsible for the first isolation of brucine in
1819.
Structural investigations of strychnine were begun by Hanssen
and Tafel and continued by Leuchs, Perkin, Robinson, Wieland
and Woodward. Finally the structure of strychnine was estab-
lished by Robinson and co-workers in 1946 and confirmed by
X-ray crystallographic analysis and total synthesis by Wood-
ward. The alkaloid has no important therapeutic use as it
is highly toxic. However, the crude drug nux-vomica is used
as a bitter tonic and stimulant (as it is official in certain
pharmacopoeias including the B. P. of 1980).
Strychnine is used as a rodenticide for destroying agricul-
tural rodents and predatory animals. Occasionally, domestic
animals and man are poisoned by this agent.
Strychnine is CNS stimulant, it stimulates the spinal cord.
It is also a powerful convulsant which produces character-
istic convulsions. Sometimes, strychnine is used for the
adulteration of street drugs.

1. Description
1.1 Nomenclature
1.1.1 Chemical Name
Strychnidin-lO-one
1.1.2 Generic Name
Strychnine
1.2 Formulae
1.2.1 Empirical
(strychnine)
C21H22N202
(strychnine sulfate)
C42H46N408S
566 FARID J. MUHTADI A N D MOHAMED S. HIFNAWY

1.2.2 Structural 5

10

11

'19

Several structures have been proposed for strychnine,

these include, Perkin and Robinson structure of 1910(1),
Perkin and Robinson structure of 1929(2), Robinson structure
of 1932 (3,4), Leuchs structure of 1932 (5). The currently
accepted structure of strychnine was finally established in
1946 by Robinson et al. (6) and was confirmed by the total
synthesis of strychnine which was carried out by Woodward
et a1 (7-9). The absolute structure was deduced by exten-
sive X-ray crystallographic studies which were achieved by
several authors (10-13 ) .
1.2.3 CAS Registry No.
[ 57-24-91 strychnine
[60-41-31 strychnine sulfate
1.2.4 Wiswesser Line Notation

T6 G656 B 7 C6 E5 D
5ABCEF AGFX MQ
- 0 VN NH-UHJ 114)
1.2.5 Stereochemistry
The absolute stereochemistry has been deduced
STRYCHNINE 567

from combinations o f massive chemical d e g r a d a t i o n s

and c o r r e l a t i o n s (15-18), X-ray c r y s t a l l o g r a p h i c
s t u d i e s (10-131 9 lH-and carbon-13 n u c l e a r magnetic
resonance s p e c t r a l d a t a (19,20). From t h e s e s t u d i e s ,
i t h a s been e s t a b l i s h e d t h a t D and G r i n g s have t h e
b o a t conformation, w h i l e E and F r i n g s have t h e c h a i r
conformation. H atom a t C 2 and carbon 7 are above t h e
p l a n e o f t h e molecule (8-atoms), H atoms a t C3,
Cis, c16 and C 1 7 l i e below t h e p l a n e of t h e molecule
(a-atoms). Hydrogens a t C3 and t h a t a t C15 are equa-
t o r i a l , w h i l e H a t C2 and t h a t a t C16 are a x i a l w i t h -
i n t h e c h a i r conformation o f r i n g E.
The conformation of s t r y c h n i n e molecule i s p r e s e n t e d
i n I . Recent s t u d i e s (20) h a s f a v o r e d a c h a i r c o n f o r -
mation f o r t h e seven-membered t e t r a h y d r o o x e p i n r i n g
F (11).

H
7\
Strychnine
9
II
I
II a

"W
1.3 Molecular Weight
334.40 (strychnine)
766.92 (strychnine s u l f a t e )
1.4 Elemental Composition
C , 75.42%; H, 6.63%; N , 8.38 %;
0 , 9.57%. (strychnine)
C , 65.78%; H , 6.05%; N , 7.31%;
0, 16.69%; S, 4.18%. ( s t r y c h n i n e s u l f a t e )
1.5 Appearance, C o l o r , Odor and Taste
Orthorhombic, s p h e n o i d a l p r i s m s (from a l c o h o l ) ( 2 1 ) ,
o r translucent c o l o r l e s s c r y s t a l s o r white c r y s t a l l i n e
powder ( 2 2 ) , o d o r l e s s and h a s a v e r y b i t t e r t a s t e .
(strychnine)
568 FARID J. MUHTADI AND MOHAMED S. HIFNAWY

Colorless c r y s t a l s o r white c r y s t a l l i n e powder,

odorless and has a ' b i t t e r t a s t e , e f f l o r e s c e s i n a
dry a i r (strychnine s u l f a t e ) .

1.6 Dissociation Constant

pK1 a t 20' : 6.0; pK2 : 11.7 (21)

1.7 PH Range
pH of a s a t u r a t e d s o l u t i o n of strychnine i s 9.5,
pH of a strychnine s u l f a t e s o l u t i o n (1:lOO)
is 5.5 (21)
2. Physical Properties

2.1 Me 1t ing Range

268-290' Depending on the speed of heating (21)

270-271O Slow heat ( 2 3 )
strychnine s u l f a t e (anhydrous) : about 200' (21).

2.2 Solubi 1i t y

One gram d i s s o l v e s i n 6400 m l water, 3100 m l b o i l i n g

water, 150 m l alcohol, 35 m l b o i l i n g alcohol, 5 m l
chloroform, 180 m l benzene. (strychnine)
One gram d i s s o l v e s i n 35 m l water, 7 m l b o i l i n g
water, 81 m l alcohol, 26 m l alcohol a t 60', 220 m l
chloroform and i n 6 m l glycerol (strychnine s u l f a t e ) .

2.3 ODtical Rotation

[ a ] D18 - 139.3' (chloroform) (21)

[a] D20 - 104' (c = 0.5 absolute alcohol)
[a] D - 109.9' (80% ethanol) (23).
( a l l above d a t a a r e f o r strychnine)
The s p e c i f i c r o t a t i o n s f o r both strychnine i n chlo-
roform and strychnine s u l f a t e i n water were d e t e r -
mined as 20 mg/ml s o l u t i o n s using a Perkin Elmer
Polarmatic model 241 MC and found:-
[a]D2' - 142.6' (strychnine)
- 25.1' (strychnine s u l f a t e )
STRYCHNINE 569

2.4 Crystal Structure

The c r y s t a l s t r u c t u r e of s t r y c h n i n e was determined

by X-ray d i f f r a c t i o n , which was achieved by s e v e r a l
a u t h o r s (10-13).
The c r y s t a l s of s t r y c h n i n e hydrobromide, C21H22N202.
H B r . 2H20 are orthorhombic w i t h space group P212121
and w i t h c e l l dimensions a=7.64, b=7.70 and c=33.20Ao
( 1 0 , l l ) . Each bromine atom is bonded t o a s t r y c h n i n e
molecule through t h e b a s i c n i t r o g e n atom (N2).
While t h e c r y s t a l s o f s t r y c h n i n e s u l f a t e p e n t a h y d r a t e
are monoclinic with space group C2 and c e l l edges
a=35.85+0.05, b=7.56+0.01, c=7.84+0.01A0 (12). I t
h a s been shown t h a t t h e r e are f o u r molecules of s t r y -
chnine i n t h e u n i t c e l l (11,lZ).
The s t u d y r e v e a l e d t h a t t h e s t r y c h n i n e molecule has
a c o n f i g u r a t i o n i d e n t i c a l w i t h t h a t deduced by o t h e r
methods.
Peerdeman (13) by r e c a l c u l a t i n g v a l u e s of t h e i n t e n -
s i t i e s of r e f l e x i o n s o f s t r y c h n i n e hydrobromide dihy-
d r i d e , h a s deduced t h e a b s o l u t e c o n f i g u r a t i o n o f
n a t u r a l s t r y c h n i n e as p r e s e n t e d i n Fig. 1.
The c r y s t a l s t r u c t u r e o f s t r y c h n i n e s u l f a t e p e n t a -
h y d r a t e is p r e s e n t e d i n Fig. 2 (12). I n t r a m o l e c u l a r
bond l e n g t h s of s t r y c h n i n e hydrobromide (11) a r e
t a b u l a t e d i n t a b l e 1 and shown on s t r y c h n i n e molecule.
570 FARID J. MUHTADI AND MOHAMED S. HIFNAWY

F i g . 1: The Absolute C o n f i g u r a t i o n o f
Strychnine.

F i g . 2: The C r y s t a l S t r u c t u r e of
Strychnine Sulfate.
STRYCHNINE
571
0
Table 1. I n t r a m o l e c u l a r Bond Lengths ( A ) of
S t r y c h n i n e Hydrogen Bromide
C - N 1-37 C2 - C3 1.34 1.56
1 1 ‘12- ‘13
c8 - 1.46 1.29 1.51
N1 C3 - C4 ‘13- ‘14
c21- N1
1-50 C4 - 1.30 1.58
c5 ‘13- ‘16
c9 - N2 1.59 c - c6 1.30 1.36
5 ‘14- ‘19
5 0 - N2 1.44 c6 - c7 1.65
1.59 ‘15- ‘16
‘15- N2 1-55 c7 - c8 1.49 1.16
‘16- ‘17
c21- 0 1.15 1.41 c17- c 18 1.54
1 - c9
‘18- ‘2 1.45 C7 - Cll 1.53 ‘19- ‘20 1.56
‘19- ‘2 - ‘8 - ‘14 1.55 c20- c21 1.49
c1 - c2 1.31 C9 - C12 1.64 N
2 - Br 3-17
‘1 - ‘6 1.40 Cl0- Cll 1.54
572 FARID J. MUHTADI A N D MOHAMED S. HIFNAWY

2.5 Spectral Properties

2.5.1 Ultraviolet Spectrum (UV)

The UV spectrum of strychnine in methanol
(Fig. 3) was scanned from 190 to 400 nm using
DMS Varian Spectrometer. It exhibited the
following UV data (Table 2).
Table 2. UV Characteristics of Strychnine
Amax. nm log E A (l%, 1 cm)
205 - -
254 4.10 377
280 3.64 131.4
290 3.54 104
Other reported W spectral data for strych-
nine in ethanol Xmax. 255 nm (E 1%, 1 cm =
377) (24 ) ; in sulfuric acid hmax. at 255 nm
(E 1%, 1 cm = 315) (24 > ; Amax. for strych-
nine in ethanol, 254, 278, 288 mu (log~4.10,
3.63, 3.53 (21 ) .
2.5.2 Infrared Spectrum (IR)
The IR spectrum of strychnine as KBr disc was
recorded on a Perkin Elmer 580 B Infrared
Spectrometer to which an infrared data stat-
ion is attached (Fig. 4).
The structural assignments have been co-
related with various frequencies (Table 3).
Table 3. IR Characteristics of Strychnine
Frequency cm-1 As signment
2950-2800 CH stretch
1672 C=O substituted
amide
1600, 1485 C=C aromatic
1150, 1110, 1055 ether 1inkage'O'
770 4-adjacent aromatic
hydrogens
 V

F i g . 3 : The W s p e c t r u m of s t r y c h n i n e i n methanol
0-
40 wavrnum&r(c~-1)3ooo 2500 2000 1CW Hoo toe0 &O coo 4

FIG. 4 . ItiE 1R SPECTRUM OF STRYCHNINE KBR. DISC

STRYCHNINE 575

The I R e x h i b i t e d t h e f o l l o w i n g o t h e r c h a r a c -
t e r i s t i c bands : - 1465, 1450, 1395, 1365,
1350, 1295, 1280, 1240, 1190, 1170, 1000,
990, 950, 925, 860, 780, 540, 470 cm-1.
Other I R d a t a f o r s t r y c h n i n e have been
r e p o r t e d ( 9 ,14,24) .
2.5.3 Nuclear Magnetic Resonance S p e c t r a

2.5.3.1 Proton S p e c t r a (PMR)

The PMR s p e c t r a o f s t r y c h n i n e i n
CDC13 and i n TFA ( t r i f l u o r o a c e t i c
a c i d ) were r e c o r d e d on a Varian T-
60A, 60 MHz NMR S p e c t r o m e t e r u s i n g
TMS ( T e t r a m e t h y l s i l a n e ) as an i n t e r -
n a l r e f e r e n c e . The s p e c t r a are shown
i n Fig. 5 and 6 r e s p e c t i v e l y . The
f o l l o w i n g s t r u c t u r a l assignments
have been made (Table 4 ) .

22

The PMR of s t r y c h n i n e i n CDC13 u s i n g

250 MHz s p e c t r o m e t e r h a s been p u b l i -
shed e a r l i e r ( 1 9 ) . The spectrum
o b t a i n e d by t h i s i n s t r u m e n t (Fig. 7 )
afforded b e t t e r resolution f o r s t r u c -
t u r a l assignment p a r t i c u l a r l y i n t h e
r e g i o n 1.2 - 4 . 3 ppm.
The r e p o r t e d PMR d a t a of s t r y c h n i n e
(19) are a l s o p r e s e n t e d i n t a b l e 4
a l o n g w i t h o u r found PMR c h a r a c t e r -
istics.
 500 400 300 200

I I I I I # I I
, * l I , , , , I , , , , l l , , , l ~ ~ ~ ~ l ~ l ~ ~ ~ ' " ' ~ ~
8.0 70 6.0 5 0 PPM(6) 4.0 3.0 2.0 1.0 0

FIG. 5. THE PMH SPECTRUM OF STRYCHNINE IN C D C L ~ ,

 T
577
Fig.7: The PMR of strychnine in CDC13 using
250 MHz spectrometer.
 ..
M
579
580 FARID J. MUHTADI AND MOHAMED S. HIFNAWY

Table 4. PMR Characteristics of Strychnine

Chemic 1 Shift (ppm)

CDC13 CDC13
Proton at Reported Found TFA
7.14,7.07,7.23 7.20(m) 7.40

C (aromatic) 8.08 8.07 (2q) 8.13

4
3.84 3.88(s)
‘8
3.10 3.08(s) 4.23
cll 2.65 2.60(s)
4.26 4.27(q) 4.76
c12
1.25 1.25(t) 1.74
‘13
3.12 -
‘14
1.43 1 .52(d) 2.03
‘15 2.33 2.76
2.35(t)

‘16 3.92 3.88(s)

1.86, 1.87 1.85 (m) 2.38

‘17
2.86 2 83(q) 3.66
‘18
3.18 3.161s)

c20 2.71, 3.69 3.6 ( s )

CZ2 (vinylic) 5.88 5.87(bt) 6.53

4.04 4.04( s ) 4.40
‘23 4.12 4.13(s)

s = singlet, d = doublet, t = triplet, m = multiplet,

q = quartet, 2q = 2 quartets, bt = broad triplet.

Some stereochemical correlations in strychnine molecule

were deduced from the coupling constants of its protons
(19,ZO).
STRYCHNINE 58 1

2.5.3.2 "C-NMR

The 13C-NMR n o i s e - d e c o u p l e d and o f f -

r e s o n a n c e s p e c t r a are p r e s e n t e d i n
Fig. 8 and Fig.9 r e s p e c t i v e l y . Both
were r e c o r d e d o v e r 5000 Hz i n CDC13
on a J o e l FX-100 NMR S p e c t r o m e t e r ,
u s i n g a 10 mm sample t u b e and t e t r a -
m e t h y l s i l a n e (TMS) as an i n t e r n a l
reference s t a n d a r d a t 20'. The car-
bon chemical s h i f t s are a s s i g n e d on
t h e b a s i s of a d d i t i v i t y p r i n c i p a l s
and t h e o f f - r e s o n a n c e s p l i t t i n g p a t -
tern (Table 5 ) .
Assignments of a l l 21 c a r b o n s of
strychnine are consistent with those

o f Wenkert e t a l . (20) and Verpoovte

e t a1 (26).

T a b l e 5. Carbon Chemical S h i f t s of S t r y c h n i n e
~~ -

Carbon Chemical S h i f t Carbon Chemical S h i f t

No [PPml No [PPml

122.17 (d) '13 48.14 (d)

124.05 (d) 31.52 (d)

c2 '14
128.39 (d) 26.77 ( t )
c3 15
c4 116.06 (d) '16 60.00 (d)
 I
I
I

d
582
 ...
c.(
a
LL
0
f
c
I-
U
w
&
wl
wl
v)
4
5
583
584 FARID J, MUHTADI A N D MOHAMED S. HIFNAWY

Carbon Chemical Shift: Carbon Chemical S h i f t

No [PPml No r
PPml
142.08 (s) 42.80 ( t )
c5 ‘17
132.68 (s) 50.25 ( t )
‘6 ‘18
c7 51.84 (s) ‘20 52.40 ( t )

60.11 (d) 140.49 (s)

‘8
‘10 169.14 (s) c22 127.04 (d)

cll 42.38 ( t ) ‘23 64.52 ( t )

77.49 (d)
‘12

M u l t i p l i c i t y symbols, s = s i n g l e t , d = d o u b l e t ,
t = triplet.
Other 1 3 C - M s p e c t r a l d a t a o f s t r y c h n i n e have been
a l s o r e p o r t e d (20,25-27).

2.5.4 Mass Spectrum

The mass spectrum of s t r y c h n i n e i s p r e s e n t e d i n
Fig. 10. T h i s was o b t a i n e d by e l e c t r o n impact
i o n i z a t i o n ( E I ) on a Finnigan - Mat 1020 by
d i r e c t i n l e t probe a t 2 7 0 ’ ~ . The e l e c t r o n energy
was 70 eV. The spectrum scanned t o mass 550 amu.
The spectrum (Fig. 10) shows a molecular i o n peak
M+ a t m / e 334 w i t h r e l a t i v e i n t e n s i t y of 100%
which corresponds t o t h e base peak.
The most prominent fragments and t h e i r r e l a t i v e
i n t e n s i t i e s are p r e s e n t e d i n t a b l e 6 .
STRYCHNINE 585

Table 6. Mass Fragments of Strychnine

m/ e Relative m/e Relative

Inten. % Inten. %

335 24.65 (M”) 130 30.50

334 100.0 (M’) 129 12.79
333 16.20 (M-l) 120 20.40
167 13.11 115 14.74
162 12.41 108 11.13
144 13.90 107 19.42
143 18.17 94 11.00
134 11.76
m/e Re 1a t ive
Inten. %

91 17.07
81 11.18
80 11.10
79 23.50
77 19.13
71 14.55
69 19.99

Other mass spectrum d a t a of s t r y c h n i n e have

been a l s o r e p o r t e d (28).
586 FARID J. MUHTADI A N D MOHAMED S. HIFNAWY

3. Isolation of Strychnine
Strychnine occurs along with brucine in several spec-
ies of Strychnos particularly S. nux-vomica and S. ignatii
( F d Z y Loganiaceael which contain varying quantities of
both alkaloids (0.5 to 5.3%).
Finely powdered nux-vomica is thoroughly moistened
with lime water and extracted with hot chloroform till
exhausion.
The alkaloids are removed from the solvent by shaking
with successive portions of diluted sulfuric acid. The
combined acid solution is concentrated. Strychnine can be
isolated from brucine by one of the following steps:-
1- The less soluble brucine bisufate crystallizes from
the acid concentrated solution first and removed by
filteration. Upon neutralization of the mother liquor,
strychnine sulfate crystallizes out, and purified.
The alkaloid is made from it by precipitation with
ammonia solution ( 2 9 ) .
2- The concentrated acid solution is rendered alkaline
with excess ammonia solution, where strychnine and
brucine precipitated together. The precipitate is
extracted with 25% ethanol which dissolves the brucine,
and leaves the strychnine as insoluble residue. The
undissolved strychnine is filtered off (30).
- Strychnine is purified by repeated crystallization
from ethanol.
4. Total Synthesis of Strychnine

The total synthesis of strychnine was achieved in 1954

by Woodward and his associates ( 7, 8 ). Before this
several attempts have been made, but these were unsuccess-
ful. Woodward’s synthesis of strychnine stands out as a
major synthetic achievement (17,31).
Woodward’s total synthesis of strychnine ( 7-9):-
Acetoveratrone [l] and phenylhydrazine are added to poly-
phosphoric acid and the mixture is warmed to give 2-
veratrylindole [ 21.
2-veratrylindole [2] is condensed with formaldehyde and
aqueous dimethylamine in dioxan and acetic acid to give
2-veratrylgramine [3]. The methiodide of [3] is conver-
ted by sodium cyanide in dimethylformamide to the nitrile,
which in turn is reduced by lithium aluminium hydride in
hot tetrahydrofuran to 2-veratryltryptamine [4]. This is
condensed with ethyl glyoxylate in warm benzene to give
STRYCHNINE 587

t h e corresponding S c h i f f ' s base [5]. The l a t t e r i s trea-

t e d w i t h p - t o l u e n e s u l f o n y l c h l o r i d e i n p y r i d i n e t o produce
t h e i n d o l e n i n e [6]. The i n d o l e n i n e [6] i s reduced by
sodium borohydride i n e t h a n o l t o t h e corresponding i n d o l i n e
which i s converted i n t o t h e N - a c e t y l - i n d o l i n e d e r i v a t i v e
[7] by t h e a c t i o n o f a c e t i c anhydride and p y r i d i n e . [7]
i s t r e a t e d with ozone i n aqueous a c e t i c a c i d ( o z o n o l y s i s )
t o y i e l d t h e t r i e s t e r [8]. The t r i e s t e r [8] i s r e a c t e d
with b o i l i n g methanolic hydrogen c h l o r i d e , cleavage o c c u r s
t o g i v e t h e pyridone ester [ 9 ] . T h i s i s t r e a t e d with h o t
h y d r i o d i c a c i d i n t h e presence of r e d phosphorus, followed
by e s t e r i f i c a t i o n , N - a c e t y l a t i o n and t r e a t m e n t w i t h sodium
methoxide i n h o t methanol t o y i e l d t h e c y c l i z e d product
[ l o ] . The l a t t e r i s t r e a t e d w i t h p-toluene s u l f o n y l c h l o r -
i d e i n p y r i d i n e t o y i e l d 0 - t o s y l d e r i v a t i v e which i s reac-
t e d w i t h sodium benzylmercaptide t o g i v e t h e 8-benzylmer-
c a p t o e s t e r . T h i s upon t r e a t m e n t with d e a c t i v a t e d Raney
n i c k e l i n h o t e t h a n o l followed by r e d u c t i o n with hydrogen
i n t h e p r e s e n c e of palladium on c h a r c o a l g i v e s t h e c i s
s a t u r a t e d ester, a l k a l i n e h y d r o l y s i s i s e f f e c t e d followed
by a c i d i f i c a t i o n t o give t h e t r a n s a c i d [ l l ] . The a c i d [ l l ]
i s r e s o l v e d with q u i n i d i n e and ref luxed w i t h acetic anhydr-
i d e and p y r i d i n e t h e n hydrolysed w i t h aqueous h y d r o c h l o r i c
and a c e t i c a c i d s t o produce t h e aminoketone [ 1 2 ] . Oxida-
t i o n of [12] with selenium d i o x i d e i n e t h a n o l y i e l d s dehy-
drostrychninone [13]. The l a t t e r i s r e a c t e d w i t h sodium
a c e t y l i d e i n t e t r a h y d r o f u r a n followed by r e d u c t i o n w i t h
hydrogen i n t h e p r e s e n c e of L i n d l a r palladium ( 3 2 ) to
g i v e t h e corresponding v i n y l compound [ 1 4 ] . T h i s i s t r a n s -
formed by l i t h i u m aluminum hydride i n e t h e r i n t o t h e b a s e
[15]. The base [15] i s s u b j e c t e d t o b o i l i n g a t 120' w i t h
hydrogen bromide i n a c e t i c a c i d i n t h e presence o f red
phosphorus followed by h y d r o l y s i s with b o i l i n g aqueous
s u l f u r i c a c i d t o y i e l d i s o s t r y c h n i n e [16]. T h i s upon
t r e a t m e n t w i t h e t h a n o l i c potassium hydroxide i s converted
i n t o s t r y c h n i n e [ 171.
588 FARID J. MUHTADI AND MOHAMED S. HIFNAWY

The Total Synthesis of Strychnine

Fischer
O a o C HOCH3
3
reaction * 0ch3

0ch3
[I1

[51

COOCH3 c =o
1 0ch3
STRYCHNINE 589

OH
590 FARID J. MUHTADI AND MOHAMED S. HIFNAWY

LiAIH4
1141

1151
i) H Br
ii) H2S04

1171 1161

Degradation
Strychnine molecule can be degraded into several pro-
ducts. The most interesting degradative products are
those obtained by alkaline degradation and by the action
of 20% nitric acid. Alkaline degradation generate seven
simple isolable products of known structures (15,16,17,
31,33,34), while 20% nitric acid treatment affords 5,7-
dinitroindole-2,3-dicarboxylic acid (35).
The degradative products are shown below.
STRYCHNINE 59 1

Products of the Alkaline Degradation of Strychnine

and the effect of 20% nitric acid.

Qjr$'.". N

\
H
N02
5,7-dinitro- Tryptamine
indole,2,3-
dicarboxylic 20% Alcoholic
acid
HN03
H/

4
- STRYCHNINE -+
B-picoline B-Collidine

Zn dust.
A

H H
3-ethylindole 3-methyl indol e
592 FARID J. MUHTADI A N D MOHAMED S. HIFNAWY

5. Biosynthesis of Strychnine
It has long been assumed that the indolic moiety of
the indole alkaloids is derived from the aminoacid "tryp-
tophan" (36-38). Thomas (39) and Wenkert (40) have indep-
endently predicted that the non-tryptophan portion of
these alkaloids is formed from two mevalonate units to
afford a cyclopentane monoterpene. Thomas (41) has fur-
ther suggested that the glucoside "loganin" could be a
key intermediate in the biosynthetic pathway to indole
alkaloids. This suggestion has been established upon
experimental evidence by several authors (42-50).
It is now known that loganin arises in the plants from
mevalonate which is transformed by a series of steps to
isopentenyl diphosphate (51) and dimethylallyl diphos-
phate (52). Combination of these leads to geraniol
(53,54), then to loganin and finally to secologanin
which condenses with tryptophan to afford intermediate
condensate to many indole alkaloids (55).
Many radioactive precursors were fed into the plants
of Stryehnos nm-vornica. Several of those were incorpora-
ted into strychnine. These include [2-14C] -tryptophan
(47), [l-15N]-tryptophan (56,57), [1-14C]-acetate, [2-14C]-
a
acetate (47 , [2-14~]-glycine(57-59), [ ~ - 1 4-meValonate
~]
( 47 ) , [2-l C] -geraniol (47), and [2-14C]-geranylpyro-
phosphate (47). While other precursors were not incorpora-
ted into strychnine and among these are [~P~H]. Weiland-
Gumlich aldehyde and [2-14C]-1-acetyl Weiland-Gumlich
aldehyde (47).
The biosynthetic pathway leading to strychnine can be
visualized from two different systems ( 55 ) . The first
system involves condensation of tryptamine and secologanin
to form a Schiff base which could be attacked intramole-
cularly by the @-position of the indole nucleus to afford
the corresponding spiroindolenins (55 ) . This system is
presented in Scheme I.
The other system involves condensation of tryptophan and
secologanin at the a-position of the indole neucleus to
afford 8-carboline system which leads to strictosidine
(55 ) . This system is presented in Scheme I1 ( 55,60).
Scheme I
Tryptomine [l] is condensed with secologanin (Mannich
condensation) [Z] to give the spiroindolenine [3]. This
undergoes ring opening to give the intermediate [4]. The
latter is transformed into the intermediate [S]. Acetate
unit is incorporated into [5] to afford [6]. Intramole-
cular cyclization gives [7], which upon further cyclization
and decarboxylation yields strychnine [8].
STRYCHNINE
593

Biosynthesis of Strychnine (Scheme I).

o*c
, CH20H

['I I H3C0
CHO
OH
594 FARID J. MUHTADI A N D MOHAMED S. HIFNAWY

Biosynthesis of Strychnine (Scheme 11)

H

OTJ- H'

OCH3

OCH3
STRYCHNINE 595

J OCH3
[91
596 FARID J . MUHTADI AND MOHAMED S. HIFNAWY

Scheme I1
Tryptophan [ l ] is condensed with secologanin [2] t o give
the condensate [3]. Cyclization of [3] y i e l d s isovinco-
s i d e ( a l s o known as s t r i c t o s i d i n e ) [4]. Further c y c l i z a -
t i o n and r i n g opening of [4] a f f o r d s pregeissoschizine [S].
This is transformed i n t o preakuammicine [6] which i s con-
verted by a r e t r o - a l d o l process t o akuammicine [7]. Oxida-
t i o n a t t h e a l l y l i c carbon of [7] a f f o r d s [8]. Incorpora-
t i o n of a c e t a t e u n i t t o [8] y i e l d s [9] which undergoes
reductive c y c l i z a t i o n t o produce strychnine [ l o ] .

6. Metabolism of Strychnine

Strychnine i s very r a p i d l y absorbed from the gastro-

i n t e s t i n a l t r a c t , through mucous membranes, i n t a c t s k i n
and from an i n j e c t i o n s i t e (22,24,61).
Strychnine t r a v e l s i n both plasma and erythrocytes and
approximately 50% of t h e agent w i l l d i f f u s e i n t o t i s s u e s
i n f i v e minutes (62).
The CNS does not contain higher concentrations than do
o t h e r t i s s u e s (61).
Strychnine i s r e a d i l y metabolised i n t h e l i v e r by micro-
soma1 enzymes (61,63), where up to 80% of t h e dose i s
oxidized and approximately 10 t o 20% of t h e dose w i l l
appear i n t h e 24 hour u r i n e unchanged (24,62),
Strychnine i s cumulative and very s t a b l e and may be found
i n cadavers exhumed many years a f t e r death (24).

Toxi c i t v

Poisoning by strychnine s t i l l occurs p a r t i c u l a r l y from

rodenticides o r from t h e a d u l t e r a t i o n of s t r e e t - d r u g s with
strychnine. Many cases of a c c i d e n t a l poisoning a r e i n c h i l -
dren who may swallow o r suck strychnine b a i t s . The f a t a l
adult o r a l dose is about 50 t o 100 mg of strychnine, but
30 mg has been found l e t h a l (61).
The main e f f e c t of strychnine is on t h e s p i n a l cord where
i t i n h i b i t s post-synaptic i n h i b i t o r y c o n t r o l , leading t o
t e t a n i c convulsions. Death occurs from c e n t r a l r e s p i r a -
tory failure.
STRYCHNINE 597

7. Methods of Analysis

7.1 Identification tests

The following c o l o r t e s t s a r e used t o i d e n t i f y

strychnine.
- I f a t r a c e (few c r y s t a l s ) of strychnine is dissolved
i n few drops of concentrated s u l f u r i c acid and s t i r -
red with a c r y s t a l of potassium dichromate, a deep
b l u i s h v i o l e t c o l o r develops, which gradually changes
t o red and f i n a l l y t o orange yellow color.
- When a t r a c e of strychnine i s t r e a t e d with Mandalin's
reagent (Ammonium vanadate i n s u l f u r i c a c i d ) , a blue
c o l o r is formed which changes first t o purple then
t o red c o l o r ( s e n s i t i v i t y 0.05 ug).
- Few drops o f fuming n i t r i c a c i d a r e added t o a t r a c e
of strychnine, a yellow c o l o r is produced. Brucine
gives with n i t r i c acid an i n t e n s e orange red color.
- Strychnine gives with V i t a l i ' s t e s t a brown c o l o r
( s e n s i t i v i t y 0.5 ug). The t e s t can be performed
as follows:
Few c r y s t a l s of strychnine a r e dissolved i n few drops
of n i t r i c a c i d and the yellow s o l u t i o n is evaporated
t o dryness on a water bath, when a l c o h o l i c potash i s
added t o the residue, a brown t o purple c o l o r i s
developed.
- When 0- Tosyl-p-phenolsulfonic acid i s added t o c e r -
t a i n a l k a l o i d s including strychnine, a p r e c i p i t a t e
i s obtained which i s c r y s t a l l i z e d from water t o give
c r y s t a l s with m.p. of 242-244' (64).
- When 3-nitro-4-chlora-5-methylbenzene s u l f o n i c a c i d
i s added t o some a l k a l o i d s including strychnine, a
p r e c i p i t a t e of the s a l t is formed, upon c r y s t a l l i z a -
t i o n from water, the c r y s t a l s melt a t 265-266' (64).
- A simple, rapid and s e n s i t i v e technique f o r t h e
d e t e c t i o n of strychnine among o t h e r a l k a l o i d s and
psychotropic amines i n physiological f l u i d s such as
s a l i v a and u r i n e was described, pot. a c i d p h t h a l a t e
b u f f e r 0.1 M, pH 4.5, CHC13, and NaOH 0.05 N were
used f o r e x t r a c t i o n and bromophenol blue 0.04% i n
MeOH f o r c o l o r production. The blue c o l o r i n a l k a l i n e
medium was very v i s i b l e . The method was recommended
f o r toxicology (65).
7.2 Microcrystal Formation
Strychnine hydrochloride was dissolved i n water (10
mg i n 10 ml), 1 t o 2 drops of t h i s s o l u t i o n was trea-
t e d with t h e reagent on a microscopical g l a s s s l i d e .
After s p e c i f i c time, the c r y s t a l s were microscopical-
l y examined. The c r y s t a l formation i s presented i n
t a b l e 7.
598 FARID J. MUHTADI AND MOHAMED S. HIFNAWY

Table 7. Microcrystal Formation of Strychnine

Plate Time Shape of t h e Crystal

1 Mayer s 2 - 3 min. minute p l a t e s i n s t a r l i k e

arrangement
2 P 1a t e n i c 2-3 min. broad p l a t e s , r o s e t t e
ch 1o r i d e arrangement
3 Mercuric 10 min. f i n e curved h a i r y with speci-
chloride f i c arrangement
4 Wagner s 15 min. f i n e s h o r t rods
5 Dragendor f f 2-3 min. rods i n c l u s t e r form
6 Marm s immediate r o s e t t s of p l a t e s , enlarged
by time
7 Sodium 10 min. long rods i n r o s e t t e shape
carbonate and f r e e arrangement
8 Sodium 10-15 min, c l u s t e r of l a r g e p l a t e s
phosphate
9 Pot a s sium immediate t h i n r a d i a t i n g rods
chromate
10 Potassi um 3-5 min. broad p l a t e s , r a d i a t i n g form
iodide

7.3 T i t r i m e t r i c Methods

The o f f i c i a l methods of determination of s t r y c h -

nine i n formulations o r i n powdered nux vomica and
i t s preparations, by t i t r i m e t r i c methods a r e described
i n t h e Egyptian Pharmacopoea (E.P.) 1963 and 1972
(66,67).
7.3.1 Aqueous T i t r a t i o n

The E.P. 1963 method f o r determination of

strychnine i n nux vomica g a l e n i c a l s is based
on t h e oxidative d e s t r u c t i o n of brucine with
HN03 and t h e a c i d base t i t r a t i o n of strychnine.
Transfer accurate volume (20 ml) of nux vomica
e x t r a c t t o a s e p a r a t o r , d i l u t e with about 20ml
H20, a c i d i f y with d i l u t e H2SO4 (about 5 ml)
and e x t r a c t with CHC13 (2x20 ml). Receive t h e
STRYCHNINE 599

Platc 1 : hlicrocrystalr of strychninc w i t h Hayer’s rcagcnt

Plate 2 : Microcrystals of strychnine with I’latinic c h l o r i d e

Plate 4 : Microcrystals of strychnine with Wagner‘s reagent

600 FARID J. MUHTADI AND MOHAMED S. HIFNAWY

Plate 5 : Microcrystals o f strychnine with Uragendorff's reagent

Plare 7 : Flicrocrystals of strychnine with Sodium carbonate

I'latc 6 : L1icrocryst;ils o f strychninc with Llnrm's rcngent

STRYCHNINE 60 1

P l a t e 8 : M i c r o c r y s t a l s of s t r y c h n i n e w i t h Sodium phosphate

P l a t c 9 : b l i c r o c r y s t a l s of s t r y c h n i n e n i t h P o t a s s i u m chromatc
602 FARID J. MUHTADI AND MOHAMED S. HIFNAWY

CHC13 i n t o another separator containing 10 m l

N H2SO4, shake, r e j e c t t h e CHC13 and add t h e
a c i d washings t o t h e first separator. Render
alkaline with ammonia and e x t r a c t the l i b e r a -
t e d a l k a l o i d s completely with successive por-
t i o n s of CHC13 (3x20 ml). Wash t h e CHC13 e x t -
r a c t with 5 m l H20, dehydrate over anhydrous
Na2S04 and d i s t i l l t h e CHC13 completely. Dis-
solve the residue i n 15 m l of 3%w/v H2SO4,
warm i f necessary, cool, then add 2 m l concd.
HNO3 and few c r y s t a l s of NaN02, allow t o s t a n d
f o r 30 min. a t ordinary temperature. Transfer
the contents t o a separator containing 30 m l of
10%NaOH, r i n s e t h e container with 2 successive
portions each of 5 m l H20, add t h e r i n s i n g t o
the separator and shake f o r about 2 min. Extr-
act strychnine with CHC13 (3x10 ml). Wash t h e
combined CHCl3 e x t r a c t with 2x10 m l H2O. Dehy-
d r a t e the CHC13 over anhydrous Na2S04. Evapo-
r a t e the CHC13, add t o t h e residue 5 m l n e u t r a l
95% alcohol, evaporate and leave t h e residue
on a b o i l i n g water bath f o r f u r t h e r 15 min.
Dissolve t h e residue i n about 2 m l CHC13, add
20 m l N/20 H2SO4, evaporate CHC13 completely,
then t i t r a t e excess a c i d with N/20 NaOH using
methyl red as i n d i c a t o r . Each 1 m l N/20 H2SO4
E 0.01672 g strychnine. The r e s u l t i s multi-
p l i e d by 1.02 t o c o r r e c t f o r the l o s s of s t r y -
chnine during oxidative d e s t r u c t i o n of brucine.
Another method based on the same p r i n c i p l e
was a l s o reported (68).
A method f o r simultaneous determination of
strychnine and brucine was presented (69). A
s u i t a b l e vol. of an aq. soln. of the 2 a l k a l -
o i d s , made a l k . with N a O H , i s e x t r a c t e d first
with E t 2 0 , then with CHC13, and t h e combined
e x t r a c t s , d r i e d with anhydrous Na2S04, is f i l t -
ered i n t o a t a r e d f l a s k . After evaporating t h e
s o l u t i o n , the combined weight of brucine and
strychnine i s determined, following which t h e
residue is dissolved i n alcohol and H20 and
t i t r a t e d with 0.01 N HC1,
Another method (70) f o r determination o f
micro amounts of strychnine using heteropoly
a c i d s was described.
T i t r a t e 20 m l of 0.001 - 0.002 M s o l u t i o n of
strychnine,containing 1 m l of 1 M HC1 (or 2 m l
of M NaOAC) with 0.00125 - 0.005 M t u n g s t o s i l -
i c i c , tungstophosphoric o r molybdophosphoric
STRYCHNINE 603

acids. The molar ratios of the stoichiometric

compounds formed in pH 1 (HC1) o r pH 7 (NaOAC)
solutions are respectively : with tungstosili-
cic acid 4, 4; with tungstophosphoric and moly-
bdophosphoric acids 3, -.
A method for determination of strychnine
nitrate by titration with silicatungstic acid
was also reported (71).
Another method (72) for rapid titrimetric
determination of strychnine with 2 1% accuracy
is based on addition of CHC13 and a buffer
solution to the base dissolved at pH 2 . 8 .
Titrate with Na dioctylsulfosuccinate (dimethyl
yellow screened with oracet blue as indicator).
The color change is from green to pink.
Satisfactory results were obtained for the
determination of strychnine nitrate by using
K-Bi complex; the liberated EDTA is titrated
with 0.01 M ZnSOq with the Nag B03 buffer (73).
A direct amperometric titration of amines
with sodium tetraphenylborate (Na B Ph4) solu-
tion, based upon anodic depolarization at a
dropping Hg electrode (74), was found suitable
for determination of strychnine.
A micro-procedure, permitting determination
of strychnine in mg rang was described ( 7 5 ) . A
small separatory funnel ($ 50 ml capacity) is
used to shake out the sample with 2 ml of 10%
NH40H, then with CHC13. The CHC13 extract is
transferred to a second small separatory funnel,
washed with H20, placed in a 50 ml (or less)
Erlenmeyer, dried, treated with 0.5 ml of 95%
alcohol and with 2.0 ml of 0.01 N H2SO4, heated
gently to aid solution and the excess acid tit-
rated against 0.01 N NaOH from a 2 ml micro-
buret, using 1 drop of methyl red solution as
indicator. Good accuracy was obtained with
strychnine - HC1 and a mixture of strychnine
salt with Na methyl arsenate.
The use of 2 , 5-dimethylbenzene sulfonic,
3, 4-dichlorobenzene sulfonic and 4-(benzene
sulfonyloxy) benzene sulfonic acids (76) and
3 methyl-6-nitrobenzene sulfonic acid, 2 , 5-
dichlorobenzene sulfonic acid (77 ) were used
for titrimetric determination of strychnine
in injection solutions, giving reproducible
results and lower mean standard deviation.
604 FARID J. MUHTADI A N D MOHAMED S. HIFNAWY

Another method depends on the p r e c i p i t a t i o n

of strychnine from an a c i d i f i e d d i l u t e s o l u t i o n
with K3 C r (SCN)6. The excess reagent hydro-
lyzed i n t h e f i l t r a t e by a l k a l i n i z a t i o n and -
SCN determined by t i t r a t i o n with Br03 The
reagent/strychnine r a t i o i n t h e ppt. was 1 : 3 ,
r e l a t i v e deviation f 0.3 - f 0 . 7 % (78).
Other method f o r t i t r i m e t r i c determination
of strychnine a t p o l a r i z a t i o n p o t e n t i a l s of up
t o 1000 mv was reported ( 7 9 )
A more recent method f o r photometric t i t r a -
t i o n of strychnine n i t r a t e was described ( 8 0 ) .
The a l k a l o i d a l s a l t i s mixed with 1 N HC1,
d i l u t e d with H20, and t i t r a t e d with tungsto-
phosphoric a c i d with photometric end p o i n t
indication.

7.3.2 Non-Aqueous T i t r a t i o n

The Egyptian Pharmacopoeia (1972) describes

the following method: Dissolve 0.5 g strych-
nine hydrochloride i n 30 m l a c e t i c anhydride.
Add 10 m l HgC12 and 20 m l dioxane. Using 2-3
drops of c r y s t a l v i o l e t reagent, t i t r a t e with
0.1 N HClO4. Carry a blank experiment and c a l -
c u l a t e the mls of HC104 consumed; l m l 0.1 N
HC104 5 0.03343 g strychnine.
Another method based on t i t r a t i o n of s t r y c h -
nine with 0.1 N of hydrochloric a c i d complex of
chloroaluminium isoproxide i n CHC13 ( 81 ) . The
deviation was f 1 % i n the range of 38-245 mg
of a l k a l o i d .
A t h i r d method f o r determination of s t r y c h -
nine n i t r a t e i n j e c t i o n s i n an anhydrous medium
was presented (82 ) . Adjust t h e pH of the i n j -
e c t i o n s o l u t i o n t o 8-9 with NaHC03. Extract 3
times with CHC13, f i l t e r the e x t r a c t and tit-
rate i n t h e presence of dimethylyellow by 0.005
N p-toluenesulfonic a c i d i n dioxane.
Alkaloids i n t i n c t u r e of nux vomica were
determined i n non aqueous media, by t h e i r l i b e -
r a t i o n with NH40H, e x t r a c t i o n with CHC13 and
t i t r a t i o n with 0.02 N HClo4 i n dioxane i n t h e
presence of methyl red i n PhCl ( 83) ; 1 m l
0.02 N H C 1 a i s equivalent t o 6.688 mg a l k a l -
oids (strychnine + brucine).
Another method f o r non aqueous determina-
t i o n of strychnine was reported ( 8 4 ) . The
STRYCHNINE 605

following a c i d s i n 0.005 N dioxane s o l u t i o n s

a r e used as t i t r a n t s : naphthalene s u l f o n i c ,
naphthalene-2-sulfonic, 5-nitro-naphthalene-6-
s u l f o n i c , and 2-propoxy~1aphthalene-6-sulfonic.
The t i t r a n t s contained 1%phOH and were standar-
dized against atropine o r brucine dissolved i n
CHC13 using 0.1% dimethyl yellow a s i n d i c a t o r .
For the determination, 5 m l of a s o l u t i o n t o be
analyzed were taken (containing about 5 mg alka-
l o i d a l s a l t ) . i t s pH adjusted t o 8-9 with s a t u r a -
t e d NaHC03 s o l u t i o n o r 5% NaOH, and then e x t r a c -
ted 4-5 times with 5-10 m l CHC13 each. The
combined e x t r a c t s a r e f i l t e r e d and t i t r a t e d ; t h e
e r r o r was <-I 1%.
T i t r a t i o n i n non aqueous media was a l s o
adopted f o r determination of strychnine and
other a l k a l o i d s i n e l i x i r t o n i c and Cola e x t r a c t
( 8 5 ) . The preparations a r e e x t r a c t e d with
CHC13 - E t O H (5:1), and the CHC13 phase i s sepa-
r a t e d by TLC on s i l i c a gel DG p l a t e s using E t O H -
CHC13 (88:12) f o r development. The strychnine
spot was i d e n t i f i e d by sequential treatment with
alcoholic KI, 25% HC1 - 95% E t O H ( l : l ) , and
Dragendorff reagent. For q u a n t i t a t i v e determi-
nation, t h e drug i s e x t r a c t e d with CHC13 - E t O H
(20:3) and the CHC13 phase was mixed with A C 2 0 ,
d i l u t e d with CgHg and t i t r a t e d with 0.1 N H C l O 4 ,
using c r y s t a l v i o l e t i n d i c a t o r , The r e l a t i v e
standard deviation i s 3%.

