Analytical Profiles

of
Drug Substances
Volume 3
Edited by
Klaus Florey
The Squibb Institute for Medical Research
New Brunswick, New Jersey

Contributing Editors

Norman W. Atwater Salvatore A. Fusari

Olenn A. Brewer, Jr. Erik H. Jensen
Lester Chafetz Boen T. Kho
Jack P. Comer Gerald J. Papariello
Bernard 2. Senkowski

Compded under the auspices of the

Pharmaceutical Analysis and Control Section
Academy of Pharmaceutical Sciences

Academic Press New York and London 1974

A Subsidiary of Harcourt Brace Jovanovich, Publishers
 EDITORIAL BOARD

Norman W. Atwater David E. Guttmaa

Glenn A. Brewer, Jr. Erik H. Jensen
Lester Chafetz Boen T. Xho
Edward M. Cohen Arthur F. Michaelis
Jack P. Comer Oerald J. Papariello
Klaus Florey Bernard Z. Senkowski
Salvatore A. Fusari Frederick Tishler
COPYRIGHT 1974, BY THEAMERICAN PHARMACEUTlCAL ASSOCIATION
ALL RIGHTS RESERVED
NO PART OF THIS BOOK MAY BE REPRODUCED IN ANY FORM,
BY PHOTOSTAT, MICROFILM, RETRIEVAL SYSTEM, OR ANY
OTHER MEANS, WITHOUT WRITTEN PERMISSION FROM
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Main entry under title:

Analytical profiles of drug substances.

Compiled under the auspices of the Pharmaceutical

Analysis and Control Section, Academy of Pharmaceutical
Sciences.
Includes bibliographical references.
1. Drug-Collected works. 2. Chemistry, Medical
and pharmaceutical-Collected works, I, Florey, Klaus,
ed. 11. Brewer, Glenn A. 111. Academy of Pharma-
ceutical Sciences. Pharmaceutical Analysis and Control
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 AFFILIATIONS OF EDITORS,
CONTRIBUTORS, AND REVIEWERS
N. W. Atwater, Searle and Company, Chicago, Illinois

W. F. Beyer, The Upjohn Company, Kalamazoo, Michigan

K. W. Blessel, Hoffmann-LaRoche, Inc., Nutley, New Jersey

R . H. Bishara, Eli Lilly and Company, Indianapolis, Indiana

C. A . Brewer Jr., The Squibb Institute for Medical Research, New

Brunswick, New Jersy

L . Chafetz, Warner-Lambert Research Institute, Morris Plains, New

Jersey

E. M . Cohen, Merck, Sharp and Dohme, West Point, Pennsylvania

J. P. Comer, Eli Lilly and Company, Indianapolis, Indiana

R . D. Daley, Ayerst Laboratories, Rouses Point, New York

J. E. Fairbrother, The Squibb Institute for Medical Research, Moreton,

Wirral, England

K. Florey, The Squibb Institute for Medical Research, New Brunswick,

New Jersey

R . I. Fryer, Hoffmann-LaRoche, Inc., Nutley, New Jersey

S.A . Fusari, Parke, Davis and Company, Detroit, Michigan

C. A . Caglia, Jr., Warner-Lambert Research Institute, Morris Plains,

New Jersey

vii
 AFFILIATIONS OF EDITORS, CONTRIBUTORS, AND REVIEWERS

D. E. Guttman, School of Pharmacy, University of Kentucky, Lexington,

Kentucky

I. J. Holcombe, Parke, Davis and Company, Detroit, Michigan

I. M. Jakovljeric, Eli Lilly and Company, Indianapolis, Indiana

E. H. Jensen, The Upjohn Company, Kalamazoo, Michigan

B. T. Kho, Ayerst Laboratories, Rouses Point, New York

E. P. K. Lau, Searle and Company,Chicago, Illinois

H. H. Lerner, The Squibb Institute for Medical Research,

New Brunswick, New Jersey

A . F. Michaelis, Sandoz Pharmaceuticals, East Hanover, New Jersey

G. J. Papariello, Wyeth Laboratories, Philadelphia, Pennsylvania

C. R. Pilla, Wyeth Laboratories, Philadelphia, Pennsylvania

E. L. Pratt, The Sterling-Winthrop Research Institute, Rensselaer, New York

B. C. Rudy, Hoffman-LaRoche, Inc., Nutley, New Jersey

B. 2. Senkowski, Hoffmann-LaRoche, Inc., Nutley, New Jersey

C. M . Shearer, Wyeth Laboratories, Philadelphia, Pennsylvania

viii
 PREFACE
Although the official compendia list tests and limits for drug substances
related t o identity, purity, and strength, they normally do not provide other
physical or chemical data, nor d o they list methods of synthesis or pathways
of physical o r biological degradation and metabolism. For drug substances
important enough to be accorded monographs in the official compendia such
supplemental information should also be made readily available. To this end
the Pharmaceutical Analysis and Control Section, Academy of Pharmaceuti-
cal Sciences, has undertaken a cooperative venture t o compile and publish
Analytical Profiles of Drug Substances in a series of volumes of which this
is the third.
Reviews and comments received so far have reinforced our belief that
the series fills a need and they have strengthened our determination to con-
tinue. The enthusiasm and cooperative spirit of our contributors have made
these profiles possible. All those who have found the profiles useful are
earnestly requested t o contribute a monograph of their own. The editors
stand ready to receive such contributions.
Beginning with Volume 2 a cumulative index has been added, t o facili-
tate the correction of errors and t o encourage the addition or relevant new
information.
The concept of analytical profiles is taking hold not only for com-
pendial drugs but, increasingly, in the industrial research laboratories. Ana-
lytical profiles are being prepared and periodically updated t o provide
physico-chemical and analytical information on new drug substances during
the consecutive stages of research and development. Hopefully then, in the
not too distant future, the publication of an analytical profile will require
a minimum of effort whenever a new drug substance is selected for com-
pendial status.

Klaus Florey

ix
ACETAMINOPHEN

John E. Fairbrother
 JOHN E. FAIRBROTHER

CONTENTS
1. Description
1.1 Name, Formula, Molecular Weight
1.2 Appearance, Color
2. Physical Properties
2.1 Spectra
2.11 Infra-red Spectrum
2.12 Ultra-violet Spectrum
2.13 Fluorescence Spectrum
2.14 N.M.R. Spectrum
2.15 Mass Spectrum
2.2 Physical Properties of the Solid
2 . 2 1 Melting Characteristics
2.22 Density
2.23 Vapor Pressure and T.G.A.
2.24 D.T.A. and D.S.C.
2.25 Crystal Characteristics
2.26 X-ray Diffraction
2.3 Powder Characteristics
2 . 3 1 Static Charge
2.32 Flow Properties
2.33 Compression Characteristics
2.34 Surface Area and Porosity
2.4 Solubility
2.41 Solubility in Aqueous Solvents
2.42 Solubility in Water Miscible
Solvents
2.43 Solubility in Solvents Immisc-
ible with Water
2.44 Rate of Dissolution
2.5 Physical Properties of Solutions
2 . 5 1 Cryoscopy
2.52 Ionisation and pH
2.53 Dipole Moment
2.54 Refractive Index
2.55 Adsorption from Solution
2.56 Partition Coefficients
3. Molecular Complexes

2
 ACETAMINOPHEN

4. Synthesis and Purification

4 . 1 Chemical Synthesis
4.11 Synthetic Routes
4.12 Purification
4.13 Impurity Profile
4.14 Reference Standards
4.2 Biosynthesis
4.21 Metabolism of Phenacetin and
Acetani1ide
4.22 Prodrugs
4.23 Microbial Biosynthesis
5. Stabi1ity
5.1 Stability to Light
5.2 Stability of Solid Acetaminophen to

5.3
-Heat
Stability of Solutions of Acetamino-
phen
5.4 Stability to Oxidation
5 . 5 Compatibility with Excipient
Materials
5.6 Compatibility with Aspirin
5.7 Physical Incompatibilities
6. Analytical Chemistry
6.1 Identity Tests
6.2 Methods of Analysis
6.20 Gravimetric Procedures
6.21 T itrimet ric Procedures
6.22 Polarographic Procedures
6.23 U.V. Spectrophotometric Pro-
cedures
6.24 Photocolorimetric Procedures
6.25 Ion-Exchange Chromatrographic
Procedures
6.26 Partition Chromatographic Pro-
cedures
6.27 Paper and Thin-Layer Chromato-
graphic Procedures
6.28 Vapor' Phase Chromatographic
Procedures
6.29 High - Pressure Liquid Chrom-
atographic and Gel Filtration
Procedures
6.3 Automated Procedures
6.4 Radiochemical Procedures

3
 JOHN E. FAIRBROTHER

6.5 Determination of Trace Impurities and

Degradation Products
6.6 7 of Acetami nophen and
Luids and
Tissues
6.61 Determination in Urine
6.62 Determination in Serum, Plasma
and Whole Blood
6.63 Determination in Tissues and
Orsans
7. Metabolic Transzormations
7.1 Metabolism in Man
7.11 Adults
7.12 Newborn Infants
7.2 Metabolism in Animals
8. Drug Availability
8.1 Pharmacokinetics
8.2 Protein Binding
8.3 Interactions with Other D,rugSub-
stances
8.4 Biopharmaceutics
9. Toxicity
References

4
 ACETAMINOPHEN

1. Description
1.1 Name, Formula, Molecular Weight
Generic names - Acetaminophen1,
Paracetamol and Acetophenum2.

Chemical names - 4 - Hydroxyacetan-

1

ilide; p-hydroxyacetanilide; p-
acetamidophenol; p-acetaminophenol;
p-acetylaminophenol; N-acetyl-p-
aminophenol.

‘gHgN02 Mol. wt. 151.16

1.2 Appearance, Color, Odor, Taste

White, odorless, crystalline powder,
possessing a bitter taste.
2. Physical Properties
2.1 Spectra
2.11 Infrared Spectrum
Infrared spectra of solid dis ersions
of acetaminophen in potassium bromide3 t 7 and in 6
Nujo16, have been recorded. In the solid state
the carbonyl stretching band appears at 1659
c m - I (1650 cm-1; ref. 3 ) , the N-H stretching
band at 3326 cm-1 and a broad 0-H stretching
band at 3162 cm-1. In solution the C=O,N-H
and 0-H stretching bands occur at higher fre-
quencies.

5
 JOHN E. FAIRBROTHER

c =o N-H 0-H
Solvent Stretching Stretching Stretching
Band Band Band
Chloroform 1686cm-1 ( 9 )
Dichloro- 1690cm 3435cm (6)3588cm-1(8
methane
1700cm-1 (8)
1,4-Dioxan 1692cm-1 (8)

Several other authors10,11,12,15,16

report infrared spectra of acetaminophen. The
infrared spectra of acetaminophenl4 in KBr and
in a mineral oil mull are presented in figures
1 and 213.
2.12 Ultraviolet Spectrum
The U.V. spectrum of acetaminophen
has been recorded in a number of solvents,
showing two bands in each. The long wave-
length band corresponds to the A l g + B2u trans-
ition while the short wavelength band corres-
ponds to the IT^ + T co
* transition1’.

6
Fig. 1. I n f r a r e d spectrum of acetaminophen (KBr p e l l e t )
Fig. 2. Infrared spectrum of acetaminophem (Mineral Oil Mull)
 ACETAMINOPHEN

TABLE 1
Absorption maxima of
acetaminophen in neutral solvents

Solvent K band B band Referenca

Me thano1 248-249mp. 3.18

Ethanol 249-250mu. about 290mi.l. 4, 8,19
(abs.)
n-Butanol 250mv. 20
iso-Propanol 250mp. 19
Cyclo- 244-245mi.l. 19
hexane
Cyclo- 278mp. 8
hexane
Ether 264mi.l. 19
Ether (dry) 247mp. about 283mi.l. 8
Water 242.5- about 283mp. 8,19,23
243.5mp.

The addition of acid to aqueous and

alcoholic solutions does not give any observable
change in the sition of the maximum of the
main band 7 I 16r~~,19r21,22* In 10-1M caustic
alkali acetaminophen ionises to give the p-
acetamidophenolate ion and the maximum of the
main band is shifted bathochromically, in aque-
ous solution from 243 mu. to about 258 mu.
19,20,22,23 anpSin methanolic solution from 248
mp. to 262 mp. .

9
 JOHN E. FAIRBROTHER

TABLE 2
Molar absorptivities ( c ) of
acetaminophen in different solvents

Solvent Wavelength -E . References

Ethanol 249 mu. 13,090 to 4,8,19,24
14,000
288 mu. 2,000 to 19,24
2,120
Me thano1 249 mu. 13 I 600 3
Ethanol/O.lN 249 mu. 13 ,750 26
Hydrochloric
Acid
Water (pH 2 to 242 mp. ca .11 ,000 25
3)
Water ( p H 7.2) 242.5mp. 10,037 8
(Clark and
Lubs Buffer)
0.1N Sodium 257 mp. 10,820 21
Hydroxide
0.01N Sodium 258 mu. 10,830 24
Hydroxide
Water (pH 10 to 258 mu. ca.10,500 25
11 1

The ultraviolet spectra of acetamino-

phen in ethanol (95%) and in 0.01N Sodium hydr-
oxide (aqueous) are presented24 in figures 3a
and 3b.

10
FIGURE 3a. Ultraviolet Spectrum of
Acetaminophen ( e t h a n o l 95%)

11
FIGURE 3b. Ultraviolet Spectrum of Acetamino-
phen (0.01N sodium hydroxide)

12
 ACETAMINOPHEN

2.13 Fluorescence Spectrum

Acetaminophen has been reported25 ,27
to exhibit fluorescence in neutral and acidic
solution (excitation at 330 mp. and emission
peak at 400 mp.) .Nang et a1.25 also observed
fluorescence in aqueous alkaline conditions
(excitation at 315 mp. and emission peak at
430 mu.). However, recent attempts 24,28 to
confirm these findings have been unsuccessful
and it has been suggested28 that the earlier ob-
servation~ ' 27
~ ~could have resulted from the
Raman emission of water in conjunction with
poorly aligned monochromator systems. Acet-
anilide is not fluorescent436 and it is unlikely
that the introduction of a para-hydroxylgroup
into the molecule would change this character-
istic.
2.14 N.M.R. Spectrum
Puar and Funke201 recorded the N M. R. .
spectrum of acetaminophen in dimethylsulphoxide
- d6 (see figure 4) and assigned the observed
chemical shifts in the following manner.

69.07 69.58 62.00

HO 5 -
\& NH

66.70d ( 9.0)
0
II
- C-CH

67.35d(9.0)
Theriault and Longfield181 used the
N.M.R. spectrum as determined in deuterated
acetone to identify acetaminophen, formed by
Amanita muscaria as a conversion product of
acetanilide.

13
c
P
 ACETAMINOPHEN

2.15 Mass Spectrum

The effects of substituents on the mass
spectral fragmentation of para-substituted
acetanilides has been studied in detail29 ,30,446
but unfortunately examination of the p-hydroxy
com ound was omitted in each case. Burtis et
al.y1 give the main peaks of the mass spectrum
of acetaminophen as m/e 151, 135, 121, 109, 95,
81 and 55. The molecular ion undergoes a mass
l o s s of 42 to give the base peak of m/e 109.
This results from the re-arrangement of a proton
of the acetyl group to the phenyl ring, followed
by cleavage of the amide bond with the loss of
CH CO (m/e 42). This metastable transition
gi6es a strong diffuse peak.

Fales, Milne and Law444 recorded the

mass spectrum of acetaminophen, reporting the
most abundant peaks as m/e 109, 151, 43, 80 and
81. The relative abundancies for m/e 40 to
152 are tabulated444.
Puar and Funke201 have also recorded
the high-resolution mass spectrum of acetamin-
ophen (see figure 5 ) and suggest the following
scheme of fragmentation:-

15
 JOHN E. FAIRBROTHER

The occurrence of all three alter-

native modes of fragmentation of m/e 109 is
supported by observation of corresponding metas-
table ions and high resolution data.
Milne, Fales and Axenrod 445 have re-
corded the isobutane chemical ionisation mass
spectrum of acetaminophen indicating the peaks
found to be m/e 152, 153 and 151.

16
 ACETAMINOPHEN

1595 A C E T A M I N O P H E N

'Ih)

L-J:
35 5
2
I

38
I

25 0 -
Z

28 2J
0
t
t
Z

1 8

d 3

MASS/CHARCE
TOTAL UNSEALED INTENSITY. -
692QN
B A S E P E A K IS '13.93 PERCENT OF T O T A L

Fig. 5. Mass spectrum of acetaminophen

17
 JOHN E . FAIRBROTHER

2.2 P h y s i c a l P r o p e r t i e s of t h e S o l i d

2.21 Melting C h a r a c t e r i s t i c s
31
The m e l t i n g p o i n t f i r s t quoted
f o r acetaminophen ( 1 7 9 O C ) a p p e a r s t o be e r r o n -
eous. Subsequent d e t e r m i n a t i o n s gave m e l t i n g
p o i n t s o f 165 t o 168OC f o r r e l a t i v e l y u n p u r i f i e d
m a t e r i a l 32 t o 39 and a m e l t i n g r a n e of 1 6 8 t o
1690C f o r p u r i f i e d m a t e r i a l 40 to 49. More
r e c e n t l y improved p u r i f i c a t i o n p r o c e d u r e s have
been developed g i v i n g m a e r i a which m e l t s i n
t h e range 1 6 9 t o 1 7 1 0 C . 4i to i8. It is t h i s
m e l t i n g r a n g e t h e r e f o r e which i s quoted i n t h e
c u r r e n t r e f e r e n c e books3 t 4 and o f f i c i a l compendia
14,21.

Kuhnert-Brandsttitter 4 9 , 5 0 has
recorded t h e melting p o i n t using a Kofler h o t
s t a g e a s 1 6 7 t o 1 6 9 O C and c a r e f u l l y d e s c r i b e s
the melting process. From 14OoC t o t h e m e l t i n g
p o i n t g r a i n s , hexagonal prisms and rhomboids
sublime. R e s i d u a l c r y s t a l s i n t h i s temp-
e r a t u r e range grow i n t o hexagonal t o p o l y h e d r a l
g r a i n s and prisms. The m e l t s o l i d i f i e s t o a
g l a s s and g i v e s u n s t a b l e columnar a g g r e g a t e s a t
l l O ° C on which r e c t a n g u l a r prisms of t h e s t a b l e
m o d i f i c a t i o n a r e induced from a b o u t 1 4 0 O C . T h i s
u n s t a b l e m o d i f i c a t i o n (11) melts i n t h e r a n g e
154 t o 156OC.

2.22 Density

Fels4' reported the s p e c i f i c

g r a v i t y o f acetaminophen a t 2 1 O C a s 1.293.

2.23 Vapor P r e s s u r e and T . G . A .

Thermogravirnetric A n a l y s i s
(T.G.A.) f a i l e d t o d e t e c t any l o s s of v o l a t i l e s
from a sample of acetaminophen N F 30. ..
J a e c k e l and P e ~ e r l e ~ ~ ~
measured
t h e dependence of t h e c o n d e n s a t i o n c o e f f i c i e n t

18
 ACETAMINOPHEN

on the partial pressure over an evaporating cry-

stal face of acetaminophen. Measurements of
the vapor pressures on single crystal faces as
functions of the partial pressure were made using
a torsion balance.
2.24 D.T.A. and D.S.C.
Differential Thermal Analysis D.T.A.)
of a sample of acetaminophen N.F. gave436 a sharp
melting endotherm at 171OC. Examinati0x-1~~~
of
a sample of B.P. grade material by Differential
Scanning Calorimetry (D.S.C.) similarly gave an
endotherm at 171OC. On cooling the sample and
rescanning a different pattern was obtained
showing the sample melting at 157OC and also an
exotherm occurred at 67OC. From the D.S.C. 431
data a value of 6.8 K cals./mol. was obtained
for the Latent Heat of Fusion.
2.25 Crystal Characteristics
Morse31 in a paper describing the first
reported synthesis of acetaminophen, recorded
that it crystallised in the form of white mono-
clinic prisms.
Kuhnert-Brandstatter49r50 has des-
cribed visual changes which take place in crys-
talline acetaminophen durin the melting process
(see Section 2.21). F e l obtained
~ ~ ~ two app-
arently isomorphous crystalline forms of acetam-
inophen on recrystallisation from ethanol. From
his optical examination of these crystals, Fels4O
assigns them to the monoclinic system with sym-
metry 2/m; C2h
a: b: c = 1.3688 : 1 : 1.5103
6 = 115O 49.5'
40
For the two isomorphous forms he
makes the following face assignments:-

19
 JOHN E. FAIRBROTHER

Form 1

and Form 2 m =

c =
f = p3

The observed a n g l e s between t h e s e f a c e s

a r e g i v e n and i n some c a s e s compared w i t h cal-
culated values. Form l i s r e p o r t e d t o be gap-
a b l e of undergoing t r a n s f o r m a t i o n ( t o 001/010/
100) b u t Form 2 does n o t .

T h i s d a t a h a s been s y s t e m a t i s e d i n t h e
Barker Index of C r y s t a l s 4 3 4 .
D i s p e r s i o n of t h e o p t i c a l a x e s i s v e r y
s t r o n g i n acetaminophen, r < v . A v e r y s t r o n g
negative birefringence i s exhibited.
F a i r b r ~ t h e r ~found~’ that crystallisa-
t i o n of acetaminophen from a wide r a n g e of s o l -
v e n t s gave e s s e n t i a l l y two t y p e s of c r y s t a l
h a b i t . Hexagonal prisms (by c r y s t a l l i s a t i o n
from a l c o h o l s , e s t e r s , k e t o n e s , water, d i o x a n
and a c e t o n i t r i l e ) and s l e n d e r rhombohedra1
n e e d l e s (by c r y s t a l l i s a t i o n from benzene, t o l u -
e n e , d i c h l o r o e t h a n e and s e v e r a l o t h e r c h l o r i n -
ated solvents). Examination of t h e s e two
c r y s t a l t y p e s by D . S . C . , i . r . and x-ray d i f f r a c -
t i o n (powder) f a i l e d t o show any e v i d e n c e of
polymorphism.
2.26 x-ray D i f f r a c t i o n
Coy and Ochs433 have r e c o r d e d t h e x-ray
powder d i f f r a c t i o n p a t t e r n f o r a sample of acet-
aminophen N.F. (see F i g . 6 and Table 3 ) .

20
 ACETAMINOPHEN

TABLE 3
X-ray Powder Diffraction Pattern
of Acetaminophen (7032-LKR-242)
Interplanar Distances Relative Intensities
-
d (8) i/i1
7.36 0.26
6.42 0.20
5.78 1.00
5.30 0.13
4.90 0.66
4.70 0.19
4.38 0.34
3.81 0.65
3.68 0.90
3.37 0.74
3.29 0.11
3.21 0.06
3.08 0.09
2.75 0.20
2.48 0.07
2.44 0.11
2.34 0.07
2.3 Powder Characteristics
2.31 Static Charge
Acetaminophen particles flowing through
a hopper acquire a negative static charge51.
This charge is reduced by the addition of tablet
lubricants and by small quantities (0.5%) of
water.
2.32 Flow Properties
The bulk density of acetaminophen gran-
ules falls with increasing water content. This
represents a rise in internal cohesion and causes
a deterioration in flow properties52r 53. Spher-
0nization~~9 of granules for tablet compression

21
Fig. 6. X-ray powder-diffraction pattern of acetaminophen
 ACETAM INOPH EN

improves granulation flow rate. The presence

of water in acetaminophen granules increases the
angle of repose52.
2.33 Compression Characteristics
Uniaxial compression of crystalline
acetaminophen ives a pressure cycle typical of
a Mohr's body5zr59 producing capping and lamin-
ating compacts. The effects moisture content
and granulation have on the com ression charac-
E
teristics have been studied 541 5,58,59,60,456.

2.34 Surface Area and Porosity

The surface area of acetaminophen
powder compacts has been studied by the Brunauer-
Emmett-Teller 1B.E.T.) low-temperature nitrogen
adsorption p r ~ c e d u r e ~ ~ .The change of this
surface area with changes in moisture content
and/or compression pressure of the compacts has
been studied57158.
2.4 Solubility
2.41 Solubility in Aqueous Solvents
The solubility of acetaminophen in
distilled water has been described by several
authors.
Temperature Solubility References
(mg./ml.)
2O0C about 11.3 61
about 14.5 3,21I 63
25OC 11.66 8,64
13.85 65
37OC about 19 66
about 20 67
l0O0C about 52 3,61,62,63
In pH 6.0 buffer solution at 37OC its
solubility has been recorded68 as 23.8 mg./ml.

23
 JOHN E. FAIRBROTHER

P a r u t a and showed t h e solub-

i l i t y p r o f i l e of acetaminophen i n dioxan-water
mixtures t o correlate i n v e r s e l y w i t h t h e polar-
i t y ( d i e l e c t r i c c o n s t a n t ) p r o f i l e of t h e s o l v e n t
f o r m i x t u r e s c o n t a i n i n g more t h a n 30% w a t e r .
A s i m i l a r study65 conducted w i t h suc-
r o s e s o l u t i o n s ( a s s o l v e n t s ) gave t h e o p p o s i t e
e f f e c t , t h e s o l u b i l i t y of acetaminophen dec-
r e a s i n g w i t h d e c r e a s i n g d i e l e c t r i c c o n s t a n t of
t h e s o l v e n t ( i . e . w i t h i n c r e a s i n g s u c r o s e concen-
tration) .
Goldberg e t a1.66 examined t h e solub-
i l i t y of acetaminophen i n aqueous u r e a s o l u t i o n s
and found a l i n e a r i n c r e a s e i n acetaminophen sol-
u b i l i t y with i n c r e a s i n g u r e a c o n c e n t r a t i o n . T h i s
i n c r e a s e d t h e s o l u b i l i t y a t 37OC from about 1 9
mg./ml. ( i n water) t o about 31 m g . / m l . ( i n 3.0
Molar u r e a s o l u t i o n ) . T h e authors66 a t t r i b u t e
t h e s o l u b i l i z i n g e f f e c t t o an i n t e r a c t i o n o c c u r r -
ing i n s o l u t i o n between urea and t h e acetamino-
phen. The s o l u b i l i t y of acetaminophen i n water
i s r e a t l y i n c r e a s e d i n t h e p r e s e n c e of phenazone
7 O f 7 1 by a p r o c e s s thought t o i n v o l v e hydro en
bonding. A s i m i l a r s i t u a t i o n is observed 435 i n
t h e presence of c a f f e i n e b u t t h e o p h y l l i n e h a s
been shown435 t o reduce t h e s o l u b i l i t y of a c e t a -
minophen.
2.42 S o l u b i l i t y i n Water M i s c i b l e
Solvents
Solvent

Ethanol 1 in 10 63
Ethanol ( 9 5 % ) 1 in 7 21
Ethanol 1 in 8 61
Methanol 1 in 10 61,63
Acetone 1 in 13 21,63
A c et o n e 1 in 20 61
Propylene Glycol 1 in 9 21
( c o n t ' d)

24
 ACETAMINOPHEN

Solvent Reference

Propylene Glycol 1 in 10 63
Propylene Glycol 1 in 50 61
Glycerol 1 in 40 21,63
Glycerol 1 in 50 61
The f o l l o w i n g s o l u b i l i t i e s have been
determined under c o n t r o l l e d c o n d i t i o n s .
Solvent Temperature Solubility Ref.
(OC) .
(mg / m l . )
Water c o n t a i n i n g
2% e t h a n o l 26.5 23.9 72
Propylene Glycol 37 156 68
Dioxan 25 90 69
2.43 S o l u b i l i t y i n S o l v e n t s Immiscible
w i t h Water
Solvent Reference

Chloroform 1 i n 50 3,61,63
Benzene Insol. 4,61,63
Ether Insol. 3,61,63
Petroleum E t h e r Insol. 4
Pentane Insol. 4

The following s o l u b i l i t i e s have been

determined under c o n t r o l l e d c o n d i t i o n s : -
Solvent S o l u b i l i t y a t 37OC Reference
.
(mg /ml 1
Cyclohexane 0.0015 67
Theobroma O i l 2.16 68
2.44 Rate of D i s s o l u t i o n
D i s s o l u t i o n r a t e s t u d i e s conducted by
Goldberg e t a 1 . 6 6 examined t h e d i s s o l u t i o n of

25
 JOHN E. FAIRBROTHER

monoparticulate layers73 of acetaminophen, alone,

and in fused and physical mixtures with urea.
The dissolution of samples of pure acetaminophen
followed pseudo-zero order kinetics over a 5 min.
period. Coarse particles (50-60 mesh) gave data
in reasonable agreement with the "cube root law"
7 4 thus representing a system requiring correc-
tion for the decrease in surface area during
dissolution. Finer material (100-120 mesh)
gave data more in agreement with a planar sur-
face dissolution model. The eutectic and phys-
ical mixtures with urea gave biphasic dissolution
curves, the rate constant of the first part being
approximately twice that for pure acetaminophen
of similar particle size, while the second rate
constant closely resembled that for pure acetam-
inophen. This suggests the urea is leached out
leaving a matrix of effective surface area com-
parable with that of the pure acetaminophen.
Mattok, McGilveray and Mainville75
studied the dissolution of eight different lots
of formulated acetaminophen tablets using the
USP XVIII - NF XI;i,jrotating basket) method and
two other methods . None of the methods
gave complete correlation with the blood and
urine profiles obtained with the same samples.
All of the recently manufactured samples re-
quired less than 15 mins. for 50% dissolution
but stored samples showed greatly diminished
dissolution rates in some cases.
Chow and studied the dissolu-
tion rate of acetaminophen and its physical
mixtures and complexes with caffeine and theo-
phylline. The anhydrous form and the mono-
hydrate of the 1:l acetaminophen-caffeine com-
plex showed more than two and a half times the
dissolution rate of acetaminophen. However,
the hexahydrate of the acetaminophen-caffeine
complex and the 1:l acetaminophen-theophylline
complex both showed a reduction in acetaminophen
dissolution rate relative to pure acetaminophen.

26
 ACETAMINOPHEN

2.5 Physical Properties of Solutions

2.51 Cryoscopy
Several eutectics of acetaminophen have
been described in the literature:-
Eutectic Eutectic Eutectic Reference
with Temperature Composition
(OC) ( % Acetaminophen)

Phenacetin 115
Benzanilide 136
Urea 115
Acetylsalic-
ylic Acid 118.2 37 85
Phenazone 83 28.5 6
104 59.5 6
The cryoscopic properties of acetamino-
phen in naphthalene have been reported by Auwers
86.
2.52 Ionisation and DH
Acetaminophen is a weak acid its
saturated aqueous solution having a pHi4 of 5.3
to 6.5 at 25OC. pKa values for acetaminophen
have been quoted betwe 9.078 and 9.580 and
also recently as 10.15995. Two papers describe
the determination of the pKa value of acetamin-
ophen b spectrophotometric procedures. Talukdar
et al.15 obtained a value of 9.35 + 0.05 (uncor-
rected for ionic strength) at 25%:
procedure described by Roth and Bunnett
Dob68 et al.79 a value of 9.55 + 0.03 (at 25OC)
using the procedure described by Albert and
Serjeant82.
2.53 Dipole Moment
The dipole moment of acetaminophen has
been determined8 in 1,4-dioxan solution using a
Dipolmeter DMOl (heterodyne beat apparatus) and

21
 JOHN E. FAIRBROTHER

the molecular dipole moment ca ulated using the

method described by Hedestrandk5 .

This result is in good agreement with the value

of 3.96D reported by Lutskii et al.84.
Tomlinson's8 value yields a Molar Ori-
entation Polarisation (P2-) of 325.4658 cm3.
Lutskii et a1.84 quote a value for 1 . 1 ~ ~
(i.e. 1.1 obs.- LI calc.) of - 0.53 D .
2.54 Refractive Index
Microscopic studies with the Kofler
hot stage49tSO showed melts of acetaminophen to
have a refractive index of 1.5403 at 174oC (for
red light) and at 181 - 182OC (for sodium light).
Using an Abbe refractometer solutions of aceta-
minophen in 1,4-dioxan and in methyl alcohol
show linear increase of refractive index with
concentration up to 3.6% w/w and 10.8% w/w res-
pectively94.
From the equation:

n (observed)=n (acetaminophen) + "(methanol) ( 1-4

(x = weight fraction of acetaminophen in solutim)
a refractive index for acetaminophen of 1.608
(21°C, white light) was calculated94 (methyl
alcohol solution).
Measurement of the nD in ethanol has
been used87 to quantitatively determine the con-
centration of acetaminophen in two component
mixtures.

28
 ACETAMINOPHEN

2.55 Adsorption from Solution

The quantitative adsorption of aceta-
minophen from 2% ethanol solution was investi-
gated for the solid adsorbents, nylon, cellulose
triacetate and cellulose by Ward and Upchurch72.
The influence of temperature, time, solubility
and solvent were examined.
Cellulose did not adsorb acetaminophen
while nylon adsorbed almost twice as much as
cellulose triacetate. Desorption studies indi-
cated that adsorption occurred through hydrogen
bond formation, the preferred mechanism being
through the amido hydrogen of the acetaminophen
and the carbonyl oxygen of the adsorbent. Brook
and Munday89 have examined the adsorption of
acetaminophen on a dextran gel (methylated
Sephadex (LH-20)) and suggest a similar mechan-
ism of hydrogen bond formation.
2.56 Partition Coefficients
Acetaminophen is preferentially extrac
ted into ether from acid and weakly alkaline
a ueous solutionsg0 g1 I 92. Brodie and Axelrod
9Yi92 examined the effect of pH on the partition
of acetaminophen between ether and aqueous sol-
ution saturated with NaC1.

PH Volume Fraction Extracted

ratio in ether phase
- (ether/water) (ref. 9 2 ) (ref. 91)
4.0 5 - 0.88
7.0 5 0.91 0.88
9.0 S 0.85 0.89
10.0 5 0.61 0.79
11.0 5 0.57 0.62
13.0 5 0.0 0.0

Partition coefficients for acetaminophen between

other organic phases and water have been des-
cribed.

29
 JOHN E. FAIRBROTHER

Organic Partition Co- Log P Hansch Hy- Ref.

Phase efficient drophobic
(PI Substituent
Constant
(TI) -
Cyclo-
hexane 0.000075 n.a. n. a. 67
Chloroform/
Ethanol about 0.44 n.a. n.a. 93
l-Octanol - 0.55 -0.61 88
l-Octanol
(partition
with pH 7.2
buffer) 6.237 + 0.795 -0.36 8
2 .o%-

Similar information may also be de-

rived from the R values obtained in specially
designed reversed phase silica gel thin layer
chromatographic systems. Tomlinson8 employed
two systems of this kind. (see Section 6.27)

3. Molecular Complexes
Acetaminophen has been reported to
interact with chloral95 and with sorbitolg6tg7r
98. Possibly these interactions may result in
molecular complexes but insufficient data is
available for any interpretation of this kind.
A molecular complex of acetaminophen with
pyramidon (1:l) has been made99 and acetamino-
phen is known to hydrogen bond onto the surfaces
of nylon72 and rayon72.

30
 ACETAMINOPHEN

Lach and CohenlOO demonstrated the solubilis-

ation of acetaminophen with alpha - and beta -
cyclodextrins (Schardinger dextrins) . The
cyclodextrins exist in the form of cyclic chains
having a relatively large open space within each
molecule ( 6 a for alpha - and 82 for beta - cyclo-
dextrin). The interaction of acetaminophen with
the cyclodextrins produces non stoichiometric
inclusion complexes of the clathrate typelol.
Beta - cyclodextrin solubilises acetaminophen to
a greater extent than alpha-cyclodextrin, the
respective slopes of the interaction isotherms
being 1.100 and 0.395.
When mixed with henazone (antipyrine),
acetaminophen was reportedyo2 to give a syrupy
mass. Ridgway and Johnson70 independently
found that phenazone solubilised acetaminophen
in water and that an equimolar molecular complex
crystallised from solution. The complex was
also obtained from alcoholic or acetone solution
and from melts7l. It showed a congruent
melting point (109.5 to 110.50C)71 and a hydrggen
bonded structure has been proposed by Dearden
for the complex.
Chow and prepared (1:l) com-
plexes of acetaminopheq with caffeine and with
theophylline by a process of crystallisation
from aqueous solution. These complexes were
shown to exist in several hydrated forms:-
Acetaminophen Degree of M.pt. K1:l Heat of
(1:1) Complex Hydration (OC) (l.mole) Solutim
with at 25OC
(K cal./
mole)
Caffeine anhydrous ca.145 59.4 6.5
Caffeine monohydrate 75-80 - 10.1
Caffeine hexahydrate 4 2 - 5 0 - -
Theophylline anhydrous 192-195 16.1 -
Theobromine does not form a complex with aceta-
minophen in aqueous solution435.

31
 JOHN E. FAIRBROTHER

4. Synthesis and Purification

4.1 Chemical Synthesis
4.11 Synthetic Routes
Acetaminophen was first synthesised by
Morse31 in 1878 by reduction of p-nitrophenol
with tin in glacial acetic acid. The p-amino-
phenol produced by the reducing action of the tin
was not isolated, being acetylated in situ by the
acetic acid. Tingle and W i l l i a m s 3 3 w e d the
Morse synthesis but found it necessary to increase
the acetic acid concentration to 100% by the add-
ition of acetic anhydride.
Vignololo3 simplified the synthesis by
employing p-aminophenol a6 his starting material
which he acetylated with acetic acid.
Friedlander32 modified this process slightly by
acetylating the p-aminophenol (from p-nitrophenol)
with acetic anhydride in place of acetic acid.
Many preparative methods have since been des-
cribed employing the acetylation of p-amino henol
with acetic acid and/or acetic anh dride33 I s 4 I 36 I
37 I 39 I 41 I 42 I 43I 44 I 48I104 I 105I106 I187 I108; in
some cases anh drous sodium acetate also has
been added34 I 3% I 37 I 41I 42. The p-aminophenol
has been produced in numerous ways includin
the electrolytic reduction of nitrobenzene4%I the
direct o alytic hydrogenation of p-nitrophe-
no143 I 10s I gG , the sulphide reduction of
p-nitrosophenolLu'.

A typical43 reaction sequence is : -

32
 0 0
ACETAMINOPHEN

NH,HCI
N02

zr1{5"to
p.s.i.g.)
3ob OH /4
Neutralise
0H to

Pd/C

0. 0
OH

NHCOCH, NH2

Acetic
Anhydride

OH OH

In some processes the p-aminophenol is

not isolated but is acetylated in situ as it is
formed45,47,109,110. An alternative to the
acetylation procedure uses the action of ketene
111 in p-aminophenol.

Other synthetic pathwa s involve the

saponification of esters36 I 112I lY3
such as 4-
acetamidophenylacetate,the hydroxylation of
anilides b chemica1114 I electrolytic115 or
enzymatic116 processes, and the decomposition
of diazo compounds such as p-AcNHC H N BFq117.
Acetaminophen has also been synt gide8 from
p-hydroxyacetophenone hydrazone 2%.
4.12 Purification
Crude acetaminophen is in most cases
furified by recrystallisation from hot water39 I
5 t 48t110. Coloured impurities are removed
during the recrystallisation by treatment with

33
 JOHN E. FAIRBROTHER

activated charcoal39'48 and oxidation is supp-

ressed the addi on of small quantities of
NaHS031f8, Na2S204" or h y d r o ~ u l p h i t e ~ ~ .One
process48 controls the pH to 6 . 5 during the re-
crystallisation by the addition of ammonia.
A number of processes seek to purify
the p-aminophenol intermediate in a similar
manner with activated carbon and Na2S204 43,44,
437,438 and in one case44 also by extraction of
the aqueous p-aminophenol with an organic sol-
vent such as benzene, toluene, hexane etc., to
remove impurities such as azoxybenzene and azo-
benzene.
A patent by Hahn and Q ~ i n ndeals
~ ~
specifically with the purification of acetamino-
phen and related compounds made from crude dis-
coloured intermediates (p - aminophenol). The
discoloured acetaminophen is dissolved in hot
water, acidified (pH 1 to 5 ) with a non-oxidising
mineral acid and kept in a non-oxidising atmos-
phere (H2,C020r S02). The solution is agitated
with activated charcoal, filtered and allowed
to crystallise in the presence of an alkaline
reducing sulphite, bisulphite, or hydrosulphite.

4.13 Impurity Profile

The following impurities have been de-
tected in acetaminophen:-
Substance Origin Amount Reference
in commercial
(pharmaceutical)
grade material
p-Nitrophenol Synthetic - 119
precursor
p-Aminophenol Synthetic t 0.025% 7,14(first
intermediate suppl) ,21
p-Ch loro Impurity +lo p.p.m. 7,14
acetanilide cont ' d

34
 ACETAMINOPHEN

Substance Origin Amount Reference

in commercial
(pharmaceutical)
grade material
0-Acetyl para- Impurity - 119
cetamol from over- none detected 120
( DAPAP ) acetylationl-1 to 1.3% 121
of para-
cetamo1
Azobenzene By-products - 44
Azoxybenzene of reduction - 44
of nitro-
benzene
(precursor)
Guinone Oxiuation Give a bluish 46
Quinonimine of p-amin- or greyish color 46
meri-Quinon- ophenol to acetaminophen 46
imine (synthetic
intermediate)
Inorganic
Chloride
Inorgallic - 4 0.02% 7,14
Su1$ate
Inorganic - Not detected 7,14
Sulphide
Water - 4 0.5% 7 I 14,21

4.14 Reference Standards

A National Formular Reference Stan-
dard exists f o r acetaminophenY 4 .
4.2 Biosynthesis
4.21 Metabolism of Phenacetin and
Acetanilide

35
 JOHN E. FAIRBROTHER

Acetaminophen is the main metabolite

of both acetanilide and phenacetin (acetophenet-
idin) in man and in animals.
Acetanilide was introduced by Cahn and
Hepp12’ as an analgesic and antipyretic in 1887.
Investigating the metabolism of acetanilide,
Mdrner (1889)122 isolated potassium p-acetamido-
phenyl sulphate as a double salt with potassium
ethyl oxalate from human urine. He also isol-
ated a glucuronide tentatively identified as a
conjugated p-acetamidophenol. Confirmation of
this work was provided by Greenberg and Lester
123,124 and shortly after by Smith and Williams
12’ Who demonstrated that in the rabbit 70% of
the administered dose was excreted in the urine
as the glucuronide conjugate of acetaminophen
and 12% as acetaminophen sulphate. The metab-
olic fate o aypanilide has since been studied
in detail12 r 1 .
In the case of phenacetin MBrner128
similarly isolated acetaminophen sulphate and
the conjugated glucuronide from human urine.
Smith and Williams130 showed that 54% of the dose
administered to rabbits was recovered in the
urine as conjugated acetaminophen, (47% glucuron-
ide and 7% sulphate). In man, Brodie and
Axelrodgl found up to 82% of the administered
dose in the urine as conjugated acetaminophen
and about 3 % as free acetaminophen. More re-
cent papers231,132 give an essentially similar
picture. A comparative study133 of the avail-
ability of acetaminophen administered orally as
such and as pnenacetin gave availability ratios
(acetarninophen/phenacetin) in two studies as
1.04 and 1.06.
4.22 Prodrugs
Prodrugs are defined134 as having
physico-chemical properties different from the
parent drag but retaining qualitatively identi-
cal pharmacologic effects and reverting to the
parent drug in the body.

36
 ACETAMINOPHEN

c t ophen forms numerous ester pro-

drugs67 ,13' Kotenko and MokhortlEj5 des-
cribe an ethoxyphenylmethylacrylamide homopolymer
and its copolymer with o-carboxyphenylmethacryl-
amide which as analogs of phenacetin may be con-
sidered as prodrugs of acetaminophen. Extensive
studv has been made of the release of acetamino-

4.23 Microbial Biosynthesis

Theriault and Longfield18' studied the
microbial conversion of acetanilide to acetamino-
phen. An unidentified Streptomyces species RJTS-
539 gave a peak yield of 405mg./litre of acetamirr
ophen from 1000mg./litre of acetanilide.
Amanita muscoria F-6 gave a mixed yield
of acetaminophen and 2'-hydroxyacetanilide.
5. Stability
5.1 Stability to Light
Acetaminophen is slightly light sensi-
tive in solution63 and may degrade by a mechanism
involving pre-dissociation of the N-C bond as in
the case of acetanilide1711172.
5.2 Stability of Solid Acetaminophen toHeat
Dry, pure, acetaminophen is very stable
at temperatures up to at least 45OC. Should it
however, be contaminated with traces of p-amino-
phenol or be exposed to humid conditions such
that hydrolysis to p-aminophenol takes place,
then further oxidative degradation of the p-
aminophenol occurs121 characterised by a gradual
color change through pink to brown and eventually
to black. This involves the breakdown of the p-
aminophenol to quinonimine and related compounds
46.

37
 JOHN E. FAIRBROTHER

5.3 Stability of Solutions of Acetaminophen

The degradation of acetaminophen in
aqueous solution appears to be both an acid cat-
alysed and a base catalysed reaction173 I 174.
It is first order with respect to the concen-
tration of acetaminophen and first order with
respect to the hydrogen and hydroxyl ion con-
centrationl73.
Koshy and proposed reaction
mechanisms for the acid and base catalysed hy-
drolysis of acetaminophen and determined the
specific reaction constants (k') over the pH
range 2 to 9.
pH k' (hours-' x -
Ea t\ at 25OC
35OC 7OoC 9ooc (K (years)
- -
2 2.52 29.13 168.3 16.69 0.78
3 -- 7.40
-
31.03 17.99 5.83
4 10.76 - 15.39
5 - - 8.37 - 19.78
6 -- 2.56
- 6.98 17.42 21.80
7 13.16 - 12.59
8 - 6.58 25.37 17.99 7.13
9 - 19.02 66.62 17.42 2.28
The above studies17 were carried out isother-
mally. Zoglio et a1.175 repeated part of the
study (pH 2 buffer) but used a nonisothermal
linear temperature programmed technique. The
results obtained were in good agreement with
those of Koshy and Lach yielding a value for Ea
of 17.0 K cal./mole and a value for k' (at
35OC) of 1.95 x 10-4 hr.-1. Zoglio et al.
calculated the activation energy by comparing
analytical data with Arrhenius model degradation
curves using a digital computer. This approach
was further improved by Kay and Simon176 who re-
calculated the data of Zoglio et al. using an
analog computer system.

38
 ACETAMINOPHEN

5.4 Stability to Oxidation

Acetaminophen is relatively stable to
aerial oxidation unlike its hydrolysis product
p-aminophenol. Acetaminophen has been used
as an antioxidant for carotene in mineral o i l
solution177, a heat stabilizer for p ~ l y a m i d e s l ~ ~
and as an antioxidant, stabilizer and short-stop-
ping agent for synthetic rubber latexesl79.
5.5 Compatibility with Excipient Materials
The compatibility of acetaminophen with
a wide ran e of excipient materials has been re-
ported156 20 170.
5.6 Compatibility with Aspirin
Acetaminophen has been formulated in
numerous commercial tablet preparations with
aspirin. In some cases a third active drug sub-
stance such as caffeine, codeine phosphate or
salicylamide is also present.
Acetaminophen is known85 to form a
eutectic product with aspirin (m.p. 118.2OC) and
there is also some evidence to suggest that the
two substances interact chemically to produce
salicylic acid and diacetyl-p-aminophenol (p-
acetoxyacetani1ide) .
Koshy et al.180 found up to 4mg./tab-
let of diacetyl-p-aminophenol (DAPAP) in commer-
cial products and studied the formation of DAPAP
in laboratory prepared mixtures of acetaminophen
and aspirin after storage for up to 1 month at
5OoC. They also noted that magnesium stearate
appeared to accelerate the formation of DAPAP.
Boggiano, Drew and Hancock12'in a
later study confirmed the formation of DAPAP in
formulations containing acetaminophen and aspirin
(compressed tablets and uncompressed mixtures)
on storage at elevated temperatures ( 6 0 O C ) . They

39
 JOHN E. FAIRBROTHER

also suggested that codeine phosphate and mag-

nesium stearate both accelerate the formation of
DAPAP.
Kalatzis12' refutes the findings of
both the previous authors180,120, and shows
DAPAP to be present as a synthetic impurity in
commercial grades of acetaminophen and conse-
quently is present in conunercial products con-
taining acetaminophen. In stability evaluation
experiments with mixtures of acetaminophen and
aspirin stored at 45OC for up to 2 months no
DAPAP was formed. Samples of the acetaminophen/
aspirin mixtures spiked with DAPAP in fact showed
a gradual decline in DAPAP content if stored
under humid conditions at elevated temperature.
5.7 Physical Incompatibilities
Acetaminophen shows physical incompat-
ibility with antipyrine, Irgapyrin, Irgaphen, 2-
phenylquinoline-4-carboxylic acid and diphenhydr-
amine hydrochloridelo2, mixtures with these sub-
stances becoming sticky on mixing.
Rheological examination68 of acetamin-
ophen in microcrystalline cellulose-carboxymeth-
ylcellulose gels shows some evidence of an inter-
action between the acetaminophen and MCC - CMC.
Under humid conditions and at elevated
temperatures acetaminophen discolours in the
presence of codeine phosphate or caffeinelzl.
6. Analytical Chemistry
6.1 Identity Tests
Acetaminophen may be identified by its
melting point50 (see Section 2.21) and its eut-
ectic temperatures with phenacetin50, benzan-
ilide50 or urea66. It may be identified by
measurement of hysical parameters such as infra-
red spectrum14rs1 or G.L.C. retention time182.

40
 ACETAMINOPHEN

Acetaminophen yields numerous deri-

vatives many of which have clearly defined melt-
ing points:-
Reagent Derivative M.p. Ref.
(OC1
Benzoyl chloride 0-Benzoy1 171 62,183
- KOH -acetaminophen
4-Nitrobenzoy1 0-(4-Nitroben- 210 21
chloride-pyridine zoyl)-acetamin-
'ophen
Succinyl chloride bis (p-acetam- 225- 135
-pyridine inopheny1) 227
succinate
Phthaloyl chloride 0-Phthaloyl 235- 137
-pyridine -acetaminophen 237
Et2S04- alkali Phenacetin 134- 106
136
Ally1 bromide 0-Allyl- 93 42
acetaminophen
1-Fluoro-2,4- p- (2,4-dinitro 197- 184
dinitrobenzene phenoxy ) -acetan-.198
ilide)
Br /C HC1 2 ,6-dibromo- 174 41
acetaminophen
conc.HN03/conc. 2-nitro- 158 185,186
H2S04 (-5oC) acetaminophen
Diazotised aniline m-acetamino- 226 34,36
- HC1 o-hydroxyazo-
benzene
l-Nitroso-2- 10-acetamido- 337.5 190
naphthol-HN03 SH-benzo- [a]
phenoxa-
zonium nitrate
Acetaminophen gives a characteristic
violet-blue color reaction with a ferric chloride
test solution3,14' 21 and may be distinguished
from phenacetin by the color formed with
Liebermann's reagent3,Zl. T h i s involves oxi-
dation of the acetaminophen with acid dichromate
to slowly give a violet coloration in contrast
to phenacetin which gives a red coloration.

41
 JOHN E. FAIRBROTHER

Feig1187 describes a spot test for

acetaminophen claimed to have a sensitivity of
lpg. The test uses a procedure involving the
nitrosylation of the amine group followed by its
hydrolysis to a diazonium group which is sub-
sequently coupled with 1-naphthol to give a red
precipitate.
Le Perdriel et al. 186 found that in
the case of acetaminophen the initial nitro-
sylation reaction proposed by Feigl did not
occur but 2-nitro-4-acetaminophen01 is being
formed instead.
Acidification of the final Feigl test
solution (containing 1-naphthol) (as applied to
acetaminophen) produces a yellow-orange coloured
solution whereas in the contrasting cases of
acetanilide, phenacetin and p-aminophenol; red,
violet and black precipitates are formed.
Paper and thin-layer chromatography
have been used extensively to separate acetamin-
ophen from other substances and the combination
of Rf value and chromogenic response to spray
reagents may be used as an identity test. Of
particular note are the papers by Gumprecht and
Schwartzenburg1B8 (paper chromatography of iso-
meric monosubstituted phenols) and by Goenechea
189 (thin-layer chromatography of analgesics re-
lated to acetanilide).
6.2 Methods of Analysis
6.20 Gravimetric Procedures
Poethke and Kdhne184 describe the
quantitative precipitation of acetaminophen with
l-fluoro-2,4-dinitrobenzene in a sodium bicarb-
onate-dimethylformamide medium to give p-(2,4-
dinitrophenoxy) acetanilide. The precipitation
is carried out over a 4 hour period and is
claimed to give a precision of + 0 . 3 % . Caffe-
ine, phenazone, 4-aminophenazone, phenacetin and
codeine phosphate do not interfere.

42
 ACETAMINOPHEN

6.21 Titrimetric Procedures

Acetaminophen may be determined by ti-
tration with sodium nitrite after prior acid
hydrolysis of the acetaminophen to p-amin p
Both visual511911440and potentiometric1 9 s ,P J S O l
end-points have been used. Ce4+ quantitatively
oxidises acetaminophen thus rendering it possible
to titrate acetaminophen with 0.1N Ce(S04)~in
an ethanolic HC1 medium1g3 I lg4.
Chatten and Orbecklg5 attempted to ti-
trate acetaminophen with perchloric acid in
various acetic anhydride based solvents but were
unable to obtain an end-point.
Acetaminophen may be successfully ti-
trated in a dimethylformamide medium with 0.1N
sodium methoxide (in benzene-methanol) .
The
end-point may be determined visually using anlg6
azo violet indicator1g2 or potentiometrically .
Laurentlg7 also using dimethylformamide
solution tit-ratedacetaminophen visually to a
thymol blue end-point employing 0.1N Me4NOH (in
benzene-methanol) as titrant. Fogg et al. 2o
employed a similar system with 0.1M Bu4NOH as
titrant, a N2 atmosphere and potentiometric end-
point detection using a calomel reference elec-
trode filled with EtqNBr saturated dimethylform-
amide.
6.22 Polarographic Procedures
The anode polarographic behaviour of
acetaminophen has been studiedl98 at the wax-
impregnated graphite electrodelgg. This system
employs a solution of acetaminophen in aqueous
ethanol/phosphate buffer (l:l), pH 7.1 and gives
a value for ES vs. S.C.E. of 333mV. Brockelt
2oo describes a cathode polarographic procedure
for the determination of acetaminophen after
nitration with 5N HNO3. The solution contain-
ing nitrated acetaminophen is treated with pot-
assium hydroxide and phosphoric acid to give a

43
 JOHN E. FAIRBROTHER

solution pH of 5 . 8 and examined polarographic-

ally (E4 versus S . C . E . - 0.38V).
Shearer et al. 441 found that with the
use of a glassy carbon electrode, acetaminophen
could be determined polarographically with a
peak potential of about + 0.5V versus S . C . E .
This procedure is capable of selectively de-
termining acetaminophen in the presence of p-
aminophenol ( E # versus S . C . E . + 0.2V) and thus
may be used as a stability-indicating assay.
The water content of the acetate-acetic acid-
methanol supporting electrolyte significantly
alters the measured peak current for a given
concentration of acetaminophen and thus has to
be limited.
6.23 U.V. Spectrophotometric Proc-
edures
The British Pharmacopoeia 1963223 and
U.S. National Formulary XI62 both adopted U.V.
spectrophotometric procedures for the determin-
ation of acetaminophen in acetaminophen tablets.
In both procedures the tablets are extracted
with an anhydrous alcoholic solvent (B.P.-
ethanol and N.F.-methanol), the extract acid-
ified with a small amount of dilute hydrochloric
acid and then further diluted with the alcohol.
The acetaminophen concentration is determined by
spectrophotometric measurement of the extinction
of the solutions(249 mu.) and its content calcul-
ated against, a standard E (1 percent 1 cm.)
( B . P . method) or, the extinction given by a
sample of the N.F. Reference Standard.
Brown and Gwilt26 challenged the
official B.P.223 method on the grounds of cost
of the solvent and the use of a standard extinc-
tion coefficient. They proposed the adoption
of an alternative procedure employing 0.01N
NaOH as both extractant and spectrophotometric
solvent (extinction measured at 257 mu). This
procedure was subsequently adopted for use in

44
 ACETAMINOPHEN

the Addendum 1964224 to the B.P. 1963 and has

been continued in the B.P. 196821.
Rogers202 examined the effects of slit-
width on the precision of the B.P.224t21 assay
and calculated that for an extinction error of
0 . 2 % a maximum half-intensity slit width of
1.7mp. may be used (calculated for Hilger and
Watts, Uvispek H.700 and Unicam SP. 700 spectro-
photometers) .
The U . S . National Formulary (XI1203
and XIII14 editions) retains the use of the
acidified methanol solvent but employs a revised
extraction procedure in which the acetaminophen
is extracted from an aqueous suspension of the
ground tablet with a mixture of chloroform and
ethanol (3:l). Ivashkiv93 has studied the
parameters of this14 procedure as applied to
the assay of Squibb Acetaminophen Tablets and
reports the (75:25) ratio of chloroform to etha-
nol to be critical. Also examined were the
partition coefficient of acetaminophen between
the solvent phases (see Section 2.56) and the
effect of the grinding procedure used, on the
extraction of the acetaminophen. The results
obtained indicate micro-milling should not be
employed in the sample preparation.
Acetaminophen has been determined
spectrophotometrically ( 2 5 0 mp.) after partition
into n-butan0120 from sodium bicarbonate solution.
This facilitates the determination of acetamino-
phen in the presence of aspirin20. In a similar
manner acetaminophen may be determined in form-
ulated tablets of the acetaminophen-phenazone
complex204 (see Section 3) by selective reten-
tion of the acetaminophen in 0.1N sodium hydrox-
ide solution after partitioning with chloroform.
The alkaline solution containing the acetamino-
phen is acidified with hydrochloric acid and the
acetaminophen determined spectrophotometrically
at 245 mp.

45
 JOHN E. FAIRBROTHER

Differential spectrophotometry has been

used to determine acetaminophen in mixtures with
other drug substances. Hdberli and Bgguin205
determined acetaminophen in mixtures with salicy-
lamide by differential spectrophotometric measu-
rement at two wavelengths (255 and 301 mu).
Shane and X~wblansky’~ used differen-
tial spectrophotometry to determine acetamino-
phen in the presence of aspirin, salicylamide
and caffeine in analgesic tablets. Their pro-
cedure is based on the observation that the sub-
traction spectrum obtained by measuring the U.V.
absorption of the p-acetamidophenolate ion
(pH 10) against p-acetamidophenol (pH6) yields
an absorption maximum near to the isobestic
point of zero absorbance for salicylamide (263.5
mu.). Under the same subtraction conditions
caffeine and aspirin (which is converted to
sodium salicylate) do not exhibit any absorbance
from 255 to 340 mu.
Routh et al. 206 employ a similar pro-
cedure for the determination of acetaminophen in
the presence of aspirin and salicylic acid.
Acetaminophen has been determined
spectrophotometrically in mixtures with other
drug substances by several procedures involving
preliminary ion-exchange (see Section 6.25) or
partition chromatographic (see Section 6.26)
separation of the acetaminophen.
6.24 Photocolorimetric Procedures
The majority of the published colori-
metric methods for the determination of acetamin-
ophen are based on one of three systems. These
are nitration, oxidation or hydrolysis to p-
aminophenol followed by diazotisation and phen-
olic coupling.
G i ~ z a r d lnitrated
~~ acetaminophen with
a mixture of nitric and sulphuric acids at -5OC

46
 ACETAMINOPHEN

to yield 2-nitro-4-acetamidophenol. Horn225

described the nitration of phenacetin with nitric
acid and Brockelt20° applied this procedure to
the nitration of acetaminophen. This involved
nitration of an aqueous solution of acetaminophen
with 5N nitric acid at room temperature for 20
minutes. Brockelt found no absorption maximum
for the nitrated acetaminophen in the range 380
to 750 my. and decided to use the color formed
in an assay procedure measuring the light absor-
ption at 428 mp. (mid-point of the straight
part of the light absorption curve).
Feig1 and also Rosenthaler226 re-
ported that amides such as acetaminophen undergo
nitrosylation with nitrous acid to yield an N-
nitroso compound which may be saponified to give
a diazonium compound suitable for phenolic
coupling. Koen5 noted that in acidic medium
with sodium nitrite, acetaminophen gives a
yellow color which changes to an orange color on
rendering alkaline and uses this color to quan-
titatively determine acetaminophen. Le Perdriel
et a1.186 found that acetaminophen did not form
a nitroso compound on reaction with sodium
nitrite and dilute hydrochloric acid but the
2-nitro-4-acetamidophenol described by Girard.
They found that this reaction could be used as
the basis of a colorimetric assay procedure. The
solution containing nitrated acetaminophen is
made alkaline with sodium carbonate solution
and the light absorption measured at the absorp-
tion maximum occurring between 440 and 445 mp.
Inamdar and Kaji227 employ a similar
procedure but do not make the final solution
alkaline. This yields a solution of nitrated
acetaminophen giving absorption maxima at 375
and 395 mp., the latter wavelength being used
for quantitative measurement.
Hanegraaff and Chastagner continued
the work of Le Perdriell86 studying the mechan-
ism of the nitration of acetaminophen and modi-
fied the spectrophotometric assay procedure.

47
 JOHN E. FAIRBROTHER

They greatly increased the concentration of acid

employing a very strong mixture of nitric and
sulphuric acids in addition to the use of sodium
nitrite and measured the extinction at 375 mu.
The method of Le Perdriel ha
thoroughly evaluated by Chafetz et al.
have attempted to optimize the reaction para-
meters and claim the procedure has an excellent
precision and is well suited to automated tech-
niques442. This procedure229 is claimed to be
specific for the determination of acetaminophen
in the presence of a range of excipient materials
and drug substances commonly found in formul-
ations containing acetaminophen.
The p-nitrobenzoyl esters of acetamino-
phen were prepared by Tin le and Williams35 and
7
by Reverdin and Cuisinier 83 in 1906. More re-
cently the absorption spectra of the 4-nitro-
benzoic acid230 and the 2,4- and 3,5-dinitro-
benzoic231 acid esters of acetaminophen (as
well as the trans-4-nitrocinnamic acid ester232)
have been thoroughly studied and may represent
useful colorimetric reagents for the determin-
ation of acetaminophen.
Oxidative reactions have been used in
the determination of acetaminophen. Basu250
hydrolysed acetaminophen with hydrochloric acid
to give p-aminophenol which was then oxidised
with 0.1N potassium dichromate to give a violet
coloured oxidation product. This dye was quan-
titatively extracted into isobutyl alcohol and
its absorbance measured spectrophotometrically
at 550 mu.
Brodie and Axelrodg2 found that p-
aminophenol from the acid hydrolysis of acetam-
inophen could be oxidised with sodium hypobrom-
ite and the oxidation product coupled with phenol
to form an indophenol dye (absorption maximum
620 to 630 mp.) This procedure has been used
to determine acetaminophen in biological material

48
 ACETAMINOPHEN

92r251 and has also been used to determine the

level of free p-aminophenol in acetaminophen621
203 ,252.

The Brodie-Axelrod procedureg2 requires

the neutralisation of an acid solution of p-amin-
ophenol with alkali and should the neutralisation
point be passed to give an excess of alkali, de-
gradation of the p-aminophenol results. This
roblem has been overcome by Murfin and Wragg253,
554 who have been able to remove the need for
preliminary hydrolysis of the acetaminophen to
p-aminophenol. In their manual procedure254
the acetaminophen solution is added to a hydro-
chloric acid-sodium hypochlorite mixture (pH
3.4; ca. 0.25% available chlorine) and the
excess hypochlorite is removed with sodium arsen-
ite. The quinone chlorimide thus formed is
then coupled with phenol in the presence of a
borate buffer (pH 9.9) to give a stable blue
indophenol dye (A max. 625 mp.). The procedure
yields results with good precision, the mean rel-
ative standard deviation obtained by the authors
being 0.36%.
N i n ~ m i y a ~oxidised
’~ acetaminophen
directly with potassium ferricyanide in sodium
hydroxide solution at OOC and then formed an
indophenol dye ( A max. 635 mu.) by coupling with
phenol. The coloured product was tentatively
identified by Ninomiya as N-(p-hydroxypheny1)-
p-benzoquinone imine.
Sakurai and Umeda2560xidised acetamino-
phen with chloramine T in the presence of 2,4-
dinitrophenyl hydrazine to give a coloured p-
benzoquinone imine 2,4-dinitrophenylhydrazone.
The color produced can probably be used in a
similar manner to that described in a further
paper by Umeda257 which describes the oxidation
of acetaminophen with ceric ammonium sulphate in
acidified ethanolic solution and is subsequently
reacted with 3-methyl-2-benzothiazoline hydrazone.
This reaction mixture on neutralisation with tri-

49
 JOHN E . FAIRBROTHER

ethanolamine yields a blue violet color (A max.

5 8 0 mp.) which facilitates quantitative spectro-
photometric measurement.
Routh et al. 206 employed a stable free
radical, diphenylpicrylhydrazyl, to abstract a
hydrogen atom from acetaminophen (in ethylene
dichloride solution) thereby promoting a process
of radical coupling. This results in a reduc-
tion of the violet color of the diphenylpicryl-
hydrazyl ( A max. 527 mp. ) with the formation of
yellow diphenylpicrylhydrazine. The decrease
in the intensity of the violet color is used to
measure the concentration of acetaminophen.
Dedicoat and Symonds443 found that in
a pH 8.0 borate buffer acetaminophen reduces
Folin and Ciocalteau's reagent to give a stable
blue color (A max. 700 mp.) .
This was best
produced by heating the reaction mixture at 100°C
for 10 minutes and could be used to determine
acetaminophen in the presence of several other
analgesic drugs.
A number of procedures are based on
the acid hydrolysis of acetaminophen to p-
aminophenol which is then coupled with a suit-
able agent. A much used procedure employs the
reaction of the p-aminophenol with - naphthol
in alkaline solution, extraction of the coloured
reaction product into n-butanol followed by
measurement of the extinction at 6 3 5 mp. This
procedy55,i54based on the work of Greenberg and
Lester and the later papers of Kosh and
Y
LachZ09 and Gwilt, Robertson and McChesney 3 3 ,
the latter authors employing an 0: - naphthol
reagent containing potassium dichromate.
Brodie and Axelrod 92 modified the
procedure and diazotised the p-aminophenol
before reacting it with alkaline - naphthol to
give a dye exhibiting an h max. at 5 1 0 mp. This
procedure was also employed by Carlo et a1.234
after slight modification.

50
 ACETAMINOPHEN

Kos 235 reacted the p-aminophenol (ob-

tained by acid hydrolysis of acetaminophen) with
alkaline B - naphthol (without diazotisation) to
give a green color having an absorption maximum
at 420 mu.
Mouton and Mason236 used trichloro-
acetic acid to hydrolyse acetaminophen to p-
aminophenol which they then diazotised and
coupled with N1-diethyl-N-1-naphthyl-propylene-
diamine. The dye formed was extracted into amyl
alcohol and the extinction determined at 590 mp.
This procedure was modified slightly by Heirwegh
and Fevery237 who substituted N- (1-naphthyl)
ethylenediamine as coupling agent ( A max. 596mp.).
I v a ~ h k i vhas
~ ~also
~ examined this procedure
critically, finding at least 4 hours incubation
at room temperature is required for complete
color development.
Other procedures have been described
where the p-aminophenol obtained has been cou led
with alkaline anisaldehyde239, acid vanillin2go
and other reagents241,247.
It is possible to couple diazotised
reagents directly with acetaminophen but they
s
react on1 s l 0 w l y 7 9 ~ ~ ~ Dobsg,
~. Stgrba and
VebeFLa79, 42 have studied the kinetics of these
reactions and propose a reaction mechanism.
Acetaminophen has also been shown to
couple with the diazotised reagents shown in
Table 4 thus presenting the opportunity for
possible spectrophotometric measurement.
Kondo et al. 246 have examined the ab-
sorption spectra (and bathochromic shift) pro-
duced by the reaction of acetaminophen in alkal-
ine methanolic solution with an p-nitrobenzene
diazonium fluoroborate reagent. Acetaminophen
has been shown to produce colors of potential
spectrophotometric use by interaction with 1-
nitroso-2-naphthol, 2-nitroso-1-naphthol and

51
 JOHN E. FAIRBROTHER

disodium-3-h dr
sulphonate 2x8 5%.-4-nitros0-2~7-naphthalenedi-
TABLE 4
Diazotised Reasents CaDable
of Coupling wit; Acetaminophen
Reagent Color of Ref,
Product
Diazotised aniline (acid) Yellow 7%
Diazotised 1,4-Napthyl-
amino-sulphonic acid (alkali) Red 243
Diazotised 2-Naphthyl- Yellow- 243
amino-8-sulphonic acid(alka1i) brown
Diazotised lf4-Amido-aceto- Red 243
naphthalide-6-sulphonic acid
(alkali)
Diazotised 4-Nitro-6-chloro- Olive 244
2-aminophenol brown
Diazotised 4,6-Dinitro- -
245
2-aminophenol

6.25 Ion-Exchange Chromatographic

Procedures
Ion-exchange column chromatography has
been used to separate acetaminophen from its
decomposition product, p-aminophenol, from
mixtures containing other medicinal agents and
from dosage forms (see Table 5) .
Street and Niyogi211f213 separated
acetaminophen from phenacetin (acetophenetidin)
phenobarbitone and salicylic acid by two dimen-
sional chromatographic development on diethyl-
aminoethylcellulose ion-exchange paper (Whatman
DE 20). Initial separation was by simple de-
velopment in a 0.2N ammonium hydroxide solvent.
This was followed by ionophoretic development
at right angles in the same solvent (5mA; 250V.).

52
 TABLE 5
Ion-Exchange Chromatographic Separation of Acetaminophen
Separation Ion-Exchange Elutrient Quantitation Reference
from medium
Elixir formulation Dowex 1 - X8, 20% glacial Titrimetry in DMF
200- 400 mesh acetic acid solution with sodium 196
(hydroxide form) in ethanol methoxide
Phenobarbitone Dowex 1 - X1 7 0 % methanolU .V. Spectro-
photometry (249mu. ) 207
0.1N HC1 in Differential U.V.
7 0 % ethanol Spectrophotometry 205

[
VI
p-Aminophenol Amber 1ite Water U.V. Spectrophoto-
metry ( 2 4 4 mv.) 208
p-Aminopheno1 U.V. Spectrophoto-
Chlorobenzoxazolinf:fyite IR-120 Water metry ( 2 4 4 mu.) 209
Sulphadimethoxine
Caffeine
Theophorin Tartrat

1
Phosphate Cation Exchange
Phenylephrine HC1 Resin, Alginic
Pyrilamine Maleate acid, Mesh 40-100
(B.D.H.)
Water U.V. Spectrophoto-
rnetry (249 mu.)
210
 JOHN E. FAIRBROTHER

6.26 Partition Chromatographic

Procedures
Koshy213 separated acetaminophen from
admixture with caffeine and aspirin by partition
chromatography employing consecutive sulphuric
acid and sodium bicarbonate impregnated Celite
columns. Caffeine was retained on the acid
column and aspirin on the alkaline column.
Acetaminophen which is not ionised under either
set of conditions passed through the columns in
the diethyl ether solvent.
Levine and Hohmann214 noted that the
above system was unable to separate acetamino-
phen from neutral or weakly acidic compounds.
To overcome this weakness they replaced the
sulphuric acid by hydrochloric acid and the
sodium bicarbonate by a sodium carbonate-sodium
bicarbonate buffer (pH 10.1). The sample con-
taining acetaminophen is incorporated into the
acid impregnated Celite before it is packed into
the first column which is placed directly above
the second alkaline column.
The two columns are washed with chloro-
form and then the acetaminophen is eluted with
ether. The acetaminophen is determined spectro-
photometrically in acid methanol solution (249mp.)
following evaporation of the ether. This pro-
cedure will successfully allow the determination
of acetaminophen in the presence of many co-
administered drug substances.
This rocedure214 was slightly modi-
fied by HohmannSlS who contained the two packing
materials in a single column as separated seg-
ments. The modified procedure shown to be satis-
factory for the determination of acetaminophen
in liquid preparations was adopted by the NFXIII
for the determination of acetaminophen in Aceta-
minophen Elixir.
Both procedures ’ 215 have been

54
 ACETAMINOPHEN

s u c c e s s f u l l y a p p l i e d 2 1 6 to 219 t o t h e d e t e r m i n -
a t i o n of a c e t a m i n o p h e n i n o t h e r f o r m u l a t e d p r o -
ducts. F u r t h e r evaluation220 of t h e s i n g l e
column p r o c e d u r e i n d i c a t e d t h a t optimum r e s u l t s
were o b t a i n e d if 1N h y d r o c h l o r i c a c i d and 0 . 5 %
e t h a n o l i n chloroform a r e used i n p l a c e of t h e
c o n c e n t r a t e d h y d r o c h l o r i c a c i d and c h l o r o f o r m .