7.3.3 Potentiometric T i t r a t i o n s

Strychnine among o t h e r a l k a l o i d s was inves-

t i g a t e d i n t h e concentration range to
M with potentiometric t i t r a t i o n ( 8 6 ) . An adso-
r p t i o n microelement Sb/gel/K C l (0.1 N ) , Hg2 C 1 2
/Hg connected with a lindemann quadrant e l e c t r o -
meter was used. The g e l layers contacting t h e
i n d i c a t i n g e l e c t r o d e with the reference one were
made of acacia gum i n 0.05 N KC1. The t i t r a t i o n
was c a r r i e d out i n petroleum e t h e r , C C 1 4 , cyclo-
hexane, CgHg, and acetone. A s a t i t r a n t , C13 -
C C02H o r p i c r i c a c i d i n t h e same solvent as t h e
t i t r a t e d a l k a l o i d was used. The experimental
conditions a r e most favorable when the d i f f e r -
ence between t h e d i e l e c t r i c constants of t h e
solvent and t h e gel adsorption l a y e r i s large.
606 FARID J. MUHTADI AND MOHAMED S. HIFNAWY

Another method described t h e determination

of strychnine n i t r a t e potentiometrically by
t i t r a t i o n with 2.5% Na tetraphenylborate using
valinomycin ion s e l e c t i v e e l e c t r o d e ( 8 7 ) .
A new e l e c t r o d e was developed which was
s e n s i t i v e and reasonably s e l e c t i v e f o r s t r y c h -
nine ( 8 8 ) . I t is based on the use of t h e
strychnine-picrolonate ion-pair complex i n n i t r -
obenzene a s a l i q u i d membrane. The e l e c t r o d e
shows Nernstian response down t o lO-5M s t r y c h -
nine s o l u t i o n , over a pH range of 2-7 with a
c a t i o n i c slope of 57 mV/concn. decade from
5x10-2 t o 5x10-5M. Direct poteniometric d e t e r -
mination of samples down t o 5 ppm shows an aver-
age recovery of 101.3% and a mean standard devi-
a t i o n of 2.4%. Poteniometric t i t r a t i o n with
e i t h e r p i c r o l o n i c a c i d o r Na tetraphenylboron
shows an average recovery of 99.1% and a mean
standard deviation of 1.2%. No i n t e r f e r e n c e s
were caused by o t h e r a l k a l o i d s and many of t h e
substances normally occurring as n a t u r a l con-
t aminant s with strychnine .
Another method (89 3 f o r the potentiometric
determination of strychnine was presented. I t
is based on the formation of insoluble a l k a l o i d
p i c r a t e s a l t using a p i c r a t e ion-selective i n d i -
c a t i n g electrode. The average e r r o r was about
4%. The method was s u c c e s s f u l l y applied t o
pharmaceutical preparations.
Determination of strychnine by a s e l e c t i v e
l i q u i d membrane e l e c t r o d e was a l s o described
( 90 ). The electrode s t r y c h n i n e - t e t r a (m-methyl
phenyl) borate was prepared from strychnine and
the borate compound. The c a l i b r a t i o n p l o t was
l i n e a r i n t h e range 1 ~ 1 0 t- o~ 5 ~ 1 0 ' ~ M and , the
lowest d e t e c t i o n l i m i t was 1x10-6M. Optimal pH
f o r t h e determination of strychnine a t 10-5 t o
10-2M was 2-6.

7.4 Compleximetric Determinations

A method f o r t h e gravimetric determination of s t r y -

chnine s u l f a t e was described ( 9 1 ) . The method based
on conversion of the a l k a l o i d a l s a l t t o t h e correspond-
ing hexabromotellurate by r e a c t i o n with hexabromotell-
u r i c acid.
A second method based on the same p r i n c i p l e was
described f o r strychnine a n a l y s i s and c o n t r o l ( 9 2 ) .
STRYCHNINE 607

The s t r y c h n i n e s u l f a t e i s d i s s o l v e d i n HC1 and p r e c i -

p i t a t e d as h e x a c h l o r o t e l l u r a t e s w i t h H2 ( T e c l g ) . The
ppt. i s washed with HC1, d r i e d a t 50°C and weighed.
The e r r o r of t h e g r a v i m e t r i c method was 2 0.75%.
Other methods f o r g r a v i m e t r i c and t i t r i m e t r i c
d e t e r m i n a t i o n s o f s t r y c h n i n e a r e p r e s e n t e d and d i s -
cussed (93).

7.5 Spectrophotometric Methods

7.5.1 Colorimetry

A c o l o r i m e t r i c procedure was d e s c r i b e d f o r
t h e a s s a y of s t r y c h n i n e n i t r a t e i n t a b l e t s con-
taining codeine base and o t h e r i n g r e d i e n t s
(94 ) . The a s s a y based on t h e i n t e r a c t i o n o f
s t r y c h n i n e reducing p r o d u c t s and HN02 y i e l d i n g
a measurable r e d c o l o r . The t a b l e t a l k a l o i d s
a r e E t 2 0 - e x t r a c t e d and s t r y c h n i n e i s d e t e r -
mined c o l o r i m e t r i c a l l y . The accuracy was 1%.
A second c o l o r i m e t r i c method f o r determina-
t i o n o f s t r y c h n i n e and b r u c i n e i n t h e i r m i x t u r e s
was p r e s e n t e d ( 9 5 ) . Strychnine was determined
a f t e r t h e s e p a r a t i o n o f b r u c i n e . Add H2SO4
(3%, 7.5 ml) t o t h e mixture a l k a l o i d s '(- 1 mg),
c o o l , add 1.5 m l o f a mixture of HNO3, d . 1.42
- H20 (1: 1) , make t h e mixture a l k a l i n e w i t h
NaOH a f t e r 10 min, e x t r a c t s t r y c h n i n e with
CHC13, (3x10 ml), e v a p o r a t e t h e CHC13 e x t r a c t
t o d r y n e s s , add 4 m l H20, 6 m l HC1 (d. 1.12),
and 1 g Zn d u s t t o t h e r e s i d u e , h e a t t h e mix-
t u r e on a b o i l i n g water b a t h f o r 20 min. and
c o o l . Add NaN02 ( l o % , 1 drop) t o t h e mixture,
d i l u t e t o 50 m l and measure t h e o p t i c a l d e n s i t y
of t h e s o l u t i o n on a p h o t o e l e c t r i c c o l o r i m e t e r
u s i n g a green f i l t e r . The e r r o r o f t h e method
was 2 0.76%.
A t h i r d method f o r s e p a r a t i o n and colorime-
t r i c e s t i m a t i o n o f s t r y c h n i n e , among o t h e r s t i -
mulants, i n doping h o r s e u r i n e was r e p o r t e d
( 9 6 ) . S t r y c h n i n e i s s e p a r a t e d from o t h e r i n g -
r e d i e n t s by TLC on s i l i c a g e l G p l a t e s u s i n g
MeOH as a developer. The corresponding s p o t
i s e l u t e d and determined c o l o r i m e t r i c a l l y . 18%
o f t h e a d m i n i s t e r e d s t r y c h n i n e , was d e t e c t e d
i n t h e u r i n e o f t h e doped h o r s e s w i t h i n 48 h
after a d m i n i s t r a t j o n .
608 FARID J. MUHTADI A N D MOHAMED S. HIFNAWY

A f o u r t h method was described f o r c o l o r i -

metric estimation of strychnine i n b i o l o g i c a l
preparations ( 9 7 ) . Strychnine i s separated
by ascending paper chromatography by using
BuOH-ACOH-H20 (4:1:5) a s a solvent and BiI3
as a developer. The a l k a l o i d i n t h e s p o t s i s
separated by a c i d i f y i n g with 0.1 N HC1 and d e t -
ermined c o l o r i m e t r i c a l l y with bromothymol blue.
After i n v e s t i g a t i n g 13 l e t h a l cases of poison-
ing, i t was found t h a t i n organs preserved with
HCOzH, the amounts of strychnine a r e f a r lower
than i n the same organs which were not preserved.
A c o l o r i m e t r i c method f o r determination of
t h e sum of strychnine and brucine i n seeds and
galenic preparations of nux vomica was discuss-
ed ( 9 8 ) . A 0.01 g sample of f i n e l y ground
seeds a r e shaken, 30 min, with 5 g CHC13, 10 g
Et20, and 2 m l 10% Na2C03. The organic solvent
evaporated t o dryness, the residue dissolved i n
phosphate b u f f e r , pH 5.5, t o make 100 m l . A
1 m l a l i q u o t shaken 5 min with 20 m l C6&, 2 m l
phosphate b u f f e r , pH 5.5, and 3 m l s a t u r a t e d
s o l u t i o n of bromothymol blue. The developed
c o l o r i n t h e organic l a y e r i s measured c o l o r i -
m e t r i c a l l y . The same procedure was used f o r
determination of t h e a l k a l o i d s i n dry e x t r a c t s
and t i n c t u r e s prepared from nux vomica.
Two o t h e r methods f o r colorimetric determi-
nation of strychnine were developed; one (99)
was a modification f o r t h e o t h e r (100). The
modification r e s u l t e d i n increasing the s e n s i t i -
v i t y of t h e assay. Evaporate 10 m l nux vomica
t i n c t u r e o r 1 m l of i t s e x t r a c t t o dryness.
Extract residue with 1%aqueous H2SO4. Transfer
the aqueous e x t r a c t i n t o separatory funnel t h r -
ough f i l t e r paper and wash w i t h 2 m l d i l H2SO4.
Add aqueous washings t o mother l i q u o r and ren-
der a l k a l i n e t o litmus with 10% NH40H s o l u t i o n
(Ca 5 ml). Shake with successive portions of
CHC13 u n t i l a l k a l o i d s a r e completely extracted.
(3x20 ml) . Evaporate combined CHC13 e x t r a c t
and d i l u t e t o 25 m l with the same solvent. Take
0.5 m l a l i q u o t s f o r t h e proposed c o l o r i m e t r i c
method a s follows: Evaporate t h e CHC13 solu-
t i o n t o dryness on a water bath, add 5 m l HC1
and 1 g granulated zinc, heat 10 min i n a b o i l -
ing water bath o r l e t stand 20 min a t room t e m -
p e r a t u r e , and then d i l u t e with 5 m l d i s t i l l e d
STRYCHNINE 609

water. Cool to room temperature, transfer

quantitatively to 25 ml volumetric flask, rinse
container several times with small portions of
distilled water, and dilute to volume with dis-
tilled water. To different aliquots of the
reduced solution (containing -1OOpg strych-
nine) add 3 ml nitroprusside-acetyldehyde reag-
ent (10% acetaldehyde in 1% sodium nitroprus-
side) and adjust the volume to 12 ml with disti-
lled water. Let mixture stand 15 min and meas-
ure color at 530 nm against blank of 9 ml water
and 3 ml nitroprunide-acetaldehyde reagent. The
percentage was calculated from a standard curve
of strychnine. The developed color obeyed
Beer's law in the range of 10-320,ug/12 ml. The
standard deviation was 1.02.
A fast identification and determination
method for strychnine in heroin specimen was
reported (101). It depends on measuring the
pink color developed after boiling with HgClZ
and Zn and addition of NaNo2.
Two spectrophotometric procedures depending
on complexation of strychnine with hexabromote-
lluric acid (91 ) or hexachlorotelluric acid
( 9 2 ) were presented. The ppt. is dissolved in
MeOH and strychnine determined photometrically
at 450 nm for the first complex and at 420 nm
for the second complex. The error of the sec-
ond process was < 1.6%.
A method forthe extractive spectrophoto-
metric determination of strychnine with Soloch-
rome Green V150 was presented (102). The method
depends on extracting strychnine 1:2 ion pair
with Solochrome Green V150 into CHC13. The
absorbance of the organic phase is measured at
520 nm (molar absorptivity = 3.7200~104). Beer's
law was obeyed for 1-300,ug strychnine; the
error is f 1.5%.
The differential acid dye method for deter-
mination of different pharmaceutical compounds
was successfully applied for the determination
of strychnine (103). The method involves extr-
action of a mixture of 5 ml pH 4.2 K H phthalate
buffer, 5 ml Methyl orange, and 3 ml of the
alkaloidal solution of a suitable concentration,
with CHC13, followed by absorbance measurement
of the pooled extract against a reference ext-
ract. It was reported that the results obtained
610 FARID J. MUHTADI AND MOHAMED S. HIFNAWY

by traditional and differential acid dye method

were comparable and the reproducibility of the
latter method was higher.
A method for spectrophotometric determina-
tion of strychnine nitrate and other alkaloids
was presented (104). Strychnine nitrate is
dissolved in acetate buffer pH 4.1-4.2, Chroma-
zurol S is added, after 30 min the reaction
mixture is extracted with CHC13 and optical
density is measured at 510 nm. Relative error
of the determination was ? 1-3%. Chromazurol S
reacted also with 34 other alkaloids.
A spectrophotometric titration method was
described for the determination of some alkal-
oids and their dosage forms using 0.005 M chlo-
ranilic acid solution in 1, 4 dioxane as the
titrant (105). The end point is determined by
measuring the change in absorbance of the sam-
ple at 535 nm. Quantitative recoveries with
good reproducibility were reported for stry-
chnine.
Comparati.ve study on various sulfonic azod-
yes for alkaloids analysis (106) showed that
Litol red was the most suitable for colorime-
tric determination of strychnine by ion-pair
extraction in CHC13.
Seven azo dyes including orange I1 were
evaluated for the spectrophotometric determi-
nation of alkaloids and compared with the two
most common dyes for such determinations, meth-
yl orange (11) and bromothymol blue (107).
Orange I1 was more sensitive for strychnine
determination in blood and urine and tolerated
greater amounts of extraneous ions (such as
Mg2+, Co2+, h 2 +F-,, C~04~-).
Another method for colorimetric determina-
tion of strychnine with chloranilic acid was
described (108): in dioxane - CHC13 medium, the
acceptor chloranilic acid forms purple 1:l mol.
complexes with the alkaloid which gave maximum
absorption at 535 nm.
Strychnine was determined in 4 pill and
powder preparations by TLC-Colorimetry (109).
As an example, a pill preparation is powdered,
extracted with CHC13-EtOH (10 : 1) and NH40H and
the extract is chromatographed on a silica gel
G plate (toluene-acetone-EtOH-NH40H, 8:6:0.5:2
as eluent). Spots are visualized with iodine
STRYCHNINE 611

vapor and e x t r a c t e d t o g i v e a f r a c t i o n , which

i s t r e a t e d w i t h bromothymol b l u e and benzene.
The o r g a n i c phase t r e a t e d w i t h KOH-EtOH i s
analyzed a t 610 nm f o r t h e d e t e r m i n a t i o n o f
s t r y c h n i n e . The c a l i b r a t i o n p l o t was l i n e a r
up t o 4 Q y g and t h e recovery was 89.84 - 93.96%;
r e l a t i v e s t a n d a r d d e v i a t i o n s were 2.59-4.80%.
A method €or s e m i q u a n t i t a t i v e d e t e r m i n a t i o n
of s t r y c h n i n e i n 10-50 y q u a n t i t i e s by t h e Weis-
z r i n g oven c o l o r i m e t r i c procedure was proposed
(110).
Another c o l o r i m e t r i c d e t e r m i n a t i o n o f s t r y -
chnine was r e p o r t e d . I t i s based on t h e d e v e l -
opment o f i n t e n s e l y c o l o r e d r a d i c a l i o n s due
t o the reaction of the alkaloid with s e l e c t e d
polyhaloquinone and polycyanoquinane n- a c c e p t o r
(111).

7.5.2 U l t r a v i o l e t Spect ropho t omet ry . (UV)

The B.P. (1973, 1980) (112,113)describe a

method o f a s s a y i n g s t r y c h n i n e i n nux vomica
s e e d s and g a l e n i c f o r m u l a t i o n s a s f o l l o w s :
Mix 0.4 g, i n f i n e powder, with 2 m l of a l c o h o l
(70%). Add 5 m l of d i l u t e ammonia s o l u t i o n and
t r a n s f e r with t h e a i d of 25 m l of H20 and 20 m l
o f CHC13 t o a 60-ml ground g l a s s , downward d i s -
placement, l i q u i d - l i q u i d e x t r a c t o r c o n t a i n i n g
60 m l of CHC13, t h e e x t r a c t o r being equipped
with a d i s t r i b u t o r of f o u r b a f f l e d i s c s and a
100-ml short-necked f l a s k . Ensure t h a t a l l
lumps a r e broken down and t h a t no powder adhe-
r e s t o t h e t o p of t h e d i s t r i b u t o r . Attach a
r e f l u x condenser and e x t r a c t f o r 4 h w i t h occ-
a s i o n a l s w i r l i n g of t h e c o n t e n t s of t h e e x t r a c -
t o r . T r a n s f e r t h e CHC13 i n t h e f l a s k t o a
s e p a r a t i n g f u n n e l and e x t r a c t w i t h 4 q u a n t i t i e s ,
each o f 20 m l , o f 0.5M H2SO4. Wash t h e combined
a c i d e x t r a c t s w i t h 10 m l of CHC13. F i l t e r t h e
aqueous s o l u t i o n through a small wet p l u g of
c o t t o n wool i n t o a shallow d i s h and w a r m g e n t l y
with s t i r r i n g t o remove t r a c e s o f CHC13. Cool
and d i l u t e t o 100 m l with N H2SO4. D i l u t e 20
m l t o 100 m l w i t h N H2S04 and measure t h e e x t i n -
c t i o n of a 1-cm l a y e r a t 262 nm and 300 nm.
C a l c u l a t e t h e % o f s t r y c h n i n e from t h e e x p r e s -
s i o n S(0.321 a - 0.467 b)/w, where a i s t h e
e x t i n c t i o n a t 262 nm, b i s t h e e x t i n c t i o n a t
612 FARID J. MUHTADI A N D MOHAMED S. HIFNAWY

300 nm, and w i s the weight of t h e powder taken.

For t h e assay of nux vomica l i q u i d e x t r a c t
The method mentioned f o r t h e powder was
adopted, e x t r a c t i n g 2.0 m l of t h e l i q u i d e x t r -
a c t , omitting the 2 m l of alcohol 70%, and t a k -
ing 5 m l of t h e acid e x t r a c t i v e f o r t h e prepara-
t i o n of t h e f i n a l s o l u t i o n . Calculate the %
w/v of strychnine from t h e expression : 20(0.321
a - 0.467 b ) / v , where a i s the e x t i n c t i o n a t
262 nm, b i s t h e e x t i n c t i o n a t 300 nm, and v
i s t h e volume of l i q u i d e x t r a c t .
For nux vomica t i n c t u r e c a r r y out t h e assay
as described under t h e powder, e x t r a c t i n g 5.0
m l of the t i n c t u r e and omitting t h e 2 m l of
alcohol 70%. Calculate the percentage w/v of
strychnine from t h e expression : S(0.321 a -
0.467 b ) / v , where a i s the e x t i n c t i o n a t 262
nm, b i s t h e e x t i n c t i o n a t 300 nm, and v i s t h e
volume of t h e t i n c t u r e .
A second method making use of the absorb-
ance a t t h e forementioned wave lengths was
presented (114). I t depends on complexation
of both strychnine and brucine with methyl
orange a t pH 5.0, and a f t e r treatment with 0.1
N NaOH, t h e l i b e r a t e d a l k a l o i d s a r e determined
spectrophotometrically. The method was c a r r i e d
out as follows : Dilute 10 m l t i n c t u r e nux
vomica (0.125% w/v) t o 100 m l with Mcllvaine's
b u f f e r , (prepared by a d d i t i o n of 48.50 m l 0.1 M
c i t r i c a c i d + 51.50 m l 0.2 M Na2HP04. 2H20 and
a d j u s t i n g pH t o 5 with a s u i t a b l e pH meter).
Accurately t r a n s f e r 2 m l d i l u t e d sample t o 50
m l separatory funnel containing 10 m l dye s o l u -
t i o n i n b u f f e r . Shake 1 min with 10 m l CHC13;
l e t l a y e r s separate completely. Transfer CHC13
layer t o another 100 m l separatory funnel con-
t a i n i n g 10 m l 0.1 N NaOH. Extract aqueous l a y e r
3 times each with 10 m l CHC13. Shake combined
CHC13 e x t r a c t s 15 s e c t o l i b e r a t e t h e dye.
Transfer CHC13 layer t o 100 m l separatory fun-
n e l containing 10 m l 0.1 N H2SO4. Shake, trans-
f e r a c i d e x t r a c t of t h e a l k a l o i d s t o 25 m l volu-
metric f l a s k , and d i l u t e t o volume. Measure
absorbance of 1 cm pathlength a t 262 and 300 nm.
Calculate concentration (mg %) of strychnine,
Ca, and brucine, Cb i n the t i n c t u r e from t h e
following equation:
STRYCHNINE 613

Ca =[(0.200 A 1 - 0.290 A2)/0.5955]X

(1/4) x (100/2) x (10)
Cb =[(0.305 A2 - 0.005 A1)/0.05955]
x (1/4) x (100/2) x (10)
where A 1 and A2 a r e absorbances of f i n a l solu-
t i o n ; 0.305 and 0.005 a r e absorbances of 1 mg
% strychnine; 0.290 and 0.200 a r e absorbances
of 1 mg % of brucine, a t 262 and 300 nm, respe-
c t i v e l y . The mean percentage recovery was
100.7+0.93 f o r strychnine.
Different o t h e r spectrophotometric proced-
u r e s have been reported f o r the UV spectropho-
tometric determination of strychnine i n crude
drugs o r formulations.
An o l d method makes use of t h e absorption
band a t 254 nm shown by strychnine i n absolute
alcohol (brucine shows maxima a t 264 nm) (115).
The a l k a l o i d e x t r a c t , containing both s t r y c h -
nine and brucine, i s t r e a t e d with 0.5 g of
potassium p e r s u l f a t e i n the presence of 3%
H2SO4 f o r 1 h, thereby brucine i s destroyed
while strychnine i s not. The s o l n . i s then
made a l k a l i n e and t h e strychnine e x t r a c t e d with
CHC13. The residue l e f t a f t e r evaporation of
CHC13 i s dissolved i n absolute a l c . and t h e
e x t i n c t i o n (E) a t 254 nm of t h e s o l n . i s measur-
ed. This E value is divided by E 1%a t 254 nm
(which i s equal t o 390) gives the concentra-
t i o n , as % of strychnine i n t h e a l c o h o l i c soln.
being measured.
Constituents which may absorb a t 254 nm, i f not
properly removed would of course i n t e r f e r e with
accurate q u a n t i t a t i v e estimation.
An ammoniacal suspension of t h e drug i s
e x t r a c t e d with CHC13 i n a downward displace-
ment l i q u i d - l i q u i d e x t r a c t o r . Strychnine and
brucine a r e then e x t r a c t e d from t h e CHC13 with
N H2SO4 and the strychnine determined spectro-
photometrically (116). I t was reported t h a t
t h e obtained r e s u l t s a r e i n agreement with t h e
o f f i c i a l methods but saving h a l f t h e determina-
t i o n time.
The two wave length procedure of a n a l y s i s
was a l s o applied f o r determination of mixtures
of strychnine and brucine a t 252 and 262 nm
(117) and a t 255 and 264 nm i n nux vomica
t i n t u r e (118). The l a t t e r method involves
614 FARID J. MUHTADI A N D MOHAMED S. HIFNAWY

p u r i f i c a t i o n by adsorption on alumina and by

c a t i o n exchange r e s i n .
A method f o r t h e q u a n t i t a t i v e determination
of strychnine i n brucine o r brucine s u l f a t e was
described (119). Brucine i s n i t r a t e d and s t r y -
chnine i s e x t r a c t e d with CHC13. The CHC13 is
then evaporated and strychnine i s determined
spectrophotometrically by d i s s o l v i n g t h e resi-
due i n 0.1 N H2SO4 and measuring absorbances
a t 254 and 275.5 nm. The d i f f e r e n c e between
these absorbances, compared with s i m i l a r d i f f -
erences a t these wave lengths f o r standard
strychnine s o l u t i o n s i s a measure of t h e s t r y -
chnine content; standard deviation was l e s s
than 0.03%.
A combined chromatographic and spectropho-
tometric method f o r determination of strychnine
t i n c t u r e was described (120). After paper
chromatography and development with the Dragen-
d o r f f reagent, t h e spot i s e x t r a c t e d from a
p a r a l l e l undeveloped chromatogram with 0.1 N
HC1 and t h e amounts of strychnine present dete-
rmined using t h e absorption of l i g h t a t 300 nm.
An u l t r a v i o l e t determination of strychnine
i n commercial b a i t formulations was developed
(121). The strychnine i s e x t r a c t e d i n 0.5%
(vol.) H2SO4 and the r e s u l t i n g e x t r a c t i s
cleaned up. Strychnine i s determined by t h e
d i f f e r e n c e i n absorbance a t 254 and 287 nm.
I t was reported t h a t t h e r e s u l t s obtained by
t h i s method c o r r e l a t e well with those obtained
by a gravimetric determination of strychnine
as t h e p i c r a t e.
Spectrophotometric determination of s t r y c h -
nine and brucine (100-500,ug) from a c i d i c s o l u -
t i o n s was c a r r i e d out a f t e r p r e c i p e t a t i o n with
the Mayer-Valser reagent. The ppt. a l k a l o i d -
H I + HgI i s dissolved i n 3 M AcOH and t h e a l k a -
l o i d determined i n d i r e c t l y over Hg-dithizon
(122).
UV spectrophotometry was a l s o used t o follow
t h e decomposition of c e r t a i n a l k a l o i d s , includ-
ing strychnine, (123) , on s t e r i l i z a t i o n of
t h e i r 1%aqueous s o l u t i o n s by autoclaving 20
min and 2,5,7 and 10 h a t 120'. Two-dimensi-
onal t h i n l a y e r chromatography i s used as a
reference check. UV spectrophotometric d e t e r -
mination was accurate with samples heated up
STRYCHNINE 615

t o 2 h ; prolonged h e a t i n g r e s u l t e d i n t h e
formation o f p p t s . i n t e r f e r r i n g w i t h t h e d e t e r -
minat i o n .
S t r y c h n i n e was determined p h o t o m e t r i c a l l y
a t 365 nm by r e a c t i o n with I2 i n C2HqC12 (124).
The r e s u l t s are r e p r o d u c i b l e and a c c u r a t e .
A method f o r t h e q u a n t i t a t i o n o f s t r y c h n i n e
depends on t h e s p e c t r o s c o p i c c h a r a c t e r i s t i c s
o f t h e i n t e r a c t i o n o f a l k a l o i d s with p i c r o l o n i c
a c i d i n s o l v e n t of low and i n t e r m e d i a t e p o l a r i t y
was s t a t e d (125). The p r e s e n c e o f a l k a l o i d s
caused a r e d s h i f t of t h e 322 nm band o f t h e
nonionized p i c r o l o n i c a c i d t o 355 nm, c o r r e s -
ponding t o t h e a n i o n i c resonance band. Consid-
e r a b l e i n c r e a s e i n a b s o r b t i v i t y which was depe-
ndent on b o t h t h e b a s i c i t y and molar c o n c e n t r a -
t i o n o f a l k a l o i d s p r e s e n t was used f o r t h e d e t e -
r m i n a t i o n of v a r i o u s a l k a l o i d a l s a l t s i n c l u d i n g
s t r y c h n i n e s u l f a t e . The method was s e n s i t i v e
t o 2,ug/ml with accuracy of 21.5% and s t a n d a r d
d e v i a t i o n k1.05-1.31.
Quantitative determination of strychnine
n i t r a t e and codeine i n p i l l s c o n t a i n i n g e x c i p i -
e n t s of p l a n t o r i g i n was r e p o r t e d (126). I t
depends on e x t r a c t i o n of t h e a l k a l o i d w i t h CHC13,
p u r i f i c a t i o n by p a s s i n g through A1203 column
and r e - e x t r a c t i o n o f s t r y c h n i n e from C H C 1 3 i n t o
0.25% H2SO4. Chromatography on s i l i c a gel
p l a t e s u s i n g CHC13 - a c e t o n e - NH3 (12:24:1)
was t h e f i n a l s t e p i n p u r i f i c a t i o n of t h e a l k a l -
o i d . The s u i t a b l e wave l e n g t h f o r d e t e r m i n a t i o n
of s t r y c h n i n e was 254 nm. S t r y c h n i n e n i t r a t e
was determined i n c a p s u l e s w i t h & 21.52% e r r o r
by UV spectrophotometry i n 0.1 N H2SO4 ( 127).
Using t h e same p r i n c i p l e , a s p e c t r o p h o t o -
m e t r i c d e t e r m i n a t i o n of s t r y c h n i n e n i t r a t e i n
p i l l s c o n t a i n i n g o t h e r i n g r e d i e n t s such a s
p h e n o b a r b i t a l , bromcamphor, Ca glycero-phosphate,
and As203 was proposed (128). To 15 m l 0 . 1 N
H2SO4 i s added powdered p i l l c o n t a i n i n g -0.002g
s t r y c h n i n e n i t r a t e , followed by 30 m l E t 2 0 s a t u -
r a t e d with 0 . 1 N H2SO4 and e x t r a c t e d f o r 3 min
The aqueous phase i s r e e x t r a c t e d , t h e e x t r a c t s
are washed with 10 m l 0 . 1 N H2SO4 a f t e r combin-
i n g , f i l t e r e d , brought t o a d e s i r e d volume and
t h e absorbance determined a t 254 nm. The prop-
osed procedure e l i m i n a t e s p h e n o b a r b i t a l and
bromcamphor i n t e r f e r e n c e .
616 FARID J. MUHTADI A N D MOHAMED S. HIFNAWY

An improved spectrophotometric determina-

t i o n of teriary amine drugs, including strych-
nine, using malonic a c i d / a c e t i c anhydride reag-
e n t was reported (129). The method involves t h e
condensation of malonic acid and AC20 under t h e
c a t a l y t i c e f f e c t of the t e r t i a r y amine function,
e i t h e r f r e e o r i n the form of s a l t . The con-
densation product has 2 prominent absorption
peaks a t 333 and 400 nm. The apparent molar
a b s o r p t i v i t y of t h e 333 nm band was i n the range
of 1.37~105t o 5 . 5 ~ 1 0 5 . Absorbance vs. concn.
was l i n e a r up t o 1.2,ug/ml of a l l drugs studied
and t h e s e n s i t i v i t y was O.l,ug/ml. The method
was reported t o be highly s e n s i t i v e and s p e c i f i c
f o r t e r t i a r y amine drugs without i n t e r f e r e n c e
from primary and secondary amines o r quater-
nary ammonium s a l t but does not d i s t i n g u i s h
between t e r t i a r y amines.
A method f o r determination of strychnine i n
nux vomica preparations, involving modification ,

of t h e French Pharmacopoeia method was d i s c u s s -

ed (130). The modifications proposed a r e :
maceration of t h e nut powder f o r 30 min i n
d i l u t e ammoniacal s o l u t i o n ; e x t r a c t i o n with
CHC13 - d i l . NH40H f o r 2 h; evaporating t h e
CHC13 e x t r a c t t o dryness, d i s s o l v i n g t h e r e s i -
due i n MeOH and examining the s o l u t i o n d i r e c t l y
with UV spectrometry.
Strychnine was assayed i n the seeds of
strychnos pierriama by dual - wavelength spectro-
scopy (131) as follows: The seeds a r e d r i e d ,
powdered and t h e powder (-0.4g) i s treated
with 2 N NaOH (1 ml) and mixed with -80 m l CHC13.
The mixture i s refluxed f o r - 1 h. cooled, f i l -
t e r e d and t h e f i l t r a t e i s evaporated, t r e a t e d
with 1 N H2SO4 on water bath and d i l u t e d with
1 N H2SO4 t o 100 m l . A 5-ml s o l u t i o n d i l u t e d
with d i s t i l l e d H 2 0 ( t o 25 ml) and analyzed by
dual-wavelength spectrophotometry a t 255 and
278 nm f o r t h e determination of strychnine
( r e l a t i v e standard deviation = 1.1%,r e c o v e r i e s
were 98.9%).
First d e r i v a t i v e spectrophotometric d e t e r -
mination of strychnine and brucine i n nux vomica
l i q u i d e x t r a c t was reported (132). Strychnine
and brucine a r e e x t r a c t e d from nux vomica e x t r -
act, d i l u t e d with H20 and NWOH by using CHC13
and a r e then back-extracted with 0.1 N H2SO4.
STRYCHNINE 617

The 1st d e r i v a t i v e s p e c t r a are measured a t

2 6 2 . 8 m f o r s t r y c h n i n e . The limits of d e t e c -
t i o n were 3,ugg/ml and t h e mean r e c o v e r i e s were
101.1%. They r e p o r t e d t h a t t h i s method was much
faster than t h e B r i t i s h Pharmacopoea method.
Other methods f o r s p e c t r o p h o t o m e t r i c a s s a y
o f s t r y c h n i n e i n d i f f e r e n t f o r m u l a t i o n s and
b i o l o g i c a l f l u i d s and t i s s u e s were a l s o d i s -
cussed (133-137).

7.S.3 InfraredSpectrophotometry (IR)

Small amounts o f s t r y c h n i n e and o t h e r orga-

n i c b a s i s , i n u r i n e could be i d e n t i f i e d by I R
(138). The b a s i s are e x t r a c t e d from u r i n e with
CHC13, t r a n s f e r r e d t o s p o t s o f d i l . H2SO4 sup-
p o r t e d on s t r i p s of Whatman 3 MM paper and
chromatographed, a f t e r n e u t r a l i z a t i o n o f t h e
a c i d s p o t , u s i n g e i t h e r BuOH-HC1 o r ISo-Bu Me
Ketone - ACOH as s o l v e n t . Detection of t h e
a l k a l o i d is achieved by an i o d o p l a t i n a t e s p r a y ,
o r a bromocresol green spray. S t r y c h n i n e may
t h e n be recovered f o r spectrophotometry by
e x c i s i n g t h e s p o t , making a l k a l i n e w i t h NH3
and e x t r a c t i n g w i t h CHC13. Spectrophotometry
is b e s t c a r r i e d o u t i n a s o l u t i o n i n an o r g a n i c
s o l v e n t i n m i c r o c e l l s w i t h N a C l windows.
Another method i n v o l v e s t h e i d e n t i f i c a t i o n
and e s t i m a t i o n o f s t r y c h n i n e by IR spectrometry
a f t e r i t s e x t r a c t i o n and s e p a r a t i o n by TLC on
s i l i c a g e l (139). The chromatogram e l u a t e s
are c o n c e n t r a t e d on K B r m i c r o p e l l e t s . S p o t s
c o n t a i n i n g about 10 Y of s t r y c h n i n e could be
e s t i m a t e d with a p r e c i s i o n o f 8-15%.

7.5.4 Nuclear Magnetic Resonance: NMR

S t r y c h n i n e was determined i n nux vomica

powder u s i n g NMR (140). The f o u r a r o m a t i c
p r o t o n s of s t r y c h n i n e e x h i b i t s i g n a l s a t 7.20
( p r o t o n s 9,10,11), and a t 8.20 ppm (proton 1 2 ) ,
t h e l a t t e r being i n f l u e n c e d by t h e carbonyl
group of t h e lactam. The 2 aromatic p r o t o n s of
b r u c i n e form well d e f i n e d s i g n a l s a t 6.73
(proton 9), and 7.88 ppm (proton 1 2 ) , t h e l a t -
t e r a l s o i n f l u e n c e d by t h e lactam carbonyl.
The h e i g h t t o t h e s t r y c h n i n e peak a t 7.17 ppm,
o v e r t h e h e i g h t t o t h e b r u c i n e peak a t 6.73
ppm, i s a f u n c t i o n of t h e p e r c e n t s t r y c h n i n e i n
618 FARID J. MUHTADI A N D MOHAMED S. HIFNAWY

t h e sample, The NMR method was checked by v o l u -

metric a n a l y s i s of t h e mixtures.
7.5.5 Atomic Absorption
An i n d i r e c t d e t e r m i n a t i o n of a l k a l o i d s by
atomic a b s o r p t i o n spectrometry was a p p l i e d f o r
d e t e r m i n a t i o n o f s t r y c h n i n e (141). Strychnine
i s r e a c t e d with Reinecke s a l t s o l u t i o n , PhN02
i s added and t h e mixture i s shaken f o r 2 min.
The PhN02 l a y e r is s e p a r a t e d and dehydrated
and t h e C r i n t h e Ph NO2 s o l u t i o n i s measured
by atomic a b s o r p t i o n spectrometry. Cu2+, Co2+
Mg2+, Ca2+, Na+, N i 2 + , K+, Pb2+, Cd2+, Fe3+,
NH4+, C 1 - , Po$-, Br-, NO3-, Na2S203, monosodium
glutamate, s t a r c h , and s u c r o s e d i d n o t i n t e r e f e r e
7.6 Chromatographic Methods
7.6.1 Paper Chromatography
R e s u l t s o f s e p a r a t i o n of i n d o l e a l k a l o i d s
i n c l u d i n g s t r y c h n i n e by paper chromatography
(Whatman No. 1) i n v a r i o u s s o l v e n t s , were given
(142). Petroleum e t h e r s a t u r a t e d w i t h forma-
mide-benzene-Et0H (100:25:25) , BuOH-HC1 (conc.)-
H20 (50:7.5: 17.5), BuOH-ACOH-HzO (4 :1:5) were
s u i t a b l e . Impregnating t h e paper w i t h formamide
+ 8% NH4 formate gave round s p o t s .
Using Whatman No. 1, b u f f e r e d by d i p p i n g
i n a 5% s o l u t i o n of sodium dihydrogen c i t r a t e ,
b l o t t i n g , and d r y i n g a t 25OC f o r 1 h , a s o l v e n t
system was given f o r t h e d e t e c t i o n o f o r g a n i c
b a s i s (143,144). For s t r y c h n i n e , a s o l v e n t com-
posed o f 4.8 g of c i t r i c a c i d i n a mixture o f
130 m l o f H20 and 870 m l of n. b u t a n o l i s used;
i t s Rf = 0.30 u s i n g UV and i o d o p l a t i n a t e s p r a y
reagent f o r location.
A method f o r q u a n t i t a t i v e s e p a r a t i o n o f s t r y -
chnine by paper chromatography on 'segment s t r i p '
and i t s c o l o r i m e t r i c e v a l u a t i o n by adduct forma-
t i o n with dyes was d i s c u s s e d (145).
7.6.2 Thin Layer Chromatography (TLC)
The s o l v e n t system, ( s t r o n g NH3 : MeOH,
1.5:100), used f o r g e n e r a l s c r e e n i n g o f n i t r o g e -
nous bases,was used f o r d e t e c t i o n o f s t r y c h n i n e
on s i l i c a g e l G l a y e r s (Rf=0.22) (146). Loca-
t i o n was under UV and w i t h a c i d i f i e d i o d o p l a t i -
n a t e spray r e a g e n t . TLC u s i n g Dragendorff's o r
t h e previous r e a g e n t was a l s o r e p o r t e d f o r
STRYCHNINE 619

separation and detection of nux vomica alkal-

oids (147,148).
A method for the identification of strych-
nine in biological material, using TLC was
presented (149). The alkaloid is extracted
with 0.02 N H2SO4 at pH 2.5-3.0. The extract
is alkalinized with 20% NaOH to pH 8-9, extrac-
ted with CHC13 and chromatographed on silica
gel KSK layers using CHC13 - Me2CO - NH3 (30:
30:2) for development.
The use of TLC layers containing adsorbents,
binders, fluorescent substances and colloidal
silica for separation and identification of
strychnine was discussed (150). C6H6-CHC13-
Et2NH (9:4:1) was the developer and spots were
visualized under W.
Application of different chromatographic
techniques (TLC, GLC and HPLC) for rapid detec-
tion of drugs, intoxicants and related compounds,
including strychnine was investigated (151).
An improved TLC method €or detection and
identification of basic drugs extracted from
tissues was reported (152). This was achieved
by a double development technique using CH2C12
: Me Et ketone: NH3 (90:10:1.5 and 30:70:2).
Identification was achieved after the traditio.
nal detection spray sequence with successive
topical applications of : Marquis reagent.
Mandelin reagent and a special NH4 molybdate
fuming H2S04 reagent.
TLC was also applied for the detection and
determination of strychnine and brucine (153).
The two alkaloids are first separated by TLC
and then determined photometrically with 0.5%
picric acid in HOAC.
Another method for the determination of
strychnine in Chinese medicines by dual wave
length TLC-densitometry was also reported (154).
7.6.3 Adsorption Chromatography
A method for separation of strychnine from
nux vomica seeds by adsorption chromatography
was described (155). A 2 g sample of powdered
seed material, made alkaline, is extracted with
CHC13 and the solvent evaporated to dryness.
The residue is dissolved in trichloroethylene
and chromatographed on alumina (log) column.
620 FARID J. MUHTADI A N D MOHAMED S. HIFNAWY

Strychnine i s then e l u t e d with 70 m l of CCl4

containing 9% acetone. The solvent i s then
removed by d i s t i l l a t i o n and t h e a l k a l o i d a l
r e s i d u e t i t r a t e d with a standard acid.

7.6.4 Ion Exchange Chromatography

A procedure was described f o r separation

of strychnine from nux vomica using ion exchange
resin followed by i t s t i t r i m e t r i c determination
0 5 6 ) . 0.3 g sample of the d e f a t t e d powdered
nux vomica seed material i s e x t r a c t e d with a
mixture of 5 g CHC13, 15 g e t h e r and 1 m l of
10% ammonia. The CHC13/Et20 e x t r a c t i s then
shaken f o r 30 seconds with 5 m l H20 and t h e
solvent l a y e r i s then evaporated t o almost
dryness, 1 m l of alcohol added, and evaporation
i s c a r r i e d out t o dryness. To t h e residue i s
then added 3 drops of 2% H2SO4, followed by
10 m l of 90% alcohol and t h e s o l u t i o n i s passed
through ion exchange r e s i n (Amberlite IR-4B)
t o remove i m p u r i t i e s , and t h e e l u a t e i s t i t r a -
t e d with 0.01 N HC1, using t h e antimony e l e c -
t r o d e f o r e l e c t r o m e t r i c t i t r a t i o n , and the
q u a n t i t y of strychnine calculated.
Another method f o r t h e separation of s t r y -
chnine from e x t r a c t s of animal t i s s u e s using
cation-exchange r e s i n s was reported (157).
Liver t i s s u e (100 g) i s macerated i n 500 m l of
H20, and100.ml 0.01N HC1 i s added. The mixture
is heated t o b o i l i n g and held i n a b o i l i n g
water bath f o r 1 h; then the mixture i s d i l u t e d
t o 1 1 with H20 and f i l t e r e d . The f i l t r a t e i s
put through a column of Dowex SOW x 12 (200-
400 mesh) cation-exchange r e s i n . Strychnine
i s e l u t e d with 8 N H C 1 a f t e r removal of o t h e r
a l k a l o i d s and the e l u a t e i s n e u t r a l i z e d by
NaHC03 before i t s q u a n t i t a t i v e determination.
A t h i r d method involves t h e use of a c a t i o n
exchange paper f o r determination of strychnine
was described (158). Although simpler than t h e
c o l o r i m e t r i c methods, but it ndeds a l a r g e r
amounts of substance f o r analysis.
In a f o u r t h a r t i c l e , s t r i p of Amberlite
SA-2 (RH-form) and Amberlite SB-2 (R OH form)
a r e used f o r determination of strychnine (159).
STRYCHNINE 621

A f i f t h method d e s c r i b e d t h e use of s y n t h -
e t i c i o n exchange r e s i n f o r compleximetric
d e t e r m i n a t i o n o f s t r y c h n i n e i n pharmaceuticals
(160). The s o l u t i o n c o n t a i n i n g a l k a l o i d s a r e
passed through cokumn packed w i t h Wofatite KPS
s a t u r a t e d with Zn2+ and Zn2' i s determined i n
t h e e l u a t e by t i t r a t i o n with e d e t i c a c i d .