H a m i 1t o n 221 h a s d e s c r i b e d a f u r t h e r
m o d i f i c a t i o n t o t h e two-column p r o c e d u r e i n
which t h e columns a r e s e p a r a t e d a f t e r t h e c h l o r o -
form wash and a c e t a m i n o p h e n i s e l u t e d f r o m t h e
a c i d column w i t h water-washed e t h y l a c e t a t e .

Koshy e t a l . have used a t h r e e

column s y s t e m ( b a s e d on t h e Levine222 s y s t e m )
t o s e p a r a t e d i a c e t y l - p - a m i n o p h e n o l from t a b l e t
f o r m u l a t i o n s c o n t a i n i n g acetaminophen, a s p i r i n
and c a f f e i n e .

6.27 P a p e r and T h i n Layer Chromato-

graphic Procedures

A number of t h i n l a y e r and p a p e r chrom-

a t o g r a p h i c methods have b e e n found s u i t a b l e f o r
t h e i s o l a t i o n and i d e n t i f i c a t i o n of a c e t a m i n o -
phen. T h e q u a l i t a t i v e a s p e c t s of t h e s e methods
a r e summarised i n T a b l e s 6 t o 11.

Reversed phase chromatography w a s used

by Tomlinson8 i n a s t u d y d e s i g n e d t o c o r r e l a t e
Rf c h a r a c t e r i s t i c s w i t h c h e m i c a l s t r u c t u r e f o r a
series of s u b s t i t u t e d a c e t a n i l i d e s including
acetaminophen. I n t h i s study8 t w o separate
s t a t i o n a r y p h a s e s were u s e d on s i l i c a g e l G
plates.

55
 JOHN E. FAIRBROTHER

Stationary Mob i1e Rf at 20 + 0.5OC

Phase Phase (av. of lo-results)
l-octanol acetone/water 0.758
(1:9) (0.740 to 0.779)
liquid acetone/water 0.713
paraffin (2:8) (0.710 to 0.716)
(B.P.)
Semi-quantitative procedures relying on
the visual comparison of sample spot size and
intensity with standards have been described by
Klutch and Bordunl31 and by Shand267. Bkh
et a1.270 described a quantitative procedure in
which the acetaminophen is acid hydrolysed to
p-aminophenol which is then separated (lO-lOOug./
spot) by thin layer chromatography on a Silica
Gel G layer. The p-aminophenol is eluted with
0.5N sodium hydroxide solution and determined
spectrophotometrically at2zj0 mu. A further
paper by the same authors employs chromato-
graphic separation of the acetaminophen (without
prior hydrolysis) on a layer of Silica Gel GF
followed by elution with methanol and spectropho-
tometric determination at 245 mu. The procedure
may be used to determine acetaminophen (60-3OOpg.
/spot) in serum with a precision of + 5%.
Cummings, King and Martin265 describe a similar
quantitative procedure that employs elution of
the acetaminophen from the silica gel with water
rather than with methanol.
6.28 Vapor Phase Chromatographic
Procedures (G.L.C., V.P .C.)
Acetaminophen presents difficulties for
quantitative G.L.C. determination as a result of
the pronounced elution peak tailing caused by
its polar hydroxyl group.
Koibuchi et al. 278 overcame this prob-
lem by acetylating the hydroxyl group to give
N,O-diacetyl-p-aminophenol (DAPAP) which gave a
good symmetrical peak after elution from a 1%

56
 TABLF: 6
Paper Chrmtographic Systems for Acetaminophen

paper Direction Solvent System F+ Value -

Use Reference

Wham 3
MM Two dimensional a) Isopropanol/aq. 0 ; 83 Characteris- 481
development, mnia/water ation in human
ascending (8:l:l) urine
b) Benzene/propionic 0.24
acid/water (loo0:
700:41)
Whatman No. 1 ascending Water 0.83 Chromatographic 188
whatmatman No.1 II
Mineral s p i r i t s study of
ln saturated with water 0.00 isomeric phenols II

4
Matman No. 1 11
Toluene saturated 0.03 It II II

w i t h water
whatmatman No. 1 ascending Phosphate buffer 0.80 Identity test 3
impregnated (pH 7.4)
with t r b u t y r i n
Wha- No. 1 ascending .
Benzene/gl acetic n. a. Isolation from 181
acid/water/n-butanol microbial cul-
(38:38: 17 :7) cultures
Grade FN1 ascending n-Butanol/lO% aq. 0.61 Separation from 2
(VEB Spezial- m n i a (2:l) other analgesics
papierf abrik)
Grade FN1 ascending Benzene/acet i c 0.04 Separation from 2
impregnated w i t l i acid/water (4:4:2) other analgesics
4% sodium bi-
carbonate (pH9)
 TABLE 7
Thin Layer Chromatographic Systems for Acetaminophen (Neutral Systems)

Absorbent solvent system Rf Value -

Use Reference

Silica Gel GF &k?thanol 0.50 Separation from phena- 259

zone
Silica Gel G Methyl ethyl ketone 0.50 Separation from 260
chlorpromzine
Silica G e l GF &k?thylethyl ketone 0.70 Separation frcan phena- 259
zone
Silica Gel HF Butanone 0.44 Identity test 2 68
Silica Gel GE' Chlorofom/diethyl ether 0.00 Separation from phena- 259
(85:15) zone
Silica G e l G Chloroform/acetone ( 90 :10) 0.09 Separation from butc- 191
barbitone
Silica G e l G Chlorofom/methanol (80: 20) 0.75 Identity test 261
Silica G e l G BenZene/acetone (20 :10) 0.33 Separation from other 262
analgesic metabolites
Silica G e l G Chlorofobzene/ 0.33 as abwe 189
acetone ( 65 :10:25)
Silica G e l GE' Acetone/n-butanol/ 0.92 Determination of 2 63
water (50:40:10) acetaminophen and
mtabolites in serum
Silica Gel G Two dimnsional develop-ent
.
a) Chloroform/acetone (90: n a. Determination of 264
10) acetaminophen and
b) Chloroform/benzene/ n.a. metabolites in urine
acetone (65 :5 :30)
 TABLE 8

Thin Layer Chromtographic Systems for Acetaminophen (Alkaline Systems)

Adsorbent Solvent system Rf Value -

Use Reference

S i l i c a Gel GF Chlorofom/95% rraethanol/ammnia 0.47 Isolation from micro- 181

(85: 15: 1) b i a l cultures
S i l i c a G e l GF chlorofom/iso-propanol/ 0.80 Determination of 263
35% aq. m n i a (45:45:10) acetaminophen and
metabolites i n serum
S i l i c a Gel GF Chloroform/iso-propanol/ -
33% aq. ammnia (80:15:5)
lower phase/methanol ( 90 :5 ) 0.79 as above 263
2 Silica G e l G Butylacetate/acetone/n-
butanol/lO% aq. m n i a
( 5 0 :40: 30: 10: ) 0.67 I d e n t i t y test 269
S i l i c a Gel c;F Cyclohexane/chloroform/ 0.05 Separation from phen- 259
pyridine (20:60:5) azone
0.06 Identity test 268
 mL
E
l 9
Thin Layer Chromtographic Systems for Acetaminophen (Acidic Systems)

Adsorbent solvent system Rf Value Use Reference

Silica Gel G Chloroform/ethanol/acetic Chrmtogratn SeparationTFom WAP 1 2 1
acid (88:10:2) diagram and other analgesics
Silica Gel G Chloroform/acetone/acetic as above as above 121,180
acid (80:18:2)
S i l i c a G e l GF &nzene/nethanol/acetic 0.58 Determination of 263
acid (45:8:4) acetaminophen and
metablites in serum
S i l i c a Gel GF Mhy1 acetate/nethanol/ 0.82 Determination of 265,266
water/acetic acid acetaminophen and
( 60 :30: 9 :1) metabclit=s in urine
m S i l i c a G e l (F Double developxent
0
a) Benzene/diethyl ether/ Chrmtogratn Separation from W A P 120
acetic acid/rraethanol illustrated and other analgesics
(120: m: 18 :1)
b) E t h y l acetate/diethyl
ether (80:20)
B r inlaMn Toluene/benzene/water/ 0.10 Separation from other 154
Al oxide GF acetic acid (2:2:1:2) roetabolites
(upper phase)
Brinlrman Chloroform/mthanol/water/ 0.35 as above 154
Al oxide GF acetic acid (20:10:20:1)
(lmer phase)
B r inlaMn Cyclohexane/n-propano 1/ 0.84 Separation of 131
A1 oxide GI? water/acetic acid (20: 20:20:1) phenacetin metabolites
(upper P h - 4 cont'd ......
 ?IABLE 9 (cont'd)
Thin Layer Chramatographic Systems for Acetaminophen (Acidic Systems)

Adsorbent Solvent system Rf Value Use

- Reference
Brinlcman Ethylene dichloride/methanol/ 0.40 Separation of 131
A1 oxide GF water/acetic acid (20:10:20:1) phenacetin metabolites
(1- phase)
minlanan Ethylene dichloride/nrethanol/ 0.16 as above 131
Al oxide GF water/acetic acid (30:5:10:3)
Silica Gel G Butyl acetate/chloroform/ 0.46 Identity test 269
85% formic acid (60:40:20)
S i l i c a Gel W Dichloroethane/ethyl acetate/ 0.65 as above 263
98% formic acid (60:20:20)
 TAEu;E 10
Reagents for Papa Chrm-aphic Visualisation of Acetaminophen

Reagent Color Sensitivity Reference

.
(pg acetaminophen)
1. U.V. - Fluorescence Blu-rey 20 2
2. U.V. - Fluorescence a f t e r Yellow 1 2
spraying w i t h 0.5% ethanolic
oxine
3. Diazotised p-nitroaniline Violet 2,258
4. 5%ethanolic f e r r i c chloride Violet 2,181
5. 15%aq. f e r r i c chloride/
1% aq. potassium ferricyanide (1:l)Deep blue < 1 2
O, 6. Ferric chloride/potassium Violet 20 2
h,
ferricyanide/Millon's reagent
(2: 2: 2)
7. Amnoniacal silver n i t r a t e (0.W) Black 2
8. Rromine/starch/potassium iodide Blue 3
9. C e r i c m n i u m n i t r a t e purple 188
 TABLE 11
Reagents for Thin Layer Chranatographic Visualisation of Acetaminophen

Reagent Color Sensitivity Reference

1. U.V. (254mp.) - fluorescence
( ug.acetaminophen)
Dark spot 0.5 267
quenclling
2. 5%aq. s i l v e r n i t r a t e Black 0.25-0.5 259, 262,267
3. 10%aq. s i l v e r n i t r a t e Black 0.2 189, 264
4. 10%f e r r i c chloride and 0.5% Dark blue 0.1 181, 267
potassium ferricyanide in w a t e r
5. 5% aq. f e r r i c chloride Grey blue < 5 189
6. Folin and Ciocalteu reagent Blue 0.5 263
o\
7. pDimethylaminabenzaldehyde/ Yellcw 1 189
w hydrochloric acid
8. Iodine vapor n.a. n.a. 180
9. Pauly reagent n.a. n. a. 131
10. Diazotised o-dianisidine n.a. n. a. 131
11. Diazotised sulphanilic acid n.a. n.a. 120
 JOHN E. FAIRBROTHER

DEGS column. The acetaminophen was acetylated

with a pyridine-acetic anhydride reagent em-
ploying strictly controlled reaction conditions
to suppress the formation of N,N,O-triacetyl-p-
aminophenol (TAPAP), a secondary product of the
reaction. Quantitation was effected by peak
height ratio measurement using an internal
standard and almost theoretical recoveries were
obtained from laboratory prepared mixtures
(standard deviation 0.4 to 0 . 5 % ) .
Prescott has more recently employed
a similar procedure for the determination of
acetaminophen in plasma (standard deviation
.
about 3 . 5 % )
cetaminophen may be readily silylated
286 to 29Q to form derivatives suitable for quan-
titative G.L.C. determination2791280,2811292.
PreScott2 used a N- tr imethy 1sily1imidazo1e
(TMSI) reagent to selectively silylate the hydr-
oxyl group. In a separate procedure he280 used
a N,O-bis (trimethylsilyl) acetamide (BSA) re-
agent which produces a di-TMS derivative by
silylating both the hydroxyl group and the amide
nitrogen. He reports280 near theoretical re-
coveries (TMSI procedure) for acetaminophen from
plasma and urine with standard deviations of 1.8
and 2 . 8 % respectively (calculation from peak
height ratio with an internal standard).
The direct G.L.C. determination of acet-
aminophen has been described27212741275but the
accuracy seems generally to be inferior to that
of the indirect procedures and the working range
for sample size is narrower as a result of poor
peak symmetry.
Table 12 gives details of the various
G.L.C. systems described for the separation,
identification and quantitative determination of
acetaminophen.

64
 TAJ3LE 12
G.L.C. (V.P.C.) Determination of A c e t a m i n o p h e n

Colunm Column Column R e t e n t i o n Detector Internal T y p of Reference

Support Stationary Temp. Time System Standard Deter-
Phase mination
C h r m s o r b W 10%E-W-982 195OC ca.1 min. F.I.D. A m i t r i p t y - Analgesic 272
(AWDE.r=S) line hydro-Preparations
80/100 (mesh) chloride
Anakr0111 AS 1%SE-30 plus 20O0C 3 min. E l e c t r o n External Metabolic 154
80/90 1%carbowax Capture Standard
20M (Sr-90)
6% QF-1-0065 16OoC ca.3 min. E l e c t r o n - Qualitative- 258
o\
60/70 Capture Clinical
VI (Tritium
Foil)
C h r o m s o r b W 2% SE-30 plus 180°C 3.5&. F.I.D. - Toxicological 273
(Awl 0.1%Triste-
arin
Gas Chror~i0 3% OV-17 165OC ca.5 min. KCl-T.I.D.AmbarbitalPhannaceutica1 2 7 4
100/120 Preparations
Aeropak 30 2% FFAP 2 4OoC 6.5 min. F.I.D. D i p h e n y l bktabolic 275
70/80 (in phthalate
silanised
Column)
Ana?-xmAS 0.5% SE-30 190°C 10 mins. E l e c t r o n External Metabolic 131
m/90 plus 0.5% Capture Standard
Carbcwax 2 0 M (Sr-90)
 TABLE: 12 (cont'd)
G.L.C. (V.P.C.) Determination of Acetamhaphen

Column Column Column Retention Detector Internal Type of Reference

SUPpo* Stationary Temp. Time system standard Deter-
Phase mination
Gas Chrom Q 1% HI-EFF-8BP 22OoC ~a.17min. F.I.D. External Pharmaceutical 182
8O/l00 plus 10% SE-52 Standard Preparations
Chrcdrosorb W 10% Apiezon L 2lOoC 2.4 rel- F.I.D. - Qualitative 3
(AWHQS) ative t o
barbitme
Cklrmsorb W 5% SE-30 or 190°C n.a. n.a. n.a. Pharmaceutical 2 7 6
6o/m 3%Neopentyl or Preparations
(silanised) glycol s u c c h a t e
poljjester 2003
Chmrmsorb G 5%Carhowax 225 C n.a. F.I.D. External Analgesic 180
70/8O 2oM Standard mixtures
Gas C h r o m Q 3% HI-EFF-8BP 22OoC 3 . m . F.I.D. N-butyryl- Metabolic 277
100/120 (as 0-acetyl p-aminoph-
acetaminophen) en01 (as 0-
acetyl deriv.)
Chrmsorb W 1% Diethylene- 180°C ca. 8 m i n . F. I.D. N,@diiso- Antipyretic 278
60/80 glycol succin- (as 0-acetyl butyryl-p- Preparations
(AW-silanised)a t e polyester acetamino- aminupherd
phen)
as cilrom Q 5%Apiezon L MOOC 9 min. (as F.I.D. n-Hexade- Pharmaceutical 279
100/120 T.M.S. cane Preparations
ether of
 TAE3LE 12 (cont’d)
G.L.C. (V.P.C.) Determination of Acetaminophen

Column Column Column Retention Detector Internal Type of Ref.

support Stationary T~IT~. Time System Standard Deter-
Phase mination -
Gas Chrom Q 5% ov-1 l55OC ca.15.6 m i n . (as F.I.D. p-Bram- Metabolic 280
80/100 B.S.A.* deriv.) acetanilide
(as J3.S.A.”
Gas Worn Q 10%OV-17 200OC ca.17.6 min. (as F.I.D.
deriv )
p-chloro-
. Metabolic 280
80/100 T.M.S. I. **) acetanilide
(as T.M.S.I.
** deriv.)
Chrmsorb W 5%OV-101 Program- 10.9 m i n . (as F.I.D. - Qualitative 11
(W 80/1m med from B.S.T.F.A.*** Pathological
100 t o deriv.)
3oooc a t
10 deg./
min.
Gas Ckrom Q 3%OV-1 160’~ n.a.(as B.S.T. F.I.D. p-Bram- Metabolic 281
F.A. *** deriv. ) acetanil ide
(as E.S.T.F.
A.*** deriv.)
Gas Chrom Q OV-17 1woc 3 . 3 min. (as F.1.D- ~ooosane (as bktabolic 447
..
H .M.D s + H.M.D.S. -k
deriv. ) deriv. )
 JOHN E. FAIRBROTHER

* B.S.A. - N,O-bis (trimethylsilyl)

acetamide
** T.M.S.I. - N-trimethylsilylimidazole
*** B.S.T.F.A. - bis (trimethylsilyl) trifluo-
roacetamide (Regisil)
-t L.M.D.S. - hexamethyl disilazane/trichloro
methyl silane
6.29 High-pressure Liquid Chrom-
atographic and Gel Filtration
Procedures
.,-
Burtis, Butts and Rainey'l first des-
cribed the use of high-pressure liquid chroma-
tography in the determination of acetaminophen
and its glucuronide metabolite in urine. Their
procedure employed a high-pressure anion ex-
change chromatographic system293 with a U . V . de-
tector and gave very long retention times in
excess of 16 hours.
Anders and Latorre295 have more re-
cently developed an improved procedure capable
of resolving acetaminophen and its glucuronide
and sulphate conjugates present in urine within
a total elution time of 40 min.
Henry and S ~ h m i t ~ described
'~ a rapid
high-pressure anion exchange chromatographic
procedure for the determination of acetaminophen
in analgesic tablets using a peak area ratio
measurement with an internal standard. A plot
of peak area versus concentration was linear for
both acetaminophen and the salicylamide3interna1
standard over a dynamic range of 5 x 10 This .
gave a possible range for acetaminophen deter-
mination of 3 mg. to about 50 pg. per sample
injection.
Stevenson and bur ti^^'^
have improved
on this procedure and describe a rapid high-pres-
sure liquid chromatographic assay for acetamino-
phen in a wide range of analgesic tablets claim-
ing a precision giving a relative % standard

68
 ACETAMINOPHEN

deviation of 0.79 (using an external standard).

Details of the procedures are given in Table 13.
Jagenburg, Nagy and Re)djer297 separated
the conjugated metabolites of acetaminophen by a
gel filtration technique using Sephadex G-10
(Pharmacia) but no mention is made of the elution
of acetaminophen.
Brook and Munday8’ studied the interac-
tion of a series of compounds including acetamin-
ophen with Sephadex G-10 and Sephadex LH-20
eluting with 0 . 1 N sodium hydroxide solution.
6.3 Automated Procedures
Ederma et a1.282 automated the Brodie
and Axelrodg2 procedure for the determination of
acetaminophen in serum.
The ether extraction of acetaminophen
from the serum and its backwashing into dilute
caustic soda remained as manual procedures but
the conversion of the acetaminophen to p-amino-
phenol and the colorimetric determination were
automated using an Auto Analyser system, A later
paper283 describes the application of basically
the same system to the determination of acetamin-
ophen in whole blood.
Shane and X ~ w b l a n s k yautomated
~~ their
differential U.V. spectrophotometric procedure
(see Section 6.23) for the determination of acet-
aminophen in the presence of aspirin, salicylamide
and caffeine. Alber and O ~ e r t o n ~also
’ ~ deter-
mining acetaminophen in the presence of salicyl-
amide and caffeine used a G.L.C. system with
automated peak height measurement and calcul-
ation. This system employed an amplified KC1
thermionic detector system with a direct feed
into the analog-to-digital converter of a PDP
1 2 A L I N C System computer. It is suggested that
the sample preparation could also be automated.
Daley, Moran and Chafetz442 automated

69
 TAFLE 13
High-Pressure L i q u i d Chrmtographic Determination of Acetaminophen

Insizxumnt Column S i z e C o l m Temp. Flw Elution Retention Ref.

and Packinq and Pressure -
Rate Tk2 -
"W-ANALYSER"
-
0.45x2OQ~1. 25uC i n m w b g
(ml.
30
/ii. 1
~AmnxliumChlo- Acetaminophen 11,
(Oak Ridge t o 6WC after ride-Acet i c 16.5 hr. 293
National Lab- Dowex 1-X8 1 6 h r . Acid Buffer pH
oratory) w i t h (5 t o 1 0 ~ ) 1-2000 p.s.i.g. 4.4 Acetaminophen
Pnotmter 0 . 0 1 5 M (38Qnl. ) glucwonide
Detector 1.OM (37cknl.) 22.4 hr.
(260 and 29Qnl.l.) 4.0M (36cknl.)
6.0M (525m1.)
-
.I
C
Du Pant We1 0.2UooOCm. T e n m a t u r e 90 Buffer (Fisher ca. 2 mins. 294
820 L i q u i d n. a. Gram-Pac) pH
Chrmatograph Zipax coated 1200 p.s.i.g. 9.2 containing
w i t h Model 410 w i t h strong 0.005M m n i u m
Photare!ter anion ex- nitrate
Detector change resin
( 2 5 4 ~1.

(cont'd.. ...)
 TABLE 13 (cont'd)
High-pressure Liquid ChrOIMtographic Determination of Acetaminophen

Instrurrent Column Size Column Temp. Flow Elution Retention Ref.

and Packing and Pressure

V a r i a n WS-1030 O.lOx25Ocm. 80°C X, 1 O . W formic Acetaminophen 295

with Photometer acid (pH3) con- 3.6 min.
Detector LSF pellicular -loo0 taining 1.OM Acetaminophen
(254 nu.) anion ex- p.s. i.g. potassium c h l o r - l g l m m i d e
change resin ide (gradient 2.7 min.
system also Acetaminophen
given) sulphate
4
r
9.5 min.
varian ~ ~ - 1 0 00
0.10xmcm. 60Oc 8.6 1.OM Tris Acetaminophen 296
with Photorreter LSF pellicular 925-1030 Buffer 649 secs.
Detector anion exchange p.s.i.9. (pH 9.0) - 1.08%)
(+
(254 nip.) resin
 JOHN E . FAIRBROTHER

the colorimetric procedure of Chafetz et al.229

(see Section 6.24).
Murf in253 has automated a colorimetric
procedure based on the chromogenic reaction of
acetaminophen with an acid hypochlorite - alka-
line phenol reagent system.
The procedure which may be used for
single tablet assay of acetaminophen, alone or
in combination with aspirin and codeine phos-
phate is based on a Technicon 25-channel system
preceded by a sampling unit and a Technicon
continuous filter. The complete procedure
from commencement of sampling to the recording
of the maximum color takes only 11 mins. and
yields results with a coefficient of variation
of about 0.4%. The sampling time takes 2 min.
15 secs. followed by a wash time of 45 secs.
thus permitting the examination of up to 20
samples per hour on a continuous basis.
6.4 Radiochemical Procedures

Davison et al. 284 described the prep-

aration of N-(114C-Acetyl)-p-aminophenol from
sodium hydrosulphite washed, p-aminophenol and
(acetyl 14C) acetic anhydride giving a product
with an activity of 0.88pC/mg. Koss et a1.285
prepared quantities of acetaminophen labelled on
the nucleus or acetyl side chain.
N-Acetyl-2,6-14C-p-aminophenol was pre-
pared by the reaction of sodium nitromalondialde-
h de monohydrate with 1,3-14C-acetone to give 2,6-
1XC-p-nitrophenol. This was then reduced and
concurrently acetylated to give the required pro-
duct.
N- ( l-14C-Acetyl)-p-aminophenol was pro-
duced by reacting p-aminophenol in peroxide free
tetrahydrofuran with l-14C sodium acetate.
The determination of radioactivity in
the or ans of Wistar-Rats dosed with either of
the lagelled compounds was carried out employing

72
 ACETAMINOPHEN

a Packard Scintillation Counter. Samples were

dissolved in a benzalkonium chloride solution,
decolorised with hydrogen peroxide and a scin-
tillator solution added which contained naphthal-
ene, PPO and POPOP.
Radiolabelled metabolites were estimated
after thin layer chromatographic separation using
a Berthold T.L.C. Radioactivity Scanner.
6.5 Determination of Trace Impurities
and Deqradation Products
The impurity profile of acetaminophen
has already been discussed (see Section 4.13).
The early compendia1 procedures for the
determination of p-arninophenol in acetaminophen
relied on colorimetric limit tests employing
either a sodium nitro-prusside reagent21 or the
phenol-hypobromite reaction62. The latter pro-
cedure was shown252 to be capable of quantit-
ative use having a precision of about + 5% for
a p-arninophenol level in acetaminophen-of 0.012%.

More recently the NF XI11l 4 I 7 has ad-

opted a thin layer ch;omatographic procedure for
the determination of traces ( 4 0.025%) of p-
aminophenol in acetaminophen. The procedure
uses silica gel (HR grade) plates and a methyl
ethyl ketone/acetic acid (9:l) solvent system.
Visualisation is achieved with an acid p-dimeth-
ylaminocinnamaldehyde spray reagent and the size
and intensity of the sample spot is compared with
a standard spot.
p-Chloroacetanilide levels in acetamin-
ophen were determined by Savidge and Wraggllg
using a thin layer chromatographic separation
which employed a solvent mixture of cyclohexane/
acetone/diisobutylketone/methanol/water (100:80:
30:S:l). This procedure was designed for a p-
chloroac tanilide limit test of 0.03%. The
1JF XIIIlg limits the level of p-chloroacetan-

73
 JOHN E . FAIRBROTHER

ilide to 10 p.p.m. and describes a thin layer

chromatographic procedure (solvent-chloroform/
benzene/acetone (65:10:25)) capable of this sen-
sitivity .
Savidge and Wragg showed their T.L.C.
procedure to be capable of separating O-acetyl-
acetaminophen (DAPAP) from acetaminophen and
using this procedure found levels of up to 0.09%
DAPAP in commercial samples of acetaminophen.
Several other T.L.C. procedures have
been described12° ,121,180 for the determination
of DAPAP in acetaminophen and quantitative det-
erminations have also been made using a G.L.C.
systemlb0. (see Section 5.6).
The limitation of the content of quin-
oniraine type oxidation products has been achieved
mainly by close control of the white color of
acetaminophen.
6.6 Determination of Acetaminophen and its
Metabolites in Body Fluids and Tissues
6.61 Determination in Urine
Tne majority of the published work
centres on the determination of free and con-
jugated acetaminophen in human and animal urine.
Lester and G ~ e e n b e r g ’determined
~~ the
metabolites of acetanilide by colorimetric re-
action with a - naphthol of the p - aminophenol
produced after acid hydrolysis. Acetaminophen
was selectively determined by the same colori-
metric procedure after extraction into ethylene
dichloriae from urine, salted out with dibasic
potassium phosphate.
Smith and Williams125 examining rabbit
urine containing acetanilide metabolites, hydro-
lysed etrier extracted urine by heating with acid
thus lherating p-aminophenol from the acetam-

14
 ACETAMINOPHEN

inophen conjugates. The p-aminophenol was de-

termined gravimetrically after isolation.
Brodie and Axelrodg2 determined free
acetaminophen in urine by a procedure involving
the extraction of the acetaminophen from sodium
chloride saturated urine into a solvent mixture
of isoamyl alcohol and diethyl-ether. The ex-
tracted acetaminophen was then acid hydrolysed
to p-aminophenol and determined colorimetrically
after diazo coupling with a - naphthol. Con-
jugated acetaminophen was calculated by differ-
ence from total acetaminophen determined as total
p-aminophenol obtained by direct acid hydrolysis
of the urine. In this case the p-aminophenol
was determined by the phenol/hypobromite colori-
metric procedure.
and Lach208 modified the Lester
and GreenEz$y24 procedure separating the p-
aminophenol from acid hydrolysed urine on an ion-
exchange column prior to colorimetric determin-
ation. Several authors91,126,251,264,298,299
have used slight modifications of the two pro-
cedures described by Brodie and Axelrodg2 for
the determination of free and conjugated acet-
aminophen in both human and rabbit urine. Levy
and Yamada3O0 used the Brodie and Axelrod92 pro-
cedure but deconjugated the metabolites by in-
cubation with an enzyme mixture rather than re-
1 ing on acid hydrolysis. Heirwegh and Fevery
2y7 retained the acid hydrolysis procedure for
deconjugation of the tabolites but substituted
the Bratt~n-Marshall~~'diazotisation procedure
for the diazo coupling with = - naphthol.
Lower, Murphy and Bryan302 employed
both enzyniic hydrolysis and the Bratton-Marshall
colorimetric procedure for the assay of acetamin-
ophen glucuronide in urine. In this case the
sample preparation involved a preliminary fract-
ionation step on a cation-exchange resin.
A third colorimetric procedure303 has
also been described involving diazo coupling of

75
 JOHN E. FAIRBROTHER

p-aminophenol (from acid hydrolysis of acetamin-

ophen and metabolites) with o-cresol.
Welch et al. 304 examining the metabolism
of acetaminophen in animals determined the con-
jugated metabolites in urine (after B - glucuron-
idase and sulphatase hydrolysis) by a colorimet-
ric procedure involving the formation of ion-
pairs of acetaminophen with methyl orange.
Acetaminophen and its conjugated meta-
bolites have been determined in urine after thin-
layer chromato raphic se aration by U.V. spectro-
photometryl31 I 363 I 265 I 267 I 270 and by measurement
of r a d i o a ~ t i v i t y ~ ~ ~ .
Vapor phase chromatography has been used
extensively to measure acetaminophen and its
metabolites in urine. Grove275 determined free
acetaminophen in urine by a direct G.L.C. pro-
cedure employing an ether extract of treated
urine. Klutch and B0rdun1~~r154 also determined
conjugated acetaminophen using a preliminary
enzymic (6-qlucuronidase) hydrolysis step.
Prescott, Steel and Ferrier292 describes
a procedure for the determination of both free
and conjugated acetaminophen in urine. Their
procedure requires the formation of the tri-
methylsilyl derivative of acetaminophen which is
then chromatographed. Conjugated metabolites
are enzymically hydrolysed to give free acetamin-
ophen suitable for trimethylsilylation. Improve
me t pproach have also been described
277,38$f2k!??f4$.

High pressure liquid chromatography has

been used to determine acetaminophen, acetamino-
phen glucuronide and acetaminophen sulphate dir-
ect without hydrolysis or derivative formation
l1thq5. Similarly gel filtration procedures297
may be used but the chromatographic separation is
tedious.

76
 ACETAMINOPHEN

S- (1-acetamido-4-hydroxyphenyl) cysteine
and 1-acetamido-4-hydroxyphenylmercapturic acid,
minor metabolites of acetaminophen have been de-
termined in urine by a gel filtration procedure
297. The cysteine compound has also been deter-
mined305 in urine following ion-exchange chroma-
tographic separation by a ninhydrin colorimetric
procedure.
6.62. Determination in Serum, Plasma
and Whole Blood
The procedures for the determination of
acetaminophen and its metabolites in blood are
essentially similar to those described above for
its determination in urine.
Lester and Greenberg124 treated both
blood and plasma samples with tungstic acid to
precipitate proteins and determined the acetamin-
ophen derivatives using the same procedure as
described for urine. Gwilt, Robertson and Mc
Chesney306 described a procedure for the deter-
mination of free and total acetaminophen in plas-
ma and in whole blood which is essential1 a
modification of the Lester and Greenberg134 pro-
cedure. In this procedure whole blood is tri-
turated with anhydrous sodium sulphate to give a
dry friable mass from which free acetaminophen
is extracted with 1.5% isopentanol in diethyl-
ether. Acetaminophen is back washed with
alkali, hydrolysed with acid to give p-aminophenol
which is coupled with alkaline a-naphthol as in
the Lester and Greenberg procedure. However,
the green solution so produced is then saturated
with potassium chloride and the chromophore ex-
tracted into butanol for spectrophotometric
measurement. This is claimed to increase the
sensitivity of the procedure 2% times.
The Brodie and Axelrod procedures91,92,
126'307 for plasma and serum are essentially as
described for urine after suitable sample prep-
aration. These procedures have also been auto-
mated282 I 283 for the determination of acetamin-

77
 JOHN E. FAIRBROTHER

ophen in blood.
The Heirwegh and F e ~ e r yprocedure
~ ~ ~
which employs the Bratton-Marshall colorimetric
system has been used for determinations in serum
as described for determinations in urine. This
procedure has also been used by Ivashkiv308 who
critically evaluated the reaction parameters.
Routh et al. 206 employed two procedures
for the determination of acetaminophen in serum
or plasma, one employing differential U.V. ab-
sorption spectrophotometry and the other the
decolorisation of diphenylpicrylhydrazyl dye.
Bdch, Pfleger and R ~ d i g e rdetermined
~ ~ ~
acetaminophen in serum by a quantitative thin-
layer chromatographic procedure, the acetaminopkn
eluted from the sample spot being quantified by a
U . V . spectrophotometric procedure. Koss et al.
2 a 5 used quantitative thin layer chromatography
to determine radiolabelled acetaminophen and its
metabolites in human serum, measurement being
made with a radio-autography scanner.
Free acetaminophen has been determined
in plasma by vapor phase chromatography by sev-
eral authors275~277,280,2a1~2g2. The chromato-
graphic procedures in each case are those des-
cribed for the determination in urine. The
samplz7Yreparation however, differs slightly.
Grove extracts the acetaminophen into ether
from plasma saturated with solid ammonium sul-
phate. Thomas and Coldwell281 also extract the
acetaminophen into ether but buffer the plasma
to pH 7.4 with phosphate buffer and then satur-
ate the solution with sodium chloride.
In all the papers by Prescott and co-
w0rkers27~I 280 1 292 the plasma is buffered to pH
7.4 with phosphate buffer and the acetaminophen
extracted into ethyl acetate. Amsel and Davison
447 also use extraction into ethyl acetate.

78
 ACETAMINOPHEN

6.63 Determination in Tissue and Organs

Brodie and Axelrodg2 determined acetam-
inophen and total conjugated p-aminophenol in
homogenised tissue (emulsified in acid1309 ess-
entially using the same procedures they described
for similar determinations in urine. Gwilt,
Robertson and McChesney306 used a very similar
procedure to Brodie and Axelrod homogenising the
tissue in 0 . 1 N hydrochloric acid, neutralising
and buffering to pH 6.6 before extracting the
free acetaminophen.
Davison et a1284 and Koss et al. 285
describe the radioassay of total acetaminophen in
tissue and organ homogenates using radiolabelled
acetaminophen and also describe the separation of
free acetaminophen and the individual conjugates
in bile by a radioautographic procedure.
7. Metabolic Transformations
7.1 Metabolism in Man
7.11 Adults
Lester and Greenberg124 and Brodie and
Axelrod911921126 established that acetaminophen
is the main metabolite of acetanilide and aceto-
phenetidin (phenacetin). Thus the main meta-
bolites excreted in the urine after administr-
ation of acetanilide, acetophenetidin, bucetin310
or acetaminophen are the glucuronide and ether
sulphate conjugates of acetaminophen124I 125 I 1 2 6 .
Acetaminophen sul hate had already been
isolated in 1889 by MBrnerls2 from the urine of
patients who had received acetanilide. Smith
and Williams1251130 isolated crude acetaminophen
lucuronide from rabbit urine and Shibaski et al.
3 6 6 purified this isolated material and also pro-
duced it synthetically.
Minor metabolites have been identified

79
 JOHN E. FAIRBROTHER

by Jagenburg and Toczko305 and by Jagenburg, Nagy

and Rddjer297. These are the cysteine and mercap-
turic acid conjugates of acetaminophen. The
recent findings of Nery311 of four new metabol-
ites of acetophenetidin suggest that the list of
acetaminophen metabolites may not et be complete
329. 5;
Focella, Heslin and Teitel4 8 identified
a metabolite of acetophenetidin isolated from dog
urine as 4-hydroxy-3-methylthioacetanilide.
This substance may also be a metabolite of acet-
aminophen. In the same ~ t u d y ~the
4 ~S - ( 1 -
acetamido-4-hydroxyphenyl) cysteine found by
Jagenburg and Toczko305 was tentatively identi-
fied more correctly as 3-[(5-acetamido-2-hydroxy-
phepyl)thio] alanine.
Burtis et al.ll described the formation
of 3-methoxy-acetaminophen (by a girl with a
neuroblastoma) after treatment with acetophene-
tidin. They11 ascribe the formation of this
metabolite and its excretion as the glucuronide
to an induced activity of the hydroxylase and
catechol 0-methyl transferase enzyme systems
caused by the high level of acetaminophen (see
also refs. 331 and 332).
The metabolic routes are summarised in
Fig. 7 .
The relative amounts of free acetamin-
ophen and its sulphate and glucuronide conjug-
ates excreted in the urine var with the indiv-
idual. Typical results265,29Y,300,312 for a
dose of 1 to 2 gm. acetaminophen show 75 to 90%
of the dose is excreted in the urine with the
acetaminophen and its metabolites distributed
(approximately) in the following manner:-
Free acetaminophen 2 to 5% (of total excreted)
Acetaminophen glucuronide 55 to 75% (but some
results are much lower)
Acetaminophen sulphate 20 to 40%
Acetaminophen 3-cysteine 0.5 to 7 % (only 3 resula
Acetaminophen 3-mercapturic acid 5 to 7% (only 3
results).

80
 ACETAMINOPHEN

HO
acetaminophen
glucuronide

OH acyinophen

NHCOCH NHCOCH3

$SC12y HC O2H @scH2c I HC02H

HNCOCH
3 OH H2
OH 3-[(5-acetamido-2-hydro-
xyphenyl) thiolalanine
l-acetamido-4-
hydroxyphenyl-mercapturic
acid
FIGURE 7 - Metabolic Pathways of Acetaminophen

81
 JOHN E. FAIRBROTHER

Patients suffering with chronic hepa-

titis and with liver cirrhosis show a decrease in
the blood serum and urine levels of acetaminophen
glucuronide and increased levels of free acetam-
inophen. This results from a corresponding de-
crease in the activity of lucuron ltransferase
in the pathologic l i v e r s 2 3 q , 3 1 3 1 3 1 ~ , 3 1 5 . 3 1 6 , 3 1 7 .
Renal insufficiency does not effect the ratio of
free to conjugated acetaminophen in the plasma
but through a decrease in glomerular filtration
it may increase the plasma level of total acet-
aminophen by as much as 4-fold318. The metabol-
ism of acetaminophen to its sulphate can be
blocked by salicylamide which competes with the
acetaminophen for sulphate3O0 I 319. This effect
can be counteracted by L-c steine, a well ab-
sorbed source of sulphate350. This may be due
to sulphate availability being the capacity-
limiting factor3001320. Salicylamide also de-
creases the excretion of acetaminophen glucuronide
possibly by a similar mechanism. Salicylic
acid has no significant effect on the formation
of acetaminophen sulphate and g l u ~ u r o n i d e ~ ~ ~ .
7.12 Newborn Infants
Vest and co-workers 307r323 found that
in newborn infants acetaminophen (produced by
the administration of acetanilide) is much more
slowly conjugated to the glucuronide than in
older children and adults. Similar results have
been obtained after the administration of acet-
amino hen324,325,3261327 and it has been sugges-
ted32g1327 that the urinary excretion and blood
levels of acetaminophen conjugates depend on the
maturity of the glucuronide-forming enzyme system
(glucuronic acid transferase and uridine dipho-
sphate glucuronic acid) and the development of
renal tubular function.
7.2 Metabolism in Animals
Clark328 demonstrated that the metabolic
pathways of acetaminophen in man and dog were

82
 ACETAMINOPHEN

similar. It has been shown in rats285 that

about30% of the dose was secreted with the bile
in 4 hr. The acetaminophen in the rat bile was
shown to be almost completely conjugated to the
glucuronide (83%) and the sulphate (14%) and
only about 2.5% free acetaminophen was still
available.

Cats dosed with acetaminophen metabolise

the drug in a different manner from man, dog,
rabbit and rat, in that less than 6% of the dose
was excggied in the urine as acetaminophen gluc-
uronide and less than 2 % as acetaminophen sul-
phate304. It has been reported3331334 that the
cat has an impaired ability to form glucuronides,
and this defect has been attributed to the lack
Q35the glucuronyl transferase enzyme in the liver.
. The cat, however, does excrete acetamin-
ophen in the urine, in a conjugated form (cap-
able of enzymic hydrolysis with 6-glucuronidase)
and it has been suggested by Welch et a1.304 that
it may be conjugated with cysteine.
These same authors304 found that 10 to
13% of the dose administered to cats appears in
the urine as an aromatic primary amine but this
does not appear to be p-aminophenol.
Acetaminophen is metabolised in the rat
and rabbit in a similar manner to that in ma
with different ratios of the metabolites125 ,Y29Yt
263,330,

8. Drug Availability
8.1 Pharmacokinetics
Many authors have described various
aspects of the harmacokinetics of acetaminophen
68 80 91 124 127,148,234,285,292,298,299,300,306 ,
325,336 t o 347

83
 JOHN E. FAIRBROTHER

G w i l t e t a l . 336 examined t h e a b s o r p t i o n o f a c e t -
amino hen i n man f o l l o w i n g o r a l a d m i n i s t r a t i o n .
They336 f o u n d t h e h i g h e s t a v e r a g e b l o o d l e v e l o f
t o t a l a c e t a m i n o p h e n was r e a c h e d a f t e r between 30
and 9 0 min. d e p e n d i n g on t h e i n d i v i d u a l . The
e f f e c t s of f o o d and s l e e p on t h e a b s o r p t i o n and
e x c r e t i o n of a c e t a m i n o en h a v e b e e n examined by
McGilveray and Mattok 4 s 2 . Koss e t a l . 2 8 5 fol-
lowed t h e a d m i n i s t r a t i o n (100 mg./kg.) of l a b -
e l l e d a c e t a m i n o p h e n i n r a t s , showing t h a t r a p i d
a b s o r p t i o n occurs i n t h e f i r s t h a l f h o u r , o n l y
a b o u t 4 0 % of t h e d o s e r e a c h i n g t h e s m a l l i n t e s t -
ine. This gradually reaches t h e l a r g e i n t e s t -
i n e where t h e c o n t i n u e d a b s o r p t i o n a p p e a r s t o
b e compensated f o r by b i l i a r y s e c r e t i o n of acet-
aminophen ( a b o u t 30% of t h e d o s e ) .

The p l a s m a h a l f l i v e s r e p o r t e d v a r y a s
shown i n T a b l e 1 5 .
TABLE 1 5
Acetaminophen Plasma Half - L i f e i n Man

Author Plasma h a l f - l i f e (tf) -

Ref.
( h o u r s1

B r o d i e and A x e l r o d 1.5 1 26
2.4
Carlo e t a l . 2.3 234
G w i l t et al. 2.7 336
Prescott e t a l . 2.03 34 7
(mean of 8 s u b j e c t s )
Heald and Evans 2.94 322
(mean of 10 s u b j e c t s )
Prescott e t al. 2 . 0 + 0.1 317
(17 scbjects)
McGilveray e t al. 3.02 + 0 . 3 339
Careddu e t a l . 2.23 - 0 . 5 315

The e l i m i n a t i o n r a t e c o n s t a n t s f o r f r e e
(unchanged) a c e t a m i n o p h e n and i t s c o n j u g a t e d
m e t a b o l i t e s f o r man h a v e been d e t e r m i n e d by s e v -
eral authors265,312,319,339,348,454.

84
 ACETAMl NOPHEN

8.2 Protein Binding

The bindin of acetaminophen to n lon72
cellulose triacetateq2 and to dextran gels84; has
been described in Section 2.56. Hansch and
Helmer88 related this work to the octanol-water
partition coefficient and ultimately to the bind-
ing of acetaminophen to natural polymers such as
proteins.
Dearden and T o m l i n ~ o nhave
~ ~ examined
the binding of acetaminophen to bovine serum
albumin (BSA) using a dynamic dialysis method
finding the association constant to be suffic-
iently low to give a fairly high free drug con-
centration in the bloodstream over a relatively
long time period.
Koss et al. 285 measured the binding of
radiolabelled acetaminophen onto serum protein
using a Sephadex filtration technique and found
that about 18% of the acetaminophen is bound to
the serum albumin over a wide acetaminophen con-
centration range. Hartshorn 349 quotes the
amount of acetaminophen bound to the plasma pro-
teins as about 25%.
8.3 Interactions with Other Drug Substances
The analgesic activity of acetaminophen
has been claimed to be enhanced by the co-admin-
istration of a number of other analgesics and
harmacologically active drug substances2t285I 300,
521,322,350-356,

Levy and Yamada300 examined the effects

of salicylamide on the pharmacokinetics of acet-
aminophen, showing that salicylamide retards the
excretion rate of acetaminophen conjugates. This
was shown to be accompanied by a competitive in-
hibition of the formation of acetaminophen and
salicylamide conjugates in the blood implying
increased therapeutic availability of free acet-
aminophen.

85
 JOHN E. FAIRBROTHER

Niwa and N a k a ~ a m a ~found

~l that acetam-
inophen and ant ipyrine (phenazone) mutually in-
hibit the metabolism of each other in the rat and
rabbit and showed that penetration of acetamino-
phen and antipyrine through excised intestine
is mutually inhibted by the other drug substance.
Heald and Evans322 determined the
effect of antipyrine (as acetaminophen-antipyrine
complex) on the plasma level of free acetamino-
phen in man (10 subjects). Their results sug-
gest that antipyrine prolongs the peak plasma
level of free acetaminophen.
Acetaminophen has been re orted to be
anta onistic to a number of drugs3251349 350,353
3551361. It has also been reported to show
synergism of anti-inflammator c iv wi h
other anti-inflammatory drugs3 5 4 t o st8 an$ 362
to 367.

8.4 Biopharmaceutics
Assessment of the bioavailability of
acetaminophen has been made using both in vitro
measurement of dissolution rate and in vivo
pharmacokinetic methods.
Goldberg, Gibaldi and Kanig66 used
dissolution rate measurement to evaluate the po-
tential increase in the bioavailability of
acetaminophen after fusing it with urea to form
eutectic mixtures. Lach and CohenlOO carried
out similar studies employing alpha and beta
cyclodextrins to increase the dissolution rate of
acetaminophen (see also Section 3).
Many authors have used the measurement
of acetaminophen plasma levels and/or urinary
levels to demonstrate ‘ts bioavailability. Mattok
and c o - ~ o r k e r s ~ ~ rI 345 have used both in vivo
339
and in vitro procedures and attempted to cor-
relate the results. Levy133 used the areas
under the acetaminophen plasma concentration vs.
time curvesI to estimate the comparative systemic

86
 ACETAMINOPHEN

availabilities of acetaminophen when administered

orally as such and when administered as aceto-
phenetidin (phenacetin).
Mattok and co-workers75,339,345 used
these techniques to compare the bioavailability
of acetaminophen in eight commercial tablet form-
ulations, a formulated elixir and a simple lab-
oratory prepared solution and showed no signifi-
cant differences between them. Gwilt et a1.336
examined the plasma levels given by seven acet-
aminophen formulations and later also examined
an eighth formulation containing acetaminophen
and sorbitol. The plasma levels of free acet-
aminophen.given by this acetaminophen-sorbitol
formulation were significantly higher than
those given by the seven other formulations.
Sorbitol was claimed336 to potentiate
the absorption of acetaminophen and thus is
claimed to enhance the antipyretic and analgesic
effects of the drug96~97198.
Walters 385 has critically examined
these claims using in vitro methods and concludes
that sorbitol does not form an absorbable com-
plex with acetaminophen and that the enhanced
activity of acetaminophen in tablets containing
sorbitol may result solely from the improved
dissolution rate.
Bloor and Morrison455 examined the
effects of solubilization of acetaminophen by
Tween4' (a polyoxyethylene sorbitan monopalm-
itate) on its rate of diffusion.
Carlo et al. 234 examined the effect on
bioavailability obtained by formulating acetam-.
inophen in an effervescent tablet form. The
effervescent formulation gave higher and more
rapidly attained plasma concentrations of acet-
aminophen than an ordinary non-effervescent form-
ulation but did not maintain plasma levels as
efficiently as the latter. Bru452 makes similar

87
 JOHN E. FAIRBROTHER

claims.
The effect of vehicle composition on
the rectal absorption of acetaminophen from
su pository formulations has been examined6812981
455.

Incorporation of enzymes having hyalur-

onidase and chrondrosulphatase activity into
acetaminophen formulations has been claimed to
enhance acetaminophen absorption from
and rectally administered dosage forms3sb!?3?6;1 ly
371~372~3731374. These claims were later refu-
ted in a study by Brustier et a1.375.
The administration of acetaminophen by
percutaneous absorption from s lution in dialkyl
sulphoxides has been reported376 .
Modification
of drug availability can be effected by formul-
ation as a sustained release (timed-release)
dosage form. Several such formulations have
been described for acetaminophen377I 3781379 1380
381,382. Timed release may also be effected by
formulation of the drug in a microencapsulated
form. This method of presentation has also been
used to mask the taste of acetaminophen383138~.
9. Toxicity
The acute and chronic oral toxicity of
acetaminophen in man and animals has been well
reported3~8r67~386 to 398. Overdosin a e -
amino hen can cause hepatic necrosis317,996,s92I
3961 go 4 0 5 and also in a few cases of heav over-
dosin renal insufficiency3181347 I 386,3961x06 to
414 421
91I 124,304,%b:s9k:?f!$? fff5e%s4%ve been discussed

88
 ACETAMINOPHEN

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87. Rapport L. I. and S o l y a n i k G.K., F a r m a t s e v t . Zh.

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111. Patent, Ger. 453,577 (see Chem. Zentr. 1928 I,

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153. R a t t i e E.S., Shami E.G., D i t t e r t L.W. and Swintosky

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196. H u n t J. , Fthcdes H.J. and B l a k e M.I., Can. J.

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200. EirockeE G. , Pharmazie 20 (3), 136-140 (1965)
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204. Hoyland D. , Squibb Private C a m n u n i c a t i o n
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206. Fauth J.I., Shane N.A., Arrendondo E.G. and Paul
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221. Hamiltm J.L., -

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246. Tatsuo Kondo and Iwao Kawashiro,. Shokuhin Eiseigaku

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255. ~ e r u oNinaniya, yakugaku Zasshi 85 (51, 394-399
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256. Hiroshi Sakurai and Masuo m a , Yakugaku Zasshi
82, 1282-1286 (1962)
257. Gsuo Umeda, Yakugaku Zasshi 83, 951-956 (1963)
258. Shinsaku Imashuku and La Brosz E.H., C l i n . Chem.
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17 (2), 122-124 (1971)
259. Fairbrother J.E. and Johnson B.A., Squibb Private
Ccmrmnication
260. Datta D.D. and Ghosh D., Indian J. P h m . - 28 (51,
133-134 (1966)
261. C i e r i U.R., J. P h m . Sci. 58 (12), 1532-1535 (1969)
262. Shun-Ichi Naito and Kazuo F&i, J. P h m . Sci.
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263. Blich H., Pfleger K. and Rildiger W. , 2. Klin. Chm.
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264. Goenechea S., 2. Klin. Chm. 7 (4), 346-349 (1969)
265. Cumnings A. J. , King M.L. , and-hartin B.K. , Brit.
J. Pharmacol. Chemther. 29 (21, 150-157 (1967)
266. Juichim Shibasaki, Etsuko Sadakane, Ryoji Konishi
and Tarmtsu Koizuni, Chm. Pharm. Bull. 2 (U),
2340-2343 (1970)
267. Shand S., Squibb Private Camnulication
268. Stahl E., "Thin Layer Chranatography" Springer,
Berlin-Gdttingm-Heidelberg, 1965.
269. Zarnack J. and Pfeifer S., Pharmazie 19, 216 (1964)
270. glich H., Hauser H., Pfleger K. and Rmger W., 2.
Klin. Chgn. 4 (6), 288-290 (1966)
271. Lindberg N.OT, Aktiebolaget Draco, Lund, Sweden,
Private Comnmication

100
 ACETAMINOPHEN

272. Nieminen E. , Bull. Narcot. 23 (l), 23-28 (1971)

273. McMartin C. and street H.V.,J. Chromatog. -
22 (21,
274-285 (1966)
274. Alber L.L. and Overton M.W., J. A s s . Offic. Anal.
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Chm. 54 (3) 620-624 (1971)
275. Grove T I J. Chranatog. 59 (2), 289-295 (1971)
276. Ryabtseva I.M., Kuleshom-M.I., Rudenko B.A. and
Kucherov V.F., Izv. Akad. Nauk SSSR, S e r . Khim.
(12) I 2676-2680 (1970)
277. Prescott L.F., J. Pharm. Pharmacol 23, 807-808 -
(1971)
278. Masanobu Koibuchi , Toshio Shibazaki, Tsutanori
Minamikawa and Yukio Nishimra, Yakugaku Zasshi
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88 (7), 877-881 (1968)
279. Kipping S.A.B., The Boots C m y Ltd., Nottingham,
Private Cammication
280. Presoott L.F., J. Pharm. Pharmacol. -
23 (21, 111-115
(1971)
281. Thanas B.H. and Coldwell B.B., J. Pharm. Pharmacol.
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24, 243 (1972)
282. Ederma H.M., Skerpac J., Cotty V.F. and Sterbenz F.,
Automation in Analytical Chemistry, Technicon
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283. Ederrna H.M. , Cotty V.F. and M e m K.J., Advan.
AutcaMt. Anal. , Technicon Int. Congr. (Chicago)
1969 (pub. 1970), 2, 179-181
284. Davison C. , Guy J.E., Levitt M. and Smith P.K., J.
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285. Koss F.W., Mayer D. , Haindn. and Kabbara T.,
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286. Reikhsfel'd V.O., Tr. Konf. po Probl. Primeneniya
Korrelyatsion. Uravnenii v Organ. Khim., Tartusk.
Gos. Univ., Tartu (1962) (11, 214-249
287. Reikhsfel'd V.O. , Prokhorova V.A. and Pmia V.A.,
Kinetika i Kataliz 6 (11, 171-176 (1965)
288. Reikhsfel'd V.O. a d Korol'ko V.V. , Reaksionnaya
Sposobnost. Organ. Scedin., Tartusk. Gos. Univ.
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289. Reikhsfel'd V.O. and Prokhorova V.A., Zh. Obshch.
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290. ReikhsGl'd V.O. , Intern. Symp. Organosilicon
Chen. Sci. Comnun., Suppl., Prague (1965) 34-40
291. Reikhsfel'd V.O., Khim. Prakt. Primen. Krerrmiiorg.
Win., Tr. Sovesch. (19661, 7-23

101
 JOHN E. FAIRBROTHER

292. Prescott L.F., S t e e l R.F. and F e r r i e r W.R., C l i n .

Pharmac01. Ther. 11 (4)I 496-504 (1970)
293. Burtis C.A. and Wzren K.S., C l i n . Chem. 14 (4) ,
2 9 e 3 0 1 (1968)
294. H e n r y R.A. and Schnit J.A., C h r c m a t c g r a p h i a - 3, 116-
1 2 0 (1970)
295. Anders M.W. and Latorre J.P., J. C h r o m t o g . - 55,
409-413 (1971)
296. Stevenson R.L. and Burtis C.A., J. ChrcaMtog.
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61, 253-261 (1971)
297. Jagenburg R., Nagy A. and RcMjer S., Scand. J.
C l i n . Lab. Invest. 22 (11, 11-16 (1968)
298. Hobel M. and TalebiK M. , Arzneimittel - Forsch.
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10, 653-656 (1960)
299. Juichiro Shibasaki, R y o j i Konishi, Yoshinori T a k e d a
and T m t s u Koizumi, Chem. P h m . B u l l . (Tokyo)
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1 9 (9), 1800-1808 (1971)
300. Levy G. and Y m d a H. , J. Pharm. Sci. - 60 (2) ,
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301. B r a t t o n A.C., Marshall E.K., Babbitt D. and
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302. Lower G.M. , Murphy S.B. and B r y a n T T . , C l i n . Chim.
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303. Tonpsez S.L. , Ann. C l i n . Biochem. - 6, (41, 81-82
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304. Welch R.M., C o n n e y A.H. and Bums J.J., Biochem.
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305. Jagenbmg O x . and Toczko K. , Biochem. J., 92, 639-
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306. W i l t J . R . , R o b e r t s o n A. and McChesney E.W., J.
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307. V e s t M.F. , S t r e i f f y . R . , A.M.A. J. D i s e a s e s of
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308. Ivashkiv E T Squibb Private C c m m n i c a t i o n
309. Brodie B.B., Udenfriend S. and Baer J . C . , J. B i o l .
Chem. 168, 299 (1947)
310. Shibasaki J., K o i z u n i T . , Tanaka T. and N a k a t a n i M.,
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311. N e r y R., Biochem. JY 122 (3) , 317-326 (1971)
312. Levy G. and -&dh C.- G., J. P h m . S c i . 9,(4)
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313. Hart'umr C.H., and P r e l l w i t z W., K l i n . W o c h e n s c h r .
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44 (17) I 1010-1014 (1966)

102
 ACETAMINOPHEN

314. V e s t M.F. and F r i t z E., J. C l i n . Pathol. -14, 482

(1961)
315. Careddu P. , Mereu T. and Apolloni T. , Minerva Pedi& .
13, 1619-1622 (1961)
316. Every J. and D e G r o o t e J. , Acta Hepato-Splenol.
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16 (1), 11-18 (1969)
317. F’rescott L.F., Roscoe P., Wright N. and Brown S.S.,
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318. Dubach U.C. I K l h . Wochenschr. - 46 (5) I 261-264
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319. Levy G. and Yamada H., J. Pharm. Pharmacol. - 22,
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320. Buech H., R m l W., Pfleger K., Eschrich C. and
T e x t e r N. , Naunyn-Schmiedebergs Arch. Pharmakol.
Exp. Pathol. 259 (31, 276-289 (1968)
321. N i w a H. and N Z y m T. , Yakugaku Zasshi, - 88 (5) ,
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322. Heald A.F. and Evans R. , Squibb Private Ccarmunic-
ation
323. V e s t M.F., 89, 102-105
Schweiz.med. hbchenschr. -
(1959)
324. Careddu P., Mereu T. and Apollonio T., Boll. SOC.
i t a l . biol. sper. 37, 359-363 (1961)
325. Careddu P. , Pacenizereni L. , Apollonio T. and
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326. Mereu T. , Apollonio T. , P a G i - S e r e n i L. and
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327. V e s t M.F. and Rossier R . , Ann. N.Y. Acad. Sci.
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328. Clark B.B., Symposium on N-acetyl-p-minophenol.
p. 23-34, I n s t i t u t e f o r t h e Study of Analgesic
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329. Klutch A., Harfenist M. and Conney A.H., J. Med.
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330. Bray HTG. , Thorpe W.V. and White K. , Biochem. J. ,
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331. Inscoe J.K., Daly J. and Axelrod J., Biochem.
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332. Axelrod J., Science 140 (3566), 499-500 (1963)
333. Wbinson D. and W i l l 5 R.T. , Biochem. J., 68,
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334. Borrell S., Biochem. J . , 70, 727 (1958)
335. Dutton G. J. and Greig C . G Y Biochem. J. , 66, 52P
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103
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336. G w i l t J.R., Robertson A., Go- L. and Blanchard

A.W., J. Phann. Pharmacol. 15, 445-53 (1963)
337. J a f f e J.M., Colaizzi J.L. a
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338. WeikerJ.H. and Lish P.M., Arch. I n t . P h a n r a d y n .
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339. McGilv=y I.M., Mattok G.L., Fooks J.R., Jordan
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340. S t r i c k e r H., Pharm. Ind. 31 (111, 794-799 (1969)
341. Dearden J.C. and Tcsnlinsm-E., J. Pharm. Pharmacol.
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342. Dearden J.C. and Tcanlinson E., J. Phann. Pharmacol.
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343. Salzn?ann K., Therapiemche 12, 1034 (1962) -
344. D i t t e r t L.W. and Adams H.J., J. Phann. Sci. 2,
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345. Mattok G.L., W i l v e r a y I.M. and Cook D., Can. J.
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346. W i k e l J . H . , J. Am. Pharm. Assoc., Sci. Ed. 2,
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347. Prescott L.F., Sansur M., Levin W., Conney A.H.,
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348. N e l s o n E. and Morioka T., J. Pharm. Sci. 52. (91,
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349. H a r t s h o r n E.A., Drug I n t e l l i g e n c e and C l i n i c a l
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350. Grotto M., D f i s t e h S. and Sulman F.G., Arch. Int.
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351. Takagi K., Tajiii-pagi I., Kayaoka S., Okabe S . ,
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352. Grosto M., H a r e f a 73 (31, 90-93 (1967)
353. Boreus L.O. and S&g F., A c t a Physiol. Scand.
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28, 266-271 (1953)
354. Botha D., Mueller F.O., Krueger F.G.M., Melnitzky
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355. Grotto M., Harokeach H a i v r i 11, 152/172-157/167
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356. Patent, U.S., 3,439,094 (15 Apr. 1969) to W l e
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357. Patent, U.S. 3,482,021 (2 D e c . 1969) t o Gosling
R.H. (Geigy Chemical Corp.)

104
 ACETAMINOPHEN

358. Patent, Ger. Offen. 2,058,893 (9 Jun. 1971) t o

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359. Lechat.P., Delaeu D. and Bunot O., Therapie - 20 (41,
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360. Lechat P., Delaeu D., Fontagne J. and m o t O.,
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Therapie, 21 (31, 565-570 (1966)
361. B u s h H., Gerhards W., Pfleger K., Rueaiger W.
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362. Patent, B r i t . 901,674 (25 J u l y , 1962) t o Johnson
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363. Patent, U.S. 3,053,737 (Sept. 11, 1962) t o
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364. Corte G. and Johnson W . J . , Proc. Soc. Exptl. B i o l .
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365. MacK&ie D.H.H. and smythe H.A., Can. Conf. Res.
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366. Levillain R., Cluzan R., David G. and Cayeux Ph.,
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367. Weichselbaum T.E. and Margraf H.W., Proc. Soc.
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368. Niwa H. and Nakaym-T., Yakugaku Zasshi 88 (71,
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369. Patent, Belg. 668,418 (Dec. 16, 1965) t o Riviere J.
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370. Patent. F r . M 4,196 (July 4, 1966) t o Riviere J.
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371. Patent, Fr. M 4,825 (Mar. 20,1967) to Riviere J.
372. Patent, Fr. M 4,519 (Nov. 21,1966) t o Riviere J.
373. Patent, F'r. M 5,575 (Jan. 2,1968) to C e n t r e d'Etudes
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374. Patent, Fr. M 5,022 (May 29, 1967) t o C e n t r e d '
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375. Brustier V., Aubert D., Amselem A., Gazz. I n t .
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376. Patent, B c 1,043,104 (Sept. 21, 1966) t o Senior
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377. Patent, U.S. 3,133,863 (May 19, 1964) t o Tansey R.P.
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378. Patent, B r i t . 1,019,146 (Feb. 2, 1966) t o Hermelin
V.M.

105
 JOHN E . FAIRBROTHER

379. Patent, B r i t . 1,021,924 (Mar. 6, 1966) t o W t h ,

K l i n e and French Laboratories
380. Patent, N e t h . Appl. 6,512,130 (Mar. 21, 1966) t o
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381. Patent, U.S. 3,279,998 (Oct. 18, 1966) to Raff A.M.
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382. Patent, Brit. 1,125,882 (Sept. 5, 1968) t o Shepard
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383. Patent, Ger. Offen. 1,917,930 (Nov. 6, 1969) t o
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384. Bakan J.A. and Sloan F.D., D r u g and Cosmetic
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386. Ebyer T. and muff S., J. Am. Med. A s s o c . - 218,
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387. Boxill G.C., Nash C.B. and Wheeler A.G., J. Am.
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388. Renault H., Rohrbach P. and D u g n i o l l e J . , Therapie
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389. z t c h f i e l d J.T. and Wilcoxon F., J. Phannacol.
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390. Burn J.H. I Finn= D.J. and Goodwin L.G., "Biological
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391. S t m r G.A., McLean S. and Thorns J., 'Ibxicol.
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392. Manolov P., Suwemenna Med. 14 (101,33-37 (1963)
393. Miller M.M., Squibb Private C&munication
394. Boyd E.M. and Bereczky G.M., Brit. J. Pharmacol.
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395. Swinyard E.A., Brown W.C. and Goo&nan L.S., J.
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396. Boyd E.M. and H o g a n S.E., can. J. Physiol. Pharmacol.
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397. &d E.M., J. C l i n . P h m c o l . 10 (4), 222-227 (1970)
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400. E i d s m D.G.D. and Eastham W.N., B r i t . M e d . J.
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401. Thomson J.S. and Prescott L.F., B r i t . Med. J.
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106
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402. Pimstone B.L. and Uys C.J., S. Afr. M e d . J., 42,

259 (1968)
403. Maclean D. , P e t e r s T.J., Brown R.A.G. , m a t h i e M.,
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406. P r e s c o t t L.F., J. P h a n n ~ P h a r m c o l .-
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407. Prescott L.F., Lancet (19651, ii, 91-95
408. C a l d e r I.C. , Funder C.C. , G r e e n C.R., Ham K.N.
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409. Smith P.K., Davison C. and Scdd M.A. , (1956) ,
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410. -
E i s a l o A. and T a l a n t i S., A c t a Med. Scand. 169,
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411. Angervall L. , Lehmnn L. and L i n c o l n K. , A c t a
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412. Angervall L. , LehsMnn L. a n T L i n c o l n K., A c t a
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413. Angervall L., LehTMnn L. a n ? L i n c o l n K. , A c t a P a t h o l .
Microbiol. Scand. 154, Suppl. 61-63 (1962)
414. Angervall L. , Lem- L. and Bengtsson U. , A c t a
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415. K i e s e M. and-zel H. , Arch. Exptl. P a t h o l .
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Pharmakol. 242, 551-554 (1962)
416. Schwerd W., Med. W l t (1962) 2348-2349
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418. S c h n i t z e r B. and Smith E.B., Arch. P a t h o l . - 81
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419. J3aader H., G i r g i s S., Kiese M., Menzel H. and
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420. L e s t e r D., J. P h a m c o l . E x p t l . Therap. - 77,
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421. D o l l G. and Hackenthal E., Arzneimittel-Forsch. - 13,
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422. Bums J.J. and Conney A.H., Proc. Eur. Soc. Study
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423. Dern R.J. , B e u t i e r E. and A l v i n g A.S. , J. Lab.
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424. C l a u s e n E. and Larsen O.A., A c t a Pharmacol. T o x i c o l .
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22 ( 2 ) , 135-140 (1965)

107
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425. b y d M. and Sheppard E.P., B r i t . J. Pharmacol.

Chemtherapy - 27 (21, 497-505 (1966)
426. D i k s t e i n S., G r o t t o M., Zor U., T m r i M. and S u h
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427. Thanas J.M., Nakaue H.S. and Reid B.L., Poultry Sci.
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46 ( S ) , 1216-1219 (1967)
428. D i k s t e i n S., Zor U., Ruah D. and S u b a n F.G.,
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429. Lloyd T.W., G c e t (1961), i, 114
430. Jacobson H., Squibb Private Comnunication
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432.
C l a r k e G., Squibb Private C m i c a t i o n
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433. Coy N.H. and Ochs Q., Squibb Private C c g n n u n i c a t i o n
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436. W i l l i a m s R.T. and Bridges J.W., J. C l i n . Pathol.
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437. Patent, G e r . ( E a s t ) 81,119 (12 Apr. 1971) t o
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438. Patent, Ger. O f f e n . 2,121,164 (11Nov. 1971) t o
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440. Elste U. and Duda H., B u t . Apth.-Ztg. 112 ( 1 9 ) ,
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441. Shearer C.M., C h r i s t e n s o n K., Mukherii A. and
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445. Mihe G.w.A., Fales H.M. and menrod T., -1.
C h m . 43 (13), 1815-1820 (1971)
446. Lageoty., L i m u z i n Y. and Maire J.C., J.
Organmtal. Chem. 38, 23-27 (1972)

108
 ACETAMINOPHEN

447. Amsel L.P.

1474-1475 (1972)
-
and Davison C., J. Pharm. Sci. 6 1 ( 9 ) ,
448. Fccella A . , Heslin P. and Teitel S. , Can. J. Chem.
50, 2025-2030 (1972)
c

449. Fairbrother J . E . , Squibb Private C o m i c a t i o n

450. Patent, G e r . 1,493,727 (31Aug. 1972) t o Koenig C.,
Schmitz P. and P e l s t e r H. (Bayer A.-G.)
451. Peters G., Baechtold-Fowler N. , Bonjour J., e t al.,
Arch. ToxFkol. 28 ( 4 ) , 225-269 (1972)
452. Patent, Fr. m-de 2,092, 893 (3 Mar. 1972) t o
Bru J.
453. R.P. Scherer Ltd., Capsule N e w s lo ( 2 ) , 3 (1972)
454. McGilveray I.J. and Mattok G.L., J. Pharm. Pharmacol
-
24, 615-619 (1972)
455. Blmr J.R. and Mrrison J.C., J. Phann. Pharmacol.
-
24, 927-933 (1972)
456. Patent, B r i t . 1,287,431 (31 Aug. 1972) t o Lakenorris
W. and Slater J.E. (Aspro-Nicholas Ltd. )

This profile a t t q t s t o cover the published l i t e r a t u r e on

acetaminophen up t o Chemical Abstracts, V o l m 77, Issue 21

109
dl-ALPHA-TOCOPHERYL ACETATE

Bruce C. Rudy and Bernard 2. Senkowski

 BRUCE C. RUDY AND BERNARD 2 . SENKOWSKI

INDEX

Analytical P r o f i l e - dl-Alpha-Tocopheryl Acetate

1. Description
1.1 Name, Formula, M o l e c u l a r Weight
1 . 2 Appearance, C o l o r , Odor
1 . 3 I s o m e r i c Forms

2. Physical Properties
2 . 1 I n f r a r e d Spectrum
2 . 2 Nuclear Magnetic Resonance Spectrum
2.3 U l t r a v i o l e t Spectrum
2.4 Mass Spectrum
2.5 O p t i c a l R o t a t i o n
2.6 M e l t i n g Range
2.7 D i f f e r e n t i a l Scanning C a l o r i m e t r y
2 . 8 Thermal G r a v i m e t r i c A n a l y s i s

3. Synthesis

4. S t a b i l i t y Degradation

5. Drug M e t a b o l i c P r o d u c t s

6. Methods of A n a l y s i s
6.1 Elemental Analysis
6.2 Thin Layer Chromatographic A n a l y s i s
6.3 Gas-Liquid Chromatographic A n a l y s i s
6.4 Direct S p e c t r o p h o t o m e t r i c A n a l y s i s
6.5 C o l o r i m e t r i c A n a l y s i s
6.6 S p e c t r o f l u o r o m e t r i c Analysis
6.7 T i t r i m e t r i c A n a l y s i s

7. Acknowledgements

8. References

112
 dl-ALPHA-TOCOPHERYL ACETATE

1. Description

1.1 Name, Formula, Molecular Weight

dl-Alpha-tocopheryl acetate is a racemic mixture
of 2,5,7,8-tetramethy1-2-(4',8',12'-trimethy1tridecy1)-6-
chromarol acetate.

Molecular Weight: 472.76

'31'52'3
1.2
Appearance, Color, Odor
dl-Alpha-tocopheryl acetate occurs as a yellow,
nearly odorless, clear, viscous oil.

1.3
Isomeric Forms
There are four possible enantiomeric pairs of
diastereoisomers which result from the three asymmetric
centers present in the alpha-tocopheryl acetate molecule
(the asymmetric centers are marked with a small circle in
the above structural formula). dl-Alpha-tocopheryl acetate
contains an equimolar mixture of the eight isomers.

2. Physical Properties

2 - 1 Infrared Spectrum
The infrared spectrum of dl-alpha-tocopheryl
acetate is presented in Figure 1 (1). The spectrum was
measured with a Perkin-Elmer 621 Spectrophotometer on a
capillary layer of the liquid between KBr discs. The as-
signments for the characteristic bands in the infrared
spectrum are listed in Table I (1).
Table I
Infrared Assignments for dl-Alpha-Tocopheryl Acetate
Frequency (cm-1) Characteristic of
2943 and 2861 CH3 stretching vibrations

113
PI
U
rd
L) 8
OD
0
- -
0 -
Ll
L)
M
a
CJY
a
al
h
rd
Ll
w
C
H
33NVlllWSNWl K
114
 dl-ALPHA-TOCOPHERY L ACETATE

2920 a n d 2850 CH2 s t r e t c h i n g v i b r a t i o n s

1752 C=O s t r e t c h i n g v i b r a t i o n s
1456 CH2 and CH3 d e f o r m a t i o n s
1363 CH3 symmetric d e f a m a t i o n s
1206 C-0-C s t r e t c h i n g v i b r a t i o n s

2.2 N u c l e a r Magnetic Resonance Spectrum (NMR)

The s p e c t r u m shown i n F i g u r e 2 w a s o b t a i n e d on a
J e o l c o 60 MHz NMR by d i s s o l v i n g 92.4 mg of d l - a l p h a -
tocopheryl acetate i n 0 . 5 m l of CDC13 c o n t a i n i n g tetra-
m e t h y l s i l a n e a s a n i n t e r n a l r e f e r e n c e ( 2 ) . The s p e c t r a l
a s s i g n m e n t s a r e g i v e n i n T a b l e I1 ( 2 ) .

T a b l e I1

NMR Assignments € o r dl-Alpha-Tocopheryl Acetate

'

Number o f Chemical S h i f t
Proton Protons (ppm) Mu1 t i p l i c i t y

a 12 0.85 Doublet (Ja-d = 5 HZ)

b 3 1.21 Singlet
c&d 21 1.0-1.6 Complex M u l t i p l e t
e 2 1.75 T r i p l e t (Je-h = 6 . 8 Hz)
f 9 1.96,2.00,2.07 T h r e e O v e r l a p p i n g singlets
g 3 2.29 Singlet
h 2 2.57 T r i p l e t (Jh-e = 6 . 8 Hz)

2.3 U l t r a v i o l e t Spectrum (UV)

When t h e UV s p e c t r u m of d l - a l p h a - t o c o p h e r y l
a c e t a t e w a s s c a n n e d from 350 t o 220 n m , two maxima and two
minima were o b s e r v e d . One maximum is l o c a t e d a t 285 n m
( E = 2.24 x l o 3 ) w i t h a s h o u l d e r a t 287 nm ( E = 2 . 2 1 x l o 3 )
and t h e o t h e r maximum o c c u r s a t 279 n m ( E = 1 . 9 8 x l o 3 ) .
The minima a r e l o c a t e d a t 281 nm and 253 nm. The s p e c t r u m
shown i n F i g u r e 3 w a s o b t a i n e d from a s o l u t i o n of 1 2 . 4 1 1 mg
of d l - a l p h a - t o c o p h e r y l a c e t a t e p e r 100 m l o f cyclohexane(3).

115
 BRUCE C. RUDY AND BERNARD 2 . SENKOWSKI

Figure 2

NMR Spectrum of dl-Alpha-Tocopheryl Acetate

l ~ ~ [ ' ~ ~ ~ l ~ ~ ~ ~ l l

240 180 I20

116
 dl-ALPHA-TOCOPHERYL ACETATE

Figure 3

Ultraviolet Spectrum of dl-Alpha-Tocopheryl Acetate

.8 -

.7 -

.6-

;
a
.5-
m
a
%
m
4-
a

1
.3-

.2 -

.I -

0-

117
 BRUCE C. R U D Y A N D B E R N A R D 2. SENKOWSKI

2.4 Mass Spectrum

The mass s p e c t r u m of d l - a l p h a - t o c o p h e r y l a c e t a t e
was o b t a i n e d u s i n g a CEC 21-110 mass s p e c t r o m e t e r w i t h a n
i o n i z i n g e n e r g y o f 70 e V . The o u t p u t from t h e mass
s p e c t r o m e t e r w a s a n a l y z e d and p r e s e n t e d i n t h e form of a
b a r g r a p h , shown i n F i g u r e 4 , by a V a r i a n 100 MS d e d i c a t e d
computer s y s t e m ( 4 ) . The mass spectrum of dl-alpha-
t o c o p h e r y l a c e t a t e i s c h a r a c t e r i z e d by t h e a b s e n c e of
numerous f r a g m e n t a t i o n p r o c e s s e s . The m o l e c u l a r i o n peak
o c c u r s a t m / e 472. The b a s e peak a t m / e 430 o c c u r s from
t h e l o s s of k e t e n e from t h e m o l e c u l a r i o n . F r a g m e n t a t i o n
of t h e non-aromatic r i n g o c c u r s , y i e l d i n g a peak a t m / e 207
( w i t h t h e a c e t a t e group p r e s e n t ) and a peak a t m / e 165
( a f t e r l o s s of t h e k e t e n e g r o u p ) . An i n - d e p t h a n a l y s i s o f
t h e mass s p e c t r a of t o c o p h e r o l s h a s r e c e n t l y been p u b l i s h e d
by Scheppele e t a l . (5).

2.5 Optical Rotation

dl-Alpha-tocopheryl a c e t a t e i s a n equimolar
m i x t u r e of t h e o p t i c a l i s o m e r s of a l p h a - t o c o p h e r y l a c e t a t e
and t h e r e f o r e e x h i b i t s no o p t i c a l r o t a t i o n .

2.6M e l t i n g Range
dl-Alpha-tocopheryl a c e t a t e is an o i l a t room
temperature. I t s o l i d i f i e s a t a t e m p e r a t u r e of -27.5OC (6).

2.7 D i f f e r e n t i a l Scanning C a l o r i m e t r y (DSC)

A DSC s c a n w a s r u n by c o o l i n g t h e head of t h e DSC
which h e l d t h e sample pan c o n t a i n i n g ;he dl-alpha-tocopheryl
a c e t a t e t o -9OOC. A f t e r h o l d i n g t h e t e m p e r a t u r e a t -9OOC
f o r 30 m i n u t e s , t h e t e m p e r a t u r e w a s i n c r e a s e d a t a r a t e o f
10°/minute. Only a n e x t r e m e l y weak e n d o t h e r m i c t r a n s i t i o n
w a s observed s t a r t i n g a t a b o u t -44OC. It a p p e a r s t h a t
when d l - a l p h a - t o c o p h e r y l a c e t a t e s o l i d i f i e s i t forms a
g l a s s i n s t e a d of a c r y s t a l l i n e m a t e r i a l ( 7 ) .

2.8 Thermogravimetric A n a l y s i s (TGA)

The TGA of d l - a l p h a - t o c o p h e r y l a c e t a t e i n a
n i t r o g e n atmosphere showed no w e i g h t l o s s from ambient t o
210OC. A s i n g l e w e i g h t l o s s c o r r e s p o n d i n g t o 100% of t h e
sample o c c u r r e d between 210' and 37OoC ( 7 ) .

118
 a,
u
(d
u
a,
U
4
rl
h
k
a,
F:
a
0
U
. 3 RI
a m
h F :
1 a
M r l
*rl 4
k l I
rl
a
w
0
 BRUCE C. RUDY A N D BERNARD Z . SENKOWSKI

3. Synthesis
dl-Alpha-tocopheryl a c e t a t e i s p r e p a r e d by t h e r e a c t i o n
scheme shown i n F i g u r e 5. Trimethylhydroquinone is con-
densed w i t h racemic i s o p h y t o l t o y i e l d d l - a l p h a - t o c o p h e r o l
which is t h e n a c e t y l a t e d ( 8 ) .

4. S t a b i l i t y Degradation
dl-Alpha-tocopheryl a c e t a t e i s p r a c t i c a l l y u n a f f e c t e d
by t h e o x i d i z i n g i n f l u e n c e o f a i r and u l t r a v i o l e t l i g h t
( 6 ) . When i t is r e f l u x e d i n a c i d i c and and b a s i c s o l u t i o n s
i n t h e a b s e n c e o f oxygen, t h e m o l e c u l e is h y d r o l y z e d t o t h e
f r e e dl-alpha-tocopherol. I f oxygen is p r e s e n t , t h e d l -
a l p h a - t o c o p h e r o l , once formed, w i l l o x i d i z e r a p i d l y t o t h e
quinone. The r a t e of o x i d a t i o n i s much f a s t e r i n t h e b a s i c
solution.

5. Drug M e t a b o l i c P r o d u c t s
dl-Alpha-tocopheryl a c e t a t e i s m e t a b o l i z e d as o u t l i n e d
i n Figur e 6 (9,lO). The e s t e r i s r e a d i l y c o n v e r t e d i n t h e
animal t o f r e e a l p h a - t o c o p h e r o l which i s t h e n f u r t h e r
m e t a b o l i z e d t o a l p h a - t o c o p h e r o l quinone and a n a l p h a -
t o c o p h e r o l dimer. The a l p h a - t o c o p h e r o l q u i n o n e may b e
reduced t o t h e c o r r e s p o n d i n g hydroquinone o r f u r t h e r
o x i d i z e d t o a l p h a t o c o p h e r o n i c a c i d (9,10,11).

6. Methods of A n a l y s i s

6.1 Elemental A n a l y s i s
The r e s u l t s from t h e e l e m e n t a l a n a l y s i s of d l -
a l p h a - t o c o p h e r y l a c e t a t e a r e l i s t e d below (12).

E l emen t % Theory % Found

C 78.76 78.55
H 11.09 11.05

6.2 Thin Layer Chromatographic A n a l y s i s (TLC)

A TLC system which c a n b e used t o s e p a r a t e d l -
a l p h a - t o c o p h e r y l a c e t a t e from i t s major m e t a b o l i t e s i s as
f o l l o w s . The s a m p l e is a p p l i e d t o a S i l i c a G e l G p l a t e and
s u b j e c t e d t o a s c e n d i n g chromatography u s i n g cyc1ohexane:di-
e t h y l e t h e r ( 4 : l ) as t h e d e v e l o p i n g s o l v e n t . A f t e r t h e
s o l v e n t h a s ascended 10 t o 1 5 c m , t h e p l a t e is a i r d r i e d ,
sprayed w i t h c o n c e n t r a t e d s u l f u r i c a c i d , and warmed i n an
oven a t 105OC f o r 5 m i n u t e s . The a p p r o x i m a t e Rf v a l u e s

120
 Figure 5
Synthesis of dl-Alpha-Tocopheryl Acetate

a 3
TRIMETHYLHYDROQUINONE RACEMIC ISOPHYTOL

Acetic Anhydride
C"3
dl - ALPHA-TOCOPHEROL

dl- ALPHA-TOCOPHERYL ACETATE

 Figure 6
Metabolic Products of dl-Alpha-Tocopheryl Acetate

ALPHA-TOCOPHERYL ACETATE

I deestwificatian

c
w
w
/
O+
ALPHA- TOCOPHEROL
(major)

% \
 dl-ALPHA-TOCOPHERY L ACETATE

are l i s t e d below (13).

a l p h a tocopherol 0.5
a l p h a t o c o p h e r y l acetate 0.7
a l p h a t o c o p h e r o l quinone 0.9

6.3 Gas-Liquid Chromatographic A n a l y s i s (GLC)

A r e c e n t c o l l a b o r a t i v e s t u d y has shown dl-alpha-
t o c o p h e r y l a c e t a t e may r e a d i l y b e s e p a r a t e d and a s s a y e d by
GLC ( 1 4 ) . The p e r t i n e n t experimental c o n d i t i o n s as w e l l a s
t h e r e t e n t i o n t i m e s a r e given i n T a b l e 111.

Table I11

GLC Method f o r dl-Alpha-Tocopheryl Acetate

Column: 4 - 8 f e e t , 2-3 mm i . d . , Pyrex

o r s t a i n l e s s steel
Support: S i l a n i z e d Diatomaceous E a r t h
S t a t i o n a r y Phase: 5-10% SE-30
Detector: Hydrogen flame i o n i z a t i o n
Temperature (OC)
Injection Port: 300
Column : 280
Detector: 300
C a r r i e r Gas Flow Rate: $40 ml/min. of n i t r o g e n
Quantities Injected: c10 mcg i n n-hexane
R e t e n t i o n Time (minutes)
alpha-tocopherol 22
alpha-tocopheryl acid
s u cc i n a t e 23
alpha-tocopheryl
acetate 26
dotriacontane
( i n t e r n a l s t a n d a r d ) 30

6.4 Direct S p e c t r o p h o t o m e t r i c Analysis

Direct spectrophotometry may b e c a r r i e d o u t on
dl-alpha-tocopheryl a c e t a t e provided t h e r e a r e no i n t e r -
f e r e n c e s p r e s e n t . The maxima f o r dl-alpha-tocopheryl
a c e t a t e a r e dependent on t h e c h o i c e of s o l v e n t used. I f
cyclohexane is used as t h e s o l v e n t , t h e maximum a t 285 nm
may b e used f o r q u a n t i t a t i o n .

I23
 BRUCE C. RUDY A N D BERNARD 2 . SENKOWSKI

6.5 Colorimetric Analysis

An i n d i r e c t method f o r t h e a n a l y s i s of dl-alpha-
t o c o p h e r y l a c e t a t e u t i l i z e s t h e Emmerie-Engle c o l o r i m e t r i c
p r o c e d u r e ( 1 5 ) . The d l - a l p h a - t o c o p h e r y l a c e t a t e is b a s e
hydrolyzed i n anhydrous e t h a n o l t o t h e f r e e t o c o p h e r o l .
The s o l u t i o n i s a c i d i f i e d t o p r e v e n t a i r o x i d a t i o n , water
i s added, and t h e d l - a l p h a - t o c o p h e r o l i s e x t r a c t e d i n t o di-
e t h y l e t h e r . The e t h e r i s e v a p o r a t e d under n i t r o g e n and
t h e r e s i d u e i s immediately d i s s o l v e d i n anhydrous e t h a n o l .
F e r r i c c h l o r i d e i s added a l o n g w i t h 2 , 2 ' - b i p y r i d i n e , b o t h
i n anhydrous e t h a n o l . The m i x t u r e i s s h a k e n v i g o r o u s l y
and timed f o r 10 m i n u t e s . The a b s o r b a n c e of t h e r e d s o l u -
t i o n i s measured a t 520 nm ( 1 6 , 1 7 ) . A review of t h e
Emmerie-Engle and c e r r i c s u l f a t e t i t r i m e t r i c methods f o r
Vitamin E was p u b l i s h e d by Lehman (18).

6.6 Spectrofluorometric Analysis

The i n t e n s e u l t r a v i o l e t f l u o r e s c e n c e e x h i b i t e d by
a l p h a - t o c o p h e r o l h a s p r o v i d e d t h e b a s i s f o r a s i m p l e and
e x t r e m e l y s e n s i t i v e method f o r t h e d e t e r m i n a t i o n of f r e e
a l p h a - t o c o p h e r o l and a l p h a - t o c o p h e r y l a c e t a t e i n plasma
( 1 9 , 2 0 ) . Two m l of plasma are d i l u t e d w i t h 2 ml of w a t e r
and 4 ml of e t h a n o l and t h e n e x t r a c t e d w i t h 8 ml of hexane.
The f r e e a l p h a - t o c o p h e r o l i s d e t e r m i n e d by d i l u t i n g a 1 ml
a l i q u o t of t h e hexane p h a s e w i t h 3 ml of e t h a n o l and mea-
s u r i n g t h e i n t e n s i t y of t h e f l u o r e s c e n c e produced a t 340 nm
by e x c i t i n g t h e sample a t 295 nm. The a l p h a t o c o p h e r y l
a c e t a t e i s determined by d i f f e r e n c e a f t e r h y d r o l y z i n g a
4 ml a l i q u o t of t h e hexane p h a s e w i t h LiAlH4 t o c o n v e r t any
acetate t o a l p h a - t o c o p h e r o l and measuring t h e i n t e n s i t y of
t h e f l u o r e s c e n c e as above. A p l o t of f l u o r e s c e n c e v e r s u s
c o n c e n t r a t i o n of a l p h a t o c o p h e r o l was l i n e a r o v e r t h e r a n g e
o f 0 . 6 ug/ml through 40 pg/ml ( 2 0 ) . The l i m i t of d e t e c t i o n
i s about 0.01 pg/ml ( 1 9 ) .

6.7 T i t r i m e t r i c Analysis
dl-Alpha-tocopheryl a c e t a t e ( a b o u t 250 mg) i s d i s -
s o l v e d i n 25 m l of anhydrous e t h a n o l , 20 m l of 5 N e t h a n o l i c
s u l f u r i c a c i d is added and t h e s o l u t i o n r e f l u x e d f o r 3
h o u r s t o e f f e c t complete h y d r o l y s i s t o t h e f r e e d l - a l p h a -
tocopherol. After t h e s o l u t i o n i s c o o l e d , i t is t r a n s -
f e r r e d t o a 200-ml v o l u m e t r i c f l a s k and d i l u t e d t o volume
w i t h 50 m l of 0.5N e t h a n o l i c s u l f u r i c a c i d and 20 m l of
water. Two d r o p s of diphenylamine T . S . a r e added and t h e

124
 dl-ALPHA-TOCOPHERY L ACETATE

s o l u t i o n is t i t r a t e d w i t h 0.01N c e r i c s u l f a t e u n t i l a b l u e
end p o i n t i s reached which p e r s i s t s f o r 1 0 seconds. A
b l a n k is r u n and any n e c e s s a r y volume c o r r e c t i o n made.
Each ml of 0.01N c e r i c s u l f a t e i s e q u i v a l e n t t o 2 . 3 6 3 8 mg
of d l - a l p h a - t o c o p h e r y l a c e t a t e ( 1 7 ) .

7. Acknowledgments
The a u t h o r s wish t o acknowledge Mrs. A. M. Ormsby f o r
t y p i n g many of t h e A n a l y t i c a l P r o f i l e s and M r s . L. B. Rubia
f o r drawing and l e t t e r i n g many of t h e f i g u r e s . The h e l p
a f f o r d e d by t h e S c i e n t i f i c L i t e r a t u r e Department and t h e
Research Records O f f i c e of Hoffmann-La Roche I n c . i n t h e
l i t e r a t u r e s e a r c h e s also is g r a t e f u l l y acknowledged.

125
 BRUCE C. RUDY AND BERNARD 2.SENKOWSKI

8. References

1. Hawrylyshyn, M., Hoffmann-La Roche Inc., Personal

Communication.
2. Johnson, J. , Hoffmann-La Roche Inc., Personal
Communication.
3, Rubia, L. B., Hoffmann-La Roche Inc., Personal
Communication.
4. Benz, W., Hoffmann-La Roche Inc. , Personal
Communication.
5. Scheppele, S . E., Mitchum, R. K., Rudolph, C. J.,
Kinneberg, K. F. , and Odell, G. V. , L i p i d s , 1,
297 (1972).
6. Merck Index, Eight Edition, p. 1115, 1968.
7. Moros, S., Hoffmann-La Roche Inc., Personal
Communication.
8. Surmatis, J., and Weber, J., U . S . Patent 2,728,278
(1955).
9. Gallo-Torres, H. E., Miller, 0. N., Hamilton, J. G.,
and Tratnyek, C., L i p i d s , 5, 318 (1971).
10. Draper, H. H., and Csallany, A . S . , "Metabolism o f
Vitamin E", DeLuca, H. F., and Suttie, J. W.
(editors) , flae Fat-SoZubZe Vitamins, The ZTniversity
of Wisconsin Press, pp. 347-353, 1970.
11. Simon, E. J., Eisengart, A . , Sundheim, L., and
Milhorat, A . T., J . B i o l . &em.. 221, 807 (1956).
12. Scheidl, F., Hoffmann-La Roche Inc., Personal
Communication.
13. Bolliger, H., and KGnig, A . , Hoffmann-La Roche Inc.,
Unpublished Data.
14. Rudy, B. C., Mahn, F. P., Senkowski, B. Z.,
Sheppard, A. J. , and Hubbard, W. D. , J . A . 0. A. C.,
November, 1972 (In press).
15. Emmerie, A., and Engel, C., Nature, 142, 873 (1938).
16. United S t a t e s Pharmacopeia XVIII, p. 914, 1970.
17. National Formulary X I I , p. 760, 1970.
18. Lehman, R. W., J . Pham. S c i . , 53, 201 (1964).
19. Duggan, D. E., Arch. Biochem. Biophys., 9, 116
(1959).
20. Hansen, L. G., and Warwick, W. J., Am. J . Clin.
Path., 46, 133 (1966).

126
 AMITRIPTYLINE HYDROCHLORIDE

Kenneth W. Blessel, Bruce C. Rudy, and Bernard Z. Senkowski

 KENNETH W. BLESSEL, BRUCE C. RUDY, AND BERNARD 2.SENKOWSKI

INDEX

Analytical Profile - Amitriptyline Hydrochloride

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 Spectrum
2.4 Fluorescence Spectrum
2.5 Mass Spectrum
2.6 Optical Rotation
2.7 Melting Range
2.8 Differential Scanning Calorimetry
2.9 Thermogravimetric Analysis
2 . 1 0 Solubility
2 . 1 1 X-ray Crystal Properties
2 . 1 2 Dissociation Constant

3. Synthesis

4. Stability Degradation

5. Drug Metabolic Products

6. Methods o f Analysis
6.1 Elemental Analysis
6.2 Phase Solubility Analysis
6.3 Thin Layer Chromatographic Analysis
6.4 Gas-Liquid Chromatographic Analysis
6.5 Colorimetric Analysis
6.6 Fluorescence Analysis
6.7 Non-Aqueous Titration

7. Acknowledgements

8. References

128
 AMlTRlPTYLlNE HYDROCHLORIDE

1. D e s c r i p t i o n

1.1 Name, Formula, Molecular Weight

A m i t r i p t y l i n e h y d r o c h l o r i d e i s lO,ll-dihydro-N,N-
dimethyl-5H-dibenzo [ a , d ] cycloheptene-A5, '-propylamine
hydrochloride.

C20H23N*HC1 Molecular Weight: 313.87

1.2 Appearance, Color, Odor

A r n i t r i p t y l i n e h y d r o c h l o r i d e i s an o d o r l e s s , o f f -
w h i t e c r y s t a l l i n e powder.

2. Physical Properties

2.1 I n f r a r e d Spectrum
The i n f r a r e d spectrum of a m i t r i p t y l i n e hydro-
c h l o r i d e is shown i n F i g u r e 1 (1). The sample w a s
d i s p e r s e d i n f l u o r o l u b e f o r t h e r e g i o n of 4000-1350 cm-I
and i n m i n e r a l o i l t o r e c o r d t h e spectrum i n t h e r e g i o n
of 1350-600 cm-1. The f o l l o w i n g assignments of bands i n
t h e spectrum have been made (1).

-
Band Assignment
3057 cm-l Aromatic CH s t r e t c h
2949 and 2825 c m - l Asymmetric and symmetric CH3
stretch
2921 and 2852 cm-l Asymmetric and symmetric CH2
stretch
2545-2428 cm-l C h a r a c t e r i s t i c of H C 1 s a l t of
t e r t i a r y amine
767 and 757 cm-l Four a d j a c e n t hydrogens on
benzene r i n g

129
 Figure 1

Infrared Spectrum of A m i t r i p t y l i n e Hydrochloride

WAVELENGTH (MICRONS)
25 3 4 5 6 7 8 9 10 12 15 20
I I I I I I I I I I I I
100 I I I I I I I 100
I

80 - - 80
c
w
0 -60

-40
a
a
k
$20- -20
OL I I I I I I I I 0
4OOO 3500 3OOO 2500 ZOO0 1700 1400 1100 800 500
FREQUENCY (CM-')
 AMlTRlPTYLlNE HYDROCHLORIDE

2.2 Nuclear Magnetic Resonance Spectrum (NMR)

The NMR spectrum of amitriptyline hydrochloride
is shown in Figure 2 ( 2 ) . The spectrum was recorded using
a solution of 54.7 mg/0.5 ml in CDC13. The spectral as-
signments are shown in Table I ( 2 ) .

Table I

NMR Spectral Assignments for Amitriptyline Hydrochloride

Proton Total No. Chemical Shift

Identification of Each (ppm) Multiplicity

N,N-dimethyl 6 2.65 Singlet

Protons on 7 -
membered ring and Multiplet
methylene protons 8 2.75-3.34 (unresolved)
Methine pro tons 1 5.8 Triplet
Aromatic protons 8 7.2 Mu1tiplet
Proton of Hydro- Singlet
chloride 1 12.5 (broad)
2.3 Ultraviolet Spectrum
The ultraviolet spectrum of amitriptyline hydro-
chloride is shown in Figure 3 ( 3 ) . The spectrum was re-
corded on a solution which contained 1.00 mg in 100 m l of
0.1N HC1. A maximum was observed at about 239 nm ( E =
1.4 x 104) and a minimum at 228-231 nm.
2.4 Fluorescence Spectrum
The excitation and emission spectra of amitrip-
tyline hydrochloride are shown in Figure 4 (4). The sample
was dissolved in methanol at a concentration of 1 mg/ml
and the spectra were recorded using a Farrand MK-1 record-
ing spectrofluorometer. Excitation at 302 nm produced
emission with a maximum at 357 nm.

2.5 Mass Spectrum

The low resolution mass spectrum of amitriptyline
is shown in Figure 5 ( 5 ) . The spectrum was recorded on a
CEC 21-110 mass spectrometer using an ionizing energy of
70 eV, which was interfaced with a Varian data system

131
L
132
 Figure 3
Ultraviolet Spectrum of Amitriptyline Hydrochloride
I .(
O.!
0.f
0.;
0.c
0
z
a
m
Iz: 0.:
0
in
m
U
0.4
0.3
0.2
0. I
0.0
210 250 300 3 50
NANOMETERS
133
 0
z
U
z
AlISN31NI
134
.7
I I I 1 I I I I I I
l a s e 8 8 8 8 8 0
AlISN31NI 3A11Vl3Y
135
 KENNETH W. BLESSEL, BRUCE C. RUDY, A N D BERNARD Z . SENKOWSKI

100 MS. The d a t a system accepted t h e o u t p u t of t h e

s p e c t r o m e t e r , c a l c u l a t e d t h e masses, compared t h e i r i n t e n -
s i t i e s t o t h e b a s e peak and p l o t t e d t h i s d a t a as a series
of l i n e s whose h e i g h t s were p r o p o r t i o n a l t o t h e i n t e n s i t i e s .
The molecular i o n w a s measured a t m / e 277 ( f r e e
b a s e ) . The c h a r a c t e r i s t i c f e a t u r e of t h i s compound is i t s
s t r o n g tendency t o l o s e N i n o r d e r t o a t t a i n a r o m a t i c i t y
and/or r i n g c l o s u r e . The main fragments are t h e loss of
N(CH3)2 and CH2N(CH3)2 from t h e s i d e c h a i n t o form m / e 233
and 219 r e s p e c t i v e l y . Each o f t h e s e s p e c i e s then l o s e s
hydrogens, g i v i n g rise t o m / e 231, 217, and 215. Complete
l o s s of t h e s i d e c h a i n g i v e s rise t o m / e 192 and m / e 85.
The b a s e peak o c c u r s a t m / e 58 and i s due t o CH2N(CH3)2
( 5 ) . A h i g h r e s o l u t i o n spectrum w a s found t o b e f u l l y com-
p a t i b l e w i t h t h e low r e s o l u t i o n scan.

2.6 Optical Rotation

A m i t r i p t y l i n e h y d r o c h l o r i d e does n o t e x h i b i t
optical activity.

2.7 Me1t i n g Range

The m e l t i n g range r e p o r t e d i n USP X V I I I i s
195-198OC when a Class I procedure i s used ( 6 ) .

2.8 D i f f e r e n t i a l Scanning Calorimetry (DSC)

The DSC curve f o r a m i t r i p t y l i n e h y d r o c h l o r i d e a t
a s c a n r a t e of 10°C/min. i s shown i n F i g u r e 6 ( 7 ) . The
curve was recorded w i t h a P e r k i n E l m e r DSC-1B under a n
atmosphere of n i t r o g e n . A s i n g l e endotherm was observed,
t h e e x t r a p o l a t e d o n s e t of m e l t i n g o c c u r r i n g a t 193.0 5
0.2OC and t h e peak a t 197.5 5 0.2OC. A l l temperatures a r e
c o r r e c t e d . The v a l u e c a l c u l a t e d f o r AHf was 6.7 kcal/mole
f o r t h e m e 1 t i n g endo therm.

2.9 Thermogravimetric Analysis (TGA)

The TGA curve f o r r e f e r e n c e s t a n d a r d a m i t r i p -
t y l i n e h y d r o c h l o r i d e e x h i b i t e d no l o s s of weight from
30-195OC. A s i n g l e weight l o s s o c c u r r e d i n t h e temperature
range of 195-317OC which accounted f o r 100% of t h e sample
weight ( 7 ) .

2.10 Solubility
The s o l u b i l i t y d a t a o b t a i n e d f o r r e f e r e n c e s t a n -
d a r d a m i t r i p t y l i n e h y d r o c h l o r i d e is l i s t e d i n Table I1 ( 8 ) .

136
 AM1TR IPTY LINE H Y DROCH LOR IDE

Figure 6

DSC Curve of A m i t r i p t y l i n e H y d r o c h l o r i d e

I I I I i

A Hf =6.7kcal / m o l e

Endothermic

t
I
4

Exothermic

I I I I I
I70 I80 190 200 210

OC

137
 KENNETH w. BLESSEL, BRUCE c. RUDY, AND BERNARD z . SENKOWSKI

Table I1

S o l u b i l i t y of A m i t r i p t y l i n e Hydrochloride

Solvent S o l u b i l i t y (mg/ml)

petroleum e t h e r (30-60°C) 0.30

diethyl ether 0.50
water >SO0 -
2 -propano1 53 *
3A a l c o h o l 313 -
chloroform >500.
95% e t h a n o l >SO0 -
benzene 5.0
methanol >500

2.11 Crystal Properties

The x-ray powder d i f f r a c t i o n d a t a f o r a sample
of r e f e r e n c e s t a n d a r d a m i t r i p t y l i n e h y d r o c h l o r i d e i s g i v e n
i n Table I11 (9). The o p e r a t i n g parameters of t h e hstru-
ment are given below.

I n s t r u m e n t a l Conditions

General E l e c t r i c Model XRD-6 Spectrogoniometer

Generator: 50 KV, 12-112 MA

Tube t a r g e t : Copper
Radiation : CU Ka = 1.542 8
optics : 0.1' D e t e c t o r s l i t
M.R. S o l l e r s l i t
3' Beam s l i t
0.0007" N i f i l t e r
4O t a k e o f f o a n g l e
Goniometer: Scan a t 0.2 2 0 p e r minute
Detector: Amplifier g a i n - 1 6 c o u r s e ,
8.7 f i n e
Sealed p r o p o r t i o n a l c o u n t e r
tube and DC v o l t a g e a t
plateau
P u l s e h e i g h t s e l e c t i o n EL -
5 volts
Eu - o u t
Rate meter T.C. 4
2000 CIS f u l l s c a l e

138
 AMlTRl PTYLINE HY DROCHLORlDE

Recorder: Chart speed 1 inch per 5

minutes
Samples: Prepared by grinding at room
temperature
Table I11
Interplanar Spacings in Amitriptyline Hydrochloride
from Powder Diffraction Data
28
- d&)* IITo% -
28
11-50 7.69 21 29.36 3.04 3
12.78 6.93 10 29.52 3.03 2
13.46 6.58 4 30.10 2.97 4
14.86 5.96 16 30.96 2.89 4
15.66 5.66 9 31.28 2.86 7
16.06 5.52 4 31.74 2.82 6
16.34 5.42 15 31.98 2.80 3
16.72 5.30 18 32.46 2.76 5
17.72 5.01 1 32.76 2.73 7
18.58 4.78 74 33.80 2.65 6
18.90 4.70 13 34.12 2.63 3
19.26 4.61 36 34.98 2.57 2
20.24 4.39 9 35.30 2.54 3
20.94 4.24 100 36.00 2.49 3
21.34 4.16 10 38.26 2.35 3
21.87 4.06 4 39.02 2.31 6
22.94 3.88 60 39.66 2.27 2
23.42 3.80 7 40.04 2.25 1
24.32 3.66 9 40.54 2.23 1
24.88 3.58 3 41.22 2.19 2
25.78 3.46 19 41.88 2.16 1
26.62 3.35 16 42.06 2.15 2
27.30 3.27 9 43.24 2.09 3
27.58 3.23 3 43.88 2.06 2
28.16 3.17 9 44.22 2.05 2
29.00 3.08 4 44.70 2.03 2
45.30 2.00 2

*d = (interplanar distance) - nh
2 Sin e

**I/ = relative intensity (based on highest

I0 intensity of 100)

139
 KENNETH W. BLESSEL, BRUCE C. RUDY, AND BERNARD 2.SENKOWSKI

2.12 D i s s o c i a t i o n Constant
The d i s s o c i a t i o n c o n s t a n t f o r a m i t r i p t y l i n e
hydrochloride was determined u s i n g a g r a p h i c a l meihod i n -
volving t h e pH dependence of t h e water s o l u b i l i t y . The
v a l u e f o r t h e pKa determined by t h i s method was 9.4 (10).

3. Synthesis
Two s y n t h e t i c r o u t e s t o a m i t r i p t y l i n e are shown i n
Figure 7. The f i r s t , r e a c t i o n sequence I (11,12), i n v o l v e s
t h e a d d i t i o n of a Grignard r e a g e n t followed by d e h y d r a t i o n
w i t h HC1 o r a c e t y l c h l o r i d e , w h i l e I1 (13) u s e s a cyclo-
propyl Grignard r e a g e n t followed by r i n g opening w i t h
dimethylamine t o form t h e d e s i r e d compound. A number of
a l t e r n a t i v e syntheses have been d e s c r i b e d i n t h e l i t e r a t u r e
(14-17).

4. S t a b i l i t y Degradation
The s t a b i l i t y of a m i t r i p t y l i n e h y d r o c h l o r i d e , i n t h e
bulk form, was s t u d i e d under c o n d i t i o n s of e l e v a t e d temper-
a t u r e o r exposure t o l i g h t (18). It was found t o b e s t a b l e
a t room temperature and a t 45OC f o r a p e r i o d i n excess of
two months. It showed some decomposition a f t e r two months
a t 100°C, and when exposed t o l i g h t , a s evidenced by t h e
formation of a brownish d i s c o l o r a t i o n of t h e powder. A 1%
aqueous s o l u t i o n was found t o be s t a b l e a t O°C, room
temperature, and 45OC, a s w e l l as f o r a p e r i o d of 60 hours
a t IOOOC.

5. Drug Metabolic Products

Hucker and P o r t e r (19) demonstrated t h a t very l i t t l e of
a dose of a m i t r i p t y l i n e i s e x c r e t e d unchanged i n humans.
Other i n v e s t i g a t o r s (20-23) have demonstrated t h e p r e s e n c e
of t h e major m e t a b o l i c products shown i n F i g u r e 8. Facino
and Corona (20) have demonstrated m e t a b o l i t e s I, 111, IV,
t h e two isomers o f V, and VI i n t h e organs of r a b b i t s .
Eschenoff and Rieder (21) demonstrated m e t a b o l i t e s I, 11,
and V and i n a d d i t i o n r e p o r t e d t h e e x i s t e n c e of t h e N-oxide
m e t a b o l i t e ( V I I ) i n s t u d i e s on rats and humans. They
r e p o r t e d t h a t t h e metabolism o f a m i t r i p t y l i n e i n t h e s e two
species i s n e a r l y i d e n t i c a l ( 2 4 ) . I n a d d i t i o n , Facino and
Corona (25) l a t e r r e p o r t e d t h e e x i s t e n c e of an a c i d i c
m e t a b o l i t e t o which they a s c r i b e d t h e s t r u c t u r e of t h e
c a r b o x y l i c a c i d which would b e formed by o x i d a t i o n deamina-
t i o n of t h e drug.

140
KENNETH W. BLESSEL, BRUCE C. RUDY, AND BERNARD Z . SENKOWSKI

Figure 8

Metabolic Products of Amitriptyline Hydrochloride

CH(CH1)zN(CHJ2
AMlTRlPTYLlNE HYDROCHLORIDE

f
-
0

t
CI

IV

142
 AMlTRl PTY LINE HYDROCHLORIDE

6. Methods o f A n a l y s i s

6.1 Elemental Analysis

The r e s u l t s o f an e l e m e n t a l a n a l y s i s of a sample
of r e f e r e n c e s t a n d a r d a m i t r i p t y l i n e h y d r o c h l o r i d e i s p r e -
s e n t e d i n T a b l e I V below (32).

Element Theory % Found %

C 76.53 76.44
H 7.70 7.79
N 4.47 4.50
c1 11.30 11.26

6.2 Phase S o l u b f l i t y Analysis

A phase s o l u b i l i t y a n a l y s i s f o r a m i t r i p t y l i n e
h y d r o c h l o r i d e is shown i n F i g u r e 9 . The s o l v e n t u s e d w a s
a c e t o n e and t h e e x t r a p o l a t e d s o l u b i l i t y w a s 17.81 mg/g
(8).

6.3 Thin Layer Chromatographic A n a l y s i s

A number of t h i n l a y e r chromatographic s y s t e m s
f o r a m i t r i p t y l i n e h y d r o c h l o r i d e h a v e been d e s c r i b e d i n t h e
l i t e r a t u r e . A r e p r e s e n t a t i v e number o f t h e s e s y s t e m s are
shown i n T a b l e V. Methods of d e t e c t i o n were n o t i n c l u d e d
s i n c e t h e s e are many times d e t e r m i n e d by t h e i n d i v i d u a l
p r e f e r e n c e s of t h e a n a l y s t o r t h e p a r t i c u l a r s e p a r a t i o n
d e s i r e d (26).
Table V

TLC Systems f o r A m i t r i p t y l i n e H y d r o c h l o r i d e

Adsorbent S o l v e n t System & Reference

Silica G e l benzene:dioxane:NH3 0.74 27

(60 :35 :5)
Silica G e l e t h a n o 1 : a c e t i c a c i d : 0.65 27
H20 (50 :30 :20)
S i l i c a Gel methanol :b u t a n o l 0.37 27
(60 :40)
Silica G e l + 0.1M NaOH cyc1ohexane:benzene: 0.72 28
d i ethylamine
(75 :1 5 :10)

143
 Figure 9
I-
z
W 25
>
J
0
v)
0
\
W 20
I-
3
-I
0
v)

LL 15 PHASE SOLU B ILI TY AN ALY S IS

0
w Sample: Amitriptyline
E
c
P - Solvent = Acetone
P
z 10 Slope : 0.04 o/o
2 Equilibration : 20 hrs at 25OC
t
v) Extrapolated Solubility * 17.81 mg/g of Acetone
0
0.
I 5
0
V
z
0 l l l r l l ~ ~ ~ ~ ~ ~ ~
I-
3 0
J
0 0 25 50 75 100
v)
SYSTEM COMPOSITION : mq OF SAMPLE PER g SOLVENT
 AMITR IPTY LINE HYDROCHLORIDE

Silica G e l + 0.1M NaOH methanol 0.50 28

S i l i c a Gel + 0.1M NaOH acetone 0.34 28
Silica G e l + 0.1M KHSO4 methanol 0.41 28
S i l i c a Gel + 0.1M KHS04 95% e t h a n o l 0.28 28

6.4 Gas-Liquid Chromatographic Analysis (GLC)

A GLC method f o r t h e 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 a m i t r i p t y l i n e has been d e s c r i b e d (29). The method w a s
developed f o r measuring t h e a m i t r i p t y l i n e c o n t e n t of
plasma. The lower l i m i t of d e t e c t i o n is 20 ng/ml. The
chromatographic c o n d i t i o n s a r e given below.

Column - 5 f t . x 114 i n . s i l a n i z e d
glass
Support - Chromosorb W (80-100 mesh)
Liquid Phase - 1% polyvinyl pyrrolidinone
and 3% Versamid 900
Detection - Flame i o n i z a t i o n
Oven Temperature - 205OC
Detector
Temperature - 240°C
Injection Port
Temperature - Maximum
C a r r i e r Gas - N i t r o g e n , 50 ml/min.

6.5 C o l o r i m e t r i c Analysis
A m i t r i p t y l i n e h y d r o c h l o r i d e can b e determined
c o l o r i m e t r i c a l l y u s i n g t h e methyl orange r e a c t i o n . The
method i n v o l v e s b u f f e r i n g t h e s o l u t i o n c o n t a i n i n g t h e com-
pound a t a pH v a l u e of 4 . 3 , adding the methyl o r a n g e and
e x t r a c t i n g t h e r e s u l t i n g complex i n t o e t h y l e n e d i c h l o r i d e .
The absorbance of t h e e t h y l e n e d i c h l o r i d e e x t r a c t i s mea-
s u r e d a t about 430 nm and t h e amount of a m i t r i p t y l i n e cal-
c u l a t e d by comparison w i t h a c a l i b r a t i o n curve prepared
from pure a m i t r i p t y l i n e . This method can b e used t o de-
termine a m i t r i p t y l i n e i n t h e p r e s e n c e of i t s N-demethylated
m e t a b o l i t e s by t h e a d d i t i o n of acetic anhydride b e f o r e ex-
t r a c t i o n s i n c e primary and secondary amines do n o t r e a c t
w i t h methyl orange i n t h e p r e s e n c e o f a c e t i c anhydride (30).

6.6 Fluorescence A n a l y s i s
A s e n s i t i v e f l u o r i m e t r i c a s s a y h a s been developed
f o r a m i t r i p t y l i n e h y d r o c h l o r i d e i n b i o l o g i c a l samples (31).
 KENNETH W. BLESSEL, BRUCE C. RUDY, A N D BERNARD 2 . SENKOWSKI

The b i o l o g i c a l material t o b e analyzed is homogenized and

a n e q u a l volume of methanol added. Then 0 . 2 g of borax,
1 5 m l of heptane and 1 m l of d i s t i l l e d w a t e r i s added t o a
1 m l a l i q u o t of t h e methanolic sample. The heptane l a y e r
a f t e r s e p a r a t i o n , is t h e n e x t r a c t e d w i t h p e r c h l o r i c a c i d .
The a c i d e x t r a c t is t h e n heated i n a b o i l i n g water b a t h f o r
10 min. and cooled. The f l u o r e s c e n c e of t h e carbonium i o n
generated by t h e h e a t i n g p r o c e s s i s t h e n measured a t 555 nm
u s i n g a n a c t i v a t i o n wavelength of 305 nm. The f l u o r e s c e n c e
i n t e n s i t y was found t o b e l i n e a r w i t h a m i t r i p t y l i n e con-
c e n t r a t i o n i n t h e range of 0.05-5.0 mcg/ml.

6.7 T i t r i m e t r i c Analysis
A non-aqueous t i t r a t i o n w i t h D e r c h l o r i c a c i d i n
acetic a c i d i s t h e p r e f e r r e d method f o r ' t h e a n a l y s i s of
bulk a m i t r i p t y l i n e hydrochloride. The sample is d i s s o l v e d
i n g l a c i a l a c e t i c a c i d . Then mercuric a c e t a t e T.S. and
c r y s t a l v i o l e t T.S. are added. The s o l u t i o n 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 t o a green end-point. Each m l of
0.1N p e r c h l o r i c a c i d i s e q u i v a l e n t t o 31.39 mg of a m i t r i p -
t y l i n e hydrochloride ( 6 ) .

7. Acknowledgments
The a u t h o r s wish t o acknowledge t h e a s s i s t a n c e o f t h e
Research Records O f f i c e and t h e S c i e n t i f i c L i t e r a t u r e
Department of Hof fmann-La Roche I n c .

146
 AMlTRlPTYLlNE HYDROCHLORIDE

8. References
1. Hawrylyshyn, M,, Hoffmann-La Roche Inc., Personal
Communication.
2 . Johnson, J. H., Hoffmann-La Roche Inc., Personal
Communication.
3. Rubia, L. B., Hoffmann-La Roche Inc., Personal
Communication.
4. Boatman, J., Hoffmann-La Roche Inc., Personal
Communication.
5. Benz, W., Hoffmann-La Roche Inc., Personal
Communication.
6. The United S t a t e s Pharmacopeia XVIII, pp. 38-40
(1970).
7. Moros, S., Hoffmann-La Roche Inc., Personal
Communication.
8. MacMullan, E., Hoffmann-La Roche Inc., Personal
Communication.
9. Hagel, R., Hoffmann-La Roche Inc., Personal
Communication.
10. Green, A. L., J . Pharm. Pharmac.,E, 10 (1967).
11. Hoffmann-La Roche, F. and Co., A . G., Belgian
Patent 577,057 (1959).
12. Merck and Co. Inc., Belgian Patent 584,061 (1960).
13. Hoffsommer, R. D., Taub, D., and Wendler, N. L.,
J. Org. Chem., 2, 1829 (1962).
14. Protiva, M., et al., J . Med. Pharm. Chern.,&, 411
(1961).
15. Villani, F. J., Ellis, C. A . , Teichman, C., and
Bigos, C . , J . Med. Pharm. Chem.,?, 373 (1961).
16. Winthrop, S . , et al., J . Org. Chern.,Z, 230 (1962).
17. Merck and Co, Inc., United States Patent 3,205,264
(1965).
18. Schmidli, B., Hoffmann-La Roche Inc., Unpublished
Data.
19. Hucker, H. B. and Porter, C. C., Federation Proc.,
-
20, 172 (1961).
20. Facino, R. M. and Corona, G. L., J . Pharm. Sci., 58,
764 (1969).
21. Eschenhof, E. and Rieder, J., ArzneimitteZ-Forsch ,
19, 957 (1969).
-
22. Hucker, H. B., PharmaeoZogis$ k, 171 (1962).
-
23. Diamond, S., Current Therap. Res., 7, 170 (1962).

147
 KENNETH W. BLESSEL. BRUCE C. RUDY, AND BERNARD 2 . SENKOWSKI

24. Eschenhof , E. and Rieder, J., Deut. Apotheker-Ztg.,

-
108, 1202 (1968).
25. Facino, R., Santagostino, G. and Corona, G., Biochem.
PharmacoZ., 2 , 1503 (1970).
26. Comer, J. P. and Comer, I., J . Pham. S c i . , 56, 413
(1967) .
27. Cochin, J. and Daly, J. W., J. PhamacoZ. ExptZ.
Therap., 139, 160 (1963).
28. Fike, W. W., AnaZ. Chem.,E, 1697 (1966).
29. Braithwaite, R. A . and Widdop, B., CZin. Chim. Acta ,
-
35, 461 (1971).
30. Silverstein, R. M., AnaZ. Chem.,g, 154 (1963).
31. Eschenhof, E. and Rieder, J. , Hoffmann-La Roche Inc. ,
Unpublished Data.
32. Scheidl, F., Hoffmann-La Roche Inc., Personal
Communication.

148
 DIGITOXIN

Ivan M . Jakovljevic
 IVAN M. JAKOVLJEVIC

CONTENTS

1. DESCRIPTION
1.1 R e g i s t e r e d Names
1.2 Chemical Name
1 . 3 Formula, S t r u c t u r e , M o l e c u l a r Weight
1 . 4 Appearance
2. PHYSICAL PROPERTIES
2.1 I n f r a r e d Spectrum
2.2 N u c l e a r M a g n e t i c Resonance S p e c t r u m
2.3 U l t r a v i o l e t Spectrum
2.4 Mass S p e c t r u m
2.5 Optical Rotation
2.6 O p t i c a l R o t a t o r y D i s p e r s i o n (ORD) a n d
C i r c u l a r D i c h r o i s m (CD)
2.7 M e l t i n g Range
2.8 X-Ray D i f f r a c t i o n P a t t e r n
2.9 Polarography
2.10 S o l u b i l i t y
3. SYNTHESIS
4. STABILITY
5. METABOLISM, P R O T E I N BINDING A N D
C L I N I C A L ASSAYS
5.1 Metabolism
5.2 P r o t e i n Binding
5 . 3 D e t e r m i n a t i o n i n Blood
5.3.1 Chemical Methods
5.3.2 P h y s i c a l Methods
5.3.3 Radioimmunoassay ( D e u t e r i u m and
T r i t i u m Labeled D i g i t o x i n )
6. METHODS O F ANALYSIS
6.1 I d e n t i f i c a t i o n Tests
6.2 Elemental Analysis
6 . 3 Chromatography
6.3.1 Column Chromatography
6.3.2 Thin L a y e r Chromatography
6.3.3 P a p e r Chromatography
6.3.4 Gas Chromatography
6.3.5 High S p e e d Liquid Chromatography

150
 DIG ITOX IN

6.4 Colorimetric Analysis

6.5 Fluorometric Analysis
6.6 Electrophoresis
6.7 Automated Assay
7. CLEAVAGE OF CARDIAC GLYCOSIDES
8. BIOLOGICAL ACTIVITY
8.1 Characteristic Structural Features
8.2 Bioassay
9 , ACKNOWLEDGMENT
10. REFERENCES

15 1
 IVAN M. JAKOVLJEVIC

1. DESCRIPTION
Digitoxin is a cardiotonic glycoside obtained
from D i g i t a l i s p u r p u r e a LinnQ, D i g i t a l i s l a n a t a
Ehrhart , and other suitable species o f D i g i t a l i s
leaves,
1.1 Registered Names -
Digitoxin is designated by the following
names:
C A R D I G I N (Nat ,Drugs) , C R Y S T O D I G I N (Li 1 ly) ,
D I G I C O R Y L (Roussel) , D I G I L O N G (Roehringer) , D I G I -
JYERCK (Merck) , D I G I P A N , D I G I S I D I N (Winthrop) , D I -
G I T A L I N E N A T I V E L L E (Varick), D I G I T O R A (Upjohn),
D I G I T O X I N (Sandoz) , D I G I T O X O S I D E (W.H.0,) , D I G I -
T R I N (Astra) , L A N A T O X I N (Beiersdorf) , P U R O D I G I N
(Wyeth) , P U R P U R E N , P U R P U R I D (Promonta),
1.2 Chemical Name -
3B-(D-Digitoxosyl-D-digitoxosyl-D-digito-
xosyl-oxy)-14~-hydroxy-5~-card-2O(2Z)-enolide,
1.3 Formula, Structure, Molecular Weight

c 4 1H6 4 0 1 3 Mol.Wt. 764.94

C18H3109 C23H3404
(tridigitoxose) digitoxigenin
THE CONFORMATIONAL ARRANGEMENT

Digitoxin belongs t o the cardenolide se-

ries, which consists of a steroid nucleus with a
5-membered unsaturated lactone ring at C-17.
As in most other glycosides, the sugar i s
present in the six-membered ring form (pyranoid

152
 DIGITOXIN

form) w i t h t h e c h a i r c o n f o r m a t i o n .
The s t e r o i d framework i s c o n s i d e r a b l y b e n t
a t e i t h e r e n d o f t h e m o l e c u l e , which i s a n impor-
t a n t s t e r i c requirement f o r c a r d i o t o n i c a c t i v i t y .
S a t u r a t i o n of t h e l a c t o n e r i n g g r e a t l y r e d u c e s t h e
c a r d i o t o n i c a c t i v i t y . The u n s a t u r a t e d l a c t o n e r i n g
must be a t t a c h e d i n t h e B - c o n f i g u r a t i o n . E p i m e r i z a -
t i o n r e d u c e s p h a r m a c o l o g i c a l a c t i v i t y by a t l e a s t
400 times. O p e n i n g t h e l a c t o n e r i n g by a l k a l i n e h y -
drolysis also results i n loss of activity',
1.4 Appearance
Very s m a l l e l o n g a t e d , r e c t a n g u l a r
p l a t e s from d i l u t e d e t h a n o l , o r m i c r o c r y s t a l l i n e
powder, w h i t e o r p a l e b u f f , o d o r l e s s , v e r y b i t t e r
taste.
2. PHYSICAL PROPERTIES
2.1 I n f r a r e d Spectrum
The i n f r a r e d ( I R ) s p e c t r u m o f d i g i t o x i n ,
USP r e f e r e n c e s t a n d a r d , i s g i v e n i n F i g . 1 . The I R
s p e c t r u m was t a k e n i n a K B r p e l l e t on a Beckman
IR-12 s p e c t r o m e t e r .
IR s p e c t r a l a s s i g n m e n t s o f d i g i t o x i n a r e
as follows2:
W a v e l e n g t h of V i bra ti on Mode 8
A b s o r p t i o n (CM-') t

3575 -OH s t r e t c h Inon-hydrogen

bonded)
3440 -OH s t r e t c h ( h y d r o g e n
bonded)
2960, 2935 CH s t r e t c h , C H 3 , CH2
stretch
1740 C E O s t r e t c h (a,B u n s a t u -
rated lactone]
1630 CH s t r e t c h [ c o n j u g a t e d
d o u b l e bond)
1448, 1403,
1 3 7 9 , 1 3 6 7 , 1348 C H d e f o r m a t i o n (CH3, C H 2 ,
C-CHJ, CH)

153
 I V A N M. JAKOVLJEVIC

1162 3O-OH d e f o r m a t i o n
1125 ZO-OH deformation
1075 C-0- s t r e t c h ( g l y c o s i d i c
ether)
1060 C-0- s t r e t c h ( c y c l i c e t h e r
oxygens )
1040 1°-OH d e f o r m a t i o n

The IR s p e c t r a o f 36 g l y c o s i d e s a n d t h e i r
a g l y c o n e s w e r e s t u d i e d . G l y c o s i d e s were c h a r a c t e r -
i z e d by a d o u b l e t i n t h e r e g i o n 1099-1031 and
1 0 6 6 - 1 0 1 3 cm-'.'
Examination of t h e I R s p e c t r a of d i g i t o -
x i n r e v e i l e d t h e p r e s e n c e o f two p o l y m o r p h s . One
o f t h e p o l y m o r p h i c c r y s t a l s was o b t a i n e d b y r e c r y s -
t a l l i z a t i o n from 85% e t h a n o l and t h e o t h e r from
t h e c o l d ethanol evaporation1 ,
2.2 Nuclear Magnetic Resonance Spectrum
The N M R s p e c t r u m o f d i g i t o x i n i s c o m p l e x
e v e n a t 2 2 0 MHz, h o w e v e r 1 7 p r o t o n s i g n a l s may b e
s e e n t o low f i e l d o f 2.5 ppm b o t h i n C D C 1 3 nnd
CD3SOCD3 a f t e r e x c h a n g e w i t h D,O t o remove t h e
s i g n a l s f o r O H , They may b e a s s i g n e d w i t h r e a s o n -
a b l e c e r t a i n t y from chemical s h i f t and c o u p l i n g
c o n s t a n t s , The 6 v a l u e s a r e l i s t e d a s f o l l o w s :
I n C D C 1 , : 2 . 7 9 ppm ( b r o a d e n e d t r i p l e t ) ,
p r o t o n a t 1 7 ; 3 . 2 0 , 3.23, 3 . 2 7 ( o y e r l a p p i n g ) ,
J a a = 1 0 . 0 Hz,
Jae
= 2.8, p r o t o n s 4 , l o ' , 16'
( n u m b e r i n g f r o m t h e a n o m e r i c p r o t o n away f r o m t h e
oxygen i n e a c h s u g a r r i n g s e q u e n t i a l l y s t a r t i n g
w i t h p o s i t i o n 3 on t h e s t e r o i d ) ; 3 . 7 7 u n r e s o l v e d
p r o t o n s 5 ' , l l ' , 1 7 ' ; 4 . 0 1 , p r o t o n 3 ; 4.10, p r o t o n
1 5 ' ; 4 . 2 2 , u n r e s o l v e d p r o t o n s 3' and 9 ' ; 4.78,
4 . 9 7 , J g e m - - 1 8 . 0 Hz, J z 1 - 2 2 = 1 . 8 , C H 2 a t 2 1 ;
4.84, 4.88, 4.91, ( o v e r l a p p i n g ) Jaa- 10, p r o t o n s
l', 7', 1 3 ' ; 5.88, p r o t o n a t 22.5 See F i g . 2 ,
P o s i t i o n s o f t h e a c e t y l groups i n p a r t i a l l y
a c e t y l a t e d c a r d e n o l i d e s were e s t a b l i s h e d b y t h e
a n a l y s i s o f NMR s p e c t r a . Chemical s h i f t s o f p r o t o n s
belonging t o s p e c i f i c a c e t y l groups d i f f e r s u f f i -
c i e n t l y t o serve as d i a g n o s t i c e h a r a c t e r i s t i c s .

154
 DIGITOXIN

FREOUENCY
3000 2500 2000 1600 1400 1200 1000 900 850 800 750 700 65
I I 1 0 1 I I

I I I I I I I I I I I I I

3 4 5 6 7 8 9 10 11 12 13 14 15
WAVELENGTH

F i g . 1 I R S p e c t r u m of D i g i t o x i n , U S P R e f e r e n c e
S t a n d a r d . K B r P e l l e t . I n s t r u m e n t : Beckman I R - 1 2
Spectrophotometer.

500 400 300 200 100

I
I I I
1

80 70 60 50 40 30 20 10
PPM ( 6 )

Fig. 2 NHR S p e c t r u m of D i g i t o x i n , U S P R e f e r e n c e
S t a n d a r d . I n s t r u m e n t : V a r i a n T-60A.

155
 I V A N M. JAKOVLJEVIC

The s i g n a l s o f t h e e q u a t o r i a l p r o t o n s o f d i g i t o x o -
se molecules a r e a l s o o f d i a g n o s t i c value a s t h e i r
p o s i t i o n c h a n g e s when t h e a d j a c e n t a x i a l g r o u p i s
acetylated6.
2.3 U l t r a v i o l e t Spectrum
The u l t r a v i o l e t c u r v e o f a s o l u t i o n i n
m e t h a n o l shows a p e a k a t 218 nm, E 17.4 x
2.4 -Mass S p e c t r u m
Mass s p e c t r o m e t r i c d a t a were o b t a i n e d
u s i n g e l e c t r o n i m p a c t a n d low r e s o l u t i o n on a
C.E.C. 110 mass s p e c t r o m e t e r . The f r a g m e n t a t i o n
pattern i s t y p i c a l of t h e s t e r o i d portion only.
The h i g h e s t s i g n i f i c a n t mass i s a t m/e 357 w h i c h
represents t h e digitoxigenin fragment.
2.5 Optical Rotation
I n v e s t i g a t o r s have determined t h e o p t i c a l
r o t a t i o n under d i f f e r e n t conditions:
[a];'= +4.8' (c31.2 i n d i o x a n e ) '

[a];'= a b o u t +18' (cp2.5 in chlor~form)~

20= a b o u t +21' (cxl.0 i n c h l o r ~ f o r m ) ~

[a];'= a b o u t +13' (cl.1.0 i n methanol)'

2.6 O p t i c a l Rotatory Dispersion (ORD) and

C i r c u l a r Dichroism (CD)
The c i r c u l a r d i c h r o i c (CD) s p e c t r a were
r e c o r d e d on a C a r y 60 s p e c t r o p o l a r i m e t e r , e q u i p p e d
w i t h Model 6 0 0 2 C D u n i t . The O R D c u r v e shows a
p o s i t i v e [ a ] of 1 3 . 1 a n d a p o s i t i v e C o t t o n e f f e c t
o f [ a ] 1 2 . 3 x l o - ' a t 254 nm a s d o many s t e r o i d s .
S e e F i g . 3. The C D c u r v e shows a p o s i t i v e e f f e c t
o f [ a ] 11.1 x 1 O - j a t 238 nm. These s t u d i e s were
done i n m e t h a n o l . 7

156
 DIGITOXIN

S t e r e o c h e m i c a l e f f e c t s a r e found i n car-
d e n o l i d e s w i t h a - k e t o l groups i n t h e 11,12 p o s i -
tion”.
Fig. 3 O R D a n d C D S p e c t r a of D i g i t o x i n , U S P
Reference Standard. Instrument: Cary 60 Spectro-
p o l a r i m e t e r , e q u i p p e d w i t h Model 6 0 0 2 C D U n i t .

.?.

200 300 350

250 nm

2.7 M e l t i n g Range
The m e l t i n g p o i n t o f d i g i t o x i n is 256 -
257°C.(anhydrous).e Digitoxigenin has a m e l t i n g
p o i n t 25OoC.”
The e f f e c t o f p r o t e c t i v e e n v i r o m e n t s s u c h
a s a ) immersing t h e s u b s t a n c e under s i l i c o n e o i l ,
b ) u n d e r an a t m o s p h e r e o f n i t r o g e n a n d c ) i n a n
e v a c u a t e d , s e a l e d c a p i l l a r y t u b e were s t u d i e d on
a m i c r o s c o p e h o t s t a g e , The m e l t i n g p o i n t s w h i c h
a r e obtained under such conditions a r e o f t e n
higher12.
2.8
-
X-Ray D i f f r a c t i o n P a t t e r n
The X-ray d i f r a c t i o n p a t t e r n o f d i g i t o x i n
conforms t o t h e f o l l o w i n g p a t t e r n ” .

dA 1/11 dA I/I’ dA I/I’

15 7 6.26 40 4 . bO 40
9.07 20 5.95 I00 4.62 40
8.01 7 5.63 50 4.32 70
7.2b 30 5.32 13 4.10 30
7.00 30 5.40 40 3.93 30

157
 I V A N M. JAKOVLJEVIC

dA 1/1 ' dA I/I' dA 1/11

3.75 30 3.06 7 2.37 3
3.62 7 2.89 7 2.20 3
3.52 7 2.75 7 2.10 3
3.39 7 2.60 3 2.05 3
3.30 20 2.52 3 2.01 3
3.23 7 2.43 3 1.95 3

2.9 Polarography

A study of the polarographic characteris-

tics of digitoxin in 50% ethanolic solution con-
taining tetraethylammonium hydroxide as electro-
lyte, showed an average half-wave potential o f
-1,965 volts. The diffusion current wave height
versus concentration graph indicated that quanti-
ties as low as 2 mcg could be determined. The
method has been successfully applied to the tinc-
ture o f d i g i t a l i ~ ' ~ 1)5 .
2.10 Solubility
Digitoxin is practically insoluble in
water (1 g dissolves in about 100 liters at 20'C).
One gram dissolves in about 40 m l chloroform, 6 0
m l ethanol, and 400 m l ethyl acetate. It is also
soluble in ether, petroleum ether, benzene and
vegetable oils.
3. SYNTHESIS
Digitoxigenin, a typical member o f t h e
cardenolide family has been synthesized using as
the starting material m e t h y l - 3 B - a c e t o x y - 1 4 B - h y -
droxy-Sf3-etinate by a seven-step sequence16. In
the last step a solution ofa,B-unsaturated ester
was treated with SeO2 by boiling under reflux for
10 hours. The filtrate was poured into water and
the product isolated with ether. Acid hydrolysis
o f digitoxigenin acetate yielded digitoxigenin
(M.p. 246-249'C and [ a ] +19' in ethanol).

158
 DIGITOX IN

4. STABILITY
The s t a b i l i t y o f two l i q u i d e x t r a c t s f r o m t h e
l e a v e s o f D i g i t a l i s p u r p u r e a was e x a m i n e d . B o t h
p r o d u c t s c o n t a i n e d d i g i t o x i n a n d g i t o x i n . The a c -
t i v i t y o f e a c h d r u g was d e c r e a s e d b y more t h a n 1 0 %
o f t h e i n i t i a l v a l u e i n less t h a n t h r e e months a t
20". The r a t e o f d e c o m p o s i t i o n was g i t o x i n > d i -
gitoxin".
S t o r a g e of d i g i t o x i n p r e p a r a t i o n s for o n e y e a r
did not s i g n i f i c a n t l y decrease t h e i r potency s t o r -
e d a t t e m p e r a t u r e s u p t o 3OoC. The p o t e n c y was
c h e c k e d by B a l j e t c o l o r i m e t r i c a s s a y a n d by t h e
b i o l o g i c a l method a c c o r d i n g t o t h e S w e d i s h P h a r -
m a c o p e i a XI
No b r e a k d o w n o f d i g i t o x i n i n t a b l e t s , i n j e c -
t i o n s o r s o l u t i o n s was f o u n d when s t o r e d f o r 5
y e a r s i n t h e d a r k up t o 3OoC. l 9
5. METABOLISM, P R O T E I N B I N D I N G A N D
CLINICAL ASSAY
5.1 Metabolism
Digitoxin i s completely absorbed follow-
i n g o r a l i n g e s t i o n and i t s f u l l e f f e c t a p p e a r s a s
r a p i d l y a s by i n t r a v e n o u s i n j e c t i o n * ' . The l i v e r
i s t h e main s i t e o f d e t o x i f i c a t i o n o f d i g i t o x i n ,
I t m e t a b o l i z e s v e r y r a p i d l y . One m e t a b o l i t e h a s
been i d e n t i f i e d : digoxigenin-di-digitoxoside, p r o -
d u c e d by h y d r o x y l a t i o n a n d t h e l o s s o f o n e m o l e -
cule of sugar.
Ten d a y s a f t e r an i n j e c t i o n , h a l f o f t h e
d o s e i s s t i l l p r e s e n t i n t h e b o d y , a n d some re-
mains a f t e r 2 0 days. Following t h e a d m i n i s t r a t i o n
o f m a i n t e n a n c e d o s e s o f 0 . 1 t o 0 . 3 mg d a i l y , 1 0 %
o f t h e dose i s e x c r e t e d unchanged.
An e x p e r i m e n t a l m e t h o d h a s b e e n d e v e l o p e d
i n order t o study t h e metabolic degradation of di-
g i t o x i n a n d d i g o x i n and c h a n g e s i n l i p i d s o l u b i l -
i t y o f t h e r a d i o a c t i v e m a t e r i a l i n p l a s m a 2 ' , The
changes occur a f t e r t h e a d m i n i s t r a t i o n o f r a d i o -
a c t i v e l y l a b e l l e d g l y c o s i d e s i n t o an i s o l a t e d p e r -
fused l i v e r system (guinea p i g ) o r t o i n t a c t r a b -
b i t s , The m e t a b o l i c d e g r a d a t i o n o f d i g i t o x i n a n d
digoxin i n t h e l i v e r r e s u l t s i n t h e formation of

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t h e mono- a n d b i s - d i g i t o x o s i d e s o f d i g o x i g e n i n a n d
o f c o n j u g a t e d p r o d u c t s , T h e s e m e t a b o l i t e s a r e more
p o l a r t h a n o r i g i n a l g l y c o s i d e s . An i n c r e a s e d p l a s -
ma-chloroform c o e f f i c j e n t i n d i c a t e s a change i n
t h e r a t i o o f p o l a r / n o n p o l a r s u b s t a n c e s i n t h e ex-
t r a c t e d medium.
5.2 Protein Binding
The p r o t e i n b i n d i n g o f d i g i t o x i n i s
thought t o account f o r t h e higher plasma l e v e l s .
The r e s u l t s were o b t a i n e d b y t h e R b E 6 u p t a k e i n -
hibition technique, suggesting its probable value
as a c l i n i c a l l y a p p l i c a b l e q u a n t i t a t i v e m e t h o d f o r
t h e d e t e c t i o n o f commonly u s e d d i g i t a l i s g l y c o s i d e s
.
i n p l a s m a . Com a r i s o n r e s u l t s b y t w o l a b o r a t o r i e s
were p r e s e n t e d 2 !
The p r o t e i n b i n d i n g c a p a c i t y o f poorly
s o l u b l e c a r d e n o l i d e s i n water is d e t e r m i n e d from
the saturation concentration of these substances
b o t h i n p r o t e i n s o l u t i o n and i n t h e i r u l t r a f i l -
t r a t e i n microscale. Figures about t h e binding o f
d i g i t o x i n , d i g o x i n e t c . t o human s e r u m p r o t e i n ,
and t h e b i n d i n g o f d i g i t o x i n t o serum p r o t e i n s o f
d i f f e r e n t s p e c i e s , a r e p r e s e n t e d . The c a r d e n o l i d e -
serum p r o t e i n b i n d i n g i s a f f e c t e d by c a l c i u m i o n s 2 3 .
5.3 D e t e r m i n a t i o n i n Blood
5.3.1 Chemical Methods
Digitoxin concentration i n the
b l o o d o f o r a l l y d i g i t a l i z e d p a t i e n t s was q u a n t i -
t a t i v e l y determined employing a combination o f
TLC and a f l u o r o m e t r i c method:
30 m l v e i n - b l l d was d i l u t e d t o
300 m l w i t h w a t e r , a n d t h e h a e m o l y s a t was e x t r a c t e d
w i t h c h l o r o f o r m , T h e c h l o r o f o r m e x t r a c t was e v a p -
o r a t e d u n d e r m i l d c o n d i t i o n (temp. n o t e x c e e d i n g
40OC.). T h e r e s i d u e was d i s s o l v e d i n 5 0 % a q u e o u s
methanol and t h e n e x t r a c t e d w i t h p e t r o l e u m e t h e r .
R e m a i n i n g m e t h a n o l was e x t r a c t e d w i t h c h l o r o f o r m ,
The r e s i d u e a f t e r c h l o r o f o r m e v a p o r a t i o n ( r e d i s -
s o l v e d i n a n e x a c t a m o u n t o f c h l o r o f o r m ) was a p p l i e d
t o K i e s e l g e l G p l a t e s . Mobil p h a s e : m e t h y l e n c h l o
ride/isopropanol/formamide, 8 0 : 1 9 : 1 . S p r a y r e a g e n t :
-
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a m i x t u r e o f chloramine and t r i c h l o r o a c e t i c a c i d ,
A f t e r s p r a y i n g t h e p l a t e s were h e a t e d a t 115OC.
f o r 1 0 min. O p t i m a l f l u o r e s c e n c e i n U V l i g h t was a t
365 nm24.
D i g i t o x i n i n b l o o d p l a s m a was d e -
t e r m i n e d b y enzyme p - e s t e r h y d r o l a s e a n d ATP-ase
i n h i b i t i o n technique2'.
5.3.2 P h y s i c a l Methods
Cardiac g l y c o s i d e s can b e d e t e r -
mined i n b i o l o g i c a l f l u i d s f r o m c o n c e n t r a t i o n s o f
1 n g / m l b y t h e i r i n h i b i t i o n o f t h e u p t a k e o f Rb
by r e d b l o o d c e l l s . The g l y c o s i d e e x t r a c t was i n -
cubated a t 37'C. f o r 2 hours w i t h dimethyl s u l f o -
x i d e , r e d b l o o d c e l l s , a n d a s o l u t i o n o f RbC1. The
Rb r e m a i n i n g i n t h e s u p e r n a t a n t i s m e a s u r e d by
atomic absorption spectrometry26,
5.3.3 Radioimmunoassay ( D e u t e r i u m and
Tritium Labeled Digitoxin)
A l l t h r e e pro-
t o n s i n t h e u n s a t u r a t e d b u t e n o l i d e r i n g can b e
exchanged i n a b a s e - c a t a l y z e d p r o c e s s . The e x -
change takes p l a c e even under v e r y mild c o n d i t i o n s
and t h e r i n g d o e s n o t o p e n . D i g i t o x i n was t r e a t e d
w i t h t r i e t h y l a m i n e and d e u t e r i u m o x i d e , and
U V , IR a n d N N R d a t a showed t h a t t h e compound
f o r m e d c o r r e s p o n d s t o t h e 21,21,22-trideuterodigi-
toxin. Similar r e a c t i o n t a k e s p l a c e w i t h t r i t i u m
o x i d e . The e x c h a n g e i s l i m i t e d t o t h e t h r e e p r o -
tons i n the unsaturated butenolide ring2'.
C l i n i c a l l y a p p l i c a b l e radioimmuno-
a s s a y t e c h n i q u e s f o r measurement o f serum d i g i t o x -
i n have been used t o determine l e v e l s o f t h i s drug
from 250 p a t i e n t s . U n l a b e l e d d r u g i n t h e p a t i e n t s 8
serum d i s p l a c e s t r i t i a t e d d i g i t o x i n (added i n v i t -
r o ) f r o m s p e c i f i c a n t i b o d y b i n d i n g s i t e s . The p r o -
cedure r e q u i r e s one hour2'.
The p h a r m a c o d y n a m i c s o f d i g i t o x i n
i n man h a v e b e e n s t u d i e d u t i l i z i n g a s e n s i t i v e
(0.2 n g / m l ) s p e c i f i c r a d i o i m m u n o a s s a y . P a t i e n t s
r e c e i v i n g 0 . 1 mg o f d i g i t o x i n d a i l y h a d a mean
s e r u m d i g i t o x i n l e v e l o f 25 n g j m l , a n d 4 4 n g / m l
w a s d e t e c t e d i n p a t i e n t s r e c e i v i n g 0.2 mg d a i l y 2 ' .