7.6.5 Gas Liquid Chromatography (GLC )

Although non v o l a t i l e , numerous methods

f o r GLC a n a l y s i s o f s t r y c h n i n e were r e p o r t e d .
A system u s i n g 1%SE-30 on 100-120 mesh Anakrom
ABS 6 f t x 4 mm I . D . b o r o s i l i c a t e g l a s s columns
was d e s c r i b e d f o r GLC a n a l y s i s o f s t r y c h n i n e
(161). The f o l l o w i n g o p e r a t i n g c o n d i t i o n s
were adopted : column temperature, 25OoC;
c a r r i e r g a s , argon; flow r a t e , 80 ml/min; d e t -
e c t o r , argon i o n i z a t i o n o r FID (Hz, 50 ml/min;
a i r , 300 ml/min). The r e l a t i v e r e t e n t i o n time
o f s t r y c h n i n e ( r e l a t e . t o codeine) is 4.83.
A method i n v o l v i n g t h e use of g l a s s columns
( 3 f t x 0.07 i n ) packed w i t h Gas Chrom. P was
d e s c r i b e d (162), f o r s e p a r a t i o n of d i f f e r e n t
a l k a l o i d s i n c l u d i n g s t r y c h n i n e . The packing
material i s washed w i t h concd. HC1, d r i e d ,
t r e a t e d w i t h h e x a m e t h y l d i s i l i z a n e and c o a t e d
with 1%o f q a n o s i l i c o n e , a p o l y e s t e r methyl
s i l i c o n e copolymer and cyclohexane dimethanol
.
succ i n a t e p o l y e s t e r
A t h i r d method f o r GLC d e t e r m i n a t i o n o f
d r u g s , i n c l u d i n g s t r y c h n i n e i n body f l u i d s was
r e p o r t e d ( 163). Four columns are used (2.5%
Marlophene 87 (M 8 7 ) , 1%Neopentylglycol seba-
cate (NGSB), 2.5% S i l i c o n Rubber SE 52 and 1%
Versamid 900 (SE 52/V 900).N2 i s used as a
c a r r i e r gas and Me m y r i s t a t e , Me stearate and
Me behenate are used as s t a n d a r d s . Best r e s u l t s
are o b t a i n e d w i t h a low t e m p e r a t u r e , a l i g h t l y
packed column, and a s e n s i t i v e d e t e c t o r .
Afourthmethod which a l l o w s GLC d e t e c t i o n
and q u a n t i t a t i v e d e t e r m i n a t i o n o f s t r y c h n i n e
i n b r u c i n e and v i c e v e r s a , and allowed s e p a r a -
t i o n of t h e a l k a l o i d b a s e s i n e x t r a c t s o f S t r y -
chnos nux vomica and S. icaja was p r e s e n t e d
(164) A FID,used f o r d e t e c t i o n , N2, a s c a r r i e r
gas (35 ml/min), and a 2 - f t s t a i n l e s s steel
column 1/8 i n d i a m e t e r , packed with Aeropak
622 FARID J. MUHTADI A N D MOHAMED S. HIFNAWY

30 (100-200 mesh) impregnated with 5% SE-52

a s s t a t i o n a r y phase. To determine strychnine
i n brucine, d i s s o l v e 100 mg of t h e sample i n a
mixture of 5 m l 5% H2SO4 and 15 m l H20. Heat
i f necessary. After cooling t o room tempera-
t u r e , added 20 m l 10% NaOH and e x t r a c t with
successive 20 m l portions of CHC13. F i l t e r
the combined CHC13 f r a c t i o n , Add 5 m l 95% E t O H ;
and evaporate t o dryness. Dissolve t h e residue
i n successive 10-ml p o r t i o n s 95% E t O H , and
d i l u t e t o 100 m l with EtOH. I n j e c t 0 . 4 ~ 1
s o l u t i o n i n t o t h e gas chromatograph system
described. Percent of strychnine = 84 A1/A1+
A2 where A 1 i s the a r e a of strychnine peak
and A2 i s a r e a of brucine peak. Retention time
of strychnine was 5.85 min with column tempera-
t u r e , 25OoC and i n j e c t o r temperature 28OoC.
A f i f t h GC method described determination
of strychnine i n b i o l o g i c a l materials (165).
Tissue homogenate a t pH 8.6 i s s t i r r e d with
acetone f o r 15 min. The mixture i s heated and
the acetone volatized. The mixture i s a c i d i f -
ied t o pH 3.3 with 5% C C13C02H, s t i r r e d f o r
30 min., and f i l t e r e d through sand. The f i l -
t r a t e i s e x t r a c t e d with CHC13 which i s concen-
t r a t e d and an a l i q u o t i s i n j e c t e d on a s t a i n -
l e s s s t e e l column packed with 30% SE-30 on
Chromosorb W a t 250'. Recovery from l i v e r
i s 90%.
Another method presented a GLC procedure
f o r determination of strychnine i s grain b a i t s
(166), using glass column (6 f t x i n . ) packed
with 5% SE-30 on Chromosorb W (DMCS) and F I D
f o r detection. The g r a i n b a i t s containing
strychnine are ground, mixed, and e x t r a c t e d by
shaking with CHC13 containing 1,3,5-triphenyl-
benzene as i n t e r n a l standard. Without f u r t h e r
cleanup, e x t r a c t f i l t r a t e s a r e i n j e c t e d d i r e c t l y
i n a gas chromatograph. Peak height r a t i o s a r e
used f o r q u a n t i t a t i o n of strychnine with 2,ug
s e n s i t i v i t y and recoveries ranging from 89.9
t o 91.7%.
The r e t e n t i o n index IR a n d d IR were con-
sidered f o r the gas chromatographic assay of
d i f f e r e n t compounds including strychnine (167).
Another method f o r GLC a n a l y s i s of s t r y c h -
nine a f t e r i t s s i l y l a t i o n with N-methyl-N-(tri-
methylsilyl) hepta fluorobutyramide I)- BuOAC
STRYCHNINE 623

(40:1:59) was d i s c u s s e d (168). An a l k a l i flame

i o n i z a t i o n d e t e c t o r (AFID), and t e m p e r a t u r e
programming w i t h lS°C/min from 150-260°C are
adopted.
A gas chromatographic d e t e r m i n a t i o n of b a s i c
d r u g s i n u r i n e o r blood and i t s a p p l i c a t i o n
f o r doping a n a l y s i s was r e p o r t e d f o r s t r y c h n i n e
( 169).
A r a p i d gas chromatography method f o r t h e
q u a l i t a t i v e detection of b a s i c drugs, includ-
i n g s t r y c h n i n e , i n postmortem blood, u s i n g a
n i t r o g e n phosphorous d e t e c t o r was d e s c r i b e d
(170). The method i n v o l v e s a 1 - s t e p e x t r a c t i o n
from 1 . 0 m l of postmortem blood b u f f e r e d t o
pH 9.0-9.5 i n t o n-Bu c h l o r i d e . No back e x t r a c -
t i o n or c l e a n up s t e p s a r e r e q u i r e d . E x t r a c t s
are i n j e c t e d on 3% OV-1 and 3% OV-17 columns
coupled t o NP d e t e c t o r s . S e n s i t i v i t y l i m i t s
are drug dependent and range from 200-500 ng/ml
f o r the various drugs l i s t e d i n the report.
Combined u s e of f u s e d s i l i c a c a p i l l a r y c o l u -
mns and nitrogen-phosphorus d e t e c t i o n f o r t h e
t o x i c o l o g i c a l a n a l y s i s of i l l i c i t h e r o i n samp-
les was s u c c e s s f u l l y a p p l i e d f o r d e t e c t i o n o f
nanogram amounts of s t r y c h n i n e (171). The
method i n v o l v e s t h e u s e of c a p i l l a r y columns
o f CP-Sil 5 o r C P - s i l 8 f u s e d s i l i c a , d e a c t i v a -
t e d w i t h a p o l y s i l o x a n e a t high t e m p e r a t u r e
and d i a c e t y l n a l o r p h i n e a s t h e i n t e r n a l s t a n d a r d
The same column was a l s o used i n t h e a n a l y s i s
o f an e x t r a c t o f t h e small i n t e s t i n a l c o n t e n t
i n an overdose c a s e (172).
Drug s c r e e n i n g v i a gas chromatography,
f u s e d s i l i c a c a p i l l a r y columns c o a t e d w i t h a
0.25,um f i l m o f SE 30, y i e l d e d r e p r o d u c i b l e
r e t e n t i o n i n d e x e s f o r s t r y c h n i n e (173). The
s c r e e n i n g was performed on a t e m p e r a t u r e prog-
rammed columns a t 100°-2950C a t S°C/min w i t h
t h e c a r r i e r v e l o c i t y o f 45 cm/s a t 100°C. The
c a p i l l a r y column p r o v i d e s s u p e r i o r r e s o l u t i o n
a n d / o r g r e a t l y reduced a n a l y t i c a l time r e l a t i v e
t o packed columns and i s t h u s well s u i t e d t o
emergency t o x i c o l o g i c a l s i t u a t i o n s i n f o r e n s i c
laboratories.
The v a r i o u s s u b s t a n c e s p r e s e n t i n s t r e e t
samples of n a r c o t i c s ( i n c l u d i n g s t r y c h n i n e )
were d e t e c t e d i n a s i n g l e a n a l y s i s by means
of s i l y l a t i o n and h i g h - r e s o l u t i o n gas chromato-
624 FARID J. MUHTADI A N D MOHAMED S. HIFNAWY

graphy 0 7 4 ) . This technique allows t h e detec-

t i o n of n a r c o t i c s , of most common a d u l t r a n t s ,
and of organic d i l u e n t s a t the same time. The
method i s highly s e n s i t i v e and has high r e s o l v -
ing power.
The use of t h e combined technique GC/MS
f o r i d e n t i f i c a t i o n of strychnine among o t h e r
compounds e x t r a c t e d from b i o l o g i c a l m a t e r i a l s
i n a v e t e r i n a r y toxicology laboratory was d i s -
cussed (1 75).
Gas chromatography employing 3%OV-1 on
chromosorb W-HP f o r r a p i d d e t e c t i o n of drugs,
i n t o x i c a n t s and r e l a t e d compounds , including
strychnine, was a l s o reported (151).
Another GLC method f o r separation of some
strychnos a l k a l o i d s was discussed (176).
An experiment was c a r r i e d out by us on t h e
e f f e c t of s i l y l a t i o n on GLC c h a r a c t e r s of s t r y -
chnine. Under our operating conditions, the
non d e r i v a t i z e d base showed a r e t e n t i o n t i n e
6.3 min. Although t h e peak was f a i r l y well
defined and sharp, but i t s base l i n e was spread
out over a period of approximately 6 min. The
s i l y l a t e d strychnine, on the o t h e r hand, showed
a sharp well defined peak a t 6.0 min r e t e n t i o n
time with a highly reduced baseline spread.
The s e n s i t i v i t y of d e t e c t i o n was much higher
(Fig. 11).
The following operating conditions were
adopted.
Column, 3% OV 1 on Gas Chrom, S O / l O O mesh;
temperature, isothermal a t 25OOC; d e t e c t o r F D
H2 flow, 16 ml/min; a i r flow, 160 ml/min);
c a r r i e r gas, N2 with a flow r a t e , 40 ml/min;
i n j e c t o r and d e t e c t o r temperature, 25OoC. Si Y-
l a t i o n was c a r r i e d out by using Tri-Sil/BSA
reagent and a small amount of pyridine as a
solvent.
The apparatus used: Gas Chromatograph -
Varian Model 3700 with Dual FID.
STRYCHNINE 625

Fig. 11.

GLC o f s t r y c h n i n e

(a) Free base

(b) S i l y l a t e d base

a b

7.6.6 High Performance Liquid Chromatography (HPLC)

HPLC o f f e r s obvious advantages by combining

a s e p a r a t i v e power s u p e r i o r t o t h a t o f TLC with
a means o f a c c u r a t e q u a n t i t a t i o n and e a s y t r a p -
ping o f e l u t e d m a t e r i a l f o r subsequent c o n f i r -
mation of s t r u c t u r e . T h i s technique i s moving
f a s t f o r t h e a n a l y s i s of many components. Beca-
u s e a l l t h e experimental work i n HPLC i s p e r f -
ormed a t ambient temperature, t h e problem o f
thermal i n s t a b i l i t y i s e l i m i n a t e d .
Using C o r a s i l I with 1.1%Poly G 300 as
a s t a t i o n a r y phase and heptane - E t O H r a n g i n g
from 100:s t o 100:10, v/v a s mobile phase,
q u a n t i t i e s i n nanogram l e v e l o f s t r y c h n i n e can
be s a t i s f a c t o r i l y analyzed with r e a s o n a b l e
accuracy ( 177).
A paper d e s c r i b i n g t h e high-speed l i q u i d
chromatography o f some a l k a l o i d s i n c l u d i n g
s t r y c h n i n e , u s i n g l i q u i d - s o l i d chromatography
was published (178). The wave l e n g t h o f d e t e c -
t i o n was 255 nm. The column was a s t a i n l e s s -
s t e e l t u b e , 30 cm x 2 mm I.D., f i l l e d with
Merckosorb S i 60 (5,um). The column tempera-
t u r e was maintained a t 20'. The r e t e n t i o n
time (RT) o f s t r y c h n i n e i n f i v e d i f f e r e n t
mobile phases and t h e i r flow r a t e s (FR) are as
626 FARID J. MUHTADI AND MOHAMED S. HIFNAWY

follows: CHC13 - MeOH (9:1),

RT, 6.9, FR 0.81; CHC13 - MeOH (8:2), RT 3.9,
FR 0.81; CHC13 - MeOH (7:3), RT 4 . 7 , FR 0.75;
E t Z O - MeOH (7:3) RT 9.4, FR 1.43; E t 2 0 - MeOH
(6:4), RT, 7.3, FR 1.34.
Using t h e same column and as mobile phases,
E t 2 0 containing 1%of diethylamine a t a flow-
r a t e of 2.0 ml/min a t 205 kg/cm2 and E t 2 0 -
MeOH ( 1 : l ) a t a flow-rate of 1.15 ml/min a t
200 kg/cm*, strychnine and o t h e r r e l a t e d a l k a l -
oids were analyzed (179). RT of strychnine
was 7.2 and 12.4 min i n the two systems res-
pectively.
A simple and r a p i d HPLC procedure i s desc-
ribed f o r t h e determination of strychnine i n
grain b a i t s (180). The b a i t s are ground, mixed,
and e x t r a c t e d by shaking with CHC13. Without
f u r t h e r cleanup, e x t r a c t f i l t r a t e s a r e i n j e c t e d
d i r e c t l y i n t o a l i q u i d chromatograph. The anal-
y s i s i s completed within 7 min and peak heights
are used f o r q u a n t i t a t i o n . Separations were
made on a 30 cm x 4 mm I . D . , s t a i n l e s s s t e e l
column packed w i t h p p o r a s i l (8-12,um s i l i c a ) .
The e l u t i n g solvent was MeOH-CHC13 (10:90) a t
a flow rate of 2 . 0 ml/min. Recovery of spiked
samples ranged from 91.5 t o 95.2%. Confirma-
t i o n of strychnine from a commercial sample was
made by high r e s o l u t i o n mass spectrometry with
mass agreement t o 1 . 2 ppm.
A procedure is described f o r t h e q u a l i t a -
t i v e d e t e c t i o n of strychnine among other conta-
minants i n an i l l i c i t diamorphine s e i z u r e s by
means of high-pressure c a t i o n exchange chrnma-
tography (181). The i l l i c i t diamorphine samp-
l e s a r e homogenised by grinding i n a mortar and
20-25 mg, accurately weighed, are dissolved i n
50% aqueous methanol, The s o l u t i o n is made up
t o 2 m l i n a volumetric f l a s k and 2 p l a r e taken
f o r an i n i t i a l q u a l i t a t i v e a n a l y s i s using t h e
following chromatographic conditions: column,
120 cm x 2.1 mm. Zipax SCX; e l u e n t s : (a)
H3B03 (0.2 M y aqueous), adjusted t o pH 9.3 with
40% NaOH, (b) H3B03 (0.2 M y aqueous) - aceto-
nitrile-n-propanol (86: 12:2) adjusted t o pH
9.8 with NaOH (40% aqueous); flow r a t e , 2 m l /
min; l i n e a r gradient, 0-100% (b) i n 6 min; UV
absorbance detector, 270 nm, 0.2 absorption
u n i t s f u l l scale. Strychnine was found i n
STRYCHNINE 627

many of the analyzed samples, r e t e n t i o n time,

6 . 3 min and r e t e n t i o n volume 12.8 m l (Fig. 12).

4 7

1 I I
0 5 10 (rnin.)
F i g . 1 2 . HPLC of some c o n s t i t u e n t s of i l l i c i t diamorphine
s e i z u r e s . For column conditions, see t e x t .
1 = Barbitone; 2 = c a f f e i n e ; 3 = morphine; 4 = monoacety-
lmorphine; 5 = strychnine; 6 = diamorphine; 7 = quinine;
8 = cocaine.
628 FARID J. MUHTADI AND MOHAMED S. HIFNAWY

The r e t e n t i o n of strychnine r e l a t i v e t o
morphine, when analyzed by HPLC among a wide
range of drugs of abuse, was 1.57 (182). The
following conditions were adopted: column,
s t a i n l e s s s t e e l , 25 cm x 4.6 mm I.D. f i l l e d
with small s i z e s i l i c a ' f i n e s ' ; solvent, MeOH-
2 N ammonia s o l u t i o n - 1 N ammonium n i t r a t e
s o l u t i o n (27:2:1); flow-rate, 1 ml/min; p r e s s -
ure, 1500 p . s . i , room temperature; d e t e c t o r ,
UV a t w , l . 278 nm. A weighed amount of sample
is made up i n e i t h e r water o r d i l u t e HC1 t o
known concentration, usually 10 mg/ml, and
d i s s o l u t i o n i s a s s i s t e d by immersion f o r about
1 min i n an u l t r a s o n i c bath. An a l i q u o t of
1-5,ul of t h i s sample i s i n j e c t e d on t o t h e
chromatographic column. Peak h e i g h t s a r e
used f o r q u a n t i t a t i o n .
A simple, rapid e x t r a c t i o n and subsequent
determination f o r strychnine, using HPLC with
a reverse-phase solvent system was described
(183 ) . Stomach contents o r g r a i n b a i t con-
t a i n i n g strychnine a r e made a l k a l i n e with NaOH
and e x t r a c t e d with CHC13. Extract f i l t r a t e s
a r e i n j e c t e d d i r e c t l y i n t o a l i q u i d chromato-
graph without f u r t h e r preparation, except f o r
d i l u t i o n , i f necessary. Peaks are resolved
within 3.5 min and peak heights a r e used f o r
q u a n t i t a t i o n . HPLC of strychnine was c a r r i e d
out on a 30 cm x 4 mm I.D. s t a i n l e s s s t e e l
column packed with Bondapak C18. The solvent
program was 0.005 M phosphate buffer-MeOH (60
+ 40) a t a flow r a t e of 1.5 ml/min. Recovery
from spiked stomach contents was 93.9%. The
d e t e c t i o n limits, using t h e 254 nm UV d e t e c t o r ,
was 5 mg. The strychnine peak was c o l l e c t e d
and subjected t o TLC with standard f o r con-
firmation.
Reversed-phase HPLC employing octadecyl-
s i l a n i z e d columns (RP 18) was described f o r
d e t e c t i o n of strychnine (151).
An HPLC method was developed f o r t h e quan-
t i t a t i o n of strychnine i n u r i n e of children
with nonketotic hyperglycinemia and o t h e r deve-
lopment d i s o r d e r s t r e a t e d with t h e a l k a l o i d
(184). Mobile and s t a t i o n a r y phases were p o l a r ,
i . e . MeOH-H20-330 g/kg NH3 ammonia (vols., 85
STRYCHNINE 629

m l + 14.2 m l + 0.8 ml) and LiChrosorb S i - 6 0 ,

7,um. Brucine was t h e i n t e r n a l s t a n d a r d .
E x t r a c t i o n was performed by t h e E x t r l u t t e c h n i -
que. A t s t r y c h n i n e n i t r a t e c o n c e n t r a t i o n s i n
u r i n e of 2 1 , 126 and 760,ug/L, recovery was
92.1, 98.1 and 102.5. A c h i l d with n o n k e t o t i c
hyperglycinemia under continued s t r y c h n i n e
t r e a t m e n t e x c r e t e d 1-13.6% o f t h e d a i l y dose
unmetabolized i n u r i n e . The method was a l s o
s u i t a b l e f o r the estimation of unreacted s t r y -
chnine i n t i s s u e e x t r a c t s .
The d e t e r m i n a t i o n of s t r y c h n i n e i n s e e d s o f
S. piersirma o r t h e i r p r e p a r a t i o n s , i n c l u d e d
e x t r a c t i o n with CHC13 - NH40H ( 9 : 1 ) , s e p a r a t i o n
of t h e o r g a n i c phase, and i n j e c t i o n of an a l i -
quot i n t o a HPLC column (4 mm diam. x 250 mm)
c o n t a i n i n g s i l i c a g e l (3-5 v, i r r e g u l a r ) and
Et20-MeOH-NHqOH (80:20:1 v/v) a s mobile phase
(flow r a t e 2 ml/min.) f o r a n a l y s i s . D e t e c t i o n
was a t 250 nm. The r e l a t i v e s t a n d a r d d e v i a t i o n
was 1.29% (measured by t h e peak h e i g h t method)
and t h e recovery was 99.3%. The c a l i b r a t i o n
p l o t was l i n e a r t o 0.75,ug. The method was
simple and r a p i d , and may be used i n r o u t i n e
a n a l y s i s (185,186).
Measurement o f s t r y c h i n e by HPLC i n b i o l o g i -
c a l media was a l s o r e p o r t e d (187). CHC13 a t
b a s i c pH was used t o extract s t r y c h n i n e and
t h e i n t e r n a l s t a n d a r d q u i n i n e from plasma. HPLC
was c a r r i e d o u t on a s i l i c a g e l column u s i n g as
e l u e n t c o n c e n t r a t e d NH4OH i n MeOH a t a flow
r a t e of 1.1 ml/min and d e t e c t i o n a t 254 nm.
Recovery from plasma was 83%, t h e lower l i m i t
o f s e n s i t i v i t y was 0 . 6 2 5 ~ g / m l , and i n t r a - a n d
i n t e r - a s s a y v a r i a n c e was 3-5% and ~ 1 0 %res- ,
pectively.
A method f o r i d e n t i f i c a t i o n and determina-
t i o n of s t r y c h n i n e and b r u c i n e i n nux vomica
e x t r a c t s and o t h e r pharmaceuticals u s i n g HPLC
was d e s c r i b e d (188 ) . The e x t r a c t s were f i r s t
p u r i f i e d by p a s s i n g through a Sep Pak C18 -
c a r t r i d g e column and HPLC c a r r i e d o u t on a
reversed-phase ODS column with MeCN-H2O (25:75)
as t h e mobile phase c o n t a i n i n g 1 - h e p t a n e s u l f -
o n i c a c i d as t h e c o u n t e r i o n . The recovery was
S98.1.
Another method f o r r a p i d e s t i m a t i o n of
s t r y c h n i n e i n t i n c t u r e nux vomica BP was r e p o r -
t e d (189 ). The method was a m o d i f i c a t i o n o f
630 FARID J. MUHTADI A N D MOHAMED S. HIFNAWY

a previous procedure (182 ) . This involves

the use of a s h o r t e r column (12.5 cm x 4.6 mm
I.D.), changing t h e s t a t i o n a r y phase t o s p h e r i -
c a l p a r t i c l e s (Hypersil), increasing the flow
rate (2 ml/min) ,and changing the d e t e c t o r wave
length from 278 t o 254 nm; t h e n e t e f f e c t being
t o decrease t h e r e t e n t i o n time of strychnine
from 14 t o 3.6 min and 6 min were s u f f i c i e n t
t o s e p a r a t e strychnine, brucine and the minor
a l k a l o i d s of nux vomica. Concentrations were
c a l c u l a t e d using e i t h e r peak a r e a s o r h e i g h t s
and comparing against an e x t e r n a l standard
(0.15% w/v strychnine base i n 45% ethanol).
Other a r t i c l e s discussing t h e a p p l i c a t i o n s
of d i f f e r e n t HPLC techniques f o r a n a l y s i s of
strychnine among drugs of f o r e n s i c i n t e r e s t ,
i n v e t e r i n a r y toxicology, i n pharmaceutical
formulations, i n mixture with o t h e r b a s i c drugs
and f o r screening of drugs and i n s e c t i d e s were
a l s o reported (190-194).

7.6.7 Electrophoresis

Electrophoretic determination of s t r y c h -
nine, brucine and quinine i n t i n c t u r e mixtures
was developed (195). The separation was done
i n 2 N HC02H s o l u t i o n (pH 1.5) a t 0 . 5 ma./cm
and 10 v./cm. The s p o t s were developed with
Dragendorff reagent, and t h e concentration
determined photometrically o r by planimetry.
Errors, were within 5-12%.

7.7 Radiometric Determinations

A radiometric t i t r a t i o n method has been developed

t o enable microdetermination of many a l k a l o i d s , includ-
ing strychnine (196 ) i n t h e form of t h e i r s a l t s (hyd-
rochloride, phosphate o r s u l f a t e ) . The method was
c a r r i e d out a s follows:
For hydrochlorides: Add a known volume of concd. HNO3
t o 0.5-3 m l of t h e a l k a l o i d a l s a l t i n a c e n t r i f u g e
test tube, t o r e s u l t i n 0.7-0.8 M HNO3. Then t i t r a t e
with 0.01 M AgN03 (approx. 3000 c.p.m./cc). After
each addition of AgN03 s o l u t i o n , a g i t a t e t o homoge-
n i z e and c e n t r i f u g e with complete sedimentation of
the ppt., t r a n s f e r a known volume of the c l e a r solu-
t i o n t o t h e Y-counter, and a f t e r counting, r e t u r n t o
the t e s t tube, and repeat sequence. I t i s necessary
STRYCHNINE 63 1

t o make a t l e a s t 2 a d d i t i o n s o f t i t r a t i n g agent
b e f o r e t h e e q u i v a l e n t p o i n t and a t l e a s t 3 a d d i t i o n s
a f t e r it. The e q u i v a l e n t p o i n t i s determined g r a p h i -
c a l l y by e x t r a p o l a t i o n , with an e r r o r o f ?0.4%. For
phosphates : b r i n g t h e a l k a l o i d a l s a l t with d i l . NaOH
t o pH 8 and t i t r a t e w i t h AgN03 as b e f o r e ; b u t s t r o n -
g e r a g i t a t i o n and b e t t e r c e n t r i f u g a t i o n are r e q u i r e d ,
t h e e r r o r being 20.8%. For s u l f a t e s : t i t r a t e i n a
pH 7 a l c o h o l i c medium i n which t h e Ag s a l t is i n s o l u -
b l e , t h e e r r o r being 0.8%. The method can be adapted
t o mixtures of a l k a l o i d a l s a l t s by t i t r a t i n g a t d i f f -
e r e n t pH v a l u e s .
Another method f o r r a p i d d e t e r m i n a t i o n o f s t r y c h -
n i n e w i t h Mayer's r e a g e n t was based on r a d i o m e t r i c
t i t r a t i o n ( 197 ) . Mayer r e a g e n t was p r e p a r e d by irr-
a d i a t i o n of 1,358 g HgC12 i n a n e u t r o n f l u x o f 1012
n e u t r o n s / s q . cm. sec. f o r 10 h and a d d i t i o n of t h e
s a l t t o 50 m l H20. A s o l u t i o n of 4.9 g K I i n 20 m l
H 2 0 was prepared and added with s t i r r i n g t o t h e above.
The s o l u t i o n was d i l u t e d t o 100 m l . D i l u t i o n o f 5 m l
of t h e s o l u t i o n t o 100 m l gave 0.0025 M o f Mayer rea-
gent. The pH o f a known volume o f t h e a l k a l o i d s o l u -
t i o n was made n e u t r a l o r s l i g h t l y a c i d i c . Mayer
r e a g e n t was added i n small increments, and t h e mix-
t u r e was s t i r r e d f o r 20-30 s e c . and c e n t r i f u g e d . A
known volume of t h e c l e a r l a y e r i n t h e c e n t r i f u g a t e
was t r a n s f e r r e d i n t o a s p i r a l surrounding a c y l i n d r i -
c a l c o u n t e r and t h e r a d i o a c t i v i t y was determined.
The procedure was r e p e a t e d s e v e r a l times t o allow a
p l o t o f c o u n t s p e r min. vs. m l o f Mayer r e a g e n t t o
be made. The equivalence p o i n t was s h a r p l y d e f i n e d .
N i t r a t e o r s u l f a t e i o n s do n o t i n t e r f e r e . S t r y c h n i n e
can a l s o be determined i n a l c o h o l i c s o l u t i o n s and i n
t h e presence o f s 0.5 g b r u c i n e .
S t r y c h n i n e and b r u c i n e , 5-30 Y each, a r e determin-
ed r a d i o m e t r i c a l l y i n t i n c t u r e of nux vomica ( c o n t a i n -
i n g 0.224% t o t a l a l k a l o i d s ) by measuring t h e p - a c t i -
x
v i t y of iodinated a l k a l o i d - I ppts, after separat-
i n g s t r y c h n i n e and b r u c i n e by TLC on p l a t e s c o a t e d
with A 1 2 0 3 without a b i n d i n g agent (198). The method
was c a r r i e d out as f o l l o w s :
Apply t h e s o l u t i o n c o n t a i n i n g s t r y c h n i n e and b r u c i n e ,
develop t h e chromatogram with Et20 - E t O H ( 9 5 : 5 ) , d r y
a t 2 S 0 , s p r a y t h e chromatogram with 1%i o d i n e i n CHC13,
mark t h e s p o t s , and remove excess i o d i n e by h e a t i n g
t h e p l a t e a t 105'. Remove t h e A1203 c o n t a i n i n g t h e
marked s t r y c h n i n e and b r u c i n e s p o t s w i t h a m i c r o p i p e t
by a p p l y i n g g e n t l e s u c t i o n . Convert t h e a l k a l o i d s
632 FARID J. MUHTADI AND MOHAMED S. HIFNAWY

t o t h e r e s p e c t i v e a c e t a t e s a l t s by warming t h e A1203
containing spot with 5 m l o f 2% HOAC, f i l t e r the
A1203 on a s h l e s s f i l t e r paper. Ppt strychnine and
brucine s e p a r a t e l y with 2 . 5 m l of a c i d i f i e d aqueous
"1 s o l u t i o n , f i l t e r t h e p p t s , measure t h e p - a c t i v i t y
of t h e p p t s , and f i n d the concentration of both a l k a l
oids from the r e s p e c t i v e c a l i b r a t i o n curve. The
,&count vs. concn. curve is l i n e a r f o r 5-30 Yof s t r y -
chnine and brucine.
A method f o r determination of strychnine i n blood
by double i s o t o p i c l a b e l i n g was described (199 ) .
Double-labeled (3H, 14C) strychnine methiodide was
prepared. For t h e assay, add strychnine stock solu-
t i o n and (or) water t o 1 m l human c i t r a t e d blood t o
make t o t a l volume of 2 m l , then add 0 . 2 m l (9600 cou-
nts/min) of a strychnine s o l u t i o n i n absolute E t O H
and 0.2 m l of absolute EtOH. Evaporate a t 60' i n
vacuo nearly t o dryness. Add 20 m l E t O H and repeat
the evaporation. Add 1 m l of methyl iodide - 3H
s o l u t i o n (absolute EtOH) (-30 c./ml), s e a l t h e
mixture i n a g l a s s tube, heat a t 60' f o r 4 h , open
t h e tube, and evaporate t o 0 . 1 m l . This was paper
chromatographed by a descending technique using
EtOAC-MeOH-28% NH40H (100:10:15); unlabeled s t r y c h -
nine and double-labelled strychnine (for quenching
corrections) were a l s o chromatographed simultaneou-
s l y with the sample, The amount of 14C and 3H i n
each spot was determined, using t h e percent recovery
of 14C t o c o r r e c t f o r l o s s e s during work up, t h e
amount of strychnine - 3H and thus t h e amount of
strychnine i n t h e o r i g i n a l sample was calculated.
I t was reported t h a t strychnine can be analyzed
more a c c u r a t e l y by t h i s method than by more conven-
t i o n a l procedures.
Another method €or determination of strychnos
a l k a l o i d s by radiometric t i t r a t i o n s with potassium
thallium iodide (K204TLI)reagent was discussed (200).
The sample s o l u t i o n containing 0.01 N acid was reac-
t e d with excess t i t r a n t within a t o t a l s o l u t i o n volu-
me of 5 m l . The ppt of (Alkaloid H) TLI4 was separa-
t e d by centrifuging and t h e p a c t i v i t y of a superna-
t a n t a l i q u o t was measured by using a Geiger counter.
The s e n s i t i v i t i e s were 5 ~ 1 0 '-~ 2x10-3 M f o r s t r y -
chnine and brucine. The e r r o r s were within +2.0%
f o r determining 4-6 mg a l k a l o i d alone and they were
within 23.2% f o r determining 7-15 rng a l k a l o i d i n
pharmaceutical preparations.
STRYCHNINE 633

The c a t a l y t i c p o l a r o g r a p h ic waves o f s t r y c h n i n e
i n b u f f e r s pH 8 were used f o r i t s d e te r m in a tio n i n
10-4 - 10-5 M i n pharmaceutical p r e p a r a t i o n s , i n
s o l u t i o n s ( 1 ml) and t a b l e t s ( a f t e r paper chromato-
g r a p h i c s e p a r a t i o n from v i t a m in s and s u g a r s (201).

ACKNOWLEDGEMENT
The a u t h o r s would l i k e t o thank M r . A m i r Shehata
(B.Pharm.) of t h e Pharmacognosy Dept. f o r performing
t h e m i c r o c r y s t a l t e s t s . Our thanks a r e a l s o due t o
Mr. Uday C. Sharma of Pharmacognosy Dept., C o lle g e
of Pharmacy, King Saud U n i v e r s i t y f o r h i s secretarial
a s s i s t a n c e i n t h e r e p r o d u c t i o n of t h e m a n u sc r ip ts.
634 FARID J. MUHTADI AND MOHAMED S. HIFNAWY

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STRYCHNINE 643

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644 FARID J. MUHTADI AND MOHAMED S. HIFNAWY

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STRYCHNINE 645

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646 FARID J. MUHTADI A N D M O H A M E D S. HIFNAWY

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 VIDARABINE

Wen-Hai Hong, 'bun Chang and Robert E. Daly

1. Introduction

1.1 History

1.2 Name, Formula, Molecular Weight

1.3 Appearance, Color, Odor

2. Physical Properties

2.1 Melting Range

2.2 Infrared Spectrum

2.3 Ultraviolet Spectrum

2.4 Nuclear Magnetic Resonance Spectrum

2.5 Optical Rotation
2.6 Solubility

2.7 pKa Value

2.8 Thermal Analysis (DSCand TGA)

3. Synthesis and Production

4. Impurity

5. Stability

ANALYTICAL PROFILES OF DRUG SUBSTANCES Copyright 0 1986

VOLUME 15 by the American Pharmaceutical Association
647 A11 rights of reproduction in any form reserved.
648 WEN-HA1 HONG ET AL.

6. Metabolism and Disposition

7. Pharmacokinetics
8. Methods of Analysis
8.1 Elemental Analysis

8.2 Identification

8.3 Loss on Drying

8.4 Specific Rotation

8.5 Ultraviolet Spectrophotometric Analysis

8.6 Thin-Layer Chromatography

8.7 High-pressure Liquid Chromatography

8.8 Microbiological Assay

9. References
VIDARABINE 649

1. Introduction
1.1 History

Vidarabine is a purine nucleoside which does not occur

naturally. It was first synthesized by Lee, et. al., as a
potential anticancer agent in 1960 (1.2). Its antiviral
activity against herpes simplex virus (HSV) and vaccinia
virus (VV) in cell culture was first demonstrated by Privat
de Garilhe and DeRudder in 1964 (3). Vidarabine has
subsequently been obtained from fermentation cultures of
Streptomyces antibioticus, tested and developed by
Warner-Lambert/Parke-Davis Company ( 4). It has been
used for the treatment of herpes simplex virus types 1 and
2, varicellazoster, and vaccinia viruses infections ( 5).

1.2 Name, Formula, Molecular Weight

Vidarabine is a U.S. Adopted Name (USAN). Its chemical

name is 9 4 8 -D-arabinofuranosy1)adenine monohydrate.
It is also called adenine arabinoside, spongoadenosine,
ara-A, vira-A and CI-673 ( Parke-Davis).

C10H13N504 H 2 0 MW: 285.27

1.3 Appearance, Color, Odor

Vidarabine is an odorless, white crystalline solid.
2. Physical Properties
2.1 Melting Range

Vidarabine melts in the range of 262O and 27OOC.

650 WEN-HA1 HONG ET AL.

2.2 Infrared Spectrum

The infrared spectrum of vidarabine presented in Figure 1

w a s taken as a 0.5% dispersion of vidarabine in potassium
bromide using a Perkin-Elmer Model 6 2 1 infrared
spectrophotometer. Table I gives the infrared
assignments which are consistent with the structure of
vidarabine.
TABLE I

INFRARED SPECTRAL ASSIGNMENTS FOR VIDARABINE

FREQUENCY (em-1) ASSIGNMENTS

3550 and 3450 NH stretch
3400 - 3200 OH stretch
1640 NH2 bending
1140 - 1040 various C-0-C stretchings

2.3 Ultraviolet Spectrum

The ultraviolet spectrum of vidarabine in water is shown
in Figure 2. Table I1 summarizes the ultraviolet spectral
data for vidarabine ( 6).

TABLE II
ULTRAVIOLETSPECTRAL DATA FOR VIDARABINE
PlI 1 MAX(nm) E

1.0 257.5 12,700

7.0 259.0 13,400
13.0 259.0 14,000

2.4 Nuclear Magnetic Resonance Spectrum

The NMR spectrum of vidarabine is shown in Figure 3.
The spectrum was obtained with a Varian Model 60 MC
spectrometer. Deuterated dimethylsulfoxide was used as
the solvent with tetramethylsilane as an internal
standard. The assignments, chemical shifts and
multiplication are described in Table I11 and are consistent
with the structure of vidarabine.
VIDARABINE 65 1

WAVELENGTH MICRONS

2.5 3 $ 5 f, 8 ?lF 1214 1822 35 3

100

Figure 1 Infrared Spectrum of Vidarabine

w
0
z
U
m
a
0
v)
m
U

220 260

WAVELENGTH(nm)

Figure 2 Ultraviolet Spectrum of Vidarabine in Water

rr
0
m
VIDARABINE 653

TABLE IIl
NUCLEAR MAGNETIC
RESONANCE ASSIGNMENTS FOR VIDARABINE

CHEMICAL SHIFTS NO. OF

ppm MULTIPLICITY PROTONS ASSIGNMENTS

8.27, 8.21 Doublet 2 Two aromatic

protons,
one at C-2,
and one
at C-8
of adenine
7.29 Singlet 2 Two protons of
the 6-
amino group
6.38, 6.31 Doublet 1 Anomeric proton
at C-1
split by the
cis
proton at
C-2 with a
J of 4.2 cps

5.63 Triplet 2 A superposition

of two doublets
due to the
hydroxyl protons
of (7-2 and Ct-3
5.18 Triplet 1 Ct-5-h ydroxyl
proton being
split by the
Ct-5 protons

4.22 Multiplet 2 Two of the five

protons at C'-2,
C'-3, (7-4 and
C'-5 being split
by the neigh-
boring protons
3.8, 3.71 Multiplet 3 Three of t h e five
protons a t C'-2,
(7-3, C'-4
and C'-5
654 WEN-HA1 HONG ET AL.

2.5 Optical Rotation

The specific rotations of a 1% (w/v) vidarabine solution in
dimethylformamide are presented in Table IV.

TABLE IV

SPECIFIC ROTATIONS OF A 1%(w/v)

VIDARABINE SOLUTION IN DIMETHYLFORMAMIDB

WAVELENGTH (nm) I
589 (NaD) -2.5'
578 -2.9'
546 -12.5'
436 -30.4'
365 -76.2'
Vidarabine Lot RxX41064 was used
The specific rotations of a 1% (w/v) solution of vidarabine
and its optical enantiomorph, 9 4 B -L-arabinofuranosyl)
adenine, in dimethylsulfoxide are presented in Table V.
As shown, the rotations for the enantiomorph are opposite
in sign. The enantiomorph was prepared according to the
procedure given by Glaudemans and Fletcher ( 7).
TABLE V

SPECIFIC ROTATIONS OF A
1%(w/v) VIDARABINE AND ITS OPTICAL ENANTIOMORPH
9-( bL-ARABINOFURANOSYL) ADENINE, SOLUTIONS IN DMSO

25
[ a1

WAVELENGTH OPTICAL
(nm) VIDARABINE ENANTIOMORPH
589 (NaD) -9.7' +9.2'
578 -10.5' +9.8'
546 -12.5' +12.0°
436 -30.4' +30.2'
365 -76.2' +76.4'
VI DAM B I N E 655

2.6 Solubility

Since vidarabine behaves as both a weak acid and a weak

base, its solubility in water depends on pH as well as other
factors such as temperature and state of hydration. The
aqueous solubility of vidarabine, expressed in terms of the
hydrate, at various pHs is shown in Figure 4. As seen,
vidarabine behaves as a neutral compound over the pH
region 5 to 10.5. Over this pH region the solubility is
invarient and equal to 0.44 mg per m l (23OC). Also, as
seen, rather acidic or basic pHs are required to
significantly increase its solubility.
Hydration influences vidarabine solubility as shown in
Figure 5. The solubility of the anhydrate has not been
accurately determined since conversion of anhydrate to
hydrate occurs very rapidly as shown in Figure 5.
However, the anhydrate is at least 5 times more soluble
than the hydrate in water.
Solubilities of vidarabine at various temperatures are
given in Figure 6. Since the van't Hoff relationship is
applicable, these data indicate the monohydrate is the
stable solid phase in equilibrium with solution at
temperatures in the range of 25' to 100°C.
In regard to nonaqueous solvents, the solubility of
vidarabine in such commonly used organic solvents as
alcohols, esters, chlorinated hydrocarbons, etc., is very
low. Polar solvents such as dimethylsulfoxide, and
dimethylformamide are good solvents for vidarabine as is
indicated by the data given in Table VI.
TABLE VI
SOLUBILITY OF VIDARABINE
IN DIMETHY LFORMAMIDE, DIMETHY ISULFOXIDE
AND DIMETHYISULFOXIDE - WATER MIXTURE AT 25OC
SOLVENT SOLUBILITY (mg/ml)
Dimethylf ormamide 20
Dimethylsulfoxide 250
DMSOI Water (v/v)
90/10 150
75/25 20.5
50150 3.4
25/75 1.2
656 WEN-HA1 HONG ET AL.

4 7

04
2
I

4
I

6
.
8 10 12 14
PH

Figure 4 Solubility Profile of Vidarabine

0
E
\
2.0 -
E"
Y

1.0-

a
I
m
A
Y
c
-
0 . I

0 10 20 30 40 50 60 70 80

TIME (min.)

Figure 5 Dissolution of Vidarabine in Water at Room Temperature

VIDARABINE 657

1OOOO( 1IT" K)

Figure 6 A van't Hoff Plot of Vidarabine Solubility Data

658 WEN-HA1 HONG ET AL.

2.7 pKa Value

Vidarabine is an amphoteric compound which can act as
both a weak acid and a weak base, The protonated form
of vidarabine has a pKa of 3.5. This value is similar to
that reported for adenosine, (3.6) as would be expected ( 8
- 12). The protonation site for vidarabine has not been
established but anology with adenosine suggests the
nitrogen in position 1 is involved ( 13).