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In another study unlabeled d i g i -

t o x i n i n t h e unknown s a m p l e c o m p e t e s w i t h a t r i -
t i a t e d d i g i t o x i n tracer f o r b i n d i n g s i t e s of h i g h
a f f i n i t y r a b b i t a n t i b o d i e s t o a human s e r u m a l b u -
m e n - d i g o x i n c o n j u g a t e . Free l a b e l e d d i g i t o x i n was
s e p a r a t e d from t h e antibody-bound f r a c t i o n by ad-
s o r p t i o n t o d e x t r a n - c o a t e d c h a r c o a l . The m e t h o d
i s s e n s i t i v e t o 2 ng/ml o r l e s s ” .
6. METHODS O F ANALYSIS
6.1 Identification Tests
D i s s o l v e about 1 m g of d i g i t o x i n i n 2 m l
o f a s o l u t i o n p r e p a r e d by mixing 0.3 m l o f a 9%
aqueous f e r r i c c h l o r i d e s o l u t i o n and 50 m l o f
g l a c i a l a c e t i c a c i d , and u n d e r l a y w i t h 2 m l of
s u l f u r i c a c i d : a t t h e zone o f c o n t a c t o f t h e two
l i q u i d s a brown c o l o r i s p r o d u c e d , and i t g r a d -
u a l l y changes t o l i g h t g r e e n , t h e n t o b l u e , and
f i n a l l y t h e entire acetic layer acquires a blue
c o l o r 3l ,
D i s s o l v e a b o u t 0 . 2 mg o f d i g i t o x i n i n
2 m l o f a f r e s h l y p r e p a r e d 1 i n 100 s o l u t i o n o f
m-dinitrobenzene i n e t h a n o l , and a l l o w t o s t a n d
f o r 1 0 min, w i t h f r e q u e n t s h a k i n g . Add 2 m l o f a
m i x t u r e o f 1 volume o f a 10% tetramethylammonium
h y d r o x i d e a n d 2 0 0 volumes o f e t h a n o l , a n d m i x : a
r e d - v i o l e t c o l o r develops s l o w l y and t h e n f a d e s ” .
6.2 Elemental Analysis
Elemental a n a l y s i s o f d i g i t o x i n as
‘4 l H 6 4’1 3 :
C -64.4%
H - 8.4%
0 -27.2%

6.3 Chromatography
6.3.1 Column C h r o m a t o g r a p h y
Aluminum o x i d e , s i l i c e o u s e a r t h ,
a n d S e p h a d e x a r e a d s o r b e n t s commonly u s e d f o r t h e
s e p a r a t i o n o f c a r d e n o l i d e g l y c o s i d e s and t h e i r
metabolites,

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The USP X V I I I e m p l o y s a column o f

s i l i c e o u s e a r t h previously cleaned with hydrochlo-
r i c a c i d , a n d t h e n a c t i v a t e d a t 500°Cc. Formamide
i s added as t h e s t a t i o n a r y phase. D i g i t o x i n i s
e l u t e d with a mixture of benzene/chloroform, 3:l.
Aluminum o x i d e d e a c t i v a t e d w i t h
3% w a t e r and p a c k e d i n a column o f 1.5 cm d i a m e t e r
t o a height o f 10 cm h a s b e e n u s e d s u c c e s s f u l l y f o r
t h e s e p a r a t i o n o f d i g i t o x i n from i t s m e t a b o l i t e s .
E l u t i o n i s a c h i e v e d w i t h 100 m l o f c h l o r o f o r m f o l -
lowed b y 35 m l o f 2 % e t h a n o l i n c h l o r o f o r m , a n d
f i n a l l y w i t h 250 m l o f 10% e t h a n o l i n c h l o r o f o r m .
D i g i t o x i n and i t s m e t a b o l i t e s a r e f o u n d i n t h a t
p o r t i o n o f e l u a t e b e t w e e n 275 and 425 m l ( t h i s
i n c l u d e s t h e chloroform prewash)12.
S e p h a d e x G-200, s w e l l e d w i t h a
m i x t u r e o f w a t e r / m e t h a n o l , 7 : 3 , was e m p l o y e d f o r
t h e s e p a r a t i o n o f d i g i t o x i n and i t s m e t a b o l i t e s .
S e p h a d e x was p a c k e d i n a column o f 1 cm d i a m e t e r
t o a h e i g h t o f 1 5 cm. The s a m p l e was e l u t e d u s i n g
t h e a b o v e m i x t u r e . C a r d e n o l i d e s were i n t h e f r a c -
t i o n between 1 0 and 2 5 m 1 3 ’ .
6.3.2 Thin Layer Chromatography
A r a p i d s e p a r a t i o n of d i g i t o x i n
from d i g o x i n and a c e t y l d i g i t o x i n c a n b e a c h i e v e d
a p p l y i n g 2p1 o f a 0 . 0 1 % s a m p l e s o l u t i o n i n c h l o -
roform/methanol, 1:l t o K i e s e l g e l G p l a t e s . A s
t h e e l u e n t a c h l o r o f o r m / m e t h a n o l , 9 : l m i x t u r e was
u s e d , D e t e c t i n g a g e n t : h y d r o c h l o r i c a c i d . The
s p o t s o f d i g i t o x i n ( R f 0 . 3 3 ) , d i g o x i n (Rf 0 . 2 4 )
a n d a c e t y l d i g i t o x i n (Rf 0 . 4 8 ) w e r e d a r k brown
a f t e r 5 min. D r y i n g a t l l O ° C . f o r 5 min a n d exam-
i n a t i o n u n d e r u l t r a v i o l e t l i g h t ( 3 6 5 nm) showed
brown s p o t s f o r d i g i t o x i n a n d a c e t y l d i g i t o x i n ,
w h i l e d i g o x i n s p o t was b l u e . The l i m i t o f d e t e c -
t i o n i s 0.05 mcg3’.
D i g i t o x i n was s e p a r a t e d f r o m d i -
g o x i n on S i l i c a Gel G p l a t e s w i t h c h l o r o f o r m / m e t h -
a n o l , 8 8 : 1 2 d e v e l o p i n g s o l v e n t . The z o n e s were
l o c a t e d by s p r a y i n g w i t h 1% i o d i n e i n c h l o r o f o r m ,
t h e n removed f r o m t h e p l a t e a n d e x t r a c t e d w i t h
chloroform/methanol, 1:l mixture. After c e n t r i -
f u g a t i o n , a 7 m l a l i q u o t o f s u p e r n a t a n t was e v a p -

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o r a t e d t o d r y n e s s . The r e s i d u e was d r i e d a n d t h e n
t r e a t e d w i t h d i x a n t h y l u r e a r e a g e n t and t h e chro-
mophor r e a d a t 5 3 5 n m 3 4 ) 3 5 .
A method f o r t h e d i r e c t q u a n t i t a -
t i v e e v a l u a t i o n o f d i g i t o x i n , d i g o x i n and a c e t y l -
d i g i t o x i n o n T L C u s i n g s p e c t r o f l u o r o m e t r y was i n -
v e s t i g a t e d . The o n l y r e a g e n t u s e d was h y d r o c h l o -
r i c a c i d , L i n e a r s t a n d a r d c u r v e s were o b t a i n e d
when t h e a r e a u n d e r t h e f l u o r o m e t r i c c u r v e was
c o r r e l a t e d w i t h t h e amount o f g l y c o s i d e s a p p l i e d .
The o p t i m a l r a n g e f o r t h e s p e c t r o f l u o r o m e t r i c d e -
t e r m i n a t i o n o f t h e s e t h r e e g l y c o s i d e s was a b o u t
0.25 mcgj6.
Separation of t h e cardiac glyco-
s i d e s d i g i t o x i n a n d d i g o x i n from t h e i r 2 0 , 2 2 - d i -
h y d r o d e r i v a t i v e s c a n b e a c h i e v e d b y m u l t i p l e TLC
on c e l l u l o s e f i l m s ’.
An u l t r a m i c r o f l u o r e s c e n t s p r a y
r e a g e n t f o r d e t e c t i o n and q u a n t i t a t i o n o f d i g i -
t o x i n a n d o t h e r c a r d i o t o n i c g l y c o s i d e s on T L C was
d e s c r i b e d . The s p r a y r e a g e n t c o n s i s t s o f a s c o r b i c
a c i d , methanol, h y d r o c h l o r i c a c i d and hydrogen
p e r o x i d e . The l i m i t s o f d e t e c t i o n were 0 . 0 1 m c g 3 ’ ,
A p p l i c a t i o n of d i f f u s i o n a n d f l u -
o r e s c e n c e t o t h e d i r e c t d e t e r m i n a t i o n of d i g i t o x -
i n was a c h i e v e d by c o n v e r t i n g d i g i t o x i n i n t o a
f l u o r e s c e n t d e r i v a t i v e b y means o f a r e a g e n t c o n -
taining p-toluenesulfonic acid, hydrochloric acid,
a s c o r b i c a c i d and h y d r o g e n p e r o x i d e . S e n s i t i v i t y :
0 . 3 - 1 rncg3’.
A T L C s y s t e m on s i l i c a g e l G
p l a t e s h a s been d e v e l o p e d u s i n g as t h e mobile
s o l v e n t a m i x t u r e of methylene chloride/methanol/
formamide, 80:19:1, and s p r a y i n g t h e p l a t e s w i t h
a c i d - f e r r i c c h l o r i d e j l . The same t e c h n i q u e c a n b e
used f o r t h e i d e n t i f i c a t i o n o f d i g i t o x i n , digoxin
and a c e t y l d i g i t o x i n , a n d f o r t h e d e t e r m i n a t i o n o f
any g i t o x i n p r e s e n t i n t h e i r drug f o r m u l a t i o n s ,
Digoxin i s n o t a c t i v a t e d t o v i s i b l e f l u o r e s c e n c e
a t room t e m p e r a t u r e b y a c i d - f e r r i c c h l o r i d e r e -
a g e n t . T h e r e f o r e a n y f l u o r e s c e n c e p r e s e n t immedi-
a t e l y a f t e r s p r a y i n g i s due t o g i t o x i n a l o n e ,
H e a t i n g t h e p l a t e a t 100°C. d e s t r o y s t h e g i t o x i n
f l u o r e s c e n c e and c o n v e r t s t h e d i g o x i n t o a f l u o -

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r e s c e n t a n h y d r o d e r i v a t i v e w h i c h may b e s e e n u n d e r
b o t h UV and v i s i b l e l i g h t " ' .
6.3.3 Paper Chromatography
After s e p a r a t i o n by p a p e r chroma-
t o g r a p h y i n formamide s a t u r a t e d m e t h y l e t h y l k e t o n e /
x y l e n e , 1 : l m i x t u r e , d i g i t o x i n a n d d i g o x i n were
d e t e r m i n e d w i t h x a n t h y d r o l ( 1 0 - f o l d excess o f re-
a g e n t i n a c e t i c a c i d / h y d r o c h l o r i c a c i d , 9 9 : l mix-
t u r e ) . I t was n e c e s s a r y t o h e a t t h e r e a c t i o n m i x -
t u r e f o r 2 0 m i n . a t 60'C. A 1 : l c o m p l e x ( A m a x 535
n m ) , s t a b l e f o r 3 h o u r s was f o r m e d . Beer's law was
obeyed o v e r t h e r a n g e 1-20 m ~ g / m l " ~ * " * .
A b u t e n o l i d e r i n g s p e c i f i c method
of q u a n t i t a t i v e paper chromatographic analysis of
d i g i t o x i n u s i n g 2,4,2',4'-tetranitrodiphenyl is
d e s c r i b e d . P a p e r : S c h l e i c h e r a n d S c h U l l 2043b i m -
p r e g n a t e d w i t h formamide. Developing s o l v e n t :
methylethylketone/xylene, 1 : l s a t u r a t e d w i t h f o r -
mamide. T h e c h r o m a t o g r a m was d r i e d f o r 1 5 min. a t
60'C. "'
6.3.4 Gas C h r o m a t o g r a p h y
Trimethylsilyl ether derivatives
o f d i g i t o x i n , d i g o x i n and g i t o x i n h a v e b e e n shown
t o b e r e s o l v a b l e on a g a s C h r o m a t o g r a p h i c column
p a c k i n g c o n t a i n i n g as a l i q u i d p h a s e 2.5% O V - 1 o r
OV-17 on C h r o m o s o r b W. Gas c h r o m a t o g r a p h y was p e r -
formed on a Barber-Colman 5000 s e r i e s i n s t r u m e n t
equipped with hydrogen flame d e t e c t o r . During i s o -
t h e r m a l o p e r a t i o n i n j e c t i o n p o r t and column b a t h
t e m p e r a t u r e s were i d e n t i c a l , D e t e c t o r t e m p e r a t u r e
was m a i n t a i n e d a t 34OoC. I n t h e c a s e o f t e m p e r a -
t u r e p r o g r a m m i n g , i n j e c t i o n p o r t t e m p e r a t u r e was
i d e n t i c a l t o t h e s t a r t i n g t e m p e r a t u r e : 240'C.""
An i m p r o v e d m e t h o d f o r t h e g a s
chromatographic i d e n t i f i c a t i o n o f d i g i t a l i s car-
d e n o l i d e s as t h e i r a n h y d r o d e r i v a t i v e s h a s b e e n
developed, r e s u l t i n g i n g r e a t l y reduced r e t e n t i o n
times and e n h a n c e d r e s o l u t i o n . R e t e n t i o n d a t a o f
18 c a r d e n o l i d e s on t h r e e l i q u i d p h a s e s a r e r e p o r t -
ed. S p e c t r a l e v i d e n c e i s p r e s e n t e d showing t h a t
t h e t e r t i a r y 14B-OH g r o u p i s n e i t h e r a f f e c t e d b y
esterification nor e t h e r i f i ~ a t i o n ~ ~ .

165
 IVAN M. JAKOVLJEVIC

6.3.5 High Speed Liquid Chromatography

High speed liquid chromatography
has been used to examine steroids and steroid con-
jugates such as digitoxin or digoxin. In these
studies reverse phase liquid partition chromato-
graphy was employed. The columns consisted of a
cyanoethylsilicone polymer (Dupont ZipaxR ANH)
and the mobile phase was a mixture containing 2.5%
methanol and 97.5% water, The compounds were de-
tected by means of a 254 nm photometerk6.
6.4 Colorimetric Analysis
The methods for the determination of car-
diac glycosides can be divided into three general
groups based on: 1- the sugar moiety, 2 - the bute-
nolide moiety, and 3 - the steroid part of the mol-
ecule.
1- A s far back as 1885, a colorimetric
methodk7 was published using equal amounts of sul-
furic acid and ethanol with the addition of ferric
chloride. Others4’ used a solution of ferric sul-
fate in concentrated sulfuric acid, or added fer-
ric chloride to a solution of glycoside in acetic
acid and then underlaid the Kiliani reagent“’.
Many methods employ xanthydrol as the reagent for
digitoxoseSo.
2 - The reagent employing picric acid in
alkaline ethanol is the most frequently used5’.
There are many modifications and applications of
this reactions2#5 3 ) 5 4 ? 5 5 ?
The official U S P XVIII method depends on
a chromatographic separation on siliceous earth in
the presence of formamide, and a reaction in the
butenolide side chain by picric acid in alkaline
solution.
The application of m-dinitrobenzene for
the quantitative determination of cardiac glyco-
sides is very successfuls6, as well as 1,3,5-tri-
nitrobenzene in alkaline medium5’. Some authors

diphenyl ’. ’‘
use 2,4-dinitrodiphenylsulfone in alkaline etha-
nol”. The other rea ents used are 2-naphtho-
quinone -4 -sul fonat e , and 2 ,2 ,4.4 -
t et ran i t ro-

166
 DIG ITOX IN

The r e a g e n t s b a s e d upon t h e r e a c t i o n i n
b u t e n o l i d e r i n g have a wide a p p l i c a t i o n i n c a r d i -
a c glycosides metabolism s t u d i e s , These r e a g e n t s
r e a c t with any glycoside o r i t s metabolite
that s t i l l contains the intact butenolide ring.
3- Methods b a s e d upon t h e r e a c t i o n i n
t h e s t e r o i d m o i e t y a r e f o r t h e most p a r t f l u o r o -
metric,
6.5 Fluorometric Analysis
M e t h o d s b a s e d upon t h e r e a c t i o n i n t h e
s t e r o i d moiety.'are m a i n l y d e h y d r a t i o n t y p e of re-
a c t i o n s such as t h a t u s i n g syrupy phosphoric
a c i d 6 2 , o r e q u a l amounts o f h y d r o c h l o r i c a c i d and
g l y c e r o l a s t h e d e h y d r a t i n g a g e n t s 6 3 . Hydro e n
p e r o x i d e , h y d r o c h l o r i c a c i d and methanol64' 8, , or
a mixture of s u l f u r i c and phosphoric a c i d s w i t h
t h e a d d i t i o n o f f e r r i c c h l o r i d e 6 6 are a l s o used.
The f l u o r o p h o r obtained with a mixture o f
acetic anhydride, a c e t y l c h l o r i d e and t r i f l u o r o -
a c e t i c a c i d , s u p p o r t s t h e t h e o r y , b a s e d on N M R ,
I R and f l u o r e s c e n c e a c t i v a t i o n s p e c t r a l d a t a , t h a t
a low y i e l d o f a h i g h l y c o n j u g a t e d f l u o r o p h o r O f
s u b s t i t u t e d 3.4-benzpyrene i s o b t a i n e d 6 7 .
6.6 ~
ElectroDhoresis
~~ ~~

D i g i t o x i n may b e d e t e c t e d and e s t i m a t e d i n
human a u t o p s y t i s s u e s b y p a p e r e l e c t r o p h o r e s i s
a c c o r d i n g t o a n a u t h o r 6 * who u s e d a m i x t u r e o f
o x a l i c a c i d , b o r i c a c i d and e t h a n o l t o d e v e l o p
d i f f e r e n t c o l o r s d e p e n d i n g upon t h e compound. The
l i m i t f o r i d e n t i f i c a t i o n was a b o u t 1 5 mcg f o r d i -
g i t o x i n , a n d 1 0 mcg f o r d i g i t o x i g e n i n . The d i g i -
t o x i n was t o t a l l y d e g r a d e d i n t h e t i s s u e s f o l l o w -
i n g p u t r e f a c t i o n f o r t h r e e months6',
6 . 7 Automated Assay
An a u t o m a t e d p r o c e d u r e u s i n g a s t a n d a r d
T e c h n i c o n a u t o m a t i c a n a l y z e r s y s t e m is d e s c r i b e d
f o r t h e u n i t dose a n a l y s i s o f d i g i t o x i n and d i -
g o x i n i n t a b l e t s 7 ' . The t e c h n i q u e i s b a s e d on t h e
f l u o r o m e t r i c measurement o f t h e d e h y d r a t i o n prod-
u c t s of t h e c a r d i o t o n i c s t e r o i d s r e s u l t i n g from
t h e i r r e a c t i o n w i t h hydrogen p e r o x i d e and hydro-

167
 I V A N M. JAKOVLJEVIC

c h l o r i c a c i d , The automated system as d e s c r i b e d i s

capable of analyzing 1 2 t a b l e t s p e r hour,
7. C L E A V A G E O F C A R D I A C GLYCOSIDES
The i s o l a t i o n o f c a r d i a c a g l y c o n e s f r o m p l a n t
m a t e r i a l i s made d i f f i c u l t by t h e l a c k o f r e l i a b l e
methods t h a t w i l l m a i n t a i n t h e g e n i n s i n t a c t
a f t e r hydrolysis of t h e i r glycosides.
A procedure i s described f o r c l e a v i n g t h e sug-
a r bond o f d i g i t o x i n i n o r g a n i c m e d i a a n d u n d e r
m i l d a c i d c o n d i t i o n s . Maximum y i e l d s were o b t a i n e d
i n 30 min. o f r e a c t i o n t i m e a t 5OoC: i n a medium
c o n s i s t i n g o f a n h y d r o u s t e t r a h y d r o f u r a n made 0 . 0 0 2
N with r e s p e c t t o p e r c h l o r i c acid. Digitoxigenin
i s s t a b l e u n d e r t h e s e condition^^^.
8. B I O L O G I C A L ACTIVITY
8.1 C h a r a c t e r i s t i c S t r u c t u r a l F e a t u r e s
From t h e s t u d i e s o f b i o l o g i c a l a c t i v i t y 7 2
four characteristic structural features of the
g e n i n s c a n be e a s i l y i d e n t i f i e d as e s s e n t i a l f o r
cardiac activity:
1- The L a c t o n e R i n g : The d o u b l e bond o f
the lactone ring is apparently necessary f o r car-
d i a c a c t i o n . The r u p t u r e o f t h e l a c t o n e r i n g r e -
s u l t s i n a loss o f c a r d i a c a c t i o n .
2 - The Hydroxy Group on C - 1 4 Atom: T h i s
hydroxy group i s i m p o r t a n t and i t s m o d i f i c a t i o n
results i n a significant loss of activity. I f t h i s
g r o u p i s removed, t h e i m p o r t a n t s t e r e o c h e m i c a l re-
l a t i o n s h i p i s d e s t r o y e d so t h a t l o s s o f a c t i v i t y
c o u l d b e d u e e i t h e r t o t h e loss o f t h e 1 4 - h y d r o x y l
group, o r t o t h e a l t e r a t i o n o f t h e c i s C / D r i n g
arrangement 3 ,
3- S u g a r s a t C-3 Atom: I n t h e c a s e o f t h e
a g l y c o n e , t h e a t t a c h m e n t o f o n e o r more s u g a r s a t
C-3 u s u a l l y r e s u l t s i n i n c r e a s e d a c t i v i t y .
4- Stereochemical Arrangements: A llcislt
f u s i o n o f t h e C and D r i n g s i s n e c e s s a r y f o r a c -
t i v i t y . Other types of n a t u r a l s t e r o i d s have t h e
" t r a n s " c o n f i g u r a t i o n . The p r e s e n c e o f two h y d r o x -
y l g r o u p s a t C - 1 2 a n d C-16 i n t h e m o l e c u l e o f d i -
 DIGITOXIN

gitoxigenin diminishes the activity hy two-thirds.

1 4 ~ ~ , 1 5 u - e p o x y - 1 4 - a n h y d r o d i g i t o x i g e n i nis practi-
cally inactive.
8.2 Bioassay
Experiments were made t o examine
the suktability of young chicks for the assay of
digitalis glycosides, The jugular vein was cannu-
lated and a volume of the test solution was in-
fused. The dose was repeated at 5 min. intervals
until cardiac arrest was noticed. The order of
susceptibility of tested animals was as follows:
pigeon > chick> rat74.
9. ACKNOWLEDGMENT
The author acknowledges the invaluable help
of Miss Adele Hoskin for her assistance in t h e
1ite rat u r e s e a r c h .

lo. REFERENCES
1. Chen,K.K.,Ann.Rev.Physiol. 7,677 (1945).
2. Kossoy,A.D. and Underbrink,F.D.,Eli Lilly and
Company, personal communication,
3. Khalique,A. et al., Sci.Res.Pakistan 3,177
(1966).
4. Takuma,O. et al., Bunseki Kagaku, 17(1),53
(1968).
5. Boaz,H.E.,Eli Lilly and Company, personal
communi cat i on.
6. Voigtlaender,H.W. et al., Arch.Pharm. 3 0 1 , 2 0 8
(1968).
7. Beasly,F.W., E l i Lilly and Company, personal
communication.
8. The Merck Index, VIII Edition, 1968.
9. British Pharmacopoeia, 1968.
10, Djerassi,C. et al., Helv.Chim.Acta - 41,250
(1958).
11, Fieser and Fieser, Steroids, Reinhold Publish.
Corp., 1959.
12. Mueller,L. , Microchem.J., z ( 1, 2)0 (1968).
 I V A N M. JAKOVLJEVIC

13. Parson et al., Henry Ford Hosp.Med.Bull.,

6,365 (1958).
14. Hilton,J.G,, Science, 110,526 (1949).
15. Hilton,J.G., J.Pharmacol,Exptl.Therap.
100,258(1950).
16. Danieli,N. et al., Tetrahedron 22,3189(1966).
17. Lutomski,J. , Herba Pol. 12,243 (1966).
18. Samuelsson,G., J.Mond,Pharm. 4,112 (1961).
19. Samuelsson,G., Acta Pharm.Suecica 1(6),227- (1964).
20. Clarke,E.G.C., Isolation and Identification
of Drugs, London, 1969.
21. Glover,A., Klin.Wochenschr. 49,1062 (1971).
22. Shapiro,W., Circulation 4O0supp1.3,l84(1969).
23. Pfordte,K. et al., Acta Biol.Med.Germ. 25,
19 (1970).
24. Seipe1,H. et al., Klin.Wochenschr. - 46,1257
(1968).
25. Bentley,J.D., Circulation 41(1),67 (1970).
26. Bourdon,R. et al., Ann.BiorClin.(Paris) 27
(10-12) ,651 (1969).
27. Haberland,G. et al. , Naturwissensch, 56(10) ,
516 1196n.
28. Smith,T.W. et al., Circulation 4O(Suppl.3),
1 8 8 n969).
29. Morrison,J. et al., Clin.Res. 18(4),668(1970).
30. Smith,T.W., J.Pharrnacol.Exptl.merap.l75(2),
352 (1970).
31. The United States Pharmacopeia, XVIII.
32. Pfordte,K. et al., Z.Med.Labortechn. 11,272 -
(1970).
33. Hoeke,M. et al., Pharm.Weekhlad 104,877 - (1969).
34. Rican-Fister et al., J-Chromatogr. 41(1),91
(1969).
35. Johnston,E. J. et a1 . , J. Pharm.Sci. -
55 ( 5 ) ,531
(1966) .
36. Frijns,J.M.G,J., Pharm.Weekblad 105,209 - (1970).
37. Rabitzsch,G., J.Chromatogr, 35,122 (1968) *
38. Jelliffe,R.W. et a l . , J.Chromatagr. 27,172 -
(1967).
39. Eide-Juergensen et al., Plants Med.98
(Suppl.5) ,112 (1971).
 DIG ITOX IN

40. Dow,M.L. et al., J.Pharm.Sci. %(2),298

(1971).
41. Dzyuba,N.P. et al.,
Pharm.Zh. (Kiev) 26(3) ,42 - f 1971).
42. Dzyuba,N.P. et al,, Khim.Farm.Zh. -
S(llj,Sl
(1971).
43. Rabitzsch,G. et al., J.Chromatogr. 4l,96
(1969).
44. Wilson,W.E. et al., Analyt.Chem. 41(6),810 - (1969).
45. Tan,L., J.Chromatogr. 45,68 (1968).
46. Henry,R.A. et al., J.Chromatogr.Sci. 9,513
(1971).
47. Lafon,P., Compt.Rend. 100,1463 (1885).
48. Kiliani,H., Arch.Pharm. 234,273 (1896).
49. Keller,C., Ber.Pharm.Ges. 5,275(1895).
50. Pesez,M., Ann.Pharm.Franc.-ED104 (1952).
51. Baljet,H., Schweiz.Apoth.Ztg. s , 7 1 , 8 4 (1918).
52. Bel1,F.K. et al., J.Am.Pharm.Assoc.,Sci.Ed.
37,297 (1948).
53. Bel1,F.K. et al., J.PharmaZl.Exptl.Therap.

54. Dyer,F.J., Quart.J.Pharm.

-
88,14 (1946).
5,172 (1932).
55. Kennedy,E.E., J.Am,Pharm.Assoc.,Sci.Ed.
39,25 (1950).
56. Morel,A., Bull.Soc.Chim.FrGce 5 ( 2 ) ,949(1935).
57. Kimura,M., J. Pharm.Soc. Japan 2 7 9 9 1 (1951).
58. Tattje,D.H.E., Pharm,Weekblad 93,245 (1958).
59. Warren,A. T. et al. , J.Am. P h a r m x s s o c . ,Sci .Ed.
37,186 (1948).
60. Doelker E. et al., Pharm.ActaHelv. 44,647
(1969).
61. Rabitzsch,G. et al. , Pharmatie a ( 5 ) ,262
(1969).
62. Petit,A. et al., B u l l . S o c . C h i m . F r a n c e - ~ , 2 8 8
(1950).
63. Jensen,K.B., Acta Pharmacol.Toxico1. 8,101
T1952).
64. Jensen,K.B., Ibid. 9,66 (1953).
65. Wells,D. et al., J.Fharm.Pharmaco1. 13,389
(1961).
66. Tattje,D.H.E., J.Pharm.Pharmaco1, 6,476(1954).
67. Jakovljevic,I.M., Anal.Chem. 35(10),1513
(1963).

171
 I V A N M. JAKOVLJEVIC

68. Dyes,H.P., Pharm.Weekblad 95,682 (1960).

69, Bors,G. et al., Farmacia (Bucharest) 15(5),
269 (1967).
70. Cullin, L.F. et al. , J.Pharm.Sci. -
59(5),697
(1970).
71. Frey,M.J. et al., Anal.Biochem. 36,78 (1970).
72. Henderson,F.G., Digitalis, G r u n e E Stratton
Publ.,New York-London 1969.
73. Wright,S.E., The Metabolism of Cardiac Glyco-
sides, Charles C. Thomas Publ. 1960.
74. Fukuda,H. et al,, J.Pharm.Soc.Japan 89(6),
866 (1969).

***********
During the preparation of these analytical profiles,
the following, most recent, papers on different topics of
digitoxin have been found:
1- Gisvold,O.
Acetyldigitoxin and acetyldigoxin from Digitalis lana-
ta.
J.Pnarm.Sci. - 6 1 , 1320 (1972).
2- suss, w.
Extraction of digitalis leaves with the aid of ultra-
sonics.
Pharmazie - 27, 615 (1972).
3- Watson, E. et al.
Identification of submicrogram amounts of digoxin, di-
gitoxin ..... ..
Isolation by chromatography.,
J. Chromatogr. - 69, 157 (1972).
4- Potter, H. et al.
TLC analysis of digitalis glycosides.
Pharmazie - 27, 315 (1972).
5- Butler, V.P.,Jr.
Assay of digitalis in the blood.
Prog.Cardiovasc.Dis. - 14, 571 (1972).
6- Bodem, G. et al.
Determination of digoxin and digitoxin in the blood.. .
Klin.Wochenschr. - 51, 57 (1973).
THE L l T E R A T U R E SEARCff WAS CONDUCTEV UP TO MAY 1 9 7 3 .

172
DIPHENHYDRAMINE HYDROCHLORIDE

Ira J. Holcomb and Salvatore A . Fusari

 IRA J. HOLCOMB AND SALVATORE A. FUSARI

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
2 -3 ultraviolet Spectrum
2 -4 Mass Spectrum
2 -5 optical Rotation
2 .6 Me Iting Range
2 -7 Differential Thermal Analysis
2 .8 Solubility
2.9 crystal Properties
2.91 optical crystal Properties
2.92 X-Ray Diffraction

2.10 Distribution Coefficients

2.11 Aggregation: Micelle Formation
2.12 PK; values
2.13 Metal Complex Formation and Binding

3. Synthesis

4. Stability - Degradation

5. Drug Metabolic Products - Pharmacokinetics

6. Identification: Microchemical Tests

7. Methods of Analysis

7.1 Elemental Analysis

7.2 Spectrophotometric Analysis

174
 DIPHENHYDRAMINE HYDROCHLORIDE

7.21 Elemental Analysis

7.22 Separation Methods Prior to
Spectrophotometric Assay
7.23 Methods Based on Conversion to
Benzophenone Prior to Spectro-
photometric Assay
7.24 Method Based on Conversion to
Chloranilic Acid Prior to
Spectrophotometric Assay

7.3 Colorimetric Analysis

7.31 Ion-Pair Extraction Methods
7.32 Ammonium Reineckate Methods
7.33 Picric Acid Method
7.34 Method Based on Molle Reaction
7.35 Miscellaneous Colorimetric Methods

7.4 Titrimetric Analysis

7 . 4 1 Direct Methods of Titration
7.42 Separation Prior to Titration
7.421 Reineckate salt Formation
7.422 Complexometric Method
7.423 Slurry Method
7.424 Ion Exchange Method
7.425 Extraction Method
7.43 Miscellaneous Titrimetric Methods

7.5 Fluorometric Analysis

7.6 Automated Analysis
7.7 Biological Assay
7.8 Gravimetric Analysis
7.9 Chromatography
7.91 Paper Chromatography
7.92 Thin Layer chromatography
7.93 Gas Chromatography

175
 I R A J. HOLCOMB A N D SALVATORE A . FUSARI

7.931 D i r e c t Methods on
N e u t r a l columns
7.932 D i r e c t Methods on
B a s i c columns
7.933 o x i d a t i o n to Benzophenone
P r i o r t o G a s Chromato-
graphy
7.94 Column c h r o m a t o g r a p h y
7.95 Electrophoresis

8.0 References

176
 DIPHENHYDRAMINE HYDROCHLORIDE

1. Description
1.1 N a m e , Formula, Molecular Weiqht
Diphenhydramine h y d r o c h l o r i d e i s 2-
(dipheny1methoxy)-N,N-dimethylethylamine hydro-
c h l o r i d e -1 S i x a d d i t i o n a l chemical names are
l i s t e d i n The Merck Index2, along w i t h twelve
t r a d e names. One a d d i t i o n a l name i s Dimedrol.

The e m p i r i c a l formula i s C17H21NO'HCl

w i t h a molecular weight of 291.82.

. HC1

The CAS R e g i s t r y Number is 58-73-1 for

2- (diphenylmethoxy) -N, N-dimethylethylamine and
f o r t h e h y d r o c h l o r i d e , 147-24-0.

1.2 Appearance , c o l o r , odor

White, o d o r l e s s , c r y s t a l l i n e powder. 1

2. Physical Properties
2.1 I n f r a r e d Spectrum
The i n f r a r e d spectrum of diphenhy-
dramine h y d r o c h l o r i d e i s p r e s e n t e d i n F i g u r e 1.
The S a d t l e r Reference Number i s 9382. The
spectrum is used f o r c o n t r o l pur o s e s . 1 i 3
S p e c t r a a r e p r e s e n t e d by de ~ o o'I4 s and W a l l a c e . 5

The i n f r a r e d band assignments a r e

g i v e n i n Table I .

177
 Y

-
C
0
.-
c

F r e q u e n c y (cm-1)

Fig. 1. I n f r a r e d Spectrum o f Diphenhydramine H y d r o c h l o r i d e , U.S.P. Parke-Davis 8 Co.

Lot No. 5 6 3 4 6 3 . Instrument: Perkin-Elmer 621, Phase: KBr, 1:300.
 c
0 u-4
-I4 0
4J
a c
& 0
h Q
ul -rl
m 3
0
m rl
a
4J
aJ
Ec, rl
aJ
tn aJa
aJ
&
U
% c
.d
G @
m u
in tn
rl
V
c
-4 F F
%4J .d
3: & c c a,
.I4 k
N a a 4J
h E: E: m
Q) al
id id n n U
I
E
0 0
k
m
Z
1
k
m
i?
u u
I
179
 IRA J. HOLCOMB AND SALVATORE A. FUSARI

2.2 Nuclear Maqnetic Resonance

In Figure 2 the nuclear magnetic
resonance spectrum of diphenhydramine hydro-
chloride is presented. The spectral peak
assignments7 are presented in Table 11. The
Sadtler NMR Reference Number is 14360.

2.3 ultraviolet Spectrum

The ultraviolet spectrum of diphenhy-
dramine hydrochloride is presented in Figure 3.
The absorptivities at 258 nm. listed in Table
111 gompare well with the literature values of
15.4 in methanol and 16.59 in an aqueous system.

2.4 Mass Spectrum, LOW Resolution11

A plot of the relative intensities vs.
mass/charge ratio is presented in Figure 4 and
summarized in Table IV. The ionization potential
is 70 electron volts.

2.5 Optical Rotation

Diphenhydramine hydrochloride is not
optically active.

2.6 Melting Range

Diphenhydramine hydrochloride melts in
the range 167O to 172°C.’ The actual range in
which the compound melts is usually less than
2OC. The melting point is affected by the rate
of heating1* as shown in Table v.

Data was obtained from melting point

scans using the Mettler FP-1.

180
c
W
c

Fig. 2. Nuclear Magnetic Resonance Spectrum of Diphenhydramine Hydrochloride, U.S.P.,

Parka-Davis & Co. l o t No. 5 6 3 4 6 3 .
Instrument: V a r i a n A-60. Solvent. D20. Sweep Offset: 0 cps.
x7 167 &i
u
Y0Y I
0
I
0
d
m
. In
?
m

m
?‘c!
182
Fig. 3. Ultraviolet Spectrum of Diphenhydramine H y d r o c h l o r i d e , U.S.P., Parke-Davis & Co.
Lot N O . 5 6 3 4 6 3 . Instrument: Cory 14.
 IRA J. HOLCOMB A N D SALVATORE A . FUSARI

Table 111. Absorptivities'O of

D iphenhydr amine Hydrochloride ,
Parke, Davis & Co., Lot N o . 563463

Aqueous Medium (pH 3):

wavelength a (l%, 1 cm.) -

e
267 nm(s) 9.3 270

263 nm(s) 12.7 370

257.5 nm(s) 16.3 476

252 nm 13.95 406

Methanol, absolute:

268 nm 7.95 232

264 nm 12.1 353

258 nm 15.5 452

252 nm 12.8 374

184
 W

BC

--
K
0
Y
70

C
8 60
-u
C
a
n
* 5 0
.-+
>
-:
0
4c

30

1
Lr/lA
I XI0 Xloo
m

lc

200 250

Fig. 4. Mass Spoctrum of Diphenhydramine Hydrochloride, U.S.P., Parke-Davis & CO. Lot NO. 563463.
lnstru men t: F i n n ig a n Q u a dr u pol e Mass Spectr o mete r, M ode I 1015.
 IRA J. HOLCOMB AND SALVATORE A. FUSARI

Table Iv
Low R e s o l u t i o n Mass Spectrum Assignments
f o r Diphenhydramine Hydrochloride

Measured Relative
Mass I n t e n s ity S t r u c t u r a l Assignments

H
256 10.67 -CH2-N ,CHCH3
3
4-

4-
183 10 - 0 ( O \ H
0 C13H110
(oyc-
+

1 67 30 .O
C13Hll

a
+

165 52.67 C13H9

152 22.00pJ-$J1+ C12H8

186
 DIPHENHYDRAMINE HYDROCHLORIDE

Table Iv( cont .)

Measured Relative
Mass Intensity S t r u c t u r a l Assiqnments

58 100 C3H8N

45 12 2H7

187
 Table V. M e l t i n g P o i n t and R a n g e 1 2 of D i p h e n h y d r a m i n e

Hydrochloride, Parke, D a v i s & co ., Lot No. 563463

S t a r t Temp. ( O c .) Heating Rate Ranqe Mid-Point

163 1OC/min. 167 -7-168-6(0-9) 168.1

167 -7-168-7(1- 0 ) 168.2

158 O 3 OC/min. 168 -7-169.3 ( 0 -6) 169 -0

168 -7-170-0(1- 3 ) 169 -4
 DIPHENHYDRAMINE HYDROCHLORIDE

2.7 D i f f e r e n t i a l Thermal A n a l y s i s
The DTA curve o b t a i n e d u s i n g a M e t t l e r
TA 2000 is shown i n F i g u r e 5 . The p e r c e n t p u r i t y
found f o r t h e sample, Diphenhydramine Hydro-
c h l o r i d e , USP, Parke, Davis & c o . , Lot N o .
593125, i s 99 .62%.13

2.8 Solubility
The s o l u b i l i t y of diphenhydramine i n
w a t e r i s 0.7 rng./m1.lo S o l u b i l i t i e s of diphen-
hydramine h y d r o c h l o r i d e have been determined14
and a r e p r e s e n t e d i n Table VI.

2.9 c r y s t a l Properties
The o p t i c a l c r y s t a l l o r a p h i c c o n s t a n t s
have been r e p o r t e d by Keenan .'? Diphenhydramine
h y d r o c h l o r i d e i s d e s c r i b e d as c o l o r l e s s , m o s t l y
s i x - s i d e d p l a t e s w i t h l e n g t h w i s e c l e a v a g e . The
n20 v a l u e s are: a , 1 . 6 0 2 ; p , 1 . 6 2 5 : and y , 1.630;
a f l 5 0.002. I n p a r a l l e l p o l a r i z e d l i g h t , ex-
t i n c t i o n is p a r a l l e l and t h e s i g n of e l o n g a t i o n
i s n e g a t i v e . S h e l l 1 6 a l s o r e p o r t e d on o p t i c a l
c r y s t a l l o g r a p h i c p r o p e r t i e s and gave t h e d e n s i t y
a s 1.189.

2.92 x-Ray D i f f r a c t i o n
The X-Ray d i f f r a c t i o n d a t a f o r
diphenh dramine h y d r o c h l o r i d e were r e p o r t e d by
Gadret''. The compound h a s been run by Krcl*
and t h e d i f f r a c t i o n p a t t e r n i s p r e s e n t e d i n
Figure 6 .

The c a l c u l a t e d I'd spacings18

I'

f o r t h e d i f f r a c t i o n p a t t e r n a r e g i v e n i n Table
VII. The 28 a n g l e s were c o r r e c t e d on t h e
d i f f r a c t i o n p a t t e r n u s i n g known v a l u e s f o r
c a l c i t e added t o a sample.
Fig. 5 Diphenhydramine H y d r o c h l o r i d e , D.T.A Curve, Parke-Davis & CO. Lot NO. 593125
Instrument: M e t t l e r T A 2 0 0 0 AHz7.495 kcal. M e l t i n g p o i n t : 1 6 8 . 3 O C .
 DIPHENHYDRAMINE HYDROCHLORIDE

Table V I . S o l u b i l i t y 1 4 of Diphenhydramine

Hydrochloride i n Various S o l v e n t s

Solubility,
Solvent mq ./ml.

Water 858

Methanol 599

A l c o h o l , 95% 408

Chloroform 3 94

Isopropyl Alcohol 35

Acetone 16

191
-2z
0
z
c
C
m
E
2
u
a
c
C
 DlPH EN H Y DRAMIN E HYDROCHLORIDE

Table VII . Diphenhydramine Hydrochloride :

calculated 'Id" Spacings and I/I, Values

Radiation: C u G , h 1.5418

Filter: Ni

d (Ao)

8.56 7 2.86 4
7.97 (1 2.78 1
7.19 10 0 2 -75 (1
6.74 2 2.70 1
5.70 (1 2.60 2
5.42 5 2.58 (1
5.30 (1 2 -53 (1
4.78 13 2.46 (1
4.59 5 2.42 (1
4.37 20 2.29 1
4.05 24 2.19 1
3.85 2 1.98 1
3.59 12
3.40 7 d 7.19 4.05 4.37 8.56
3.35 l1 1/11 100 24 20 7
3 -33 6
2.98 4
2.91 3

193
 IRA J. HOLCOME AND SALVATORE A . FUSARI

2.10 Distribution Coefficients

Doyle” h a s determined t h e d i s t r i b u t i o n
b e h a v i o r o f - diphenhydramine h y d r o c h l o r i d e i n
ch1oroform:water and e t h e r :water. A l o g a r i t h m i c
d i s t r i b u t i o n diagram i s p r e s e n t e d i n t h e s t u d y
f o r s e l e c t i o n of p a r t i t i o n chromatographic
systems . I 9 The d i s t r i b u t i o n of diphenhydramine
h y d r o c h l o r i d e between ch1oroform:water as a
f u n c t i o n of aqueous p - t o l u e n e s u l f o n i c a c i d w a s
a l s o s t u d i e d by Doyle .20 The s o l v e n t composition
e f f e c t on t h e p a r t i t i o n of amines, i n g e n e r a l ,
h a s been s t u d i e d . 2 1

Konyushko22 examined t h e e f f e c t of pH
on t h e d i s t r i b u t i o n of diphenhydramine between
water and chloroform. The e x t r a c t i o n of diphen-
hydramine w i t h chloroform i n p r e s e n c e of W F ,
KF, K C 1 , KBr, K I and KSCN a s s a l t i n o u t a g e n t s
a t 20° and pH 3 h a s been r e p o r t e d . 29

Aggregation - M i c e l l e Formation

’‘
2.11
Diphenhydramin h y d r o c h l o r i d e forms
aggregates i n s o l u t i o n . Attwood2’ h a s deter-
mined t h e c r i t i c a l m i c e l l e c o n c e n t r a t i o n u s i n g
s c a t t e r i n g a t a n a n g l e of 9 0 ° t o t h e i n c i d e n t
beam and determining t h e i n f l e c t i o n p o i n t s i n
t h e p l o t v e r s u s t h e molal c o n c e n t r a t i o n .

2.12 PKA v a l u e s
AndrewsLo determined t h e i o n i z a t i o n
c o n s t a n t of diphenhydramine h y d r o c h l o r i d e a t 0 O ,
p d = 9.67, and 25 O , pKA = 9.12 i n w a t e r . These
v a l u e s compare w e l l w i t h t h o s e o b t a i n e d by
L ~ r d oi f ~pK~ A = 9.00 i n water. deRoos28 h a s
determined t h e p& a t 2 0 ° t o b e 9.06 i n w a t e r .

194
 DIPHENHYDRAMINE HYDROCHLORIDE

The pK4 of Diphenhydramine H d r o c h l o r i d e ,

USP, Lot 563463, h a s been determined2S; i n a
water:methanol (1:1) system t o be 8 . 4 . Since
t h e p& v a r i e s s l i g h t l y w i t h t h e a l c o h o l c o n t e n t ,
the value obtained i s acceptable.

2.13 Metal complex Formation and Binding

Diphenhydramine h y d r o c h l o r i d e forms
complexes26 w i t h v a r i o u s m e t a l i o n s such as
Cu++, co++, and N i + + .

Evidence f o r i n t e r a c t i o n of diphenhy-
dramine h y d r o c h l o r i d e w i t h s t rene-maleic30 and
sodium carboxymethylcelluloseYl h a s been r e p o r t e d .

3. Synthesis
The f i r s t method p a t e n t e d f o r t h e
32
s y n t h e s i s of diphenhydramine w a s by R i e v e s c h l
i n 1947, a s s i g n e d t o Parke, Davis & C o . The
g e n e r a l method i n v o l v e s t h e r e a c t i o n of bromo-
diphenylmethane w i t h t h e a p p r o p r i a t e d i a l k y l a m i n o
a l c o h o l i n t h e p r e s e n c e of anhydrous sodium
c a r b o n a t e . The d i a l k y l a m i n o a l c o h o l used i s
dimethylamino e t h a n o l (see F i g u r e 7 ) . The
diphenhydramine b a s e t h a t i s formed i s t h e n
converted t o t h e H C 1 s a l t .

A v a r i e t y o f s y n t h e t i c methods have
appeared i n t h e l i t e r a t u r e . 3 3 , 34, 35 I n t h e
m a j o r i t y o f methods, t h e b a s e , diphenhydramine
i s formed f i r s t and t h e n c o n v e r t e d t o t h e hydro-
chloride. I n some i n s t a n c e s , diphenhydramine
h y d r o c h l o r i d e may be formed d i r e c t l y by re-
arrangement of a q u a t e r n a r y ammonium s a l t 3 6 , 37
( s e e F i g u r e 8 ) . The f r e e b a s e can a l s o be
formed as t h e r e s u l t of a d e c a r b o x y l a t i o n
reaction38 (Figure 9 ) .

195
 IRA J. HOLCOMB AND SALVATORE A. FUSARI

QCHZ + Brz
I
- hv

Q
H C - O C H ~ C H ~-
I
7
HCI
H C - 0 -cH~-cH~-N',
CH3

(b2 150-165')

Fig. 7. Synthesis of Diphenhydramine H y d r o c h l o r i d e .

196
 u
2
\ /
U
I
u
N

z
u
I
m
I97

-
?
U
Fig. 8. Synthesis of D i p h e n h y d r a m i n e Hydrochloride: R e a r r a n g e m e n t R e a c t i o n
 \ 230° -300° c
CHzC~-O-C-COzNa
/
A

AH3
HC-0-CHZCHZ-N HCI
/ ‘CY

Fig. 9 . Synthesis of Diphenhydramine Hydrochloride : Decarboxylation Reaction

 DIPHENHY DRAMINE HYDROCHLORIDE

4. stability - Deqradation
The earliest published detailed work on
the stability - decomposition o diphenhydramine
hydrochloride is that of Nogami59 in 1961. The
kinetics of the decomposition was examined in
an acidic and alkaline medium. In an acidic
medium, diphenhydramine undergoes fairly rapid
decomposition, whereas the compound is fairly
stable in an alkaline solution. The decomposition
in an acid medium is due to hydrolysis of the
ether linkage. The rate determining step is
first order and catalyzed by hydrogen ion. The
principle degradation products are benzhydrol
and 2- (dimethylamino) ethanol.

Earlier observations on the decomposition

of diphenhydramine hydrochloride were in relation
to the effect of h drogen peroxide4 0 t 41and
ultraviolet light43 on the compound. The de-
composition products with hydrogen peroxide are
toluene, benzophenone, benzyl alcohol, benzoic
acid and phenolic substances in addition to
dimethylaminoethanol. The benzhydrol under the
conditions used undergoes further reactions.
Under ultraviolet irradiation, the principle
decomposition products are benzhydrol and di-
methylaminoethanol .

The work by Nogami3’ on the stability

of diphenhydramine hydrochloride was confirmed
in part by d e R ~ o sin~ 1963
~ in a study on the
stability of the ether bond in a series of
benzhydryl ethers.

199
 I R A J. HOLCOME A N D SALVATORE A. FUSARI

5. Drug M e t a b o l i c P r o d u c t s - Pharmacokinetics
I n t h e i n i t i a l work by Glazko and co-
workers 44, 45 on t h e m e t a b o l i c f a t e o f diphen-
hydramine h y d r o c h l o r i d e , r a t s and g u i n e a p i g s
w e r e examined a t d e f i n i t e t i m e s a f t e r sub-
cutaneous i n j e c t i o n s . The h i g h e s t c o n c e n t r a t i o n s
of diphenhydramine w e r e found i n t h e l u n g s , w i t h
lower c o n c e n t r a t i o n s i n t h e s p l e e n , l i v e r and
muscles. Peak c o n c e n t r a t i o n s occurred i n about
one hour w i t h a f a i r l y r a p i d drop o v e r a s i x
hour p e r i o d . Diphenhydramine w a s demonstrated
i n human u r i n e i n s m a l l amounts by e x t r a c t i o n
and u l t r a v i o l e t a b s o r p t i o n .

The r e s u l t s o b t a i n e d u s i n g r a d i o a c t i v e
carbon i n c o r p o r a t e d i n t o t h e ci p o s i t i o n of t h e
benzhydryl group of diphenhydramine a g r e e w i t h
t h e chemical a n a l y s i s . 4 5 I n r a t s t h e maximum
r a t e of e x c r e t i o n occurred i n t h e f i r s t seven
h o u r s . Radioautographs p r e p a r e d from u r i n e
samples showed a t l e a s t s i x d i f f e r e n t r a d i o -
a c t i v e compounds p r e s e n t , one of which was
d iphe nh yd r a m i n e .
K i k k a ~ ai d~e~n t i f i e d benzhydrol and dimethyl-
aminoethanol a s m e t a b o l i c p r o d u c t s i n v i t r o and
i n v i v o . An a c i d i c compound w a s a l s o d e t e c t e d ,
b u t not i d e n t i f i e d .
D r a ~ h 48 ~ ~u,s i n g t r i t i u m l a b e l e d
diphenhydramine i n r h e s u s monkey plasma found
t h e major m e t a b o l i t e t o be a deaminated c a r -
b o x y l i c a c i d d e r i v a t i v e of diphenhydramine,
(diphenylmethoxy) a c e t i c a c i d . The a c i d , t h e
mono- and d i - d e a l y k y l a t e d d e r i v a t i v e s of diphen-
hydramine and t h e N-oxide d e r i v a t i v e w e r e
i d e n t i f i e d c h r o m a t o g r a p h i c a l l y . The diphenyl-

200
 DIPHENHY DRAMINE HY DROCHLORI DE

methoxyacetic a c i d i s e x c r e t e d a s t h e g l u t a m i n e
conjugate.

Kinke14’ examined plasma l e v e l s of

diphenhydramine a f t e r s i n g l e dose o r a l adminis-
t r a t i o n of diphenhydramine h y d r o c h l o r i d e c a p s u l e s
a t d i f f e r e n t l e v e l s i n human v o l u n t e e r s . Peak
plasma l e v e l s were o b t a i n e d 2 t o 3 h o u r s follow-
i n g t h e dose. I n a m u l t i p l e dose s t u d y , a
r e l a t i v e l y c o n s t a n t plasma l e v e l i s o b t a i n e d
a f t e r t h r e e days.
50
A d d i t i o n a l work h a s been done on t h e
m e t a b o l i t e s i n r e l a t i o n t o d i f f e r e n c e s observed
between s p e c i e s . The major m e t a b o l i t e i n a l l
s p e c i e s s t u d i e d , e x c e p t t h e r a t , w a s (diphenyl-
methoxy) a c e t i c a c i d . T h i s m e t a b o l i t e i s con-
j u g a t e d w i t h glutamine i n t h e monkey and g l y c i n e
i n t h e dog.

6. I d e n t i f i c a t i o n : Microchemical T e s t s
The f i r s t c o l l e c t i o n o f microchemical
t e s t s f o r d e t e c t i o n and i d e n t i f i c a t i o n of
diphenhydramine w a s d e s c r i b e d by ~ a l e y ’ l i n 1948.
The r e a c t i o n s and r e s u l t s are summarized i n
Table V I I I .

~ o l l dee s~c r ~
i b e d an a d d i t i o n a l c o l o r
r e a c t i o n i n 1950 i n which t h e r e a g e n t , H2SO4,
90%, and HNO3, lo%, r e a c t s t o g i v e a r e d - v i o l e t
c o l o r changing slowly t o y e l l o w . The r e s u l t i n g
mixture i s d i l u t e d w i t h w a t e r t o g i v e an orange-
yellow c o l o r and t h e n t h e t u r b i d m i x t u r e becomes
a violet-rose. chloroform i s added and mixed
w e l l : the s e p a r a t e d chloroform layer i s v i o l e t
and t h e aqueous l a y e r becomes c o l o r l e s s . The
c o l o r r e a c t i o n i s due t o b e n z h y d r o l .

201
 Table V I I I . Identification of Diphenhydramine

Hydrochloride: Microchemical Tests 51

Observation

Definite crystalline form

Mandelin reagent Red oily globules

Marquis reagent Canary yellow, reddish-orange then

h)
0 chocolate brown
h)

Mecke reagent canary yellow then reddish-orange

Frohde reagent Canary yellow to orange-red

H2SO4, conc. orange

K2Cr207- H2SO4 Yellow

Resorcinal - H2SO4 Orange then reddish-orange and wine color

on dilution

Furfural, 1%, over H2S04 Orange brown and yellow-green on shaking

 Table VIII (cont -)
Reagent observation

chromic a c i d , 5% orange-red precipitate

Foucery r e a g e n t Cherr y-r e d

Name Reagent Compositions:

Mandelin r e a g e n t Ammonium v a n a d a t e , 1 g . i n LOO m l .

N
0
concentrated s u l f u r i c aci d
W

Marquis r e a g e n t s u l f u r i c acid-formaldehyde. Two m l . of a

40% s o l u t i o n of formaldehyde mixed w i t h
45 m l . of w a t e r and 5 5 ml. of c o n c e n t r a t e d
sulfuric acid

Mecke r e a g e n t .
Selenous a c i d , 0 . 5 g , i n s u l f u r i c a c i d , 100
ml
a

Frohde r e a g e n t Ammonium molybdate, 0 .l% i n c o n c e n t r a t e d

s u l f u r i c acid

Foucery r e a g e n t Quinone, 1 g . in acetic a c i d : a l c o h o l ( S :100)

 IRA J. HOLCOMB A N D SALVATORE A. FUSARI

A d d i t i o n a l microchemical tests have been

described by A ~ t e r h o f f ~ 0 ~~, 0 and 1 ~N ~e ~ h o f *f ~ ~
Clarke56 i n 1957 gave, i n a d d i t i o n t o observa-
t i o n s , t h e s e n s i t i v i t y of t h e t e s t s used (Table
1x1.

Clarke57 h a s l d e s c r i b e d a method f o r t h e
r a p i d d e t e c t i o n of b a s i c drugs i n u r i n e t h a t
u t i l i z e s many o f t h e r e a c t i o n s d e s c r i b e d i n
procedures t h a t require a m i n i m u m of equipment.

A d d i t i o n a l r e a g e n t s which form c r y s t a l -
l i n e p r e c i p i t a t e s w i t h diphenhydramine are
f l a v i a n i c acid58, 4 , 4 I -dibromodibenzenesulf on-
amide59, and 8 h droxy-7-iodoquinoline-5-
s u l p h o n i c a c i d-68
.

7. Methods of A n a l y s i s

7.1 Elemental A n a l y s i s
The e l e m e n t a l a n a l y s i s of Diphenhydramine
Hydrochloride, USP, Lot 563463, i s p r e s e n t e d
below :
Element % calculated ReportedG1
C 69.97 69.99
H 7.60 7.56
N 4.80 4.84
c1 12.15 12.09

7.2 S p e c t r o p h o t o m e t r i c Assay

7.21 D i r e c t Methods
Methods i n which t h e sample i s ~

d i l u t e d t o the proper c o n c e n t r a t i o n for absorbance

r e a d i n g i n t h e u l t r a v i o l e t r e g i o n have been
r e p o r t e d by S e t n i k e r 6 2 and D e m i r 6 3 . Corrections
using orthogonal functions f o r i r r e v e l a n t

204
 T a b l e IX. Microchemical Identification Tests

and Sensitivity 5 6

Reagent Observations Sensitivity

Gold bromide/HCl Needles, some curved 0.1 pg

Potassium triiodide plates 0.1 p g

Formaldehyde-sulfuric
acid (Marquis) yellow color 0.1 pLJ

Ammonium vanadate ye 1l o w 0.1 w

Ammonium molybdate ye 1low 0.1 pg

Selenium dioxide yellow 0.1 pg

 I R A J. HOLCOMB AND SALVATORE A. FUSARI

absorption in tablets64 has been applied with a

recovery of 99.2 2 1.8%.

7.22 Separation Methods Prior to

Spectrophotometric Assa
column chromatography 0 : alumina65
thin layer chromatography with an alumina layer66,
direct extraction from a basic solution67, ion
exchange chromatography68, and extraction from a
5% hydrochloric acid ~ o l u t i o n ~7o
~ rhave been
used prior to the spectrophotometric assay.

7.23 Methods Based on Conversion to

~enzophenonePrior to Spectro-
photometric As say
Diphenhydramine hydrochloride is
oxidized to benzophenone b either dichromate in
a sulfuric acid medium’’, y2 or permanganate in
a basic medium73. The benzophenone is either
steam distilled or separated by extraction into
hexane or heptane and determined spectrophoto-
metrically.

7.24 Conversion to Chloranilic Acid

Prior to Spectrophotometric Assay
Diphenhydramine has been deter-
mined in drugs by conversion to chloranil with
FeC13 in hydrochloric acid and hydrogen peroxide74 .
The chloranil formed is extracted and hydrolyzed
to chloranilic acid (2,5-dichloro-3,6-dihydroxy-
p-benzoquinone) with potassium hydroxide. The
absorbance measured at 331 nm. The conversion
to chloranilic acid is constant, but not 100%.

206
 DIPHENHY DRAMlNE HYDROCHLORIDE

7.3
Colorimetric Assay
7.31 lon-Pair Extraction Methods
The method commonly used for
routine control procedures is based on the
extraction of diphenhydramine with methyl orange
into chloroform. The procedure was initially
studied by Dill and G l a z k ~for
~ ~use in the
determination of diphenhydramine in body tissues.
A recent modification involves the addition of
methanol after the complex is completely ex-
tracted into chloroform to prevent adsorption of
the methyl orange-diphenhydramine ion-pair onto
the walls of the f l a ~ k 7 ~ .

Other dyes that have been used

for the colorimetric assay are bromocresol
green77 bromocresol p ~ r p l e 7 ~bromothymol
I , blue78,
er iochrome blue SE79 I tetrabromophenolphthalein
ethyl ester*O and eosin811 8 2 .

7.32Ammonium Reineckate Methods

Bandelin83 separated diphenhy-
dramine as the reineckate salt followed by
solution of the salt in acetone and colorimetric
estimation at 525 nm. A very comprehensive
paper on the identification and determination
of nitrogenous bases with ammonium reineckate was
presented by K U ~ n - T a t tin~ ~ which the mole com-
position of diphenhydramine reineckate is given
. salt decomposes at 178 -
as ~ 2 1 ~ 2 8 c r ~ 7 O S qThe
180O .
7.33
Picric Acid Method
Picric acid has been used for the
colorimetric determination of diphenhydramine in
the urine of rabbits and man85.

207
 IRA J. HOLCOMB AND SALVATORE A. FUSARI

7.34 Method based on Molle R e a c t i o n

Horn86 examined s e v e r a l d i f f e r e n t
procedures f o r t h e d e t e r m i n a t i o n of diphen-
hydramine h y d r o c h l o r i d e , one o f which i s based
on t h e M O l h r e a c t i o n d e s c r i b e d e a r l i e r . The
compound i s r e a c t e d w i t h a mixture o f s u l f u r i c
a c i d and n i t r i c a c i d ( 9 : 1 ) , d i l u t e d w i t h w a t e r
and t h e c o l o r e d compound formed e x t r a c t e d w i t h
chloroform. The absorbance of t h e chloroform
l a y e r w a s t h e n determined w i t h a f i l t e r t y p e
instrument.

7.35 Miscellaneous C o l o r i m e t r i c Methods

D iphe nh ydr a m i n e h a s been d e t e r -
mined by e x t r a c t i o n of a chloroform s o l u b l e
complex w i t h c o b a l t t h i o c y a n a t e 8 7 , 88, *9.
Diphenhydramine reacts i n a 2 : l mole r a t i o and
i n chloroform i s measured i n t h e r e g i o n 590 t o
625 nm.

Diphenhydramine can be e x t r a c t e d
from an acetate b u f f e r , p H 5 , c o n t a i n i n g i o d i d e
w i t h a 0.5% I 2 s o l u t i o n i n e t h y l e n e d i c h l o r i d e g O .

c o n d i t i o n s f o r t h e complex forma-
t i o n of diphenhydramine w i t h H ( T 1 Br4) have been
examined w i t h subsequent d i s p l a c e m e n t b y
b r i l l i a n t green”.

The c o l o r r e a c t i o n w i t h d i e t h y l
o x a l a t e g 2 and t h i o b a r b i t u r i c g 2 acid w a s used t o
determine diphenhydramine i n p i l l s g 3 .

7.4 T i t r i m e t r i c Analysis
7.41 D i r e c t Methods of T i t r a t i o n
The o f f i c i a l methodl f o r t h e
a s s a y of diphenhydramine h y d r o c h l o r i d e i s by

208
 DIPHENHYDRAM INE HYDROCHLORIDE

nonaqueous t i t r a t i o n w i t h 0.1N p e r c h l o r i c a c i d i n
t h e p r e s e n c e of mercury (11) a c e t a t e u s i n g c r y s t a l
violet as indicator.

Work on t h e nona ueous t i t r a t i o n

method w a s r e p o r t e d by Ekablad" and a c e t o n i t r i l e
w a s examined as a s o l v e n t by Mainvi1leg5.
S e v e r a l a c i d s a l t s of diphenhydramine were
t i t r a t e d w i t h p e r c h l o r i c acid i n g l a c i a l a c e t i c
a c i d using t h e glass-glass retarded potentio-
m e t r i c method f o r d e t e c t i o n o f t h e e n d p o i n t g 6 .
The endpoint h a s been d e t e c t e d conductimet-
r i c a l l y g 7 . Diphenhydramine and a c i d s a l t s can
be t i t r a t e d d i r e c t l y i n anhydrous p r o p i o n i c
acid with p e r c h l o r i c a c i d using glass-calomel
electrodes98.

Diphenhydramine can a l s o be d e t e r -
mined u s i n g a 0.004M s o l u t i o n of sodium l a u r y l
s u l f a t e o r sodium d i o c t y l s u l f o s u c c i n a t e a s t h e
t i t r a n t g g , 100. The r a t i o of t h e a n i o n i c s u r f a c e -
a c t i v e agent t o t h e base i s not i n t e g r a l , but
approximate and c o n s t a n t .

7.42 separations prior t o Titration

7.421 Reineckate S a l t Formation
The r e i n e c k a t e of diphenhydramine
may be decomposed by h e a t i n g i n an a l k a l i n e
medium and a Volhard t i t r a t i o n performed t o deter-
mine t h e t h i o c y a n a t e c o n t e n t
#' ' ' l o 2 . The s a l t
formed may a l s o be determined b r o m a t ~ m e t r i c a l l y ~ ~ ~ ,
r e s u l t s are about 5% low. The chromium (111)
c o n t e n t c a n b e determined w i t h v e r y ood a c c u r a c y
f 0.5% u s i n g a c h e l a t o m e t r i c method 184 .

209
 IRA J. HOLCOMB A N D SALVATORE A. FUSARI

7.422 Complexometric Method

Diphenhydramine forms an
i n s o l u b l e s a l t w i t h bismuth which, from t h e
r e a g e n t r e p a r e d , releases an e q u i v a l e n t amount
of EDTA1g5 . The l i b e r a t e d EDTA i s t i t r a t e d w i t h
0.lM ZnSO4 a t pH 9.1 u s i n g eriochrome b l a c k T
as i n d i c a t o r .

7.423 S l u r r y Method
c 1a ir l u b and c h a t t e n l O 7
p r e s e n t e d s l u r r y methods f o r t h e s e p a r a t i o n of
a n t i h i s t a m i n e s f r o m t a b l e t o r c a p s u l e material.
The sample i s simply s l u r r i e d w i t h c h l o r o f o r m
and f i l t e r e d . The f i l t r a t e i s t i t r a t e d w i t h
acetous perchloric acid a f t e r g l a c i a l a c e t i c
a c i d i s added. Tuckermanlo8 used a m i x t u r e of
magnesium o x i d e and s i l i c e o u s e a r t h for pre-
t r e a t m e n t of a n aqueous i n j e c t i o n followed by
washing w i t h warm chloroform i n t o g l a c i a l a c e t i c
a c i d . T h e base is then t i t r a t e d w i t h 0.1N per-
c h l o r i c a c i d u s i n g p-naphtholbenzein as i n d i c a t o r .

7.424 Ion Exchanqe Method

Ion exchange columnsl09, 110
have been used i n t h e d e t e r m i n a t i o n o f a n t i -
h i s t a m i n e s w i t h subsequent t i t r a t i o n of t h e
effluent .
7.425 E x t r a c t i o n Method
A c o l l a b o r a t i v e s t u d y on
t h e e x t r a c t i o n method was r e p o r t e d by H e i m l l l .
The f r e e base i s e x t r a c t e d w i t h e t h e r and d e t e r -
mined by t i t r a t i o n . R e c o v e r i e s w e r e 99-101%.

Miscellaneous T i t r i m e t i c Methods
7.43
p-Toluenesulfonic a c i d i n c h l o r o -
form112 and m e t h a n e s u l f o n i c a c i d i n g l a c i a l

210
 DIPHENHYDRAMINE HYDROCHLORIDE

a c e t i c a c i d l l 3 have been used as t i t r a n t s f o r

diphenhydramine w i t h v i s u a l i n d i c a t o r s In
aqueous s o l u t i o n , s i l i c o t u n g s t i c a c i d l i 4 w i t h
m e t a n i l yellow or con o r e d as i n d i c a t o r w a s
recommended by ~ramml” and t h e compound h a s
a l s o been t i t r a t e d w i t h 0 . W n i t r a n i l i c a c i d
.
u s i n g a p o l a r o raph f o r d e t e c t i o n of t h e endpoint
i n 0.01~K C 111%

7.5 Fluorometric Analysis

Weak f l u o r e s c e n t i n t e n s i t y w a s observed
f o r diphenhydramine when t r e a t e d w i t h 3% H202 1 1 7 ,
b u t no a n a l y t i c a l u s e w a s made of t h i s observa-
t i o n . Martinll8 treated a residue containing
diphenhydramine w i t h c o n c e n t r a t e d s u l f u r i c a c i d
and perchloric acid t o o b t a i n f l u o r e s c e n c e a t
525 nm. w i t h e x c i t a t i o n a t 3 7 5 nm. L i m i t of
d e t e c t i o n observed w a s 0.02 w./ml.

Glazko119 used f l u o r e s c e n t dyes t o

e x t r a c t diphenhydramine as an ion-pair and
i n c r e a s e d the s e n s i t i v i t y o f d i r e c t e x t r a c t i o n
methods s e v e r a l hundred f o l d over t h e use of
methyl orange and c o l o r i m e t r y .

7.6 Automated A n a l y s i s
Robertson120 has p r e s e n t e d an automated
method of a n a l y s i s f o r amine drugs based on
acid-dye methods. B r O m O C r e S O l p u r p l e i s used
f o r diphenhydramine. The automated and manual
method a g r e e q u i t e w e l l w i t h a 0.4% l a b e l c l a i m
d i f f e r e n c e . F u s a r i h a s p r e s e n t e d an u l t r a v i o l e t
method f o r c o n t e n t u n i f o r m i t y of diphenhydramine
samplesl21.

21 1
 I R A J. HOLCOMB A N D SALVATORE A . FUSARI

7.7 Biological Assay

Chen122 used isolated guinea pig ileum
for the assay of histamine and diphenhydramine
in vitro. A linear relationship is observed
between dose and effect.

7.8 Gravimetric Analysis

U ~ e n used
o ~ ~the
~ picrate method to
determine diphenhydramine gravimetrically. The
picrate is filtered, washed with water and ether,
dried and weighed.

7.9 Chromatography
7.91 paper chromatography
The results of chromatography on
paper are summarized in Table X for diphenhy-
dramine hydrochloride.

7.92 Thin Layer Chromatography

An excellent review of the thin
layer chromatography methods for diphenhydramine
was presented by Comer130 in 1967. Additional
mobile phases used on silica gel prior to 1967
are presented by Kampl31,
(1) CC14:BUOH:MeOH:25% W O H (40:30:30:1), and
(2) Petroleum ether:ether:EtzNH (20:80:1) and
by F ~ w a l ~ ~ , :MeOH:NqOH (98:1:1) Diphen-
CHC13 .
hydramine has also been chromatographed on thin
layers of alumina using
@H:EtOH (9:1) or (9:1.5)l33;
6H:EtOH :HOAc ( 3 :1.2 :0 - 5 ) 133;
CHC13:BuOH (98:2)134;
CHC13:Me2CO and
6H:EtOH (9:1)134.

212
 Table x. Paper Chromatoqraphy of

Diphenhydramine Hydrochloride

Mobile Phase Rf
L.
Pretreatment Ref crence

n-BuOH:HOAc:H20 (40:10: 5 0 ) 0.85 none 124

n-BUOH:HCl, 0.5N (90:30) 0-68 none 124

I sopropyl alc. :HCl, 0.5N
h, (90:30) 1.00 none 124
+
w
EtOH:H2O:QOH (55:43 :2) 0-31 Impregnated with s o h . of 125
petroleum (180-215O C ) and
petroleum ether

EtOH:H20:NH40H (95:3:2) 0-76 Impregnated with s o h . of 125

petroleum (180-215Oc) and
petroleum ether

n-BUOH sat.. with 1N HC1 0-60 none 126

C6H6:HOAC:H20 (4:4:1) 0 -70 none 126
n-BuOH sat. with pH 3 buffer 0.63 Treated with p H 3 buffer 127
Table x( cont .)

Mobile Phase -
Rf Pretreatment Reference

n-BUOH s a t . with pH 5 b u f f e r 0.63 Treated w i t h pH 5 b u f f e r 127

n-BuOH s a t . with pH 6 . 5
buffer 0.67 Treated with pH 6 . 5 b u f f e r 127

n-BUOH s a t . with pH 7 - 5
buffer 0.91 Treated w i t h pH 7.5 b u f f e r 127

n-BUOH:H20 (50:50) w i t h
1 g . c i t r i c a c i d (use Dipped i n 5% sodium 128
upper l a y e r ) - dihydrogen c i t r a t e

n-BUOH s a t . with 1 N HC1 0 -96 Whatman No. 4 129

 DIPHENHYDRAMINE HYDROCHLORIDE

Additonal systems for the thin

layer chromatographic examination and detection
of diphenhydramine are presented in Table XI.

The reverse phase paper chromato-

graphic s stem developed by Vecerkova has been
modifiedlT2 to run on thin layers of silica gel
in about 3 hours and will separate diphenhydramine
from bromodiphenhydramine.

Thin layer chromatography has

been used prior to assays by the spot-area
method143 and by the acid-dye reaction after
elution from the plate.144

7.93 Gas chromatography

7.931 Direct Methods on Neutral
columns
The majority of published
gas chromatographic systems for diphenhydramine
hydrochloride involve injection of the free
base on columns differing in polarity.

MacDonald 14’ used a 6 ft .

column of 1% SE-30 on 100-120 mesh Gas Chrom P.
Retention time for diphenhydramine was 6.3 min. :
.
column temperature 173 Oc ; injection port, 256 Oc;
argon flow rate 60 ml./min. using an argon @-ray
ionization detector.
146
Kazpk presented
additional data on 1% SE-30 columns at different
temperatures.
147
MacDonald compared four
columns in 1964 and found 0.08% PDEAS on a 120/
170 glass bead column to be most successful for

215
 T a b l e XI. Thin Layer chromatography
of Diphenhydramine Hydrochloride
Mobile Phase Sorbent Rf - Reference

CHC13 :Me2CO (9:1) Kieselgel G w/ 0.08 El Gendi135

fluorescent
indicator
MeOH I1 0.22

CHC13:EtOH (9:1) I1 0.31 II

N CHC13:EtOH (8:2) I1 0.33 I1

c
m
EtOAc:MeOH:QOH(85:10:5) Silica Gel G 0.90 Davidow 136

CHC13 :MeOH (9:1) Silica Gel G 0.76 Bastos 137

1 sopropyl ether :EtOH (8:2) 0 -11 I1

MeOH :NH40H ( 100 :1 -5) 0.77 II

ISOprOpyl ether:Me2CO(l:l) Kieselgel G 138

0.55 s Eiden
or GF 0.48 f
 Table XI. (cont .)

Mobile Phase Sorbent -

Rf Reference

Isopropanol Kieselgel c7 0.50 s

or GF 0.50 f Eiden

Benzene :dioxane :HOAc 0.07 s II

I1
(50:40 :10)

Cyc10hexane:EtOAc:Et~NH 0.51
z
4 (65:30 :5)

Et0Ac:cyclohexane:dioxane: Gelman silica

MeOH :H2 0: OH g e l glass micro- 0.71 Kaistha 139
(50:50:10 :lo: 1.5 :O .5) fiber s h e e t s

Et0Ac:cyclohexane: Gelman silica g e l

NH40H :MeOH :H20 glass microfiber 13 9
(70:15:2:8:0.5) sheets 0 -86 Kaistha

EtOAc :cyclohexane :MeOH:

NH4OH I1
(70:15: 10: 5) 0.91
 3.r
k
0
c,
m
al
9
0
i?
Y
X o m
0
c s E
5:
x
dn
** N
00
9' x **
003
4Jm
w-
218
 DIPHENHY DRAMINE HYDROCHLORIDE

the antihistamines. Diphenhydramine has a

retention time of 2.5 min. on a 6' column at
175OC. with an argon flow rate of 60 ml./min.

Jain148 used 1% Hi-Eff-8B

on 100/120 mesh silanized Gas Chrom P. Retention
times of diphenhydramine given are relative to
methapyrilene; 0.43 at 160Oc. and 0.46 at 190".
Gas chromatography was used for the determination
of diphenhydramine in blood after an extraction
with acetone-ether .
A mixture of Hi-Eff-8BP,
1%, and 10% SE-52 on Gas Chrom Q was used by
~ a d e r lin~ application
~ to single and multiple
component drugs. The column temperature was
22OOc. Relative retention time was 0.83 to
pentobarbital.

presented a
general article on gas chromatography in which
diphenhydramine was chromatographed on 3% Phenyl
Methyl Silicone (OV 17) on Gas Chrom Q at 175O
(6 ft., 4 mm. I.D.).

A rapid, direct analysis

of antihistamines was reported by Reiss151 in
which the sample is dispersed in water, diluted
to volume, filtered and injected. Relative
retention times to chlorpheniramine maleate are
.
reported on two columns both 4 ft x 0.25 in.
0.d. glass: 2% SE-30 and 2% Carbowax 20M on
80/100 mesh DlatOpOrt S, 0.52; and 10% silicone
oil DC-200 on 60/80 mesh Diatoport S, 0.63.
column temperature was 210 OC.

219
 I R A J. HOLCOMB AND SALVATORE A. FUSARI

7.932 D i r e c t Methods on B a s i c
Columns
S t e e l e reported152 on t h e
use of a column w i t h 5% Apiezon L and 4.5%
potassium hydroxide. column t e m p e r a t u r e w a s
138O f o r t h e f i r s t 6 min. a f t e r i n j e c t i o n and
t h e n r a i s e d a t 6Oc./min. t o 275°C. R e t e n t i o n
t i m e f o r diphenhydramine w a s 3.64 min.

7.933 Oxidation t o Benzophenone

P r i o r t o G a s Chromatoqraphy
Oxidation w i t h 0.033M
Cr2O3 f o r 60 min. c o n v e r t s diphenhydramine t o
benzophenone which can b e determined i n nanogram
amounts w i t h a p r e c i s i o n of 1.5% u s i n g e l e c t r o n
capture gas c h r ~ m a t o g r a p h y l ~ ~ .

7.94 Column chromatography

~ e v i n e l b 4r e p o r t e d on t h e par-
t i t i o n i n g of diphenhydramine between 2 N H C 1 and
chloroform on a C e l i t e column. The diphenhy-
dramine i s e l u t e d w i t h a mixture of 90 m l .
chloroform c o n t a i n i n g 1 m l . of a c e t i c a c i d
a f t e r a prewash o f t h e column w i t h d i e t h y l e t h e r .

Doyle’’ h a s examined d i s t r i b u t i o n
diagrams and s e l e c t e d C e l i t e p a r t i t i o n chromato-
g r a p h i c systems f o r v a r i o u s s e p a r a t i o n s on t h e
b a s i s o f t h e diagrams. The e f f e c t s of s o l v e n t
composition on t h e column p a r t i t i o n chromato-
graphr5gf a m i n e s h a s a l s o been examined by
Doyle and some i n f o r m a t i o n on diphenhydramine
was p r e s e n t e d .

7.95Electrophoresis
The e l e c t r o p h o r e s i s of diphen-
hydramine h a s been carried o u t by B a r u f f i n i 124

220
 DIPHENHY DRAMINE HYDROCHLORIDE

i n d i f f e r e n t pH b u f f e r s a t 7 volts/cm. f o r 3
h o u r s . M i g r a t i o n i s o p t i m a l a t low pH w i t h 8 2
mm. d i s p l a c e m e n t toward t h e cathode a t pH 2 .l.

Der likowski 156 examined t h e

e l e c t r o p h o r e s i s of diphenhydramine i n 1 9 6 5 .
zones w e r e detected w i t h a Dragendorff r e a g e n t .

The l i t e r a t u r e h a s been reviewed through

1972.

Acknowledgment

The most c a p a b l e a s s i s t a n c e of M r s . L u c i l l e
Kelly, I n f o r m a t i o n S p e c i a l i s t , parke, Davis &
.
C o , i s g r a t e f u l l y acknowledged. The a u t h o r s
a l s o wish t o thank M i s s Beverly Jozwiak f o r h e r
p a t i e n c e i n t h e p r e p a r a t i o n and c o r r e c t i o n of
t h i s manuscript.

22 1
 IRA J. HOLCOMB AND SALVATORE A. FUSARI

8. References

1. USP XVIXI, p . 207 (1970)

2. The Merck Index, p . 386, Eighth Edition,
Merck and Co., Inc., Rahway, N . J . (1968)
3 . deRoos, A. M., pharm. Weekbl., - 102,
.
1071-7 (1967). C A. 68, 6240d (1968)
4. deRoos, A. M., ReCl.TraV. Chim. Pays-Bas
-
87, 1368-71 (1968). C. A. 70, 72303 u
(1969)
5. Wallace, J. E., BiggS, J. D., Dahl, E.V.,
Anal. Chem. - 38, 831-4 (1966)
6. Schoeb, E. J., parke, Davis & co.,
Personal Communication
7. Scott, R. B., Parke, Davis & co.,
Personal Communication
8. Kracmar, J., Kracmarova, J . , Cesk. Farm.
.
15, 16-24 (1966) c. A. 64, 19323a (1966)
-
.
9. Friedlaender, A. S , Friedlaender, S , .
Vandenbelt, J. M., J . Allergy - 20, 229
(1949)
10. Vandenbelt, J. M., Parke, Davis & Co.,
personal Communication
11. Okerholm, R. A., Scott, R. B., Parke,
Davis & Co., Personal Communication
12. Luers, R. B., Parke, Davis & co.,
Personal Communication
13. Quan, J. H., parke, Davis & Co., Personal
Communication
14. Terhalle, M., parke, D a v i s & Co.,
personal Communication
15. Keenan, G. L., J. Am. Pharm. ASSOC.,
-
S c i . Ed. 36, 281-2 (1947)
16. Shell, J. W., Witt, It. F., Poe, c. F.
-
Mikrochim. Acta, 1960, 31-7. c . A.
-
58, 2649g (1963)

222
 DIPHENHYDRAMINE HYDROCHLORIDE

17. .
G a d r e t , M., Bregeve, C B u l l . SOC Pharm. -
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232
ECHOTHIOPHATE IODIDE

Raymond D. Daley
 RAYMOND D. DALEY

1. Description
1.1 Name, Formula, Molecular Weight
1 . 2 Appearance, Color, Odor

2. Physical Properties
2 . 1 Infrared Spectra
2.2 Nuclear Magnetic Resonance Spectra
2.3 U l t r a v i o l e t Spectra
2 . 4 Mass Spectra
2.5 D i f f e r e n t i a l Thermal Analysis
2.6 S o l u b i l i t y
2.7 C r y s t a l Properties
2.8 Melting Point

3. Synthesis

4. Stability -- Degradation
5. Drug Metabolic Products

6. Methods of Analysis
6 . 1 Elemental Analysis
6 . 2 U l t r a v i o l e t Spectrophotome t r i c Analysis
6 . 2 1 Direct U l t r a v i o l e t Absorption Measurement
6.22 I n d i r e c t U l t r a v i o l e t Absorption Method
6.3 Titrimetric Assay Method
6 . 4 Thin Layer Chromatography
6 . 5 Other Tests

234
 ECH OTH I OPHATE I OD I DE

1. Description

1.1 Name, Formula, Molecular Weight

The Chemical Abstracts name for echothiophate

iodide is 2-[ (diethoxyphosphiny1)thiol -N,N,N-trimethyl
ethanaminium iodide, starting with Volume 76 (1).
Previously the Chemical Abstracts name was (2-mercapto-
ethy1)trimethylauunonium iodide S-ester with 0,O-diethyl
phosphorothioate. The CAS Registry No. is [ S U - l O - O ] .
The British Pharmaceutical Codex lists the compound as
ecothiophate iodide (2), and The Merck Index lists several
other names (3).

C 9H23IN03PS Mol. Wt.: 383.23

1.2 Appearance, Color, Odor

White crystalline pwder, with a slight

mercaptan-like odor.

2. Physical Properties

2.1 Infrared Spectra

Figure 1 is an infrared spectrum of one of the

crystalline forms of echothiophate iodide. This form will
be designated as Form I in Section 2.7, Crystal Properties.
The spectrum was run in several sections: (a) from 200
to 540 cm” as a mineral oil mull on polyethylene; (b)
from 470 to 1360 cm” as a mineral oil mull between
potassium bromide plates, with the 900 to 1055 cm’ and
1220 to 1275 cml regions run in two thicknesses; (c)
from 1360 to 4000 cm” as a perfluorinated oil mull between
potassium bromide plates. The spectrum was obtained with
a Beckman IR-12 spectrophotometer.

Some of the absorption bands can be assigned as

follows ( 4 ) :

235
Figure 1. Infrared spectrum of echothiophate iodide, perfluorinated and
mineral o i l mull.
 ECH OTH I OPH ATE I 0 0 I DE

3000 cm' C-H Stretching

1240 Cm" P=O Stretching
1157 cm' P-0-Ethyl Vibration
975 cm' P-0-C (Alkyl) Vibration

2.2 Nuclear Magnetic Resonance Spectra

' b o proton magnetic resonance spectra of

echothiophate iodide are shown in Figures 2 and 3 . These
were run in D 0 and in CDCl , on a Varian A-60A 60 MHz
NMR spectraneger, with a te2ramethylsilane reference ( 5 ) .
The NMR spectra of organic phosphorus compounds
are complicated by coupling of the proton signals with
that of phosphorus. This coupling causes readily
observable splitting of the lines from methylene protons
in the groups P-0-CH and P-S-CH2, with JpH coupling
constants of 9 Hz ( 6 f .

The proton NMR spectral assignments are given in

Table I (5).

A phosphorus NMR scan indicates a chemical shift

of about -28 ppm for the phosphorus in echothiophate
iodide in aqueous solution, relative to a phosphoric acid
reference ( 7 ) . This is consistent with literature values
for this structure ( 8 ) .

2.3 Ultraviolet Spectra

Figure 4 shows the ultraviolet absorption

spectrum of echothiophate iodide, run on a Cary Model 14
spectrophotometer. The sample was dissolved in water.
The maximum at 226 nm has an absorptivity of 1.34 x 104
liters per mole cm. This absorption is essentially that
of the iodide ion (the ultraviolet spectrum of a
potassium iodide solution exhibits a
with an absorptivity of about 1.35 x
cm). 3
imum at 226 nm
liters per mole

2.4 Mass Spectra

Attempts to obtain the mass spectrum of echothio-

phate iodide were unsuccessful; the compound apparently

237
N
w
00

Figure 2. Proton NMR spectrum of echothiophate iodide, D20 solution.

bJ
w
W

Figure 3. Proton NMR spectrum of echothiophate iodide, CDC13 solution.

 N N
X X
.
v)
hl
I-
a
In
hl
I-
.
?. B ?
cb a
n
ON) r, hl r,@
N N
X X
9
I-
9
I-
a a
0 hl
s
4
hl
U
n
rl
h
9
w
VJ
+Z
U lhl
X
+zI I
hl
m u
I X
m x
U
X
U U V I
a
cb
\o Q I h l
240
Figure 4 . Ultraviolet spectrum of echothiophate iodide i n aqueous solution
vs water, 1 an c e l l s ; 25.0 mcg/ml, 250 mcg/ml, 2.50 mg/ml.
 RAYMOND D. DALEY

decomposed i n the heated i n l e t of the mass spectrometer

(5).

2.5 D i f f e r e n t i a l Thermal Analysis

Figure 5 shows the d i f f e r e n t i a l thermal a n a l y s i s

curve of echothiophate iodide, run a t 10°C per minute on a
Dupont Model 900 instrument. The only thermal event below
2OO0C is the melting point, which occurs a t 122OC on t h i s
scan. When the m a t e r i a l was run a t 2OoC p e r minute, the
melting endotherm was observed a t 125.5OC ( 9 ) .

2.6 Solubility

The s o l u b i l i t y of echothiophate iodide a t room

temperature is as follows:
Approximate
Solvent Solubility, wI m l

Water > 500

Methanol > 250
Ethanol (952) > 120
2-Propanol 4
Ace t oni t r i l e 25
Chlorof orm 250
Acetone 8
Die thy1 Ether < 1
Petroleum Ether < 1
Benzene < 1
Ethyl Acetate < 1

2.7 Crystal Properties

Two c r y s t a l forms of echothiophate iodide have

been observed. The x-ray powder d i f f r a c t i o n patterns a r e
given i n Table 11. These were obtained with a Norelco
diffractometer, using n i c k e l - f i l t e r e d copper IUr radiation.

2.8 Melting Point

The following melting points have been reported:

124-4.5OC (10)
138 (11)

242
N
P
w

Figure 5. Differential thermal analysis scan of echothiophate iodide.

 RAYMOND D. DALEY

TABLE I1

X-Ray Puwder Diffraction Data

for Echothiophate Iodide

Form I Form I1
-
d , A'
-
1/11 -
d, A'
-1/11

10.54 23 10.00 64
8.00 9 6.79 24
6.14 99 6.68 15
5.90 66 5.40 100
5.49 22 4.99 40
5.30 41 4.64 47
5.01 10 4.45 56
4.86 100 4.37 56
4.75 52 4.21 22
4.49 10 4.06 28
4.33 25 4.00 17
4.21 56 3.95 92
4.16 66 3.81 24
4.10 8 3.73 20
4.01 46 3.60 30
3.94 13 3.41 35
3.82 60 3.34 30
3.69 21 3.28 4
3.61 2 3.22 24
3.54 70 3.14 13
3.49 92 3.07 12
3.45 22 2.96 24
3.40 33 2.92 13
3.28 11 2.86 7
3.24 6 2.81 24
3.20 22 2.77 6
3.16 4 2.71 21
3.12 15 2.67 7
3.06 9 2.43 13
3.01 3 2.34 11
2.97 8
2.95 13
2.92 15
2.89 12
2.82 31

244
 ECHOTHIOPHATE IODIDE

TABLE I1 (Cont'd.)

-
d , A'
-
1/11 -
d , A'
-
1/11

2.74 10
2.70 16
2.67 25
2.58 13
2.53 4
2.50 6
2.48 4
2.44 14
2.40 3
2.37 4
2.31 4
2.27 7
3. Synthesis

Two methods f o r preparing echothiophate iodide have

been published. The r e a c t i o n s a r e shown i n Figure 6.

I n the f i r s t method ( l o ) , the sodium s a l t of

dimethylaminoethyl mercaptan is prepared by t r e a t i n g
dimethylaminoethyl mercaptan hydrochloride with sodium.
The product is treated with diethylchlorophosphate t o
y i e l d 0,O-diethyl-S-$ -dimethylaminoethyl thiophosphate.
This m a t e r i a l is t r e a t e d with methyl iodide t o make
echothiophate iodide.

I n the second method ( l l ) , a mixture of diethylchloro-

phosphate, dimethylaminoethyl mercaptan, and triethylamine
i n e t h e r is refluxed. The mixture is f i l t e r e d t o remove
the insoluble triethylammonium chloride and d i s t i l l e d
t o obtain the 0,O-diethyl-S-6 -dime thylaminoe thy1
thiophosphate. This material is t r e a t e d with methyl
iodide t o make echothiophate iodide.

4. Stability -- Degradation
Hussain e t a 1 (12) have shown t h a t echothiophate
iodide decomposes by a t l e a s t two mechanisms. I n the pH
range 2.4 t o 5, the major r e a c t i o n i s hydrolysis of one
of the C-0 bonds t o form ethanol and the monoethyl e s t e r .

245
 1. (a)
+
(CH3l2NH -CH2CH2-SH + 2Na *(CH3)2N-CH2CH2S- + 2Na+ + H2
0 0

(b) (CH3)2N-CH2CH2S-
+
+ Cl-P(OC2H5)2-
9
(CH3)2N-CH2CH2-S-P(OC2H5)2 + C1-
0 0
(c)
f
( c H ~ > ~ N - c H ~ c H ~ - s - P ( o+cC~HH ~ I
)~
+
I: (cH~)~N+cH~cH~-s-P(oc~H~)~-J
I-

N
P
rn
2. (a) (CH3)2N-CH2CH2-SH
t
0
+ C1-P(OC2H5)2 + (C2H5)3N
0
-
9
( c H ~ ) ~ N - c H ~ c H ~ - s - P ( o c+~ [H(~ )
c ~~ H ~ ) ~ N + ~ c ~ -

(b C H ~ ) ~ N - C H ~ C H ~ - S - + ( -+
C ~CHH~~)I~- 0
I: ( c H ~ )+~CNH ~ C H ~ -tS - P C O C ~ HI-~ ) ~ J

Figure 6. Synthetic Methods for Echothiophate Iodide.

 O
hl
X
+
X I
r-l
Om hl
n
m
.
xhl
u
+
O+pr
3
W 0)
a
..
.
d
0 a
U 0
+ W
0)
Y
hl
X
I
ON
+:
ON X
X hl
+ h

m
+ m
X
m -X& n u
n
hl u
W
hl
n
v
U
LA LA
L
2- .
N
“T
xN r(
0 I
0
v
W rr)
O f $ 0th QI
I
rn rn X
hl
2 a
X X-
uhl
3
+: +:
X
m m
n n
m m
X X
u
v v
V
U U
n n
0 P
W W
241
 RAYMOND D. DALEY

In the pH range 9.5 to 12, the major reaction is

hydrolysis of the S-P bond to yield (2-mercaptoethyl)
trimethylammonium iodide and diethylphosphoric acid.
These reactions are shown in Figure 7. At intermediate
pH, both react ions occur.

5. Drug Metabolic Products

No metabolic products have been reported.

6. Methods of Analysis

6.1 Elemental Analysis

The elemental composition of echothiophate iodide

is as followst
Element - 'K Theory
-
Carbon 28.21
Hydrogen 6.05
Iodine 33.11
Nitrogen 3.65
Oxygen 12.52
Phosphorus 8.08
Sulfur 8.37

6.2 Ultraviolet Spectrophotometric Analysis

6.21 Direct Ultraviolet Absorption Measurement

Although the ultraviolet absorption at

226 nm has been used in hydrolysis studies (12), it was
useful only because it increased as the echothiophate
iodide hydrolyzed. The iodide ion is the principal
absorbing species at this wavelength in echothiophate
iodide solutions (see Section 2.3), so that this maximum
can be used only indirectly to measure the echothiophate
cation concentration.

6.22 Indirect Ultraviolet Absorption Method

An ultraviolet assay for echothiophate

cation is possible, using hydrolysis to thiocholine,
followed by reaction with 4,4'-dithiopyridine to form an

248
 ECHOTHIOPHATE lODl DE

ultraviolet absorbing material (13). The method is based

on that of Grassetti and Murray (14). The ultraviolet
quantitation is essentially an alternative to the
titration described in Section 6.3, but requires less
sample.
The echothiophate iodide is hydrolyzed
quantitatively to thiocholine in 20 minutes in pH 12.0
phosphate buffer. The hydrolyzed sample is then treated
with a solution of 4,4'-dithiopyridine in pH 2.3
phosphate buffer. The final solution has a pH of 6.2,
and the 4.4I-dithiopyridine reacts with thiocholine to
form 4-thiopyridone. 4-Thiopyridone has an absorption
maximum at 323 nm. A blank is prepared by mixing a
portion of the original echothiophate iodide solution,
before hydrolysis, with a solution of 4,4'-dithiopyridine.
Echothiophate iodide assayed by the titration procedure
is used as a standard (13).
6.3 Titrimetric Assay Method
The USP method for assay of raw material and
dosage forms is iodimetric titration of the thiocholine
formed by hydrolysis of the echothiophate iodide. In
the USP XVIII procedure, the sample is hydrolyzed with
sodium hydroxide (15). It has been shown recently that
greater specificity is obtained when hydrolysis is
conducted with a pH 12 buffer (16). It is necessary for
the pH to be as high as 12 in order that the hydrolysis
be completed in 20 minutes. On the other hand, too high
a pH increases interference from possible impurities (16).
6.4 Thin Layer Chromatography
The following systems have been found useful for
separating echothiophate iodide from possible degradation
products: (a) Silica Gel G (E. Merck) with methanol-
water-concentrated ammonium hydroxide (2: 2: 1) developing
solvent and iodine vapor detection (17); (b) Cellulose
F (E. Merck) with butanol-acetic acid-water (4:1:5)
developing solvent and iodine vapor detection (18).

249
 RAYMOND D. DALEY

6.5 Other Tests

A microscopic identity test for echothiophate
iodide has been reported. Echothiophate iodide in
aqueous solution forms a crystalline precipitate with
amnonium reineckate (19).
7. Acknowledgments
The writer wishes to thank Dr. B. T. Kho for his
review of the manuscript, Dr. G . Schilling of Ayerst
Research Laboratories and Dr. W. E. Krueger of the State
University of New York at Plattsburgh for their NMR data
and interpretation, the library staff for their literature
search, the numerous other contributors who provided
information for this profile, and Mrs. Kay Mannan for
typing the profile.

250
 ECHOTHIOPHATE IODIDE

REFERENCES

1. C. A. 76, 2946 (1972), Chemical Abstracts Index Guide.

2. British Pharmaceutical Codex 1968, Supplement 1971,
The Pharmaceutical Press, London, 1971.
3. The Merck Index, 8th Ed., Merck and Co., Inc.,
Rahway, N. J., 1968.
4. L. J. Bellamy, The Infra-red Spectra of Complex
Molecules, 2nd Ed., John Wiley and Sons, Inc., New
York, 1958.
5. G. Schilling, Ayerst Research Laboratories, personal
communi ca tion.
6. H. Babad, W. Herbert, and M. C. Goldberg, Anal. Chim.
Acta 41, 259-68 (1968).
7. W. E.Krueger, State University of New York at
Plattsburgh, personal communication.
80 V. Mark, C. H. Dungan, M. M. Crutchfield, and
J. R. Van Wazer, Top. Phosphorus Chem. 2, 227-457
(1967).
9. F. Q. Gemmill, Ayerst Laboratories, Inc., personal

. communica ti on.
10 H. M. Fitch, U. S. Patent 2,911,430; C. A. 54,
4386h.
11. L-E. Tammelin, Acta Chem. Scand. ll, 1340-9 (1957).
12 A. Hussain, P. Schuman, V. Peter, and G. Milosovich,
J. Pharm. Sci. 57, 411-8 (1968).
13. N. G. Nash and F.DiBernardo, Ayerst Laboratories,
Inc., personal communication.
14. D. R. Grassetti and J. F. Murray, Jr., Arch. Biochem.
Biophys. 119, 41-9 (1967).
15. Echothiophate iodide monographs, Pharmacopeia of the
United States of America, 18th Revision, Mack
Printing Co., Easton, Pa., 1970, pp. 220-1.
16. C. Warner, F. DiBernardo, A. B y l w , A. Hussain, and
B. T. Kho, J. Pham. Sci. 60, 1548-9 (1971).
17. A. Bylow, Ayerst Laboratories, Inc., personal
communication.
18. G. R. Boyden, Ayerst Laboratories, Inc., personal
communication.
19. L. G. Chatten, A. C. Napper, and P. J. Barry, J.
Pharm. Sci. 56, 834-8 (1967).

The above references cover the literature through 1972.

25 1
 ETHYNODIOL DIACETATE

Edward P. K. Lau and John L . Sutter

 EDWARD P. K. LAU AND JOHN L. S U T E R

Contents

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 Spectrum
2.4 Mass Spectrum
2.5 Optical Rotation
2.6 Melting Range
2.7 Differential Scanning Calorimetry
2.8 Thermogravimetric Analysis
2.9 Solubility
3. Synthesis
4. Stability and Degradation
5. Drug Metabolic Products and Phannacokinetics
6. Methods of Analysis
6.1 Phase Solubility
6.2 Spectrophotometric Analysis
6.3 Colorimetric Analysis
6.4 Fluorometric Analysis
6.5 Titrimetric Analysis
6.6 Chromatographic Analysis
6.61 Column Chromatography
6.62 High Pressure Liquid Chromatography
6.63 Thin Layer Chromatography

7. Acknowledgments
8. References

254
 ETHYNODIOL DIACETATE

1. Description
1.1 Name, Formula, Molecular Weight
Ethynodiol Diacetate is 19-Nor-17a-pregn-4-en-
20-
yne-3B, 17-diol Diacetate.

Weight: 348.52

1.2 Appearance, Color, Odor

Ethynodiol diacetate is a white to off-white, es-
sentially odorless powder.
2. Physical Properties
2.1 Infrared Spectrum
The infrared absorption spectrum of an ethynodiol
diacetate reference standard compressed in a KBr pellet
is shown in Figure 1. The compound exhibits essentially
the same infrared spectrum in chloroform solution. Tne
following assignments have been made for absorption bands
in Figure 1 ,'
Qn. -1 Assignment
3315 C r . ( 3 I : Acetylenic C-H
stretching
1740 C=O : Acetate Carbonyl
stretching
1670 C=C : Ethylenic stretching

255
w
 ETHYNODIOL DIACETATE
n
I1
1275, 1028 CH - C - 0 - : Acetate C-0
3 stretching
2.2 Nuclear Magnetic Resonance Spectrum
The NMR spectrum of ethynodiol diacetate in deuterated
chloroform is shown in Figure 2. Spectral assignments
are as follows:2
Chemical Shift
(PPm) Type Ass ignment
5.00-5.41 Broad Singlet Protons at C-3 and
c-4
2.58 Singlet CGCH : Ethynyl
proton
0
2.03 Sing1et -0-C-CH : Acetyl
methgl protons
0.90 Sing1et -a : C-18
Aethyl protons

2.3 Ultraviolet Spectrum

Ethynodiol diacetate does not absorb between 420 nm and
210 nm. A peak is observed at 204 nm which is not con-
venient for quantitative determination.
The USP XVIII assay procedure involves acid hydrolysis
of the compound in methanolic 0.7 N HC1, for 10 minutes
on a steam bath. The resulting soTution o f diene exhib-
its the absorption spectrum sham in Figure 3, with max-
ima at about 229 nm, 236 nm and 244 nm. The peak at 236
nm is used for quantitative determination.3

2.4 Mass Spectrum

The low resolution mass spectrum of ethynodiol diacetate
shown in Figure 4 was obtained with an AEI Ihdel M5-30

251
259
FIG. 3: ULTRAVIOLET SPECXRLIM OF ETHYNODIOL DIACETATE

h)
w
UY

10
0
0

PJ
4

0
0
N
N

N
In
0

CI)
N

N
0

co
0
m
0

N
m
0
Wavelength (mp)
H
n
A
'LNI ' 1 3 M I
 ETHYNODIOL DIACETATE

mass spectrometer. A molecular ion was observed at m/e

384. The base peak in the spectrum was at m/e 43, COT-
responding to (31 a+. Structure assignments are sum-
marized below: '

m/e Assignment % Relative Intensity

384 M'f 1.0

369 M- CH3' 1.4

342 M- m2C0 0.2

324 M- C H 3 0 € I 8.5
30 9 M- (QI + M3COOH) 1.6
3
282 M- (CHzCO + M3COOH) 2.2

2 64 M- (2 CH3cooH) 4.0

249 M- ( 2 M3COCH + M,') 1.4

43 c)13co+ 100.0

2.5 Optical Rotation

The following specipc rotation values in chloroform
have been reported.

261
 EDWARD P. K. LAU AND JOHN L. SU7TER

2.6 Melting Range

"lie melting range given i n the USP XVIII is 126'to
132OC.

2.7 Differential Scanning Calorimetry

The DSC tliermogram of ethynodiol d i a c e t a t e obtained a t
a heating rate of S°C/minute is shown in Figure 5 .
Tne endothermic change observed a t about 126OC corres-
ponds t o the melting of the drug. The decomposition
temperature is 228OC. '
2.8 Thennogravimetric Analysis
"lie TGA spectrum of ethynodiol diacetate i n Figure 6
was produced under a nitrogen sweep a t a heating rate
of 10°C/minute. A rapid weight loss was observed
from about 21OoC t o 26OOC. Another Fapicl weight
loss was seen s t a r t i n g a t about 400 C.

2.9 Solubility
S o l u b i l i t i e s i n various solvents a t 25OC are given in
the following table:

Solvent Solubility, mg ./d.

Water 0.0014

Methanol >so
Ethanol >50
Chloroform )SO

Heptane 18

262
 ETHYNODIOL DIACETATE

80 100 120 140 160

TEMPERATURE oc

263
 EDWARD P. K. LAU AND JOHN L. SUTTER

FIG, 6:
TGA SPECIRJM OF FI1NNODlC)L DIAEI'XTE

100

80

60
s.-.
9
0
"

40

20

100 200 300 400 500

TEGW?ATJRE OC

264
 ETHYNODIOL DIACETATE

3. Synthesis
Ethynodiol diacetate has been synthesized by routes
utilizing both estradiol 3-methyl ether (I) and 38-
hydroxyandrost-5-en-17-one (11) as starting materials.
In the former method, 9 9 l o , l'outlined in Figure 7 ,
estradiol 3-methyl ether (I) is reduced by the Wills-
Nelson modification of the Birch procedure12,togive
the 1,4-dihydro derivative (111). Oppenauer oxidation
of (111)13 yields the 17-ketone (IV), which is then
ethynylated,14 giving the enol ether intermediate, (V).
Reaction of (V) with dilute acetic acid produces
.
norethynodrell (VI) Treatment of either (V) or (VI)
with aqueous mineral acid gives norethindrone (VII),
which is then converted to ethynodiol (VIII) by reduc-
tion with sodium borohydridelO, 16. The diol is then
diacetylated with acetic anhydride and pyridine, yielding
ethynodiol diacetate (IX) .
Alternatively, as shown in Figure 8, peracid treatment of
3fi-hydroxyandrost-5-en-17-one(11) yields the 5,6
a-epoxide (X). Perchloric acid cleavage of (X) re-
sults in the 5,6-diol (XI); acetylation then gives the 3,
5,6-triacetate (XII) , which reacts selectively with bi-
carbonate to give the 3fi,68-diol-5a-acetate(XIII) ,
Selective acetylation at C-3 followed by lead tetraacetate
and iodine functionalization of C-19 then yields the 68,
19-oxide (XIV). Bicarbonate hydrolysis of (XIV) followed
by chromic acid oxidation of the resulting alcohol affords
the key intermediate (XV), which, when treated with zinc
and zinc chloride in methanol gives 19-hydroxyandrostene-
dione (XVI). Treatment of (XVI) with chromic acid affords
the acid (XVII), which on heating in pyridine is decarboxy-
lated to give the 5 (10)-dehydro derivative (XVIII).
Selective ketalization of (XVIII) at C-3 is accomplished by
treatment with weak acid in methanol, yielding (XIX).
Ethynylation at C-17 then gives the 3-dimethyl ketal of
norethynodrel (XX), Weak acid cleavage of (XX) gives
norethynodrel (VI) , while more vigorous acid treatment gives
norethindrone (VII) , Conversion of (VII) to ethynodiol
diacetate (IX) is accomplished as previously described,

265
 EDWARD P. K. LAU AND JOHN L. SUTTER

FIG. 7: SYNTHESIS OF E"ODI0L DIACETAE

a3 && I 013

d
I11

m=c - &3 ;&

Q13 V

IV
I/

ca-&-;

VI VI I
a - & ; : & @' d

HO
VIII

266
 ETHYNODIOL DIACETATE

FIG. 8: SyN?HESIS OF ETHYNODIOL DIACETATE

0 X
I1

XI XI I
I(

H
Ac6
Aco OH XIV
XI11

267
EDWARD P. K . LAU A N D JOHN L. SUTTER

FIG. 8: (CONT.)

XVI I WIII

268
 ETHYNODIOL DIACETATE

4. S t a b i l i t v and Demadation
Ethynodiol diacetate appears to be very s t a b l e as a solid.
The degradation of ethynodiol diacetate in both a c i d i c and
basic alcoholic solutions is s h m i n Figure 9. I n the
acidic alcohol solution, t h e primary degradation product
was found to be the diene ( I ) . In basic alcohol solution,
the primary degradation product was found t o be the d i o l
(11). l 9

5. Drug hletabolic Products and Pharmacokinetics

The major metabolites of ethynodiol diacetate in urine

a r e shown i n Figure 10. These metabolites were ;$en-
t i f i e d by Kishimoto, Kraychy, Ranney and G a n t t , The
metabolism of ethynodiol diacetate by rat and human
l i v e r was reported by Freudenthal, Cook, Forth, Rosen-
f e l d and Wall, *' They found t h a t the biotransformation
-
reactions involved i n the in v i t r o metabolism include
deacetylation, saturation 3 r i n g A, aromatization of
ring A, formation of 3-ketone and an6-bond formation.
A method of analysis of very low levels of t h e metabolite
norethindrone has been developed by Freudenthal , Cook
and Wall. 2 2 The principle of t h i s method is t o convert

3,17- B-diol . e
the cold noreth' rone by enzyme reduction i n the pres-
ence of NADF'H-4 t o t r i t i a t e d 17-a-ethynylestrane-

The pharmacokinetic p r o f i l e of the t o t a l tritium label

and metabolic composition i n the plasma a t e r an o r a l ad-
\
ministration of ethynodiol diacetate-6,7- I t o a human
subject was studied by Karim, Ranney, Cook and B r e s ~ l e r . ~ 3
The ab orption rate constant (k) of the t o t a l label was
0.79% % per hour, the peak plasma level being attained
a f t e r 3 hours. The elimi a t i o n r a t e constant (K) of the
t o t a l label was 0.0276% % per hour ( h a l f - l i f e 25 hours).
The volume o f d i s t r i b u t i o n (V) was found t o be 33L and
the metabolic clearance rate (MCR) 21.9L per day. On
chloroform extraction of the pooled plasma, 20% of the
radioactivity was obtained as a f r e e f r a c t i o n which on
TLC analysis gave two major spots tentatively identified
as saturated dihydroxy metabolites and norethindrone.
Eighty percent of the pooled plasma radioactivity was pres

269
FIG. 9: CEGRADATION OF ETHYNODIOL DIACETATE IN ACIDIC F7 BASIC SOLUTION
 ETHYNODIOL DIACETATE

FIG. 10: MAJOR METABOLITES OF

ETHYNODIOL DIACETATE IN URINE

27 1
 EDWARD P. K. LAU AND JOHN L. SUTTER

ent as water-soluble conjugates which on acidic hydrolysis

furnished two major aglycones having chromatographic mo-
b i l i t i e s similar t o the two major spots. In a plasma sam-
p l e taken one hour after administration of the labeled
drug, 58% of the radioactivity was associated with the con-
jugated metabolites, 12.5% with the spot identified as
saturated dihydroxy metabolites and 19.7% with the spot
identified as norethindrone.

6 . Methods of Analysis

6.1 Phase Solubility

Phase s o l u b i l i t y analysis can be carried out by
equilibrating the drug substance i n hexane a t 25%.
Figure 11 shows the phase s o l u b i l i t y diagram of a
reference standard run.
6.2 Spectrophotometric Analysis
Ethynodiol diacetate does not have a useful spectrum
f o r d i r e c t U.V. analysis. The solution of diene re-
s u l t i n g from acid treatment has an absorbance maximum
a t about 236 nm. The USP XVIII assay is based on t h i s
reaction.
6.3 Colorimetric Analysis
A variety of colorimetric methods have been de-
veloped t o detect and t o determine ethynodiol
diacetate.
6.31 Reaction of ethynodiol diacetate with anti-
mony t r i c h l o r i d e in dry chloroform contain-
ing 1%acetic anhydride produces a v i o l e t
color. The absorbance of the solution a t
565 nm. is l i n e a r w i t h ethynodiol concen-
t r a t i o n over a range of 5-60 mcg./5 ml.
Tne method has been adapted f o r the analysis
of ethynodiol dosage forms.25 None of the
other steroids comonly found in o r a l estro-
gen-progestin combination dosage forms in-
t e r f e r e . A chloroform solution of a n t h n y

212
 ETHYNODIOL DIACETATE

FIG. 11:
PHASE SOLUBILITY

& = Q s -
n
V
-
W

WIPE: ETHYNODIOL DIACETAE

SOLW: H E M
SLOPE: 0.0%
EQUILIBRATICN: 24 hrs. at 2S°C
EXTRAPOLATED S O ~ I L I T Y :27.9 mg./g. solvent

u 20 40 00 llu 100 120

27 3
 EDWARD P. K. LAU AND JOHN L. S U l T E R

trichloride has also been proposed as a

spray reagent for ethynodiol diacetate in
quantitative thin layer chromatography.
6.32 Reaction of ethynodiol diacetate with 52%
sulfuric acid for 5 minutes at room temp-
erature yields a solution having an absor-
bance maximwn at 484 nm. This method
may be used for quantitative determina-
tion of ethynodiol diacetate, provided that
a preliminary separation from other ster-
oids is made,
6.33 Reaction of ethynodiol diacetate with hy-
droxylamine hydrochloride and ferric
chloride produces a deep red color which
serves to distinguish the compound from
steroids having no ester group, The color
is not sufficiently stable for use in a
quantitative determination. *
6.4 Flwrometric Analysis
Ethynodiol diacetate can be quantitatively de-
termined by flwrometry in 65%sulfuric acid
solution, with an activation wavelength of about
458 nm. and measuring fluorescence at about 520
nm. Separation from other steroids is neces-
sary due to their interference. The limit of sen-
sitivity of the method is 4 mcg./lOO d.”
6.5 Titrimetric Analysis
6.51 Ethynyl Titration - ethynodiol diacetate
reacts stoichiometrically with silver ni-
trate in tetrahydrofuran. The nitric acid
produced can be titrated with sodium hy-
droxide, either potentiometrically o r using
phenolphthalein as indicator. One equiv-
alent of the compound is titrated.
6.52 Ester Saponification - ethynodiol diacetate
may be saponified with a h o r n amount of
standardized alcoholic potassium hydroxide.

274
 ETHYNODIOL DIACETATE

The excess base is then titrated with hydro-

chloric acid, either potentiometrically or
using phenolphthalein as indicator.2 9 One
equivalent of the compound is saponified.

6.6 Chromatographic Analysis

6.61 Column Chromatography - the quantitative
separation of ethynodiol diacetate and mes-
tranol in dosage f o m on Sephadex LH-20
has been reported.
6.62 H i g h Pressure Liquid Chromatography -
ethynodiol diacetate can be separated from
its possible degradation products and quan-
titatively determined by reverse-phase high
pressure liquid chromatography, using a
W o n t ODs column and methanol-water eluants. 3 1
6.63 Thin Layer firomatography - TLC systems and
corresponding Rf values of ethynodiol di-
acetate are summarized in the following
table:

Thin Layer Chromatography of Ethynodiol Diacetate

Solvent System Adsorbent Detection 3 Reference

cyclohexane: SG 1, 2 0.40 32
isopropanol
(97:3)
benzene :methanol SG 1, 2 0.77 33
(95:s)

benzene:acetone SG 3, 4 0.68 33
(80:20)

chloroform: SG 3, 4 0.76 33
methanol
(90: 10)

27 5
 EDWARD P. K. LAU A N D JOHN L. SUlTER

methylene chlor- SG 3, 4 0.84 33

ide:methanol:water
(150:9:0.5)

SG = Silica gel.
Detection: 1. Spray with 50%H2S04,
heat at 8OoC for 10 minutes.
2. Spray with phosphomolybdic
acid.
3. Spray with concentrated
H SO4; heat at 100°C for
38 mmutes.
4. Observe under short wave U.V.

7. Acknowledgments
The authors wish t o express their appreciation to
Dr. N. W. Atwater, Dr. R. Bible, Dr. F. Colton, Mr.
A. J. Damascus and Dr. J. Hribar for their help in pre-
paring sections of the manuscript. The expert secre-
tarial assistance of Miss Mia Mulder is also grate-
fully acknowledged, as is Mrs. Lorraine Wearley's aid in
preparing the figures,

276
 ETHY NOD1OL D IACETATE

8. References

1. Damascus, A. J., Searle Laboratories, personal

communication.

2. Bible, R., Searle Laboratories, personal cmuni-

cation.

3. Searle Laboratories Method of Analysis No, RS-

24-817.

4. Hribar, J., Searle Laboratories, personal com-

munication.

5. Damascus, A. J., Searle Laboratories, personal com-

munication.
6. "The United States Phannacopeia" XVIII, p. 259
(1970).

7. Carey, S . and Anthony, G . , Searle Laboratories,

personal c o m i c a t i o n .
8. Chow, A. and Marshall, S., Searle Laboratories,
personal c o m i c a t i o n .
9. Colton, F. B., U.S. Pats, 2,691,028 (1954);
2,725,389 (1955).

10. Colton, F. B., U.S. P a t , 2,843,609 (1958).

11. Klimstra, P. D. and Colton, F. B., Steroids -

10
4 1 1 (1967).
12. Birch, A. J. and Smith, H., J. Chem. SOC. -'
1951
1882.

13. -
Oppenauer, R. V., Org. Syn. 21, 18 (1941),
14. Stavely, H. E., J. Am. Chem. SOC. -
61, 79 (1939).

15. Colton, F . B., U.S. Pats, 2,655,518 (1952);

2,691,028 (1954); 2,725,378 (1955).

277
 EDWARD P. K. LAU AND JOHN L. S U l T E R

16. Sondheimer, F, and Klibansky, Y., Tetrahedron 5,

1 5 (1959)
17. Pappo, R. and Nysted, L . , U.S. Pat. 3,176,014 (1965).

18. Hagiwara, H., Noguchi, S., and Nishikawa, M., &em.

Pharm. Bull. (Tokyo), -
8, 84 (1960).

19. Bollweg, M. and Baier, M., Searle Laboratories,

personal comunication.
20. Kishimoto, Y., Kraychy, S., Ranney, R. E. and Gantt,
C. L., Xenobiotica -
2, 237-52 (1972)

21. Freudenthal, R. I., Cook, C. E., Forth, J.,

Rosenfeld, R. and Wall, M. E , , J. Phannacol. Exp.
Ther. -
177, 468-73 (1971).

22. Freudenthal, R. I . , Cook, C. E. and Wall, M, E.,

"Progress Report No. 2", RTI Project No. CN-385,
Research Triangle I n s t i t u t e , Research Triangle
Park, N. C., 1971.
23. Karin, A,, Ranney, R. E., Cook, C. E. and Bres-
sler, R., "Pharmacokinetics and Plasma Metabol-
i t e s of SC-11800 (Ethynodiol Diacetate) in a
Human Subject", Searle Laboratories Progress Re-
port.
24. Root, A. and Chow, R., Searle Laboratories, per-
sonal c o m i c a t i o n .
25. Pasini, R. and Gavazzi, G., J. Pharm. Sci. -'
58
872-4 (1969).
26. Keay, G. R., Analyst -
93, 28 (1968).
27- Seul, C. J., Searle Laboratories, personal corn-
munication.
28. Jack, M., Searle Laboratories, personal comuni-
cat ion.
29. Brown, V., Searle Laboratories, personal communi-
cation.

278
 ETHYNODl OL DIACETATE

30. Fernandez, A. L., and Noceda, V. T., J. Pharm.

Sci. -958 740 (1969).
31. Wood, N . , Searle Laboratories, personal comuni-
cation.

32. Smith, B., Searle Laboratories, personal comuni-

cat ion.
33. Simard, M. B. and Lodge, B. A., J. Chromatog. -
51,
517 (1970).

279
FLUDROCORTISONE ACETATE

Klaus Florey
 KLAUSFLOREY

CONTENTS

1. Description
1.1 Name, Formula, Molecular Weight
1.2 Appearance, color, Odor
2. Physical Properties
2.1 Infrared Spectra
2.2 Nuclear Magnetic Resonance Spectrum
2.3 Ultraviolet Spectrum
2.4 Mass Spectrum
2.5 Optical Rotation
2.6 Melting Range
2.7 Differential Thermal Analysis
2.8 Solubility, Dissolution, Partition
Coefficient
2.9 Crystal Properties
3. Synthesis
4. Stability, Degradation
5. Drug Metabolism
5.1 Pharmacokinetic
5.2 Metabolic Products
5.3 Microbiological Transformations
6. Methods of Analysis
6.1 Elemental Analysis
6.2 Direct Spectrophotometric Analysis
6.3 Colorimetric Analysis
6.4 Polarographic Analysis
6.5 Chromatographic Analysis
6.51 Paper
6.52 Thin Layer
6.6 Bioassay
6.7 Other
7. Determination in Body Fluids and Tissues.
a. Determination in Pharmaceutical Preparations
9. References

282
 F LUDROCORTISON E ACETATE

1. Description
1.1 Name, Formula, Molecular Weight
Fludrocortisone Acetate is 9a-fluoro-
llf3, 17a,21-trihydroxy-4-pregnane-3,2O-dione, 21-
acetate: also 9a-fluorohydrocortisone acetate:
9a-fluoro-17-hydroxycortisone 21-acetate;ga-
fluorocortisol 21-acetate; fluodrocortisone
21-acetate; fluohydrisone, 21-acetate, fluohydro-
cortisone 21-acetate, SQ 9321.
21 HzOCOCH3
L O

2' 3H3 lF06 M.W. 422.48

1.2 Appearance, Color, Odor

Fludrocortisone Acetate is a white
crystalline, odorless substance.

2. Physical Properties
2.1 Infrared Spectra
Mesley' has reported four polymorphic
forms and their infrared spectra. ~

Form A - as received from the British

Pharmacopoeia.
Form B - evaporation of chloroform
solution at room temperature followed by heating
at looo for 15 minutes.
-
Form C - (amorphous) usually obtained
by evaporation of chloroform or acetone solution
 KLAUS FLOREY

a t room t e m p e r a t u r e .
Form D - may be o b t a i n e d by s p o n t a n e o u s
c r y s t a l l i z a t i o n from f o r m C.
T h e s e forms g i v e t h e f o l l o w i n g
c h a r a c t e r i st i c a b s o r p t i o n peaks ( c m - l )
Form A:
1412 1339 1274 1239 ( s h ) 1022 958 940 819 777 680
Form B:
1418 1339 1272 1226 1195 1020 958 945 868 782 678
Form C:
1418 ( s h ) 1267 1236 1198 1023 959 936 870 782 680
Form D:
1406 1344 1267 1232 1198 1020 955 942 869 - 678
The i n f r a r e d f r e q u e n c i e s of m o d i f i c a t i o n s a n d
s o l v a t e described by Kuhnert-Brandstaetter
and G a s s e r 2 (see a l s o S e c t i o n 2.9) a r e p r e s e n t e d
i n T a b l e 1.
Table 1
9a-Fluorohydrocortisone Acetate

Frequencies ( c m - l )
Form OH CIO and C l C
Modification I 3440 1738,1716,1651,1629 (w)
3350
I1 3500 (sh) 1756,1720,1650,1617
3460
V 3510 1760,1745,1730,1721
3370 1650,1611 ( w )
3300 ( s h )
VI 3525 1761,1750,1730,1647
3500 (w)
3350
Methyl a c e t a t e
solvate 3510 1761,1748,1738,1722
3320 1643
3230 ( s h )
Ethyl a c e t a t e 3515 1759,1738,1725,1652
solvate 3360

284
 FLUDROCORTISONE ACETATE

Benzene s o l v a t e 3500 1761,1749,1736,1721,

1644
3320
D i m ethylforma-
mide s o l v a t e 3360 1740,1721,1660,1624
3320 ( s h )

The i n f r a r e d spectrum of S q u i b b House S t a n d a r d

( b a t c h #48004-001) i n f i g u r e 1 r e p r e s e n t s
m o d i f i c a t i o n I. (Form A ) 3

2.2 N u c l e a r Magnetic Resonance Spectrum

The NMR spectrum of f l u o d r o c o r t i s o n e
a c e t a t e is presented i n f i g u r e 2. The
s t r u c t u r a l d a t a p r e s e n t e d in T a b l e 2 a g r e e w i t h
t h e a s s i g n e d s t r u c t u r e 4 . The two h y d r o x y l
p r o t o n s a t C 1 1 and C 1 7 exchange w i t h d e u t e r i u m .

Table 2
NMR S p e c t r a l Assignments of SQ 9321a

C h e m ic a 1
Proton a t S h i f t , 6 (ppm)
c4 5.64 s
c-11 4.10 b
C- 18 0.77 s
c-19 1.49 s
c-21 4*78 ABq;J=17.0 H z
c-21 5.06

C-21 Acetate 2.10

Ox(Exchangeab1e) 5.00 b, 5 . 4 3 s
a = DMs0-d~ s= s i n g l e t b= b r o a d
ABq = AB q u a r t e t

285
Figure 1. Infra Red Spectrum of Fludrocortisone Acetate (Squibb House
Standard batch 48004-001) from KBr/Chloro€orm. 1nstrument:Perkin Elmer 21.
Figure 2. NMR Spectrum of Fludrocortisone acetate (batch 76682) in
deuterated DMSO (1nstrument:Varian XL-100).
 KLAUS FLOREY

2.3 U l t r a v i o l e t Spectrum
F r i e d a n d Sabo5 r e p o r t e d max 238 nm;
c C = 16,800 i n e t h a n o l .
2 . 4 Mass Spectrum
The l o w - r e s o l u t i o n mass s p e c t r u m o f
SQ 9 , 3 2 1 (see f i g u r e 3 ) shows t h e e x p e c t e d M+ a t
m/e 422. C o r t i c o s t e r o i d s g e n e r a l l y show fragmen-
t a t i o n p a t t e r n s r e s u l t i n g from t h e loss o f D-ring
s u b s t i t u e n t s (cf A n a l y t i c a l P r o f i l e s , Triamcino-
lone, Triamcinolone Acetonide, Triamcinolone 16,
17-diacetate) . I n a d d i t i o n , f l u o r i n a t e d steroids
a l s o h a v e f r a g m e n t a t i o n pathways i n v o l v i n g the
loss o f HF. Thus, t h e f r a g m e n t a t i o n pathways
shown below d e p i c t t h e losses o f t h e s e g r o u p s .

m / e 422 M+

m/e 363 -C0CH20Ac m/e 362

m/e 292
m/e 301
m/e 303
1-HF
m/e 283
IH2
m / e 344
34 2

The base p e a k of m/e 42 i s from t h e a c e t y l

p o r t i o n of t h e 21-acetate. Although n o t a s
i n t e n s e a s t h o s e from A-ring d i e n o n e s , t h e m / e
121-123 (CgHg-110)and t h e m / e 135-137 (C Hll-130)
i o n s s u p p o r t t h e p r e s e n c e o f t h e A - r i n g enone
group. The mass s p e c t r u m i s c o n s i s t e n t w i t h t h e
proposed s t r u c t u r e 6 .

288
Figure 3. Low r e s o l u t i o n mass s p e c t r u m of F l u d o r c o r t i s o n e Acetate.
( S q u i b b s t a n d a r d batch 48004-001) I n s t r u m e n t : AEI-MS-902.
 KLAUS FLOREY

2.5 Optical Rotation

Ldi~ Solvent -
Ref
+143O Chloroform 5
+127O Acetone 5
+149O Dioxane 7
+145- 15O0Dioxane 8

2.6 M e l t i n q Range

233-234'
-
Ref
5
230° ( decomp. ) 7
220-233O ( decomp. ) 8

I t was n o t e d 5 t h a t o c c a s i o n a l samples s t a r t e d t o
m e l t a t 2 0 5 - 2 0 8 ° , r e s o l i d i f i e d and e v e n t u a l l y
m e l t e d a t 226-228 (See s e c t i o n 2 . 1 0 ) . The
m e l t i n g b e h a v i o r by h e K o f l e r method h a s been
described a s follows : 5
A t 210° d r o p l e t s s t a r t t o form. The r e s i d u a l
c r y s t a l s grow t o g r a i n s , s q u a r e s , and hexagons
t h a t f i n a l l y a g g r e g a t e t o a mosaic. Three o r
f o u r d i f f e r e n t forms a r e produced a t 1600 i n t h e
glassy solidified m e l t . The b u l k c o n s i s t s of
long s t a l k e d s p h e r u l i t e s of Form I1 t h a t m e l t a t
208-212O and l e a f y , p a r t i a l l y f a n l i k e r a d i a t e
s p h e r u l i t e s o f Form I11 t h a t m e l t a t 205-208O and
e x h i b i t low-order i n t e r f e r e n c e c o l o r s . Form I V
appears only r a r e l y a s fibrous-twisted
s p h e r u l i t e s . The m e l t becomes brown i n c o l o r .
The e u t e c t i c t e m p e r a t u r e w i t h p h e n o l p h t h a l e i n i s
202O. (For t h e m e l t i n g b e h a v i o r o f p o l y m o r p h i c
forms see a l s o s e c t i o n 2.9. )

2.7 D i f f e r e n t i a l Thermal A n a l y s i s
Squibb S t a n d a r d ( b a t c h 48004-001)
e x h i b i t s a s h a r p endotherm a t 23OoC1O.

290
 FLUDROCORTISONE ACETATE

2.8 Solubility''
I n water: 0 . 0 4 mg/ml; i n acetone 5 6 mg/ml;
i n chloroform 20 mg/ml; i n ether 4 mg/ml.
The d i s s o l u t i o n b e h a v i o r of c r y s t a l l i n e
f l u d r o c o r t i n s o n e a c e t a t e and i t s p e n t a n o l and e t h y l
a c e t a t e l g o l v a t e s were s t u d i e d by Shef t e r and
Higuchi . The i n i t i a l d i s s o l u t i o n rates of t h e
s o l v a t e w e r e s i g n i f i c a n t l y h i g h e r t h a n t h e non-
s o l v a t e d form. Flynn determined t h e p a r t i t i o n
c o e f f i c i e n t between e t h e r and w a t e r a s 45.7.i3
2.9 C r y s t a l Properties
The o p t i c a l c r y s t a l l o g r a p h i c p r o p e r t i e s
of f l u d r o c o r t i s o n e a c e t a t e (probably m o d i f i c a t i o n
A) and f l u d r o c o r t i n one i t s e l f have been p r e s e n t e d
as f o l l o w s by B i l e s T4 .

System Crystal Optic Axial

Habit S iqn
Fludrocortisone Orthorhombic Columnar +
Fludrocortisone
acetate Tetraqonal Columnar - 00
Refractive Indexes
optic Orientation a (w) B (5) Y
Fludrocortisone xxll c 1.575 1.588 1.646
~ al l
2 2 I1 b
Fludrocortisone
acetate w II a 1.604 1.538 --
t IIC
Photomicrographs of the two crystals also were presentedl4.

29 1
 FLAUS FLOREY

The existance of several polymorphs has already

been reported in section 2.1. A s many as six may
exist, according to Kuhnert-Brandstaetter and
Gasser15 but classification of the polymorphic
conditions proved very difficult since except for
modification I (m.p. 225-233OC), all other modi-
fications show only very slight differences in
their melting temperatures which are in the
205-215O range. Modification I11 and IV were
not obtained in pure form. A further complica-
tion is the formation of solvates from a variety
of solvents (see section 2.8). The powder X-ray
diffraction pattern of fludrocortisone acetate
(Form A ) is presented in table 3l6:
Table 3
Relative Re lative
.d- Intensity** d Intensity
12.40 0.07 3.77 0.23
9.10 0.16 3.70 0.16
8.70 0.10 3.56 0.16
7.40 0.15 3.53 0.13
6.80 0.28 3.44 0.12
6.50 0.59 3.29 0.32
6.30 0.65 3.20 0.13
6.20 0.98 3.10 0.18
5.78 0.40 3.03 0.18
5.62 1.00 2.89 0.13
5.49 0.17 2.81 0.18
5.15 0.35 2.69 0.13
4.80 0.35 2.55 0.15
4.62 0.35 2.45 0.12
4.50 0.63 2.39 0.09
4.33 0.15 2.32 0.20
4.20 0.18 *d= (interplanar distance)ni'L
4.12 0.29 Asin 0
-
4.02 **based on highest intensit
3.94 0.12 3
of 1.00 Radiation: k a l an
3.83 0.17 Ka, Copper
1nstrument:Phillips

292
 F LUDROCORTISON E ACETATE

3. Synthesis
F l u d r o c o r t i s o n e A c e t a t e (Fig. 4 ) was f i r s t
s y n t h e s i z e d by F r i e d and Sabo’ by t r e a t m e n t of
t h e epoxide 111 w i t h hydrogen f l u o r i d e . Compound
VII h y d r o c o r t i s o n e a c e t a t e ) was found a s a
byproduct of t h e r e a c t i o n 5 , 7 9 4 2 . Other
approaches r e p o r t e d a r e i n t r o d u c t i o n of t h e
4-double bond v i a bromination ( I V and V ) , a l b e i t
i n low y i e l d 1 7 , and osmium t e t r o x i d e o x i d a t i o n
of t h e A17(20) p r e c u r s o r ( V I ) 1 8 . I t can be
p u r i f i e d from V I I v i a t h e benzene adduct19. A
method f o r t h e p r o d u c t i o n of dense c r y s t a l h a s
been p a t e n t e d z 0 . I t can be d e a c e t y l a t e d t o
f l u o r o h y d r o c o r t i s o n e (11)5. I t can s e r v e a s
s t a r t i n g m a t e r i a l f o r 9a-fluoro-prednisolone
(cf. r e f . 2 1 ) . For m i c r o b i o l o g i c a l c o n v e r s i o n
t o t r i a m c i n o l o n e see s e c t i o n 5 . 2 .

4. Stability-Degradation
F l u d r o c o r t i s o n e a c e t a t e i s very s t a b l e a s a
s o i i d . I n aqueous and a l c o h o l i c s o l u t i o n s t h e
a - k e t o l s i d e c h a i n , a s i n a l l such c o r t i c o s t e r o i d q
i s prone t o o x i d a t i v e rearrangement and degrada-
t i o n a t a l k a l i n e pH*s.

I t h a s been r e p o r t e d 2 2 t h a t h y d r o c o r t i s o n e
and p r e d n i s o l o n e , when exposed t o u l t r a v i o l e t
l i g h t or ordinary fluorescent laboratory lighting
i n a l c o h o l i c s o l u t i o n s , undergo p h o t o l y t i c
d e g r a d a t i o n of t h e A-ring. Since fludrocortisone
a c e t a t e h a s t h e same A-ring a s h y d r o c o r t i s o n e i t
p r o b a b l y a l s o i s l a b i l e under t h e s e c o n d i t i o n s .

5. Drug Metabolism
5 . 1 Pharmocokinetics
The d i s t r i b u t i o n i n r a t t i s s u e s and
o r g a n s was s t u d i e d w i t h t r i t i u m l a b e l e d f l u d r o -
cortisone23. The k i n e t i c s of metabolism were
determined i n man, dog, r a t , monkey, and g u i n e a

293
 CH2OCOCH3
H20COCH3
I CH2OCOCH3
c=o I CH20COCH3
H20COCH3
c=o CH20COCH3

I11
I11
VI
w VI
\D
P
w C H OCOCH3
\D
P
L O C H OCOCH3
L O

Br 0
Br 0
I R=COCH3
Br I R=COCH3
V I1 R=H VI I
Br
V I1 R=H VI I
Figure 4
Figure 4
 FLUDROCORTISONE ACETATE

pig after I . V . and intraduodenal administration.

Depending on species,50% or more of the
steroid remained unchanged 30 minutes after
adminis tration24. Fludrocortisone and it' s
acetate had the same pharmocokinetic profile in
dogs. The blood level reached a peak between 4
and 8 hours25. Silber26 found that introduction
of fluorine at position 9 prolonged the plasma
half-life and depressed urinary excretion after
oral and I . V . administration to dogs as compared
to hydrocortisone. Disappearance of fludro-
cortisone acetate after incubation with rat
liver slices27-31 or perfusion of rat liver32
was a l s o studied.

5.2 Metabolic Products

After incubation of fludrocortisone
W ith rat liver slices S ~ h r i e f e r sidentified
~~
9a-f luoro-5@-pregnan-l1@, 17a, 21-trihydroxy-
3,20-dione and 9a-fluoro-5@-pregnan-3@, ll@, 17a,
21-tetrahydroxy-20-one. There was no evidence
for 5a-or 20-hydroxy metabolites. Bush and
Mahesh3' identified the following metabolites in
human urine:
9a-Fluoro-3a,lip, 17a,20,21 pentahydroxy-5@-
pregnane
9a-Fluoro-3a,ll~,17a,20,21 pentahydroxy-58-
pregnane
9a-Fluorotetrahydrocortisol
9a-Fluoroallotetrahydrocortisol
9a-Fluoro-20,20-dihydrocortisol
9a-F 1uorocortiso1
9a-Fluoro-ll@-hydroxyetiocholanolone
9a-Fluoro-11B-hydroxyandrostanone

Bush and M a h e ~ hnoted

~ ~ the far greater
proportion of 5a-(H) steroids than found with the
halogen-free parent steroid. The expected
11-ketone steroids were completely absent.

295
 KLAUS FLOREY

5.3 Microbiological Transformation

The f o l l o w i n g m i c r o b i o l o g i c a l t r a n s -

genation 9
l a t i ~ n(see
',
f o r m a t i o n s o f f l u d r o c o r t i s o n e and i t ' s a c e t a t e
have been r e o r t e d : 1 - h y d r o x y l a t i on35, l-dehydro-
6 -hydroxy 1a t i o n 38, 16-hydroxy-
~ ~a l s o under T r i a m c i n o l o n e ,
A n a l y t i c a l P r o f i l e s o f Drug S u b s t a n c e s , V o l . l ) ,
and 20-carbonyl r e d u c t i o n t o 20-hydroxy140. For
t r a n s f o r m a t i o n by mixed c u l t u r e s see r e f . 41.

6. Methods of A n a l y s i s
6 . 1 Elemental A n a l y s i s r

Element % Theory Repor t e d 3

C 65.39 65.32
H 7.39 7.26
F 4.52 4.50

6.2 Direct Spectrophotometric Assay

The u l t r a v i o l e t a b s o r p t i o n band a t
238 nm ( s e e 2 . 3 ) i s due t o t h e a , @ u n s a t u r a t e d
k e t o n e of t h e A-ring. The a b s o r b a n c e i s u s e f u l
a s a measure of p u r i t y from e x t r a n e o u s m a t e r i a l s
and h a s been s o used8, a l b e i t a t 242 nm.