Vidarabine + H+

Vidarabine's second pKa ( 11.4) also approximates that

reported for adenosine ( 12.34). This ionization is likely
associated with a hydroxyl group (position unknown)
contained in arabinofuranosyl moiety.
2.8 Thermal Analysis (DSC and TGA)
A differential scanning calorimetry curve was obtained
(Figure 7) on a Perkin-Elmer DSC-2C differential
calorimeter. Using nitrogen as the purge gas, the scan
was performed at a rate of 10°C/minute from 50' to
300OC. The DSC curve reveals a broad endothermic
dehydration peak (onset 123.1OC) and a single sharp
endothermic melting peak (onset 263.9OC) followed by
decomposition of the sample.
 5.00
sample wt : 2.69 mg

scan rate : 10.00 degfmin

3.25

0
\
0
Q
2.50
-m
m
\

0
E
max:140.91

125
I

0
50.0 80.0 110.0 140.0 170.0 2OOx) 230.0 260.0 290.0

TEMPERATURE ('GI

Figure 7 Differential Scanning Calorimetry Curve of Vidarabine

660 WEN-HA1 HONG ET AL.

A thermogravimetric analysis curve was obtained using a

Perkin-Elmer TGS-2 thermogravimetric analyzer. The
analysis was performed at a rate of lO'C/minute from
35.8' to 230'C. The thermogravimetric analysis curve
(Figure 8) shows a weight loss of 6.32% from 30' t o
llO°C, which conforms the presence of one mole of
water.
3. Synthesis and Production
Vidarabine has been synthesized by two different methods.
The first involves a cleavage reaction of 94 2,3-anhydro- I3 -D-
lyxofuranosyl)adenine, which is derived from 9- B -D-
xylofuranosyladenine ( 1). The second is a condensation of
benzoyladenine with 2,3,5-tri-O-benzyl- 6 -D-arabinosyl-
chloride ( 7). Both methods involve laborious condensation of
adenine with a sugar derivative. Ikehara $ & (14) synthesized
vidarabine from naturally occurring adenosine employing a
selective cleavage reaction of 8,2'-anhydroadenosine by
hydrogen sulfide, and desulfurization of the resulting 6-amino-
9- I3 -D-arabinof uranosylpurine-8-thiol,

Production of vidarabine by fermentation was conducted until

the late 1960's by Parke Davis and Company using a strain of
Streptomyces Antibioticus (4). The harvested beer was
filtered, concentrated under reduced pressure and cooled at 2
- l0'C to effect optimal precipitation of vidarabine. The
deposited crystals were filtered off and recrystallized from
boiling water. The thrice crystallized vidarabine was filtered
off, washed with cold water, and cold acetone, and then dried
in vacuo at room temperature.
4. Impurity
The impurity most likely in vidarabine is the deaminated
analog, 94 B -D-arabinofuranosyl) hypoxanthine. This impurity
can be detected by TLC using silica gel plate F-254 with the
solvent system ethylacetate-isopropyl alcohol-water
(195:70:30) or chloroform-methanol (100:30). The Rf values
will vary depending on temperature, TLC plates, etc., but t h e
separation of the two components is consistent. The
chloroform-methanol system provides the most reproducible
results.
VIDARABINE 66 1

Rf VALUE
SOLVENT SYSTEM VIDARABINE DEAMINATED ANALOG
Ethylacetate-isopropyl
alcohol-w ater 0.42 - 0.58 0.33 - 0.49
Chlorof or m-methanol 0.38 - 0.39 0.14 - 0.17

sample wt : 2.8917 mg

scan rate : 10.00 deglmin

TEMPERATURE ( "C)

Figure 8 Thermogravimetric Analysis Curve of Vidarabine

662 WEN-HA1 HONG ET AL.

Inspection of the developed plates under short wavelength UV

light reveals blue spots for either vidarabine or the
deaminated analog; a minimum of 0.25 pg of either compound
per spot is necessary t o detect the purine derivatives. Elution
of each spot or zone from the TLC plate with aqueous
methanol (50%) and examination of each eluate in the UV
spectrophotometer gave the respective UV spectra for
vidarabine (1 max at 260 nm) or the deaminated analog (A max
at 249 nm). A minimum of 6 pg of material is required to
obtain the spectrum.
5. Stability

The solution stability of vidarabine in 0.1 N hydrochloric acid

a t 100°C was followed by spectrophotometric and polarimetric
methods (15). Both methods gave the same results indicating
the hydrolysis only involved the cleavage of the N-glycoside
linkage without racemization or other side reactions. This
result was also confirmed by TLC analysis.
6. Metabolism and Disposition
The disposition of vidarabine has been studied in laboratory
animals and in man following parenteral administraiton of
tritium labeled drug.
Vidarabine is rapidly and extensively deaminated to the less
active metabolite, ara-hypoxanthine (ara-Hx) ( 16-18) followed
by subsequent oxidation of the purine ring (19) (Figure 9). A
small fraction of vidarabine is phosphorylated by cellular
enzymes to yield ara-ATP (20). Ara-Hx represents the major
metabolite in plasma, urine, erythrocytes, and tissues after
the administration of tritium labeled drug.
7. Pharmacokinetics
Animal Studies
Following intravenous administration of tritium labeled
vidarabine to dogs and monkeys, plasma nonvolatile tritium
(NV-H3) declined rapidly with estimated half life of 30
minutes and 2 to 4 hours, respectively. Loss of the tritium
label resulted from further oxidation of ara-Hx which was
evident from the presence of tritiated water in plasma and
urine. Application of a specific HPLC assay revealed that
greater than 90% of the plasma NV-H3 could be accounted for
by ara-Hx. Only traces of vidarabine was detected 5 minutes
after iv injection. Existing evidence also indicate that
vidarabine or metabolites equilibrated rapidly between plasma
and erythrocytes followed by slow incorporation of drug
VIDARABINE 663

Ara-A
Adenine
I
I
I
Ara-AMP I
c
Ara-ADP
6
Ara-ATP
arabiise H
Ara-Hx Hypoxanthine
Nucleotides I
I
I I
I
I I
I

I HO
~ i n o s e
I
H

( * 3H) Ara-X Xanthine

Figure 9 Pathways of Metabolic Disposition of Vidarabine

664 WEN-HA1 HONG ET AL.

related tritium label into the erythrocytes. Urinary excretion

accounted for 20% and 90% of the administered dose in dogs
and monkeys, respectively, with less than 1% of the dose
recovered in feces. Chronic once daily iv doses failed to show
any evidence of drug (or metabolite) accumulation (17).
Absorption following intramuscular administration of
vidarabine suspension was slow and prolonged resulting in
lower plasma levels in both animal species. Half life values of
12 to 24 hours were reported (17).

Vidarabine w a s extensively distributed to tissues. Following

parenteral administration of 3H-ara-A, highest concentration
of tritium were found in the kidneys with progressively lower
concentrations in the spleen, liver, heart, skeletal muscle,
lung, brain, and blood plasma ( 17).
Clinical Studies
Glazko et al (17) reported that following iv administration of
tritium labeled vidarabine, the plasma NV-3H half life ranged
from 3 to 5 hours. Among infants and children receiving
unlabeled iv doses of vidarabine, the maximal plasma level was
on t h e order of 1-2 pg/ml and the half life was about 1.5-2.5
hr, shorter than in the adult. In adults receiving im
vidarabine, the maximal plasma level was about 0.2-0.3 pg/ml
and the plasma half-life about 10-16 hr.
With iv administration of vidarabine to humans, the maximum
urinary excretion rate of 3H occurred in the first 4 hr. after
dosing with mean recovery of about 45 percent within 24 hr.
Again as in animals, maxima were delayed after intramuscular
adm inistra t ion.
LePage et al (21) studied the excretion of 3H-labeled
vidarabine and ara-Hx in human patients receiving rapid
intravenous infusions ( 1/2-2 hr), continuous intravenous drips
( 2 4 hr), or intramuscular injections. They reported that in the
two patients receiving rapid infusions, 90 percent of the drug
was recovered in the urine in 24 hr. In the two patients
receiving im injections, about 5 percent was recovered in 24
hr.

Kinkel and Buchanan (22) studied the plasma, cerebrospinal

fluid, erythrocyte, and urine levels of vidarabine and ara-Hx
following iv infusion in seven patients. Vidarabine was
administered by either iv drip or by a constant rate infusion
pump over a range of 4-10 days.
VIDARABINE 665

The levels of vidarabine appearing in the plasma were just

barely above the limits of the HPLC assay. The levels of ara-
Hx, however, rose promptly during the infusion period and then
declined promptly when infusion w a s stopped. The elimination
half lives in the patients observed were between 3 1/2 and 4
hr. The levels found on day 1 and day 5 were similar,
indicating no accumulation or change in metabolism,
distribution, or excretion over that period.
8. Methods of Analysis
8.1 Elemental Analysis
A typical elemental analysis of a sample of vidarabine lot
RxX41064 is presented in Table VII.
TABLE M

ELEMENTAL ANALYSIS FOR VIDARABINE

% AS MONOHYDRATE % AS ANHYDRATE
ELEMENT THEORY FOUND THEORY FOUND
C 42.10 42.76 44.94 45.10
H 4.60 4.64 4.90 4.89
N 24.55 25.09 26.21 26.46
H20 6.32 5.19 0.00 0.00

8.2 Identification
The identity of vidarabine is established by infrared
spectroscopy. The infrared absorption spectrum of a 0.5%
dispersion in potassium bromide exhibits maxima only at
the same wavelengths as that of a similar preparation of
USP Vidarabine Reference Standard.
Ultraviolet spectroscopy is also used to identify
vidarabine. The spectrum of a sample dissolved in water
(approximately 15 pg per ml) scanned from 320 - 220 nm
compares qualitatively to that of a vidarabine standard
similarly prepared.
8.3 Loss on Drying

Dry about 100 rng of vidarabine in vacuum a t 100°C and

at a pressure not exceeding 5 m m of mercury for 4 hours;
i t loses between 5.0% and 7.0% of its weight (23).
666 WEN-HA1 HONG ET AL.

8.4 Specific Rotation

Using a solution containing 10 mg of anhydrous vidarabine

per m l in dimethylformamide and a polarimeter tube 1.0
decimeter in length, determine the specific rotation at
25'C at 365 nm. The value lies between -56.0' and 65.0'
(23).

8.5 Ultraviolet Spectrophotom etric Analysis

Vidarabine exhibits a UV absorption band with a maximum

near 260 nm in water. Quantitation of the vidarabine
content is based on the net absorbances at max of the
assay preparation as compared to that of the standard
preparation. The method is not stability indicating since
adenine, a hydrolytic product, possesses a similar UV
spectrum. However, separation of adenine from
vidarabine prior to UV analysis can be achieved on an
anionic ion exchanger using 0.1 M pH 10 borate buffer as
the eluent ( 15).
8.6 Thin-Layer Chromatography

A number of thin-layer chromatographic systems have

been reported for the identification of vidarabine and for
the separation from its mostly likely impurities, adenine
and 9- B-D-arabinofuranosylhypoxanthine ( 24,25). The
systems are described below and their respective Rf
values are tabulated.
System I
Plate: Silica Gel GF 254 plate, 250 p m
Mobile phase: isopropyl alcohol/ammonia/water ( 65:
10:25)

Detection: shortwave UV
System II
Plate: Silica Gel GF 254 plate, 250 p m
Mobile phase: lower phase of chloroform/methanol/
3% acetic acid (3:2:1)

Detection: shortwave UV
VIDARABINE 667

8.6 Thin-Layer Chromatography (continued)

System I11

Plate: Silica Gel GF 254 plate, 250 p m

Mobile phase: Lower phase of chloroform/
methanol/l5% ammonia (3:2:1)

Detection: shortwave UV

System IV

Plate: Plastic s h e e t s (20 x 20 c m ) coated with 10

p m of PEI-impregnated MN300 cellulose with
fluorescent indicator

Mobile phase: 0.1 M boric acid

Detection: shortwave UV

TABLE MI

THIN-LAY ER CHROMATOGRAPHIC
SEPARATION OF MDARABINE,
9- 8-D-ARABINOPURANOSYLHYPOXANTHINE AND ADENINE
Rf VALUE
9- B-D-ARABINOFURANOSYL
SYSTEM YIDARABINE HYPOXANTHINE ADENINE
I 0.67 0.60 0.74
I1 0.26 0.16 0.43
I11 0.44 0.18 0.60
IV 0.57 0.49 0.40

8.7 High-pressure Liquid Chromatography (HPLC)

The following systems have been reported f o r t h e analysis

of vidarabine in dosage formulations and biological fluids.

System 1(23,26)

Column: 30 cm x 4 mm i.d., c 1 8 column 5 or 10 p m

Mobile phase: Docusate sodium/glacial acetic acid/
methanol (2.2:10:500), 4.s. to 1000 with water.
668 WEN-HA1 HONG ET AL.

8.7 High-pressure Liquid Chromatography (HPLC) (cont.)

System I (23,26) (continued)
Temperature: ambient
Detection: 254 nm
Flow rate: 1.5 ml/ml
System I1 (17)
Column: 60 c m x 1.8 mm i.d., Aminex A-28 column

Mobile phase: 0.2 M pH 7.4 acetate buffer

Temperature: 65OC
Detection: 254 nm
Flow rate: 1 5 - 20 ml/hr.
System I11 (27)
Column: 1 5 c m x 3.7 mm i.d., Aminex A-28 column
Mobile phase: 0.01 M pH 6.3 borate buffer
Temperature: 6OoC
Detection: 254 nm
Flow rate: 0.5 ml/min.
System IV (28)
Column: 15 cm x 4.6 mm i.d., Ultrasphere ODS 5
p m column
Mobile phase: 0.01 M potassium dihydrogen
phosphate (Solution A) and 30% methanol in 0.01 M
potassium dihydrogen phosphate ($elution B). Linear
gradient of 10 - 30% of Solution B in Solution A,
achieved in 1 5 minutes.
Temperature: Ambient
Detection: 254 nm
Flow rate: 1.0 ml/min.
VIDARABINE 669

8.7 High-pressure Liquid Chromatography ( HPLC): (cont.)

System V (29)
Column: 25 c m x 4.6 mm i.d., Li-Chrosorb RP-8
column
Mobile phase: 20 m l of acetonitrile in 480 m l of
0.005 M sodium pentanesulfonate buffer, pH 7.2

Temperature: 4OoC
Detection: 250 nm

Flow rate: 1.0 ml/min.

System VI (30)
Column: 25 c m x 4.6 m m i.d., Whatman Partisil
10/ODS-3 column, preceded by a Brownlee C-18
guard catridge system
Mobile phase: 0.01 M potassium dihydrogen
phosphate, pH adjusted to 3.25 -
+ 0.005 with acetic
acid/acetonitrile (95:5)
Temperature: Ambient
Detection: 254 nrn
Flow rate: 1.0 ml/min.
8.8 Microbiological Assay
Vidarabine is assayed microbiologically with Pichia
membranae-faciens as the test organism using the agar
diffusion method. The potency of a sample of vidarabine
is then obtained by comparing the zone of inhibition with
those of the standard preparation (31). The sensitivity of
this method is approximately 20 vg per ml of the test
solution.
670 WEN-HA1 HONG ET AL.

9. References

1. W.W. Lee, A. Benitez, L. Goodman, and B.R. Baker, J-

Am. Chem, Soc., 82, 2648 (1960).

2. E.J. Reist, A. Benitez, L. Goodman, B.R. Baker, and W.W.

Lee, J. Ow. Chem., 27, 3274 (1962).

3. M. Privat de Garilhe and J. DeRudder, C. R. Acad. Sci.

(D), (Paris), 259, 2725 (1964).

4. Brit, Pat. 1,159,290 (1969 to Parke Davis Company).

5. Physicians Desk Reference Medical Economics Company,

38th Edition, p 1530, 1532 (1984).

6. The Merck Index, Merck ti Co., Inc. 10th Edition, 9779

1983).
7. C.P.J. Glaudeman and H.G. Fletcher, Jr., J. Org. Chem.,
x 3 0 0 4 (1963).
8. H.A. Sober ed., l'Handbook of Biochemistryf1, The
Chemical Rubber Company, Cleveland, Ohio, 2nd edition,
5-65, 1970.

9. E.P. Serjeant and B. Dempsey "Ionization Constants of

Organic Acids in Aqueous Solutionf1, p. 561, Pergamon
Press, 1979.
10. R.B. Martris and Y.H. Mariam, Met. Ions Biol. Syst., 8, 57
(1979)
11. A.E. Martell and J. Schwarzenbach, Helv. Chim. Acta,
39,653 (1956).
12. K. Wallenfels and H. Sund, Biochem. Z., 329, 41 (1957).
13. N.C. Gonnella, H. Nakanishi, J.B. Holtwick, D.S.
Horowitz, K. Kanamori, N.J. Leonard, and J.P. Roberts,
J. Am. Chem. Soc., 105, 2050 (1983).

14. M. Ikehara, Y. Ogiso, T. Maruyama, and M. Kaneko, Nucl.

Acid Chem., 2, 485 (1978).
15. W.H. Hong and D.H. Szulczewski, J. Pharm. Sci., 68, 499
(1979).
16. G.A. LePage and I.G. Junfa, Cancer Res. 25, 46 (1965).
VIDARABINE 67 1

9. References (continued)

17. A.J. Glazko, T. Chang, J.C. Drach, D.R. Mouger, P.E.

Borondy, H. Schneider, L. Croskey, and E. Maschewske,
In Adenine Arabioside: An Antiviral Agent, pp 111-133,
Pavan Langston, D. Buchanan, R.A., and Alford, C.A., Jr
.,
(eds). Raven Press, N.Y (1975).

18. F.A. Miller, G.J. Dixon, J. Ehrlich, B.J. Sloan and I.W.
McLean, Jr., Antimicrob. Ag. Chemother., 8, 136-147
(1968).

19. M.E. Cowen, T.R. Oegema and J.C. Drach, Fed. Proc. 33,
471 (1974).

20. J.C. Drach, J.S. Bus, S.K. Schultaz and J.N. Sandberg,
Biochem. Pharm., 23, 2261 (1974).

21. G.A. LePage, A. Khaliq and J.A. Gottlieb, Drug Metab.

Dispos. 1, 756 (1973).

22. A.W. Kinkel and R.A. Buchanan, In Adenine Arabinoside:

An Antiviral Agent, pp 197-204, Pavan-Langston, D.,
Buchanan, R.A., and Alford, C.A., Jr (eds), Raven Press,
N.Y. (1975).

23. USP XXI, p. 1115 (1985), 21 CFR 436, 200 (e).

24. J.C. Drach and J.M. Novack, Anal. Biochem., 52, 633
(1973).

25. P.M. Schwartz and J.C. Drach, Nucl. Acid Chem., 2, 1061
(1978).

26. 21 CFR 436, 325.

27. H.G. Schneider and A.J. Glazko, J. Chrom., 139, 370

(1977).

28. R.P. Agarwal, J. Chrom., 231, 418 (1982).

29. D.B. Bowman and R.E. Kauffman, J. Chrom., 229, 487

( 1982).

30. W. Schroeder 111, T.L. Cupps, and L.B. Townsend, J-

Chrom., 254, 315 (1983).

31. Dr. S. W. Mamber, Warner-Lambert/Parke-Davis,

Personal Com -m unica t ion.
672 WEN-HA1 HONG ET AL.

ACKNOWLEDGEMENT

We thank Mrs. Patricia A. Estwin for excellent secretarial

assistance.
 ZOMEPIRAC SODIUM

Mladen iiniE, Josip Kuftinec, Hrvoje Hofman,

Franjo KajfeZ, Zlatko Meit

1. Foreword, History, Therapeutic Category

2. Description
2.1. Name, Formula, Molecular Weight
2.2. Appearance, Color, Odor
3. Synthesis
4, Physical Properties
4.1. Spectra
4.1.1, Infrared
4.1.2. Ultraviolet
4.1.3. Mass
4.1.4. f3oton Magnetic Resonance
4.1.5. C-Nuclear Magnetic Resonance
4.2. Solid Properties
4.2.1. Melting Characteristics
4.2. X-Ray Diffraction
4.3. Solution Properties
4.3.1. Solubility
4.3.2. Acidity (pKa)
5. Methods of Analysis
5.1. Elemental Analysis
5.2. Chromatographic Methods
5.2.1. Thin Layer
5.2.2. Gas
5.2.3. High Performance Liquid
5.3. Titrat ion
6. Stability and Degradation
7. Drug Metabolism, Pharmacokinetics, Bioavailsbilitp
ANALYTICAL PROFILES O F DRUG SUBSTANCES Copyright 0 1986
VOLUME 15 by the American Pharmaceutical Association
673 All rights of reproduction in any form reserved.
674 MLADEN 2INIC ET AL.

8. I d e n t i f i c a t i o n and D e t e r m i n a t i o n i n Body F l u i d s and

Tissues
9. D e t e r m i n a t i o n i n PharmaceiiticRls
l o . References
ZO M E PI RAC S OD1U M 675

l * Foreword, History, Therapeutic Category

Zomepirac is a benzoylpyrrole acetic acid derivative
which is chemically closely related to the non-steroidal
anti-inflammatory agent tolmetin /I/. It is a potent anal-
gesic with a predominantly peripheral action mediated
through prostaglandin synthetase inhibition. Zomepirac so-
dium has been shown to be effective in many chronic and
acute pain conditions; it is at least as effective as usu-
al therapeutic doses of aspirin, codein alone or with as-
pirin, phenacetin and caffein, dextropropoxyphene with pa-
racetamol or orally administered pentazocine. Additional-
ly, zomepirac sodium may provide analgesia comparable to
that of standard doses of intramuscular morphine. However,
serious side effects have been found during the therapy
with zomepirac sodium which resulted in withdrawal pf this
drug by the manufacturer (McNeil Lab. USA) in 1983. It is
expected that zomepirac sodium will be again available but
only for those patients where treatment with other non-
-steroidal anti-inflammatory agents is not successfull.

2. Description
2.1. Name, Formula, Molecular Weight
Chemical Name: sodium salt of 1,4-dimethyl-5-( p-chlo-
robenzoyl)-1H-pyrrole-2-acetic acid.
Generic name: Zomepirac sodium
Trade name: Zomax

C H ClNNaO
15 13 3
I
Mol. wt. 313.56
CH3
2.2. Appearance, Color, Odor
Zomepirac is marketed as the sodium salt dihydrate;
pale yellow crystalline, odorless powder. Free acid appe-
ars as white, odorless crystalline powder.

3. Synthesis
The first description of a synthesis of zomepirac
I) was published by Carson and Wong /1,2/ in
( see Scheme
1973. The first step in this procedure is a modified
Hantzsch pyrrole synthesis from chloroacetone ( I), diethyl
3-oxoglutarate I1 and 40% aqueous methylamine which ga-
ve the diester 111. Saponification of this product gave
 40% a q . C H 3 h J ~ ! 5 c 0 0 E t 1. NaOH/ H2yv:H)y OOHEtOH/ HCL,
CHzCOOEt 2. H+/H20
CHzCOOEt I I
I I1 CH3 111 CH3 IV

SCHEME 1.
ZOM EPIRAC SODIUM 677

the diacid IV which was selectively esterified to give

the monoester V. The latter was decarboxylated by heating
at 190-2oo0C. The resulting pyrrole derivative VI was
aroylated with N,N-dimethyl-p-chlorobenzamide (VII) and
phosphoryl chloride ( Vilsmeier aroylation) to give com-
pound VIII, which was finally saponified to zomepirac.
The overall yield is about 6%.
Recently, two modifications of this synthetic route
appeared in the literature describing selective one pot
decarboxylation /3/ and decarbethoxylation /4/ of inter-
medate 111 to VI.
The syntheses of deuterium /5/ and tritium /6/ label-
led zomepirac were also published.
4. Physical Properties

The IR absorption spectra of zomepirac and its sodi-

um salt dihydrate are shown on Fig. 1. Spectra were re-
corded from KBr-pelleted samples on the Pye-Unicam SP-3
infrared spec trophotometer.
On the spectrum of zomepirac ( A ) , 4pe following
characteristic bands should be noted (cm ) : 3100 ( C - H
stretching, phenyl and pyrrole rings), 2500-3000 (carbo-
xylic 0-H stretching), 1720 and 1700 [ketone and carboxy-
lic C=O strzfching respectively). Further bands at 1604
and 1475 cm can be attributed to the pyrrole ring ske--l
leton vibrations, and the strong bands at 760 and 750 cm
to the pyrrole C-H out-of-plane vibrations.
On the spectrum of zomepirac sodium salt dihydrate
(BJ1 the 0-H stretching vibration band appears at 3500
cm , due to the pre ence of crystal water. Strong bands
at 1580 and 1370 cm-f are caused by symmetric and asym-
metric stretching vibrations of the carboxylate anion.

4.1.2. Ultraviolet
Fig. 2 shows the UV spectra of zomepirac measured
in methanol, 0.1 M hydrochloric acid and 0.1 M sodium
hydroxide solution. Slight shift of the longer wavelength
maxima toward higher values should be noted by the change
of the medium from acidic (321 nm) over neutral (322 nm)
to basic (328 nm). Shorter wavelength maxima at 252 nm
is also shifted to 258 nm in basic solution and 261 nm
in hydrochloric acid.
4.1.3. Mass
The mass spectra of zomepirac Fig.(3A) and its so-
dium salt dihydrate (Fig. 3B) were recorded on a Kratos
Fig. 1. A- Infrared spectrum of zomepirac i n KBr p e l l e t .
Instrument: Pye-Unicam SP 3-200 spectrophotometer.
04
3500 3000 2500 2000 1600 1200 cm
-f 800

Fig. 1. B- Infrared spectrum of zomepirac sodium salt dihydrate in KBr pellet.

Instrument: Pye-Unicam SP3-200 spectrophotometer.
680 MLADEN ZINIC ET AL.

0.7

0.6

0.5

0.4
Q,
0
c
5 0.3
0
v)
a
4
0.2

0.1

Fig. 2. Ultraviolet spectra of zomepirac in:

......
------
0.1 M sodium hydroxide,
0.1 M hydrochloric acid, and
methanol solution.

Instrument: Pye-Unicam sP8-loo.

ZOMEPIRAC SODIUM 68 1

60-

60-

20-

0 II I ’ I * llrrrllllllrllrl
40 60 80 100 120 160 160
mle

Fig. 3. A- Mass spectrum of zomepirac.

Instrument: Kratos MS-25.
682 MLADEN ZINIC ET AL.

100 -
80 -
60-
-
4 0-
-
$20-
.CI
h -
I-

.-a 0 0
.-5a> 80--
d

E -
60-

40 -

20-

Fig. 3. B- Mass spectrum of zomepirac sodium salt

dihydrate. Instrument: Kratos MS-25.
ZOMEPIRAC SODIUM 683

MS-25 s p e c t r o m e t e r l i n k e d t o DS 50s d a t a system u s i n g d i -

r e c t i n s e r t i o n probe.
I n t h e mass spectrum of zomepirac, t h e m o l e c u l a r i o n
a p p e a r s a t m/e 291 w i t h 51% of r e l a t i v e i n t e n s i t y . The
b a s e peak a t m/e 246 i s a p p a r e n t l y formed by d e c a r b o x y l a -
t i o n . Other prominent peaks a r e m/e 139 ( C H 4 0 C l ) and m/e
111 ( C H C 1 ) formed by f r a g m e n t a t i o n of t h z c a r b o n y l gro-
up. .Smhft p e a k s a t m/e 276 and m/e 256 r e s u l t e d from N-
- d e m e t h y l a t i o n and t h e c l e a v a g e of c h l o r i n e atom i n t h e
p - p o s i t i o n of t h e phenyl r i n g r e s p e c t i v e l y .
The main c h a r a c t e r i s t i c s of t h e mass spectrum shown
on Fig. 3 B i s t h e a b s e n c e of m o l e c u l a r i o n . The b a s e peak
a t m/e 247 r e s u l t s from t h e d e c a r b o x y l a t i o n p r o c e s s . Fur-
t t e r f r a g m e n t a t i o n can+be i n t e r p r e t e d a s f o l l o w s : m/e 231,
(M - C O +O), m/e 212, ( M - C O + C 1 ) , m/e 184, (M+- C 0 2 + C 1 + C O ) ,
m/e 136 ( C 7 H 4 0 C 1 ) and m/e lfl ( c ~ H1. ~ c ~
4.1.4, P r o t o n Magnetic Resonance
'H-NMR spectrum of zomepirac o b t a i n e d from t h e d e u t e -
r a t e d DMSO s o l u t i o n i s shown on Fig. 4. The spectrum was
r e c o r d e d on JEOL FX-loo s p e c t r o m e t e r u s i n g TMS a s i n t e r -
n a l s t a n d a r d . Assignements, c h e m i c a l s h i f t s and r e l a t i v e
i n t e n s i t i e s of s i g n a l s a r e p r e s e n t e d on Table 1.

Table 1. 'H-NMR spectrum of zomepirac -

chemical s h i f t s ,
r e l a t i v e i n t e n s i t i e s and a s s i g n e m e n t s
1 L

0
II 3
Cl C HzCOOA
1 2

_-I.
cH3
chem. s h i f t
i n t ens. multiplicity assignement

1.67 3 H singlet 1
30 67 3 H singlet 2
3.74 2 H singlet 3
5.97 1 H singlet 4
7.59 4 H mult i p l e t 5
12.66 1 H broad 6
 I

I 3

I
'I 1
I
12 10 8 6 4 2 PPm 0
Fig. 4. Proton magnetic resonance spectrum of zomepirac in DMSO-d6. Instrument: Jeol FX-loo.
ZOMEPIRAC SODIUM 685

4.1.5, ,"C-Nuclear Magnetic Resonance

&'C-NMR spectrum of zomepirac shown on Fig. 5 was
obtained from the deuterated DMSO solution using TMS as
internal standard. It was recorded on JEOL FX-loo spectro-
meter at 25.05 MHz. Chemical shifts and coupling constants
are given in Table 2.
The assignements for the two methyl, methylene, car-
boxyl and carbonyl carbons are straightforward because of
their well defined regions of chemical shifts. On the ot-
her hand, resonances due to benzene and pyrrole rings gi-
ve a rather complicated pattern in the appropriate regi-
on. The additivity rules for substituted benzenes /7/ have
Table 2, 13C-NMR c$eEical shifts '
6 and 13C-'H coupling
constants JCH for zomepirac

C-atom 1 2 3
6 (PPd JC H JCH JC H
- 7.9 c-2 -
2' 136.25 s 8.i c-3
3' 112.15 a 171.2
- - 4.3
4' 127.89 3.3 7.1
5' 128.62 s
s
- - -
2 32.11 t 129.4 - -
1 170.78 s - 7.6 -
N-CH 32.80 q 139 8 - -
CH 14.13 q 127. o 2.4 -
C=a 185.03 s
- -
3.7
1.8
-
1'I 134.81 s
21' 130.30 d 164.8 5.5 -
3 128.45 d 168.5 4,o -
4I f 139.34 6 1.0 6.1
a &in ppm downfield from the internal TMS. Off-resonan-
ce multiplicities are given an a second dolumn.
J in HZ.
 m
I
0
-0
hl
m
r cy
u
v
i -0
U
- 0
(D
.o
aD
0
'I!
i..
0
-e
-5
0
-z
i:'
0
0 -2
I1
0
0
.#-I
ZOMEPIRAC SODIUM 687

h e l p e d t o a s s i g n t h e s i g n a l s f o r C - l " , C-2", C-3I1 and C-

-411 a t 134.81, 130.30, 128.45 and 139.34 ppm, r e s p e c t i v e -
l y . The o f f - r e s o n a n c e spectrum, and t h e comparison w i t h
t h e r e l a t e d spectrum of p-chloroacetophenon /8/, c o n f i r -
med t h i s assignement. However, t h e p y r r o l e "C-resonances
were n o t e a s y t o a s s i g n b e c a u s e o f t h e complex s u b s t i t u -
e n t e f f e c t s . I n a g a t e d decoupled spectrum t h e s i g n a l a t
112.33 pfm was a d o u b l e t w i t h a c h a r a c t e r i s t i c f i r s t - o r -
der C- H c o u p l i n g c o n s t a n t a t 171.2 Hz. The o t h e r t h r e e
c a r b o n s were a s s i g n e d from t h e i n t r i c a t e long-range coup-
l i n g e f f e c t s . It should be n o t e d t h a t t h e C-5' r e s o n a n c e
i s , s u r p r i s i n g l y , s i t u a t e d a t h i g h e r f i e l d (128.62 ppm)
t h a n s h o u l d be e x p e c t e d f o r a p y r r o l e r i n g carbon c a r r y -
i n g a c a r b o n y l group /9/.

4.2. S o l i d P r o p e r t i e s
4.2.1. M e l t i n g C h a r a c t e r i s t i c s
Zomepirac can be c o n v e n i e n t l y c r y s t a l l i z e d from i s o -
propanol. The c r g s t g l l i n e p r o d u c t n e e d l e s m e l t s and de-
composes a t 178-179 C .
The sodium s a l t of zomepirac c r y s t a l l i z e s a s t h e d i -
a
h y d r a t e f r m a n i s o p r o p a n o l / w a t e r mixture. The s a l t m e l t s
a t 295-296 C.

4.2.2. X-Ray D i f f r a c t i o n
X-Ray d i f f r a c t i o n p a t t e r n s f o r zomepirac and i t s so-
dium s a l t - d i h y d r a t e a r e - g i v e n i n T a b l e s 3 and 4, r e s p e c -
t i v e l y . D i f f r a c t i o n s p e c t r a were prod ced by monochromatic
r a d i a t i o n from t h e CuK, l i n e (1.542 Y? ) which wa6 o b t a i n e d
by e x c i t a t i o n a t 35 kV and 20 mA. Recording c o n d i t i o n s
were a8 fo11ows. O p t i c s : d e t e c t o r s l i t 0.2 , M.R. s o l l e r
s l i t 3 , beam s l i t O . O O G ? " , I I i f i l t e r 3' t a k e o f f a n g l e .
Goniometer: s c a n a t 2 , 2o/min. D e t e c t o r : a m p l i f i e r g a i n
0

16 c o a r s e , 9.1 f i n e . S c i n t i l l a t i o n c o u n t e r t u b e and DC
v o l t a g e a t p l a t e a u . P u l s e l i g h t s e l e c t i o n EL 9V, En o u t .
Rate m e t e r : T.C. 1.0, 1000 c p s f o r zomepirac and 1.0,
2000 f o r sodium s a l t .

4.3. Solution Properties

3.3.1. Solubilitx
Zomepirac i s f r e e l y s o l u b l e i n methanol, e t h a n o l and
h o t i s o p r o p a n o l . It i s s o l u b l e i n a c e t o n e and c h l o r o f o r m ,
s l i g h t l y s o l u b l e i n e t h e r and p r a c t i c a l l y i n s o l u b l e i n
m e t h y l e n c h l o r i d e , t o l u e n e and hydrocarbon s o l v e n t s .

4.3.2. Acidity pK
pK Value of &mepirac was determined by p o t e n t i o -
m e t r i c ' E i t r a t i o n u s i n g a n a u t o m a t i c b u r e t t e , Model ABU 13,
688 MLADEN ZINIC ET AL.

Table 3. X-Ray d i f f r a c t i o n d a t a of zomepirac

interplanar d i s t a n c e rel. i n t e n f i t y
0" da ( 8 ) I/I,
4.16 10.63 6
5913 8.62 7
5.08 7.40 15
7.30 6.07 50
8.24 5.38 57
10.33 4.30 75
10.63 4.18 loo
11.32 3.93 75
11.60 3.63 49
12.40 3.59 16
13-7 1 3- 50 21
13.08 3.41 19
13.31 3*53 51
13.98 3.19 25
14.32 3.12 9
15.99 2.80 6
16.17 2.77 lo
16.58 2.70 17
17-38 2.58 12
18.42 2.44 14
19.79 2.28 8
21.77 2.08 lo
~~

a
a=nh/2 sin 0
Based on t h e h i g h e s t i n t e n s i t y which i s s e l e c t e d a s u n i t y .
coupled w i t h t h e r e c o r d i n g u n i t T i t r i g r a p h SBR-2C of a T i -
t r a t o r TTT2 ( a l l equipment from Radiometer-Copenhagen).
The g l a s s e l e c t r o d e G-202 c , was used a g a i n s t a calomel K
401 r e f e r e n c e e l e c t r o d e . Zomepirac a c c u r a t e l y weighed and
d i s s o l v e d i n an ethanol/water m i x t u r e 1:1(v / v ) , was t i t r a -
t e d w i t h 0.1 M NaOH s o l u t i o n . P o t e n t i o m e t r i c c u r v e s were
recorded between pH 3.45-12.0 and t h e pKa v a l u e of 4.75
was obtained by c a l c u l a t Y o n a c c o r d i n g t o r e f . /lo/.

5. Methods o f A n a l y s i s
5.1. Elemental A n a l y s i s
P e r c e n t a g e s of e l e m e n t a l c o n t e n t of a zomepirac mo-
l e c u l e and i t s sodium s a l t d i h y d r a t e a r e given i n Table 5.
ZOMEPIRAC SODIUM 689

Table 4. X-Ray d i f f r a c t i o n d a t a of zomepirac sodium s a l t

dihydrate
interplanar distance rel. i n t e g s i t y
0" da ( 8 ) I/Io

6.07 7.29 11
7.00 6.33 32
7.64 5.80 84
8.10 5.48 65
8.34 5-31 28
9-11 4.87 31
9.89 4.49 32
10.50 4.23 71
lo. 92 4.07 loo
11.64 3.82 20
11.85 3-75 30
12.68 3.51 44
13.90 3.21 48
14.23 3.14 30
15.37 2.91 16
15.60 2.87 12
16.45 2.72 19
16.06 2.64 lo
17-31 2.59 20
17 59 2-55 21
20.00 2,15 14
21.41 2.11 13
21.90 2-07 25
24.91 1.83 14
~~

a'b See f o o t n o t e s i n Table 3.

Table 5. Elemental c o m p o s i t i o n of zomepirac and i t s s o d i -

um s a l t d i h y d r a t e

Mol. formula
96 of t h e element ( t h e o r . )
H C1 N Na 0
zomepirac C15H14C1N03 61.79 4.89 12.16 4.80 - 16.41

3.2. Chromatographic Methods

5.2.1. Thin Layer
R - v a l u e s and s o l v e n t s y s t e m s f o r e l u a t i o n of zome-
p i r a c &n Merck F s i l i c a g e i p l a t e s a r e g i v e n i n Table
6. S p o t s a r e 1oc&d under a n UV lamp.
254
690 MLADEN ZINIC ET AL.

T a b l e 6. R v a l u e s and s o l v e n t systems f o r TL chromato-

f
graphy of zomepirac

s o l v e n t system
Rf
Me OH 0.78
C H C1 /conc. HOAc 3:l 0.80
~ t 6I c; f. conc. HOAc 6 : l 0.82
ether/conc. HOAc 1 o : l 0.84
C H C l /conc. HOAc 1 o : l 0.62
cyclahexane/conc. IIOAc 3:l 0.12

5.2.2. Gas
Zomepirac w a s chromatographed on c o i l e d g l a s s column
1 m x 4 mm i . d . f i l l e d w i t h G a s Chrom Q 80-100 mesh i m -
p r e g n a t e d w i t h 3% 0V;jlol. Temperature maintenagce wafi a s
f o l l o w s : oxen a t 200 C , i n j e c t i o n b l o c k a t 3 0 0 C , d e t e c -
t o r a t 300 C. The g a s r a t e s (ml/min) were: c a r r i e r g a s
( N ) and hydrogen 40, a i r 400, The column e f l u e n t was mo-
n i $ o r e d w i t h a f l a m e - i o n i s a t i o n d e t e c t o r . Zomepirac was
i n j e c t e d i n t h e form of i t s methyl e s t e r which was o b t a -
i n e d by t h e f o l l o w i n g p r o c e d u r e : l o mg o f zomepirac was
mixedowith 0.3 m l of 10% m e t h a n o l i c BF s o l u t i o n , h e a t e d
a t 60 C f o r l o min and allowed t o c o o l ? The r e s u l t i n g li-
q u i d was mixed w i t h 3 m l o f hexane, t r a n s f e r e d t o a s e p a -
r a t o r y f u n n e l and washed t h r e e t i m e s w i t h a s a t u r a t e d
aqueous sodium s u l p h a t e . The d r y l i q u i d was used d i r e c t l y
f o r GC. Gas chromatogram of zomepirac methyl e s t e r i s
shown on Fig. 6.
A l t e r n a t i v e l y , zomepirac can b e e s t e r i f i e d b y d i a z o -
methane s o l u t i o n ; i f i t i s a n a l y s e d w i t h o u t e s t e r i f i c a t i -
on d e c a r b o x y l a t i o n t a k e s p l a c e d u r i n g t h e GC a n a l y s i s /ll/.

5.2.3. High Performance L i q u i d

Zomepirac can be a n a l y s e d by HPLC on a column f i l l e d
w i t h S u p e l c o s i l LC-8 ( 5 p m ) . A s a mobile p h a s e t h e mix-
t u r e of a c e t o n i t r i l e , 0.05 M KH PO and phosphoric a c i d
(85%, d = 1 . 7 1 ) , 60, 40 and 2 m l , 2 r e i p e c t i v e l y , was used;
t h e m i x t u r e had pH v a l u e of 3.5. The mobile phase was
f o r c e d t h r o u g h t h e column a t 40-50 b a r ; t h i s p r e s s u r e
m a i n t a i n e d a flow r a t e of 1.7 ml/min. The e f l u e n t was mo-
n i t o r e d a t 323 nm. HPL chromatogram i s shown on Fig. 7.

5.3. T i t r a t i o n
T h i r t y mg of zomepirac was weighed (20.1 mg) i n t o a
t i t r a t i o n v e s s e l an(? d i s s o l v e d i n a m i x t u r e of 1.0 p.1 o f
e t h a n o l and l o m l of water. The r e s u l t i n g s o l u t i o n was
ZOMEPIRAC SODIUM 69 1

1 ' 1 1 1 . 8 1

0 2 4 6 8 10 min.
Fig. 6. Gas chromatogram of zomepirac methyl ester (peak
21 and internal standard methyl heptanoate,(pe-
ak 1). Instrument: Pye-Unicam 204 chromatograph.
692 MLADEN ZINIC ET AL.

F i g . 7. HPL chromatogram of
zomepirac.
I n s t r u m e n t : Pye-
-Unicam LC-3-XP.

J N

t l l l l
6 4 2 0 min.
ZOMEPIRAC SODIUM 693

t i t r a t e d p o t e n t i o m e t r i c a l l g u s i n g t h e g l a s s and calomel
e l e c t r o d e p a i r . The c o n t e n t of zomepirac was c a l c u l a t e d
from t h e q u a n t i t y o f consumed 0.1 M NaOH s o l u t i o n u s i n g
t h e e q u a t i o n (1):

c o n t e n t of zomepirac % = V x Nf x E x l o o ( 1)
where V = volume of 0.1 M N a O H s o l u t i o n consumed ( m l )
f = n o r m a l i t y f a c t o r of 0.1 M NaOH s o l u t i o n
E = 29.16 -
e q u i v a l e n t of zomepirac f o r 1 m l of
0.1 M NaOH s o l u t i o n
W = mass of t h e sample (mg)

Using t h e above p r o c e d u r e , t h e s o l u t i o n of 50 mg of
zomepirac sodium s a l t d i h y d r a t e i n 20 m l of w a t e r was t i -
t r a t e d w i t h 0.1 M h y d r o c h l o r i c a c i d s o l u t i o n . The c o n t e n t
of t h e zomepirac sodium s a l t d i h y d r a t e was c a l c u l a t e d
u s i n g t h e e q u a t i o n (1) where V = volume of 0.1 M hydro-
c h l o r i c a c i d consumed ( m l ) , f = n o r m a l i t y f a c t o r of 0.1
M h y d r o c h l o r i c a c i d s o l u t i o n and E = 33.96, e q u i v a l e n t
of zomepirac sodium s a l t d i h y d r a t e f o r 1 m l of 0.1 M
h y d r o c h l o r i c acid s o l u t i o n .

6. S t a b i l i t y and D e g r a d a t i o n
Zomepirac and i t s sodium s a l t d i h y d r a t e , powdered,
and t a b l e t s c o n t a i n i n g sodium s a l t d i h y d r a t e were k e p t
a t 40-50 Sh r e l s t i v e humidity, a t t e m p e r a t u r e s w i t h i n t h e
r a n g e of 45-50 C f o r seven days. The samples were a n a l y -
s e d by HPLC. T a b l e t s and powdered s a l t d i h y d m t e d i d n o t
show any change b u t 7.6% o f n o n i d e n t i f i e d .i.mpurity appe-
a r e d i n t h e powder of zomepirac.