6.3 C o l o r i m e t r i c Methods
A number of c o l o r i m e t r i c methods f o r
i d e n t i f i c a t i o n , d i f f e r e n t a t i o n from o t h e r
s t e r o i d s and q u a n t i t a t i o n have been a p p l i e d t o
fludrocortisone acetate. Based on r e a c t i o n w i t h
t h e A-ring a r e t h e i ~ o n i a z i d k ~ ~max
( 382 nm i n
e t h a n o l ) and 2,4-dinitrophenylhydra~ine~~methods.
Based on r e d u c t i o n of t h e d i h y d r o x a c e t o n e
sidechain a r e the blue tetrazoliumYO, Porter-
S i l b e r 3 O , and Nessler' s r e a g e n t 4 4 methods. A
b l u e chromogen ( k m a x 625 nm) i s produced b y
reacting fludrocortisone acetate with
2,6-di-tert-butyl-p-cresol i n a l k a l i n e s o l u t i o n45 .
R e a c t i o n s w i t h a p h e n o l , hydroquinone,phosphoric-
s u l f u r i c a c i d m i x t u r e (amber c o l o r ) 4 6 , p - n i t r o so-

296
 FLUDROCORTISONE ACETATE

d i m e t h y l a n i l i n e ( 2max 650 nm) *’ and a s u l f u r i c

acid, f r u c t o s e , c y s t e i n e m i x t u r e ( 2 max 548 nm)
have been d e s c r i b e d . The l a s t r e a c t i o n h a s a l s o
been u s e d t o d e t e r m i n e s g s i d u a l f l u d r o c o r t i n s o n e
i n fermentation broths. Chromo ens are a l s o
formed i n c o n c e n t r a t e d s u l f u r i c 53 and p h o s p h o r i c
acids. 5 1
6.4 Polarpgraphic Analysis
CohenJL s u b j e c t e d f l u d r o c o r t i s o n e t o
p o l a r a g r a p h i c r e d u c t i o n i n dime t h y 1f ormamide
and found two r e d u c i n g waves:

Wave 1 Wave 2
E 1/2 (Volts vs.
mercury p o o l anode) 1.66 2.10
Id (Diffusion
current constant) 1.4 1.8
n (Apparant number
of e l e c t r o n s t r a n s f e r r e d 0.0 3 0.69

6.5 Chromatographic A n a l y s i s

6.51 P a p e r Chromatographic A n a l y s i s
For p a p e r chromatographic
s y s t e m s , see Table 4 .

297
 Table 4

System # Solvent System Developing Time Rf Values Ref.

(hrs)
1 Formamid/Chloroform 18 - 33
2 Methanol/Water/Benzene 1:1:2 4 - 33
3 Methanol/Water/Ethylacetate/
Benzene 25:25:2.5:47.5 4 - 33
4 Propylene glycol/Toluene 96 - 29
5 Benzene/Formamide -- - 53
6 Toluene/Heptane, Methanol/Water
5:5:7:3 -- 0.27 53
7 Benzene/Methanol/Water 2:l:l -- 0.9 53
8 Petroleum ether (b.p. 100-120°)
Toluene/Methanol/Water
67:33 :85: 15 -- - 53
9 Benzene/Ethanol/Water 2:1:2 5 0.9 54
10 Toluene/Petr. ether (b.p. 30-60°),
Methanol/Water 12:8:13:7 2- 1/2 0.35 54
11 Benzene/Petr. ether (b.p. 90-looo),
Methanol/Water 5:5:7:3 2-1/2 0.18 54
12 Methyl isobutyl ketone/Formamide
20:l 2-1/2 0.87 55
 FLUDROCORTISONE ACETATE

The following detection systems have been

reported:
Detection Systems Ref.
u.v. 33
Tetrazolium 33,34
Phosphoric acid fluorescence 33
2,4 Dinitrophenylhydrazine 53
Tollen's Reagent 53
Isonicotinic acid hydrazide 55

System #12 can be used to separate fludro-

cortisone acetate (Rf 0 . 8 7 ) from fludrocortisone
(Rf 0.68) and 16a-hydroxyfludrocortisone
(Rf 0 . 3 0 ) . It can be used for the quantitative
determination of fludrocortisone acetates5 by
dissolving the ground tablets in dimethylforma-
mide, spotting approx 100 mcg. on filter paper
impregnated with formamide-methanol 20:80,
developing with methyl isobutylketone-formamide
20:1, elution, reaction with isonicotinic acid
hydrazide and determination of the absorbance at
4 1 5 nm against a standard, usinz6the genera1
procedure of Roberts and Florey .
6.52 Thin Layer Chromatographic
Analysis
Experience with the thin layer
chromatography of fludrocortisone acetate is
summarized in Table 5.

299
 Table 5
Rf or "running distance" values
(for explanation of individual values, see below)
Sys tem 1 A B C D E a b C
Fludrocortisone
Acetate 0.87 1.031.14 1.16 2.6 0.62 0.48 0.42 0.31
Fludrocortisone - 0.24 0.33 0.63 0.70 0.01 -- -- --
S y s tem I I1 I11 IV V VI
Fludrocortisone
Acetate -- -- -- -- 0.55 0.90
Fludrocortisone 0.49 0.46 0.29 0.71 -- 0.76

w
0
System 157: Kieselguhr G plate; Dichloroethane/methylacetate/water 2: 1: 1:
Spray reagent:Alkaline 2,5-diphenyl-3(4-styrylphenyl)tetrazolium solution;
''Running distances" values related to cortisone acetate = 1 . 0 0 ;
Systems A-E58:Kieselguhr GF 254 plates; Spray reagent:Tetrazolium blue:
"Running distance" values: A,B,C,E related to hydrocortisone acetate=1.00
D related to hydrocortisone = 1.00
Solvent systems: A- 1,2-Dichloroethane:methanol:water 95:5:0.2
B- 1,2-Dichloroethane:2-methoxyethyl acetate:water 80:20:1
C- Cyc1ohexane:ethylacetate:water 25:75:1
D- Stationary phase: 20% v/v formamide in acetone
Mobile phase:Chloroform:ether:water 80:20:0.5
E- Stationary phase: 25% v/v formamide in acetone
Mobile phase:Cyclohexane:tetrachloroethane:water
50:50:0.1
 FLUDROCORTISONE ACETATE

S y s t e m s a-c59: K i e s e l g u h r G p l a t e s : S p r a y
r e a g e n t : T e t r a z o l i u m b l u e . V a l u e s g i v e n a r e Rf
values.

S o l v e n t systems: a -
methylene c h 1 o r i d e : t o l u e n e
60:40
b - methylene c h l o r i d e : t o l u e n e
50:50
c - c h l o r o f o r m : t o l u e n e 25: 75

Systems I - 1760:
S i l i c a g e l p l a t e s , Spray reagent: Vanillin-
p e r c h l o r i c a c i d sprayed over tetrazolium reagent.
V a l u e s g i v e n a r e Rf v a l u e s .
S o l v e n t system I - E t h y l a c e t a t e
I1 - Methylene ch1oride:dioxan:
water
I11 - C h l o r o f o r m - e t h e r - w a t e r
(80:25:0.5) on formamide
plate
I V - A m y l a c e t a t e - a c e t o n e 1:1
V - Ether

System ~ 1 ~ 5 ,
S i l i c a g e l GF P l a t e , U.V. d e t e c t i o n o r e l u t i o n
and r e a c t i o n w i t h N y d r a z i d .
S o l v e n t : Ether-dimethylformamide, a c e t o n e ,
m e t h a n o l 88:8:2:2. I n t h i s system A8~14-hydro-
c o r t i s o n e a c e t a t e h a s an R v a l u e of 0 . 8 3 i n
relation t o fludrocortisone acetate.

6.6 Bioassay
A s e n s i t i v e b i o a s s a y i s b a s e d on t h e
u r i n a r y Na+/K+ r a t i o , e x p r e s s e d a s p e r c e n t o f t h e
c o n t r o l v a l u e a f t e r i n j e c t i o n of f l u d r o c o r t i s o n e
a c e t a t e i n t o adrenalectomized rats61.

30
 FLAUS FLOREY

6.7 Other
Bismuth oxidation to the corresponding
17-ketosteroid has a l s o been used as the basis
for an analytical method62.

7. Determination in Body Fluids and Tissues

References mentioned earlier, can be
summarized as follows:

References :
Thin Layer Chromatography 2
Paper Chromatography 29,33,34
Colorimetric 25,30,31,49
Bioassay 61

8. Determination in Pharmaceutical Preparations

The following references specifically
mention analysis in pharmaceuticals.

References :
Paper Chromatography 55
Colorimetric 8,45,47

302
 FLUDROCORTISONE ACETATE

9. References

1. R. J. M e s l e y , S p e c t r o c h i m i c a A c t a 2 2 , 8 8 9
(1966).
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76, 1 4 5 5 ( 1 9 5 4 ) a n d i b i d . 79, 1130 ( 1 9 5 7 ) .
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2,957,013 (1960).

303
 KLAUS FLOREY

21. J. F r i e d , K. F l o r e y , E . F. Sabo, J. H. Herz,

A. R. Restivo,A. Borman and F. M. S i n g e r ,
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27. G. M. Reaven, E n d o c r i n o l o g y 57, 5 8 0 ( 1 9 5 5 ) .

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-
26,974 ( 1 9 6 1 ) .

304
 F LUDROCORTISONE ACETATE

39. R. W. Thoma, J. F r i e d , S. Bonanno, and

P. Grabowich, J. Am. Chem. SOC. 79,
4818 ( 1 9 5 7 ) .
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-
79,4476 ( 1 9 5 7 ) .
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2 3 960 ( 1 9 5 8 ) .
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48, 528 ( 1 9 5 9 ) .
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305
 KLAUS FLOREY

J. S. Wragg, J. Pharm. Pharmacol. S u p p l .

-
16, 11T (1964).
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L i t e r a t u r e s u r v e y e d t h r o u g h July 1972.

306
 FLURAZEPAM HYDROCHLORIDE

Bruce C. Rudy and Bernard Z. Senkowski

Chemistry reviewed by R. I. Fryer.

 BRUCE C. RUDY A N D BERNARD Z . SENKOWSKI

INDEX

Analytical Profile - Flurazepam Hydrochloride

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
2.21 Proton Spectrum
2.22 19F Spectrum
2.3 U1 traviolet Spectrum
2.4 Fluorescence Spectrum
2.5 Mass Spectrum
2.6 Optical Rotation
2.7 Melting Range
2.8 Differential Scanning Calorimetry
2.9 Thermal Gravimetric Analysis
2.10 Solubility
2.11 X-ray Crystal Properties
2.12 Dissociation Constant
3. Synthesis
4. Stability Degradation
5. Drug Metabolic Products
6. Methods of Analysis
6.1 Elemental Analysis
6.2 Fluorine Analysis
6 . 2 1 Organically Bound Fluorine Analysis
6.22 Free Fluoride Analysis
6.3 Thin Layer Chromatographic Analysis
6.4 Gas-Liquid Chromatographic Analysis
6.5 Polarographic Analysis
6.6 Direct Spectrophotometric Analysis
6.7 Colorimetric Analysis
6.8 Fluorimetric Analysis
6.9 Titrimetric Analysis
7 . Acknowledgement
8. References

308
 F LURAZEPAM HYDROCHLORIDE

1. D e s c r i p t i o n

1.1 Name, Formula, Molecular Weight

Flurazepam h y d r o c h l o r i d e is 7-chloro-1-(2-
[diethylamino] e t h i l ) -5~(o-fluorophenyl)-1,3-dihydro-2_TI-l,4-
benzodiazepin-2-one d i h y d r o c h l o r i d e .

C21H23C1FN30.2HC1 Molecular Weight: 460.83

1.2 Appearance, Color, Odor

Flurazepam h y d r o c h l o r i d e o c c u r s a s a n o f f - w h i t e
t o yellow, n e a r l y o d o r l e s s , c r y s t a l l i n e powder.

2. Physical Properties

2.1 I n f r a r e d Spectrum
The i n f r a r e d spectrum of flurazepam h y d r o c h l o r i d e
is p r e s e n t e d i n F i g u r e 1 (1). The spectrum was measured
with a Perkin-Elmer 621 Spectrophotometer i n a KBr p e l l e t
c o n t a i n i n g 1 . 0 mg of flurazepam hydrochloride/300 mg of
KBr. The assignments f o r t h e c h a r a c t e r i s t i c bands i n t h e
i n f r a r e d spectrum a r e l i s t e d i n Table I ( 1 ) .

Table I
I n f r a r e d Assignments f o r Flurazepam Hydrochloride
Frequency (cm-l) Characteristic of

3066 a r o m a t i c CH s t r e t c h i n g
vibrations
2500 h y d r o c h l o r i d e of t e r t i a r y
amine
1683 C-0 s t r e t c h i n g v i b r a t i o n s
1560 and 1483 aromatic r i n g
748 4 a d j a c e n t H ' s on phenyl r i n g

309
&I
&I-
s-
m-
cu--
0-
a--
a-
Ic--
a--
m--
t--

?:
-
m-
-
310
 FLURAZEPAM HYDROCHLORIDE

2.2 Nuclear Magnetic Resonance Spectrum (NMR)

2.21 P r o t o n Spectrum
The p r o t o n NMR s p e c t r a shown i n F i g u r e 2
were r u n on a J e o l c o 60 MHz NMR u s i n g 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 ( 2 ) . The flurazepam h y d r o c h l o r i d e
spectrum, F i g u r e 2A, was o b t a i n e d by d i s s o l v i n g 59.0 mg of
sample i n 0.5 m l of methanol-d4. The s p e c t r a l a s s i g n m e n t s
are l i s t e d i n T a b l e I1 ( 2 ) . The s o l v e n t peak f o r methanol-
d4 o c c u r s a b o u t 3.27 ppm and i n t e r f e r e s w i t h t h e a s s i g n -
ments i n t h a t r e g i o n . T h e r e f o r e , t h e spectrum of f l u r a z e -
Pam b a s e (54.2 mg/0.5 m l CDC13), shown i n F i g u r e 2B, w a s
determined and t h e s p e c t r a l assignments p r e s e n t e d i n T a b l e
I1 ( 2 ) .

2.22 19F Spectrum

The 1yF spectrum shown i n F i g u r e 2C w a s
o b t a i n e d w i t h a J e o l c o C-60 HL i n s t r u m e n t w i t h a 19F module
c r y s t a l modified t o a f r e q u e n c y of 56.446 MIz. Two hundred
mg of flurazepam h y d r o c h l o r i d e were d i s s o l v e d i n 0 . 5 m l of
methanol c o n t a i n i n g CC13F as t h e i n t e r n a l r e f e r e n c e ( 2 ) .
The spectrum c o n s i s t s of a q u i n t e t a t -108 ppm. The c h o i c e
of CC13F as t h e i n t e r n a l r e f e r e n c e a l o n g w i t h t h e a s s i g n -
ment of -108 ppm i s i n accordance w i t h Bovey ( 3 ) .

T a b l e I1

NMR Assignments f o r Flurazepam and Flurazepam Hydrochloride

No. of Chemical S h i f t
Proton Protons (ppm) Multiplicity

Flurazepam Hydrochloride
a 6 1.40 Triplet
(JH,-H~ = 7Hz)
b 6 ~3.42 Mu 1t i p 1et
C 2 '~4.50 Triplet

311
 BRUCE C. RUDY AND BERNARD 2. SENKOWSKI

Figure 2

A. NMR Spectrum of Flurazepam Hydrochloride

B. NMR Spectrum of Flurazepam Base
C. 19F NMR Spectrum of Flurazepam Hydrochloride

312
 FLURAZEPAM HYDROCHLORIDE

No. o f Chemical S h i f t
Pro t o n Pro t o n s (pprn) Mu1t i p 1 i c i t y

d 2 ~4.50
e 2* 4.92 Sing1et
f 7 7.30-8.15 Mu1t i p l et

Flurazepam Base
a 6 0.95 Triplet
(J H ~ - H ~ =7.5Hz)
b 6 2.50 Quartet
( J H ~ - H ~7 =. 5 ~ 2 )
C 2 3.47-4.55 Mu 1t i p l e t
d 2 3.76-4.85 Two sets of
doublets
(J = 11 Hz)
f 6.95-7.80 Mu1t i p l e t

I9F Spectrum of Flurazepam Hydrochloride -108 Q u i n t e t

*Also any H20 p r e s e n t i n methanol-d4

2.3 U l t r a v i o l e t Spectrum (UV)

When t h e W spectrum of flurazepam h y d r o c h l o r i d e
w a s scanned from 450 t o 210 nm, t h r e e maxima and t h r e e
minima were observed. The maxima a r e l o c a t e d a t 362 nm
(E = 3.7 x lO3), 284 nm (E = 1.2 x 1041, and 239 nm (E =
2.8 x lo4). The m i n i m a o c c u r at 333 nm, 263 nm, and 219 nm.
The spectrum shown i n F i g u r e 3 was o b t a i n e d from a s o l u t i o n
of 1.006 mg of flurazepam h y d r o c h l o r i d e f 1 0 0 m l of a c i d i f i e d
methanol (2.8 m l of c o n c e n t r a t e d H2SO4 d i l u t e d t o 100 m l
w i t h anhydrous methanol) (4).

2.4 F1uorescenc.e Spectrum

The e x c i t a t i o n and emission s p e c t r a f o r f l u r a z e -
Pam h y d r o c h l o r i d e (1 mg/ml of methanol) a r e shown in F i g u r e
4 (5). One maximum appears i n t h e e x c i t a t i o n spectrum a t
378 nm and one maximum i n t h e e m i s s i o n spectrum a t 492 nm.

2.5 Mass Spectrum

The mass spectrum of flurazepam h y d r o c h l o r i d e w a s
o b t a i n e d u s i n g a CEC 21-110 mass s p e c t r o m e t e r w i t h an
i o n i z i n g energy of 7 0 ev. The o u t p u t from t h e mass s p e c t m -
meter was analyzed and p r e s e n t e d i n t h e form of a b a r

313
314
 tn
a
w
I-
w
I
0
z
4
z
1S N3lN I
A1
315
 7
t
si
R
I
P
fl
ar
a
.?I
k
A
0
H
E
w
8
0
rw
0
316
 F LURAZEPAM HYDROCHLORIDE

g r a p h , shown i n F i g u r e 5 , by a V a r i a n 1 0 0 MS d e d i c a t e d com-
p u t e r system. Due t o t h e e x t r e m e i n t e n s i t y of t h e b a s e
peak a t m / e 86, t h e r e l a t i v e i n t e n s i t y of t h e peaks a t t h e
h i g h e r mass u n i t s a r e v e r y weak. T h e r e f o r e , t h e peaks from
m / e 1 5 0 up were s u b j e c t e d t o a t e n f o l d s c a l e e x p a n s i o n
( F i g u r e 5 - i n s e r t ) ( 6 ) . The p a r e n t peak (+)a t m / e 387 is
due t o t h e free f l u r a z e p a m b a s e . The b a s e peak a t m / e 8 6
i s d u e t o t h e (C2H5)2NCH2 f r a g m e n t . The o t h e r m a j o r p e a k s
can b e a t t r i b u t e d t o s t e p w i s e f r a g m e n t a t i o n o f t h e p a r e n t
i o n ; i . e . , M+ - (C2H5)zN = 315, 315 - CH2N = 287 ( 6 ) .

2.6 Optical Rotation

Flurazepam h y d r o c h l o r i d e e x h i b i t s no o p t i c a l
activity.

2.7 M e l t i n g Range
Flurazepam h y d r o c h l o r i d e m e l t s w i t h d e c o m p o s i t i o n
w i t h i n a 5 O r a n g e between 208' and 218OC when t h e USP X V I I I
Class Ia p r o c e d u r e i s u s e d ( 7 ) .

2.8 D i f f e r e n t i a l Scanning C a l o r i m e t r y (DSC)

The t h e r m a l p r o p e r t i e s of f l u r a z e p a m hydrochloride
i n t h e m e l t i n g r e g i o n a r e v e r y dependent on p r e v i o u s
t h e r m a l h i s t o r y . Using a t e m p e r a t u r e program of 20°C/min.,
t h e e x t r a p o l a t e d o n s e t of a n e n d o t h e r m i c t r a n s i t i o n
o c c u r r e d a t 215 . a 0 C f o l l o w e d immediately by t h e e x o t h e r m i c
t r a n s i t i o n d u e t o d e c o m p o s i t i o n a t 229.5OC ( F i g u r e 6 ) . A t
a s c a n r a t e o f 10°C/min., a small endothermic t r a n s i t i o n
o c c u r s a t 203.3OC f o l l o w e d by sample d e c o m p o s i t i o n a t
217.5OC. Due t o t h e sample i n s t a b i l i t y i n t h e r e g i o n o f
t h e m e l t , t h e AHf was n o t d e t e r m i n e d ( 8 ) .

2.9 Thermal G r a v i m e t r i c A n a l y s i s (TGA)

A TGA performed a t a s c a n r a t e of 10°C/minute
showed l i t t l e w e i g h t loss f o r f l u r a z e p a m h y d r o c h l o r i d e from
ambient t o 190°C. A w e i g h t l o s s amounting t o a b o u t 70% of
t h e sample o c c u r r e d between 190 and 345OC (8).

2.lo Solubility
The s o l u b i l i t y d a t a f o r f l u r a z e p a m h y d r o c h l o r i d e
o b t a i n e d by e q u i l i b r a t i o n f o r 20 h o u r s a t 25OC are g i v e n i n
T a b l e 111 ( 9 ) .

3 17
 Figure 6

DSC Scan for Flurazepam Hydrochloride

d
240 230 220 210 200 190 I80 170 I
2 3
TEMPERATURE 'C

318
 FLURAZEPAM HYDROCHLORIDE

T a b l e I11
S o l u b i l i t y P r o f i l e f o r Flurazepam H y d r o c h l o r i d e
Solvent S o l u b i l i t y (mg/ml)
3A al c o h o l 28.3
benzene 0.4
chloroform 11.1
95% e t h a n o l 2 60
diethyl ether 0.2
m e thano 1 340
p e t r o l e u m e t h e r (3Oo-6O0) 0.2
2-propanol 14.6
water >500
2.11 X-ray C r y s t a l P r o p e r t i e s
The x-ray powder d i f f r a c t i o n p a t t e r n of f l u r a z e -
Pam h y d r o c h l o r i d e i s p r e s e n t e d i n T a b l e I V (10). The
i n s t r u m e n t a l c o n d i t i o n s a re g i v e n below.
Instrumental Conditions
General E l e c t r i c Model XRD-6 S p e c t r o g o n i o m e t e r
Generator : 50KV-12-1/2 MA
Tube t a r g e t : Copper
Radiation : Cu K, = 1 . 5 4 2 1
optics: 0.1' D e t e c t o r s l i t
M.R. S o l l e r s l i t
3' B e a m s l i t
0.0007 i n c h N i f i l t e r
4' t a k e o f f a n g l e
Goniometer : Scan a t O.Zo 28 p e r m i n u t e
Detector: Amplifier g a i n - 1 6 c o u r s e ,
8.7 f i n e
Sealed proportional counter
t u b e and DC v o l t a g e a t
plateau
P u l s e h e i g h t s e l e c t i o n EL -
5 v o l t s ; EU - o u t
Rate meter T . C . 4
2000 C I S f u l l s c a l e
Recorder: C h a r t Speed - 1 i n c h p e r 5
minutes
Samples p r e p a r e d by g r i n d i n g a t room t e m p e r a t u r e .

319
 BRUCE C. RUDY AND BERNARD 2. SENKOWSKI

Table I V
X-ray Powder D i f f r a c t i o n P a t t e r n of
Flurazepam H y d r o c h l o r i d e

-
20 d (8) * I/TE -
28 d (8) * I/TE
8.32 10.6 3 31.40 2.85 1
9.04 9.78 20 32.36 2.77 38
11.62 7.62 37 32.92 2.72 7
12.49 7.09 15 33 - 3 0 2.69 8
12.86 6.88 11 33.88 2.65 6
15.50 5.72 62 34.24 2.62 7
16.40 5.40 11 34.87 2.57 11
16.78 5.28 9 35.20 2.55 14
17 38 2.10 7 35.77 2.51 5
17.94 4.94 5 36.18 2.48 10
18.65 4.76 50 37.28 2.41 5
19.16 4.63 100 37.72 2.38 11
20.05 4.43 26 38.17 2.36 11
20.38 4.36 20 38.78 2.32 19
20.75 4.28 19 39.47 2.28 3
21.66 4.10 12 40.18 2.24 3
23.14 3.84 82 40.86 2.21 5
23.61 3.77 39 41.28 2.19 2
24.32 3.66 40 41.86 2.16 5
24.50 3.63 36 42.20 2.14 8
25.18 3.54 98 42.94 2.11 9
25.96 3.43 12 43.58 2.08 9
26.18 3.40 33 44.24 2.05 4
26.40 3.38 24 44.70 2.03 5
26.96 3.31 12 46.06 1.97 7
27.44 3.25 15 47.00 1.93 4
27.94 3.19 18 47.52 1.91 2
28.46 3.14 5 48.32 1.88 5
28.78 3.10 5 49.08 1.86 3
29.96 2.98 19 50.00 1.82 3
30.54 2.93 4 50.26 1.82 3
31.00 2.88 15 50.84 1.80 4

*d - (interplanar distance) nX
2 Sin 0

**I/Io= r e l a t i v e i n t e n s i t y (based on h i g h e s t i n t e n s i t y
of 1.00)

320
 FLURAZEPAM HYDROCHLORIDE

2.12 Dissociation Constant

The pKa's for flurazepam were determined spectro-
photometrically and found to be 1.90 5 0.05 and 8.16 5 0.05
(11). The apparent pKa2 has also been determined from the
titration curve in a 2-propano1:water (1:l) mixture and
found to be 7.0 5 0.1 (11). In water, the trialkylamino
type compounds are stronger bases, on the average, by 0.9
pK units (11,12). Therefore, the estimated pKa2 in water
would be 7.9 which is in good agreement with that found
spectrophotometrically.

3. Synthesis
Flurazepam hydrochloride may be prepared by the re-
action scheme shown in Figure 7. 7-Chloro-5-(o-fluoro-
phenyl)-1,3-dihydro-2H-l,4-benzodiazepin-2-one is reacted
with diethylaminoethyl chloride in the presence of sodium
methoxide to yield flurazepam which is then converted to
flurazepam hydrochloride by the addition of hydrochloric
acid (13). A complete review of the chemistry of benzo-
diazepines by Archer and Sternbach presents several path-
ways to arrive at the basic benzodiazepine ( 1 4 ) .

4. Stability Degradation
When sealed amber ampuls with dilute solutions of
flurazepam hydrochloride in 0.1N HC1, water, and 0.1N NaOH:
3A alcohol (1:l) were heated in a boiling water bath for
one hour, the degradation products shown in Figure 8 were
observed by thin layer chromatography (15). In 0.1N HC1
solution the main hydrolysis product was 5-chloro-2-(2-
diethylaminoethylamino)-2'-fluorobenzophenone hydrochloride.
In aqueous solution the main degradation product was 7-
chloro-5-(o-fluorophenyl)-ly3-dihydro-2H-l,4-benzodiazepin-
2-one. Finally, in the 0.1N NaOH:3A alcohol (1:l) solu-
tion, the main degradation products were 5-chloro-2-(2-
diethylaminoethylamino)-2'-fluorobenzophenone ,and 2-chloro-
l0-(2-diethylaminoethyl)-9-acridone. When a solution of
flurazepam hydrochloride in water in irradiated with light
from a high pressure U.V. lamp for 3 hours, some hydrolysis
to 5-chloro-2-(2-diethylaminoethylamino)-2'-fluorobenzo-
phenone occurs (16). Flurazepam hydrochloride, when stored
in well closed, light resistant containers, is quite
stable.

321
 Figure 7

S y n t h e s i s f o r Flurazepam Hydrochloride

H
CH2CH3
CI
+ ClCH CH N(
* CH2CH3
Naoc%
_____)

7-CHLORO-5-(&FLUORO- DIE THYLAMINO-

PHENYL )-I ,3-DI HYDRO- 2 H-1.4- ETHYLCHLORIDE
BENZODIAZEPINE- 2-ONE

CIK T=C
" H

FLURAZEPAM FLURAZEPAM HYDROCHLORIDE

 Figure 8

Major D e g r a d a t i o n P r o d u c t s f o r Flurazepam H y d r o c h l o r i d e
i n Acid, Basic, and Aqueous S o l u t i o n s

&\ FLURAZEPAM HYDROCHLORIDE

nw
6
$H2CHZ"C2H32

C
&?.ti2

bf
CIa-
6
CH2CH2NlC2H&,
I
+ fJ?n
CI

8
CH CH N(C2H5)2
1 2 2

BENZOPHENONE 7-CHLORO-54 O-FLUOR& BENZOPMENONE ACRIDONE

PHENYLkt3-DIHYDRO -2 H-
I.4-BENZWUZEPIN-2-ONE
 BRUCE C. RUDY AND BERNARD 2. SENKOWSKI

5. Drug Metabolic Products

The metabolic pathways of flurazepam in dog and man are
shown in Figure 9 (17-22). Initially, five metabolites
plus the intact drug were found in the urine of dogs. Four
of these metabolites were identified as monodesethyl-
flurazepam, didesethylflurazepam, flurazepam-N1-ethanol,
and N1-desalkyl-3-hydroxyflurazepam (17,ZO). The fifth
metabolite was tentatively identified as a phenolic deriva-
tive of N1-desalkyl-flurazepam by mass spectrometry (17).
The major metabolite found in the dog urine is flurazepam-
N1-acetic acid (17,19). In human urine, the flurazepam-
N1-ethanol was the major metabolite along with smaller
quantities of the mono- and didesethyl-flurazepam and N1-
desalkyl-3-hydroxyflurazepam (17-22).

6. Methods of Analysis
6.1 Elemental Analysis
The results from the elemental analysis are
listed in Table V (23).

Table V

Elemental Analysis of Flurazepam Hydrochloride

Element X Theory % Found

C 54.74 54.73
H 5.47 5.46
N 9.12 9.11
F 4.12 4.22
c1 7.69 7.86
Cl-(ionic) 15.38 15.42

6.2 Fluorine Analysis

6.21 Organically Bound Fluorine Analysis

There are several methods available to
determine the amount of carbon-bonded fluorine. One of the
earlier methods employed the Schoniger Combustion technique
followed by thorium nitrate or cerous chloride titration
using sodium alizarin sulfonate or murexide as the indi-
cator (24).
With the advent of good specific ion

324
 Figure 9

Metabolic Products of Flurazepam Hydrochloride

H

NI-DESALKYL- FLURAZEPAM
FLURUEPAY Nl-ETHAWL

- \ CHpCOOH

ac=;.I.
I
N-C-0
Cl &o
:" cgfa a

&F
N,-DESALKYL-3-
OH

N I -DESALKYL
8"
FLURAZEPAY
HYDROXYFLURAZEPAM FLURAZEPAM PHENOL NI-ACETIC ACD
 BRUCE C. RUDY AND BERNARD 2.SENKOWSKI

e l e c t r o d e s , methods were developed t o l i b e r a t e t h e bound

f l u o r i n e and d i r e c t l y measure t h e f l u o r i d e c o n c e n t r a t i o n .
The r e a g e n t , s o d i u m b i p h e n y l , followed by o x i d a t i o n w i t h
hydrogen p e r o x i d e , is used t o l i b e r a t e t h e o r g a n i c a l l y
bound f l u o r i n e i n flurazepam h y d r o c h l o r i d e . A f l u o r i d e i o n
s p e c i f i c e l e c t r o d e i s used, i n t h e p r e s e n c e of a high-
i o n i c - s t r e n g t h b u f f e r s o l u t i o n , f o r d i r e c t measurement of
t h e l i b e r a t e d f l u o r i d e (25).
The l a s t method t o b e p r e s e n t e d f o r t h e
a n a l y s i s of t h e carbon-bonded f l u o r i n e i n flurazepam hydro-
c h l o r i d e i s 19F Nuclear Magnetic Resonance s p e c t r o m e t r y (2).
A flurazepam h y d r o c h l o r i d e r e f e r e n c e s t a n d a r d and an i n t e r -
n a l s t a n d a r d , r e a g e n t g r a d e o-fluorobenzoic a c i d , a r e d i s -
solved i n methanol and t h e I 9 F spectrum o b t a i n e d and i n -
t e g r a t e d . From t h i s d a t a an i n t e r n a l s t a n d a r d f l u o r i n e
conversion f a c t o r c a n b e c a l c u l a t e d and used t o d e t e r m i n e
t h e amount of f l u o r i n e p r e s e n t i n a sample of flurazepam
h y d r o c h l o r i d e t h a t i s r u n i n a s i m i l a r manner (26).

6.22 F r e e F l u o r i d e Analysis
The d e t e r m i n a t i o n of any f r e e f l u o r i d e
p r e s e n t i n flurazepam h y d r o c h l o r i d e b u l k samples i s c a r -
r i e d o u t by d i r e c t measurement u s i n g a f l u o r i d e s p e c i f i c
i o n e l e c t r o d e . The measurements a r e made i n an a c e t a t e
b u f f e r s o l u t i o n (pH 5 . 3 ) . The e l e c t r o d e r e s p o n s e w a s
found t o b e l i n e a r throughout t h e working range of 0.08 t o
0.20 mg of F-/100 m l of s o l u t i o n ( 2 7 ) .

6.3 Thin Layer Chromatographic Analysis (TLC)

S e v e r a l TLC systems f o r t h e s e p a r a t i o n of
flurazepam h y d r o c h l o r i d e from its m e t a b o l i t e s and s i m i l a r
s t r u c t u r e d compounds are g i v e n i n Table V I . I n each c a s e
t h e sample i s s p o t t e d on a s i l i c a g e l GF p l a t e * which is
allowed t o develop i n a s a t u r a t e d t a n k u n t i l t h e s o l v e n t
f r o n t h a s ascended a b o u t 15 cm. The p l a t e is t h e n removed,
a i r d r i e d , and viewed under shortwave and longwave U.V.
radiation.
*I f t h e sample s o l u t i o n is t o o a c i d i c , an a r t i f a c t a p p e a r s
a t t h e p o i n t of a p p l i c a t i o n due t o t h e quenching of t h e
phosphor i n t h e s i l i c a g e l GF p l a t e by t h e a c i d .

326
 FLURAZEPAM HYDROCHLORIDE

Table VI
Rf Values for Flurazepam in Various Developing Solvents
Solvent System R Value Reference
-
diethyl ether:diethylamine (75:2) 0.6 28
methylene ch1oride:ethyl ether:
methano1:conc. ammonium hydroxide
(240:360:8:3) 0.2 28
ethyl acetate:conc. ammonium
hydroxide (200:1) 0.14 20
ethyl acetate: ethanol:conc. ammonium
hydroxide (100:10:0.3) 0.38 20
benzene:methanol:glacial acetic acid
(9:1:1) 0.26 20
chloroform:acetone (17 :3) 0.00, 0.05* 20
*When the plate is developed two times in the same system

6.4 Gas-Liquid Chromatographic Analysis (GLC)

The acid hydrolysis of blood extracts containing
flurazepam and its metabolites has been used by deSilva
et al. (29) to prepare the respective benzophenones as
volatile derivatives for gas chromatography. This method
is an adaptation of the method developed for GLC of diaze-
pam and its metabolites (30). When the benzophenones were
chromatographed at 21OoC on a 2 feet x 114 inch column
containing 2% Carbowax 20M-TPA, they showed an excellent
response to detection by electron capture which was linear
between 10 and 40 ng. The main disadvantage of hydrolysis
to the benzophenone is the lack of specificity for a given
benzodiazepine.
A method recently published by Sine et al. (31)
for chromatographing flurazepam directly, utilizes a
3 feet x 2 mm glass column packed with 3.8% SE-30 on
Chromosorb W (AW-DMCS, 80-100 mesh). The GLC is equipped
with a hydrogen flame ionization detector and the column
temperature is about 23OoC. The patient's serum is
adjusted to pH 7.4 and extracted with chloroform. The
chloroform is evaporated, the residue is dissolved in
acidic methanol (1 ml HCl/liter methanol) and chromato-
graphed.

327
 BRUCE C. RUDY AND BERNARD 2. SENKOWSKI

This method w i l l s e p a r a t e flurazepam from diazepam and

chlordiazepoxide.

6.5 P o l a r o g r a p h i c Analysis
P o l a r o g r a p h i c a n a l y s i s of flurazepam hydro-
c h l o r i d e h a s been c a r r i e d o u t i n Britton-Robinson B u f f e r
a t pH 4.4. The halfwave p o t e n t i a l o c c u r s a t -0.78 V.versus
a s i l v e r / s i l v e r c h l o r i d e r e f e r e n c e e l e c t r o d e and is pro-
p o r t i o n a l t o c o n c e n t r a t i o n . T h i s wave is a t t r i b u t e d t o the
r e d u c t i o n of t h e azomethine (,C=N) f u n c t i o n a l group and
v a r i e s w i t h pH ( 3 2 ) .

6.6 Direct Spectrophotometric Analysis

Direct s p e c t r o p h o t o m e t r i c a n a l y s i s is used t o
determine t h e q u a n t i t y of flurazepam h y d r o c h l o r i d e p r e s e n t
i n capsules. A q u a n t i t y of t h e c a p s u l e c o n t e n t s is at-
c u r a t e l y weighed and t h e flurazepam is e x t r a c t e d i n t o
a c i d i f i e d methanol ( s e e s e c t i o n 2 . 3 ) . The methanol s o l u -
t i o n is f i l t e r e d and a p p r o p r i a t e s u b d i l u t i o n s made t o y i e l d
a f i n a l s o l u t i o n c o n t a i n i n g 1 . 0 mg of flurazepam hydro-
c h l o r i d e p e r 100 m l of a c i d i f i e d methanol. The absorbance
of t h i s s o l u t i o n along w i t h a s o l u t i o n of flurazepam hydro-
c h l o r i d e r e f e r e n c e s t a n d a r d s i m i l a r l y prepared is measured
v e r s u s a c i d i f i e d methanol a t t h e 239 NO maximum. From t h i s
d a t a t h e c o n c e n t r a t i o n of flurazepam h y d r o c h l o r i d e i n t h e
c a p s u l e s is c a l c u l a t e d ( 3 3 ) .

6.7 C o l o r i m e t r i c Analysis
Flurazepam h y d r o c h l o r i d e forms a i o n - p a i r complex
w i t h bromocresol green i n a pH 5 . 3 b u f f e r . This c o l o r e d
complex is e x t r a c t e d i n t o chloroform and i t s absorbance
measured a t t h e 415 nm maximum. A p l o t of c o n c e n t r a t i o n
v e r s u s absorbance is l i n e a r from 0 t o 2.5 mg of flurazepam
h y d r o c h l o r i c p e r 100 m l of chloroform ( 3 4 ) .

6.8 F l u o r i m e t r i c Analysis
A f l u o r i m e t r i c a n a l y s i s f o r t h e d e t e r m i n a t i o n of
flurazepam h y d r o c h l o r i d e and i t s m e t a b o l i t e s i n blood and
u r i n e h a s been d e s c r i b e d by d e S i l v a and S t r o j n y ( 2 0 ) .
This a s s a y i n v o l v e s s e l e c t i v e e x t r a c t i o n i n t o d i e t h y l e t h e r
from blood b u f f e r e d t o pH 9 o r u r i n e made b a s i c w i t h NaOH,
then back-extracted i n t o 4N H C 1 , and hydrolyzed t o t h e
r e s p e c t i v e benzophenones. The benzophenones are t h e n
c y c l i z e d t o t h e 9-acridone d e r i v a t i v e s i n dimethylformamide

328
 FLURAZEPAM HYDROCHLORIDE

i n t h e p r e s e n c e of K2CO3. These d e r i v a t i v e s a r e s e p a r a t e d
by TLC, e l u t e d from t h e s i l i c a g e l , and t h e i r f l u o r e s c e n c e
determined i n methanol:O.lN HC1 ( 4 : l ) . T h i s method a l l o w s
q u a n t i t a t i o n i n t h e r a n g e of 0.003 t o 10.0 mcg of compound/
ml of blood o r u r i n e ( 2 0 ) .
6.9 T i t r i m e t r i c Analysis
Flurazepam h y d r o c h l o r i d e i s assayed by d i s s o l v i n g
about 0 . 6 gm of sample i n g l a c i a l a c e t i c a c i d , adding ex-
c e s s mecuric acetate, and t i t r a t i n g w i t h 0.1N 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 . The end-point is determined
p o t e n t i o m e t r i c a l l y u s i n g a g l a s s - c a l o m e l e l e c t r o d e system.
Each m l of 0.1N p e r c h l o r i c a c i d is e q u i v a l e n t t o 23.04 mg
of C21H23C1FN30*2HC1.

7. Acknowledgment
The a u t h o r s would l i k e t o thank D r . P. S o r t e r and t h e
S c i e n t i f i c L i t e r a t u r e Department as w e l l as t h e Research
Records Department of Hoffmann-La Roche I n c . f o r t h e i r h e l p
i n t h e l i t e r a t u r e search f o r t h i s Analytical Profile.

329
 BRUCE C. R U D Y A N D BERNARD Z . SENKOWSKI

8. References

1. Hawrylyshyn, M., Hoffmann-La Roche Inc., Personal

Communication.
2 . Johnson, J. H., Hoffmann-La Roche Inc., Personal
Communication.
3. Bovey, F. A . , NucZear Magnetic Resonance
Spectroscopy, Academic Press, New York City, pp.
211-214.
4 . Rubia, L. B., Hoffmann-La Roche Inc., Personal
Communication.
5. Boatman, J . , Hoffmann-La Roche Inc., Personal
Communication.
6. Benz, W., Hoffmann-La Roche Inc., Personal
Communication.
7. United S t a t e s Pharmacopeia XVIII, 935 (1970).
8. Moros, S . , Hoffmann-La Roche Inc., Personal
Communication.
9. MacMullan, E., Hoffmann-La Roche Inc., Personal
Communication.
10. Hagel, R. B., Hoffmann-La Roche Inc., Personal
Communication.
11. Toome, V. and Raymond, G., Hoffmann-La Roche Inc.,
Unpublished Data.
12. Gutbezahl, B. and Grunwald, E., J . Amer. Chem. Soc.,
75, 559 (1953).
-
13. Sternbach, L. H., Archer, G. A., Earley, J . V.,
Fryer, R. I., Reeder, E., Wasyliw, N., Randall, L.,
and Banziger, R., J. Med. Chem., 8_, 815 (1965).
14. Archer, G. A. and Sternbach, L. H., Frem. Review.,
68, 747 (1969).
15. Senkowski, B. Z., Hoffmann-La Roche Inc.,
Unpublished Data.
16. Fryer, R. I., Hoffniann-La Roche Inc., Unpublished
Data.
17. Schwartz, M. A . , Vane, F. M., and Postma, E., J .
Med. Chem., 11,770 (1968).
18. Usdin, E., PsychopharmaeoZ. BUZZ., 5, 4 (1970).
19. Schwartz, M. A. and Postma, E., J . .?harm. S c i . , 59,
1800 (1970).
20. deSilva, J . A . F. and Strojny, N., J . Pharm. S e i . ,
60, 1303 (1971).
- I
21. Randall, L. O., I n t . Symp. Benzodiazepines, Sum.,
Milan, Italy, 1971:Z.

330
 F LURAZEPAM HYDROCHLORIDE

22. S c h w a r t z , M. A . , I n t . Symp. Benzodiazepines, Sum.,

M i l a n , I t a l y , 1971:3.
23. S c h e i d l , F., Hoffmann-La Roche I n c . , P e r s o n a l
Communication.
24. Steyermark, A , , Quantitative Organic MieroanaZysis,
2nd Ed., Academic Press, New York, N. Y., 1 9 6 1 ,
pp. 326-332.
25. J o n e s , B. C . , Heveran, J . E . , and Senkowski, B . Z.,
J . Pharm. S c i . , 60, 1036 (1971).
26. Rudy, B. C. and Senkowski, B. 2. " A n a l y t i c a l
P r o f i l e of F l u o r o u r a c i l " , a c c e p t e d f o r p u b l i c a t i o n
i n AnaZyticaZ ProfiZes of Drug Substances, V o l . 2,
1972.
27. J o n e s , B. C . and Heveran, J . E . , Hoffmann-La Roche
I n c . , Unpublished Data.
28. Hochhauser, L . , Hoffmann-La Roche I n c . ,
Unpublished Data.
29. d e S i l v a , J. A . F . , Bader, G . , and Kaplan, J . ,
Hoffmann-La Roche I n c . , Unpublished Data.
30. d e S i l v a , J. 4. F . , S c h w a r t z , M. A . , S t e f a n o v i c , V.,
Kaplan, J . , and D'Arconte, L . , AnaZ. Chem., 36,
2099 (1964).
31. S i n e , H. E . , McKenna, M. J . , Law, M. R., and
Murray, M. H . , J . Chromatog. S c i . , 10 297 ( 1 9 7 2 ) .
32. L e v i n , M. , Hof fmann-La Roche I n c . , Unpublished
Data.
33. G u a s t e l l a , J . , Hoffmann-La Roche I n c . , Unpublished
Data.
34. Houghton, R. E . , Hoffmann-La Roche I n c . ,
Unpublished Data.

331
 IODIPAMIDE

Hyam Henry Lerner

 HYAM HENRY LERNER

Table of Contents

1. Description

1.1 Name, Formula, Molecular Weight

1.2 Appearance, Color, Odor

2. Physical Properties

2.1 Spectra

2.11 Infrared Spectra

2.12 Nuclear (Proton) Magnetic Resonance
2.13 U l t r a v i o l e t Spectra
2.14 Mass Spectrometry
2.2 Crystal Properties

2.21 Differential Thermal Analysis

2.22 Thermal Gravimetric Analysis
2.23 Melting Range
2.24 X-Ray Powder Diffraction

2.3 Solution Data

2.31 Solubility
2.32 Apparent Molecular Weight i n Solution
2.33 Isotonicity
2.34 pKa
2.35 pH
2.36 Index of Refraction
2.37 Phys icochemical Data

3. Synthesis

4. Stability

5. P u r i f i c a t i o n and Analysis f o r Impurities

5.1 Gel F i l t r a t i o n
5.2 Complexometric Methods of Separation
5.3 Countercurrent Distribution
5.4 Free Iodine and Free Halide
5.5 Free Amino Compounds

334
 IODIPAMIDE

5. (Cont'd.)

5.6 Free Adipic Acid

5.7 Determination of Water and Conditions f o r
Drying

6. Methods of Analysis

6.1 Elemental Analysis

6.2 I d e n t i f i c a t i o n Tests
6.3 Direct Spectrophotometric Analysis
6.4 Organically Bound Iodine
6.5 Polarograpliy
6.6 Chromatographic Analysis

6.61 Paper Chromatography

6.62 Thin-Layer Chromatography
6.63 E l e c t r o p h o r e t i c Analysis

6.7 X-Ray and B-Particle Dispersion Methods

6.8 Flame Photometry

7. Drug Metabolism
8. References

335
 HYAM HENRY LERNER

1. Description
1.1 Name, Formula, Molecular Weight
Iodipamide is N,N'-adipyl bis (3-amin0-2~4~6tri-
.
iodobenzoic acid) Chemical Abstract listings are under
the heading benzoic acid, 3,3' (adipyldiimino) bis [2,4,6
triiodol. Other derived chemical names are adipic acid di-
(3-carboxy-2,4,6 triiodoanilide; N,N'-di- (3-carboxy-2,4,6-
triiodopheny1)-adipamide and 3,3'- (adipoy1diimino)-bis t2,
4,6-triiodobenzoic acid].
Among the generic and trivial names for this com-
pound are iodipamic acid and adipiodon. Common trade names
are Biligrafin and Cholografin.

Iodipamide was officially recognized in "National

Formulary XI." United States Pharmacopoaeia XVIII continues
this name in a monograph for Meglumine Iodipamide Injection.

COOH YOOH

C20H1416N206 Mol. Wt. 1,139.7

1.2 Appearance, Color, Odor
Iodipamide is a white, odorless and tasteless
crystalline powder1*11,12. The disodium salt has a sweet,
metallic taste followed by a bitter aftertastd2.

336
 IOD I PAMI DE

2. Physical Properties

2.1 Spectra

2.11 Infrared Spectra

The s p e c t r a of iodipamide i n Figures l a and

l b were determined on a Perkin-Elmer Model 621 g r a t i n g i n -
f r a r e d spectrophotometer. Samples of iodipamide were d i s -
persed i n a potassium bromide p e l l e t or i n mineral

The following s p e c t r a l assignments were made

by ToeplitzS0 on t h e spectrum obtained from t h e sample d i s -
persed i n mineral o i l (Figure l b ) :

-
cm-1 Assignment

3200 N-H
2500, 1900 N-H and OH of amido acid
1690 C=O o f carboxyl group
1610 C=O o f amide
1530 secondary amide
1280 C-OH of carboxyl group

The s p e c t r a shown i n Figures l a and are

dissimilar. An explanation was advanced by Toeplitzi’, who
suggested t h a t iodipamide might be r e a c t i n g with potassium
bromide.

Herrmannl published an i n f r a r e d spectrum ob-

t a i n e d on a potassium bromide dispersion t h a t agrees quali-
t a t i v e l y with t h e spectrum i n Figure la. Neudert and RUpke3
published an i n f r a r e d spectrum t h a t does not agree with t h e
spectrum i n e i t h e r Figure l a or Figure lb.

2.12 Nuclear (Proton) Magnetic Resonance Spectrum

The MlR spectrum of iodipamide i n Figure 2
w a s determined on a Varian XL-100 NMR spectrometer2 at ambi-
e n t probe temperature (z. 31O). The sample was dissolved
i n deuterated dimethylsulfoxide containing tetramethylsilane
as an i n t e r n a l reference (Me4Si = 0 ppm). Spectral assign-
ments of t h e peaks are recorded i n Table I.

337
w
w
m

Figwe la. Infrared Spectnxn of Iodipamide, Squibb Iot 03122, from KBr pellet.
Instrun-ent: PE -1 621 Infrared spectm&oixm=@r
 WAVELENGTH m)

Figure lb, Infrared Spectrum of Iodipamide, Sguibb Lot 03122, from m i n e r d l o i l mull.
Instnarent: PE -1 621 Infrared Sp-olxmeter
zdw
I
1000

f
4

Figure 2. NMR Spectrum of Iodipamide, Squibb Lot 03122 in lXSO-%.

Instrumnt: V a r i a n - m 0 0 NMR Speetrmeter
 IODIPAMIDE

In deuterated water and deuterated sodium

hydroxide, the peak at 69.86 w a s absent, indicating ex-
change of the amine proton. The carboxylic acid protons
were not located, probably because of hydrogen bonding.
High-field methylene resonance indicated the absence of
other groups attached t o the methylene groups.

Table I

NMR Spectral Assignments

Chemical S h i f t
Assignment (Ppm , 6) No. of Protons
-cI12-(312- 1.78 (s) 4
fi
-C-CHz- 2.36 (m)
aromatic 8.33 (3)
-NH 9.86 (s)
-COW not located

s = singlet; m = nultiplet

2.13 Ultraviolet Spectra

The following u l t r a v i o l e t s p e c t r a l data have

been reported f o r iodipamide :

Solvent Amax, nm -E Reference

0.01N NaOH 238 70,700 48

0.1N-NaOH 236 72,000 48
0.lT NaOH 236 71,800 1
0,lR KOH 237 72,400 4
Metbol 238 68,000 8
Methanol 239 71,800 4
0.15M N a C l 238 73,200 5
0.lSR Phosphate 238 73,200 5
Bufrer (pH 5.8)
Neudert and RtJpke3 reported the E value of
the disodium s a l t i n methanol, a t the 239
nm maximum, t o be
74,700. Ostrow and LevyS reported t h e i r data i n terms of
absorbance per micromole of iodine. Sodium iodide, which
peaks a t 226 NB, has the same absorbance p e r rnicromole of

341
 H Y A M HENRY LERNER

iodine as does iodipamide, which suggests t h a t t h e u l t r a -

.
v i o l e t absorption of iodipanide r e s u l t s from t h e presence
of iodine chromophores

2.14 Mass Spectrometry

No w l e c u l a r ion is observed f o r iodipamide
because of i t s low v o l a t i l i t y and because of t h e thermal
degragation of t h e compound. Per-trimethyl s i l y l a t i o n by
Funke yielded a compound with a molecular ion of m/e 1428,
consistent with t h e replacement of four protons by four
trimethyl s i l y l groups. The s t r u c t u r e and major fragmen-
t a t i o n p a t t e r n are depicted below:
COOH YOOH

NH-c -(C%),- C -HN

I 0 0 I

Other fragments t h a t have been found are due t o l o s s o f I

o r ti1 and include:

m/e 1428+m/e 1301 + I

m/e 7 4 2 4 m/e 614 + HI
m/e 728 3 m/e 601 + I
m/e 728+m/e 600 + HI
2.2 Crystal Properties

2.21 D i f f e r e n t i a l Thermal Analysis

Valenti7 determined t h e DTA of iodipamide

on a Du Pont 900 Themnoanalyzer a t a temperature rise of
15O per min. A s i n g l e ondothem a t 308O and a s i n g l e

342
 1001PAM I DE

exotherm a t 314" were detected. The thermogram is repro-

duced i n Figure 3.

2.22 Thermal Gravimetric Analysis

Valenti' determined t h e TGA o f iodipamide

on a Du Pont Thennogravimetric Analyzer. When t h e compound
was heated a t a rate of 15" per minute under nitrogen
sweep, no weight l o s s w a s observed below 250°,

2.23 Melting Range

W i l l i reported
~ ~ ~ a melting range f o r i o d i -
pamide of 306.5 -308", with decomposition, as determined
on a Thomas-Hoover Ca i l l a r y Melting Point apparatus.
Priewe and Rutkowskilj reported t h e meltin! range t o be
306O - 308*, with decomposition. Herrmann reported t h a t
t h e compound decomposes above 280O.

Hoevel-Kestermann and Muhlemng determined

the melting range on a Kofler Microblock (Reichert) and re-
ported t h e melting range t o be 289 -290°, with decomposi-
tion. This l a t t e r value appears t o be i n e r r o r , when com-
pared with t h e previously c i t e d DTA d a t a (Section 2.21) and
measurements made with t h e c a p i l l a r y melting point appara-
tus.

2.24 X-Ray Powder Diffraction

OchslO obtained t h e X-ray powder d i f f r a c -
t i o n spectrum of iodipamide on a P h i l l i p s X-Ray Powder D i f -
fractometer, a t a voltage o f 45 kv and a current of 15 ma.
The sample w a s i r r a d i a t e d by a copper source a t 1.54A.
Diffraction d a t a f o r Squibb Lot 03122 are recorded i n Table
11.

343
P

T. OC (CHROMEL: ALUMEL)* W E IlS,""CIIO* I

.*
".
/ roll I C l l l COIIIIILIId.

figure 3. IICA Themxqram of IOaipami.de, Squibb Iat 03122.

Instrumnt: Du Pant 900 ThermDanalyzer
 IODIPAMIDE

Table I1
~

X-Ray Powder Diffraction Pattern o f Iodipamide,

Squibb Lot 03122
d (Ao)* Relative I n t e n s i t y * *
8.80 0.25
7.40 0.18
5.70 0.13
5.5 0.19
5.10 0.19
4.46 0.34
4.40 0.60
4.29 0.51
4.23 0.39
4.16 0.34
4.09 1.00
3.93 0.69
3.80 0.15
3.67 0.25
3.64 0.18
3.47 0.16
3.44 0.16
3.37 0.10
3.30 0.15
3.25 0.56
3.17 0.28
3.14 0.39
3.10 0.40
2.99 0.29
2.96 0.43
2.92 0.20
2.90 0.20
2.84 0.14
2.66 0.22
2.61 0.17
2.56 0.25
2.51 0.17
2.43 0.13
2.32 0.13
*d = ( i n t e r p l a n a r d i s t a n c e ) nX
2 sin 0
where X = 1.539A
** Based on h i g h e s t i n t e n s i t y of 1.00
345
 HYAM HENRY LERNER

2.3 Solution Data

2.31 Solubility

The following data were reported f o r t h e

s o l u b i l i t y of f r e e acid of iodipamide a t room temperature:

Solubility (mrg /lo0 m l )

Solvent -
Ref.12 Ref.8 a t 20° -
Refa7
Acetone
Ethanol, 95%
200
-
-- <20
<20
Ether
-
100 -- <20
<20
Chloroform
Methanol
Water
800
ins o lub 1e
440
46
-
<20
0.1N sodium
- - 5,240
hyxroxi de
n-hexane - - <20
Benzene
Propylene glycol
insoluble
- -- <20
<20
0.1N hydrochloric
acrd
Ethylene glycol
Tet rahydro f uran
Tet rahydrofurfury1
a1coho1 - 8,200 -
Neudert and R8pke8 a l s o reported t h e solu-
b i l i t y of iodipamide i n acetamide, urethan and phenol, a t
the melting point of the solvents, t o be 3 g, 0.5 g, and
0.3 g per 100 g of solvent, respectively.

The s o l u b i l i t y a t 20° of t h disodium and

dilithium salts of iodipamide were reported : Q

346
 IODIPAMIDE

S o l u b i l i t y (g/100 ml)

Disodium Dilithium
Solvent Salt Salt

Water 350 450

Methanol 14 65
Tetrahydrofuran 4 3
Tetrahydro f u r f u r y l alcohol 4 -
2.32 Apparent Molecular Weight i n Solution

According t o Neudert and R(lpke8, iodipamide

forms micelles and has soap-like properties. Colloidal so-
l u t i o n s contain molecular aggregates with an apparent mole-
cular weight 16 times that of the empirical formula of
iodipamide.

2.33 Isotonicity

A 14.6% (w/v) s o l u t i o n of t h e disodium s a l t

of iodipamide (0.1233M) is i s o t o n i c with physiological s a l t
solutionl2*24.

2.34 pKa
The pKa of t h e f r e e a c i d of iodipamide w a s
rep~rted~ t or be
~ ~3.5. This value may be a composite of
pKa1 and pKa2, s i n c e both d i s s o c i a t i o n constants can be ex-
pected t o be similar i n value.

2.35 pH
The pH of a 1%suspension of iodipamide w a s
Herrmannl proposed limits f o r a sat-
reportedg t o be 3.95.
-
urated s o l u t i o n o f 3.5 3.9.
Iodipamide n e u t r a l i z e d with sodium hydroxide
w a s reported8 t o have a pH of 7.4.
2.36 Index of Refraction

The refractive index of iodipad.de at 21,5O,

i n methanol12, is given i n Table 111. Neudert and R1Spke8
reported t h e refractive index of the disodium s a l t , a t a

341
 H Y A M HENRY LERNER

concentration of 35 e l l 0 0 i n water, and measured with t h e

D-line of sodium, t o be 1.4016.
Table I11

Refractive Index of Iodipamide a t 21.5OC i n Methanol12

$/lo0 m l
-
"D

0.0 1.3278
0.116 1.3283
0.263 1.3288
0.348 1.3292
0.445 1.3294

2.37 Physicochemical Data

The freezing-point depression (-AT) , degree

of d i s s o c i a t i o n (a), and osmotic pressure (Po) of t h e di-
sodium salt of iodipamide i n aqueous s o l u t i o n were re-
ported8*12, and are recorded i n Table IV.

Table IV

Physicochemical Data8,12 f o r t h e Disodium S a l t of

Iodipamide :

Freezing-Point Depression (-AT), Degree of Dissociation

(a), and Osmotic Pressure (Po)

&/lo0 m l Molarity -
-AT -a -
Po
2.0 0.0170 0.095 1.00 1.13
5.0 0.0422 0.226 0.94 2.69
10.0 0.0844 0.425 0.85 5.04
14.6 0.1233 0.557 0.72 6.64
21.6 0,1825 0.720 0.56 8.56

3. Synthesis

Iodipamide is prepared by t h e r e a c t i o n of 2,4,6-

triiodo-3 aminobenzoic acid with adipic a c i d dichloride13.
The former is dissolved i n chlorobenzene and heated t o
110 -130%. The a d i p i c acid dicliloride is added dropwise,

348
 IODIPAMIDE

r e s u l t i n g i n t h e evolution of hydrochloric acid. When t h e

evolution of HC1 has ceased, t h e p r e c i p i t a t e d crude product
is f i l t e r e d , with suction, while s t i l l hot. The crude pre-
c i p i t a t e is washed with chlorobenzene, and t h e r e s i d u a l
chlorobenzene is extracted by b o i l i n g with methanol. The
p r e c i p i t a t e is dissolved i n c a u s t i c methanol, f i l t e r e d
through charcoal, and p r e c i p i t a t e d with d i l u t e hydrochloric
acid.

An a l t e r n a t e solvent f o r t h e reaction o f 2,4,6-tri-

iodo-3 aminobenzoic a c i d with a d i p i c acid d i c h l o r i d e is
toluenel4.

-
COOH

2 'elz +
0
C1-C-(CH,),-C-Cl
II II
* 110"-130°
C,H,Cl
2 HCl +
i
COOH COOH

NH - C -(CH2),- C -HN
I I
4. Stability

Iodipamide is chemically s t a b l e a t room temperature.

By extrapolation t o room temperature of t h e d a t a f o r a c t i -
vation energy and frequency constant of the reaction be-
tween 70 and l l O o , t h e decomposition at t h e N-acyl bond
w a s calculated t o be 0.1% i n 50 yr.33 Evolution of iodine
a t 90° from very pure iodiparnide i n s o l u t i o n at pH 9 is
less than 3% i n 75 hr. The presence of impurities such as
under-iodinated compounds , enhances d e c o m p ~ s i t i o n ~ ~ .

Formulated neutralized (pli 7.2) solutions containing

iodipamide (192 mg/ml) sodium citrate (3.2 mg/ml) a and
sodium e d e t a t e (0.28 mg/ml), show l i t t l e o r no l o s s of po-
tency a f t e r storage f o r 5 yr. at room temperaturel5.

5. P u r i f i c a t i o n and Analysis f o r Impurities

P u r i f i c a t i o n procedures used f o r analysis a r e de-

scribed under Cliromatographic Analysis (Section 6.6).

349
 HYAM HENRY LERNER

5.1 Gel F i l t r a t i o n

Ostrw and Levys attempted t o i s o l a t e and p u r i f y

iodipamide on 1.0- x 12.5-cm columns prepared from dextran
gel (Sephadex G-10 and G-25, Pharmacia) and polyacrylamide
gel (Bio-Gel P-2, Bio-Rad). Samples o f 0.2 m l , containing
0.3 t o 8.6 pmoles o f iodipamide, were e l u t e d with 0.15M so-
dium phosphate b u f f e r (pi4 5.8). The e l u t e d f r a c t i o n s rere
moasured spectrophotometrically at 226, 238, and 255 nm.
Results are shown i n Table V. Due t o adsorption of iodipa-
mide on t h e gel, recoveries were not q u a n t i t a t i v e . Con-
taminants c l o s e l y r e l a t e d t o iodipamide were shown, by
paper chromatography o f t h e iodipamide f r a c t i o n s , t o be
present even after chromatography on Sephadex Gel G-10.
Table V

Gel F i l t r a t i o n of Iodipamide

P r o f i l e on Three Different Gels, with pH 5.8 Phosphate

Buffer as Elution Solvent3

Sephadex Gel Sephadex Gel Bio Gel

Component G-25 G- 10 P-2

Iodipamide 8-32 m l e l u t e d a t void 8-50 m l

(peak a t 20 ml) volume - tail- (peak a t
i n g till 50 m l 16 ml)

Sodium Iodide 10-24 m l 28-50 m l 11-25 m l

(peak a t 15 ml) (peak a t 37 ml) (peak a t
18 ml)

5.2 Complexometric Methods of Separation

Hentrich and P f e i f e r d 4 described methods f o r the
p r e c i p i t a t i o n o f ten c o n t r a s t agents as t h e metallic s a l t s
o r metallic complex salts. Iodipamic a c i d can be p r e c i p i -
t a t e d q u a n t i t a t i v e l y by s i l v e r n i t r a t e , cadmium s u l f a t e
with thiourea, cadmium s u l f a t e with pyridine, copper s u l -
fate with thiourea, and copper s u l f a t e with pyridine.
Chelatometric methods are a l s o described f o r t h e t i t r a t i o n
of excess p r e c i p i t a n t , after separation of t h e p r e c i p i t a t e d
salt by f i l t r a t i o n . The formulas of t h e p r e c i p i t a t e d salts
and complexes, t h e i r molecular weights, melting ranges, and

350
 IODIPAMIDE

t h e equivalent weight o f t h e iodipamide p r e c i p i t a t e d by 1

m l of 0.1M s o l u t i o n of t h e inorganic p r e c i p i t a n t are given
i n Table m.
Table VI

S a l t Complexes of Iodipamic Acid34

Equ iv. Weight

t o 1 m l of
Precip- Mol. Formula M.W. of Melting 0.1M
itant of Salt Salt Range Preciprtant

CdS04- Cd[(NH2)2CSl4*- 1,554.7 237O-238O 0.1140 g

Thiourea C20H1216N206

Cd[(CsH N)4]*- 1,586.6 290 O 0.1140 g

ig%ne C2oH12f6N206

CsSO4- <Cu[(NH2)2C- 1,569.4 167O 0.0570 g

Thiourea S l 2 ) 'C20H12-
IgN2'6
CuSO - [CU(C~HN)2]'- 1,359.5 202O-203O 0.1140 g
Pyrijine ~20~1256~206

5.3 Countercurrent D i s t r i b u t i o n

--
S t r i c k l e r e t a120 separated iodipamide and o t h e r
c o n t r a s t agents from sera by countercurrent d i s t r i b u t i o n .
They used a solvent system composed o f sec-butanol: - dilute
aqueous ammonia (1:l). Both a 30-tube manual procedure and
a 200-tube automatic procedure are described.

5.4 Free Iodine and Free Halide

Free i o d i n e can be d e t e c t e d by b o i l i n g iodipamide

with water f o r 2 min, f i l t e r i n g , and observing a blue c o l o r
a f t e r treatment with s t a r c h . After a c i d i f i c a t i o n o f
another p o r t i o n of f i l t r a t e with d i l u t e n i t r i c a c i d and
treatment of it with s i l v e r n i t r a t e test s o l u t i o n , t h e
presence o f free h a l i d e ions can be detected by t h e

35 1
 HYAM HENRY LERNER

occurrence of opalescence o r turbidity1 a2 '.

Hartmann and RUpke33 quantitated free iodide by
-
t i t r a t i n g potentiometrically with 0.001 N s i l v e r nitrate,
under a protective cover of nitrogen.

5.5 Free Amino Compounds

Hartmann and RUpke33 determined iodinated impuri-
t i e s having a free amino group, by a k i n e t i c method based
on t h e more rapid r e a c t i v i t y of t h e s e impurities with ele-
mental bromine i n acetic acid solution than is shown by
iodipamide. Under these conditions, t h e free amino com-
pounds q u a n t i t a t i v e l y s p l i t o f f iodine, which is then oxi-
dized t o iodate by t h e bromine. After destruction of t h e
excess bromine, t h e i o d a t e is reduced t o iodine and ti-
t r a t e d with sodium t h i o s u l f a t e , t o a s t a r c h endpoint.
Free-iodide compounds a l s o react with bromine and would
give p o s i t i v e r e s u l t s by t h i s method.

Hoevel-Kestermann and hluhlemann9 described a

Bratton-Marshall colorimetric reaction f o r t h e detection of
f r e e amino groups. Hartmann and R8pkd3 used the Eratton-

.
Marshall reaction t o q u a n t i t a t e f r e e amino compound impuri-
ties

5.6 Free Adipic Acid

Hoevel-Kestermann and Wlemanng described a

thin-layer chromatographic procedure f o r adipic acid, after
its cleavage from i o d i p m i c acid. This procedure is de-
scribed i n Section 6.2 and can be adapted t o determine free
a d i p i c acid.

5.7 Determination of Water and Conditions f o r h y i n g

Herrmannl dried iodipamide at 1 0 5 O f o r 4 hr.
Hoevel-Kestermann and Muhlemanng d r i e d iodipamide over
phosphorus pentoxide f o r 24 hr. In t h e "National Formulary
XI,'@ water is determined by t h e Karl Fischer titrimetric
method.

352
 lODlPAMlDE

6. Methods of Analysis

6.1 Elemental Analysis

Element % Theory % Reported25

C 21.075 21-16
n 1.238 1.35
I 66.806 67.28
N 2.458 2.30
0 8.422 -
6.2 I d e n t i f i c a t i o n Tests

Infrared (Section 2.11) , paper chromatography

(Section 6-61), and thin-layer chromatography (Section
6.62) have been used t o i d e n t i f y iodipamide.

S h a m o t i e n k ~i ~d e~n t i f i e d iodipamide by b o i l i n g it
with t r i c h l o r o a c e t i c a c i d and a 5% aqueous s o l u t i o n of
chloramine. Iodipamide gives a cloudy yellow s o l u t i o n with
a yellow p r e c i p i t a t e .

The evolution of intense v i o l e t fumes o f l i b e r -

ated iodine can be observed by heating a sample of iodipa-
mide over an open flame1*9a11.

I d e n t i f i c a t i o n of t h e bound m i n e group can be

made by f i r s t a s c e r t a i n i n g t h e absence of free amino groups
(Section 5.6) and then cleaving t h e molecule by refluxing
i n base and repeating t h e Bratton-Marshall reactiong.
Iodipamide may be hydrolyzed by refluxing with
hydroiodic acid t o l i b e r a t e a d i p i c acid. The adipic acid
can be extracted with e t h e r and chromatographed on s i l i c a
gel G plates. The solvent system benzene:dioxane:acetic
acid (65:25:29) w a s used. Adipic acid (Rf 0.64) can be de-
t e c t e d v i s u a l l y by spraying t h e developed p l a t e with bromo-
c w s o l greeng.

6.3 Direct Spectrophotometric Analysis

The strong u l t r a v i o l e t band of iodipamide a t 238
2 2 nm i n b a s i c s o l u t i o n and methanol (Section 2.13) can be
used f o r d i r e c t spectrophotometric analyses4. The

353
 H Y A M HENRY LERNER

absorption band is s a i d t o r e s u l t from t h e i o d i n e chromo-

#ores5. Detection and q u a n t i t a t i o n of e l u a t e s from
chromatographic s e p a r a t i o n s are e a s i l y accomplished by
using this band f o r analyses.

6.4 Organically Bound Iodine

Under r e f l w conditions, t h e i o d i n e i n iodipamide

can be reduced and replaced by hydrogen, generated by t h e
r e a c t i o n of powdered z i n c and sodium hydroxide. The iodide
is determined by t i t r a t i o n with standardized s i l v e r n i t r a t e
i n a c i d s o l u t i o n i n t h e presence of tetrabromophenol-
phthalein e t h y l ester i n d i c a t o r solution1

Ates and Ama126 decomposed iodipamide with alka-

l i n e p e n ~ a n g a n a t ,28.
e ~ ~ After decoloration of t h e pennan-
ganate with sodium n i t r i t e and a c i d , they t i t r a t e d t h e
-
l i b o r a t e d i o d i n e with 0.1N sodium t h i o s u l f a t e .

Yakatan and TuckenaanZg reviewed f o u r methods f o r

decomposing c o n t r a s t agents t o l i b e r a t e o r g a n i c a l l y bound
iodine: A. Parr bomb (fusion with sodium peroxide); B. al-
k a l i n e permanganate reduction; C. zinc-sodium hydroxide re-
duction; and D. oxygen f l a s k (Schbniger) combustion. Meth-
od D is recommended as a general technique f o r a l l i o d i -
nated organic compounds because of i t s r e p r o d u c i b i l i t y ,
s i m p l i c i t y , and r a p i d i t y . For compounds t h a t have a l l t h e
iodine atoms ortho o r p a r a t o t h e e l e c t r o n e g a t i v e carbox-
y l i c acid on t h e aromatic ring, e g iodipamide, Method C
(zinc-sodium hydroxide reduction$ is recommended.

Krasnova’O recornended a modification of th oxy-

gen-flask combustion method of Yakatan and hickermann 39 .
Hoevel-Kestermann and hhlemann9 reviewed t h r e e
methods f o r l i b e r a t i n g o r g a n i c a l l y bound iodine i n c o n t r a s t
agents: A. Parr banb (fusion with sodium peroxide); B.
c a t a l y t i c reduction with sodium borohydride; and C. zinc-
sodium hydroxide r e d u c t i o n . They recommend t h e sodium boro-
iiydride reduction because of i t s s i m p l i c i t y and s h o r t assay
time. The sodium borohydride reduction w a s o r i g i n a l l y pro-
posed by EgliS1.

354
 IOD1PAM I DE

6.5 Polarography

V a s k e l i s et al.32 determined c o n t r a s t agents

polarographically irOm potassium chloride containing
gelatin. Iodipamide y i e i d s a s i n g l e waveD with a half-wave
p o t e n t i a l of - 1 . 2 ~ vs SCE. Results were q u a n t i t a t e d from
prepared c a l i b r a t i o r c u r v e s .

6.6 Chromatographic Analysis

6.61 Paper Chromatography

Many descending paper chromatographic

methods have been found s u i t a b l e f o r t h e i s o l a t i o n and de-
t e c t i o n of iodipamide. These are summarized i n Table VII.
Some of t h e references c i t e d give sample preparation tech-
niques and methods of e l u t i n g t h e drug from t h e developed
chromatogram t o permit q u a n t i t a t i o n by u l t r a v i o l e t spectro-
photometry (Section 2.13) o r o t h e r means. Pileggi e t a1.22 --
described a method f o r separating 19 organic iodide com-
pounds from blood serum.

Table VII

Paper Chromatographic Systems f o r Iodipamide

Method of
Solvent Sys t em Paper Detection Reference

I Whatman No. 1 A 35
I1 Whatmen No. 4 A 36
I11 Whatman No. 1 B 5
IV Whatman No. 3 B#C 26 39
V Whatman No. 3 B #C 26 39
VI Whatman No. 3 D 22

Solvent Systems

I- n-butanol:lc ammonium hydroxide:ethanol (5:2:1)

I1 - H20:n-butano1:ethanol (5:4:1) ;upper phase used for
development lower phase t o e q u i l i b r a t e chamber.

355
 HYAM HENRY LERNER

Table VII (Cont’d)

I11 - n-butanol:O.SN-
(20:20:2:1);
ammonium hydroxide:ethanol:H20
upper phase used f o r development, lower
phase + 20 m l o f upper phase used t o e q u i l i b r a t e
chamber.

IV - Ethanol:25% ammonia ( r a t i o o f s o l v e n t s not given)

V - Methanol:2N- acetic a c i d ( r a t i o of solvents not given)
VI --sec-butano1:enmronia 4% (3:l)

Methods of Detection

A. Long-wave u l t r a v i o l e t l i g h t .

9. Spray with 10% ceric s u l f a t e and 5% sodium arse-

-
n i t e , both prepared i n 1 N s u l f u r i c acid37.

C. Short-wave u l t r a v i o l e t l i g h t .

D. Spray with mixture o f c e r i c ammonium s u l f a t e and

arsenious acid, followed b spraying with 0.5%
s o l u t i o n o f methylene bluei 2 .
6.62 Thin-Layer Chromatography

Thin-layer chromatographic methods found

s u i t a b l e f o r t h e separation and detection of iodipamide are
summarized i n Table VIII. Some of t h e references c i t e d
present sample preparation techniques and methods for e l u t -
ing t h e drug from t h e developed p l a t e t o p e r m i t q u a n t i t a -
t i o n by u l t r a v i o l e t spectrophotometry (Section 2.13) o r
o t h e r means.

Hol lingsworth e t a l .41 separated iodoamino

acids and r e l a t e d compounds o n T e m u l o s e p l a t e s , with a

form (376:70:60).
-
solvent system composed o f tert.-butanol:2N ammnia:chloro-
They d i d not report usiiig t h i s technique
on iodipamide. However, it seems reasonable t o expect t h a t
t h i s system w i l l s e p a r a t e iodinated c o n t r a s t agents. S t a h l
and Pfeifle38 reported on s i x systems used t o s e p a r a t e 17
iodinated compounds and Hocvel-Kestermann and Muhlemann’
separated 8 c o n t r a s t agents with one system.

356
 IODIPAMIDE

Table VIII
Thin-Layer Systems for Separation of Iodipamide
Solvent Detect ion
System Plate Syst em -
Rf Reference
I A a,b 0.09 9
I1 BDC a, C not given 26,39
I11 BBC a,c not given 26 ,39
IV A a,b 0.27 38
V A a Bb 0.33 38
VI A aD b 0.33 38
VI I D a 0.20 40
Solvent Systems
- Ethyl acet8te:isopropyl alcoho1:ammonia
I 25% (11:7:4)

I1 - Methanol:amrPonia, 25% (2:l)

I11 - Methanol:2N- acetic acid (1O:l)
IV - Acetone:isopropyl alcohol:ammonia, 25% (2:2:1)

V - Isopropyl alcohol:ammonia, 25% (4:l)

VI - Ethy1acetate:methanol :diethylamine (5:4: 2)
VII - n-butano1:ethanol:lg ammonia (5:1:2)

-
Plate

A. 30 g of silica gel HF254, 70 ml of H20 and 0.5 g

of starch.
B. Silica Gel G (Merck).
C. Silica Gel H254-366 (blerck).

D. Eastman tThromogramtg#6061 silica gel, plastic

plates.

351
 HYAM HENRY LERNER

Table VIII (Cont'd)

Detection System

A. Short-wave u l t r a v i o l e t l i g h t (254 m).

B. Spray with 50% solution of acetic acid, followed

by i r r a d i a t i o n a t 254 MI f o r 10 lain t o give blue-
v i o l e t spots. Spraying and i r r a d i a t i o n may be re-
peated t o increase i n t e n s i t y of t h e spots.

C. Spray with 1:l solution of 10% ceric s u l f a t e and

-
5% sodium arsenite, both i n 1 N s u l f u r i c acid3'.
6.63 Electrophoretic Analysis

Ardoino and P a ~ o n ereported

~~ on t h e elec-
t r o p h o r e t i c analysis of c o n t r a s t agents- i n b i o l o g i c a l secre-
t i o n s from t h e l i v e r and kidney. Iodipamic acid migrates at
the speed of albumin and with albumin.

6.7 X-Ray and B-Particle Dispersion Methods

Holynska and J a n k i e w i c ~used ~ ~ X-ray fluorescence

and absorption techniques t o determine iodine i n various
contrast agents. I n t h e X-ray fluorescence work, e x c i t a t i o n
was obtained by i r r a d i a t i o n of t h e sample, from a 2 4 1 h
source of 5 mCi a c t i v i t y . Energy of e x c i t a t i o n w a s 60 keV,
The fluorescence of t h e K series of iodine (28.5 keV) was
measured with a s c i n t i l l a t i o n counter having a 6-mm t h i c k
NaI/T1 c r y s t a l . The c h a r a c t e r i s t i c r a d i a t i o n of iodine was
separated by means of a single-channel, pulse-amplitude ana-
l y z e r covering t h e t o t a l width of t h e K-peak iodine. t4ea-
surement time was 1 min. A c a l i b r a t i o n curve of t h e whole
range of iodine contents investigated w a s made from standard
samples of i o d i c acid (HI03).

I n t h e absorption method t h e X-ray source w a s

241Am and t h e energy w a s 60 keV. The d e t e c t o r w a s a scin-
t i l l a t i o n counter with a 6-mm thick NaI/T1 c r y s t a l . Absorp-
t i o n measurements were made by means of a single-channel,
pulse-amplitude analyzer i n t h e energy channel of 5 keV
covering 60 keV. Calibration curves were again prepared
from s u i t a b l e concentrations of i o d i c acid. The authors
claim t h e fluorescence technique is t h e method of choice.

358
 l OD1PAMI DE

Analyses take less than 5 min, and t h e r e l a t i v e e r r o r is

claimed t o be 1-3%, depending on iodine content.

Mikolajek - e tT a1,44 used t h e method of retrograde

persion of b e t a p a r t i c l e s t o assay c o n t r a s t media.
%l, with about 3 V C i a c t i v i t y deposited on a r i n g was
used as t h e source of radiation. A c a l i b r a t i o n curve show-
ing t h e number o f s c a t t e r e d e l e c t r o n s VS. concentration w a s
established. Results by t h i s method are i n good agreement
with determinations made by conventional metliods.

6.8 Flame Photometry

S h m ~ o t i e n k odetermined
~~ sodium iodipamide i n
pharmaceutical preparations by measuring t h e sodium content
by flame photometry. P r i o r t o analysis, t h e iodipamic a c i d
w a s p r e c i p i t a t e d by a c i d i f y i n g t h e s o l u t i o n with 2N hydro-
c h l o r i c acid, and w a s then separated by filtration: De-
terminations f o r t h e f i l t r a t e were evaluated from a c a l i -
bration curve of sodium chloride i n t h e range of 4.5
mg 0 .
- 8.5

This procedure lacks s p e c i f i c i t y , s i n c e it is de-

pendent on t h e content of an atom not associated with t h e
a c t i v i t y of iodipamide. Many formulations are a l s o pH ad-
j u s t e d with sodium hydroxide and contain o t h e r sodium com-
sodium c i t r a t e , sodium e d e t a t e , as excipi-
"%'
ents. In t ose cases, t h i s method would give erroneously
high values,

7. Drug Metabolism

Iodipamide has been demonstrated t o be excreted large-

l y i n t h e unchanged form12r16,17. Langecker e t a1.12 re-
covered 70% of t h e unchanged compound i n t h e K l r o f rab-
b i t s within 6 h r after dosing. Because of i t s low pKa
(3.5)24 and high molecular weight, i o d i amide is not reab-
sorbed after its excretion i n t h e b i l e J*19. Strickler
--
et al. 2o reported evidence f o r t h e metabolic conversion of
2 1.
iodipamide t o an u n i d e n t i f i e d product. Hydro1 sis of t h e
amide linkago was postulated as a p o s s i b i l i t y

Deiodination of iodipamide w a s s t u d i e d i n man21 and

was found t o be less than 1%of t h e given dose. Pileggi

359
 HYAM HENRY LERNER

e t a1.22 described a papor chromatographic screening t e s t

F r - & . e m i n a t i o n of iodipamide and i o d i d e i n sera (Section
6.61). A method f o r t h e removal o f iodipamide from blood
sera by Craig countercurrent d i s t r i b u t i o n w a s described by
--
S t r i c k l e r e t a1.20 (Section 5.3).

M ~ C h e s n o yreviewed
~~ t h e literature through February
1968 i n a chapter e n t i t l e d , l"llie Biotransforrnation of Iodi-
nated Itadiocontrast Agents.

(1) C. tlermann, Drug Stand., 2, 169 (1957).

(2) M. Puar, Squibb I n s t i t u t e , personal communica-

tion.

(3) W. Neudert and H. Rbpke , s.E.,89, 845

(19%).

(4) T. Pomazonska and W. Zyzynski, Diss. Pharm. , 17,

319 (1965).

(5) J. D. Ostrow and R. P. Levy, Gastroenterology,

-
54, 1085 (1968).

(G) P. Funke, Squibb I n s t i t u t e , personal comunica-

tion.

(7) V. Valenti, Squibh I n s t i t u t e , personal comuni-

cation.

(8)
(1954).
-
W. Neudert and H. Hbpke, Chem. -Ber
*I -87, 659

(9) kl. tloevel-Kestermann and H. Muhlemann, Pharm.

Acta Helv -*47 394 (1972).
--*'
(10) Q. Ochs, Squibb I n s t i t u t e , personal communica-
tion.
(11) National Formulary X I , p. 176 (1960).

360
 IOOIPAMIDE

H. Langecker, A. Harwart and K, JUnkmann, Arch.

(12)
- 220, 195 (1953).
E x p o Pathol. Pharmakol., -
(13) H. Prime and R. Rutkowski, U. S. Patent
2,776,241 (1957).

(14) H. Casselbaum and R. Drux, German (East) Patent

-
33738 (1964); Chem. -.’-’
Abstr 63 11441 b (1965). -
(15) P. Kallos , Squibb Corp. , personal communication.

(16) E. Clerc, Aerztl. Wochenschr., 10, 1156 (1955).

&.
-N. -Y. -Acad, =.,E, 319 (1963).
(17) B. H. B i l l i n g , Q. Maggione and M. A. Carter,

(18) H, W. Fischer, Radiology, 84, 483 (1965).

(19) K. H. Kimbel, W. Bbrner and E. Heise, Fortschr.

Roentgenprax.,

(20) H. S t r i c k l e r , E. S a i e r , E. Kelvington, J. Kempic,

E, Campbell and R. Graven, 2. Clin. ocrinol.
Metab
-*’ -’
24 15 (1964).

(21) H. Langecker, A. Hartwart, K. H. Kolb and M.

Kramer, Arch. 9, Pathol. Pharmakol., 247, 493
(1964).

(22) V. J. P i l l e g g i , K. J. Henry, M. Segalove and G.

C. klami11, C l i n . Chem., 2, 647 (1962).

(23) E. W. McChesney i n t g I n t e n a t i o n a l Encylopedia of

Pharmacology and- Therapeutics P. K. Knoefel,
Ed., Pergamon Press, New York, 1971, Vol. I ,
Sect, 76, pp. 147-163.

(24) P. K. Knoefel, Wadiopaque Diagnostic Agents , I ’

Charles C. Thomas, Springfield, Ill., 1961, pp.
41-44.

(25) J, Hydro, Squibb I n s t i t u t e , personal communica-

tion.

361
 HYAM HENRY LERNER

-
0. Ates and H. Amal, Istanbul Univ. E c z a c i l i k .
66,
-
Fak. Mecm.,
-
11889-1965i).
--
2, 82 (1966) ; Chem. Abstr.,

National Formulary X I , p. 173 (1960).

United S t a t e s Pharmacopoaeia XV, p. 356 (1955).

G. J. Yakatan and M. M. Tuckerman, J. Pham. --

- -
Sci., 55, 532 (1966).

bi. A.Krasnova, Farmatsi a (Moscow), 18, 57

-
(1969); Chem. -*’-
Abstr
*24-(196v. -
---
R. E g l i , Z. Anal. Chem., 3,39 (1969).

A. Vaskelis, Y. Gaule and S. Chausovskii,

Farmatsiya (Moscow 17 54 (1968); Chem. Abstr.,
- - (m’
70, 6 0 8 9 0 ~ -’
--
---
E. Hartmann and H. Rbpke, 2. Anal. Chem., 232,
268 (1967).

-
K. Hentrich and S. P f e i f e r , Pharmazie, 21, 296
-- -
(1966); Chem. Abstr., 65, 8672f (1966).

T. Soh, Squibb Corp., personal communication.

L. Chow, Squibb Corp., personal communication.

----
S. L i s s i t z k e y , Bull. SOC. Chim. Biol.,
(1955).
37, 89

---
E. Stahl and J. P f e i f l e , Z. Anal. Chem.,
377 (1964).
200,

0. Ates and H. Amal, Istanbul Univ. Eczacilik.

Fak. Mecm., 4 , 36 (1968);m=str.,
co886-9 - 697.
-- 70, -
T. Soh, Squibb Corp. , personal communication.

D. R. Hollingsworth, M. D i l l a r d and P. K. Bondy,

-J. -
Lab. - Med
Clin. -*’-’ 62 346 (1963).

362
 IODlPAMlDE

(42)
Biophys, 1 81, 25 (1959).
-
R. H. Mandl and R. J. Block, Arch. Biochem.

(43) B. Holynska and J. Jankiewicz, Chem. Anal.

-
2=(1969).
--
(Warsaw), 14, 219 (1969); Chem. Abstr., 2,

(44) E. Mikolajek, J. Kormicki and 2. Kawalczyk,

Pharm. Pharmecol., 2, 523
e.
(1966); Chem. Abstr.,
6449b-m.
-2 -
(45) G. D. Shamotienko, Khim. Farm. Zh., L, 32 (1968);
-
Chem. Abstr -’
-0, 69 S m y W8)T

(46) L. A. Ardoino and M. Pavone, Boll. Soc. Ital.

Biol.
m 9 (IYOU J

(47) G. D. Shamotienko,
(1970); Chem.

(48) S, Willis, Squibb I n s t i t u t e , personal communica-

tion.

(49) A. Hoffmann, Squibb I n s t i t u t e , personal communi-

cation.

(50) B. Toeplitz, Squibb I n s t i t u t e , personal c o m n i -

cat ion.

3 63
METHADONE HYDROCHLORIDE

Rafik H. Bishara
 RAFlK H. BISHARA

CONTENTS

1. Description
1.1 Nomenclature
1.2 Formula
1.3 Molecular Weight
1.4 Structure
1.5 Appearance, Color, Odor, and T a s t e
1.6 Proprietary Names
2. Physical Properties
2.1 M e l t i n g Range
2.2 Solubility
2.3 Optical Rotation
2.4 pH Range
2.5 D i s s o c i a t i o n C o n s t a n t (pKa)
2.6 Partition Coefficient
2.7 D i f f e r e n t i a l Thermal A n a l y s i s
2.8 Thermogravimetric A n a l y s i s
2.9 O p t i c a l and C r y s t a l l o g r a p h i c P r o p e r t i e s
2 . 1 0 X-Ray Powder D i f f r a c t i o n
2 . 1 1 U l t r a v i o l e t Spectrum
2.12 I n f r a r e d Spectrum
2.13 Nuclear Magnetic Resonance Spectrum
2.14 Mass Spectrum

3. S y n t h e s i s , S t r u c t u r e and R e s o l u t i o n
3.1 S y n t h e s i s and C o n f i r m a t i o n o f S t r u c t u r e
3.2 Resolution
4. R e a c t i v i t y and S t a b i l i t y
5. Drug M e t a b o l i c P r o d u c t s and P h a r m a c o k i n e t i c s
5.1 Absorption
5.2 Distribution
5.3 Metabolism
5.4 Excretion

6. Identification
7. Microchemical R e a c t i o n s

366
 METHADONE HYDROCHLORIDE

8. Methods of A n a l y s i s
8.1 Elemental A n a l y s i s
8.2 T i t r a t ion
8.2.1 Non-Aqueous T i t r a t i o n
8 . 2 . 2 Direct T i t r a t i o n
8.3 Chloride Determination
8.4 Ultraviolet Analysis
8.5 Fluorometric Analysis
8.6 Infrared Analysis
8.7 Colorimetric A n a l y s i s
8.8 Polarography
8.9 Bioassay
8.10 S p i n Immunoassay
8.11 R a d i o t r a c e r T e c h n i q u e s
8.12 Column Chromatography
8.13 P a p e r Chromatography
8.14 T h i n L a y e r Chromatography
8.15 Gas Chromatography
8.16 Combined G a s Chromatography-Mass
Spectrometry
8 . 1 7 High P r e s s u r e L i q u i d Chromatography
9. E x t r a c t i o n from B i o l o g i c a l F l u i d s
10. Determination i n Tissues

11. Bibliography

12. Acknowledgements

13. References

367
 RAFlK H. EISHARA

1. Description

1.1 N o m e n c l a t u r e
6-Dimethylamino-4,4-diphenyl-3-
heptanone h y d r o c h l o r i d e .
1 , l - D i p h e n y l - l - (2-dimethylaminopropy1)-
2- b u t a n o n e h y d r o c h l o r i d e.
4,4-Diphenyl-6-dimethylamino-3-
heptanone h y d r o c h l o r i d e .
6-Dimet hylamino-4,4-diphenylheptan-3-
one h y d r o c h l o r i d e .
1.2 --
Formula
CBlH2,NO*HCl

1.3 Molecular Weight

43.92

1.4 Structure

1.5 --
ApEearance, Color, Odor, a n d T ---aste
WhiFe, e s s e n t i a l l y o d o r l e s s powder.
B i t t e r t a s t e f o l l o w e d by s t i n g i n g s e n s a t i o n .

1.6 - Proprietary Names

dl-Form:
-
Adanon h y d r o c h l o r i d e ; A l g i d o n ;
A l g o l y s i n ; Amidon h y d r o c h l o r i d e ; AN-148;
B u t a l g i n ; D e p r i d o l ; Diadone; Diaminon
h y d r o c h l o r i d e ; D o l o p h i n e h y d r o c h l o r i d e ; Eptadone;
Fenadone; Heptadon h y d r o c h l o r i d e ; Hoechst 10,820;
K e t a l g i n h y d r o c h l o r i d e ; Mecodin; Mephenon;

368
 METHADONE HYDROCHLORIDE

Miadone; Moheptan; Phenadone h y d r o c h l o r i d e ;

P h y s e p t o n e h y d r o c h l o r i d e ; Polamidon
h y d r o c h l o r i d e ; Symoron; Z e f a l g i n .
1-Form: Levadone; L e v o t h y l .
2. Physical Properties

2.1 M e l t i n Range
8Idk--e- 235.0'C1
mp 232.5 - 233.0°C2
mp 233.0 - 236.0°C3
mp 236.0 - 236.5"C4
1-Form: mp 241.O'Cl
mp 245.0 - 246.OoC4

2.2 S o l u b i l i t ~ ~ ~ ~ ~ ~ ~ ~
=-metha= h v d r o c h l o r i d e is v e r v
s o l u b l e i n water (12 g/100 mi), s o l u b l e i n
a l c o h o l ( 8 g/100 ml), i n i s o p r o p a n o l ( 2 . 4 g/
100 m l ) , and i n c h l o r o f o r m ; p r a c t i c a l l y
i n s o l u b l e i n e t h e r and i n g l y c e r i n e .
The 1-form o f methadone h y d r o c h l o r i d e
h a s s i m i l a r s o l u b i l i t y t o t h e racemic form i n
a l c o h o l , i n c h l o r o f o r m and i n e t h e r .

2.3 ----
O p-t i c a l R o t a t i o n
No o p t i c a l r o t a t i o n is o b s e r v e d w i t h
t h e racemic methadone h y d r o c h l o r i d e . F o r t h e
1-form t h e f o l l o w i n g o p t i c a l r o t a t i o n s a r e
reported : 1
-145' (C = 2 . 5 )
and
[a], 2 0 -169" (C = 2 . 1 i n a l c o h o l ) 1 9 4 .
2.4 pH Range
T h e m o f a 1% s o l u t i o n is between 4 . 5
and 6 . 5 . 1 , 3 , 5
2.5D
-- --
i s s o c i a t i o n C o n s t----
a n t (pKa)
L e v i e t al.8 r e p o r t e d t h e pKa of
methadone h y d r G h E r i d e , i n water a t 2OoC., t o

369
 RAFlK H. BISHARA

be 8.25. O t h e r data on t h e d i s s o c i a t i o n c o n s t a n t
of methadone are r e p o r t e d by Marshall' and
Beckett. *
2.6 Partition Coefficient
F m o n coef f icGZs of d l - m e t hadone
i n heptane/p]C! 7.4 b u f f e r and chloroform/pH 7.4
b u f f e r a t 25 C . a r e 0.84 and 1fb56
r e s p e c t i v e l y . 9 Misra a n d Mule r e p o r t e d 57.3
and 28.3 t o be t h e p a r t i t i o n c o e f f i c i e n t s of t h e
1- and d-isomers, r e s p e c t i v e l y , i n o c t a n o l /
pH 7.4 b u f f e r . No e x p e r i m e n t a l d e t a i l s are
g i v e n t o e x p l a i n t h i s d i f f e r e n c e between t h e two
o p t i c a l isomers.
2.7 D i f f e r e n t i a l Thermal
TZTfferentialhermal
of methadone h y d r o c h l o r i d e was performed- u s i n g a
DuPont 900 D i f f e r e t t i a l Thermal A n a l y z e r a t a
h e a t i n g r a t e of 20 C. p e r min. a n d a n i t r o g e n
a t m o s p h e r e . T h e o t h e r m o g r a m ( F i g u r e 1) shows a n
endotherm a t 235 C . , w h i c h appears t o be a m e l t ,
f o l l o w e d immediately by d e c o m p o s i t i o n .
2.8 Thermogravimetric Analysis"
A-GagravTmetric-anaysis, TGA , of
methadone h y d r o c h l o r i d e w a s performed u s i n g a
DuPont 950 T h e r m o g r a v i m e t r i c A n a l y z e r a t a
h e a t i n g r a t e of 5 C . p e r min. a n d a n i t r o g e n
atmosphere. The thermogram ( F i g u r e 2) s h o w s a
w e i g h t loss b e g i n n i n g a t 156.C. and c o n t i n u i n g
through decomposition.
2.9 0 t i c a l and C r y s t a l l o g r a p h i c P r o p e r t i e s
T h E E of methadone hydrocklEFTaE
p r e p a r e d by Huback and 3ones12 f o r o p t i c a l
examinat i o n and i d e n t i f i c a t i o n were o b t a i n e d by
c o o l i n g a w a r m s a t u r a t e d aqueous s o l u t i o n of t h e
compound. The s i n g l e c r y s t a l s were mounted onl a
s t a g e g o n i o m e t e r t o measure t h e p r i n c i p a l
r e f r a c t i v e i n d i c e s . A rotatory s t a g e w a s used t o
measure a l l a n g l e s . Diamond-shaped c r y s t a l s
r e s t i n g on a n end face a r e r e p o r t e d . T h e s e
c r y s t a l s show s y m m e t r i c a l e x t i n c t i o n , g i v e a n

370
 I I I I I I I
20 40 59 79 98 117 137 157 177 197 217
217 236 256 276
T, "C (CHROMEL: ALUMEL)

Figure 1. lYl?A-thenrogram of methadone hydro&loride taken on a W o n t 900

D i f f e r e n t i a l Thermal Analyzer
 * 80-
I--
I
1004
90 -

!2
5 70-

60 -

50 -
I 1 I I I I I I I I

Figure 2. TGA-themogram of mthadone hydrochloride taken on a DuPont 950

Thenrcgravk&xic Analyzer
 METHADONE HYDROCHLORIDE

i n t e r f e r e n c e f i g u r e t h a t shows t h e o b t u s e
b i s e c t r i x a t o n e Zdge o f t h e f i e l d , and t h e i r
a c u t e a n g l e o f 62 is r e l i a b l e f o r c h a r a c t e r i s -
t i c d i a g n o s i s . T a b l e 1 summarizes t h e o p t i c a l
and c r y s t a l l o g r a p h i c d a t a o f racemic methadone
hydrochloride .
The s i n g l e c r y s t a l d a t a f o r d l -
methadone h y d r o c h l o r i d e , t a b u l a t e d by B a r n e s and
Forsyth,13 i n d i c a t e t h a t t h e space group of t h i s
compound is Cc or C2/c. The a u t h o r 9 l i s t e d
a = 16.26, b = 9.76, and c = 25.74 9. The
m o n o c l i n i c a n g l e was measured as 74 ( i . e . , I3 =
106'). The s p a c e g r o u p e x t i n c t i o n o f C2/c is
f a v o r e d by t h e p r e s e n c e of e i g h t m o l e c u l e s p e r
c e l l (z = 8 m o l e c u l e s / c e l l ) . A density of
1.178 g./ml. ( a v e r a g e o f 8 measurements) w a s
o b s e r v e d f o r c r y s t a l s from d i f f e r e n t p r e p a r a -
t i o n s ( P c a l c d . = 1 . 1 7 1 g./ml.).
2.10 X-Ray Powder D i f f r a c t i o n
The x - r a y d i f ? r x t I o n - p o w d e r d a t a o f
dl-methadone h y d r o c h l o r i d e o b t a i n e d by B a r n e s
and Sheppard14 u s i n g f i l t e r e d CoKa (A = 1 . 7 9 0 A )
r a d i a t i o n a r e i n v e r y good agreement w i t h t h o s e
o b t a i n e d by Hubach and J o n e s , 1 2 f o r a s a m p l e
from a d i f f e r e n t commericgl s o u r c e , w i t h
f i l t e r e d CuKa (A = 1.542 A ) r a d i a t i o n . With
c o p p e r r a d i a t i o n , t h e s p a c i n g s of t h e 3
s t r o n g e s t l i n e s , o f t h e p a t t e r n a r e 7 46 A
(v.v.s.), 4.55 A (v.v.s.), A
and 6 . 4 5 (v.s.)
w i t h cobalt r a d i a t i o n thg 3 s t r o n g e s t pacings
o f t h e p a t t e r n are 4 . 5 7 A (1001, 7.50 1
and 6 . 4 8 A (701, t h u s merely i n t e r c h a n g i n g t h e
(901,
" f i r s t " and "second" l i n e s .
B a r n e s and Sheppard14 d r e w t h e a t t e n -
t i o n t o t h e f a c t t h a t while t h e pattern of t h e
dl-methadone h y d r o c h l o r i d e is n o t t h e same a s
t h a t of t h e d- and t h e 1- isomers, t h e p a t t e r n s
of t h e f r e e base i n d-, 1-, and d l - forms a r e
i d e n t i c a l . The x-ray powder d i f f r a c t i o n d a t a o f
dl-methadone h y d r o c h l o r i d e r e p o r t e d by these
a u t h o r s 1 4 are shown below:

373
 TABLE 1
OPTICAL AND CRYSTALLOGRAPHIC PROPERTIES OF
RACEMIC METHADONE HYDROCHLORIDE*

C r y s t a l system Monoclinic, C l a s s ?, o n l y a p l a n e of symmetry;

a c u t e a n g l e B = 74

Optic orientation t3 v i b r a t i o n d i r e c t i o n is p a r a l l e l t o
c r y s t a l l o g r a p h i c axis b. P l a n e o f symmetry
contains axial plane. a d i r e c t i o n is a c u t e
b i s e c t r i x w h i c h is n e a r l y p e r p e n d i c u l a r t o
c r y s t a l l o g r a p h i c axis c.

5 Refractive indices, a = 1.5713 0.0005, B = 1.6232 f 0.0005,

P 5893A. ; 25.C. y = 1.6360 * 0.0005, a' = 1.5760 f 0.0005
from c r y s t a l s r e s t i n g on a n e n d f a c e

Optic axial angle

Observed 2E = 90° f 1. by c a l i b r a t e d micrometer
eyepiece

C a l c d . from s i n V = 2V' = 52.

sin E
1.623
Observed 2V = 52. by r o t a t i n g f r o m o n e o p t i c a x i s t o
t h e o t h e r on goniometer
(continued ) . ..
 0
fi
c,
0
P
n E
a 0
2
0
0 a,
W cu 3
.d
Lo
d c,
II cd
*
cu
M
' C
0
d
v)
v)
*rl
tr
Ea,
a, a
L)
0 h
cd P
tr
a
*cd0 a,
0
5
r( a
cd 0
0 fi
d a
*a IEr:
a,
0 I *
375
 RAFlK H. BISHARA

d (1)
- -
1/11 -
(A)
d -
I/I 1

12.4 25 3.10 30
8.25 10 3.03 3
7.87 5 2.97 10
7.50 00 2.92 3
6.48 70 2.84 5
6.20 1 2.75 20
5.92 20 2.68 15
5.70 5 2.60 3 B
4.72 30 2.53 20
4.57 100 2.48 2
4.34 40 2.30 5
4.20 3 2.23 1
4.14 20 2.16 2
4; 00 20 2.11 8
3.87 20 2.08 3
3.71 5 BB 2.04 15
3.49 20 B 1.99 2
3.33 5 1.92 3
3.20 25 1.67 2
3.14 1 B

2.11 U l t r a v i o l e t Spectrum
A scan of methadone-hydrochloride
i n e t h a n o l a t a c o n c e n t r a t i o n o f 0.27 mg./ml.,
(7.8 x 10'' M) on a Cary 1 5 spectrophotometer,
from 400 t o 210 nm. ( F i g u r e 3) shows maxima a t
254, 250, 265, and 203 nm. The corresponding
(e
molar a b s o r b t i v i t i e s of t h e s e maxima are
410, 485, 460, and 470 r e s p e c t i v e l y .
Hubach and Jones12 r e p o r t e d that a n
a l c o h o l i c s o l u t i o n of methadone h y d r o c h l o r i d e
exhibited t w o characteristic electronic
a b s o r p t i o n bands a t hmax = 294 nm. ( E = 460)
and t h e aromatic band a t Amax = 259 nm.
(6 = 480). In water s o l u t i o n , t h e long wave-
l e n g t h maximum is s h i f t e d t o 292 nm. and t h e
molar a b s o r p t i v i t y is i n c r e a s e d t o E. = 520.
The spectrum of t h e f r e e base, methadone, u s i n g
e t h a n o l or hexane as a s o l v e n t , is e s s e n t i a l l y
t h e same i n t h e r e g i o n o f t h e 294 nm. band,

376
 METHADONE HYDROCHLORIDE

20 -

18 -

16 -

14 -
N

52
;12-
k
5
t
g
v)
10-
m
4 A max 254 (t = 410)
a
5
0
8- Amax 2 5 9 ( c = 485)
I A max 265 ( e = 460)
X m a x 293 ( e = 470)
6-

0
200 250 300 350 400
WAVELENGTH. nm

F i g u e 3. Ultraviolet spectrum of mthadone hydrochloride

i n ethanol taken on a Cam 15 Spectroeotcmter

377
 RAFlK H. BISHARA

However, t h e molar a b s o r p t i v i t i e s a r e E = 7 6 0
i n a l c o h o l a n d E = 830 i n h e x a n e .
The u l t r a v i o l e t a b s o r p t i o n s p e c t r a
data o f Mule15 o n methadone i n 0 . 1 N
h y d r o c h l o r i c a c i d showed Xmax a t 292 nm,
( E = 5 5 4 ) , Xmin a t 2 7 5 nm. ( E = 3 7 2 ) . Data
o b t a i n e d i n e t h y l e n e d i c h l o r i d e c o n t a i n i n g 25%
i s o b u t a n o l (v/v) are Xmax a t 2 9 5 nm. ( 6 = 4 3 3 )
and Xmin a t 2 8 0 nm. ( E = 3 9 0 ) .
Ultraviolet absorption s p e c t r a a t
r e d u c e d t e m p e r a t u r e s of methadone n i t r i l e a n d
i s o m e t h a d o n e n i t r i l e are p r e s e n t e d by
Sinsheimer - et a--l . l 6
2.12 I n f r a r e d Spectrum
Ascanof-methaa&e h v d r o c h l o r i d e i n a
p o t a s s i u m bromide p e l l e t on a Beckman IR-12
s p e c t r o p h o t o m e t e r is shown i n F i g u r e 4 .
Underbrink" a s s i g n e d t h e f o l l o w i n g b a n d s t o t h e
methadone h y d r o c h l o r i d e I R s p e c t r u m :
a. 710-770 c m . ' l character is t i c f o r
aromatic c a r b o n -
hydrogen o u t of p l a n e
bending.
b. 900-1200 cm." f i n g e r p r i n t region;
due t o s k e l e t a l
f r e q u e n c i e s and
aromatic c a r b o n -
hydrogen i n p l a n e
bending .
C. 1300- 1500 c m .- characteristic for
methylene and methyl
bending .
d. 1450, 1490, characteristic for
1580, and aromatic r i n g
1600 c m . - 1 frequencies .
e. 1 7 0 8 cm." c h a r a c t e r is t i c f o r
carbony 1 s t r e t c h i n g .
378
100 n

o l , , l ~ ~ ~ ~ ~ ~ ~ ~
4000 3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 1900 1800 1700 1600 1500 1400 1300 1200 1100 10.00 900 800 700 600 500 4nl:
WAVENUMBEA CM-’

Figure 4. Infrared spectrum of methadone hydrochloride taken in a KE

3r pellet on a
BeduMn IR-12 Spectrqhotorneter
 R A F I K H. BISHARA

-1
f. 2400 c m . characteristic for
t e r t i a r y amine
hydrochloride

g. 2810-3000 cm." characteristic for

a l i p h a t i c carbon-
hydrogen s t r e t c h i n g .

h. 3000-3080 c m . -' characteristic for

aromatic c a r b o n -
hydrogen s t r e t c h i n g .

2.13 N u c l e a r M a g n e t i-
c--R e s o n a n c e S p e c t r u m

The NMR s p e c t r u m o f m e t h a d o n e
h y d r o c h l o r i d e i n CDC1, c o n t a i n i n g
t e t r a m e t h y l s i l a n e as i n t e r n a l s t a n d a r d on a
V a r i a n Associates HA-100 is shown i n F i g u r e 5 .
The s p e c t r a l a s s i g n m e n t s 1 * are summarized i n
T a b l e 2 . The c h e m i c a l s h i f t s a r e m e a s u r e d i n
p.p.m. d o w n f i e l d f r o m t e t r a m e t h y l s i l a n e . The
m u l t i p l i c i t y of t h e peaks, and t h e a p p r o x i m a t e
c o u p l i n g c o n s t a n t s (J) a r e g i v e n i n Hz w h e r e
appropriate.
The m e t h y l e n e a n d m e t h i n e p r o t o n s o f
Group 3 a n d Group 4, r e s p e c t i v e l y , were
i d e n t i f i e d by d e c o u p l i n g . The t w o m e t h y l e n e
p r o t o n s of Group 6 are n o n e q u i v a l e n t g r o b a b l y
because o f c o n f o r m a t i o n a l e f f e c t s . "9 Their
chemical s h i f t s are a s s i g n e d a t approximately
2 . 3 0 a n d 3.15 p.p.m. by t h e p r o c e s s o f e l i m i n a -
t i o n a n d i n t e g r a t i o n . The p r o t o n s o f t h e
N-methyl g r o u p s a r e n o n e q u i v a l e n t d u e t o t h e
r e l a t i v e p r o x i m i t y of e a c h methyl g r o u p t o t h e
d e s h i e l d i n g cone o f t h e phenyl g r o u p and a p p e a r
as a p a i r o f d o u b l e t s (J" 4 Hz) c e n t e r e d a t
2 . 7 5 p.p.m.
D i s c u s s i o n o f t h e NMR a n d PMR o f
methadone a n d some r e l a t e d s u b s t a n c e s , a n d t h e
a p p l i c a t i o n of t h i s t e c h n i q u e f o r s t e r i o c h e m i c a l
and o p t i c a l i s o m e r i s m p r o b l e m s a r e f o u n d i n t h e
l i t e r a t u r e . 19-23

3 80
381

figure 5. Nuclear magnetic resomce spectnm of mthadone hydrochlori& in CDCl3

taken on a varian Associates HA-100 Spectmmter
 TABLE 2
NMR SPECTRAL ASSIGNMENTS OF METHADONE HYDROCHLORIDE
Group Multiplicity Chemical Shift (p.p.m.1 J(H=)
1. %-CH- Doublet 0.70 6.5
I
N (CH31 2

2. CH3-CH2-C-
II Triplet 0.83 7

Approx. 2.30

N ( C H1
~
I
4. CHs-CJ-CH2- Mult iplet Approx. 3.04
5. CH3-CH- Mu 1t i p let 2.75
1
N (CH3 2

(continued . ..
 n l
n
P
a , a ,
d C E
0 0 0
C
0
0
W
c,
a,
.I4
4 u1
E
P
(d
0
h
m
383
 R A F l K H. BISHARA

2.14Mass S e c t r u m
-f--
T h e r e a t i v e mass f r a g m e n t a t i o n p a t t e r n
of methadone was o b t a i n e d 2 4 u s i n g a n LKB-9000
combined gas chromatograph-mass s p e c t r o m e t e r
(GCMS). A f o u r - f o o t s i l i c o n i z e d glass column
(2.5 mm. I . D . ) packed w i t h 1%W-98 m e t h y l v i n y l
s i l i c o n gum r u b b e r on 80-100 mesh G a s Chrom Q
w a s employed a s t h e O K column. The column
t e m p e r a t u r e was 1 7 0 C . a n d t h e c a r r i e r gas
( h e l i u m ) flow w a s 4 0 ml./min. An e l e c t r o n
e n e r g y of 7 0 e V w a s u s e d f o r i o n i z a t i o n . The
c o m p u t e r i z e d mass f r a g m e n t a t i o n p a t t e r n shown i n
F i g u r e 6 was o b t a i n e d by u s i n g a H e w l e t t P a c k a r d
2100 c o mp u t e r i n t e r f a c e d d i r e c t l y t o t h e E M S .
The a s s i g n m e n t s a n d c o m p o s i t i o n s a r e as follow:
m/e
c- -
Assignment C o m D o s it i o n

3 09 M+ c2 l H 2 7NO

294 (M-CH3 >+ c2 O H 2 4NO

26 5 (M-N ( C H ~) ) +
c1g H 2 lo
223 (M-CHz-CH-CH,
+ ClgH150
I
N(CH3 ) 2

165 C13H9

72 C 4 H 1 ON

57 c3 H5O
44 C2H6N

29 C2H5

384
 r
12
I

I I I

-
0
z
294
I E

10 120 110 220 210

mle

Figure 6. Relative mass fragmntation pattern of mthadone abtained by using an

.
LKB-9000 ccxbined gas chrangogra&-mass s p e c t r c m t e r ((336) mta
courtesy of Sullivan, H. R.
 RAFlK H. BISHARA

The above data are i n good agreement

w i t h t h e h i g h r e s o l u t i o n mass s p e c t r u m 2 5 of
methadone h y d r o c h l o r i d e o b t a i n e d by u s i n g a
CEC21-11OA mass spectrometer w i t h p h o t o p l a t e
r e c o r d i n g . The a s s i g n m e n t s of t h e prominent
i o n s are g i v e n i n Table 3.
The mass s p e c t r u m of methadone, t h e
most a b u n d a n t p e a k s and t h e metastable i o n s are
r e p o r t e d by Fales e t a1.26 The i d e n t i f i c a t i o n
of methadone by i s s u s n e chemical i o n i z a t i o n
mass s p e c t r o m e t r y is p r e s e n t e d by Milne e t -- -
al.27
3. -
S y n t h e s i s , S t r u c t u r e and R e s o l u t i o n
3.1
b
--
S n t h e s i s and C o n f i r m a t i o n of S t r u c t u r e
t of t h e U n i t e d States D e p a r t -
ment of Commerce28 a b o u t Amidone (methadone),
t h e new German a n a l g e s i c d r u g no. 10820,
i n c l u d e s t h e method g i v e n by t h e German chemists
f o r its s y n t h e s i s . I n t h i s method, 1 - c h l o r o - 2 -
p r o p a n o l is added t o a n aqueous s o l u t i o n of
d i e t h y l a m i n e and sodium h y d r o x i d e t o p r e p a r e
1-dimethylamino-2-propanol [l]. To [ 1 3 a s o l u -
t i o n of t h i o n y l c h l o r i d e i n b e n z e n e is added t o
form 1-dimethylamino-2-chloropropane [2 1.
Compound [ 2 1 is t h e n dropped on a cool m i x t u r e
of sodamide and d i p h e n y l a c e t o n i t r i l e and t h e
t e m p e r a t u r e is allowed t o rise. The s o l u t i o n is
r e f l u x e d f o r 15 m i n u t e s , cooled, poured on water,
and t h e water is removed. The b e n z e n e s o l u t i o n
is a c i d i f i e d w i t h h y d r o c h l o r i c a c i d , t h e a q u e o u s
acidic l a y e r is made a l k a l i n e w i t h sodium
h y d r o x i d e , and t h e p r o d u c t ,
1-d imet hy laminopropy 1-2-d ipheny lacet o n i t r i l e [3 1,
is d i s s o l v e d i n x y l e n e and a d d e d t o a s o l u t i o n
of ethylmagnesium bromide, h e a t e d , a n d poured
o v e r a c i d i f i e d water t o s e p a r a t e t h e hydrobromide
of t h e k e t o n e . The l a t t e r compound is d i s s o l v e d
i n w a r m water and made a l k a l i n e t o y i e l d a n o i l y
base, methadone, which is c r y s t a l l i z e d from
methanol. The h y d r o c h l o r i d e of methadone is
p r e p a r e d by d i s s o l v i n g t h e base i n alcohol,
alcoholic hydrogen c h l o r i d e is added, and upon
c o o l i n g , t h e material c r y s t a l l i z e s .

386
 TABLE 3

HIGH RESOLUTION MASS SPECTRUM ASSIGNMENTS OF METHADONE HYDROCHLORIDE

C a l c u l a t e d Mass T h e o r e t i c a l Mass Emperical Formula
C
I
H
- N
- 0
-
3 09.2061 309.2093 21 27 1 1
294.1854 294.1858 20 24 1 1
265.1586 265.1592 19 21 0 1
236.1433 236.1439 17 18 1 0
223.1121 223.1123 16 15 0 1
179.0846 179.0861 14 11 0 0
178.0779 178.0782 14 10 0 0
w
W
4
165.0699 165.0704 13 9 0 0
117.0711 117.0704 9 9 0 0
115.0543 115.0548 9 7 0 0
91.0552 91.0548 7 7 0 0
85.0896 85.0892 5 11 1 0
72.0814 72.0813 4 10 1 0
71.0745 71.0735 4 9 1 0
70.0653 70.0657 4 8 1 0
 R A F l K H. BISHARA

I t was n o t e d 2 8 , 2 9 t h a t t h e above-
m e n t i o n e d s y n t h e s i s would n o t be e x p e c t e d t o l e a d
t o methadone [41, b u t t o t h e f o r m a t i o n of t h e
isomeric s t r u c t u r e of i s o m e t h a d o n e [5]:

S c h u l t z e t a l . 2 9 e s t a b l i s h e d and p r o v e d
s t r u c t u r e [41 f o r T e n a d o n e . In t h e i r s y n t h e s i s ,
when d i p h e n y l a c e t o n i t r i l e is r e a c t e d w i t h
1-dimethylamino-2-chloropropane [2 1 e i t h e r i n t h e
p r e s e n c e of s o d a m i d e 2 8 or p o t a s s i u m t - b u t o x i d e ,
t h e p r o d u c t is a m i x t u r e of a p p r o x i m a t e l y e u a l
amounts of t h e isomeric n i t r i l e s [3] and [ 6 7.
The 2 , 2 - d ipheny l-4-d i m e t h y l a m i n o p e n t a n e n i t r i l e
[6] r e a c t s s m o o t h l y w i t h e t h y l m a g n e s i u m bromide
t o g i v e methadone [41. T r e a t m e n t o f 2,2-
diphenyl-3-methyl-4-dimethylaminobutanenitrile
[ 3 ] w i t h t h e G r i g n a r d ' s r e a g e n t does n o t g i v e
t h e methadone isomer C5l b u t a d i b a s i c p r o d u c t
C71 w h i c h a p p e a r s t o be t h e c o r r e s p o n d i n g
k e t i m i n e . A l t h o u g h t h e k e t i m i n e s t r u c t u r e is
s u p p o r t e d by a n a l y t i c a l d a t a , t h e o r d i n a r y
c o n d i t i o n of h y d r o l y s i s f a i l s t o g i v e t h e
k e t o n e . 3 0 The s t r u c t u r e of t h e isomeric
n i t r i l e s , a n d h e n c e t h e s t r u c t u r e o f methadone,
were e s t a b l i s h e d by d e c o m p o s i t i o n of t h e
q u a t e r n a r y b a s e s d e r i v e d from t h e m e t h i o d i d e s
of t h e n i t r i l e s by t r e a t m e n t w i t h s i l v e r o x i d e .
A summary of t h i s s y n t h e s i s is i l l u s t r a t e d i n
F i g u r e 7.
B r o d e and H i l l 3 1 r e p o r t e d t h e
r e a r r a n g e m e n t of t h e isomeric 1 , 2 -
dimethylaminochloropropanes, d e r i v e d from t h e
c h l o r i n a t i o n of 1-dimethy lamino-2-propanol [ 8 )
and 2-dimethylamino-1-propanol [91, t h r o u g h t h e

388
 METHADONE HYDROCHLORIDE

C H /CH3
5‘CH-CN + CI-CH-CH2-N
C6H5/ I

KOCaHS-t

CH CH
5‘C-CN VC-CN
C&l’
CH2-CH-N
+ C6H5’1 /CH3
CH-CH2-N
@ I ‘CH3 1 ‘CH3
CH3 0
C2HglYlgBr

Figure 7 . Synthesis of
TFadone according to
S c f i u l t z et a L
--
389
 R A F l K H. BISHARA

e t h y l e n e immonium i o n . 3 2 T h e i r d a t a a l s o show
t h e c o n v e r s i o n o f [81 t o C91 i n l o w y i e l d s .
To a v o i d t h e t e c h n i c a l d i f f i c u l t i e s d u e
t o t h e formation of t h e isomeric a m i n o n i t r i l e s ,
a new s y n t h e s i s w a s d e v e l o p e d by E a s t o n e t a l . 3 3
i n w h i c h d i p h e n y l a c e t o n i t r i l e is c o n d e n s e d a t h
p r o p y l e n e o x i d e i n t h e p r e s e n c e o f sodium amide
t o y i e l d 3,3-diphenyl-5-methyltetrahydro-2-
furanoneimine [lo]. When [ l o ] is t r e a t e d w i t h
p h o s p h o r u s t r i b r o m i d e , t h e p r o d u c t is 4-bromo-
2,2-diphenylpentanenitrile [111* On c o n d e n s i n g
[111 w i t h d i m e t h y l a m i n e , Compound [6 1 is f o r m e d .
Methadone [ 4 1 is p r e p a r e d from [ 6 ] by t h e a c t i o n
of e t h y l m a g n e s i u m b r o m i d e a s d i s c u s s e d
p r e v i o u s l y . 2 8 The y i e l d s of t h e a m i n o n i t r i l e
from t h e h a l o n i t r i l e is below 107;. The major
p r o d u c t a l w a y s formed is a n u n s a t u r a t e d
n i t r i l e , presumably 2,2-diphenyl-3-pentene-
n i t r i l e [ 1 2 ] or a m i x t u r e of [ 1 2 ] and 2 , 2 -
diphenyl-4-pentenenitrile 113 1.
The s t r u c t u r e o f methadone e s t a b l i s h e d
--
b y S c h u l t z e t a1.29 w a s c o n f i r m e d by t h e
f o l l o w i n g s e r i e s of r e a c t i o n s . Compound [ 6 3 is
d e g r a d e d by e x h a u s t i v e m e t h y l a t i o n ( m e t h y l
i o d i d e , s i l v e r o x i d e , and h e a t i n g ) t o a n
u n s a t u r a t e d n i t r i l e [121, [131 o r a m i x t u r e
w h i c h is h y d r o l y z e d w i t h o u t p u r i f i c a t i o n t o
y i e l d t h e l a c t o n e of 2,2-diphenyl-4-hydroxy-
p e n t a n o i c a c i d [14]. The h y d r o l y s i s o f [13]
f o r m s t h e same l a c t o n e [141. Long s t a n d i n g o f
t h e h y d r o c h l o r i d e o f Compound [ l o ] i n a q u e o u s
s o l u t i o n g i v e s t h e l a c t o n e [141. T h e s e f a c t s
a r e a c c o u n t e d f o r by t h e s t r a i g h t s t r u c t u r e of
t h e a m i n o n i t r i l e [ 6 ] . F i g u r e 8 shows t h e
s y n t h e s i s and c o n f i r m a t i o n o f s t r u c t u r e
according t o Easton e -t a- l.33
The p r e p a r a t i o n of some isomers,
a n a l o g s , 3 5 - 3 9 and r e l a t e d s u b s t a n c e s 4 0 - 4 4 t o
methadone is r e p o r t e d i n t h e l i t e r a t u r e .
T o l b e r t e t a l . 4 5 s y n t h e s i z e d dl-methadone
I -

l a b e l e d w i t h 1 4 C - i n e i t h e r t h e 1 or 2 p o s i t i o n .
 METHADONE HYDROCHLORIDE

C6H5 /O\
‘CH-CN + CH2-CH-CH3
C6H5
/

Figure 8. Synthesis and confirmation of mthadone

s t r u c t u r e according t o Easton e
- al,33
t -

39 1
 R A F l K H. BISHARA

3.2 -_--
R esolution
Methadone h a s o n e a s s y m e t r i c c a r b o n atom
and t h e r e f o r e c a n e x i s t a s d e x t r o or l e v 0 f o r m s
or as racemic m i x t u r e . The o p t i c a l r e s o l u t i o n of
methadone is r e p o r t e d by Brode a n d H i l l , 4 e L a r s e n
et e.,47 and T h o r p -et - a l . 4 8 through t h e use of
d - t a r t a r i c a c i d . Howe a n d S l e t ~ i n g e r a, n~d~ Howe
and T i s h l e r 5 ' r e s o l v e d d l - m e t h a d o n e , o r i t s
h y d r o c h l o r i d e , by f o r m i n g t h e e a s i l y p u r i f i e d ,
w a t e r - i n s o l u b l e d-a-bromocamphor-n-sulfonate o f
t h e d- isomer. P u r e d-met hadone is p r e c i p i t a t e d
by slow a d d i t i o n o f water. The 1-form is
o b t a i n e d , f r o m t h e m o t h e r l i q u o r , by f o r m i n g t h e
d - t a r t r a t e s a l t . When t h e 1-methadone is
d e s i r e d , t h e d-isomer is removed f r o m a s o l u t i o n
i n b u t y l a l c o h o l as t h e p-nitrobenzoyl-l-
glutamate. The u s e o f a-bromocamphor-n-sulfonic
a c i d and p-nitrobenzoyl-L-glutamic a c i d a s t h e
resolving agent reduces t h e excessive
c r y s t a l l i z a t i o n time and s u b s t a n t i a l l y i n c r e a s e s
t h e y i e l d s . ZauggS1 p a t e n t e d a s p e c i a l a p p a r a t u s
t o p r o v i d e a new p h y s i c a l method f o r s i m u l t a n e o u s
r e s o l u t i o n o f b o t h o p t i c a l isomers o f d l -
methadone. Zaugg e x p l a i n s t h a t " t h i s i n v e n t i o n
is based o n t h e knowledge t h a t a seed c r y s t a l of
t h e d e x t r o - r o t a t o r y isomer w i l l a t t r a c t t h e
d - i s o m e r i n s a t u r a t e d s o l u t i o n , and when t h e
d e g r e e of s a t u r a t i o n of t h e s o l u t e i n t h e
s o l u t i o n is i n c r e a s e d , t h e d - i s o m e r w i l l t e n d t o
c r y s t a l l i z e o u t o n t h e d - i s o m e r seed c r y s t a l .
A t t h e same t i m e , a p o r t i o n o f t h e 1-isomer w i l l
t e n d t o c r y s t a l l i z e o u t o n t h e 1 - i s o m e r seed
c r y s t a l . T h i s p r o c e s s w i l l c o n t i n u e s o l o n g as
t h e s o l u t i o n is s u p e r s a t u r a t e d w i t h t h e composi-
t i o n o r s o l u t e and s e e d e d c r y s t a l s w i l l grow t o
s u b s t a n t i a l size. A t t h e conclusion of t h e
o p e r a t i o n , i t w i l l be f o u n d t h a t r e l a t i v e l y p u r e
c r y s t a l s o f t h e d-isomer and 1-isomer w i l l h a v e
b e e n grown o n t h e seed c r y s t a l s . "

me------
4. R e a c t i v i t y and S t a b i l i t y
relatively l o w r e a c t i v i t y of t h e c a r b o n y l
g r o u p of methadone [11 is i n d i c a t e d by n o t g i v i n g
t h e semicarbazone under t h e u s u a l c o n d i t i o n s and-

392
 METHADONE HYDROCHLORIDE

r e s i s t i n g r e d u c t i o n w i t h aluminum i s o p r o p o x i d e
or s o d i u m amalgam.36 The c o r r e s p o n d i n g c a r b i n o l
[ 2 ] is formed w i t h p l a t i n u m o x i d e . A c e t y l a t i o n
o f [ 2 ] y i e l d s t h e 0 - a c e t y l d e r i v a t i v e [3-a].
Reaction of [ 2 ] w i t h c h l o r i n a t i n g a g e n t s
( t h i o n y l c h l o r i d e or phosphorus p e n t a c h l o r i d e )
leads t o t h e f o r m a t i o n of a m i x t u r e o f
6-d i m e t hy l a m i n o - 4,4- d ipheny l-2- h e p t e n e [4 1 a n d
3-chloro-6-dimethylamino-4,4-diphenylheptane [51.
Alkaline cleavage of t h e e t h y l k e t o group
r e s u l t s i n t h e formation of 3-dimethylamino-1,l-
d i p h e n y l b u t a n e [6 1. Compound [ 6 ] is a l s o formed
by r e f l u x i n g 4 - d i m e t h y l a m i n o - 2 , 2 -
d i p h e n y l p e n t a n e n i t r i l e [7] w i t h potassium
hydroxide and t r i e t h y l e n e g l y c o l . Hydrogenation
of t h e r e s u l t i n g o l e f i n C91 from t h e Hofmann
d e g r a d a t i o n o f [8], t h e m e t h i o d i d e o f [6l (which
is a l s o formed by a l k a l i t r e a t m e n t o f t h e
m e t h i o d i d e o f [ 111, g i v e s 1 , l - d i p h e n y l b u t a n e
[ l o ] . The l a t t e r compound is a l s o p r e p a r e d f r o m
e t h y l butyrate [ll] v i a 1,l-diphenyl-1-butanol
[12] w h i c h is h y d r o g e n a t e d t o [ l o ] w i t h
p a l l a d i u m - c h a r c o a l o r p a l l a d i u m - b a r ium s u l f a t e
c a t a l y s t i n t h e p r e s e n c e of a c e t i c a c i d
c o n t a i n i n g traces o f p e r c h l o r i c a c i d . A l k a l i
t r e a t m e n t o f a,a-diphenylvaleronitrile [131
g i v e s a l o w y i e l d o f [ l o ] a l o n g w i t h a,a-
d i p h e n y l v a l e r i c a c i d [14]. F i g u r e 9 shows t h e s e
r e a c t i o n s . I n a l a t e r r e p o r t by May a n d
P e r ~ - i n et h~e~ s t r u c t u r e s of [41 a n d [53 were
p r o v e n t o be 6-d i m e t hylamino-3,4-d i p h e n y l - 3 -
h e p t e n e [15 I, a n d 4-chloro-6-dimethylamino-3,4-
d i p h e n y l h e p t a n e [16], r e s p e c t i v e l y . T h i s is d u e
t o t h e Wagner's r e a r r a n g e m e n t of [21 t o g i v e
6-dimethylamino-3,4-diphenyl-4-heptanol [ l 7 ] ,
[151 and [161 d e p e n d i n g upon t h e r e a c t i o n
conditions.
I r r a d i a t i o n w i t h gamma r a y s ( c o b a l t - 6 0 1 ,
u l t r a v i o l e t , or t h e r m a l n e u t r o n s h a r d l y a f f e c t s
t h e m e l t i n g p o i n t o f m e t hadone
h y d r o c h l o r i d e . 5 3 , 5 4 However, t h e i r r a d i a t i o n
p r o d u c e s a brown color, c h a n g e s o f pH i n s o l u -
t i o n , decreases s p e c i f i c r o t a t i o n , and m o d i f i e s
t h e i n f r a r e d spectrum. Additional s p o t s appear

393
w
394

W
P

I I I
C3H7C0ZCZH5 t PhzC(OH)CH2CHzCH3 PhzCC3H7 -Ph2CC3H7

0 0 63 0
Figure 9 . S o m reactions of nethadone. 36 Reproduced by permission.
 METHADONE HYDROCHLORIDE

on t h e t h i n l a y e r chromatogram of t h e
i r r a d i a t e d sample. I r r a d i a t i o n is less d e s t r u c -
t i v e t o t h e s o l i d material t h a n t o t h e a q u e o u s
solution.
P h o t o l y s i s a n d r a d i o l y s i s of methadone
h y d r o c h l o r i d e s o l u t i o n r e s u l t in t h e formation
of 3 , 3 - d i p h e n y l - 2 - e t h y l i d e n e - 5 -
-
m e t hy 1t e t r a hyd r o f u r a n a n d 3 d i m e t hy l a m i n o - 1,l-
d i p h e n y l b u t e n e , r e s p e c t i v e l y . " 9 56
S t o r a g e of $n o r g a n i c s o l u t i o n of methadone
free base a t 30 C . shows t h e f o r m a t i o n of
methadone-N-oxide b y t h i n l a y e r chrom atography
a n d combined gas chromatography-mass
s p e c t r o m e t r y a n a l y s e s . 5 7 The r e l a t i v e concen-
t r a t i o n of t h e chemical o x i d a t i o n p r o d u c t ,
methadone-N-oxide, i n c r e a s e s w i t h t i m e of
storage.
5. Drug M e t-----
a b o l i c P r o d u c t s a n d-------.--P- h a r m a c o k i n e t i c s
5.1 Absorption
A b s o r p t i o n o f methadone is r e l a t i v e l y
p r o m p t . E x p e r i m e n t s w i t h "C-methadone show
a p p r e c i a b l e c o n c e n t r a t i o n s of 1 4 C i n plasma58
a n d b i l e 5 9 w i t h i n 10 m i n u t e s a f t e r s u b c u t a n e o u s
i n j e c t i o n of t h e labeled d r u g . F o l l o w i n g
s u b c u t a n e o u s i n j e c t i o n of methadone i n r a t s , 47%
of t h e dose r e m a i n s a t t h e i n j e c t i o n s i t e a f t e r
1 h o u r , 6 o 10-15$ a f t e r 2-3 h o u r s , 3% a f t e r
5 h o u r s , a n d v i r t u a l l y n o n e is p r e s e n t a f t e r
24 h o u r s . 6 1 S e v e n t y p e r c e n t of a methadone dose
a d m i n i s t e r e d by stomach t u b e t o f a s t e d rats
d i s a p p e a r s w i t h i n 2 h o u r s from t h e gastro-
i n t e s t i n a l t r a c t .'j2
5.2 Distribution
Methadone m a i n l y l o c a l i z e s i n t h e l i v e r ,
k i d n e y s , l u n g s , a n d s p l e e n . B l o o d , heart, b r a i n ,
a n d m u s c l e show o n l y l o w level^.^'-^^ Methadone
is a l s o c o n c e n t r a t e d i n t h e a d r e n a l s and
t h y r o i d . 58,61965 Using s e n s i t i v e tracer
t e c h n i q u e s , 6 5 methadone l e v e l s of 0.6 t o 0.9
p g . / g . of v a r i o u s s e g m e n t s of t h e c e n t r a l
n e r v o u s s y s t e m are f o u n d 30 m i n u t e s a f t e r

395
 RAFlK H. BISHARA

s u b c u t a n e o u s a d m i n i s t r a t i o n of 3 mg./kg. This
correlates w e l l w i t h t h e i n t e n s i t y and d u r a t i o n
of t h e a n a l g e s i c e f f e c t a s d e m o n s t r a t e d by t h e
r e s c t i o n t i m e of t h e r a t t a i l t o t h e r m a l
stimulus. Elliott e -t a- l . 6 1 r e p o r t e d h i g h concen-
t r a t i o n of methadone i n t h e g a s t r o i n t e s t i n a l
t r a c t a f t e r s u b c u t a n e o u s a d m i n i s t r a t i o n of t h e
d r u g . C o n s i d e r a b l e amounts of r a d i o a c t i v i t y a r e
found i n t h e p l a c e n t a e and f e t u s e s of t h e
p r e g n a n t r a t a f t e r t h e a d m i n i s t r a t i o n of 1 4 C
l a b e l e d methadone.61 The methadone c o n c e n t r a -
t i o n i n t h e b r a i n of t h e f e t u s is 2-3 times t h e
c o n c e n t r a t i o n found i n t h e m a t e r n a l b r a i n . "
Methadone appears t o be f i r m l y bound
t o t i s s u e p r o t e i n . 6 7 However, a c c u m u l a t i o n of
t h e d r u g does n o t o c c u r t o a n y great e x t e n t .
A large p a r t of t h e methadone p r e s e n t i n t h e
whole a n i m a l is f o u n d i n t h e carcass m a i n l y
t h e s k e l e t o n , mu s c l e , a n d bone. '9 61'
A d i s t r i b u t i o n s t u d y of methadone i n
man6' shows t h a t t h e blood c o n c e n t r a t i o n of t h e
d r u g is l e s s t h a n b i l e a n d u r i n e c o n c e n t r a t i o n s .
The k i d n e y a n d l i v e r c o n c e n t r a t i o n s are
a p p r o x i m a t e l y e q u i v a l e n t . B r a i n t i s s u e is t h e
p o o r e s t s o u r c e of methadone and l u n g t h e
r i c h e s t . B i n d i n g of me t h a d o n g 9to human plasm a
a l b u m i n is reported by O l s e n .
5.3 Metabolism
!i%6 i n d i c a t i o n t h a t t h e f i r s t two
c a r b o n atoms of methadone a r e n o t removed by
o x i d a t i o n w a s d e m o n s t r a t e d by E l l i o t t e t a l . ' l
No 1 4 C 0 2 is e l i m i n a t e d by r a t s g i v e n m Z h a d o n e
labeled w i t h l 4 C i n p o s i t i o n 2 . C o n t r a r y t o
these e a r l y data, t h e r e c e n t work by S u l l i v a n 2 4
shows t h a t a b o u t 1s of t h e dose of 14C l a b e l e d
methadone i n p o s i t i o n 2 is e l i m i n a t e d as 1 4 C 0 2 .
The p r e s e n c e of 4-dimethylamino-2,2-
d i p h e n y l v a l e r i c a c i d i n u r i n e of humans is a l s o
a n i n d i c a t i o n of t h e s i d e c h a i n o x i d a t i o n , 5 7
The l i v e r a p p e a r s t o be t h e o r g a n c h i e f l y
r e s p o n s i b l e f o r t h e metabolism of
methadone. 9 O- 73

396
 METHADONE HYDROCHLORIDE

The major metabolites of methadone i n

humans are shown i n F i g u r e 10.57,74 The primary
metabolite o f methadone [11 is formed by
N-demethylation t o y i e l d t h e u n s t a b l e
N-desmethylmethadone [2 3 which i s ~ y c l i z e dt o~ ~
1,5-dimethyl-3 3 - d i p h e n y l - 2 - e t h y l i d e n e
p y r r o l i d i n e [3>. F u r t h e r N-demethylation o f [3 1
forms 2 - e t hy 1-5-met hy l-3,3-d ipheny 1-1-p y r r o l i n e
[el. Both [3] and [ 4 ] and t h e i r c o r r e s p o n d i n g
r i n g hydroxylated analogs, 2-ethylidene-l,5-
d i m e t hyl-3- (p-hydroxyphenyl - 3 - p h e n y l p y r r o l i d i n e
[:5 3 and 2- et hy 1-5-me t hy 1-3- (p- hy d r oxy p heny 1 -3 -
p h e n y l - 1 - v g r r o l i n e [S 1, are detected i n human
u r i n e .76-
I n a minor pathway, t h e k e t o group of
methadone is e n z y m a t i c a l l y r e d u c e d S B t o form
methadol [ 7 ] which is N-demethylated t o y i e l d
normethadol [81 which is excreted i n t h e u r i n e . 8 0
I n a r e l a t i v e l y minor pathway t h e s i d e c h a i n o f
methadone is o x i d i z e d t o form 4-dimethylamino-
2 , 2 - d i p h e n y l v a l e r i c a c i d 191 w h i c h s u b s e q u e n t l y
N-demethylates, i n p a r t , t o a n o n - i s o l a t e d
i n t e r m e d i a t e , 4-met hy lamino-2,2- d ipheny l v a l e r i c
acid [lo]. Ring c l o s u r e ( c y c l i z a t i o n ) of t h e
i n t e r m e d i a t e [lo] y i e l d s 1,5-dimethyl-3,3-
d ipheny l-2- p y r r o l idone [ 111.
In a d d i t i o n t o t h e p r e v i o u s l y mentioned
p h e n o l i c metabolites [ 5 3 and c61, r i n g
h y d r o x y l a t e d methadone [12] is a l s o found i n t h e
u r i n e of s u b j e c t s m a i n t a i n e d o n methadone.57
Methadone N-oxide [13] is found i n u r i n e from
s u b j e c t s r e c e i v i n g a s i n g l e dose o f methadone,
i n u r i n e from a d d i c t s b e i n g treated w i t h t h e
drug, 74 a n d in u r i n e of r a t s . 1 0 However, t h e
work o f S u l l i v a n and Due57 i n humans i m p l i e s
t h a t t h e r e is some q u e s t i o n a s t o whether t h e
methadone N-oxide is a t r u e m e t a b o l i t e o r a n
a r t i f a c t caused by o x i d a t i o n ,
5.4 Excretion
--I--

Way and Adler" i n d i c a t e d t h a t less t h a n

10% of methadone is e x c r e t e d unchanged i n t h e
u r i n e and i n t h e feces. U r i n a r y e x c r e t i o n
s t u d i e s show v a r i o u s c o n c e n t r a t i o n s o f methadone

397
 RAFlK H. BISHARA

Figure 10. Major mtabolites of mthadone in h

-
. 57,74

398
 METHADONE HYDROCHLORIDE

i n u r i n e . Recovery of 4 s t o 35% o f t h e
a d m i n i s t e r e d dose is r e p o r t e d . 8 2 - 8 5 Twenty-four
h o u r s a f t e r a d m i n i s t r a t i o n of methadone t o r a t s ,
t h e unchanged d r u g found i n u r i n e and feces is
4-11s and 19-24s r e s p e c t i v e l y from t h e
a d m i n i s t e r e d dose.6a However, Way e t g . , 8 2
u t i l i z i n g c o u n t e r - c u r r e n t t e c h n i q u e s , showed
t h a t t h e p r e v i o u s v a l u e s are h i g h and
recommended a f a c t o r of 0.8 a n d of 0.25 f o r t h e
c o r r e c t i o n of t h e u r i n a r y and f e c a l e x c r e t i o n
respectively.
B i l a r y e x c r e t i o n is r e p o r t e d t o be a n
i m p o r t a n t avenue f o r t h e e l i m i n a t i o n of
methadone and its b i o t r a n s f o r m a t i o n
p r o d u c t s , 5 9 ~ 6 3 - 65 ~ 8 2
The r e s u l t s of Baselt and Casarett'
demonstrated t h a t , i n man, r e n a l e l i m i n a t i o n may
become t h e major e x c r e t o r y pathway a f t e r d a i l y
d o s e s g r e a t e r t h a n 55 mg. S i x t y p e r c e n t of a
160 m g . dose of methadone p e r day is e x c r e t e d a s
unchanged d r u g i n u r i n e . T h e s e r e s u l t s are i n
c o n f l i c t w i t h t h o s e o f S u l l i v a n and Due5' who
r e p o r t e d t h a t a r e l a t i v e l y small p o r t i o n of a n
8 0 mg. dose of methadone w a s found unchanged i n
t h e u r i n e of h e r o i n maintenance s u b j e c t s .
U r i n a r y methadone e x c r e t i o n is markedly
enhanced by a c i d i f i c a t i o n of t h e u r i n e . ' A sex
d i f f e r e n c e i n t h e p a t t e r n o f e x c r e t i o n of
methadone and its metabolites is found and is
r e l a t e d t o t h e r a t e of b i o t r a n s f o r m a t i o n of t h e
drug.
Methadone, metabolites C33 and L43 (See
F i g u r e 101, are p r e s e n t i n s u f f i c i e n t l y h i g h
c o n c e n t r a t i o n i n human sweat t o s u g g e s t t h a t
sweat may be a s i g n i f i c a n t r o u t e of e l i m i n a t i o n
of t h i s d r u g . 8 6
The mean a p p a r e n t h a l f - l i f e of orally-
a d m i n i s t e r e d methadone is 15 hours." Following
i n t r a m u s c u l a r a d m i n i s t r a t i o n , t h e h a l f - l i f e is
7.3 hours.88 S u b j e c t s of a methadone
maintenance program who r e c e i v e a large o r a l
dose of 100 or 120 m g . show a mean a p p a r e n t
h a l f - l i f e of methadone t o be 2 5 hours.''