7. Drug Metabolism, F h a r m a c o k i n e t i c s , B i o a v a i l a b i l i t y
Because of i t s l i p o p h i l i c i t y , zomepirac may owe a t
l e a s t p a r t of i t s a n a l g e s i c a c t i v i t y t o an i n c r e a s e d a b i -
l i t y t o p e n e t r a t e i n t o t h e s p i n a l c o r d and b r a i n and sub-
s e q u e n t l y t o t h e i n h i b i t i o n of p r o s t a g l a n d i n s y n t h e s i s
w i t h i n t h e c e n t r a l n e r v o u s system /l.2/.
On t h e b a s i s of u r i n a r y r e c o v e r y d a t a , zomepirac i s
a l m o s t absorbed a f t e r o r a l a d m i n i s t r a t i o n of d o s a g e s of
25-200 mg t o h e a l t h y s u b j e c t s . Mean peak plasma concen-
t r a t i o n s of 2.47, 4.42 and 7.94,&g/ml were a t t a i n e d 44,
57 and 80 min a f t e r a s i n g l e o r a l dose of 50, l o o o r 200
mg, r e s p e c t i v e l y . B i o a v a i l a b i l i t y of zomepirac i s t h e sa-
me a f t e r i n g e s t i o n of t a b l e t s , c a p s u l e s o r a n aqueous so-
l u t i o n . Zomepirac i s e x t e n s i v e l y bound t o plasma albumin
(98.5%) i n man. Maximal plasma c o n c e n t r a t i o n s ( 4000 ng/ml)
a r e r e a c h e d i n 1 hour a t d o s e of l o o mg.
T i s s u e c o n c e n t r a t i o n s o f r a d i o l a b e l l e d zomepirac i n
0
I
0
Q
d
0
d
0
t /
I
0
Q 0
E"
0
%
I
N
m
-r
d
u
ZOMEPIHAC SODIUM 695

r a t s a r e i n t h e stomach, k i d n e y s , i n t e s t i n e , and l i v e r ,
2.9, 2.2, 1.7, and 0.5 t i m e s , r e s p e c t i v e l y , plasma con-
c e n t r a t i o n 6 h o u r s a f t e r o r a l a d m i n i s t r a t i o n of a 6 mg/
/kg dose. The r a t plasma c o n c e n t r a t i o n s of zomepirac and
m e t a b o l i t e s a r e about 5 t i m e s t h e t i s s u e c o n c e n t r a t i o n s
of h i g h l y p e r f u s e d o r g a n s such a s t h e h e a r t and l u n g s ,
and about 50 t i m e s t h a t o f t h e b r a i n . A f t e r 48 h o u r s
a b o u t 0.3% of t h e dose r e m a i n s i n t h e c a r c a s s of t h e r a t ,
0.1% of t h e d o s e b e i n g c o n c e n t r a t e d i n t h e l i v e r /13/.
Zomepirac i s d e t e c t e d i n t h e c e r e b r o s p i n a l f l u i d o f c a t s
l o m i n u t e s a f t e r i n t r a v e n o u s a d m i n i s t r a t i o n of a 3 mg/kg
dose. C e r e b r o s p i n a l c o n c e n t r a t i o n s were a p p r o x i m a t e l y 7%
of plasma c o n c e n t r a t i o n s 24 h o u r s a f t e r d o s i n g /14/.
The e l i m i n a t i o n h a l f - l i f e of zomepirac i s a b o u t 4
h o u r s f o l l o w i n g a s i n g l e dose, b u t may be i n c r e a s e d f o l -
lowing m u l t i p l e d o s e s . Plasma c l e a r a n c e a f t e r d o s e s of
40 mg/kg i n r h e s u s monkeys i s d e p r e s s e d t o l e s s t h a n one
h a l f t h a t observed a f t e r d o s e s of 5 and l o mg/kg. A u t h o r s
consider t h a t the reason f o r t h i s nonlinear k i n e t i c s is
s a t u r a t i o n o f m e t a b o l i c c o n j u g a t i o n /15/.
Zomepifac i s e x c r e t e d a l m o s t e x c l u s i v e l y i n t h e u r i -
n e , t h e major m e t a b o l i t e b e i n g t h e g l u c u r o n i d e c o n j u g a t e ,
which a c c o u n t s f o r a b o u t 57% of r a d i o a c t i v i t y a f t e r a 25
mg dose. Hydroxyzomepirac and 4-chlorobenzoic a c i d a r e
minor m e t a b o l i t e s /Fig. 8/ and t h e y a r e a p p r o x i m a t e l y
200-300 t i m e s l e s s a c t i v e t h a n zomepirac a s i n h i b i t o r s
of human p l a t e l e t a g g r e g a t i o n i n v i t r o . About 22% of t h e
d o s e i s e x c r e t e d i n u r i n e unchanged.

8. I d e n t i f i c a t i o n and D e t e r m i n a t i o n i n Body F l u i d s and

Tissues
The d e t e r m i n a t i o n of zomepirac i n t h e b l o o d , plasma
and u r i n e i s p o s s i b l e by HPL and g a s chromatographic me-
t h o d s /11,16,17/. HPL chromatographic d e t e r m i n a t i o n of
zomepirac i n t h e human plasma a l l o w s i t s d e t e r m i n a t i o n
a t c o n c e n t r a t i o n s down t o l o ng/ml f o r 2 m l plasma samp-
l e s and 5 0 ng/ml f o r 1 m l plasma samples /16,17/. Schutz
and Suphachearabhan /11/ r e p o r t e d t h a t UV photometry se-
emed t o b e t h e b e s t method f o r t h e u r i n e s c r e e n i n g . The
d e t e r m i n a t i o n of zomepirac and i t s m e t a b o l i t e s by R con-
t i n o u s body f l u i d m o n i t o r i n g system based on HPLC was
d e s c r i b e d by Muller and Z u l l i g e r /la/. T h e i r method a l -
lows a d e t e r m i n a t i o n of zomepirac down t o 50 ng/ml i n t h e
50 s / m l plasma samples; i n t h e c o n c e n t r a t i o n r a n g e o f
0.5-100 P g / m l o f t h e plasma a f u l l l i n e a r i t y was a c h i e -
ved w i t h a r e c o v e r y of 97% f o r t h e plasma samples a n d
95% f o r t h e u r i n e samples.
I n t h e r e f e r e n c e s /11/ and /19/ r e v e r s e d phase HPLC
696 MLADEN ZINIC ET AL.

was used under t h e f o l l o w i n g o p e r a t i n g c o n d i t i o n s :

Column: S p h e r i s o r b O D s , 5,&m (125 m m x 4.9 mm),
Kontron AG
Mobile p h a s e : a c e t o n i t r i l e , water, o r t h o p h o s p h o r i c
a c i d 50 : 49.5 : 0.5 ml, r e s p e c t i v e l y .
Flow r a t e : 2 ml/min.
D e t e c t o r : UV, 313 nm.
Sensitivity: variable
P. more s e n s i t i v e and p r e c i s e method f o r t h e d e t e r m i n a t i o n
of zomepirac i n t h e human plasma i s GC /20/. I n t h i s me-
thod, zomepirac m u s t be t r a n s f o r m e d i n i t s p e n t a f l u o r o -
b e n z y l e s t e r p r i o r t o GC a n a l y s i s . The u s e o f a n e l e c t r o n
c a p t u r e d e t e c t o r ( E C D ) a l l o w s t h e d e t e r m i n a t i o n of zome-
p i r a c p e n t a f l u o r o b e n z y l e s t e r even i n t h e Dicogram range.
The l o w e s t a c c u r a t e c o n c e n t r a t i o n i s 5 ng/ml f o r 2 m l p l a -
sma samples and 25 ng/ml f o r 0.1 m l plasma samples. Column
f i l l i n g and o p e r a t i n g maintenance were a s f o l l o w s :
Column: 122 cm x o.It cm i . d . , s i l i l a t e d g l a s s f i l l e d
w i t h a Gas Chrom 8, (60-83 mesh) impregnated
w i t h 3% OV-17. Temp. 230 C.
C a r r i e r gas: a r g o n w i t h 546 o f mgthane.
D e t e c t o r : ECDb 63 N i . Temp. 295 C.
I n j e c t o r : 200 C.
A combination of GC and MS a l l o w s i d e n t i f i c a t i o n and de-
t e r m i n a t i o n o f zomepirac and i t s m e t a b o l i t e s i n body f l u -
i d s /11,20/.

9. D e t e r m i n a t i o n i n P h a r m a c e u t i c a l s
P r e p a r i n g a n a n l y t i c a l s o l u t i o n : t e n t a b l e t s , each
c o n t a i n i n g 119.89 mg of zomepirac sodium s a l t d i h y d r a t e ,
a r e powdered i n a m o r t a r and a mass of powder e q u i v a l e n t
t o 150 mg of zomepirac i s weighed i n t o a l o o m l v o l u m e t r i c
f l a s k . F i f t y m l of w a t e r a r e added, t h e c o n t e n t shaken
f o r 30 min, and t h e volume made up t o mark. An a l i q u o t of
15 m l i s c e n t r i f u g a t e d ( 3 0 0 0 g, l o m i n ) , and t h e s u p e r n a -
t a n t taken f o r a n a l y s i s ( s o l u t i o n A ) .
Standard s o l u t i o n : i n t o a l o m l v o l u m e t r i c f l a s k 18.0
mg of zomepirac sodium s a l t d i h y d r a t e a r e weighed i n , d i s -
s o l v e d i n w a t e r and t h e volume made up.
Work up and measurement: 2 m l of c l e a r s o l u t i o n A a r e
p i p e t t e d i n t o a l o m l t e s t tube. To each t u b e a r e added:
1 m l of 0.1 N H C 1 s o l u t i o n and 5 r n l of e t h e r c o n t a i n i n g
0.5 mg/ml of methyl h e p t a n o a t e a s a n i n t e r n a l s t a n d a r d .
Both t u b e s a r e s t o p p e r e d , shaken f o r 5 min. and l e f t t o
s t a n d f o r a n o t h e r 5 min. T h e r e a f t e r 2 m l of t h e e t h e r e a l
l a y e r s were t r a n s f e r e d i n t o two c l e a n t e s t - t u b e s and t h e
e t h e r was c o m p l e t e l y e v a p o r a t e d i n a s t r e a m o f a i r . To
e a c h r e s i d u e were added 2 m l of 10% m e t h a n o l i c boron tri-
ZOMEPIRAC SODIUM 697

f l u o r i d e s o l u t i o n . Both t u b e s were t h e n p l a c e d i n block-

- t h e r m o s t a t , k e p t a t b o i l i n g t e m p e r a t u r e f o r 5 min, t h e n
l e f t t o c o o l down t o room temperature. A f t e r a d d i t i o n of
8 m l of water and 1 m l of hexane t h e methyl e s t e r of zo-
mepirac i s e x t r a c t e d i n t o t h e hydrocarbon l a y e r and t h e
e x t r a c t i n j e c t e d i n t o a g a s chromatograph. D e t e r m i n a t i o n
of t h e d e t e c t o r r e l a t i v e weight r e s p o n s e (RWR) : o n e p of
t h e hexane e x t r a c t from t h e s t a n d a r d was i n j e c t e d i n t o
t h e chromatograph. The RlrlR v a l u e was c a l c u l a t e d a c c o r d i n g
t o equation ( 2 ) :
RWR =
*Z cI.s.
(2)
AI.s. cz
where A z = peak a r e a of zomepirac methyl e s t e r , A =
= peak a r e a of i n t e r n a l s t a n d a r d methyl h e p t a n o a h : Cz=
= c o n c e n t r a t i o n 0 % zomepirac methyl e s t e r i n m g / m l , cI. s. - -
= c o n c e n t r a t i o n of methyl h e p t a n o a t e i n mg/ml
Content of zomepirac i n one t a b l e t : t h e hexane e x t r a c t
of t h e sample s o l u t i o n was i n j e c t e d i n t o t h e g a s chromato-
graph and t h e d e s i r e d c o n t e n t was c a l c u l a t e d u s i n g equa-
tion (3): X 50 X Wa
mg of zomepirac p e r t a b l e t =
AZ cI.s. (3)
AI.s.
x RWR x W
where AZ, A and C I have t h e same meaning as i n eq.
2 , WA is &ig'average*m&ss of a t a b l e t a n d W = mass of
t h e powdered sample /21/.

Acknowledgement The a u t h o r s a r e t h a n k f u l t o d r A. Nagl,

L a b o r a t o r y of General and I n o r g a n i c Chemistry, F a c u l t y of
S c i e n c e , U n i v e r s i t y of Zagreb, f o r X-ray d i f f r R c t i o n data.

lo. References
1. J.R. Carson and S. Wong, J. Med. Chem. 16, 172 /1973/.
2. J.R. Carson, U.S. Pat. 3,752,826 /1973/I-
3 . G.P. S t a h l y , E.M. Marlett and G.E. Nelson, J. Org.
Chem. 48, 4423 /1973/.
4. M. i i n i 6 , J. K u f t i n e c , H. Hofman, F. K a j f e i and Z.
Mei6, J. Org. Chem.. 2,697 /1985/.
5. T.H. Sun, F.S. Abbott, R. Burton and J. O r r , J. Label-
l e d , Compd. Radiopharm. 2,1043 /1982/.
6. D.C. Hoerr, A.T. S c h a r f , J.R. Carson and L.E. Weaner,
J. L a b e l l e d Compd. Radiopharm. 20, 1383 /1983/.
7. E. P r e t s c h , T. C l e r c , J. S e i b l and W. Simon, "Tabellen
z u r S t r u k t u r a u f k l a r u n g o r g a n i s c h e r Verbindungen m i t
s p e k t r o s k o p i s c h e n Methoden" Springer-Verlag, B e r l i n
1976, p C120.
8. K.S. Dhami and J.B. S t o t h e r s , Can. J. Chem. 9, 479
698 MLADEN AINIC ET AL.

/1965/.
9. E B r e i t m a i e r and V o e l t e r , tr13C NMR Spectroscopy",
,
Zhd ed. V e r l a g Chemie, Weinheirn 1978, pa 199.
lo, C. Tenford and S. Wawzanek i n l f P h y s i c a l Methods of
Organic Chemistry", A. Weissberger e d i t o r , I n t e r s c .
Publ. Co., v o l . I, Bd. 4, p. 2915.
11. H, Schutz and Suphachearabhan, Arzneim.-Forsch. Drug
Ri&. 2, 293 /1984/.
12. P.A. Morley, R.N. Brogden, A.A. Carmine, R.C. Heel,
T.M. Speght and G.S. Avery, 2,250 /1982/.
13. J.M. G r i n d e l , P.J. O ' N e i l l , K.A. Yorsey, M.H.
Schwartz, L.A. Mckown, B.H. Migdalof and W.N. Wu, Drug
Metab. and Disp. 8, 343 /1980/.
14. S. Divinetz-Romero, T.L. Yake, L,D. Muschek and P.J.
O ' N e i l l , Ann. Meet. SOC. Neurosci. L.A. USA, Abs. -9286
1s /1981/.
15. P.J. O ' N e i l l , J. Pharm. S c i . 70, 818 /1981/.
16. K.T. Ng and T. Snyderman, J. Chromatog. 178, 241
/1979/*
17. C.L. Welch, T.M. Annesley, H.S. L u t h r a and T.P. Moyer,
C l i n . Chem. 28, 481 /1982/.
18. H, Miiller and H.W. Z u l l i g e r , Arzneim.-Forsch. ( Drug
Res.) z, 152 /i985/.
19. P.J. O ' N e i l l , K.A. Yorsey and L.A. Mckown, J. Pharma-
c o l . Exp. Ther, 219, 665 /1981/.
20. K.T. Ng and J.J. Kalbron, J. Chromatog.
/1983/.
*, 311

21. M. i i n i 6 , J. K u f t i n e c , H. Rofman, B. B e l i n , F. KajfeZ,

z,
11982/ .
N. B l a i e v i 6 and 2. Meit, Acta Pharm. Jugosl. 281
PROFILE SUPPLEMENTS
This Page Intentionally Left Blank
 CHLORAMPHENICOL

AbduReah A. M-Badh and Hurneida A. El-Obeid

1. D e s c r i p t i o n

1.1 Nomenclature

1.2 Formulae

1 . 3 Molecular Weight

1 . 4 Elemental Composition
1.5 Appearance, Color, Odor and Taste

2. Physical Properties

2.1 Melting P o i n t
2.2 Solubility

2.3 Spectral Properties

3. Synthesis

4. Methods of A n a l y s i s

4.1 I d e n t i f i c a t i o n Tests

4.2 Q u a n t i t a t i v e Analysis

ANALYTICAL PROFILES OF DRUG SUBSTANCES Copyright Q 1986

VOLUME 15 by the American Pharmaceutical Association
701 All rights of reproduction in any form reserved.
702 ABDULLAH A. AL-BADR AND HUMEIDA A. EL-OBEID

5. Pharmacokinetics

5.1 Absorption and Distribution

5.2 Excretion

5.3 Half-life

5.4 Metabolism
Acknowledgement

References
CHLORAMPHENICOL 703

1. Description

1.1 Nomenclature

1.1.1 Chemical Names

D(-)-threo-2-Dichloroacetamido-l-p-nitro-
phenyl-1,3-propanediol.
D( - ) threo-N-Dichloroacetyl-1-p-nit rophenyl-
2-amino-1,3-propanediol.
D(-) threo-2,2-Dichloro-N-[B-hydroxy-a-
(hydroxymethyl)-p-nitrophenethyl]acetamide.
D( - ) threo-2,2-Dichloro-N- [ 2-hydroxy-l-
( hydroxymethyl) -2-( 4-nitrophenyl)ethyl] aceta-
mide.

1.1.2 Generic Names

Chloramphenicol, Chloramfenicol, Chlorampheni-

c o l m , Chloramphenicolo, Laevomycetin.

1.1.3 Trade Names (1)

Alficetyn, Ambofen, Amphicol, Anacetin,

Aquamycetin, Bemacol, Berlicetin, Biocetin,
Biophenicol, Cafenolo, Cebenicol, Chemicetina,
Chemyzin, Chlomin, Chloramex, Chloramol,
Chloramphenicol-POSY Chlorasol, Chlora-tabs,
Chloricol, Chlornitromycin, Chlorocid,
Chloromycetin, Chloronitrin, Chloroptic,
Chloro-25 Vetag, Chlorsig, Cloramidina,
Clorbiotina, Clorof'enicina, Cloromicetin,
Cloromycetin, Clorosintex, Comycetin,
Cylphenicol, Desphen, Detreomycine, Devamyce-
tin, Dextromycetin, Doctamicina, Duphenicol,
Econochlor , Erbaplast , Ert ilen, Farmicetina,
Fenicol, Globenicol, Glorous, Halomycetin,
Hortfenicol , Isicetin, Ismicetina, Isopheni-
col, Isopto Fenicol, Kamaver, Kemicetin,
Kemicetine, Kloromisin, Labamicol, Leukomycin,
Levomycetin, Lomecitina, Loromisin, Mamma-
phenicol, Pledichol, Micochlorine, Misetin,
Mycetin, Mychel, Mycinol, Neocetin, Novochlo-
rocap, Nova-Phenicol, Novophenicol, Oftaklo-
ram, Oftalent , Oleomycetin, Opclor, Ophta-
phenicol, Ophtochlor, Oralmisetin, Otachron,
Otomycin, Otophen, Pantovernil, Paraxin,
 CH,- OH
I YH2 OC 6 H9 7 CH OH ,
H,N@ VH-CH-NHCOCHCl, O,N-@ FH-CH-NHCOCHC1 o,N@ FH-;H-NH-CCOOH
11
OH OH
I OH 0
FH2OH
dog. O,N@ FH-CH-NHCOCH2 OH
OH
CH, OH
CHO O,N@ FH-CH-NH,
I

OH
yH2 OC 6 H9 7
0
0
O2N
-0- FH-CH-NHCOCHC12
OH
II
FH2OH
H-C-CH,NHCOCHC~,--€ yH2OH
H,N @-F H - c H - ~ ~ o c H c ~ ,
, O,N -@ YH-CH-NHCOCHC1, 0
OH
yH2OH
f--+ OH-CH,CH,-NHCOCHCl, OH
CH,?HN -
@
:
C
H
-
NO
C
H
~
,
yH20H R a t hepato- - .*

yH2 OC 6 H9 7
02N@ ~H-CHNHCOCH2C1 0,N -@:H-CH-NIICOCHCl
OH OH
CH, OH
0,N -@FH-CH-NH, I

OH
Scheme 4. Metabolites o f chloramphenicol i n i n vivo and i n v i t r o s t u d i e s .
CHLORAMPHENICOL 705

1.2.3 CAS R e g i s t r y No.

[ 56-75-7 1
1.2.4 Wiswesser Line Notation

WNR DYQYIQ MVYGG ( 2 ) .

1.3 Molecular Weight

323.13, 322.01 ( 2 )

1.4 Elemental Composition

C 40.88%, H 3.74%, C 1 2-1.95%, N 8.67%, 0 24.76%.

1 . 5 Appearance, Color, Odor and T a s t e
Fine white t o greyish white o r yellowish white
c r y s t a l s , n e e d l e s o r e l o n g a t e d p l a t e s from water o r
e t h y l e n e d i c h l o r i d e w i t h very b i t t e r t a s t e .

2. Physical Properties

2.1 Melting P o i n t

2.2 Solubility
S o l u b l e (25') i n water : 2.5 mg/ml , i n propylene
g l y c o l : 150.8 mg/ml, v e r y s o l u b l e i n methanol,
ethanol, butanol, e t h y l a c e t a t e , acetone. F a i r l y
s o l u b l e i n e t h e r , i n s o l u b l e i n benzene, petroleum
e t h e r , v e g e t a b l e o i l s . S o l u b i l i t y i n 50% acetamide
s o l u t i o n i s 5%. Aqueous s o l u t i o n s are n e u t r a l .
N e u t r a l and a c i d s o l u t i o n s are s t a b l e on h e a t i n g .

2.3 Spectral Properties

2.3.1 U l t r a v i o l e t Spectrum

The u l t r a v i o l e t a b s o r p t i o n spectrum o f
chloramphenicol i n n e u t r a l methanol w a s
obtained on a Cary 219 spectrophotometer. The
spectrum, shown i n F i g . 1, i s c h a r a c t e r i z e d
by a maximum a t 274 nm and a minimum a t
235 nm. The spectrum o f chloramphenicol i n
706 ABDULLAH A. AL-BADR AND HUMEIDA A. EL-OBEID

w
u
z
4
m
L
0
In
m
4

1- * - - . - - * . - ~'
220 230 240 250 260 270280290 300 30 320330340
WAVELENGTH

Figure 1. Ultraviolet epectnuP of chlormphenicol

in neutral amthmol.
CHLORAMPHENICOL 707

water showed a maximum a t 278 nm (E 1%,

1 cm 2 9 8 ) ; i n 0.1N NaOH, a maximum a t 276 nm
(E l%, 1 cm 2 0 0 ) ; i n 0.1N H SO4, a maximum
at 278 nm ( E 1%, 1 cm 284) 73).

2.3.2 I n f r a r e d Spectrum

The i n f r a r e d a b s o r p t i o n spectrum o f chloram-

p h e n i c o l o b t a i n e d from a potassium bromide
d i s p e r s i o n i s shown i n F i g u r e 2. The spec-
trum w a s recorded on a Pye Unicam SP 1025
i n f r a r e d spectrophotometer. The c h a r a c t e r i -
s t i c bands o f t h e spectrum w i t h t h e a s s i g n -
ments are l i s t e d below:

Frequency ( cm-l) Assignment

3230, 3520 Broad H-bonded OH and NH

stretch

3100 Aromatic C-H s t r e t c h

1700, 1570 C = 0 s t r e t c h amide 1

band, amide I1 band.

1530, 1360 N 0 s t r e t c h (ArN02)

1070 C - 0 s t r e t c h (primary
alcohol)

850 C -N s t r e t c h (ArN02)

The p r i n c i p a l peaks as r e p o r t e d by Clarke

( 3 ) are 1682, 1061 and 1351 or 1526 cm'l
I
2.3.3 H-Nuclear Magnetic Resonance Spectroscopy

H
' NMR s p e c t r a o f chloramphenicol i n DMSO-d6
( F i g u r e 3 ) and i n DFlso-dg i n t h e p r e s e n c e of
D20 ( F i g u r e 4) are o b t a i n e d on a Varian-T6OA
NMR s p e c t r o m e t e r . The band assignments are
r e f e r e n c e d t o TMS and are l i s t e d below:
 f
WAVEL NGTH Urn4
5 6 7 8 9 10 11 12 U 14151 i
100
90
80

$'
70
60
50
40
30
20
10
01 - 0
3500 3WO 2500 2000 1800 1600 1400 1200 lo00 800 62
WAVENUMBER
Figure 1. Infrared e p e c t w of chloramphenicol from IEBr dime.
CHLORAMPHENICOL 709

500 100 0

TM!

,......... 1. . . . . . . , . . . . I ........... . . . a . . . . *
8.0 7.0 6.0 5.0 co 30 2.0 1.0 0
PPM ( 6 )
Ii#uro 3. 'H-NMR apectrum of chloramphenicol i n DWO-de.

500 400 300 100 0

patcr

TM!

I
, . . - . . " . . ~ . . . . . . ' . . . . ' . . . ' ' . . . . ' . . . . . . .
8.0 7.0 ' 6.0 50 GO 3.0 2.0 1.0 0
PfW ( 8 )
Figure 4. lH NHU spectrum of chloramphenicol in WSO-d, and D1O.
710 ABDULLAH A. AL-BADR AND HUMEIDA A. EL-OBEID

Chemical s h i f t * Proton No. of

Multiplicity
(6) Assignment Protons

a. 3.43
M u l tiplet -CH20H 2
b. 3.47
a. 3.97
M u l tiplet Cg-N- 1
b. 3.98
a. 4.93 Triplet -CH2-Og 1

b. Exchanged - - -
a. 5.12 )
) Triplet Cg-OH 1
b. 5.08 )
a. 5.97 Doublet CH-OK 1

b. Exchanged - - -
a. 6.47 )
Singlet -cocgc1 1
b. 6.40 )
a.

b.

a.
7.60 )

7.58

8.15 )
1
)
1
Doublet
OpN q 2

1
Doublet 2
)
b. 8.13 )
-H
a. 8.25 Doublet -NH 1

b. Exchanged - -
~

* a, i n DMSO-d6 ; b , i n DMSO-d6 + D20.

CHLORAMPHENICOL 711

2.3.4 1 3 C Nuclear Magnetic Resonance ( 1 3 C NMR)

The 1 3 C NMR s p e c t r a of chloramphenicol a r e

obtained i n DMS-d6, containing a drop of
CDC13, a t ambient temperature with 'H-
decoupling (Figure 5 ) and off-resonance
(Figure 6 ) . The s p e c t r a are recorded using
TMS as i n t e r n a l standard on a JEOL FXlOO MHz
instrument. The chemical s h i f t s , m u l t i p l i -
c i t i e s and s p e c t r a l assignments a r e given
below:

Chemical s h i f t Carbon
Multiplicity assi nment*
(6)
56.71 Doublet
c2
60.18 Triplet
c3
66.35 Doublet

68.93 Doublet

122.76 Doublet
c21
127.17 Doublet
c31
146.31 Singlet

151.12 Singlet
c4'
163.33 Singlet

*Refer t o s t r u c t u r e i n Figure 5 f o r carbon

numbering.

2.3.5 Mass Spectrum

The combined gas-chromatographic/mass-

spectrophotometric technique w a s used f o r t h e
i d e n t i f i c a t i o n and a n a l y s i s o f chlorampheni-
c o l i n aqueous s o l u t i o n s ( 4 ) and f o r chloram-
phenicol and i t s metabolites i n animal t i s s u e s
and body f l u i d s ( 5 , 6 ) . Becker et a1 ( 7 )
and Krueger ( 8 ) s t u d i e d t h e s p e c t r a of non-
v o l a t i l e substances by f i s s i o n fragment
712 ABDULLAH A. AL-BADR AND HUMEIDA A. EL-OBEID

NH c"0 6H CI,
4N

Figure 5. Proton decoupled 13C lsyR rpwtrwn of chlorunphoniool in

DYBO-dg + a drop of CDClg.

DMSO

Figure 6. Off-resonance 13C NMR spectrum of chlorsmphenicol In DYBO-dg +

a drop of CDC13.
CHLORAMPHENICOL 713

d e s o r p t i o n mass spectrometry. In t h e
s p e c t r a o f chloramphenicol b o t h quasimole-
c u l a r i o n s are p r e s e n t e d w i t h t h e i r t y p i c a l
two-chlorine i s o t o p i c p a t t e r n . The p o s i t i v e
i o n s p e c t r a show v a r i o u s d i f f e r e n t subgroups.
Although t h e r e i s v e r y l i t t l e f r a g m e n t a t i o n
i n t h e n e g a t i v e i o n spectrum, a s t r o n g peak
group arises n e a r m/e 152.

E l spectrum o f chloramphenicol w a s shown (2)

t o p o s s e s s t h e f o l l o w i n g peaks: 153(100%),
60(99%), 70(85%) 15508%), 170(70%),
106(46%), 77(40%).
P r e s e n t e d i n F i g u r e 7 i s t h e 70 e V e l e c t r o n
impact ( E I ) mass spectrum o f chloramphenicol
o b t a i n e d on Varian MAT 311 mass s p e c t r o m e t e r
u s i n g i o n s o u r c e p r e s s u r e o f 10-6 T o r r , i o n
s o u r c e t e m p e r a t u r e of 18OoC and a n emission
c u r r e n t o f 300 PA. No molecular i o n i s
d e t e c t e d and t h e spectrum i s dominated by
m / e 153 i o n (base peak) r e s u l t i n g from t h e
l o s s o f 02NC6H4CHO and H20. A proposed
mechanism o f f r a g m e n t a t i o n and t h e mass/
charge r a t i o s o f t h e major fragments i s
given i n Scheme 1. The chemical i o n i z a t i o n
(CI) spectrum ( F i g u r e 8) w i t h methane g a s as
a r e a g e n t i s o b t a i n e d on a Finnigan 4000
mass s p e c t r o m e t e r w i t h i o n e l e c t r o n energy
o f 100 eV, i o n s o u r c e p r e s s u r e o f 0.3 T o r r ,
i o n s o u r c e t e m p e r a t u r e o f 150°C and emission
c u r r e n t of 300 PA. The spectrum shows no
p a r e n t molecular i o n b u t a pronounced peak
r e s u l t i n g from t h e l o s s o f water (MH+-18)
c o n s t i t u t e t h e b a s e peak a t m / e = 305. A
quasi-molecular i o n ( M + 1) i s a l s o prominent.
Two peaks a p p e a r i n g at m / e = 351 and m / e 363
are a t t r i b u t e d t o t h e t r a n s f e r o f c a r b o c a t -
i o n s from t h e c a r r i e r g a s . The mass s p e c t r a l
assignment o f t h e prominent i o n s under CI
c o n d i t i o n s i s g i v e n i n Table I .
lW.0-

a.0-
€0
-
70 170 .

Sl

1W.O- - 28416.

a.0- -

200 220 2 40 2w 280 300 320

Figure 7. Yaee spectrum of chlorunphenicol (EI).

SO
I

MIL 2M 200 $00 PO YO 960 300 LOQ 420 440

Figure 8 . Haem epectrum of chloramphenicol (CI).

02N@
CH20H
YH-CH
OH
$5
I - NH-C-CHClp --* [02N@
CH2 OH
I
FH-CH - NH-C E
OH
3 +-+ 02N-@!€$H
, 0 -HCH20H +I
-NHC=g

m/e 322 (0%) m/e 239 m/e 239

NHCOCHC12
I
1 J
m/e 152 m/e 77

CH,= CHNHCOCH = C1+

m/e 118
CH, = CH-NH-C + +

1
0 CH = C1
1
c1
+
CH, = CH-NH-C = O m/e 83

m/e 70
Scheme 1. Mechanism of chloramphenicol fragmentation.
 Scheme I (continued)
CH2 OH
1
~

+. 0 2 . 9 -2P+
CH-OH
+
HOCH,CH = NH-CO-CC1,
02N y-&-NH-COCHClp d U
OH +

0,N

m / e 152
CH = OH
+

HOCH2CH =
+
NH2
i + O=C=C
/

\
c1

c1
m / e 60

m / e 106

+ r

L OH
m / e 291
J
CHLORAMPHENICOL 717

Table 1. Mass S p e c t r a l Assignments of

Chloramphenicol Using C I with
Methane as Reagent Gas.

m/e Species

363 [M + C3H51+
351
+
[M + C2H51
32 3 MH+

305 [MH - H20]+

287 [MH - HCl]'

27 5 [MH - (H20 + CH2 = O)]'

3. Synthesis

a. The a n t i b i o t i c w a s i n i t i a l l y i s o l a t e d from c u l t u r e s
o f v a r i o u s Streptomyces s t r a i n s . The s t r u c t u r e
s i m p l i c i t y of chloramphenicol made it amenable t o
p r e p a r a t i o n by t o t a l s y n t h e s i s both i n t h e l a b o r a t o r y
and on commercial s c a l e . One method o f s y n t h e s i s
involves a base-catalysed condensation of benzalde-
hyde with n i t r o e t h a n o l t o a f f o r d t h e a l d o l product
as a mixture of stereoisomers (Scheme 2 ) . C a t a l y t i c
r e d u c t i o n g i v e s an aminodiol whose threo-isomer i s
s e p a r a t e d and resolved i n t o t h e o p t i c a l isomers. The
( - ) isomer i s t r e a t e d with d i c h l o r o a c e t y l c h l o r i d e
followed by treatment w i t h base t o remove t h e 0-
a c y l a t e d products t o a f f o r d t h e amide. The hydroxyl
groups are t h e n p r o t e c t e d by means of a c e t i c anhyd-
r i d e . The product i s n i t r a t e d t o produce t h e p-
n i t r o d e r i v a t i v e . Removal of t h e p r o t e c t i n g groups i s
achieved by treatment with a base t o g i v e chloram-
phenicol ( 9 ) .

b. 'H-Chloramphenicol and i t s erythro-diastereomer with

high s p e c i f i c a c t i v i t i e s were prepared by o x i d a t i o n
of t h e nonlabeled a n t i b i o t i c t o i t s 0x0 d e r i v a t i v e
(Scheme 3 ) , which upon reduction with 3 H - s o d i ~boro-
hydride w a s converted t o t h e corresponding d i a s t e r e o -
mers. The diastereomers were s e p a r a t e d by HPLC. 2H-
Chloramphenicol diastereomers can b e s y n t h e s i s e d
s i m i l a r l y using 2H-sodium borohydride ( 1 0 ) .
 8
X"
u
I
N
!3-E
I
I
8-3 B-3
8
x"
u
1
x 0"
V-Z
I
8-3
k
6 0
X
0
+
718
 O2N a OH
CH2OH
YH-CH-NHC-CHC12
I

It
0
I
I Oxidation

-4
c
ED
0
It
C - CH - NH - C - CHC12

Scheme 3 . Synthesis of labeled chloramphenicols.

720 ABDULLAH A. AL-BADR AND HUMEIDA A. EL-OBEID

4. Methods of Analysis

4.1 I d e n t i f i c a t i o n Tests

Dissolve about 10 mg i n 1 ml of alcohol (50%),

add 3 m l o f a 1% of calcium c h l o r i d e and 50 mg
o f zinc powder, heat on a water b a t h for 1 0
minutes, cool and f i l t e r ; t o t h e f i l t r a t e add
100 mg of anhydrous sodium a c e t a t e and 2 drops
o f benzoyl c h l o r i d e , shake f o r 1 minute and
t h e n add 0.5 ml of f e r r i c c h l o r i d e s o l u t i o n and
3 ml o f d i l u t e hydrochloric a c i d and mix; a
reddish-violet or purple color i s produced. No
such c o l o r i s produced when t h e t e s t i s repeated
without zinc powder (11).

To 5 ml of 0.1% s o l u t i o n add a few drops of

s i l v e r n i t r a t e s o l u t i o n , no p r e c i p i t a t e i s pro-
duced. Heat about 50 m g with 2 ml of alcoholic
potassium hydroxide s o l u t i o n on a water bath for
1 5 minutes, add a s m a l l q u a n t i t y o f decolorizing
charcoal, shake, f i l t e r and t o t h e f i l t r a t e add
s i l v e r n i t r a t e s o l u t i o n ; a white p r e c i p i t a t e i s
produced which i s i n s o l u b l e i n n i t r i c a c i d but
s o l u b l e a f t e r washing with water, i n d i l u t e
ammonia s o l u t i o n . (11) .
Dissolve about 1 0 mg i n 2 m l of alcohol ( 5 0 % )
add 4.5 m l o f d i l u t e s u l f u r i c a c i d and about
50 mg zinc powder, allow t o stand f o r 10 minutes
and decant t h e supernatant l i q u i d ; cool t h e
supernatant l i q u i d i n i c e ; add 0.5 ml sodium
n i t r i t e s o l u t i o n , allow t o stand f o r 2 minutes
and t h e n add 1 gm of urea followed by 1 ml of
2-naphthol s o l u t i o n and 2 ml of sodium hydro-
x i d e s o l u t i o n ; a red c o l o r i s produced (11).

The n i t r o group i s reduced t o an amino group by

zinc-HC1 and t h e amine i s caused t o r e a c t with
dimethylaminobenzaldehyde , t h e r e s u l t i n g Schif f
base gives a colored s a l t i n a c i d medium. The
method can be applied as a spot t e s t . Chloram-
phenicol i n ointments i s e x t r a c t e d i n t o 96%
ethanol. Riboflavine does not i n t e r f e r e i n t h e
d e t e c t i o n o f chloramphencol (12).
CHLORAMPHENICOL 721

An orange-red c o l o r i s produced and ammonia i s

evolved when chloramphenicol i s heated with 50%
NaOH s o l u t i o n ( 3 ) .

Chloramphenicol g i v e s a p o s i t i v e r e a c t i o n t o
Fujiwara's T e s t ( 3 ) as follows: a l i t t l e s o l i d
o r drop of a t e s t s o l u t i o n i s added t o a mix-
t u r e of p y r i d i n e (1m l ) and 20% NaOH s o l u t i o n
(2 ml) . The mixture i s heated on a b o i l i n g
water b a t h f o r 3-5 minutes with vigorous p e r i o -
d i c shaking. A c o n t r o l t e s t must be c a r r i e d
o u t . A red c o l o r appears i n t h e p y r i d i n e l a y e r .

Ammonium molybdate t e s t for micro q u a n t i t i e s

g i v e s f a i n t b l u e color ( 3 ) .

4.2 Q u a n t i t a t i v e Analysis

4.2.1 B i o l o g i c a l Methods

4.2.1.1 Microbiological Methods

Chloramphenicol bioassay have been

r e p o r t e d by Bannatyne and Cheung
( 1 3 ) . The a u t h o r s described an
accurate p l a t e diffusion bioassay
f o r t h e drug, i n which t h e f a s t -
r e p l i c a t i n g Beneckea n a t r i e g e n s and
1.5% s a l t a g a r are used. Zone of
i n h i b i t i o n were w e l l defined a f t e r
3 hours and t h e l i m i t of s e n s i -
t i v i t y o f t h e method w a s around
2 Llg/ml.

Fabiansson and Rut egaerd ( 1 4 ) have

reviewed t h e b i o l o g i c a l methods i n
c u r r e n t use f o r t h e d e t e c t i o n of
a n t i b i o t i c r e s i d u e s i n s l a u g h t e r ani-
m a l s and reported a modified method
i n which t h e c o n d i t i o n s f o r t h e
c o n t r o l were standardized. The
standardized c o n d i t i o n s i n c l u d e t h e
use of a s p o r u l a t i n g organism,
B a c i l l u s s u b t i l i s , an inoculum s i z e
of 0 . 5 x 105 spores/ml medium, add
5 m l o f medium (pH 6.0) p e r p l a t e .
A preincubation d i f f u s i o n t i m e of
722 ABDULLAH A. AL-BADR AND HUMEIDA A. EL-OBEID

1 hour at room temperature i s

recommended before incubat ion.

Horng and KO (15) have reported sys-

tematic a n a l y s i s of a n t i b i o t i c s
via agar g e l electrophoresis and
antimicrobial spectrum of methodo-
logy. A n t i b i o t i c s are detected i n
food and drugs, by a method which
employed both agar g e l electrophore-
sis and antimicrobial spectrum.

Horng e t a 1 (16) have also reported

systematic a n a l y s i s of a n t i b i o t i c s
via agar g e l electrophoresis and
antimicrobial spectrum-candidacy f o r
detecting residual a n t i b i o t i c s i n
foods. The method i s a modification
of t h e method of Horng and KO (15)
t o increase i t s r e s o l u t i o n power and
s e n s i t i v i t y . Modification included
t h e amount of agar g e l and t h e
height of t h e t e s t organism s t r i p
and t h e width of sample s l i t and
enabled t h e a n a l y s i s of a n t i b i o t i c s
at food residue concentrations
(1 pg/ml).
4.2.1.2 Enzymatic Methods

Smith and Smith (17) have reported

an improved enzymic assay of
chloramphenicol. R-factor-incoded
chloramphenicol a c e t y l t r a n s e f e r a s e ,
from an Escherichia & mutant t h a t
was highly r e s i s t a n t t o chlorampheni-
c o l was p a r t l y p u r i f i e d and used f o r
t h e assay. Only a n t i b i o t i c a l l y
a c t i v e chloramphenicol i s attached
by t h e enzyme. C r y s t a l l i n e chloram-
phenicol i s d r i e d t o constant weight
a t 60" and dissolved i n serum, and
5 0 - ~ 1portions (as standards) a r e
a r e s t o r e d at -70'. Serum samples
and standards a r e heated a t 60° f o r
1 5 minutes bef re addition of 1 0 pl
t o 50 p1 of ['&I acetylcoenzyme A
CHLORAMPHENICOL 723

s o l u t i o n (pH 7.8) and 25 p 1 o f

enzyme source ( 0 . 1 spectrophotome-
t r i c u n i t ) . A f t e r incubation a t
37' f o r 60 minutes, t h e d i a c e t y l a t e d
product i s s e l e c t i v e l y absorbed on
micropore f i l t e r s and t h e n assayed
by s c i n t i l l a t i o n counting.
Robison e t a1 (18) have developed a
s i m p l i f i e d radio-enzymatic assay
f o r chloramphenicol , by elminat i n g
t h e need for cumbersone e x t r a c t i o n
procedure. A f t e r t h e a c e t y l a t i o n
of chloramphenicol w i t h 14C-labeled
a c e t y l CoA i n t h e presence of
chloramphenicol ac e t y l t r a n s f e r a s e
t h e r e a c t i o n mixture w a s added t o a
toluene-based s c i n t i l l a t i o n f l u i d .
Since 14C-ac e t y l a t ed c h l o r amphenicol
i s more s o l u b l e t h a n 14C-labeled
a c e t y l CoA i n t o l u e n e , t h e radio-
a c t i v e product could be counted
directly.
Detection and q u a n t i t a t i o n of chlo-
ramphenicol by competitive enzyme-
l i n k e d immunoassay w a s r e p o r t e d by
Campbell e t a1 ( 1 9 ) . The a s s a y f o r
t h e drug i n meat involves competi-
t i v e i n h i b i t i o n , by free chlorampheni-
c o l i n t h e sample, of t h e binding
of s p e c i f i c r a b b i t antibody t o solid-
phase-bound chloramphenicol. The
antibody not d i s p l a c e d w a s measured
by using a commercially a v a i l a b l e
enzyme-linked a n t i - r a b b i t 1 gm pre-
p r a t ion and added s u b s t r a t e . Enzyme
a c t i v i t y , measured spectrophotometri-
c a l l y , w a s inversely proportional
t o t h e c o n c e n t r a t i o n of chlorampheni-
c o l i n t h e sample.

4.2.2 Chemical Methods

4.2.2.1 T i t r i m e t r i c Methods

Navik and Polyakova ( 2 0 ) have

r e p o r t e d t h e a n a l y s i s of some multi-
724 ABDULLAH A. AL-BADR AND HUMEIDA A. EL-OBEID

component w a t er-alcohol mixtures

where aqueous ethanol s o l u t i o n of
chloramphenicol was determined by a
tit rimet r i c method.

El-Sebai e t a 1 (21) have described

new i n t e r n a l i n d i c a t o r s f o r t h e
determination of primary aromatic
amines (chloramphenicol y i e l d s an
amino group on reduction with zinc
dust and hydrochloric a c i d ) . Var-
ious diazo- compounds were synthe-
s i z e d and t e s t e d a s an i n d i c a t o r s
i n t h e t i t r a t i o n of such amines with
NaN02 solution.

Talegaonkar e t a1 (22) have des-

cribed an a l k a l i m e t r i c determination
of chloramphenicol i n dimethylforma-
mide. A solution of t h e drug i n
dimethylformamide i s t i t r a t e d with
sodium methoxide s o l u t i o n i n
benzene-methanol 4:1, with 2 drops
1% 2-nitroaniline s o l u t i o n i n ben-
zene a s i n d i c a t o r , t h e color change
i s from yellow t o red. The r e a c t i o n
involved i n t h e t i t r a t i o n i s r e p l a c e
ment of one chlorine atom i n t h e
drug by a methoxy group.

Koka (23) has described an iodi-

metric method f o r t h e determination
of chloramphenicol i n some medicinal
mixtures. The sample i s boiled with
sodium hydroxide s o l u t i o n , cooled,
d i l u t e d , t r e a t e d with 0.1N- iodine,
s e t a s i d e for 1 0 t o 1 5 minutes i n
t h e dark, and then t r e a t e d with
potassium iodide s o l u t i o n and
d i l u t e sulphuric acid, and t h e
l i b e r a t e d iodine i s t i t r a t e d w i t h
sodium thiosulphate s o l u t i o n using
starch a s indicator.

Koka and Koltun (24) have reported

another iodimetric method f o r t h e
determination of chloramphenicol i n
CHLORAMPHENICOL 725

some o t h e r medicinal forms.

4.2.2.2 Polarographic Methods

Chloramphenicol [ i n milk] w a s d e t e r -
mined by Fossdal and Jacobsen ( 2 5 )
p o l a r o g r a p h i c a l l y . The e l e c t r o n -
r e d u c t i o n o f t h e drug w a s s t u d i e d by
polarography of 0.5 mM s o l u t i o n of
v a r i o u s e l e c t r o l y t e s and of 1.7 mM
t o ~.IM s o l u t i o n i n 0.5M-acetate
b u f f e r a t pH 4.7 and by c y c l i c
v o l t ammetry chronopot ent iomet r y and
coulometry; t h e r e a c t i o n s involved
a r e discussed. A well-defined
polarographic wave was obtained
which even i n t h e presence of 50%
of milk i s a n a l y t i c a l l y u s e f u l over
t h e range of 0.3 t o 60 pg of a n t i -
b i o t i c per m l .

Chloramphenicol and it h y d r o l y s i s
product 2 amino-1-( p-nitropheny1)-
1,3-propanediol were determined
p o l a r o g r a p h i c a l l y i n pharmaceutical
formulations ( 2 6 ) a f t e r s e p a r a t i o n
by t h i n - l a y e r chromatography on
s i l i c a g e l GF254 using a 4 : l : l n-
but anol-acet i c ac id-wat e r or 2 :2 :4
ac et one-benz ene-pet roleum e t h e r
solvent mixtures. The s e p a r a t i o n
of t h e above two compounds by high-
p r e s s u r e l i q u i d chromatography
(Perkin-Elmer r e v e r s e phase C l 8
column with 45:55:1 methanol-water-
a c e t i c a c i d o r 30:70:1 isopropanol-
water-acetic a c i d ) made p o s s i b l e
simultaneous determination o f t h e
above two compounds.

Polak e t a1 ( 2 7 ) have determined

chloramphenicol i n body f l u i d s by
d i f f e r e n t i a l p u l s e polarography .
Reduction of t h e drug a t a dropping-
mercury e l e c t r o d e a t -0.4 V ( v s t h e
s . c . e ) a t pH 4.5 ( B r i t t o n Robinson
b u f f e r ) i s used f o r t h e determination
726 ABDULLAH A. AL-BADR AND'HUMEIDA A. EL-OBEID

of t h e drug i n such samples a f t e r

p r e c i p i t a t i o n of p r o t e i n s w i t h
a c e t o n i t r i l e or methanol. The
c o e f f i c i e n t of v a r i a t i o n w a s 4%
f o r determination of 1 5 pg/ml of
chloramphenicol i n serum. Conven-
t i o n a l d.c. plarography i s s u i t a b l e
f o r determining t h e drug only i n
urine.

4.2.2.3 Colorimetric Methods

Several colorimetric methods f o r t h e

determination of chloramphenicol
have been reported i n t h e l i t e r a t u r e .

Plourde and Braun (28) have des-

cribed a colorimetric procedure f o r
t h e determination of t h e drug i n
t a b l e t s and capsules. For t a b l e t s ,
powder t h e m a t e r i a l and dissolve
a sample containing 20 mg of chlor-
phenicol i n 50 ml of 1,2-dichloro-
ethane a t 60°, cool and d i l u t e t o
100 m l w i t h dichloroethane. F i l t e r
t h e s o l u t i o n and r e j e c t t h e f i r s t
few m l s . For s o f t capsules, s e c t i o n
longitudinaly, and dissolve i n
dichloroethane (150 ml) at 60°,
cool and d i l u t e t o 250 ml w i t h
dichloroethane. F i l t e r , r e j e c t t h e
first few m l s . and d i l u t e an a l i -
quot containing 20 mg of chloram-
phenicol t o 100 ml w i t h dichloro-
ethane. To t h i s d i l u t e s o l u t i o n
(4.5 m l ) add dichloroethane ( 6 . 5 r n l )
and 'piperidine-8-hydroxyquinoline
vanadate' reagent ( 4 m l ) and l e a v e
f o r 30 minutes a t room temperature.
Extract excess reagent w i t h M-NaOH
( 1 0 m l ) for 30 seconds, s e t aside
f o r 30 seconds and f i l t e r t h e
organic phase over Na2S04 i n t o 1 m l
of 5% dichloroacetic a c i d s o l u t i o n
i n a c e t i c a c i d then measure t h e
extinction of the r e s u l t i n g blue
s o l u t i o n a t 625 nm within 2.5 hours.
CHLORAMPHENICOL 727

A c o l o r i m e t r i c method using p-
dimethylaminobenzaldehyde i s des-
c r i b e d ( 2 9 ) f o r t h e determination of
chloramphenicol and o t h e r compounds.
I n an a c i d medium p-dimethylamino-
benzaldehyde produced r e a c t i o n pro-
d u c t s varying i n c o l o r from yellow
t o deep r e d . The r e a c t i o n products
showed maximud absorption spectrum
peaks between 425 and 450 nm.

Ivakhnenko - et - a1 ( 3 0 ) described a
procedure f o r absorbtiometric d e t e r -
mination of chloramphenicol. D i l u t e
t h e r e d u c t i o n product of 0.5 gm of
chloramphenicol t o 100 ml. To 1 ml
of t h e s o l u t i o n add 5 ml of N-HC1,
2 ml of 0.01M - NaNO and, a f t e r 4
t o 5 minutes, 5 m l of 0.3% s o l u t i o n
of a diaminoacridine reagent
( e t h a c r i d i n e l a c t a t e or p r o f l a v i n e ) ;
after a f u r t h e r 2 minutes d i l u t e t h e
s o l u t i o n t o 50 ml and measure t h e
e x t i n c t i o n of t h e r e s u l t i n g diazo-
compound a t 508 nm for t h e f i r s t
reagent o r a t $84 nm for t h e o t h e r
a g a i n s t water.

The determination of chloramphenicol

i n pharmaceuticals ( s u p p o s i t o r i e s
and coated t a b l e t s ) have been
r e p o r t e d by Cieszynski e t a1 ( 3 1 )
using colorimetry. The product of
t h e r e d u c t i o n of chloramphenicol
r e a c t s w i t h guaiacol i n a l k a l i n e
medium (pH 9.6) t o form a b l u e com-
plex, which i s s t a b l e f o r up t o 5
hours, with measurement of t h e
e x t i n c t i o n , a t 610 nm, 30 minutes
a f t e r t h e s o l u t i o n s a r e mixed.

Przyborowski ( 3 2 ) described a method

for t h e determination of t h e drug
and i t s p a l m i t a t e i n pharmaceuticals.
The method involves t h e h y d r o l y s i s
of chloramphenicol by NaOH i n a
medium containing hydroxylammonium
c h l o r i d e , and r e a c t i o n of t h e result-
728 ABDULLAH A. AL-BADR AND HUMEIDA A. EL-OBEID

ing 2,2-dichloroacetohydroxamic a c i d
with Fe3+. The coloured complex
produced i s determined by spectro-
photometry at 505 nm. A modified
method f o r t h e determination of
chloramphenicol palmitate and t h e
contents of chloramphenicol and
chloramphenicol palmitate i n s e v e r a l
preparation i s a l s o given.

Krezk and Lechniak (33) have

reported t h e a p p l i c a t i o n of copper
(11) t o t h e colorimetric determina-
t i o n of chloramphenicol i n ointments.
The method i s based on t h e formation
of a complex a f t e r mixing methanolic
s o l u t i o n s o f chloramphenicol, copper
(11) and methanol; t h e p r e c i p i t a t e
of Cu(OH)2 i s f i l t e r e d and t h e
absorbance o f t h e f i l t r a t e i s
measured a t 550 nm. The composition
o f t h e complex corresponds t o copper:
chloramphenicol molar r a t i o = 1:2.

Catechol and iodine have r e c e n t l y

been used f o r t h e spectrophotometric
determination of aromatic m i n e s
( 34). The method, involves mixing
15 m l of potassium a c i d p h t h a l a t e
buffer s o l u t i o n (pH 3.1) , 1 m l of
aqueous 0.1% catechol, 1 ml of 0.01N-
iodine and 1.5 ml of t h e m i n e
s o l u t i o n , d i l u t i o n of t h e mixture
t o 25 m l with water, and, after 5
t o 30 minutes (depending on t h e
m i n e ) , spectrophotometry a t 500 t o
520 nm ( v s a reagent blank). A
modification of t h i s procedure i s
described f o r compounds t h a t y i e l d
primary arylamino-groups on
reduction (chloramphenicol) .
Yang ( 3 5 ) has described a simul-
t aneous determination of chloram-
phenicol and i t s metabolite D-threo-
2-amino-1-( 4-nitrophenyl )propane-l,3-
d i o l i n i n j e c t i o n s . A 1 0 m l sample
CHLORAMPHENICOL 729

i s d i l u t e d t o 25 ml w i t h anhydrous
ethanol and a p o r t i o n i s sampled
f o r s p e c t r o p o l a r i m e t r i c determina-
t i o n of t h e s p e c i f i c o p t i c a l r o t a -
t i o n a t 418 and 589 nm for t h e drug,
and i t s m e t a b o l i t e s , r e s p e c t i v e l y .

Divakar e t a1 ( 3 6 ) have used b r u c i n e

and sodium metaperiodate f o r t h e
c o l o r i m e t r i c e s t i m a t i o n of chloram-
phenicol. A 10-ml p o r t i o n o f t h e
t e s t s o l u t i o n was b o i l e d under r e f -
l u x f o r 45 minutes w i t h 1 0 ml of
2M-HC1, and t h e excess of H C 1 was
removed i n vaccu. The r e s i d u e was
d i s s o l v e d i n 20 m l of w a r m water,
and t h e s o l u t i o n was d i l u t e d t o
50 m l with water. This s o l u t i o n i n
t e s t tubes w a s then t r e a t e d with
3 ml of 5 mM-brucine, 1 . 5 m l of
5 mM-NaIO4 and 2 m l of 2.3 M-H2S04
and t h e s o l u t i o n w a s d i l u t e d t o 1 0 m l
with water.

The t e s t t u b e s were shaken and

heated on a boiling-water b a t h f o r
15 minutes. A f t e r cooling t h e con-
t e n t s of each t u b e were d i l u t e d t o
25 m l w i t h water and t h e absorbance
of t h e s o l u t i o n w a s measured at
500 nm.

4.2.2.4 U l t r a v i o l e t Spectrophotometric
Methods

Chloramphenicol w a s determined i n
pharmaceutical p r e p a r a t i o n s contain-
ing b o r i c a c i d , g l y c e r i n , sodium
c h l o r i d e , zinc s u l p h a t e , belladonna
e x t r a c t , s t r e p t o c i d , glucose, or
r esorc i n o l by u l t r a v i o l e t spectro-
photometric a n a l y s i s a t 278 or 315 nm.
The drug w a s determined w i t h a
r e l a t i v e e r r o r of f 3.46 ( 3 7 ) .

Buryak ( 3 8 ) r e p o r t e d t h e a n a l y s i s
of multicomponent medicines w i t h t h e
a i d of a computer. The sample i s
730 ABDULLAH A. AL-BADR AND HUMEIDA A. EL-OBEID

b o i l e d f o r 5 minutes w i t h 95%
ethanol, and t h e e x t r a c t i s f i l t e r e d
and d i l u t e d as necessary with etha-
n o l . The absorbance i s measured
a t various wavelengths between 226
and 330 nm vs ethanol. The concent-
r a t i o n of individual components are
c a l c u l a t e d from a matrix of equations
r e l a t i n g t h e t o t a l absorbance t o t h e
sum of t h e p a r t i a l absorbances of
t h e components. The method i s s u i t -
a b l e f o r lanolin-based ointment s
containing chloramphenicol.

4.2.2.5 I n f r a r e d Spectrophotometric Methods

I n f r a r e d spectroscopy had been used

for t h e determination o f t h e a n t i -
b i o t i c s t a b i l i t y ( 3 9 ) . The e f f e c t
of e x t e r n a l f a c t o r s such as h e a t ,
a c i d i t y and hydrolysis on c r y s t a l -
l i n e a n t i b i o t i c s was examined
spectroscopically. Chloramphenicol
r e s i s t e d dry heat at 80' f o r 24 hrs,
but was unstable at 100' f o r 2 h r s .

Namigohar e t a1 (40) reported an

i n f r a r e d spectrophotomet r i c deter-
minat ion of chloramphenicol. The
drug was e x t r a c t e d from capsules,
creams and eye drops, with ethanol
or e t h y l acetate-chloroform and
chloramphenicol palmitate was
extracted with chloroform. Chloram-
phenicol and chloramphenicol palmi-
t a t e were then determined by
i n f r a r e d spectrophotometry, i n KBr
and i n chloroform, r e s p e c t i v e l y .

4.2.2.6 Proton Magnetic Resonance Spectro-

metric Methods

Chloramphenicol i n pharmaceutical
preparation has been determined using
proton magnetic resonance spectro-
metry (41). The drug was dissolved
i n dimethyl sulfoxide containing
CHLORAMPHENICOL 731

maleic a c i d as i n t e r n a l standard.
The n.m.r spectrum o f t h e s o l u t i o n
was recorded, and t h e peaks f o r t h e
aromatic protons o f chloramphenicol
at 7.6 and 7.5 ppm and t h e v i n y l i c
protons of maleic a c i d a t 6.25 ppm
were i n t e g r a t e d ; t h e amount of
chloramphenicol w a s c a l c u l a t e d from
t h e i n t e g r a t i o n r a t i o and t h e known
amount of maleic a c i d . For t e n
samples c o n t a i n i n g 100 t o 150 mg of
pure chloramphenicol, t h e average
recovery w a s 100.22% ( standard
d e v i a t i o n 1.37%). The method has
been a p p l i e d t o t h e a n a l y s i s of
commercial capsules and o r a l suspen-
s i o n of chloramphenicol p a l m i t a t e ;
it i s r a p i d and simple and can a l s o
b e used t o check t h e p u r i t y of t h e
drug.

4.2.2.7 Mass Spectrometric Methods

Mass spectrometric methods have been

described f o r t h e a n a l y s i s of
chloramphenicol i n aqueous s o l u t i o n s
(4) and i n animal t i s s u e s and body
f l u i d s ( 5,6 ,52).
4.2.3 Chromatographic Methods

A multitude of t h i n l a y e r , paper, column,

gas and l i q u i d chromatographic methods have
been developed f o r t h e d e t e c t i o n and d e t e r -
mination of chloramphenicol i n pharmaceutical
formulations, b i o l o g i c a l f l u i d s and animal
t i ssues .
4.2.3.1 Thin Layer, Paper and Column
Chromatography

Various t h i n l a y e r , paper and column

chromatographic methods used for t h e
a n a l y s i s of chloramphenicol a r e
o u t l i n e d i n Table 2.
732 ABDULLAH A. AL-BADR AND HUMEIDA A. EL-OBEID

4.2.3.2 Gas Chrmatographic Methods

Gas chromatographic methods have
been used for the determination of
chloramphenicol in dosage forms and
biological fluids and tissues.
Table 3 summarises some of these
methods.

4.2.3.3 High Performance Liquid Chromato-

graphy-(HPLC)

HPLC has been extensively usea f o r

the determination of chloramphenicol
in pharmaceutical formulations and
biological fluids as well as for
the detection and determination of
the drug residues in animal tissues.
Some of these methods are outlined
in Table 4.
Table 2. Thin Layer, Paper and Column Chromatographic Methods for t h e A n a l y s i s o f Chloramphenicol.

Support Solvent System Detect i o n Ref.

Silanized s i l i c a Mixture of s o l v e n t s e .g . Dioxane , a c e t o n e , - 42

g e l (reverse i s o p r o p y l a l c o h o l , methanol, t e t r a h y d r o f u r a n
phase) or e t h y l methyl k e t o n e w i t h c i t r a t e - p h o s -
p h a t e b u f f e r (pH 3, 5 or 7 ) .

Silica gel C H C l -methanol-2.5% a q . NH3 (60:6:1) Spray with SnC12 solu- 43

3 t i o n , h e a t t o llO'for
7 min and s p r a y w i t h
4-dimethylaminobenzal-
dehyde .
S i l u t o l W 254 Ethyl ether. Fluorescence quenching 44
Sheet

S i l i c a gel CH C 1 - e t h y l a c e t a t e ( 1 : b ) .
2 2
15% SnC12 i n aq. H C 1 , 46
t h e n W.

High p e r f o r - Chloroform-heptane-methanol ( 4 :2: 1) W a t 280 nm. 46

mance TLC con-
t a c t spotter
n-Butanol-acetic acid-water ( 4 :1:1) 26
S i l i c a g e l GF
254 or
Acetone-benzene, petroleum e t h e r ( 2 : 2 : 4 )
Table 2 (Continued)

Support Solvent System Detect i o n Ref.

Whatman No. 1 2.5% Acetic a c i d i n butanol water ( 2 2 : 3 ) . 4-Dimethylaminobenzal- 47

Paper dehyde .
17.5 cm x 2 cm Ethanol-ethylacetate-aq. NH3 (50: 50:l) Biological assay 48
column of neut-
ral alumina
 (u m
In Ln
735
 Table 4. HPLC Methods f o r t h e Determination of Chloramphenicol

Column Mobile Phase Detect or Ref.

Micropak CN Hexane-CH C 1 -methanol. W at 254 nm

2 2
A r e v e r s e phase A c i d i f i e d ethanol-water w 55
column

DVB-MCL-0 Methanol-ammonia. w 56
RP-2 (10 m) 0.01M K2HP04 -methanol ( 29 :21 ) . w 57
4 P a r t i s i l - 1 0 ODs 50 mM KH2P04 (pH 4.5). W a t 254 m 58
0
6,
1.1 Bondapak phenyl 0.05M H P O - a c e t o n i t r i l e (3:l) 59
3 4
1-1 Bondapak C18 20% A c e t o n i t r i l e i n 0.05M N a a c e t a t e W at 278 nm 60
b u f f e r (pH 5.3).

Sep-Pak C18 Ethyl e t h e r and e t h a n o l . w 61

Nucleosil C18 Wat er-methanol ( 7 :3 ) . W at 278 and 350 nm 62
( 5 Fun).
Hypersil H ODS Wat e r - a c e t o n i t r i l e - a c e t a t e b u f f e r W a t 276 nm 63
5 (80:20:1).

1-1 Bondapak C
18 Aqueous methanol . w 64
 Table 4. ( Continued)

Column Mobile Phase Detector Ref.

1-1 Bondapak C A c e t o n i t r i l e - p h o s p h a t e b u f f e r (1:3 ) W a t 278 52

18
RP-18 35 t o 40% a q . methanol c o n t a i n i n g w 45
1 0 0 mg/L o f K2HP04.

Radial-PAK c18 Methanol-0.75% a c e t i c a c i d ( 3 : 7 ) W at 280 nm 65

w i t h RCSS Guard- a d j u s t e d t o p H 5.5 w i t h t r i e t h y l a m i n e .
PAK precolumn.
4
w
4
c18 Varian Micro- Phosphate b u f f e r (pH 3.25)-methanol- w at 278 nm 66
pak MC H ( 1 0 urn) a c e t o n i t r i l e (27:9:4).

Perkin-Elmer Methanol-water-acetic a c i d ( 4 5 : 5 5 : l ) Polarographic 26

Reverse Phase c18 o r Isopropyl alcohol-water-acetic a c i d
column. (30:70:1).

1 ~ - Bondapak C18 Wat er-methanol-acet i c a c i d w a t 280 67

738 ABDULLAH A. AL-BADR A N D HUMEIDA A. EL-OBEID

5. Pharmacokinet i c s

5.1 Absorption and D i s t r i b u t i o n

Oral doses o f 1 gm chloramphenicol produce peak
l e v e l s of 10-20 pg/ml at 2 t o 4 h r . (68, 69). The
serum l e v e l peak following o r a l a d m i n i s t r a t i o n i s
approximately t h e same as t h a t o b t a i n e d following
I V a d m i n i s t r a t i o n , although peak l e v e l s are
reached slower by t h e former r o u t e . Yogev e t a1
( 7 0 ) conducted a s t u d y i n which 39 c h i l d r e n w i t h H
i n f l u e n s a e m e n i n g i t i s were t r e a t e d f o r 5 days w i t h
o r a l chloramphenicol. All p a t i e n t s responded w e l l
t o t h e r a p y and no r e l a s p s e developed. The r o u t e of
a d m i n i s t r a t i o n had l i t t l e impact on t h e p e d i a t r i c
p a t i e n t s t r e a t e d and t h e serum l e v e l s a f t e r o r a l
a d m i n i s t r a t i o n w a s equal t o o r g r e a t e r t h a n I V
f o r m u l a t i o n s . Chloramphenicol p a l m i t a t e adminis-
t e r e d t o c h i l d r e n between t h e age o f 2 months and
14 y e a r s o r a l l y every 6 h r i n doses o f 60-70 mg/kg/day
r e s u l t e d i n a serum c o n c e n t r a t i o n ( a t s t e a d y s t a t e )
r a n g i n g from 1 5 . 5 t o 29.0 pg/ml w i t h a mean o f
20.2 ug/ml a f t e r 90 min from a d m i n i s t r a t i o n (71).
Chloramphenicol s u c c i n a t e a d m i n i s t e r e d every 6 h r by
IV r o u t e t o 18 c h i l d r e n between t h e age o f 2 months
and 14 y e a r s i n doses o f 60 t o 109 mg/kg/day r e s u l t e d
i n a serum c o n c e n t r a t i o n ( a t s t e a d y state) ranging
from 1 2 . 5 t o 43.1 pg/ml a f t e r 90 min. from administ-
r a t i o n (71). Lower blood l e v e l s are produced by 1M
chloramphenicol sodium s u c c i n a t e t h a n by i d e n t i c a l
I V doses ( 7 2 ) and about 50% t h e serum l e v e l o b t a i n e d
a f t e r i d e n t i c a l doses given by o r a l r o u t e (5-6 g m / m l )
( 6 9 ) . Oral a d m i n i s t r a t i o n o f chloramphenicol
r e s u l t e d i n about 75-90% a b s o r p t i o n w i t h peak l e v e l s
o c c u r i n g 0 . 5 t o 2 h r f o l l o w i n g a d m i n i s t r a t i o n (73,
7 4 ) . The p a l m i t a t e ester, however, must be hydro-
l y s e d b e f o r e a b s o r p t i o n . Hydrolysis may be inade-
q u a t e i n newborns, i n f a n t s and c h i l d r e n and absorp-
t i o n delayed and u n r e l i a b l e ( ~ 5 ~ 7 6 )Chloramphenicol
.
b a s e a d m i n i s t e r e d o r a l l y produces peak serum l e v e l s
equivalent t o o r higher than I V administration (77,
78). Peak l e v e l s o f 10-13 pg/ml are o b t a i n e d i n
about 2 h r a f t e r t h e a d m i n i s t r a t i o n o f 1 gm o r a l d o s e ,
and s u s t a i n e d a d m i n i s t r a t i o n every 6 h r provides
cumulative e f f e c t w i t h somewhat h i g h e r peak l e v e l s
(68,79). Chloramphenicol sodium s u c c i n a t e , u s i n g
1 gm I V dose, produces similar blood pe& l e v e l s
CHLORAMPHENICOL 739

o n l y o c c u r i n g immediately. Blood l e v e l s o f t h e same

o r d e r are o b t a i n e d i n c h i l d r e n w i t h an e q u i v a l e n t
s i n g l e o r a l or I V dose ( 7 9 ) . I n newborn i n f a n t s 2 , h ,
or 1 2 h r a f t e r chloramphenicol s u c c i n a t e administ-
r a t i o n ( 1 2 . 5 mg/kg, I V ) serum chloramphenicol concent-
r a t i o n s were < 1 0 mg/L a t each t i m e s t u d i e d . A f t e r
l o a d i n g dose o f 20 mg drug/kg t h e mean serum drug
c o n c e n t r a t i o n s were h i g h e r i n i n f a n t s I 2 day old
t h a n i n i n f a n t s 2 3 days old ( 8 0 ) .

The e f f e c t of dosing methods on chloramphenicol

a b s o r p t i o n was s t u d i e d (81) u s i n g young y e l l o w t a i l s .
The a b s o r p t i o n i s found t o b e g r e a t e r i n p r p o r t i o n
t o t h e i n c r e a s e i n t h e drug c o n t e n t i n t h e f e e d . Of
s e v e r a l d i e t s t e s t e d with varying concentration of
t h e drug, a d i e t c o n t a i n i n g 50% o f t h e drug and 0.2%
of a b i n d e r produced t h e h i g h e s t drug l e v e l i n t h e
fish.

The a b s o r p t i o n o f chloramphenicol w a s s t u d i e d ( 8 2 )
i n 6 normal v o l u n t e e r s by v a r i o u s r o u t e s of administ-
r a t i o n . Peak serum l e v e l s were maximum w i t h t h e o r a l
r o u t e o f a d m i n i s t r a t i o n . With t h e 1M r o u t e t h e
average peak l e v e l w a s o n l y 70% of t h a t of t h e o r a l
r o u t e . The a b s o r p t i o n of t h e drug w a s minimum and
v a r i a b l e a f t e r r e c t a l a d m i n i s t r a t i o n . Animal s t u d i e s
were conducted for improving t h e r e c t a l a b s o r p t i o n
of chloramphenicol ( 8 3 ) . The drug r e c t a l a b s o r p t i o n
was found t o b e s t r o n g l y species-dependent. In
r a b b i t s t h e drug a b s o r p t i o n w a s g r e a t l y i n c r e a s e d
a f t e r r e c t a l a d m i n i s t r a t i o n o f chloramphenicol -
erythromycin m i x t u r e and chloramphenicol - oleando-
mycin m i x t u r e , compared t o a b s o r p t i o n a f t e r administ-
r a t i o n of o t h e r chloramphenicol m i x t u r e o f t h e drug
e s t e r s . The i n c r e a s e i n a b s o r p t i o n w a s not observed
i n people, r a t s and g u i n e a p i g s . Chloramphenicol
e s t e r s e . g . chloramphenicol p a l m i t a t e must be hydro-
l y s e d by p a n c r e a t i c l i p a s e s i n t h e duodenum b e f o r e
a b s o r p t i o n t a k e s p l a c e . Accordingly t h e r a t e o f
h y d r o l y s i s o f t h e p a l m i t a t e i s a major f a c t o r i n
determining t h e u l t i m a t e blood l e v e l s achieved. That
t h e a b s o r p t i o n o f t h e p a l m i t a t e i s slower has been
shown by Weiss e t a1 ( 7 5 ) i n t h e i r s t u d i e s i n newborn
i n f a n t s . I n o l d e r c h i l d r e n , it i s a l s o r e p o r t e d (76),
up t o 50% o f an a d m i n i s t e r e d dose o f p a l m i t a t e may be
l o s t i n t h e f e c e s . The dose of t h e p a l m i t a t e must be
h i g h e r t h a n w i t h t h e c r y s t a l l i n e chloramphenicol base
(100-200 mg/kg/day) . This i s i n agreement w i t h t h e
r e s u l t s o f e a r l i e r s t u d i e s (84, 8 5 ) .
740 ABDULLAH A. AL-BADR AND HUMEIDA A. EL-OBEID

The slow h y d r o l y s i s r e s u l t i n g i n lower blood l e v e l s

r e p o r t e d by some i n v e s t i g a t o r s i s due t o t h e s e p a r a t e
polymorphic s t a t e s i n which t h e p a l m i t a t e can e x i s t .
One c r y s t a l l i n e form i s s u b s t a n t i a l l y more hydrolysed
t h a n t h e o t h e r and t h e blood l e v e l s observed are
d i r e c t l y r e l a t e d t o t h e p r o p o r t i o n o f t h a t form
which i s p r e s e n t i n t h e p r e p a r a t i o n ( 8 6 ) . Subse-
quent t o t h e s e r e p o r t s , however, Park-Davis Company
has i n d i c a t e d t h a t t h e l e s s h y d r o l y s a b l e polymorph
of t h e p a l m i t a t e h a s been removed from t h e prepara-
t i o n and t h e a b s o r p t i o n o f chloramphenicol from t h e
p a l m i t a t e i s now complete and r e l i a b l e , producing
blood l e v e l s e q u i v a l e n t t o I V a d m i n i s t r a t i o n ( 7 8 ) .
A p r o s p e c t i v e , randomized e v a l u a t i o n o f o r a l chloram-
p h e n i c o l a d m i n i s t r a t i o n f o r completion of t h e r a p y o f
H i n f l u e n z a e t y p e b m e n i n g i t i s i s conducted (87) i n
44 c h i l d r e n : 21 p a t i e n t s r e c e i v e d t h e drug o r a l l y
a f t e r t h e second day of t h e r a p y , t h e remainder c o n t i -
nued t o r e c e i v e t h e drug v i a I V r o u t e . There was
e q u i v a l e n t b i o a v a i l a b i l i t y o f chloramphenicol. I n
43 p a t i e n t s t h e r e s o l u t i o n o f c l i n i c a l m a n i f e s t a t i o n s
and CSF a b n o r m a l i t i e s of m e n i n g i t i s w a s e q u i v a l e n t
with b o t h r o u t e s . These f i n d i n g s i n d i c a t e t h a t
higher t h a n normal doses of chloramphenicol p a l m i t a t e
a r e not n e c e s s a r y . A 6 h o u r l y devided dosage o f
1 0 0 mg/kd/day w i l l produce blood l e v e l s e q u i v a l e n t t o
t h o s e o b t a i n e d by I V o r o r a l a d m i n i s t r a t i o n o f t h e
base. Adequate CSF l e v e l w i l l be maintained s i n c e
c o n c e n t r a t i o n s o f chloramphenicol i n t h e CSF r e a c h
l e v e l s as high as 50% of t h a t o b t a i n e d i n t h e blood
and a r e w e l l above t h e M I C f o r H i n f l u e n z a e . The
a b s o r p t i o n o f chloramphenicol a f t e r o r a l a d m i n i s t r a -
t i o n i n s e v e r e l y malnourished c h i l d r e n was found t o
be e r r a t i c . T h i s r o u t e should b e avoided i n such
patients (88).

The b i o a v a i l a b i l i t y o f t h e o r a l chloramphenicol
p a l m i t a t e salt w a s i n v e s t i g a t e d ( 7 1 ) and compared
t o t h a t o f I V s u c c i n a t e s a l t i n 18 c h i l d r e n , age 2
months t o 14 y e a r s . The b i o a v a i l a b i l i t y of t h e o r a l
dose w a s found t o b e g r e a t e r t h a n t h e I V p r e p a r a t i o n .
The r e l a t i v e b i o a v a i l a b i l i t y o f t h e s u c c i n a t e com-
pared t o t h e p a l m i t a t e w a s 7 0 % . T h i s can be
explained by t h e prominent i n t e r p a t i e n t v a r i a t i o n i n
t h e e x t e n t of h y d r o l y s i s of t h e I V s u c c i n a t e s a l t
t o t h e b i o l o g i c a l l y a c t i v e chloramphenicol when com-
pared t o t h e o r a l s a l t form. I n t h i s s t u d y a mean of
36% o f t h e a d m i n i s t e r e d I V dose w a s e x c r e t e d
CHLORAMPHENICOL 74 1

unchanged i n u r i n e . T h i s v a l u e probably a c c o u n t s
f o r t h e 30% r e d u c t i o n i n b i o a v a i l a b i l i t y of t h e I V
dosage form when compared t o t h e o r a l form which i s
more completely hydrolysed t o t h e a c t i v e form i n t h e
GIT .
Simultaneous a d m i n i s t r a t i o n o f v i t a m i n s w i t h t h e
a n t i b i o t i c has been found t o reduce chloramphenicol
blood l e v e l s ( 8 9 ) .

Chloramphenicol i s widely d i s t r i b u t e d i n t h e body

w i t h t h e r a p e u t i c l e v e l s o c c u r i n g i n most body c a v i -
t i e s , t h e eye and CSF. Chloramphenicol i s 60 t o 80%
p r o t e i n bound ( 7 3 ) . Other s t u d i e s , however, suggest
t h a t it i s about 36% p r o t e i n bound ( 9 0 ) .

There i s no l i t e r a t u r e r e p o r t on complete pharmacoki-

n e t i c p r o f i l e o f chloramphenicol i n t h e c e r e b r o s p i n a l
f l u i d . Although many s t u d i e s have documented t h e
achievement o f t h e r a p e u t i c l e v e l s i n t h e CSF, o n l y
one has measured s e q u e n t i a l CSF l e v e l s over an e n t i r e
dosing p e r i o d ( 9 1 ) . A male a d u l t p a t i e n t w i t h H
i n f l u e n z a e m e n i n g i t i s r e c e i v e d o r a l chloramphenicol
1 2 . 5 mg/kg every 6 h r . Serum and CSF l e v e l s were
measured a f t e r t h e 7 t h dose t o e n s u r e s t e a d y s t a t e
k i n e t i c s . The CSF l e v e l s were 5.4 pg/ml a t 0 h r ,
5.7 at 30 min, 6 . 3 a t 1 h r , 7.4 a t 2 h r , 7 . 2 a t 3 h r
and 7.9 a t 6 h r . The mean CSF/serum r a t i o w a s 36%.
R e s u l t s o f i s o l a t e d experiments i s i n c l u d e d i n
Table 5.

The r e p o r t e d results emphasise t h e consid.erable i n d i -

v i d u a l v a r i a t i o n s , probably due t o v a r y i n g d e g r e e s
o f miningeal inflammation, t h e drug a p p e a r s t o pro-
duce v e r y good CSF l e v e l s when s u f f i c i e n t doses are
a d m i n i s t e r e d . With I V a d m i n i s t r a t i o n t h e CSF/serum
r a t i o s range from 22.5-99%, w i t h s t e a d y s t a t e CSF
l e v e l s from 4-23.3 pg/ml. Oral dosages produce s i m i -
lar and p o s s i b l y h i g h e r CSF l e v e l s w i t h CSF/serum
r a t i o from 20-60% and CSF l e v e l s o f 4-32 pg/ml.

A comparative s t u d y ( 1 0 0 ) o f t h e s t e a d y - s t a t e CSF
l e v e l s a f t e r I V or o r a l doses of 1 0 0 ug/kg/day o f
chloramphenicol i n 1 4 p a t i e n t s showed t h a t serum
l e v e l s a f t e r I V a d m i n i s t r a t i o n occured a t 45 min.
With mean corresponding t o CSF l e v e l s of 4.2 pg/ml.
Peak serum l e v e l s r e s u l t i n g from o r a l dosing occured
 Table 5. Summary of CSF Levels Reported i n I s o l a t e d Determinations *

Dose CSF/Serum
Dosage form Ref.
(mg/kg/D) r a t i o (%)

7 - 15 Oral 0 4 - 32 0 92
7 - 15 4 20- 64 6 - 20
9 - 45 8 32- 40 20-40
50 32 128 25

28 - 66 Rectal 0 2 - 3.6 0 93
50 - 68 Or& ( s i n g l e ) 4.- 10.3 12.2 - 34 25-42
4 18 - 26 Or& ( m u l t i p l e ) o - 16.7 1 - 42 0 - 39
;P unknown I M (single) 0 - 4.2 5 . 1 - 23 o - 28
18 - 72 IM 3.2 - 9.9 24.8 - 74.4 6 - 27

66 O r a1 2- 8.9 3 - 21.8 50 94

50 IV 14 25 56 95

25 - 35 IV mean 14.1 - 54.4 50 - 87 96

23.3 +/- 7.7

"Through Drugdex, Microfiche System, Micromedex, I n c . , Englewood, Colorado, U . S . A .

 Table 5 (Continued) ...
Dosge Serum l e v e l CSF/Serum
Dosage form Ref.
(mg/kg/D) (U d m l ) ratio (%)

60 IV eak a f t e r 1 6 . 5 ( peak a f t e r 22.5

4(p 3 hr) 30 m i n )
75 11.5(peak a f t e r 2 O ( peak a f t e r 57.5 97
3 hr) 30 m i n )
(Ventricular
fluid)
~

12.5 - 25 IV 4 - 18 8 - 40 45 - 99 98
(neonates)
-4
0
40 - 100
(Older
c h i 1d r en )

100 IV 5.5 - 1 3 10 - 29.5 23 - 85 99

100 IV mean 4.2 mean peak mean 65

(1 - 7 . 5 ) 1 5 a t 45 min. 100
100 Oral mean 6.6 mean peak mean 60
(1.5 - 1 1 . 5 ) 81.5 a t 2-3 hr.
744 ABDULLAH A. AL-BADR A N D HUMEIDA A. EL-OBEID

a t 2-3 h r w i t h CSF l e v e l s o f 6.6 pg/ml.

Four premature i n f a n t s under 5,500 gm were t r e a t e d

w i t h p a r e n t e r a l chloramphenicol f o r c e n t r a l nervous
system i n f e c t i o n due t o organisms r e s i s t a n t t o t h e
p e n i c i l l i n s . Serum, c e r e b r o s p i n a l f l u i d (CSF) and
v e n t r i c u l a r f l u i d c o n c e n t r a t i o n of t h e drug were
measured f r e q u e n t l y d u r i n g t h e r a p y and were used t o
m a i n t a i n drug dosages i n t h e safe and t h e r a p e u t i c
range. Concentration of t h e drug i n t h e lumbar CSF
and v e n t r i c u l a r f l u i d had a mean o f 23.3 vg/ml,
c o n s i s t e n t l y g r e a t e r t h a n 45% of peak serum l e v e l s
( 9 6 ) . The d a t a show t h a t chloramphenicol e n t e r s t h e
CSF i n b o t h v e n t r i c u l a r and lumbar r e g i o n s i n t h e r a -
p e u t i c c o n c e n t r a t ' o n s when a d m i n i s t e r e d I . V . The
d i s t r i b u t i o n of ltC-labeled D( -)-threo-chlorampheni-
c o l was s t u d i e d ( 1 0 1 ) i n newborn p i g s by whole-body
autoradiography . The amount o f r a d i o a c t i v i t y i n t h e
l u n g , l i v e r , a d r e n a l c o r t e x , kidney, myocardium,
Pancreas, t h y r o i d , s p l e e n and s k e l e t a l muscles w a s
higher t h a n t h a t i n t h e blood s h o r t t i m e a f t e r t h e
i n j e c t i o n and remained h i g h e r upto 8 r . A f t e r 4
and 8 h r t h e b r a i n c o n c e n t r a t i o n o f 1CC was a l s o
h i g h e r t h a n t h a t o f t h e blood. I n t h e bone marrow,
however, t h e c o n c e n t r a t i o n d i d not r e a c h t h a t o f t h e
blood d u r i n g t h e whole experiment. I n t h e organs
> 90% of t h e r a d i o a c t i v i t y was r e p r e s e n t e d by
unchanged chloramphenicol; t h e e x c r e t o r y o r g a n s ,
t h y r o i d s and a d r e n a l s being e x c e p t i o n s . I n t h e s t u d y
(81) u s i n g c u l t u r e s y e l l o w t a i l , S e r i o l a q u i n q u e r a d i d a ,
t h e d i s t r i b u t i o n o f chloramphenicol f o l l o w s t h e
o r d e r : l i v e r > muscle > b l o o d . The volume o f
d i s t r i b u t i o n f o r chloramphenicol was r e p o r t e d t o b e
about 40 l i t r e s ( 7 4 ) .

5.2 Excretion

Chloramphenicol i s r e p o r t e d t o b e 5 - 15% e x c r e t e d
unchanged (73,102) w i t h r e p o r t e d r e n a l c l e a r a n c e o f
13-36 ml/min ( 7 3 ) . Renal l e v e l s may b e i n a d e q u a t e
t o treat urinary t r a c t i n f e c t i o n s e s p e c i a l l y i n t h e
presence o f moderately t o s e v e r e l y impaired r e n a l
f u n c t i o n ( 1 0 3 ) . Some nom-al p a t i e n t s and p a t i e n t s
w i t h impaired r e n a l f u n c t i o n e x h i b i t impaired f r e e
drug e l i n i n a t i o n ( 1 0 4 ) . The recovery o f f r e e drug
from t h e u r i n e i s d i r e c t l y p r o p o r t i o n a l t o c r e a t i n i n e
c l e a r a n c e . With c r e a t i n i n e c l e a r a n c e s o f l e s s t h a n
CHLORAMPHENICOL 745

20 ml/min, l e s s t h a n 1% o f t h e a d m i n i s t e r e d doses
t h a t are recovered i n t h e i n e r t i n a c t i v e form.
With c r e a t i n i n e c l e a r a n c e s of < 40 m l / m i n , u r i n a r y
c o n c e n t r a t i o n s o f t h e drug a r e g e n e r a l l y not h i g h
enough t o t r e a t s u s c e p t i b l e organisms ( 1 0 5 ) . The
s t e a d y s t a t e k i n e t i c s o f t h e o r a l p a l m i t a t e vers’is
t h e I V s u c c i n a t e s a l t w a s s t u d i e d (71). No s i g n i -
f i c a n t c o r r e l a t i o n between t h e dose of chlorampheni-
c o l s u c c i n a t e and serum c o n c e n t r a t i o n s o f ”free”
chloramphenicol o r t h e average u r i n a r y c o n c e n t r a t i o n
were found. However, t h e average u r i n a r y concentra-
t i o n and serum c o n c e n t r a t i o n of o r a l chloramphenicol
p a l m i t a t e c o r r e l a t e s w e l l w i t h dose i n d i c a t i n g more
complete and p r e d i c t a b l e h y d r o l y s i s . V a r i a b l e
f r a c t i o n s of t h e dose ( a mean of 36%) w a s e x c r e t e d
i n u r i n e unchanged and w a s t h e r e f o r e n o t b i o a v a i l a b l e
i n a c t i v e form. Both v a r i a b l e h y d r o l y s i s and r e n a l
e l i m i n a t i o n o f t h e nonhydrolyzed chloramphenicol
s u c c i n a t e seems t o reduce t h e b i o a v a i l a b i l i t y of t h e
a n t i b i o t i c and a p p e a r s t o c o n t r i b u t e s u b s t a n t i a l l y
t o t h e wide v a r i a t i o n s i n serum c o n c e n t r a t i o n s
produced f o l l o w i n g an I V dose.

I n premature i n f a n t s , a n i n c r e a s e d b i o a v a i l a b i l i t y
o f chloramphenicol was a result o f decreased r a t e of
clearance of t h e succinate salt causing a g r e a t e r
f r a c t i o n o f t h e s a l t dose t o be hydrolysed t o
chloramphenicol (106). With t h e d a t a c u r r e n t l y avail-
a b l e , chloramphenicol i s not a d v i s e d d u r i n g t h e
b r e a s t f e e d i n g p e r i o d . Chloramphenicol i s e x c r e t e d
i n t o breast m i l k , i n some i n s t a n c e s i n c o n c e n t r a t i o n s
which are 50% of blood l e v e l s ( 1 0 7 ) . S i n g l e 1 gm
o r a l doses produce peak milk l e v e l s at 3 h r , which
are u n d e t e c t a b l e a t 6 h r ( 1 0 8 ) . S i n g l e dose of
500 mg o r a l l y given every 6 h r produced serum l e v e l s
of 0.98 - 3.5 pg/ml i n b r e a s t milk ( 1 0 9 ) . Most
i n f a n t s do not have developed h e p a t i c c o n j u g a t i o n
system f o r g l u c u r o n i d a t i o n which could r e s u l t i n
t o x i c i t y . Although chloramphenicol milk l e v e l s a r e
not s u f f i c i e n t t o induce t h e grey-baby syndrome,
t o x i c i t y t o t h e bone marrow may occur ( 1 1 0 ) . Toxic
e f f e c t s i n i n f a n t s had been r e p o r t e d (111) d u r i n g t h e
b r e a s t feeding period.

5.3 Half-Life

I n normal, o t h e r w i s e h e a l t h y a d u l t s , t h e h a l f - l i f e o f
chloramphenicol r a n g e s from 1.6 - 3 . 3 h r w i t h an
746 ABDULLAH A. AL-BADR AND HUMEIDA A. EL-OBEID

average v a l u e o f 2.7 h r (102,112). I n r e n a l and

l i v e r f a i l u r e s t h e h a l f - l i f e i s a p p r e c i a b l y prolonged.
I n i n f a n t s and c h i l d r e n between t h e age o f 1 month
and 11 y e a r s , a mean apparent h a l f - l i f e w a s r e p o r t e d
(113) t o b e 5.94 h r . A n a l y s i s o f v a r i a n c e r e v e a l e d
a sample v a r i a n c e o f 21.85 i n c h i l d r e n l e s s t h a n
4 months o f age compared t o 5.87 i n c h i l d r e n g r e a t e r
t h a n 4 months o f age r e f l e c t i n g a h i g h l y v a r i a b l e
h a l f - l i f e i n t h e younger i n f a n t s . A s t u d y ( 1 1 4 ) o f
t h e pharmacokinet i c parameters o f chloramphenicol
s u c c i n a t e i n i n f a n t s and young c h i l d r e n r e v e a l e d t h a t
h a l f - l i f e o f serum chloramphenicol s u c c i n a t e d i d not
c o r r e l a t e with t h e h a l f - l i f e o f serum chloramphenicol.
Chloramphenicol s u c c i n a t e i s l o s t i n t o t h e u r i n e i n
s i g n i f i c a n t q u a n t i t y and t h i s u r i n a r y loss must b e
t a k e n i n t o account i n t h e e s t i m a t i o n o f chlorampheni-
c o l pharmacokinetic parameters. One d u r a t i o n o f
i n f u s i o n o f chloramphenicol s u c c i n a t e does not a f f e c t
t h e amount e x c r e t e d i n t h e u r i n e .

5.4 Metabolism

I n e a r l y s t u d i e s by Glazko e t a1 (73,115,116) d a t a
on t h e f a t e o f chloramphenicol i n d i f f e r e n t s p e c i e s
were produced. A major m e t a b o l i c r o u t e i n v o l v i n g
g l u c u r o c o n j u g a t i o n as w e l l as t h e r e d u c t i o n o f
t h e n i t r o group by t h e i n t e s t i n a l f l o r a and conjuga-
t i o n o f t h e r e s u l t i n g m i n e s were d e s c r i b e d i n r a t s ,
guinea p i g and dog. I n man, 90% o f a s i n g l e dose o f
o f t h e drug a p p e a r s i n t h e u r i n e w i t h i n 24 h r , c h i e f l y
as chloramphenicol-3-glucuronide ( 1 1 7 ) . The l i v e r
i s t h e main s i t e o f g l u c u r o n i d a t i o n . With t h e
development o f more s e n s i t i v e a n a l y t i c a l t o o l s o t h e r
m e t a b o l i t e s of chloramphenicol have been sugggest ed
(Scheme 4 ) . I n t h e i r s t u d y o f chloramphenicol meta-
bolism i n i s o l a t e d rat h e p a t o c y t e s S i l i c i a n o e t a1
(118) chloramphenicol-3-glucuronide w a s found t o b e
t h e major m e t a b o l i t e t o g e t h e r w i t h a minor m e t a b o l i t e
b e l i e v e d t o b e D( -) threo-2-amino-l-( p-nitropheny1)-
1,3-propanediol, The formation o f t h e 3-glucuronide
w a s l i n e a r w i t h r e s p e c t t o b o t h t h e c e l l concentra-
t i o n and t o t h e t i m e o f t h e f i r s t hour o f i n c u b a t i o n .
The Ic, and Vmax v a l u e s f o r t h e g l u c u r o n i d a t i o n o f
chloramphenicol were 6.4 x 10-6 M and 420 pmol/min/l08
c e l l s r e s p e c t i v e l y . The k i n e t i c s o f t h e glucuronida-
t i o n r e a c t i o n i n r a t hepatocytes suggest a low hepa-
t i c e x t r a c t i o n r a t i o o f chloramphenicol ,
 CH,- OH
I YH2 OC 6 H9 7 CH OH ,
H,N@ VH-CH-NHCOCHCl, O,N-@ FH-CH-NHCOCHC1 o,N@ FH-;H-NH-CCOOH
11
OH OH
I OH 0
FH2OH
dog. O,N@ FH-CH-NHCOCH2 OH
OH
CH, OH
CHO O,N@ FH-CH-NH,
I

OH
yH2 OC 6 H9 7
0
0
O2N
-0- FH-CH-NHCOCHC12
OH
II
FH2OH
H-C-CH,NHCOCHC~,--€ yH2OH
H,N @-F H - c H - ~ ~ o c H c ~ ,
, O,N -@ YH-CH-NHCOCHC1, 0
OH
yH2OH
f--+ OH-CH,CH,-NHCOCHCl, OH
CH,?HN -
@
:
C
H
-
NO
C
H
~
,
yH20H R a t hepato- - .*

yH2 OC 6 H9 7
02N@ ~H-CHNHCOCH2C1 0,N -@:H-CH-NIICOCHCl
OH OH
CH, OH
0,N -@FH-CH-NH, I

OH
Scheme 4. Metabolites o f chloramphenicol i n i n vivo and i n v i t r o s t u d i e s .
748 ABDULLAH A. AL-BADR AND HUMEIDA A. EL-OBEID

The u s e o f chloramphenicol h a s been shown t o cause

bone marrow d e p r e s s i o n . T h i s t o x i c i t y i s u s u a l l y
r e v e r s i b l e i f t h e drug i s d i s c o n t i n u e d , b u t i n r a r e
c a s e s (1 i n 20,000) p a t i e n t s develop a p l a s t i c anemia
a bone marrow d i s e a s e which i s o f t e n i r r e v e r s i b l e
and f a t a l ( 1 1 9 ) . Many mechanisms have been suggested
(120-126) t o account f o r t h i s chloramphenicol-induced
t o x i c i t y , but have not been unequivocally e s t a b l i s h e d .
T h i s rare i n c i d e n c e o f chloramphenicol t o x i c i t y
suggested t o Pohl and Krishna (127) t h a t a minor
a c t i v e m e t a b o l i t e may be involved i n i t s i n d u c t i o n .
Using a cytochrome P-450 enzyme system i n l i v e r
rnicrosomes o f r a t s , t h e y s t u d i e d t h e mechanism o f t h e
metabolic a c t i v a t i o n o f chloramphenicol by measuring
t h e c o v a l e n t b i n d i n t o microsomal p r o t e i n s of s p e c i -
,f
f i c a l l y l a b e l l e d [l C ] and [ 3 H ] d e r i v a t i v e s o f
chloramphenicol. The l a c k o f b i n d i n g o f d i c h l o r o -
a c e t i c a c i d , chloramphenicol base (2-amino-l-( p-
nitrophenyl)-1,3-propanediol), and t h e acetamido and
t r i f l u o r o a c e t amido d e r i v a t i v e s of chloramphenicol
i n d i c a t e s t h a t t h e dichloroacetamido group is required
f o r a c t i v a t i o n . The b i n d i n g o f dichloroacetamide
support t h i s c o n c l u s i o n . Moreover, t h e C-H bond o f
t h e dichloromethyl carbon of chloramphenicol a p p e a r s
t o be broken i n t h e a c t i v a t i o n p r o c e s s s i n c e t h e
hydrogen i s l o s t i n c o v a l e n t b i n d i n g . Accordingly,
a mechanism (Scheme 5 ) i s proposed i n which chloram-
p h e n i c o l i s a c t i v a t e d by h y d r o x y l a t i o n o f t h e
dichloracetamido group followed by spontaneous de-
h y d r o c h l o r i n a t i o n t o a n oxamyl c h l o r i d e which a c y l a t e s
microsomal p r o t e i n s . R e c e n t l y , it has been shown t h a t
cytochrome P-450 i s t h e predominant p r o t e i n i s l i v e r
microsomes t h a t i s a c y l a t e d by t h e oxamyl c h l o r i d e
( 1 2 8 ) . Moreover , t h e c o v a l e n t l y modified cytochrome
P-450 a p p e a r s t o b e i r r e v e r s i b l y i n a c t i v a t e d as a
mixed-function o x i d a s e ( 1 2 9 , 1 3 0 ) .

Morris e t a1 ( 1 3 1 ) have f u r t h e r c h a r a c t e r i z e d t h e
o x i d a t i v e metabolism o f chloramphenicol and have
found a new pathway f o r t h e o x i d a t i v e metabolism of
chloramphenicol i n a d d i t i o n t o t h e o x i d a t i v e dehalo-
g e n a t i o n r e a c t i o n o u t l i n e d above. According t o t h e s e
a u t h o r s , when chloramphenicol was incubated w i t h r a t
l i v e r microsomes, four p r e v i o u s l y u n i d e n t i f i e d meta-
b o l i t e s were d e t e c t e d and i d e n t i f i e d . They i n c l u d e
chloramphenicol aldehyde, p-nitrobenzyl a l c o h o l , N-
(2-oxoethyl) d i c h l o r o a c e t a m i d e , and N-( 2-hydroxyethyl)
0,@TI
OH
CH, OH
I
Nucleophil i c
fH-CH-NHCOCONu-P 0, N a
P r o t e i n ( P-Nu)
H: -
CH, OH
I -
CH
OH p H C 1 OH
CH, OH
I

CH,OH OH
I I
O,N -@ CH-CH- NHCO-C-C1
I I
OH c1
P-450, NADPH,

1 02NaFH-!!H
OH
CHzOH 1-

- NHCOCHCl,

I Retroaldol
OH Reduction condensat i o n
Reduct i o n
ozN*EH2 02N@ CHO + OHC-CH2NHCOCHC12 +
-* HO-CH2CH2NHCOCHC1,

Scheme 5. Mechanism o f chloramphenicol o x i d a t i v e pathways by r a t l i v e r microsomes (127, 131).

750 ABDULLAH A. AL-BADR AND HUMEIDA A. EL-OBEID

dichloroacetamide. The formation of t h e s e metabo-

l i t e s was dependent upon t h e presence of NADPH and
02 and w a s uninhibited when SKF' 5258 or CO/O2
(8 : 2, v / v ) were present i n t h e r e a c t i o n mixture.
Moreover, t h e metabolites were formed by l i v e r
microsomes from phenobarbital-treated rats but not
by microsomes from untreated rats or rats t r e a t e d
with 6-naphthoflavone. The formation of t h e s e meta-
b o l i t e s i s c o n s i s t e n t with a mechanism t h a t involves
an i n i t i a l oxidation of chloramphenicol t o chloram-
phenicol aldehyde by cytochrome P-450. The metabolite,
being a 0-hydroxyaldehyde, can chemically undergo
a retro-aldol cleavage t o p-nitrobenzaldehyde and
N( 2-oxoethyl)dichloroacetamide. Enzymatic reduction
of t h e s e aldehyde intermediates would y i e l d p-nitro-
benzyl alcohol and N-( 2-hydroxyethyl) dichloro-
acetamide, r e s p e c t i v e l y (Scheme 5 ) . I n t h e above
study by Pohl and Krishna (127) it w a s observed t h a t
only a 58% decrease i n covalent binding of chloram-
phenicol metabolites t o microsomal p r o t e i n occurred
when r e a c t i o n s were conducted i n an atmosphere of
nitrogen. A t t h e time, it was f e l t t h a t t h e reason
t h e covalent binding w a s not decreased t o an even
g r e a t e r extent w a s because of i n s u f f i c i e n t deoxygena-
t i o n of t h e incubation mixtures. I n a f u r t h e r study
by Morris et a1 (132), however, it i s shown t h a t a t
low oxygen t e n s i o n , chloramphenicol i s a c t i v a t e d by
reductive dechlorination pathways of metabolism.
Thus, when chloramphenicol was incubated with rat
l i v e r microsomes anaerobically, it was metabolized
predominently t o deschloro-chloramphenicol and
products t h a t become i r r e v e r s i b l y bound t o microsomal
p r o t e i n . Cytochrome P-450 induced by phenobarbital
appeared t o c a t a l y s e t h e s e r e a c t i o n s 'most e f f e c t i v e l y .
Glutathione increased t h e formation of deschloro-
chloramphenicol by 13%and decreased t h e amount of
i r r e v e r s i b l y bound product by 18%. Only small amount
of t h e nitroaromatic-reduced product, chloramphenicol
m i n e , w a s detected by HPLC. The r e s u l t s a r e consis-
t e n t with t h e drug being biotransformed and a c t i v a t e d
by cytochrome P-450 anaerobically through predomi-
nently reductive dechlorination. A proposed mechanism
f o r t h e reductive dechlorination i s shown i n Scheme 6.

Bories e t a1 (133) developed a simple and ion-pair

reverse phase high performance l i q u i d chromatographic
separations combined with s e l e c t i v e e x t r a c t i o n i n
 L-450, Fe ++

pc1-
NHZCOCH
Covalent b i n d i n g 4
.
- 02N@CH-CH-CH20H
I
I
OH

Scheme 6. A proposed mechanism for t h e r e d u c t i v e d e c h l o r i n a t i o n o f c h l o r m p h e n i c o l by rat

l i v e r microsomes ( 1 3 2 ) .
75 2 ABDULLAH A. AL-BADR AND HUMEIDA A. EL-OBEID

order t o achieve an improved a n a l y t i c a l t o o l for

chloramphenicol metabolic p r o f i l i n g . Q u a n t i t a t i o n
w a s achieved by summation of r a d i o a c t i v i t y values
f o r f r a c t i o n s belonging t o t h e same peak. Metabo-
l i t e s of chloramphenicol were i d e n t i f i e d by electron-
impact and chemical i o n i z a t i o n mass spectrometry.
The study l e a d t o t h e confirmation 'of previously
suggested chloramphenicol metabolites as well as t h e
i d e n t i f i c a t i o n of new metabolites.

Acknowledgement

t h e authors would l i k e t o thank M r . Tanvir A . Butt for typing

this manuscript.
CHLORAMPHENICOL 753

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 LIDOCAINE AND LIDOCAINE HYDROCHLORIDE

Michael F. Powell

1. Introduction

2. Description
2.1 Nomenclature, Formulas, M o l e c u l a r Weights and CAS
Numbers

3. P h y s i c a l , Chemical and S p e c t r a l P r o p e r t i e s
3.2 Solubility
3.3 D i s s o c l a t l o n Constant
3.5 I n f r a r e d A b s o r p t i o n Spectra
3.6 N u c l e a r Magnetic Resonance S p e c t r a
3.9 X-ray C r y s t a l S t r u c t u r e
3.10 X-ray D i f f r a c t i o n
3.11 Hygroscopicity
3.1 2 Self Association

6. Stability
6.2 P h y s i c a l S t a b i l i t y i n P a r e n t e r a l and I V S o l u t i o n s
6.3 Photochemical S t a b i l i t y

7. Pharmacokinetics
7.1 Plasma C o n c e n t r a t l o n s a f t e r D i f f e r e n t Routes o f
Admi n i s t r a t ion
7.3 B l o t r a n s f o r m a t i o n and E l i m i n a t i o n

8. Methods o f A n a l y s i s
8.1 Reverse Phase HPLC
8.2 Normal Phase HPLC
8.3 T h i n Layer Chromatography

10. References
ANALYTICAL PROFILES OF DRUG SUBSTANCES Copyright Q 1986
VOLUME 15 by the American Pharmaceutical Association
761 All rights of reproduction in any form reserved.
762 MICHAEL F. POWELL

1. I nt roduct 1on
Lidocaine i s widely used as a local anaesthetic and for the
management of cardiac arrhythmas, particularly those
assoclated with acute myocardlal infarction.
The following supplement contains updated Information
pertalning to the analytical chemlstry of lidocaine free
base and lldocalne hydrochloride. A llterature survey was
conducted and i s complete up to December 1985. The numbering
system for topics dlscussed i s the same as that I n the
original profile1 (see Volume 14, pages 207-243).

2. Description
2.1 Nomenclature. Molecu'lar Weights and CAS Numbers
The structural formula for lldocalne i s given below.

A plethora of names for lldocalne have been used In the

recent literature, for example: llgnocalne, 2-(diethyl-
am1no)-N-( 2,6-dlmethylphenyl) -acetamlde, 2-(diethylamino)-
2',61-acetoxyidide, N,N-diethyl-(2,6-xylylcarbamoyl)-
methylamine and 2-diethylaminoaceto-2 I ,6 I-xyl idide. The
hydrochloride salt i s named similarly but with the added
suffix I'hydroch 1 or1 dell or "hydrochl or1 de monohydrate".
Trade names for lidocaine are: Lidocitin, Leostesin,
Xylocalne, Xylotox, Xylestesin, Xylocitln, Rucaina,
Duncalne, Islcalne and Anestacon.
Table 1. Descrlptlon o f Lldocalne and its Hydrochloride Salts

FORM Free Base Salt Salt Monohydrate

Formula C14H22N20*HC 1
1' 4H22N20
MW 234.34 270.80 288.81
%C,H,N 71.75,9.46,11.76 62.09,8.56,l0.34 58.22.8.72.9.70
CAS Number 137-58-6 73-78-9 61 08-05-0
LIDOCAINE AND LIDOCAINE HYDROCHLORIDE 763

3. P h y s i c a l and Chemical P r o p e r t i e s

3.2 Solubility

The s o l u b i l i t y o f l i d o c a i n e f r e e base i n aqueous s o l u t i o n i s

unusual i n t h a t i t decreases as t h e temperature i n c r e a s e s
( T a b l e 2) . 2 T h i s i n v e r s e temperature - s o l u b i l i t y
r e l a t i o n s h i p has a l s o been v e r i f i e d by others.3-5 The pH
dependence on t h e aqueous s o l u b i l i t y o f l i d o c a i n e can be
c a l c u l a t e d f r o m t h e e q u a t i o n ST = S t b ( 1 + lO(PKa-PH)) where
ST and Sfb a r e t h e t o t a l and f r e e base s o l u b i l i t i e s ,
r e s p e c t i v e l y . 6 The c a l c u l a t e d s o l u b i I i t y o f l i d o c a i n e a t
14.9", 25" and 37°C a r e shown i n F i g u r e 1. The s o l u b i l i t y
o f l i d o c a i n e and l i d o c a i n e h y d r o c h l o r i d e 7 i n v a r i o u s s o l v e n t s
a r e g i v e n i n Table 3.

Table 2. Free Base S o l u b i l i t y and pK, Values o f L i d o c a i n e

a t Various Temperatures

2
Temperature ( " C ) S o l u b i l i t y (mg/mL)

10.0 8.24
14.9 4.33 0.12
25.0 7.92 3.81 + 0.02
34.5 3.42 2 0.02
37.0 3.36 5 0.02
38.0 7.57

I I I I I 1 I I I 4
7.5 0 0.5 9 9.5
PH
F i g u r e 1. C a l c u l a t e d pH dependence o f t h e aqueous s o l u b i l i t y
o f l i d o c a i n e a t 1 4 . 9 " , 25" and 37°C. A t pH 7.4, t h e
c a l c u l a t e d s o l u b i l i t i e s o f l i d o c a i n e a t 25" and 37°C a r e
16.4 and 8.7 mg/mL, r e s p e c t i v e l y .
764 MICHAEL F. POWELL

Table 3. S o l u b i l i t y of Lidocalne Free Base and Lldocaine

Hydrochloride a t 25°C i n Various Solvents

so Iu b i 1 it y (mg/mL)
So 1 vent
Lidocaine HC I Free Base

Methanol 67 >500
Ethanol 11 >500
Acetone 1.8 >500
Chloroform 4
Ether <o. 01 -500
Carbon t e t r a c h l o r i d e 0.01 >so0

3.3 D i s s o c i a t i o n Constant

The pKa o f l i d o c a i n e has been measured by p o t e n t i o m e t r i c

t i t r a t i o n a t several temperatures a t i o n i c s t r e n g t h 0.05 M
(KC1),8 and t h e data are shown i n Table 2. The i o n i c
strength, when adjusted w i t h KC1 from 0.005 t o 0.077 M,
showed l i t t l e e f f e c t on t h e a c i d i t y c ~ n s t a n t . ~The pH o f
a 5% (w/v) s o l u t i o n o f l i d o c a i n e hydrochloride i s
approximately 4.0 t o 5.5.

3.5 I n f r a r e d Absorption Spectra

The I R spectra o f l i d o c a i n e and l i d o c a i n e h y d r o c h l o r i d e were

measured i n chloroform using a Sargent Welsh Model S-200 I R
spectrometer (Figure 2 ) . Note t h a t most o f t h e s t r e t c h i n g
frequencies are s h i f t e d upwards than those obtained i n K B r
discs’. The s t r u c t u r a l assignments have been c o r r e l a t e d
w i t h t h e f o l l o w i n g band assignments (Table 4).

Table 4. I R Band Frequencies (cm-’) f o r Lidocaine and

Lidocaine Hydrochloride Measured i n CDC13.

Assignment Lidocaine L i d o c a i ne Hydrochloride

N-H S t r e t c h 3290 3180

C-H S t r e t c h 2820 -
C-H S t r e t c h 2970 2978
Amide I,C=O 1670 1682
Amide 11, C-N 1495 1525
Fingerprint 81 2 949
region 900 978
LIDOCAINE AND LIDOCAINE HYDROCHLORIDE 765

3000 2500 2000 1600 1200 800

Wave Number, cm - l

I I T I I
B

3000 2500 2000 1600 1200 800

Wave Number, cm - l

F i g u r e 2 . I R s p e c t r a o f a) l i d o c a i n e and b) l i d o c a i n e
h y d r o c h l o r i d e i n CHC13.
766 MICHAEL F. POWELL

3.6 Nuclear Magnetic Resonance Spectra

The 90 MHz p r o t o n and 75.5 MHz 13C-NMR s p e c t r a f o r l i d o c a i n e

h y d r o c h l o r i d e i n CDC13 a r e shown i n F i g u r e 3. A sumnary
o f t h e p r o t o n chemical s h i f t s as r e f e r e n c e d t o TMS a r e g l v e n
i n Table 5 and t h e 1%-chemical s h i f t s and r e l a x a t i o n times
a r e g i v e n i n Table 6. The numbering i s as shown e a r l i e r i n
t h e s t r u c t u r a l formula. The 14N-NMQR spectrum has a l s o
been reported.

Table 5. Summary o f Proton Chemical S h i f t s and Assignments

f o r Lidocaine H y d r o c h l o r i d e i n CDC13a
~~

Assignment Chemical S h i f t (ppm)

C3-H 7.02 (m)

C4-H 7.02 (m)
Ar-Cb 2.21 ( s )
N-tj 10.24 (s-broad)
N-C( 0) -CHz-N 4.22 ( 5 )
N-CHz-CHs 3.60 ( 9 ) J=7.20 HZ
N-CHz-CH3 1.42 (t) 5-7.20 HZ

as = s i n g l e t , t = t r i p l e t , q = q u a r t e t , m = m u l t i p l e t

Table 6. Surmary o f 13C-NMR Data f o r L i d o c a i n e H y d r o c h l o r i d e

Re1a x a t i on Timea Chemlcal S h i f t

Assignment (s) (PPN
N-C( 0) -CHz-N 11.8 162.9
c2, c6 20.1 135.1
C1 20.1 133.1
c3, c 5 1.4 128.1
c4 1 .o 127.5
N-C( O)-CHz-N 1 .o 51 .O
N-CH2CH3 1.2 49.1
Ar-CH3 3.1 18.7
N-CHzCH3 2.0 10.0

a L i d o c a i n e i n CDC'l3 a t 32°C I n undegassed s o l u t i o n ,

Reference 11.
LIDOCAINE AND LIDOCAINE HYDROCHLORIDE 767

10 8 6 4 2 0
PPm

I
I I I I I I
150 120 90 60 30 0
PPm

Figure 3 . Proton NMR and 13C-NMR spectra o f lidocaine

hydrochloride in C D C 1 3 .
768 MICHAEL F. POWELL

3.9 X-Rav Crvstal Structure

The crystal structure of lidocalne free base was determjned
from a single crystal grown from dlmethylformamlde - water
solution at -2OOC.12 Cell dimensions were a = 13.24 A,
b = 14.06 A, c = 19.25 A and U = 123.7" at 20°C. Both of
the independent molecular conformations observed were I n the
trans-amide configuration.
X-ray crystal analysis of lidoca ne hydrochloride was
determined from a single crystal grown in acetone-
ethy1a~etate.l~ The translucent prisms were of high
mosaicity and gave the followlng cell parameters: a = 8.49 A ,
b = 7.11 A, c = 27.58 A and U = 06.87".

3.10 X-Ray Diffraction

The X-ray powder diffraction patterns of lidocalne and
lidocaine hydrochloride are shown in Figures 4a and 4b,
respectively, and a sunmary of the scannlng angles and
relative Intensities are given in Table 7. The data were
collected uslng a Nlcolet X-ray dlffractometer equipped with
a fine focus X-ray tube and a diffracting beam mono-
chromator. The scanning angle was from 3" to 30" 28 at
O.O5"/second.

Table 7. X-Ray Powder Dlffractlon Data - Scanning Angles and

Relative Intensities for Lidocaine and Lidocaine Hydrochloride

Lldocai ne Lidocaine Hydroch lorlde

Degrees 28 Relative Intensity Degrees 28 Relative Intensity

10.5 100 20.2 100

12.7 100 13.5 97
12.5 46 16.5 92
10.0 37 25.4 66
8.0 13 27.1 55
14.3 12 6.7 49
15.1 12 14.3 44
LIDOCAINE AND LIDOCAINE HYDROCHLORIDE 769

I I I I I
5 10 15 20 25
Degrees 20

I I I I I
5 10 15 20 25
Degrees 20
Figure 4. X-ray diffraction pattern of a) lidocaine and
b ) lidocaine hydrochloride.
770 MICHAEL F. POWELL

3.11 Hysroscopicity

L i d o c a i n e f r e e base i s n o t hygroscopic. F o r example,

l i d o c a i n e f r e e base does n o t absorb w a t e r even a t 92% RH a t
room temperature. The anhydrous HC1 s a l t however, does
adsorb 1 mole o f w a t e r p e r mole o f l i d o c a l n e below 80%
r e ' l a t l v e h u m i d i t y a t 25°C. A t 93% r e l a t i v e h u m i d i t y , more
t h a n two moles a r e a d ~ 0 r b e d . l ~L i m i t e d h y g r o s c o p i c i t y
d a t a f o r l i d o c a i n e h y d r o c h l o r i d e f r o m 22% t o 90% r e l a t i v e
hum1d i t y has a 1 so been r e p o r t e d .

3.12 Self Absociation

L i d o c a i n e i n s o l u t i o n depresses o n l y s l i g h t l y t h e s u r f a c e
t e n s i o n o f w a t e r and thus, does n o t f o r m mice'l'les t o any
measurable e x t e n t . 1 6 However, l i d o c a i n e has been r e p o r t e d
t o f o r m charge t r a n s f e r complexes, f o r example w i t h
t r i n i t r o b e n z e n e o r ch l o r o p r o m a ~ i n e . ~ ~

A c o n t r o v e r s y r e g a r d i n g i n t e r versus i n t r a m o l e c u l a r hydrogen
bonding i n l i d o c a i n e emerged i n t h e e a r l y 7 0 ' s and appears
now t o be r e s o l v e d I n f a v o r o f t h e l a t t e r . An e a r l i e r
p u b l i c a t i o n l a p o s t u l a t e d t h e a c y l i c cis-amide c o n f i g u r a -
t i o n based on I R s t u d l e s , however, subsequent I R s t u d i e s 1 9
and NMR r e s u l t s 2 0 r e b u t t e d w i t h s t r o n g evidence f o r i n t r a -
m o l e c u l a r trans-amide hydrogen bonding as shown i n F i g u r e 5.

N
E< 'Et CIS TRANS

F l g u r e 5. Cis- and trans-amide c o n f i g u r a t i o n s o f l i d o c a i n e .

LIDOCAINE AND LIDOCAINE HYDROCHLORIDE 77 1

6. Stability

6.2 P h v s l c a l S t a b l l l t y I n P a r e n t e r a l and I V S o l u t i o n s

Some I i d o c a i n e p r e p a r a t i o n s a r e p h y s i c a l l y u n s t a b l e r e s u l t i n g
i n s o l u t i o n c l o u d i n e s s , p r e c i p i t a t i o n o f drug, o r l o s s o f
d r u g potency i n s o l u t i o n . P r e c i p i t a t i o n o r c l o u d i n e s s i s
u s u a l l y caused by o t h e r a d d i t i v e s which may complex w i t h
l i d o c a i n e o r change t h e s o l u t i o n pH. Loss o f d r u g potency
i n s o l u t i o n i s n o t due t o chemical I n s t a b i l i t y , b u t i s
caused by a d s o r p t i o n o f l l d o c a i n e t o t h e c o n t a l n e r s u r f a c e ,
e s p e c i a l l y when p a r e n t e r a l o r I V s o l u t l o n s a r e s t o r e d i n
p l a s t i c containers (Table 8).

Table 8. S t a b i l i t y of Lidocaine i n Parenteral Solutions

So 1 u t ion Container Time Temp. % Reference

(days) ("C) Remaining

5% d e x t r o s e plastic 120 4 99.0 2 1.6 21

plastic 120 30 100.2 2 1 . 7 I1

0.9% sodium glass 14 25 100.9 5 0.8 22

chloride plastic 14 25 101.9 5 0.8 II

i n j e c tiona

0.45% sodium glass 14 25 94.3 2 0.3 II

c h l o r i d e b and plastic 14 25 98.6 2 0.9 I1

5% d e x t r o s e b

Lactated r i n g e r s glass 14 25 101.9 2 0.9 II

solutiona plastic 14 25 101.9 2 1.7 II

5% d e x t r o s e and glass 14 25 94.7 f. 0.8 I1

lactated ringers plastic 14 25 101.9 2 0.9 II

solutiona

Cardioplegic glass 21 22 90.1 f. 2.0 23

solutionb plastic 21 22 25.6 5 1.1 I1
II
glass 21 4 100.0 f. 2.2
plastic 21 4 89.1 2 5.8 I1

aAdmixture w i t h a m l n o p h y l l i n e , b r e t y l ium t o s y l a t e , c a l c i urn

gluconate, d i g o x l n , dopamine h y d r o c h l o r i d e , r e g u l a r I n s u l i n ,
p h e n y t o l n sodium and procainamide h y d r o c h l o r i d e .
bWith l i d o c a i n e h y d r o c h l o r l d e , K C I , NaHC03, d e x t r o s e and NaCl.
772 MICHAEL F. POWELL

The f i r s t comprehensive paper on t h e c o m p a t i b l l l t y o f

l i d o c a i n e i n glass and p l a s t i c containers c o n t a i n i n g 5%
dextrose i n s a l i n e , normal s a l i n e , o r 'lactated r i n g e r s
s o l u t i o n reported a s l i g h t loss o f l i d o c a i n e concentration
a f t e r o n l y twenty-four hours; t h i s decrease was a t t r i b u t e d
t o experimental error.24 Subsequent r e p o r t s showed t h a t
lldocaine i s stable i n parenteral solutions21 providing
c e r t a i n admixtures are n o t includedZ2.

It has a l s o been demonstrated r e c e n t l y t h a t adsorption t o

p o l y v i n y l c h l o r i d e bags may occur, e s p e c i a l l y a t room
temperature. 21

6.3 Photochemical S t a b i l i t y

The weak UV-visible absorption o f l i d o c a i n e a t A = 262 nm

has o n l y a small molar a b s o r p t i v i t y o f 470 t4-l cm-l and
so l i d o c a i n e i s n o t photoreactlve. In our l a b o r a t o r y ,
Rayonet i r r a d i a t i o n o f a 5 x loW5 H aqueous s o l u t i o n of
l i d o c a i n e f o r 10 days r e s u l t e d I n o n l y 5% l o s s o f drug.
Since t h e Rayonet r e a c t o r (Rayonet Model No. RPR-100) has
been shown t o a c c e l e r a t e most r e a c t i o n s by a f a c t o r of
100-200 times over i n d i r e c t window ' l i g h t , t h e c a l c u l a t e d
s h e l f - l i f e o f l l d o c a i n e exposed t o i n d i r e c t window l i g h t I s
approximately e i g h t years.
7. Pharmacokinetics

7.1 Plasma Concentrations a f t e r D i f f e r e n t Routes o f

Admi n i s t r a t 1on

Lidocaine e x h i b i t s r a p i d and r e l a t i v e l y h i g h absorption

a f t e r o r a l a d m i n i s t r a t i o n . However, i t I s n o t c l i n i c a l l y
u s e f u l when taken o r a l l y due t o extensive f i r s t - p a s s
metabolism and I t s s h o r t b i o l o g i c a l h a l f - l i f e . I t was
reported t h a t a f t e r o r a l a d m i n i s t r a t i o n o f l i d o c a i n e t o
r a t s , dogs and man, o n l y 0.2%, 2.0% and 2.8%, r e s p e c t i v e l y ,
was recovered unchanged i n t h e urine25 (Table 9 ) . The
mean apparent o r a l absorption o f l i d o c a i n e i n normal
subjects was approximately 35%,26 i n c l o s e agreement w i t h
t h e reported hepatic e x t r a c t i o n r a t i o . 2 7 This i s c o n t r a r y
t o an e a r l i e r r e p o r t i n which t h e c o l o r i m e t r i c method used
f o r a n a l y s i s may n o t have been s p e c i f i c f o r unmetabolized
lidocaine.28 A p r e d i c t i o n o f t h e b i o a v a i l a b i l i t y using
o r a l clearance data has a l s o been offered.29 Plasma
l e v e l s obtained from i n g e s t i n g 500 mg o f l i d o c a i n e appeared
t o be less than t h e l e v e l s r e q u i r e d t o cause an
antiarrhythmic e f f e c t .
LIDOCAINE AND LIDOCAINE HYDROCHLORIDE 773

Table 9. T i s s u e D i s t r i b u t i o n A f t e r L i d o c a i n e p.0. Dosing o f

10 mg/kg t o Rats.

Total Radioactivity Unchanged L i d o c a i n e a

Tissue
0.5 h 2 h 24 h 0.5 h 2 h 24 h

Stomach 13.7 3.3 0.0 14.1 2.7 0.0

Intestine 27.9 45.3 6.9 2.4 0.3 0.0
B 1 ood 2.8 2.1 1.6 0.2 0.0 0.0
Feces 0.0 0.2 0.5 n.d. n.d. n.d.
Brain 0.6 0.3 0.1 0.0 0.0 0.0
Liver 11.1 3.4 1.1 0.3 0.0 0.0
Spleen 0.4 0.0 0.0 0.1 0.0 0.0
Kidney 2.0 0.8 0.2 1.3 0.1 0.0
Heart 0.2 0.1 0.5 0.0 0.0 0.0
Lung 1.1 0.3 0.3 0.2 0.0 0.0
Carcass 40.4 24.2 20.8 b b b
U r i ne 6.1 24.6 73.0 0.3 0.5 0.3

aUnchanged l i d o c a i n e as c a l c u l a t e d f o r t h e h y d r o c h l o r i d e
s a l t . h o t determined.

Intravenous a d m i n i s t r a t i o n o f l i d o c a i n e t o r a t s r e s u l t s i n
r a p i d uptake by t h e h i g h l y p e r f u s e organs such as t h e l i v e r
(which i s c o n s i d e r e d t h e p r i m a r y m e t a b o l i c s i t e ) , h e a r t ,
lungs, b r a i n and kidneys ( T a b l e 9 ) .25 The e l i m i n a t i o n
h a l f - l i f e o f l i d o c a i n e i n r a t s i s 30 minutes and i n dogs i s
45-60 minutes.

A f t e r I V i n j e c t i o n , t h e mean h a l f - l i f e i n plasma o f normal

s u b j e c t s I s a p p r o x i m a t e l y 6 minutes and t h e e l i m i n a t i o n
h a l f - l i f e is -100 minutes.30 A r a p i d I V i n j e c t i o n of
160 mg ' l i d o c a i n e i n t o normal s u b j e c t s f o l l o w e d by a 4 mg/min
i n f u s i o n r e s u l t e d i n a mean i n i t i a l plasma l e v e l of'
2.6 ug/mL which decreased t o a steady s t a t e plasma l e v e l
o f 2-4 ug/mL a f t e r a p p r o x i m a t e l y 20 minutes. It has been
shown u s i n g averaged d a t a f r o m s e v e r a l p a t i e n t s t h a t an
i n i t i a l b o l u s o f 125 mg and an i n f u s i o n o f 0.8 mg/mL
m a i n t a i n e d plasma l e v e l s o f 1 pg/mL i n a 70 kg man. When
i n f u s i o n was g i v e n alone, plasma l e v e l s o f l i d o c a l n e
i n c r e a s e d u n t i l a p l a t e a u was reached, 1.e. when i n p u t
equaled o u t p u t . T h i s u s u a l l y t o o k t h r e e t o f o u r h a l f - l i v e s
o r f r o m f i v e t o seven hours. E f f e c t i v e c o n c e n t r a t i o n s o f
l i d o c a i n e f o r l o c a l anaesthesia were achieved f o r
a p p r o x i m a t e l y one h o u r by a s i n g l e 200 mg i n t r a m u s c u l a r
i n j e c t i o n . 31
774 MICHAEL F. POWELL

L i d o c a i n e proved t o be s l i g h t l y more b i o a v a i l a b l e when

a d m i n i s t e r e d r e c t a l l y r a t h e r t h a n o r a l l y . 3 2 T h i s drug I s
a l s o absorbed w e l l through i n t a c t mucous membranes, s k i n and
damaged t i s s u e . 3 3 The s k i n p e r m e a b i l i t y was g r e a t l y
enhanced i n i n - v i t r o experlments by t h e a d d i t i o n o f
N ,N-di ethyl-m-to1 uamide .34

A r e v i e w o f t h e c l i n i c a l harmacokinetics o f l i d o c a i n e has
been p u b l i s h e d r e ~ e n t l y ,and ~ ~ a synopsls o f e f f e c t s w i t h
r e s p e c t t o pregnancy and t h e neonate appears r e g u l a r l y I n
M a r t i n d a l e , The E x t r a P h a r m a ~ o p o e i a . ~

7.3 B l o t r a n s f o r m a t i o n and E l i m i n a t i o n

Two o f t h e f i r s t - f o r m e d m e t a b o l i t e s , e t h y l g l y c l n e x y l i d i d e
and g l y c i n e x y l i d i d e , showed 83% and 10% of t h e
.
a n t i a r r h y t h m i c a c t i v i t y o f 1i d o c a i ne, r e s p e c t i v e l y 36 The
a r y l - h y d r o x y l a t e d analogues o f these compounds were a I s 0
formed and were u s u a l l y found as acld-hydro1 zab'le
' c o n j u g a t e s ' .25 It was o r l g l n a l l y proposed3r t h a t
N-deethyl a t 1on preceded hyd r o x y l a t l on b u t r e c e n t stud1 es i n
r a t s have shown t h a t 1) b o t h N,N-diethyl and N-monoethyl
g l y c l n e a r e formed, 11) l i d o c a i n e and m o n o e t h y l g l y c l n
e x y l l d l d e show c o m p e t i t i v e and, Ill)t h e
3 - h y d r o x y l a t l o n and N - d e e t h y l a t i o n a r e c a t a l y z e d by
d i f f e r e n t P450-dependent enzymes ,39940 The m e t a b o l i c
p r o d u c t d i s t r l b u t i o n s i n r a t s , guinea p i g s , dogs, and man
a r e shown i n Table 10.

Table 10. Summary o f M e t a b o l i c Products o f L i d o c a i n e

% Dose Recovered I n 24 h Urinea

Compound
Rat G. P i g Dog Man

Lidocalne 0.2 0.5 2.0 2.8

Ethylglycinexylldlde 0.7 14.9 2.3 3.1
Glyclnexylidide 2.1 3.3 12.6 2.3
3-Hydroxy-1 i d o c a i ne 31.2 0.5 6.7 1.1
3-Hydroxy-ethylglyclnexylidide 39.6 2.0 3.1 0.3
2,6-Xyl i d i n e 1.5 16.2 1.6 1.0
4-Hydroxy-2,6-xy.l i d i n e 12.4 16.4 35.2 72.6
Total 85.0 53.8 65.5 83.8

".O. doses g l v e n t o r a t , guinea p i g , dog and man were 20,

20, 10 and 3 mg/kg, r e s p e c t i v e l y . Because of' t h e r e ' l a t l v e l y
l a r g e d i f f e r e n c e s i n m o l e c u l a r weights o f t h e ' l i d o c a i n e
m e t a b o l i t e s , doses and r e c o v e r i e s were determined as molar
c o n c e n t r a t i o n s o f t h e f r e e bases.
LIDOCAINE AND LIDOCAINE HYDROCHLORIDE 77s

8. Methods o f A n a l y s i s

8.1 Reverse Phase HPLC

Although t h e a n a l y s i s o f i o n i c and s t r o n g l y b a s i c drugs by

RP-HPLC i s n o t w i t h o u t problems such as 'long s o l u t e
r e t e n t i o n o r p e a k - t a i l i n g , i t was e s t i m a t e d some t i m e ago
t h a t a p p r o x i m a t e l y e i g h t y p e r c e n t o f a'I1 l i q u i d chroma-
tography analyses a r e c a r r i e d o u t u s i n g r e v e r s e phase
methods.41 The f o u r examples o f f e r e d h e r e were chosen
because o f t h e i r a b i l i t y t o separate ' l i d o c a i n e f r o m i t s
d e g r a d a t i o n p r o d u c t s o r common pharmaceuticals under a
variety o f conditions.

System 142
Column : 30 cm x 4 mn I D p-Bondapak-CN,
10 urn
Detection: Dual channel, f i x e d wavelength,
Waters Model 440
Temperature: Ambient
Flow Rate: 2 mL/min
M o b i l e Phase: 0.01 M O c t a n e s u l f o n i c a c i d sodium
s a l t , 0.01 M e d e t a t e disodium, 2%
( v / v ) a c e t i c a c i d , 2% aceto-
n i t r i l e and 1% methanol i n
d i s t i l l e d water
R e t e n t i o n Time: 7 min
Sepa r at 1 on : From d e g r a d a t i o n p r o d u c t s and
e p i n e p h r i ne

System 1143
Column: Nuc 1 e o s i 'I 100, 1Opm
Detection: F i x e d wavelength, 254 nm
Temperature : 25.0 _t_ 0.1"C
M o b i l e Phase: Sodium phosphate b u f f e r , pH 2.2,
p = 0.1 and 1% ( v / v ) l - p e n t a n o l
R e t e n t i o n Time: 1 min
Sepa r a t 1on : From o t h e r l o c a l a n a e s t h e t i c s

System 11144
Column: L i Chromsorb RP-18
Detection: F i x e d wavelength, 254 nm
Temperature: 25°C
Flow Rate: Not g i v e n
M o b i l e Phase: 0.01 M Tetramethylammonium
bromide, 20% water, 80% methanol,
pH 3.6 w i t h H3PO4
Separation: From pharmaceuticals h a v i n g b a s i c
f u n c t i o n a l groups
776 MICHAEL F. POWELL

System IV45
Columns : p-Bondapak C1 , p-Bondapak
!
Phenyl, V-Bon apak-CN,
p-Bondagel, Chromegabond C8
and Chromegabond C6H11
Detection: UV-Vis d e t e c t o r
Flow Rate: 1.5 mL/min
Mobile Phase: Methano 1 :water:acetic a c i d
(29:50:1) c o n t a i n i n g .005 M

Retentlon Time:
-
heptane s u l f o n i c a c i d sodium
s a l t , pH 4
Retention volumes f o r t h e various
columns given i n Reference 45
Sepa r a t ion : From o t h e r common pharmaceuticals

8.2 Normal Phase HPLC

System 146
Columns : 125 o r 250 x 4.9 mm I D column
packed w i t h Spherisorb 5 5 W
s i l i c a , S y l o i d 74 s i l i c a
Detection: 215 nm
Temperature : Ambient
Flow Rate: 2 mL/min
Mob1 l e Phase: Methanol c o n t a i n i n g 1 .85 mM
HC 104
Retention Time: 7 min
Separation: From o t h e r l o c a l anaesthetics

8.3 Thin Laver ChromatonraPhv (TLC)

TLC and h i g h performance TLC are o f t e n used f o r drug

a n a l y s i s because they are r a p i d , Inexpensive, and o n l y
r e q u i r e small amounts o f sample. Lidocalne i s i d e n t i f i e d on
t h e TLC p l a t e by e i t h e r s h o r t wavelength UV l i g h t o r by a
p o s i t i v e t e s t w i t h a c i d i f i e d l o d o p l a t i n a t e spray. For
example, t h e f o l l o w i n g systems have been used:

System 147
Plate: S l l l c a g e l GF-254, 0.25 mm
Temperature: Ambient
Detection: uv
Mob1l e Phase: CHC13/ether/MeOH/conc. NH40H
(15:25: 5 : l )
Rf : 0.80
LIDOCAINE AND LIDOCAINE HYDROCHLORIDE 777

System 1147
Plate: S i l i c a g e l GF-254, 0.25 mm
Temperature : Ambient
Detection: uv
M o b i l e Phase Ethyl acetateh-propanol/conc.
NHqOH (40:30:3)
Rf : 0.82

System 11147
Plate: S l l l c a g e l GF-254, 0 . 2 5 m
Temperature: Ambient
Detection: uv
M o b i l e Phase: MeOH/conc . NH40H ( 100 :1 .5)
Rf : 0.84

System IV47
Plate: S i l i c a g e l GF-254, 0.25mm
Temperature: Ambient
Detection: uv
M o b i l e Phase: Alcohol USP/acetic a c i d / w a t e r
(60: 30: 10)
Rf : 0.60

System ~ 4 8
Plate: S i l i c a g e l G60 F254 ( u n t r e a t e d )
Temperature: Ambient
Detection: uv
M o b i l e Phase: 0.01 M K B r I n methanol
Rf : 0.84 (0.54, p l a t e t r e a t e d w i t h
0.1 M KHS04)

Acknowledgements

T h i s p r o f i l e supplement was made p o s s i b l e w i t h t h e h e l p o f

Drs. M. Mattox and L. P a r t r i d g e , and t h e i r colleagues a t t h e
I n s t i t u t e o f Organic Chemistry a t Syntex, and by my colleagues
i n t h e P r e f o r m u l a t l o n Department a t Syntex, D r . D . Johnson and
W. Tamraz.
778 MICHAEL F. POWELL

10. References

1. K. Groningsson e t a l . Anal. P r o f i l e s Drug. Sub. l 4 , 207

(1984).
2. N.I. Nakano. J . Pharm. S c i . 68, 667 (1979).
3. I.S t e t n i k a . J. Pharm. S c i . 55, 1190 (1966).
4. A. B r o d i n e t a l . J. Pharm. S c i . 73, 481 (1984).
5. N.I. Nakano e t a'l. Chem. Pharm. B u l l . 26, 936 (1978).
6. The D e t e r m i n a t i o n o f I o n i z a t i o n Constants by A. A l b e r t
-
and E.P. S e r j e a n t . ChaDman and H a l l Ltd.. London
(1971).
7. M a r t i n d a l e . The E x t r a PharmacoDoeia. Ed. by J.E.F.
Reynolds. The Pharmaceutical Press (1982).
8. H. Kamaya fi &IAnesth.
. Analg. 62, 1025 (1983).
9. R.H. Levy and M. Rowland. J. Pharm. Pharmacol. 24, 841
( 1 972).
10. M.L. Buess and P.J. Bray. Org. Mag. Reson. 22, 233 (1984).
11. S.P. Singh e t a l . Spectros. L e t t . l2, 95 (1979).
12. A.W. Hanson and D.W. Banner. Acta C r y s t . 830, 2486,
(1974).
13. A.W. Hanson and M. R o h r l . Acta C r y s t . 828, 3567 (1972).
14. H.M. Koehler and J . J . H e f f e r r e n . J. Pharm. S c i . 53,
1126 (1964).
15. M.H. Lannung. Arch. Pharm. Chem. 72, 703 (1965)
16. K. Thoma and C.D. H e r z f e l d t . Expo. Congr. I n t .
Technol. Pharm. 1, 157 (1977).
17. G. E c k e r t and F. Gutman. E l e c t r o a n a l . Chem.
I n t e r f a c i a l Electrochem. 6 2 , 267 (1975).
18. G.A. N e v i l l e and D. Cook. J. Pharm. S c i . !XI, 636 (1969).
19. R.L. Jones. J . Pharm. S c i . 63, 1170 (1974).
20. J.P. Chupp. J. Pharm. S c i . 53, 1524 (1970).
21. F.M. Smith and N.O. Nuessle. Am. J . Hosp. Pharm. ja,
1745 (1981).
22. H.L. Kirschenbaum e t a l . Am. J . Hosp. Pharm. 39, 1013
(1982).
23. T.E. Lackner e t a l . Am. J . Hosp. Pharm. 40, 97 (1983).
LIDOCAINE AND LIDOCAINE HYDROCHLORIDE 779

24. S.L. M i l l s e t a l . I R C S Med. S c i . L i b r . Compend. 3, 592

(1975).
25. J.B. Keenaghan and R.N. Noyes. J. Pharm. Exp. Ther.
-
180, 454 (1972).
26. R.N. Boyes e t a l . C l i n . Pharmacol. Ther. l 2 , 105
(1971).
27. R.E. Stenson e t a.1. C i r c u l a t i o n 40, 195 (1969).
28. P . 1 . Parkinson e t a l . B r i t . Med. J . 2, 29 (1970).
29. A. Somogyi e t a l . Eur. J . C l i n . Pharmacol. 22, 85
(1 982).
30. M. Rowland. Ann. N.Y. Acad. S c i . 179, 383 (1981).
31. V . B e r n s t e i n e t a l . J. Amer. Med Ass. 219, 1027 (1972).
32. A.G. de Boer e t a l . C l i n . Pharmacol. Ther. 26, 701
(1979).
33. J. Thomas e t a l . B r . J. Anaesth. 4 l , 442 (1969).
34. T . L o f t s s o n e t a l . I n t . J . Pharmacol. 11 345 (1984).
35. N.L. Benowitz and W. M e i s t e r . C l i n . Pharmacokinet. 3,
(1 978).
36. R.G. Burney e t a l . Am. H e a r t J. 88, 765 (1974).
37. S.D. Nelson e t a l . J. Pharm. S c l . 66, 1180 (1977).
38. T. Suzuki G gl-. J. Pharm. S c l . 73, 136 (1984).
39. C. von Bahr e t a l . Acta Pharmacol. T o x i c o l . 4 l , 39
(1977).
40. R . Kawai e t a l . J. Pharm. Dyn. 6 , 5-79 (1983).
41. C. Horvath and W . Melander. Amer. Lab. lo, 17 (1977).
42. S. M . Waraszkiewicz e t a l . J . Pharm. S c i . 7 0 , 1215
(1981).
43 * J. Crommen. J . Chromatog. m,
705 (1979).
44. M. Wolff c. Pharmazie 38, 891 (1983).
45. R.G. A r c h a r i and J.T. Jacob. J. L i q u i d Chrom. 9, 81
(1 980).
46. R.J. Flanagan e t a l . J. Chromatog. 247, 15 (1982).
47. A.N. Masoud. J. Pharm. S c i . 65. 1585 (1976).
48. E.G. Sundholm. J. Chromatog. 265. 285 (1983).
This Page Intentionally Left Blank
 SODIUM NITROPRUSSIDE

Auke B u l t , Oscar R. Leeuwenkamp and Wouter P. van Bennekom

1. History
2. Description
2.1 Name, Formula, Molecular Weight
3. Physical P r o p e r t i e s
3.1 13C-NMR Spectrum
3.2 Massbauer Spectrum
3.3 Mol ecul a r Orbital Diagram
4. S t a b i l i t y and Degradation
5. Pharmacology and In-Vitro Degradation
5.1 Pharmacology
5.2 In-Vitro Degradation
6. Methods o f Analysis
6.1 I d e n t i f i c a t i o n Tests
6.2 P u r i t y Tests
6.3 Colorimetry
6.4 High-Performance Liquid Chromatography
6.5 Pol arography
6.6 Application of N i t r o p r u s s i d e as Analytical Reagent
6.7 Miscellaneous Methods o f Analysis
References

ANALYTICAL PROFILES OF DRUG SUBSTANCES Copyright 0 1986

VOLUME 15 by the American Pharmaceutical Association
781 All rights of reproduction in any form reserved.
782 AUKE BULT ET AL.

1. HISTORY
N i t r o p r u s s i d e was f i r s t described i n 1849 by P l a y f a i r
(1). The compound has a t t r a c t e d considerable i n t e r e s t a t
v a r i o u s periods i n chemical h i s t o r y . A t present, t h e r e a r e
s t i l l c o n t r o v e r s i e s on v a r i o u s p o i n t s o f i t s physical and
(photo)chemical p r o p e r t i e s .
The pharmaceutical i n t e r e s t i n n i t r o p r u s s i d e i s due t o
i t s a p p l i c a t i o n s as a n a l y t i c a l reagent and as s t r o n g hypo-
t e n s i v e agent. Boedeker ( 2 ) i n t r o d u c e d i n 1861 n i t r o p r u s s i d e
as reagent f o r t h e d e t e c t i o n o f s u l p h i t e . L a t e r on, Legal
( 3 ) extended i t s use as reagent t o t h e d e t e c t i o n o f ketone
bodies i n u r i n e o f d i a b e t i c p a t i e n t s . Simon ( 4 ) and Rimini
( 5 ) employed t h e substance f o r d e t e c t i o n o f secondary and
primary a1 i p h a t i c amines, r e s p e c t i v e l y .
The blood-pressure l o w e r i n g e f f e c t o f n i t r o p r u s s i d e was
already reported i n 1887 by Davidsohn ( 6 ) , w h i l e t h e f i r s t
c l i n i c a l t r i a l was described i n 1928 by Johnson (7).

This p r o f i l e i s supplementary t o t h e p r o f i l e o f Rucki

(1 i t e r a t u r e was surveyed through October 1976) (8).

2. DESCRIPTION
2.1 Name, Formula, Molecular Weight
Generic name - Sodium n i t r o p r u s s i d e
Nomenclature - The f o l l o w i n g nomenclature i s used i n
Chemical Abstracts: F e r r a t e ( 2 - ) ,
pentaki s (cyano-C) n i t r o s y l , d i sodi urn
-
( OC-6-22) C144O2-89-21
Synonyms - Sodium n i t r o f e r r i c y a n i d e , sodium
n i t r o p r u s s i a t e , d i sodi um
pentacyanonitrosyl f e r r a t e ( I I )
d ihyd r a t e
Trade Names -
-- N i pride. (Roche) , Nipruss. (Cedona)
2-
0
111,
N

I
.Fe''-
2+
.2H2O
I '-*
c
111
N

CgN60FeNa?*%H20 Mol ecul a r Weight : 297.95

SODIUM NITROPRUSSIDE 783

3. PHYSICAL PROPERTIES

3.1 13C-NHR Spectrm

The 13C-NMR spectrum o f n i t r o p r u s s i d e i n D20 i s simple.
The e q u a t o r i a l and a x i a l CN appear w i t h chemical s h i f t s o f
104.2 and 102.2 ppm r e f e r r e d t o t - b u t a n o l , r e s p e c t i v e l y
(9,lO). These s h i f t s a r e about 40 ppm l o w e r as compared t o
Fe(CN)5X compounds (X = NH3, H20, CN-) and i n d i c a t e s a
s t r o n g ds-backdonation t o t h e NOt l i g a n d (10).

3.2 Mssbauer Spectrum

Data on t h e MZissbauer spectrum o f n i t r o p r u s s i d e were
presented by Danon (11). The isomer s h i f t ( u ) and quadrupole
c o u p l i n g ( A E ) have v a l u e s o f -0.012 cm s - l and 0.185 cm s - l ,
respectively.

3.3 Molecular Orbital Diagram

The MO diagram o f n i t r o p r u s s i d e i s i m p o r t a n t f o r t h e i n -
t e r p r e t a t i o n o f i t s p h y s i c a l and photochemical p r o p e r t i e s .
F i g . 1 p r e s e n t s t h e p a r t i a l MO-energy l e v e l diagram as c a l -
c u l a t e d w i t h t h e SCCC-MO method ( 1 2 ) . A l l d - e l e c t r o n s a r e
p a i r e d and t h e compound i s t h e r e f o r e diamagnetic. The d i s -
t r i b u t i o n o f t h e e l e c t r o n s i m p l i e s t h a t t h e n i t r o s y l group
has a formal charge t 1 and i r o n +2 (d6). The p o s i t i v e charge
o f NO e x p l a i n s t h e r e l a t i v e l y h i g h v a l u e of v(N0) = 1940
cm-l and t h e r e a c t i v i t y o f t h i s group towards a wide v a r i e t y
o f n u c l e o p h i l i c agents as w e l l (13). The MO diagram i s a l s o
i l l u s t r a t i v e f o r t h e assignment o f t h e U V - V I S spectrum as
presented i n Table 1 ( 1 2 ) .
R e c e n t l y , a r e - i n t e r p r e t a t i o n o f t h e MO diagram based on
Scaled INDO c a l c u l a t i o n s has been p u b l i s h e d (14). Now bands
I t o 111 t r a n s i t i o n s a r e c o n s i d e r e d t o be due t o d-d and
i n t e r n a l t r a n s i t i o n s i n t h e l i g a n d s , w h i l e band I V i s an
a1 lowed c h a r g e - t r a n s f e r band.

8e (TI*- CN I 3

7e (n*-NO, d y z ,d,, I

_ld_ 2bz(dxy) occupied

6e ( d y z , d , , , ~ f N O )

Fig. 1. Energy l e v e l diagram o f t h e n i t r o p r u s s i d e i o n (12).

784 AUKE BULT ET AL.

Table 1. Assignment o f t h e U V - V I S spectrum

A remarkable phenomenon o f n i t r o p r u s s i d e i s t h a t ex-

c i t a t i o n w i t h a s u i t a b l e l a s e r a t T < 150 K r e s u l t s i n t h e
formation o f an extremely l o n g - l i v i n g metastable s t a t e (15).

4. STABILITY AND DEGRADATION

Two important pathways o f photochemical degradation o f

nitroprusside are ( 8 ) :
[Fe(CN)5N0J2- t H20 - hv [FeI1(CN)5H20l3- t NOt
[:A 1
A nd

[Fe(CN)5N072- t H20 - hv
[FeIII(CN)tjH20]2-

Recently t h e mechanism o f photodecomposition has been

t NO r B1
re-examined w i t h continuous and f l a s h p h o t o l y s i s (16,17).
The conclusions are: 1 ) band I i s p h o t o - i n a c t i v e , 2) band
I 1 i r r a d i a t i o n r e s u l t s i n pathway r A ] a n d 3 ) bands I V and V
i r r a d i a t i o n g i v e s pathway r B 3 ( f o r bands see Table 1). Owing
t o t h e o v e r l a p o f band I 1 1 w i t h bands I 1 and I V , b o t h path-
ways can occur simultaneously. The conclusions are c o n s i s t -
e n t w i t h t h e MO diagram o f Golebiewski and Wasielewska (14).

For c l i n i c a l use sodium n i t r o p r u s s i d e has t o be recon-

s t i t u t e d w i t h a s t e r i l i z e d aqueous s o l u t i o n r e s u l t i n g i n a
concentrated stock s o l u t i o n (ca. 25 g 1-1 i n 0.94: s a l i n e o r
5% glucose), which i s d i l u t e d t o an i n f u s i o n s o l u t i o n (ca.
50-200 mg 1-1 i n 5% glucose). Both s o l u t i o n s have been
s t u d i e d f o r thermal and photochemical s t a b i l i t y .
Protected from l i g h t , t h e concentrated s o l u t i o n i s
s t a b l e a t room temperature and 4°C f o r more than two years
(18). Autoclaving (15 min, 121OC) o f n i t r o p r u s s i d e s o l u t i o n s
i n water and i n 0.9% s a l i n e (50 mg 1 - l ) gives e s s e n t i a l l y no
degradation, whereas s t e r i l i z a t i o n o f n i t r o p r u s s i d e i n 5%
dextrose s o l u t i o n r e s u l t s i n about 40% l o s s (19). N i t r o -
SODIUM NITROPRUSSIDE 785

prusside s o l u t i o n s i n l i g h t - p r o t e c t e d g l a s s o r p l a s t i c con-
t a i n e r s and i n p l a s t i c i n f u s i o n s e t s remain s t a b l e f o r a t
l e a s t two days (20).
I n s o l u t i o n n i t r o p r u s s i d e i s h i g h l y 1 i g h t - s e n s i t i v e and
decomposes r a p i d l y ( 8 ) . Modern photodegradation s t u d i e s are
based on " s t a b i l i t y - i n d i c a t i n g " assay methods f o r n i t r o -
prusside, v i z. c o l o r i m e t r i c measurement o f i n t a c t n i t r o -
prusside w i t h sulphide (18) and HPLC (19-22). The r a t e o f
degradation upon exposure t o d a y l i g h t and t o l i g h t o f 350 nm
i s e s s e n t i a l l y t h e same i n water, i n 0.9% s a l i n e and i n 591
dextrose s o l u t i o n s (19). The degradation i s a n o n - l i n e a r
process, i.e. an i n i t i a l r a p i d l o s s i s f o l l o w e d by a phase
o f slower decomposition (19). Th n o n - l i n e a r i t y i s due t o

as a l i g h t - f i l t e r . a
t h e f o r m a t i o n o f [Fe(CN) H2OI5-, a y e l l o w compound a c t i n g
I n a d i t i o n , t h e back-formation o f
n i t r o p r u s s i d g from i t s degradation products n i t r i t e and
-
CFe(CN)5H201 occurs according t o ( 1 9 ) :

[Fe(CN)5H20l3- t NOp- t 2 Ht- [Fe(CN)rjNOl2- t 2 H20

The degradation y i e l d s n i t r i t e , n i t r a t e , CFe(CN)5H20I2-,

CFe(CN)5H20l3-, Fe(CN)63- and Fe(CN)64- (19). Contrary t o
previous r e p o r t s , t h e a d d i t i o n o f c i t r i c a c i d o r sodium ede-
!i
t a t e ( b o t h 0.25 mg 100 m l ) t o 5% dextrose s o l u t i o n o f n i t r o -
prusside (50 mg 1 - ) does not improve t h e s t a b i l i t y , w h i l e
cyanocobalamin (10 mg 1 - l ) s i g n i f i c a n t l y increases t h e
p h o t o - s t a b i l i t y (19). Before t h e i n t r o d u c t i o n o f t h e s t a -
b i l i t y - i n d i c a t i n g assay o f n i t r o p r u s s i d e , t h e degradation
s t u d i e s were based on t h e spectrophotometric measurement o f
t h e increase i n absorbance a t 395 nm. However, many degrada-
t i o n products o f n i t r o p r u s s i d e e x h i b i t h i g h molar absorp-
t i v i t i e s i n t h a t region, r e s u l t i n g i n c o n t r a d i c t o r y i n t e r -
p r e t a t i o n s o f t h e r e s u l t s (23-26). Very r e c e n t l y a study on
t h e photodegradation o f n i t r o p r u s s i d e was pub1 ished ' n which
t h e release o f HCN and t h e f o r m a t i o n o f Fe(CN)6 3-
were
measured c o l o r i m e t r i c a l l y w i t h d e t e c t i o n l i m i t s o f 10-12 M I
and 2-3 IM, r e s p e c t i v e l y (27). Dimethyl s u l f o x i d e (10% v / v )
was l a t e l y found t o be an e f f e c t i v e p h o t o p r o t e c t i v e agent
f o r s o l u t i o n s o f n i t r o p r u s s i d e (28,29).

5. PHARUACOLOGY AND IN-VITRO DEGRADATION

5.1 Pharmacology
The main pharmacological and t o x i c o l o g i c a l f e a t u r e s o f
n i t r o p r u s s i d e have been discussed b e f o r e (8). Recent r e p o r t s
i n t h i s f i e l d are i n d i c a t i v e f o r t h e continued i n t e r e s t i n
t h i s compound (30-37).
786 AUKE BULT ET AL.

5.2 In-Vitro Degradation

I n - v i t r o s t u d i e s have revealed t h a t cyanide i s released
from n i t r o p r u s s i d e on i n c u b a t i o n w i t h serum, plasma, whole
blood, 1 iver homogenate , haemogl o b i n and e r y t h r o c y t e s (30).
For instance, n i t r o p r u s s i d e incubated w i t h blood releases
50% o f t h e t o t a l amount o f cyanide w i t h i n 20 min and more
than 90% over 2 h (38). Upon i n f u s i o n , i n - v i v o cyanide
l e v e l s up t o 3.2 IIM and 45.5 IIM were found i n plasma and
blood o f p a t i e n t s , r e s p e c t i v e l y (26).
The number o f methods s u i t a b l e f o r measurement o f low
concentrations o f i n t a c t n i t r o p r u s s i d e and i t s degradation
products (cyanide, n i t r i c o x i d e / n i t r i t e ) i n body f l u i d s i s
very l i m i t e d (26,39-41). Arnold e t a l . (26) described an
i n d i r e c t c o l o r i m e t r i c method, based on a d i a z o coup1 i n g
r e a c t i o n , f o r measurement o f n i t r i c oxide released from
n i t r o p r u s s i de.
The cyanide d e t e r m i n a t i o n i s based on c o l o u r f o r m a t i o n
w i t h pyridine/pyrazolone (39). This method i s s u i t a b l e f o r
t h e determination o f f r e e cyanide (released from n i t r o -
prusside) and i n t a c t n i t r o p r u s s i d e v i a q u a n t i t a t i v e con-
v e r s i o n t o cyanide by i n c u b a t i o n w i t h cysteine. The detec-
t i o n l i m i t amounts t o about 3 pg m l - l o f n i t r o p r u s s i d e (39).
Recently Alkayer e t a l . ( 4 0 ) and Leeuwenkamp e t a l . (41)
developed pol arographic methods f o r t h e d i r e c t d e t e r m i n a t i o n
o f nitroprusside i n i o l o g i c a l m a t r i es w i t h d e t e c t i o n
l i m i t s o f 450 ng m l - ) and 15 ng m l - ? , r e s p e c t i v e l y ( f o r
d e t a i l s see Section 6.5). E s p e c i a l l y t h e last-mentioned
procedure enables measuring o f therapeut 'c l e v e l s o f n i t r o -
prusside, ranging from 100 t o 1000 ng ml-! (41).
Leeuwenkamp ( 4 2 ) employed t h e polarographic method i n a
study o f t h e i n - v i t r o degradation o f n i t r o p r u s s i d e (200
ng m l - l ) i n v a r i o u s media a t 37"C, v i z . s o l u t i o n s o f albu-
min, cysteine, g l u t a t h i o n e and (met)haemoglobin; human p l a s -
ma, e r y t h r o c y t e suspensions and blood, and i n 100,000 g
crude a o r t i c - s o l u b l e f r a c t i o n s . A h a l f - l i f e t i m e ( t l 2) of
about 2.6 min was found i n t h e s o l u b l e f r a c t i o n s , w i c h i s
comparable t o t h e i n - v i v o t l 2. The t 1 / 2 i n human blood was
I
6
about 15 min and i n t h e o t e r mentioned media considerably
h i g h e r values o f t 1 / 2 were observed.

6. METHODS OF ANALYSIS

6.1 I d e n t i f i c a t i o n Tests
The i d e n t i f i c a t i o n t e s t s f o r sodium n i t r o p r u s s i d e and
i t s dosage form " s t e r i l e sodium n i t r o p r u s s i d e " are t h e same
i n USP X X I as i n USP X I X (8). Recently a new p r e c i p i t a t i o n
reagent f o r t h e n i t r o p r u s s i d e anion has been described (43).
SODIUM NITROPRUSSIDE 787

6.2 P u r i t y Tests
The p u r i t y t e s t s i n USP X X I f o r sodium n i t r o p r u s s i d e
comprise c h l o r i d e and sulphate; b o t h t e s t s a r e based on
t u r b i d i m e t r i c measurements. The i m i t t e s t f o r d e t e r m i n a t i o n
I
o f t h e hexacyanoferrates Fe(CN)6 - and Fe(CN)64- a t 0.059:
l e v e l can be performed by chromatographic s e p a r a t i o n o f
sodium n i t r o p r u s s i d e f r o m t h e s e substances, followed by
spectrophotometric measurement a t 415 nm (44). Moreover,
t h e chromatographic procedure d e s c r i b e d by Leeuwenkamp e t
a l . (21) enables o n - l i n e d e t e r m i n a t i o n o f b o t h hexacyano-
ferrates .
6.3 Colorimetry
Determinations o f n i t r o p r u s s i d e , based on a q u a n t i t a t i v e
r e l e a s e o f n i t r i c o x i d e and c o l o r i m e t r i c measurement o f t h e
n i t r i t e formed by a d i a z o c o u p l i n g r e a c t i o n have a l r e a d y
been described i n S e c t i o n 5.2 (26). S i m i l a r procedures have
r e c e n t l y been r e p o r t e d (45,46).

N i t r o p r u s s i d e d e t e r m i n a t i o n , based on a q u a n t i t a t i v e
re1 ease o f cyanide and subsequent c o l o r i m e t r i c measurement
o f cyanide has been d e s c r i b e d i n S e c t i o n 5.2 (39).
Burger (47) r e p o r t e d t h e d e t e r m i n a t i o n o f c y a n o f e r r a t e
compounds, e.g. n i t r o p r u s s i d e , by complete d e g r a d a t i o n o f
these compounds i n a m i x t u r e o f phenanthrol i n e / a s c o r b i c
acid/mercury( I I ) c h l o r i d e / a c e t a t e b u f f e r (pH = 3.5) and
q u a n t i t a t i o n o f t h e r e l e a s e d HCN by t i t r a t i o n w i t h s i l v e r
n i t r a t e and c o l o r i m e t r i c measurement o f Fe2+ as f e r r o i n
a t 510 nm.

6.4 High-Performance Liquid Chromatography

Up t o now t h r e e HPLC procedures have been described i n
.
t h e l i t e r a t u r e (20-22). Baaske e t a1 ( 2 2 ) used a reversed-
phase i o n - p a i r system w i t h a s t a t i o n a r y phase o f m i c r o p a r -
t i c u l a t e 10 Ilm phenyl-bonded s i l i c a gel and a m o b i l e phase
c o n s i s t i n g o f acetonitrile/phosphate/tetra-n-butylammonium
hydroxide b u f f e r o f pH = 7.1 ( 3 0 t 7 0 ) ; UV-detection a t 210
nm; l i n e a r c a l i b r a t i o n c u r v e i n t h e c o n c e n t r a t i o n range:
10-50 p g m l - 1 ; c o e f f i c i e n t o f v a r i a t i o n (CV) < 3.1%. The
system separates n i t r o p r u s s i d e from i t s d e g r a d a t i o n p r o d u c t
Fe(CN)64-, b u t n i t r o p r u s s i d e and Fe( CN)63- a r e n o t separ-
ated.
A s i m i l a r system was d e s c r i b e d by Leeuwenkamp e t a1
(21). They used p-Rondapack phenyl-bonded pel 1 i c u l a r s i l i c a
.
g e l (10 p m ) as s t a t i o n a r y phase and a m o b i l e phase con-
s i s t i n g o f water-methanol (65+35) c o n t a i n i n g 5 mM t e t r a -
n-butyl-ammonium phosphate, 1.1 mM n-octylamine and 6.5 mM
potassium dihydrogen phosphate (pH = 7.0); UV-detection a t
220 nm; l i n e a r c a l i b r a t i o n c u r v e i n t h e c o n c e n t r a t i o n range:
788 AUKE BULT ET AL.

10-120 pg m l - 1 . This system enables t h e separation of

n i t r o p r sside from ‘ t s photodegradati n products NOp-, NO -
Fe(CN)$, 4
Fe( CN)6 -, [Fe( CN)5H201a- and [Fe(CN)5H20j3’
(19).
Most r e c e n t l y an HPLC procedure has been published (20)
i n which P a r t i s i l 10-SAX anion-exchange m a t e r i a l i s used as
s t a t i o n a r y phase w i t h a m o b i l e phase c o n s i s t i n g o f 0.5 M po-
tassium dihydrogen phosphate b u f f e r (pH = 3.0) ; UV-detection
a t 230 nm; l i n e a r c a l i b r a t i o n curve i n t h e c o n c e n t r a t i o n
range 1-200 119 m l - 1 ; CV < 1%. However, w i t h t h i s system no
degradation products were detected.

6.5 Pol arography

The key f e a t u r e s o f t h e pol arographic behaviour o f
n i t r o p r u s s i d e have been discussed b e f o r e ( 8 ) . I n t h e pH
range 4-9 a t t h e dropping mercury e l e c t r o d e , t h r e e r e d u c t i o n
processes (1-111) were observed w i t h d i f f e r e n t i a l p u l s e
pol arography (DPP) and high-performance d i f f e r e n t i a l p u l s e
polarography (HPDPP) w i t h peak p o t e n t i a l s a t -360 ( I ) , -610
(11) and -1500 (111) mV vs. s a t u r a t e d sodium c h l o r i d e c a l o -
mel e l e c t r o d e (SSCE) (48). A t pH > 10 t h e polarographic
d i f f u s i o n - c u r r e n t d creases most probably because o f forma-
f
t i o n o f [Fe(CN) NO21 - (49). A t pH = 6 peak I 1 s t a r t s t o
7
s h i f t anodical y and increases i n i n t e n s i t y w i t h a decrease
i n pH (48). Peak I 1 superimposes peak I a t pH < 1.5 and t h e
r e s u l t i n g peak s h i f t s a n o d i c a l l y i n a l i n e a r way down t o
pH = 0 (48). A t pH = 0 (1 M HCl04) t h e combined r e d u c t i o n
peak appears a t -270 mV vs. SSCE and maximum s e n s i t i v i t y i s
obtained ( l i n e a r c l i b r a t i o n curve i n t h e c o n c e n t r a t i o n
f
range 4-1000 ng m l - ) (48).

An extensive a n a l y s i s o f t h e r e d u c t i o n processes i s
g i v e n e l sewhere (23,48-51). L a t e l y , t h e one-el$ctron reduc-
t i o n product o f n i t r o p r u s s i d e , v i z . [Fe(CN)5N0l3- ( I ) , has
a t t r a c t e d considerable a t t e n t i o n because o f t h e r a p i d equi -
l i b r i u m (50-53):

The r e d u c t i o n product I decomposes t o a r e a c t i v e penta-

coordinate complex (11) due t o r e l e a s e o f cyanide, w h i l e t h e
unpaired e l e c t r o n t r a n s f e r s from t h e n i t r o s y l N atom t o t h e
i r o n atom. Compound I 1 forms t h e hexacoordinate complex
CFe(CN)3(chel)NOJ- w i t h s u i t a b l e b i d e n t a t e l i g a n d s ( c h e l ) ,
e.g. 2,2’ - b i p y r i d i ne, 1,lO-phenanthrol ine (54).

Polarography i s used t o q u a n t i t a t e n i t r o p r u s s i d e i n
dosage form (USP XXI) and i n body f l u i d s (40,41). Alkayer e t
SODIUM NITROPRUSSIDE 789

a l . (40) developed a d e t e r m i n a t i o n method f o r n i t r o p r u s s i d e

i n human serum based on phase-sensitive sine-wave p o l a r o -
graphy a t pH = 7.4 a f t e r p r o t e i n e l i m i n a t i o n w i t h p e r c h l o r i c
a c i d (1 i n e a r c o n c e n t r a t i o n range 2-24 119 ml-1). Leeuwenkamp
e t a l . (41) based t h e i r method on HPDPP and DPP a t pH = 0
(1 M p e r c h l o r i c a c i d ) . The l i n e a r c a l i b r a t i o n range i n
plasma, serum and blood ( p r o t e i n e l i m i n a t i o n by p e r c h l o r i c
a c i d ) i s 30-1000 ng m l - l .

6.6 Application o f Nitroprusside as Analytical Reagent

N i t r o p r u s s i d e i s a v a l u a b l e reagent f o r t h e d e t e c t i o n
and determination o f a wide v a r i e t y o f n u c l e o p h i l i c agents,
e.g. primary and secondary a1 i p h a t i c amines, aldoximes,
amino acids, a n i l i n e s , i n d o l s , ketones, n i t r i l s , phenols,
p y r r o l e s , quinones, s u l p h i t e , t h i o l s , t h i o u r e a s and u r a c i l s
(13,23,55-58). I t s r e a c t i v i t y i s based on t h e p o s i t i v e
n i t r o s y l group, r e a c t i n g w i t h n u c l e o p h i l i c species ( m o s t l y
i n a l k a l i n e medium) (13). Most a d d i t i o n compounds formed are
h i g h l y coloured and s u f f i c i e n t l y s t a b l e t o be used as a
b a s i s f o r spectrophotometric determination. The r e a c t i o n
mechanisms are (sometimes) r a t h e r complicated (23).

6.7 Miscellaneous Methods o f Analysis

Low concentrations o f n i t r o p r u s s i d e ( d e t e c t i o n 1i m i t ca.
3 ng ml-1) have been assayed by v i r t u e o f i t s c a t a l y t i c
e f f e c t on t h e f o r m a t i o n o f indophenol b l u e (Am 620 nm)
from ammonia, phenol and h y p o c h l o r i t e i n a l k a q i n i medium
( r e a c t i o n o f B e r t h e l o t ) (59).

N i t r o p r u s s i d e can be determined by f l o w - i n j e c t i o n analy-

s i s w i t h amperometric d e t e c t i o n ( o x i d a t i o n a t a g l a s s y -
carbon e l e c t r o d e o r r e d u c t i o n a t a sessi e mercury drop
~ C V < 2%
e l e c t r o d e ) ; c o n c e n t r a t i o n range 10-6 t o 5 0 1 0 - M;
(60)

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60, 51 (1978).
59 A. Sanchez Perez, J. Hernandez Mendez and
J. Montero Garcia, Cienc. Ind. Farm. 1, 114 (1982).
60 A.G. Fogg, M.A. Ferngndez-Arciniega aiid R.M. Alonso,
Analyst 110,345 (1985).
 CUMULATIVE INDEX

Bold numerals refer to volume numbers

Acetaminophen. 3, I ; 14, 551 Cephradine. 5, 21

Acetohexamide. 1, I ; 2, 573 Chloral hydrate, 2, 85
Allopurinol, 7, 1 Chloramphenicol. 4,47. 518; 15, 701
Alpha-tocopheryl acetate, 3, I I I Chlordiazepoxide. 1, 15
Amantadine, 12, I Chlordiazepoxide hydrochloride, 1, 39; 4, 518
Amikacin sulfate, 12, 37 Chloroquine, 13, 95
Amiloride hydrochloride, 15, I Chlyroquine phosphate, 5, 61
Aminoglutethimide, 15, 35 Chlorpheniramine maleate. 7, 43
Aminophylline, 11, I Chlorprothixene, 2, 63
Aminosalicylic acid. 10, I Chlortetrdcycline hydrochloride, 8, 101
Amitriptyline hydrochloride. 3, 127 Chlorthalidone, 14, I
Amoxicillin. 7, 19 Cholecalciferol, see Vitamin D,
Amphotericin B. 6, I; 7, 502 Cimetidine. 13, 127
Ampicillin. 2, I ; 4, 518 Cisplatin. 14, 77: 15, 796
Ascorbic acid, 11, 45 Clidinium bromide. 2, 145
Aspirin. 8, I Clindamycin hydrochloride. 10, 75
Atenolol. 13, I Clofibrate, 11, 197
Atropine, 14, 32 Clonazepam, 6, 61
Azathioprine. 10, 29 Clorazepate dipotassium. 4, 91
Bacitracin, 9, I ClotrimaLole, 11, 225
Baclofen, 14, 527 Cloxacillin sodium, 4, I13
Bendroflumethiazide. 5, I ; 6, 597 Cocaine hydrochloride. 15, I S 1
Benperidol 14, 245 Codeine phosphate, 10, 93
Benzocaine. 12, 73 Colchicine. 10, 139
Benzyl benzoate. 10,55 Cyanocobalamin. 10, 183
Betamethasone dipropionate, 6, 43 Cyclizine. 6, 83; 7, 502
Bretylium tosylate, 9, 71 Cycloserine, 1, 53
Bromocriptine methanesulfonate. 8,47 Cyclothiaz.ide.1, 66
Caffeine, 15, 71 Cypropheptadine. 9, 155
Calcitriol, 8, 83 Dapsone, 5, 87
Camphor. 13, 27 Dexamethasone, 2, 163: 4, 519
Captopril. 11, 79 Diatrizoic acid. 4, 137; 5, 556
Carbamazepine, 9,87 Diazepam. 1, 79; 4, 518
Cefaclor. 9, 107 Dibenzepin hydrochloride, 9 , 181
Cefamandole nafate. 9, 125; 10, 729 Dibucaine and dibucaine hydrochloride. 12, 105
Cefazolin, 4, I Diflunisal, 14, 491
Cefotaxime, 11, 139 Digitoxin. 3, 149
Cefoxitin. sodium, 11, 169 Digoxin, 9 , 207
Cephalexin, 4, 21 Dihydroergotoxine methanesulfonate. 7, 81
Cephalothin sodium. 1, 319 Dioctyl sodium sulfosuccinate. 2, 199; 12, 713

793
794 CUMULATIVE INDEX

Diperodon. 6 , 9 9 Imipramine hydrochloride, 14, 37

Diphenhydramine hydrochloride, 3, 173 Indomethacin, 13, 21 I
Diphenoxylate hydrochloride, 7, 149 lodamide, 15, 337
Disopyramide phosphate. 13, 183 lodipamide. 2, 333
Disulfiram. 4, 168 lopanoic acid, 14, 181
Dobutamine hydrochloride, 8, 139 Isocarboxazid, 2, 295
Dopamine hydrochloride. 11, 257 Isoniazide, 6, 183
Doxorubicine, 9, 245 Isopropamide, 2, 315; 12, 721
Droperidol, 7, 171 lsoproterenol, 14, 391
Echothiophate iodide, 3, 233 lsosorbide dinitrate, 4, 225; 5, 556
Emetine hydrochloride, 10, 289 Kanamycin sulfate, 6, 259
Ephedrine hydrochloride. 15, 233 Ketamine. 6, 297
Epinephrine, 7, 193 Ketoprofen, 10,443
Ergonovine maleate, 11, 273 Ketotifen, 13, 239
Ergotamine tartrate, 6, I13 Khellin, 9, 371
Erythromycin, 8, 159 Leucovorin calcium, 8, 315
Erythromycin estolate, 1, 10 I ; 2, 573 Levallorphan tartrate, 2, 339
Estradiol. 15, 283 Levarterenol bitartrate, I, 49; 2, 573; 11, 555
Estradiol valerate. 4, 192 Levodopa, 5, 189
Estrone, 12, 135 Levothyroxine sodium, 5, 225
Ethambutol hydrochloride, 7, 231 Lidocaine base and hydrochloride, 14, 207: 15, 761
Ethynodiol diacetate, 3, 253 Lithium carbonate. 15, 367
Etomidate, 12, 191 Lorazepam, 9, 397
Fenoprofen calcium 6, 161 Maprotiline hydrochloride. 15, 393
Flucytosine. 5, I15 Mefloquine hydrochloride, 14, 157
Fludrocortisone acetate, 3, 281 Melphalan. 13, 265
Flufenamic acid, 11, 313 Meperidine hydrochloride, 1, 175
Fluorouracil, 2, 221 Meprobamate, 1, 209; 4, 520; 11,587
Fluoxymesterone, 7, 251 6-Mercaptopurine, 7, 343
Fluphenazine decanoate, 9, 275; 10, 730 Mestranol. 11, 375
Fluphenazine enanthate, 2, 245; 4, 524 Methadone hydrochloride, 3, 365; 4, 520; 9,601
Fluphenazine hydrochloride, 2, 263; 4, 519 Methaqualone, 4, 245. 520
Flurazepam hydrochloride, 3, 307 Methimazole, 8, 351
Gentamicin sulfate, 9, 295; 10, 731 Methotrexate, 5, 283
Glibenclamide, 10, 337 Methoxsalen, 9,427
Gluthethimide. 5, 139 Methyclothiazide, 5, 307
Gramicidin, 8, 179 Methylphenidate hydrochloride, 10, 473
Griseofulvin, 8, 219; 9, 583 Methyprylon, 2, 363
Guanabenz acetate. 15, 319 Metoprolol tartrate, 12, 325
Halcinonide, 8, 251 Metronidazole, 5, 327
Haloperidd, 9, 341 Minocycline, 6, 323
Halothane, 1, 119; 2,573; 14,597 Moxalactam disodium, 13, 305
Heparin sodium, 12, 215 Nabilone, 10,499
Heroin. 10, 357 Nadolol, 9,455; 10, 732
Hexestrol, 11, 347 Nalidixic acid, 8, 371
Hexetidine, 7, 277 Naloxone hydrochloride. 14,453
Hydralazine hydrochloride, 8, 283 Natarnycin, 10, 513
Hydrochlorothiazide. 10, 405 Neomycin, 8, 399
Hydrocortisone, 12, 277 Nitrazepam, 9,487
Hydroflumethiazide. 7, 297 Nitrofurantoin. 5, 345
Hydroxyprogesterone caproate. 4, 209 Nitroglycerin, 9, 519
Hydroxyzine dihydrochloride, 7, 319 Norethindrone, 4, 268
 CUMULATIVE INDEX 795

Norpestrel. 4, 294 Secobarbital sodium, 1, 343

Nortriptyline hydrochloride. 1, 233: 2, 573 Silver sulfadiazine, 13, 553
Noscapine, 11, 407 Sodium nitroprusside. 6, 487; 15, 781
Nystatin, 6, 341 Spironolactone, 4, 431
Oxazepam. 3,441 Strychnine. 15. 563
Oxyphenbutazone. 13, 333 Succinycholine chloride. 10, 691
Oxytocin. 10, 563 Sulfadiazine. 11, 523
Penicillamine, 10, 601 Sulfamethazine. 7, 401
Penicillin-G benzothine, 11, 463 Sulfamethoxazole, 2, 467: 4, 521
Penicillin G. potassium. 15, 427 Sulfasalazine, 5 , 515
Penicillin-V, I, 249 Sulfisoxazole, 2, 487
Pentazocine, 13, 361 Sulindac. 13, 573
Phenazopyridine hydrochloride, 3,465 Sulphamerazine, 6, 515
Phenelzine sulfate, 2, 383 Terpin hydrate, 14, 273
Phenformin hydrochloride, 4, 319; 5,429 Testolactone, 5 , 533
Phenobarbital. 7, 359 Testosterone enanthate, 4, 452
Phenoxymethyl penicillin potassium, 1, 249 Tetracycline hydrochloride, 13, 597
Phenylbutazone, 11, 483 Theophylline, 4, 466
Phenylephrine hydrochloride, 3, 483 Thiostrepton, 7, 423
Phenylpropanolamine hydrochloride, 12, 357; 13, Tolbutamide, 3, 513; 5, 557; 13, 719
77 I Triamcinolone. I, 367: 2, 571; 4, 521, 524; 11,
Phenytoin, 13, 417 593
Pilocarpine, 12, 385 Triamcinolone acetonide, 1, 397, 416; 2, 571; 4,
Piperazine estrone sulfate, 5, 375 521; 7, 501; 11, 615
Piroxicam, 15, SO9 Triamcinolone diacetate, 1, 423; 11, 651
Primidone, 2, 409 Triamcinolone hexacetonide, 6, 579
Probenecid, 10, 639 Triclobisonium chloride, 2, 507
Procainamide hydrochloride, 4, 333 Trifluoperazine hydrochloride, 9, 543
Procarbazine hydrochloride. 5 , 403 Triflupromazine hydrochloride, 2, 523; 4, 521; 5,
Promethazine hydrochloride, 5 , 429 557
Proparacaine hydrochloride. 6, 423 Trimethaphan camsylate, 3, 545
Propiomazine hydrochloride, 2, 439 Trimethobenzamide hydrochloride, 2, 551
Propoxyphene hydrochloride, I, 301; 4, 520; 6, Trimethoprim, 7, 445
598 Trimipramine maleate, 12, 683
Propylthiouracil. 6,457 Trioxsalen. 10, 705
Pseudoephedrine hydrochloride, 8,489 Tripelennamine hydrochloride, 14, 107
Pyrazinamide, 12, 433 Triprolidine hydrochloride. 8, 509
Pyridoxine hydrochloride, 13, 447 Tropicamide, 3, 565
Pyrimethamine, 12,463 Tubocurarine chloride, 7, 477
Quinidine sulfate, 12, 483 Tybamate, 4,494
Quinine hydrochloride, 12, 547 Valproate sodium and valproic acid, 8, 529
Ranitidine, 15, 533 Vidarabine. 15, 647
Reserpine, 4, 384: 5,557; 13, 737 Vinblastine sulfate. 1, 443
Rifampin, 5 , 467 Vincristine sulfate. 1, 463
Rutin, 12, 623 Vitamin D,, 13, 655
Saccharin, 13, 487 Warfarin. 14, 243
Salbutamol, 10, 665 Xylometazoline hydrochloride, 14, 135
Salicylamide, 13, 521 Zomepirac sodium, 15, 673
ERRATUM TO
K. Florey ( E d . ) . (1985). Analytical P r o f i l e s of Drug Substances,
vol. 1 4 .

Page 82, T a b l e 1:
The molar e x t i n c t i o n c o e f f i c i e n t a t 285 sun i s 109, not 190.

796