399
 RAFlK H. EISHARA

A n a l y t i c a l procedures for i s o l a t i o n ,
d e t e r m i n a t i o n , a n d i d e n t i f i c a t i o n of methadone
and its metabolites from b i o l o g i c a l f l u i d s a n d
t i s s u e s i n c l u d e colorimetric a n d p h o t o m e t r i c
techni ues following i n t e r a c t i o n with indicator
3
’
6 e 2 , 6 4 , 6 7 9 70, 8 2 - 8 4 p a p e r chromato
72,90,91
raphy, column chromato r a hy, 79,8i g a s
c h r o ma t o g r a p h y , 9, 6 8 , 7 4 9 7 6 - 7 8 , 9i2 t h i n l a y e r
c h r o m a t o g r a p h y , 9, “ 9 54, “ 9 “9 7 0 repeated
c o u n t e r - c u r r e n t t r a n s f e r , 8 1 radiotracer
methods, 4 5 9 5 8 - 6 1 , 659 72, 8 0 i n rare 76 8 1 , 9 2
n u c l e a r m a g n e t i c r e s o n a n c e , 75, 76, g h , 95 and
combined gas chromatography-mass
s p e c t r o m e t r y . 579 7 4 9 7 7 - 8 0
6. Identification
E m a d o n e h y d F o c h l o r i d e c a n be i d e n t i f i e d by
v i r t u e of its c h a r a c t e r i s t i c x - r a y powder
d i f f r a c t i o n p a t t e r n , W , I R , a nd NMR s p e c t r a
(See 2.10, 2.11, 2.12, a n d 2 . 1 3 ) . The charac-
t e r 5 s t i c m e l t i n g p o i n t of a b o u t 160 C . , or a b o u t
180 C . of t h e c r y s t a l s formed by p i c r o l o n i c a c i d
and methadone is a l s o u s e f u l as a n i d e n t i t y
te~t.3,A ~ d d i t i o n of 2 m l . of m e t h y l o r a n g e
test s o l u t i o n 5 t o a 0.5% methadone h y d r o c h l o r i d e
s o l u t i o n forms a y e l l o w p r e c i p i t a t e . The
m e l t i n g p o i n t of t h e water-washed r e s i d u e ,
formed by a d d i n g excess s o d i u m h y d r o x i d e s o l u -
t i o n 3 t o a 5% s o l u t i o n of methadone, is a b o u t
76’C. Methadone h y d r o c h l o r i d e reacts p o s i t i v e l y
t o t h e chloride characteristic test w i t h s i l v e r
n i t r a t e . 3 ~5
7. ---
Microchemical React i o n s
The microchemical i d e n t i f i c a t i o n of methadone
t h r o u g h t h e f o r m a t i o n of c r s t a l s w i t h c e r t a i n
r e a g e n t s h a s b e e n r e p o r t e d . ?12,93-97 Hubach and
J o n e s 1 2 l i s t e d s e v e n r e a g e n t s w h i c h produced
c h a r a c t e r i s t i c m i c r o s c o p i c c r y s t a l s from d i l u t e
a q u e o u s m e t hadone h y d r o c h l o r i d e s o l u t i o n s .
E q u a l d r o p s of t h e r e a g e n t and t h e methadone
s o l u t i o n a r e mixed on a microscopic s l i d e and
a l l o w e d t o s t a n d u n t i l c r y s t a l s d e v e l o p or a l l
l i q u i d e v a p o r a t e s . The c r y s t a l s are t h e n

400
 METHADONE HY DROCH LORlDE

o b s e r v e d and examined m i c r o s c o p i c a l l y . The

r e a g e n t s u s e d and t h e lowest c o n c e n t r a t i o n o f
methadone h y d r o c h l o r i d e from which c r y s t a l s
a r e o b t a i n e d are 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 t h e d e s c r i p t i o n of t h e formed c r y s t a l s .
F i v e m o d i f i e d c o b a l t (11) t h i o c y a n a t e s o l u -
t i o n s g i v e t u r q u o i s e color w i t h m e t h a d o n e . @ 8
A s o l u t i o n c o n s i s t i n g of 0.8% w/v c o b a l t (11)
t h i o c y a n a t e i n a ( 2 : 3 ) v/v m i x t u r e of m e t h a n o l
and 1%o r t h o p h o s p h o r i c a c i d ( s p . g r . 1.75) g i v e s
a c o l o r r e s p o n s e w i t h i n 5 s e c o n d s from t h e
a d d i t i o n of t h e r e a g e n t .

8. Methods of A n a l y s i s

8.1 E l e m e n t a l A n a l y s i s (As C21H2,NO*HC1) -

z Determined 47,459
E l e me n t
----- ---
Theory 1 -$' ----------

C 72.91 73.14 72.77, 73.06,

72.90 72.95

H 8.16 8.03 7.98, 8.23,

8.36 7.99

N 4.05 3.96 3.88 4.07

0 4.63

c1 10.25

8.2 T i t r a t ion
------
8.2.1 Non-Aqueous T i t r a t i o n
_I--------

The t e r t i a r y amine g r o u p of
methadone h y d r o c h l o r i d e is d i r e c t l y t i t r a t e d i n
non-aqueous medium, g l a c i a l a c e t i c a c i d , i n t h e
p r e s e n c e of m e r c u r i c a c e t a t e t o t i e up t h e
c h l o r i d e i o n . The t e c h n i q u e of t h e non-aqueous
t i t r a t i o n of t h e d r u g u s i n g v i s u a l i n d i c a t o r s ,

40 1
 R A F l K H. BISHARA

TABLE 4

MICROCHEMICAL CRYSTALLIZATION OF
METHADONE HYDROCHLORIDE
Concentra-
t i o n of
Reagent Methadone HC1 - Reaction
P o t a s s i u m i o d i d e , 5% 1:1,000 Colorless
c r y s t a 1s
Potassium ferrocyanide, 1:500 Colorless
5% ( f r e s h s o l u t i o n ) crystals

Potassium f e r r i c y a n i d e , 1:500 Yellow

5% ( f r e s h s o l u t i o n ) crystals

Marme' s r e a g e n t 1:10,000 Colorless

(fresh solution) c r y s t a 1s
Mayer's r e a g e n t 1:20,000 Colorless
crystals
Wagner's r e a g e n t 1 : 1,000 Pale
brown
crystals
Lanthanum n i t r a t e , 20% 1:20 Color less
crystals

402
 METHADONE HYDROCHLORIDE

c r y s t a l v i o l e t 3 j 5 or m e t h y l v i o l e t j 6 h a s b e e n
d e s c r i b e d i n d e t a i l . Each 1 m l . of 0 . 1 N
p e r c h l o r i c a c i d , i s e q u i v a l e n t t o 34.59 m g . of
C21H2,NO*HC1. P o t e n t i o m e t r i c e n d p o i n t detec-
t i o n has also been used. 9 9
8.2.2 Direct T i t r a t i o n
J o h n s o n a n d K i n g l o o developed a
r a p i d d i r e c t t i t r i m e t r i c method, u s i n g a n
e x t r a c t i v e end p o i n t , f o r d e t e r m i n a t i o n of
methadone i n p h a r m a c e u t i c a l p r e p a r a t i o n s .
C h l o r o f o r m is a d d e d t o t h e o r g a n i c base
d i s s o l v e d i n pH 2 . 8 a c e t a t e b u f f e r s o l u t i o n s o
t h a t t h e r a t i o of chloroform t o t h e a q u e o u s
p h a s e is a b o u t 3 t o 1. T i t r a t i o n is p e r f o r m e d
u s i n g sodium d i c o t y l s u l f o s u c c i n a t e a n d D i m e t h y l
Y e l l o w s c r e e n e d w i t h Oracet B l u e as i n d i c a t o r .
The c h a n g e of c o l o r of t h e c h l o r o f o r m p h a s e from
g r e e n t o p i n k i n d i c a t e s t h e end p o i n t . The
a c c u r a c y of t h e method is f 1%.
8.3 _-----___ ________-_
C h l o r i d e Determination (Mercuric
Nitrate T i t r a t i o -
AsErnxeoPmeZkHaone hydrochloride
c o n t a i n i n g a t l e a s t 2 mg. of c h l o r i n e is
d i s s o l v e d i n 80 m l . of w a t e r - m e t h a n o l ( 2 0 : 6 0 )
m i x t u r e a n d t h r e e d r o p s of d i p h e n y l c a r b a z o n e
s o l u t i o n ( 5 mg./ml. m e t h a n o l ) are a d d e d . The
s a m p l e s o l u t i o n is t h e n t i t r a t e d w i t h s t a n d a r d
0.5 N m e r c u r i c n i t r a t e s o l u t i o n t o t h e first
s i g n of rose c o l o r , u s i n g a one-ml.
microburette.
percent chlorine =

____--- _--------
mL m e r c u r i c n i t r a t e x n o r m a l i t y
mg. s a m p l e
X 35.5 X 100

p e r c e n t p u r i t y of m e t hadone h y d r o c h l o r i d e =

__-___ h l o r i n e-
p e r c e n t c _--- - f o u n d X 100
-
I
-.
.
._-

percent chlorine-theory

403
 R A F l K H. BISHARA

8.4 Ultraviolet Analysis

--
Wallace e t a l . 1 0 1 oxidized methadone
w i t h b a r i u m p e r o x i d e t o benzophenone w h i c h was
e x t r a c t e d i n h e p t a n e a n d measured s p e c t r o -
p h o t o m e t r i c a l l y a t 247 nm., E = 18,713. T h i s
r e p r e s e n t s a n i n c r e a s e i n molar a b s o r b a n c e of
a p p r o x i m a t e l y 34 times o v e r t h a t of methadone i n
0.1 N h y d r o c h l o r i c a c i d , g = 554, a t 292 nm.
The method is s u c c e s s f u l l y u s e d f o r d e t e r m i n i n g
methadone i n b i o l o g i c a l s p e c i m e n s , namely u r i n e ,
l i v e r , l u n g s , k i d n e y s , stomach, and i n t e s t i n e s .
8.5 --l--u-o--r o m e t r i c A n a l y s i s
F
The f o r m a t i o n of a f l u o r o p h o r e when
methadone is h e a t e d i n a s o l u t i o n of form aldehyde
and c o n c e n t r a t e d s u l f u r i c a c i d followed by
a d d i t i o n of water is reported by McGonigle. 1 0 2
The f l u o r e s c e n c e is recorded a t a n e x c i t a t i o n
w a v e l e n g t h of 270 nm. a n d a n e m i s s i o n w a v e l e n g t h
of 450 nm. The a q u e o u s f l u o r o p h o r e is s t a b l e
f o r a t l e a s t 1.5 h o u r s a t room t e m p e r a t u r e .
T h i s p r o c e d u r e is s u i t a b l e f o r m e a s u r i n g
microgram q u a n t i t i e s of methadone. Morphine,
heroin, codeine, and c o c a i n e do n o t i n t e r f e r e .
However, p r i o r s e p a r a t i o n of amphetamine,
m e p e r i d i n e , and q u i n i n e is r e q u i r e d s i n c e t h e y
f l u o r e s c e u n d e r t h e c o n d i t i o n s of t h e a s s a y and
i n t e r f e r e w i t h t h e d e t e r m i n a t i o n of methadone.
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 of t h e methadone
a s s a y is 2.2%.
A p p l i c a t i o n of f l u o r e s c e n c e and gas
c h r o m a t o g r a p h y t o mass d r u g s c r e e n i n g is
p r e s e n t e d by S a n t i n g a . l o 3 The smallest
d e t e c t a b l e q u f B 4 i t y of methadone is 1 p g . / m l .
Lawler et a l . used spectrophotofluorometry
for t h e d e t e r m i n a t i o n of methadone i n t i s s u e s .
8.6 -
I n f r a r e d A n a l y s i s !3
The sample of methadone hydrochloride
s o l u t i o n is made a l k a l i n e w i t h (1:l) sodium
h y d r o x i d e i n water. The p r e c i p i t a t e d base is
e x t r a c t e d w i t h chloroform. The e x t r a c t s are
d r i e d o v e r anhydrous sodium s u l f a t e a n d t h e
chloroform is e v a p o r a t e d u s i n g steam heat a n d

404
 METHADONE HYDROCHLORIDE

a c u r r e n t of a i r . The r e s i d u e is d i s s o l v e d i n
chloroform a n d t h e a b s o r b a n c e , v e r s u s a
chloroform b l a n k , is d e t e r m i n e d a t 5.87 using
0.1 mm. c e l l s a n d a Beckman IR spectrophotometer.
The a b s o r b a n c e of t h e s t a n d a r d is d e t e r m i n e d i n
t h e same manner a n d t h e m g . methadone
h y d r o c h l o r i d e is c a l c u l a t e d from t h e r e l a t i v e
r a t i o . U l t r a v i o l e t i r r a d i a t i o n of a m e t h a n o l i c
s o l u t i o n of methadone h y d r o c h l o r i d e , f o r
1 . 5 h o u r s , c a u s e s 74% d e t e r i o r a t i o n a s shown by
t h i s method.

8.7 Colorimetric A n a l y s i s
D r ug s c o n t a i n i n g b a s i c g r o u p s form a
colored complex w i t h s u l f o n i c a c i d i n d i c a t o r
d y e s s u c h as Methyl Orange a n d Bromcresol
G r e e n . lo' The colored complex is t h e n s e p a r a t e d
from t h e e x c e s s dye by e x t r a c t i o n i n t o
chloroform o r a s u i t a b l e o r g a n i c s o l v e n t a n d
t h e q u a n t i t y of t h e complexed d r u g is estimated
spectrophotometrically.
The methadone-dye complex is formed
u s i n g bromthymol b l u e , 83 bromcresol g r e e n , 8 4
bromphenol b l u e , 8 4 b r o m c h l o r o p h e n o l b l u e , 8 4
c h l o r o p h e n o l r e d , 8 4 bromcresol p u r p l e , 8 4 , 1 0 7
a n d m e t h y l o r a n g e . 6 2 9 8 2 With these dyes, a n y
basic a m i n e w h i c h c a n form a n o r g a n i c s o l v e n t -
s o l u b l e d y e complex would react as methadone,
h e n c e p r e c a u t i o n s n e e d t o be t a k e n t o e l i m i n a t e
i n t e r f e r i n g substances. For the determination
of methadone i n t i s s u e s , R i c k a r d s e t al.GO--
l i b e r a t e d t h e compound by d i s i n t e g r a t i o n of t h e
t i s s u e w i t h s t r o n g a l k a l i , ether e x t r a c t i o n ,
n i t r a t i o n of t h e p h e n y l r a d i c a l s i n methadone,
c o l o r d e v e l o p me n t w i t h e t h y l m e t h y l k e t o n e a n d
m e a s u r i n g t h e c o l o r a t 565 nm. The
r e p r o d u c i b i l i t y a n d s e n s i t i v i t y are f 5% a n d
1 p g . of methadone. However, i t is t o be
n o t i c e d t h a t a n y methadone metabolic f r a g m e n t
r e t a i n i n g t h e p h e n y l a n d a mi n e g r o u p s would
react a s t h e p a r e n t s u b s t a n c e . 8 i N i t r a t i o n of
methadone w i t h a m i x t u r e of HN03/H2S04 (1:l) a n d
t h e s u b s e q u e n t 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 t h e

405
 RAFlK H . BISHARA

colored d e r i v a t i v e u s i n g f i l t e r S42 (428 nm.) is

p r e s e n t e d by S k o r a . l 0 * The method is used f o r
d e t e r m i n a t i o n of methadone i n a mpoules, t a b l e t s ,
and b i o l o g i c a l media (blood, l i v e r a n d u r i n e ) .
An a u t o ma t e d method f o r t h e d e t e r m i n a -
t i o n of methadone h y d r o c h l o r i d e i n d i s s o l u t i o n
s a m p l e s w a s d e v e l o p e d by B e c h t e l a n d
B r i c k l e y . l o 9 The d i s s o l u t i o n s a m p l e s a r e
s e q u e n t i a l l y s a mp l e d by a n A u t o Analyzer a n d
i n j e c t e d i n t o a n a i r - s e g m e n t e d stream of pH 1 . 2
b u f f e r ( U . S . P . ) w h i c h is pumped t h r o u g h a g l a s s
mixing c o i l , Bromcresol p u r p l e (BCP) dye
s o l u t i o n is t h e n a d d e d t o t h e stream of t h e
methadone s a m p l e a n d t h e s o l u t i o n s are mixed i n
a T e f l o n c o i l f o r t h e f o r m a t i o n of t h e dye-
complex. The methadone-dye complex is extracted
i n t o e t h y l e n e d i c h l o r i d e and t h e color i n t e n s i t y
is measured a t 4 2 0 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 (R.S.D.) a n d t h e r e l a t i v e error (R.E.)
are f 1.48% and + 0.05% r e s p e c t i v e l y .
The s p e c i f i c a s s a y of basic d r u g s i n
u r i n e by C M - c e l l u l o s e column c h rom atography
u s i n g c o n t i n u o u s d r u g - d y e complex e x t r a c t i o n as
a d e t e c t i o n s y s t e m is d e s c r i b e d by McMartin
-
e t a- l , lo6
8.8 ---
P olarography
The k e t o g r o u p of methadone is n o t
polarographically r e d u c i b l e because t h e double
bonds are n o t c o n j u g a t e d . T h e r e f o r e , a n
e l e c t r o a c t i v e n i t r o d e r i v a t i v e is prepared.
S k o r a l o 8 n i t r a t e d methadone u s i n g a m i x t u r e of
n i t r i c a c i d / s u l f u r i c a c i d ( 1 : l ) . N i t r a t i o n is
c o m p l e t e d i n 30 m i n u t e s a f t e r h e a t i n g on a
b o i l i n g water b a t h . The m i x t u r e is t h e n cooled,
d i l u t e d w i t h 5 m l . d i s t i l l e d water and made
a l k a l i n e , pH 10, w i t h 5 N NaOH. T h r e e d r o p s of
0.5% g e l a t i n s o l u t i o n are a d d e d a n d t h e s o l u t i o n
is p o l a r o g r a p h e d , a f t e r d e o x y g e n a t i o n , i n a
p o t e n t i a l r a n g e of - 0 . 4 t o -1.2 V . The h a l f -
wave p o t e n t i a l of t h e n i t r a t e d methadone is
- 0 . 6 4 V r e l a t i v e t o a s a t u r a t e d calomel
e l e c t r o d e . A s t r a i g h t l i n e is o b t a i n e d when
t h e c o n c e n t r a t i o n of t h e n i t r a t e d methadone

406
 METHADONE HYDROCHLORIDE

d e r i v a t i v e is p l o t t e d v e r s u s t h e d i f f u s i o n c u r -
r e n t ( h e i g h t of t h e r e d u c t i o n w a v e ) f o r concen-
t r a t i o n s of 2 0 , 30, 40, 50, and 60 p g . / m l .
C o n c e n t r a t i o n s lower t h a n 5 Ccg./ml. c a n be
d e t e r m i n e d . The r e d u c t i o n wave of methadone
n i t r a t e is d i s t i n c t and t h i s method is u s e d f o r
a s s a y i n g methadone i n ampoules and t a b l e t s .
A p p l i c a t i o n of t h e method t o a s s a y f o r t h e d r u g
i n blood, l i v e r and u r i n e is d i s c u s s e d .
C a t h o d e r a y p o l a r o g r a p h y of methadone
N-oxide i n W a l p o l e ' s a c e t a t e b u f f e r pH 5 g i v e s a
r e d u c t i o n wave w h i c h h a s a peak p o t e n t i a l of
- 1 . 2 1 V . R e d u c t i o n of t h e same s o l u t i o n w i t h
T i C l , / H C l g i v e s methadone. 74

8.9 --Bioassay
S ~ h a u m a n nr ~e D
~ o r t e d a bioassay
p r o c e d u r e f o r methadone ;sing i s o l a t e d g u i n e a -
p i g g u t . The method is s e n s i t i v e t o methadone
a t a c o n c e n t r a t i o n as low as lo-* M.
8.10 S p i n Immunoassay
F r e e - r a d i c a l t e c h n o l o g y is combined
w i t h immunoassay t o y i e l d t h e new method of
" s p i n immunoassay" used f o r d e t e c t i o n and a s s a y
of small m o l e c u l e s i n b i o l o g i c a l f l u i d s . l l 0 - 1 1 2
An a n t i b o d y is made a g a i n s t t h e h a p t e n e t o be
a s s a y e d . The a n t i g e n is first p r e p a r e d by
c o u p l i n g t h e h a p t e n e t o b o v i n e serum a l b u m i n and
immunizing r a b b i t s or g o a t s . Ammonium s u l f a t e
is used t o p r e c i p i t a t e t h e y - g l o b u l i n f r a c t i o n
of t h e s e r u m , The h a p t e n e is s p i n l a b e l e d u s i n g
a s t a b l e n i t r o x i d e r a d i c a l . The s p i n of t h e
u n p a i r e d e l e c t r o n of t h e l a b e l e d compound pro-
d u c e s a m a g n e t i c moment w h i c h is detected and
measured i n a n e l e c t r o n s p i n r e s o n a n c e (ESR)
spectrometer. Very broad s p e c t r a l p e a k s a r e
o b s e r v e d when t h e complex of s p i n - l a b e l e d
h a p t e n e w i t h a n t i b o d y i n a c a p i l l a r y t u b e is
placed i n t h e c a v i t y of t h e ESR spectrometer.
T h i s r e f l e c t s t h e i m m o b i l i z a t i o n of t h e f r e e
r a d i c a l a t o r n e a r t h e a n t i b o d y s i t e . Sharp
p e a k s r e s u l t from t h e d i s p l a c e m e n t of t h e s p i n -
l ab eled h a p t e n e by f r e e h a p t e n e (as by methadone

407
 RAFlK H. BISHARA

i n u r i n e or s a l i v a ) . The a m p l i t u d e s of t h e
s h a r p p e a k s m e a s u r e q u a n t i t a t i v e l y t h e number of
t h e f r e e r a d i c a l molecules tumbling f r e e l y i n
s o l u t i o n a n d is a d i r e c t m e a s u r e of t h e h a p t e n e
c o n c e n t r a t i o n , A c o n c e n t r a t i o n greater t h a n
5 X M o f methadone h y d r o c h l o r i d e or
methadone c y c l i c metabolites is r e q u i r e d t o
p r o d u c e s p i n immunoassay r e s p o n s e s e q u i v a l e n t t o
0.5 p g . / m l . (1.8 X M ) o f m o r p h i n e . The
advantages, d i s a d v a n t a g e s and comparison o f t h i s
t e c h n i q u e t o t h i n l a y e r c h r o m a t o g r a p h y are
--
d i s c u s s e d b y L e u t e e t al.''O
8.11 Radiotracer Techniques
l 4 C and 3H a r e a - = l a b e l methadone.
The 1 4 C a c t i v i t y is a s s a y e d a s a t h i n l a y e r of
b a r i u m c a r b o n a t e mounted on aluminum d i s c s a n d
c o u n t e d u n d e r a b e l l c o u n t e r u s i n g a t h i n mica
window5' and a G e i g e r - M u l l e r c o u n t e r . In
r e c e n t y e a r s , l i q u i d s c i n t i l l a t i o n c o u n t e r s have
b e e n u s e d f o r d e t e r m i n i n g t h e r a d i o a c t i v i t y of
l 4 C a n d 3 H l a b e l e d samples.10,66,114-116

8.12 Column
----------_- Chromatography
A p p l i c a t i o n of C M - c e l l u l o s e column
c h r o m a t o g r a p h y f o l l o w e d by c o n t i n u o u s dye
complex e x t r a c t i o n is u s e d f o r t h e a s s a y of
methadone i n human u r i n e . '06
S a m p l e s c o n t a i n i n g methadone a r e
i n t r o d u c e d o n Amberlite XAD-2 r e s i n column a n d
ammonia (300: 51, '' '"
e l u t e d w i t h m e t h a n o l , 7 9 9 '17, methanol-
chloroform- i s o p r o p a n o l
(3:1), ' ' 9 and waterso p r i o r t o f u r t h e r a n a l y s i s .
The XAD-2 r e s i n is a s t y r e n e - d i v i n y l b e n z e n e
copolymer and h a s t h e c a p a b i l i t y of a d s o r b i n g
many w a t e r - s o l u b l e o r g a y & compounds p r i n c i p a l l y
by Van d e r Waal forces.
The e f f e c t i v e n e s s of r e c e n t l y
d e v e l o p e d r e s i n s BRX-SM-1, -2, -4, a n d P o r a p a k
t y p e Q a r e compared w i t h XAD-2 r e s i n by Bastos
et -
-- a1.12* The r e c o v e r y o f 3H-methadone is
somewhat s i m i l a r f o r e a c h r e s i n a n d r a n g e s from
4 5 . 4 t o 56.0%.

408
 METHADONE HYDROCHLORIDE

Methadone is r e s o l v e d from o p i a t e s a n d
d i l u e n t s i n i l l i c i t n a r c o t i c m i x t u r e s by u s i n g a
column of SE-Sephadex C-25 i o n - e x c h a n g e r . 12'
D i s p o s a b l e c h r o m a t o g r a p h i c columns a r e
u s e d t o e x t r a c t methadone from u r i n e . 1 2 2
8.13 P a p e r Chromatograph
8
Paperchromatograp y o n b u f f e r e d
Whatman N o . 1 is u s e d by A x e l r o d g o t o p r o v e t h e
N-demethylat i o n of methadone.
T a b l e 5 summarizes t h e p a p e r chroma-
t o g r a p h y s y s t e m s for methadone.
8.14 T h i n L a y e r Chromatography (TLC)
A c o m p a r i s o n of t h e t h i n laser chroma-
t o g r a p h y of methadone on s e v e n c o m m e r c i a l l y
a v a i l a b l e s i l i c a g e l c o a t e d f i l m s a n d sheets
w i t h s i l i c a gel coated glass p l a t e s u s i n g
chloroform/n-butanol/ammonium h y d r o x i d e
(70:40:5), a n d benzene/dioxane/ethanol/ammonium
h y d r o x i d e ( 5 0 : 5 0 : 5 : 5 ) is p r e s e n t e d by Schweda. l Z 9
The h a n d - c o a t e d s i l i c a g e l l a y e r on t h e g l a s s
p l a t e s is t h e most v u l n e r a b l e l a y e r . The f i l m s
are s u p e r i o r t o i t . The s i l i c a g e l s h e e t s
r e q u i r e c a r e f u l h a n d l i n g . Ho e t a l . 1 3 0 u s e m i n i
t h i n l a y e r p l a t e s (3 X 3 c m . ) to detect t h e
p r e s e n c e of methadone, 100 p g . / m l . , in an
u n h y d r o l y z e d u r i n e s a m p l e . The d e v e l o p i n g t i m e
is u s u a l l y 1.5 min. Copenhaver a n d B l o s e l 3 l u s e
s l i d e s t o prepare t h i n layer microplates for
f a s t d e t e c t i o n of methadone a n d o t h e r d r u g s of
abuse i n urine. Guptal32 u s e s d i s p o s a b l e p l a s t i c
bags t o r u n t h e t h i n l a y e r chromatography o f
methadone, m e t h a d o l , n o r m e t h a d o l , a n d
acetylmethadol. I d e n t i c a l r e s u l t s are obtained
from p l a t e s d e v e l o p e d i n t h e p l a s t i c bag or i n a
g l a s s t a n k . Dole - et - a1.133-135 u s e i o n - e x c h a n g e
p a p e r e x t r a c t i o n p r i o r t o TLC. Two-dimensional
TLC is u s e d f o r i d e n t i f i c a t i o n of t h e m e t a b o l i t e s
of methadone.80 F i s h e r - e t al.136 c h r o m a t o g r a p h
methadone on p r e c o a t e d , f l e a b l e t h i n l a y e r
s h e e t s . The s e n s i t i v i t y of TLC f o r d e t e c t i o n o f
methadone is d i s c u s s e d by G o r o d e t z k y . l 3 7

409
 TABU 5

PAPER CHROMATOGRAPHY SYSTEMS FOR METHADONE

R€
-
S o l v e n t System Paper Detection* ( x 100) Reference

tert. -Amy1 alcohol/ Schleicher & IP 123

n-butyl ether/water S c h u l l , #591-C,
(80: 7: 1 3 1 pH 3.0 35
pH 4 . 0 49
pH 5.0 22
pH 6.0 56

f Butanol/formic acid/ Schleicher & BCG 78 124

0 water (12:1:7 ) S c h u l l , #2045b

D i c h l o r o e t hane/ 75 91, 124

g l a c i a l acetic acid/
water (20:8:2)
n-Butanol/c i t r i c a c i d / Whatman No. 1 D 74 1 25
water (50: 1:50)' d i p p e d i n 5%
sod i urn hy d r og e n
c i t ra t e

(cont h u e d ...)
 TABLE 5 ( c o n c l u d e d )

Rf
S--o
- -l v e n t System Paper D e t e c t i o-
n * ( x 100) -
Reference
2
Acetate b u f f e r , Whatman No. 3 IP
pH 1.00 impregnated 86' 93 126
with tributyrin 95" 88 126
pH 3.30 (1% v/v i n 86" 76 126
pH 4 . 5 8 acetone) 86" 75 126
95" 67 126
95" 59 128
pH 7 . 4 0 86O 1 126
95" 2 126

M/15 P h o s p h a t e 86" 0 128

b u f f e r , pH 7 . 4 0

'Upper layer was u s e d

2 127
A c c o r d i n g t o Vogel

*Key - BCG : bromcresol green, D : Dragendorf f , IP : iodoplatin a t e

 TABLE 6
THIN LAYER CHROMATOGRAPHY SYSTEMS FOR METHADONE
Rf
Solvent System
--- Adsorbent*
-- - Detection* --
( X 100) Reference
Benzene/ethyl acetate/ SG RS 58 10
methanol/ammonium hydroxide SG IP 68 123
(80:20:1.2 :0.1)
Et hy 1 acetate/me t hano1/ SG DPNA 95 138
ammonium hydroxide (85:10:5) SG IP 96 117
SG IP 65 118
SG IP 91 130
SG IP 96 133
SG IP 77 134,135
SG IP 82 136
SG IP 96.9 i37
SG IP 99 139
SG BCG 84 140
Dioxane/ethanol/pyridine/ C IP 34 15
water (25:50 :20:5)
Ethanol/glacial acetic acid/ C IP 59 15
water (60:30:10) SG D 46 76
(continued .. .)
 TABLE 6 (Continued)
Rf
Solvent System
- -- * Detect ion*
Adsorbent (X 100) Reference
Benzene/dioxane/ethanol/ C IP 99 15
ammon ium hydroxide ( 50:40:5 :5 SG D 75 76
Benzene/n-butanol/methanol/ C IP 17 15
water (10 :15 :60:15) SG D 15 76
tert .-Amy1 alcohol/n-butyl SG IP 17 76
ether/water (80:7:13
$ n-Butanol/glacial acetic acid/ SG IP 55 76
water (4:1:2) SG IP 64 141
.
n-Butanol/conc hydrochloric C IP 62 15
acid, saturated with water SG IP 57 141
(90:10)
Ethyl acetate/methanol/ SG IP 96 142
ammonium hydroxide (85:10:3
Ethanol/hexane (8:92) SG IP 31 142
Ethyl acetate/methanol/ SG IP + D 94 143
ammonium hydroxide (85:lO:l)
(continued ...)
 TABLE 6 ( C o n t i n u e d )

Rf
S o l v e n t System
--- A d s o r b e n t * Detect i o n * ( X 1 0 0 )
-- Reference
Carbon t e t r a c h l o r i d e SG IP 11 144'
Isopropyl e t h e r SG IP 27 144'
Benzene SG IP 42 144'
Ethylene dichloride SG IP 37 144 '
Met h y l e n e c h l o r i d e SG IP 58 144'
2
P
Chloroform SG IP 55 144 '
Ethyl ether SG IP 67 144'
E t h y l acetate SG IP 73 144 '
n-Butyl a l c o h o l SG IP 61 144 '
Isopropy1 a l c o h o l SG IP 63 144'
Acetone SG IP 79 144'
M e t hano 1 SG IP 77 144 '
(continued ...
 n
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7 a,
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rl
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415
 TABLE 6 (Continued)
Rf
Solvent System -
Adsorbent* Detection* (X 100) Reference
Ethyl acetate/cyclohexane/ SG SA 83 150
p-dioxane/methanol/water/
ammonium hydroxide
(50:50:10:10:1.5 :0.5)
Ethyl acetate/cyclohexane/ SG IP 91 150
p-dioxane/methanol/water/
ammonium hydroxide
(50:50:10:10:0.5:1.5)
5
m
Ethyl acetate/cyclohexane/ SG ASA 94 150
methanol/water/ammonium
hydroxide (70:15:8:0.5:2)
Ethyl acetate/cyclohexane/ SG BCG 98 150
ammonium hydroxide (50:40:0.1)
Benzene/cyclohexane/ SG IP 99 123
diethylamine (15:75:10)
Ethyl acetate/dimethylformamide SG IP 99 123
(3:l)
tert.-Amy1 alcohol/n-butyl SG IP 55 123
ether/water (14:7:1)
(continued ...
 TABLE 6 (Continued)
Rf
Solvent System Adsorbent *
- Detection* ( x 100) Reference

tert.-Amy1 alcohol/n-butyl SG IP 86 123

ether/wa ter ( 8 0 :7:13
Chloroform/methanol/ammonium SG3 IP 80 151
hydroxide (85:10:1)
Ethyl acetate/methanol/ SG3 IP 67 151
ammonium hydroxide (85:10:1.5)
Chloroform/methanol (9:l) SG IP 17 146
z
4
SG IP 32 152
Acetone SG IP 20 146
Acetone/ammonium hydroxide (99:l) SG IP 59 146
Methanol SG IP 16 146
Chloroform/methanol (50:50) SG IP 20 146
Chloroform/methanol/ammonium SG IP 80 146
hydroxide (47.5 :47.5 :5)
Chloroform/glacial acetic acid/ SG IP 54 146
methanol (47.5:5:47.5)
(continued ...
 TABLE 6 (Continued)
Rf
Solvent System -
Adsorbent* Detection* -( x 100) Reference
Benzene/dioxane/ethanol/ SG IP 98 153
ammonium hydroxide
(50:40:5 :5)
Methanol/l2 N ammonium SG IP 42 153
hydroxide (100:1.5) SG IP 53 152
SG IP 37 128
SG D + IP 97 154
n-Butanol/e thy 1 acetate/ SG IP 71 133
E ethanol/ammonium hydroxide
O0 (2:28:14:0.4)
Benzene/ether (10:l) A IP 20 155
Dichloromet hane/e t her ( 10 :21 A IP 35 155
Benzene/diethylamine/dioxane/ SG IP 91 156
ethanol (50:5 :40:51
Dimethylformamide/ethyl SG IP 86 157
acetate (1:3)
Ethanol/isopropyl ether (20:80) SG IP 11 152
(conth u e d .. .
 TABLE 6 (Continued)
Rf
Solvent System
- Adsorbent
--- * Detection* (X 100) Reference
Acetoacetic ester/chloroform cc S W + D 88 158
(1:1)
Acetone/chloroform/formic A D 59 159
acid (4:16:1)
Cyclohexane/diethylamine (9:1) SG D 71 159
Benzene/chloroform/ SG D 88 159
diethylamine ( 6 :3:1)
f
\b
Benzene/l,4-dioxane/ethanol/ SG D + IP 97 154
ammonium hydroxide
(100:80:10:11)
Acetic acid/chloroform/ SG S W 53 160
methano1 (10:35 :651
Benzene/ethyl acetate/ SG S W 75 160
ammonium hydroxide (35:60:5)
Acetone/chloroform/ SG D 72 92
diethylamine (2:88:10)
(continued ...
 TABLE 6 (Continued)
Rf
Solvent
- System
- Adsorbent*
-- Detection* (X 100) Reference

Benzene/diethylamine/methanol SG D 74 92
(75:10:15)

Benzene/n-butanol/methanol/ SG D 79 92
water/ammonium hydroxide
(10:15:60:10:5)
Ethanol/ethyl acetate/ SG D 73 76
ammonium hydroxide (50:45:5)
$ Chloroform/dioxane/ethyl SG I, D, PP 73 161
0 acetate/ammonium hydroxide
(25:60:10:5 )

lIn an ammonium hydroxide atmosphere

2Top layer was used

3
Silica gel GF with 1% CaSO4.&H2O

*Key - A: alumina, ASA: ammonical silver nitrate and heat, BCG:

bromcresol green, C : cellulose, D: Dragendorff, DPNA : diazotized
(continued . ..
 TABLE 6 (Concluded)
p-nitroaniline, I: 1% iodine, IP: iodoplatinate, PP: 0.1%
potassium permenganate, RS: radioscanning, SA: 0.5% sulfuric acid,
SG: silica gel, S U V : short ultraviolet light
42 1
 RAFlK H. BISHARA

T a b l e 6 c o n t a i n s t h e most commonly u s e d t h i n
l a y e r chromatography s y s t e m s f o r methadone.
8.15 ----_-
G a s Chromatography --- (GC)
G a s chromatography s y s t e m s f o r
m e t hadone a r e r e p o r t e d - i n - T a b l e 7. A f l a m e
i o n i z a t i o n detector is used i n a l l r e f e r e n c e s
u n l e s s otherwise i n d i c a t e d .
8.16 ---
Combined G a s Chromatography-Mass
Spectroscopy ~ G C I ~ E J - - - - ~
The GCMS c o n d i t i o n s and t h e mass
f r a g m e n t a t i o n p a t t e r n have been p r e v i o u s l y
d i s c u s s e d (See 2 . 1 4 ) . T h i s t e c h n i q u e is u s e d
f o r t h e i d e n t i f i c a t i o n o f methadone
m e t a b o l i t e ~ ~ 9 , 7 ~ , 7and 7 - ~f~o r t h e s c r e e n i n g and
i d e n t i f i c a t i o n of t h e d a n g e r o u s d r u g s of
a b u s e . 171, 1 7 2
8.17 High P r e s s u r ----
e L i q u i d Chromatography
(HPU:
-- 1
High p r e s s u r e l i q u i d chromatography is
used by Lorenz1T3 f o r t h e d e t e r m i n a t i o n o f
isomethadone i n methadone. A r e v e r s e p h a s e
s y s t e m is u s e d , The column, 1 meter l o n g x
2 mm. i . d . is packed w i t h DuPont Permaphase ETH
p a c k i n g material. The m o b i l e l i q u i d c o n s i s t s of
1% r e a g e n t ammonium h y d r o x i d e , 15%methanol, and
84% water. The column is o p e r a t e d a t a f l o w
r a t e of 5 0 m l . / h r . A 254 nm. d e t e c t o r is u s e d
f o r m o n i t o r i n g t h e column e l u e n t . The r e t e n t i o n
volumes t o e l u t e isomethadone and methadone a r e
6 m l . and 17 m l . r e s p e c t i v e l y .

9. E x t r a c t i o n from B i o l o g i c a l F l u i d s
Most methods u s e d f o r t h e d e t e r m i n a t i o n of
d r u g s i n b i o l o g i c a l f l u i d s i n v o l v e three s t e p s ,
namely s o l v e n t e x t r a c t i o n , c o n c e n t r a t i o n , and
d e t e c t i o n o r a s s a y i n g . Each of these s t e p s is
time-consuming and d r u g losses due t o
a d s o r p t i o n on t o g l a s s w a r e , i n c o m p l e t e t r a n s f e r
of s o l v e n t s and e v a p o r a t i o n of v o l a t i l e
compounds may lower t h e r e c o v e r y and hence t h e
s e n s i t i v i t y . Ramsey and Campbell163 d e s c r i b e d

422
 TABLE 7
GAS CHROMATOGRAPHY SYSTEMS FOR METHADONE
Carrier Flow Rate Colum: R e t e n t i o n Refer-
--
Column --
Gas ml./min. Temp.
- C. --
T i m e , min. ence

3 f t . long x 0.125 i n . N2 30 190 7.40 68

O.D., 3% OV-17 on 100/
120 mesh G a s Chrom Q

6 f t . l o n g x 3 mm. I . D . , A 47 215 3.32 15

2% SE-30 on 80/100 mesh
G a s Chrom S
P
W
h, 6 f t . l o n g X 2 mm. I.D., He 32 2 00 2.80 162
3% SE-30 on 8 0 / l O O mesh
G a s Chrom Q

2 M long X 0.125 i n . O.D., N2 30 2 00 5.20 163

2.5% E - 3 0 1 on 80/100
mesh Chromosorb G
4.75 f t . long x 0.128 i n . - - - 14.8 149
d i a m e t e r , SE-30 on 70/80
mesh DMCS

1.22 M l o n g X 5.5 mm. N2 50 240 7.7 164

I . D . , 3% OV-17 on 100/
200 mesh G a s Chrom Q (continued . .. I
 TABLE 7 (Continued)

Carrier Flow R a t e Column R e t e n t i o n Refer-

Column Gas
-- ml./min.
-- Temp. OC. Time, m i n . ence
3% ov-1 - - 220 2.6 165

3% OV-17 - - 22 0 1.7 165

3% ov-210 - - 220 1.8 165

5 f t . l o n g X 0.125 O.D., N2 30.7 23 0 4.7 166

5% SE-30 on 60/80 mesh
C h r omosorb W
P
w
+ 6 f t . l o n g X 4 mm. I . D . , A 65 180 12.1 167l
1%SE-30 on 100/200 m e s h A 56 2 00 4.9 167
Anakrom ABS A 70 210 3.3 167

2% OV-225 on G a s Chrom Q N2 35 190 2 153

1 . 2 M long X 3 mm. I . D . , N2 35 23 5 1.2 168

3 . 5 % UCW98 on 80/lOO
m e s h C hr omosorb W -A W- DMCS

1 . 2 M l o n g X 4 mm. I . D . , He 75 2 10 4 160
3 % SE-30 on 8 0 / l O O mesh
G a s Chrom Q
(continued ...
 TABLE 7 (Continued)

Carrier Flow R a t e Colum$ Retention Refer-

Column Gas --
ml./min. Temp. C. Time, min. ence

4 ft. long X 2 . 5 mm. I.D., He 60 165 4 57,78

1%W-98 on 8 0 / l O O mesh
G a s Chrom Q

2 M long X 0 . 2 5 i n . O.D., N2 65 195 12 92

3% OV-17 on 60/80 mesh
a c i d washed, DMCS t r e a t e d
G a s Chrom Q

’ 1 M long X 0.125 i n . O.D.,

2% Carbowax on 80/100
mesh a c i d washed, DMCS
N2 36 180 12 92

t r e a t e d Chromosorb G
6 f t . long X 0.25 i n . , 5% Nz 50 23 5 3.3 9
OV-1 on 100/120 mesh
Chromosorb W
1.3 M l o n g X 6 . 2 5 mm. O.D., - - 190 6.4 77
3.8% UC-W98 on D i a t o p o r t S

2 M long x 0.25 i n . O.D., N2 16 180 14.2 76

2% SE-30 on SO/lOO mesh
Chromosorb G (continued ...
 TABLE 7 (Continued)
Carrier Flow R a t e Columt Retention Refer-
Column Gas --
ml./min. --
Temp. C. - --ence
Time, min.

1 M long x 0.125 i n . O.D., Nz 14 185 7.9 76

5% KOH and 2% Carbowax
20 M

4 f t . long X 3 mm. I . D . , N2 80 175 5.4 68

3.8% W98 on 80/100 mesh
Diatoport S

6 f t . long x 3 mm. I . D . , NZ 80 185 4.6 68

fi 1%c y c l o h e x a n e d i m e t h a n o l
s u c c i n a t e on 100/120 mesh
D i a t o m i t e CQ

2 f t . l o n g x 4 mm. I . D . , N2 40 2 00 1.7 169

2.5% SE-30 on S O / l O O m e s h
Chromosorb G

6 f t . long X 0.25 i n . NZ 60 255 1.3 170

I.D., 3% OV-1 on S O / l O O
mesh Chromosorb W H P

(continued ...
 TABLE 7 ( C o n c l u d e d )

Carrier Flow Rate Columt R e t e n t i o n Refer-

Column -G-
as ml./min. -
Temp. C. -
Time, min. ence

6 f t . l o n g X 0.25 i n . N2 40 2 05 5.25 145

O . D . , 3% SE-30 on loo/
200 mesh G a s C h r o m Q

'Strontium 90 a r g o n i o n i z a t i o n d e t e c t o r
 R A F l K H. BISHARA

a r a p i d method f o r e x t r a c t i o n of methadone i n
which t h e c o m p l e t e a n a l y s i s is carried o u t i n
one v e s s e l and o m i t t i n g t h e e v a p o r a t i o n s t e p , t o
overcome t h e v o l a t i l i t y problems. L y o p h i l l i z a -
t i o n and l i q u i d - s o l i d e x t r a c t i o n a re used t o
detect d r u g s of a b u s e i n u r i n e . l r 2
R e l i a b i l i t y of i d e n t i f i c a t i o n t e c h n i q u e s
f o r d r u g s of a b u s e i n a u r i n e s c r e e n i n g program
and d r u g e x c r e t i o n d a t a is p r e s e n t e d by K a i s t h a
and J a f f e . 1 7 4 The d r u g is a b s o r b e d on c a t i o n -
exchange r e s i n l o a d e d p a p e r and t h e i o n p a p e r is
e x t r a c t e d a t p H 1 w i t h CHC13 and t h e n
chromatographed on t h i n l a y e r p l a t e s . The
c o n c e n t r a t i o n of methadone i n t h e u r i n e by i o n
exchange paper p r o v i d e s a c l e a n e r e x t r a c t t h a n
by d i r e c t e x t r a c t i o n of t h e u r i n e . 1 4 7 Dole
--
et a -l . d e s c r i b e b u f f e r e l u t i o n of t h e i o n
exchange paper135 and t h e a p p l i c a t i o n of
A m b e r l i t e IR-120 c a t i o n exchange paper p r i o r t o
t h i n l a y e r chromatography.l34 Comparison of
three u r i n e e x t r a c t i o n t e c h n i q u e s i s r e p o r t e d
by K a i s t h a and Jaf fe. 175
"Clean-up" p r o c e d u r e s f o r u r i n e and blood
for t h e d e t e r m i n a t i o n of t h e d r u g s of a b u s e are
r e v i e w e d by Sohn e t a l . l 7 6
Kaistha - et - a1.177 r e c e n t l y e v a l u a t e d t h e
d r u g a b u s e s c r e e n i n g programs, d e t e c t i o n
p r o c e d u r e s , development costs, s t r e e t - s a m p l e
a n a l y s e s and f i e l d t e s t s .
10. D etermination i n Tissues
-_-_-----_---_----__-
The p r e v i o u s methods emphasize t h e deter-
m i n a t i o n of methadone i n b l o o d s a m p l e s , u r i n e
samples and u r i n e s c r e e n s . However, L a w l e r
e t a-
-- l . 1 0 4 s u r v e y t h e q u a n t i t a t i v e a s s a y s of
methadone i n t i s s u e s . The d r u g i n t h e t i s s u e is
examined b y s p e c t r o p h o t o f l u o r o m e t r y f o l l o w i n g
formaldehyde t r e a t m e n t and u s i n g e x c i t a t i o n a t
270 nm. and e m i s s i o n a t 4 5 0 nm. Gas liquid
chromatography is a l s o used, a f t e r s i l y l a t i o n ,
on a 3% OV-17 column packed on 100/120 mesh G a s
Chrom Q and h e a t e d a t 22OoC.

428
 METHADONE HYDROCHLORIDE

11. Bibliography
A comprehensive b i b l i o g r a p h y o f "Methadone,
1929-1971" has been p u b l i s h e d i n t w o
parts,l78,l79 L a n g r o d l s o compiled a b i b l i o g -
r a p h y o f methadone m a i n t e n a n c e t r e a t m e n t o f
h e r o i n a d d i c t i o n . The reader is r e f e r r e d t o
t h e s e t h r e e lists of r e f e r e n c e s f o r c o m p l e t e
l i t e r a t u r e a b o u t methadone.
The l i t e r a t u r e s e a r c h f o r p r e p a r i n g t h i s
p r o f i l e w a s c o n d u c t e d t h r o u g h A p r i l of 1973.
12. Acknowledgments
Theauthorwishes t o t h a n k t h e Merck and
Co., Inc., Rahway, N, J., f o r g r a n t i n g t h e
p e r m i s s i o n t o u s e some of t h e i n f o r m a t i o n a b o u t
methadone h y d r o c h l o r i d e from t h e Merck Index,
8 t h e d i t i o n . Thanks are a l s o due t o
Mr. C . E. Hubach and A n a l y t i c a l C h e m i s t r y , and
t o D r . E . L. May and T h e m 5 6 T O r g a n i c -
Chemistry f o r t h e i r permission t o reproduce
Table 1 and F i g u r e 9 r e s p e c t i v e l y .
The a u t h o r e x p r e s s e s h i s a p p r e c i a t i o n t o
D r . A . D. Kossoy, M r . C . D. Underbrink,
D r . D. E. Dorman, Mr. H. R . S u l l i v a n , a n d
M r . J. L. Occolowitz o f E l i L i l l y and Company
f o r a s s i s t a n c e i n p r e p a r a t i o n and d i s c u s s i o n
o f t h e i n t e r p r e t a t i o n of v a r i o u s s p e c t r a l
d a t a i n t h i s monograph.
S p e c i a l t h a n k s go t o Miss Adele Hoskin,
Mrs. N e l l V. Ward, and Mrs. JoAnn S p e n c e r f o r
t h e i r h e l p i n t h e s e a r c h o f t h e voluminous
literature.
The a u t h o r is v e r y g r a t e f u l t o
Mrs. J a n i s A . Brown f o r h e r i n v a l u a b l e
secretarial help i n developing t h e format
o f t h i s p r o f i l e and t y p i n g t h e m a n u s c r i p t .
The a u t h o r e x t e n d s h i s s i n c e r e a p p r e c i a -
t i o n t o h i s c o l l e a g u e s a t E l i L i l l y and
Company, w i t h a s p e c i a l r e f e r e n c e t o
Mr. C . D. Wentling, f o r t h e i r h e l p , c r i t i c i s m ,
and s u g g e s t i o n s t o improve t h i s a n a l y t i c a l
profile.

429
 RAFlK H. BISHARA

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436
 METHODONE HYDROCHLORIDE

131. Copenhaver, J . H., Blose, I . L., and

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431
 R A F l K H. BISHARA

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165. Goldbaum, L. R., S a n t i n g a , P., and
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438
 METHADONE HYDROCHLORIDE

167. Kazyak, L., and Knoblock, E . C., A n a l .

Chem., 35, 1448 ( 1 9 6 3 ) .
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44, 385 (1972).
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-
B o n e l l i , E . J;, J. Chromatogr. S c i . , 10,
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(1970).

439
 OXAZEPAM

Charles M . Shearer and Caesar R . Pilla

 CHARLES M. SHEARER AND CAESAR R . PILLA

CONTENTS
1. Description
1.1 Name, Formula, Molecular Weight
1.2 Appearance, C o l o r , Odor
2. Physical Properties
2.1 Infrared Spectra
2.2 Nuclear Magnetic Resonance S p e c t r a
2.3 U l t r a v i o l e t S p e c t r a
2.4 Mass S p e c t r a
2.5 Melting Range
2.6 D i f f e r e n t i a l Thermal A n a l y s i s
2.7 Solubility
2.8 C r y s t a l P r o p e r t i e s
2.9 D i s s o c i a t i o n C o n s t a n t
3. Synthesis
4. Stability
5. Metabolism
6. Methods of A n a l y s i s
6.1 Elemental A n a l y s i s
6.2 G r a v i m e t r i c A n a l y s i s
6.3 D i r e c t S p e c t r o p h o t o m e t r i c A n a l y s i s
6.4 C o l o r i m e t r i c A n a l y s i s
6.5 F l u o r o m e t r i c A n a l y s i s
6.6 T i t r i m e t r i c A n a l y s i s
6.7 P o l a r o g r a p h i c A n a l y s i s
6.8 Chromatographic A n a l y s i s
6.81 Paper Chromatography
6.82 Thin Layer Chromatography
6.83 Gas Chromatography
6.84 Column Chromatography
7. References

442
 OXAZEPAM

1. Description

1.1 Name, Formula, Molecular Weight

The name used by Chemical A b s t r a c t s and t h e

N a t i o n a l Formulary XI11 f o r oxazepam i s 7-chloro-1,3-
dihydro-3-hydroxy-5-phenyl-2&1,4 benzodiazepin-2-one.

C H C1N202 Mol. W t . : 286.72

15 11
1 . 2 Appearance, C o l o r , Odor

Oxazepam i s a creamy w h i t e t o p a l e yellow powder

having p r a c t i c a l l y no odor.

2. Physical Properties

2.1 Infrared Spectra

An i n f r a r e d a b s o r p t i o n spectrum of a potassium
bromide d i s p e r s i o n of oxazepam (NF R e f e r e n c e Standard
m a t e r i a l ) i s p r e s e n t e d i n F i g u r e 1. This spectrum a g r e e s
w i t h p u b l i s h e d s p e c t r a l , 2 * 3 b The s p e c t r a l band a s s i g n -
ments a r e l i s t e d i n Table I.

Table I
I n f r a r e d S p e c t r a l Assignments of Oxazepam
Wavelength, u V i b r a t i o n Mode Reference
3.05 t o 3.20 OH, NH s t r e t c h 4
5.79 and 5.86 C=O s t r e t c h 5
6.19 C=N-s t r e t c h 5
6.35 and 6.72 Aromatic C=C d e f o r m a t i o n s 6
12.07 Out of p l a n e CH 6
deformation of 1 , 2 , 4
s u b s t i t u t e d aromatic
13.39 and 14.35 Out of p l a n e CH 6
deformation of mono
s u b s t i t u t e d aromatic

443
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 OXAZEPAM

2.2 Nuclear Magnetic Resonance

The 60 MHz NMR spectrum, shown i n F i g u r e 2 , was
o b t a i n e d by d i s s o l v i n g oxazepam (NF Reference Standard
material) i n deutero dimethylsulfoxide containing t e t r a -
m e t h y l s i l a n e a s 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 l a s s i g n -
ments a r e l i s t e d i n Table 11.
Table 11
NMR S p e c t r a l Assignments of Oxazepam
Chemical S h i f t ( & I Protons Splitting
4.82 a l i D h a t i c C-H Doublet
6.35 0-H' Doublet
7.52 a r o m a t i c CH Mu1 t i p 1e t
10.78 N-H Singlet
-
Both t h e hydroxyl and amino p r o t o n s exchanged w i t h D 0.
2
Sadee8 r e p o r t e d a s i n g l e t f o r t h e a l i p h a t i c C-H proton,
however, a s i n g l e t was observed i n t h e s e l a b o r a t o r i e s
only a f t e r D 0 exchange.
2
2.3 U l t r a v i o l e t Spectra
The u l t r a v i o l e t s p e c t r a of oxazepam i n 0.1Nhydro-
c h l o r i c a c i d , 0.1N sodium hydroxide, and i n pH 7 b u f f e r
a r e p r e s e n t e d i n F i g u r e 3. The spectrum of oxazepam i n
a l c o h o l i s p r e s e n t e d i n F i g u r e 4. The spectrum and absorp-
t i v i t i e s of oxazepam i n a l c o h o l a g r e e w i t h p u b l i s h e d
v a l u e ~ l , ~ , The ~ - a b s o r p t i v i t i e s and maximum wavelengths
a r e p r e s e n t e d i n Table 111.
Table I11
Ultraviolet Spectral Characteristics
Solvent max (nm) Absorptivity
0.1N HC1 236 111
284 41
362 13

PH 7 230 131
315 9
0.1N NaOH 236 111
342 11
alcohol 230 126
318 9

445
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 OXAZEPAM

0.7 1

0.6

0.5

w 0.4
u
z
3 0.3
am
Q 0.2

0.1

0 .o

WAVE LENGTH (nrn)

F i g . 3 - U l t r a v i o l e t S p e c t r a of Oxazepam (NF R e f e r e n c e
S t a n d a r d ) Solvent-0.1N HCl--- pH 7 b u f f e r , 8 I t 0,lNNaOfi.

WAVE LENGTH (nrn)

Fig. 4 - U l t r a v i o l e t Spectrum of Oxazepam (NF R e f e r e n c e

Standard) Solvent-alcohol.

447
 CHARLES M. SHEARER AND CAESAR R. PlLLA

2.4 Mass S p e c t r a

The mass spectrum of oxazepam (NF Reference Stan-

dard m a t e r i a l ) w a s o b t a i n e d by d i r e c t i n s e r t i o n of t h e sam-
p l e i n t o t h e MS-902 double f o c u s i n g , h i g h r e s o l u t i o n mass
spectrometer. The sample w a s run a t 200°C. and 1.0 x 10-6
t o r r w i t h t h e i o n i z a t i o n e l e c t r o n beam energy a t 70 e.v.
The high r e s o l u t i o n d a t a was compiled and t a b u l a t e d w i t h
t h e a i d of t h e PDP-8 D i g i t a l computer. A l i n e graph of
t h e mass spectrum i s shown a s F i g u r e 5 and t h e major high
r e s o l u t i o n d a t a i n Table IV 10

Table IV
Mass Spectrum of Oxazepam
Measured Mass C a l c u l a t e d Mass Formula
286.0484 286.0509 C H O N C 1
1 5 11 2 2
270.0372 270.0321 C15H902NC1
269.0474 269.0481 C H ON C 1
15 10 2
268.0436 268.0402 C H ON C 1
15 9 2
267.0319 267.0325 C H ON C 1
15 8 2
259.0427 259.0399 C H 0 NC1
14 10 2
257.0450 257.0481 C H ON C 1
14 10 2
241.0461
241.0420 15H10''
239.0379 239.0376
14H8N
233.0715 233.0714
'1 gH90N2
229.0526 229.0532
C13H10N2C1
205.0738 205.0765
C14H9N2
194.0849 194.0844
C13H10N2
11
This spectrum i s i n agreement w i t h t h a t p r e s e n t e d by Sadee.

The molecular i o n was a t r n / = 286. The base peak, m/g

257, i s g e n e r a t e d by loss of a formyl r a d i c a l , which may
be preceeded by a h y d r i d e m i g r a t i o n from C-3 t o C-5. Addi-
t i o n a l loss of CO g e n e r a t e d an i n d a z o l e a t m / e 229, which
then e l i m i n a t e d C 1 50g i v e m/e 194. OthFr predominant
peaks a r e m/g 269(M - OH) and m/e 259(M -CHN).

448
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 CHARLES M. SHEARER AND CAESAR R. PILLA

2.5 Melting Range

The following m e l t i n g p o i n t temperatures have been

reported:
OC. Reference
203 - 204 5
205 -
206 12
20 6 13,14

2.6 D i f f e r e n t i a l Thermal A n a l y s i s

A s t u d y by d i f f e r e n t i a l thermal a n a l y s i s (DTA)
r e v e a l s t h a t be m e l t i n g endotherm i s a f u n c t i o n of t h e
heating r a t e 15. This i s i l l u s t r a t e d i n Table V. A DTA
curve of oxazepam (NF Reference Standard m a t e r i a l ) o b t a i n e d
a t a h e a t i n g r a t e of 10°/min. i s i n c l u d e d a s F i g u r e 6.

Table V
D i f f e r e n t i a l Thermal A n a l y s i s of Oxazepam

heating r a t e -
OC.

min.
m e l t i n g endotherm OC.

5 188
10 193
20 201

2.7 Solubility

The following s o l u b i l i t y d a t a were o b t a i n e d a t

u n c o n t r o l l e d room temperature:
4.5 mg./ml. i n 95% e t h a n o l
0.03 mg./ml. i n w a t e r
4 mg./ml. i n chloroform
These v a l u e s a g r e e w i t h t h e s o l u b i l i t i e s given i n t h e
National Formulary XIII.

2.8 Crystal Properties

The X-ray d i f f r a c t i o n p a t t e r n of oxazepam (NF

Reference Standard m a t e r i a l ) was o b t a i n e d w i t h a P h i l i p s
d i f f r a c t o m e t e r using Cu KG r a d i a t i o n 15. The c a l c u l a t e d d
spacings of t h e d i f f r a c t i o n p a t t e r n a r e p r e s e n t e d i n Table
VI.

450
 1

0 50 100 150 201) 250 300 350 400 450

T. OC (CORRECTED FOR CHROMEL ALUMEL THERMOCOUPLES)
Fig. 6 - DTA Spectrum of Oxazepam (NF Reference S t a n d a r d )
 CHARLES M. SHEARER AND CAESAR R . PILLA

Table V I
X-Ray-Powder, D i f f r a c t i o n P a t t e r n
Sample: Oxazepam: NF Reference Standard
Source: C u K S
28 d - 2& d 20- d
*6.6 13.4 >G5.3 4T3 74 31.0 2T885
12.4 7.13 21.1 4.210 31.4 2.849
13.2 6.70 21.5 4.132 32.2 2.780
14.8 5.98 22.6 3.934 33.3 2.690
16.6 5.34 23.3 3.817 34.0 2.636
17.4 5.096 24.3 3.662 34.4 2.607
17.7 5.010 25.4 3.506 36.8 2.442
18.5 4.796 26.5 3.363 38.7 2.326
19.4 4.576 27.2 3.279 39.0 2.310
39.8 4.482 29.8 2.998 *Most i n t e n s e peaks
20.0 4.440 30.4 2.940
~ ~~ ~~ ~

2.9 D i s s o c i a t i o n Constant

The pKa's o f oxazepam i n aqueous s o l u t i o n s were

determined s p e c t r o p h o t o m e t r i c a l l y t o be 1.8, a t which a
proton i s g a i n e d ; and 11.1, a t which a p r o t o n is l o s t .

3. Synthesis

One s y n t h e t i c r o u t e f o r t h e p r o d u c t i o n of oxazepam i s
given i n F i g u r e 7. The q u i n a z o l i n e 3-oxide can be p r e p a r e d
by r e a c t i n g c h l o r a c e t y l c h l o r i d e w i t h 2-amino-5-chloro-
benzophenone oximel6. The r i n g expansion s t e w i t h sodium
hydroxide was d e s c r i b e d by Bell and coworkersq7. Treat-
ment of t h e r e s u l t i n g benzodiazepin -2-one 4-oxide w i t h
a c e t i c anhydride and h y d r o l y s i s of t h e e s t e r w i t h b a s e
y i e l d s oxazepam5. Other s y n t h e t i c r o u t e s and t h e chemistry
of benzodiazepines i n g e n e r a l a r e d i s c u s s e d i n review
articles18,19,20,21.

4. Stability

Oxazepam i s s t a b l e a s a s o l i d o r i n a n e u t r a l s o l u t i o n19 .
Acid h y d r o l y s i s produces 2-amino-5-chlorobenzophenone 22.
The itnino-carbinol s t r u c t u r e l e a d s t o a number of r e a r r a n e-
ments ( F i g u r e 8 ) upon t r e a t m e n t w i t h a c e t i c a c i d 5 o r b a s s g .

452
 OXAZEPAM

'gH!5

2-amino- 5- chloro- 2-amino- 5-chloro- 6- chloro- 2-

benzophenone benzophenone oxime chloromethyl-
4-pheny l- quin-

\1
a z o l i n e 3 oxlde

a$
NaOH

H 0 H
II

c1

c6H5
c6H5
3-acetoxy-7-chloro-5-phenyl-l,3- 7- chloro- 5-phenyl-
dihydro- 2E- 1,4-benzodiazepine-3 one 1,3-dihydro-2&-1,
4-benzodiazepine- 2
one 4 oxide

NaOH

6H5
oxa zep am

Figure 7 - S y n t h e t i c Route € o r Oxazepam

453
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454
 OXAZEPAM

5. Metabolism

The major metabolite (greater than 95%) of oxazepam in

man has been determined to be oxazepam glucuronide22.
Minor metabolites are as follows: 6-chloro-4-phenyl-241H)-
quinazolinone; 2- amino-5-chlorobenzophenone; 2'-ben~oyl-4~-
chloro-2,2-dihydroxyacetanilide; 2'-benzoyl-4khloro-2-
hydroxy-2-ureidoacetanilide; 7-chloro-I, 3-dlhydro-Shydr~xy-
5-(phydroxyphenyl)-2&1,4-benzodiazepin-2-one; and 7-
chloro-l,3-dihydro-3-hydroxy-5-[ 3 (or 4)-hydroxy-4-(or 3)
methoxyphenyll-2~-1,4-benzodiazepin-2-one. The first two
minor metabolites exist only in the unconjugated state in
urine while the othe 8 are present as conjugates as well
as in the free form25

6. Methods of Analysis

6.1 Elemental Analysis

The following data were obtained on NF Reference

Standard material7:
Element % Theorv % Found
C 62.94 62.71
H 3.85 3.99
N 9.79 9.62
c1 12.41 12.47

6.2 Gravimetric Analysis

Oxazepm,can be precipitated out of a very dilute

acidic solution with silicotungstic acid. The precipitate
is collected, washed with water, dried at 7OOC. and weighed
This method has been applied to dosage forms2.

When a solution of Reinecke's salt is added to a

dilute solution of oxazepam, a bright rose-violet colored
precipitate is formed. The precipitate is then washed,
dried, and weighed 2;Z4.
6.3 Direct Spectrophotometric Analysis

Salim and coworkers' described an ultraviolet

spectrophotometric method of analysis which was applicable
to capsules of oxazepam. In this method a sample equiva-

455
 CHARLES M. SHEARER AND CAESAR R. PlLLA

l e n t t o about 50 mg. oxazepam is e x t r a c t e d w i t h a l c o h o l

through a s i n t e r e d g l a s s funnel. This s o l u t i o n i s d i l u -
t e d t o o b t a i n a f i n a l c o n c e n t r a t i o n of about 4 mcg./ml.
i n alcohol. The absorbance i s r e a d on a spectrophotometer
a t 229 nm. u s i n g a l c o h o l a s a blank and compared w i t h t h e
absorbance of a s t a n d a r d s o l u t i o n of oxazepam. This i s
a l s o t h e method s p e c i f i e d i n t h e N a t i o n a l Formulary XIII.
A completely automated system c a p a b l e of d i s i n t e -
g r a t i n g a whole t a b l e t or c a p s u l e , d i s s o l v i n g t h e a c t i v e
c o n s t i t u e n t , f i l t e r i n g i t , d i l u t i n g a p o r t i o n of t h e clear
f i l t r a t e t o a d e s i r e d volume an o b t a i n i n g a complete UV-
v i s i b l e scan, h a s been reportedg5. The accuracy of t h e
automated method i s comparable t o t h a t of t h e manual spec-
t r o p h o t o m e t r i c method f o r oxazepam.
6.4 Colorimetric Analysis
When a s o l u t i o n of R e i n e c k e ' s s a l t i s added t o a
d i l u t e s o l u t i o n of oxazepam a b r i g h t r o s e - v i o l e t c o l o r e d
p r e c i p i t a t e i s formed. This can be i s o l a t e d and t h e n d i s -
solved i n a c e t o n e and i t s
p h o t o m e t r i c a l l y a t 525 nm2yt.c e n t r a t i o n determined s p e c t r o -

Oxazepam can be hydrolyzed w i t h h y d r o c h l o r i c a c i d

t o form g l y c i n e and 2-amino-5- chlorobenzophenone. The
aromatic amine can be d i a z o t i z e d w i t h n i t r o u s a c i d and
coupled t o naphthylene diamine26, N-alpha naphthy1-N-
d i e t h y p r ~ p y l e n e d i a m i n e ~N-(l-naphthyl)ethylenediamine.2
~,
HC122138, o r a l p h a naphthol29. The c o n c e n t r a t i o n of t h e
oxazepam i n t h e r e s u l t i n g c o l o r e d s o l u t i o n can be d e t e r -
mined s p e c t r o p h o t o m e t r i c a l l y i n t h e v i s i b l e region.
6.5 Fluorometric Analysis
Walkens t e i n and coworkers22 a p p l i e d f luorometry
t o t h e d e t e r m i n a t i o n of oxazepam i n b i o l o g i c a l f l u i d s .
Oxazepam w a s e x t r a c t e d from the body f l u i d s by e t h y l e n e
d i c h l o r i d e and t h e f l u o r e s c e n c e measured on a f l u o r o m e t e r
equipped w i t h a high p r e s s u r e mercury lamp, a 200-400 m p
broad band primary f i l t e r and a B540 (520-660 m p ) broad
band secondary f i l t e r .

Braun and coworkers30 developed a f l u o r o m e t r i c

method f o r determining oxazepam which had a s e n s i t i v i t y of

456
 OXAZEPAM

0.005 pg./ml. I n t h i s method an e t h a n o l i c s o l u t i o n of

oxazepam i s h e a t e d i n phosphoric a c i d , producing a v e r y
i n t e n s e f l u o r e s c e n c e . The f l u o r e s c e n c e i s measured a t
an e x c i t a t i o n wavelength of 360 nm. and an emission wave-
l e n g t h of 475 nm. T h i s method has a l s o been used f o r
dosage form a n a l y s i s 3 1 .

S t e i d i n g e r and S ~ h m i ddeveloped
~ ~ two methods f o r
determining oxazepam, combining t h i n l a y e r chromatography
and fluorometry. I n one method, t h e oxazepam i s s c r a p e d
o f f t h e developed TLC p l a t e and e x t r a c t e d w i t h methanol.
P e r c h l o r i c a c i d i s added t o t h i s s o l u t i o n and i t is heated.
The r e s u l t i n g f l u o r e s c e n c e can then be determined w i t h a
f l u o r o m e t e r . The o t h e r method, a l s o r e p o r t e d by L a u f f l e l ,3
i s t o treat the i n t a c t p l a t e with t r i c h l o r o a c e t i c acid,
h e a t t h e p l a t e , and determine t h e f l u o r e s c e n c e d i r e c t l y by
u s e of a t h i n l a y e r chromatographic p l a t e scanner.
6.6 T i t r i m e t r i c Analysis

Oxazepam can be d i s s o l v e d i n dimethylformamide,

and t i t r a t e d w i t h 0.1N tetrabutylammonium hydroxide [ p r e -
p a r e d i n benzene:methanol (9: 1 ) 1 t o a p o t e n t i o m e t r i c
e n d p o i n t u s i n g glass vs. calomel electrodes1.

Beyer and Sadee34 determined oxazepam by d i s s o l -

ving i t i n g l a c i a l a c e t i c a c i d and t i t r a t i n g w i t h 0.05N
p e r c h l o r i c acid. The e n d p o i n t w a s determined e i t h e r poten-
t i o m e t r i c a l l y o r v i s u a l l y , u s i n g c r y s t a l v i o l e t a s an i n d i -
c a t o r . A c e t i c anhydride w a s a l s o used as a s o l v e n t f o r
oxazepam, g i v i n g comparable r e s u l t s .

6.7 Polarographic Analysis

The p o l a r o g r a p h i c b e h a v i o r of oxazepam has been

d i s c u s s e d i n many papers. F a z z a r i and Riggleman350btained
w e l l - d e f i n e d c a t h o d i c waves a t t h e dropping-mercury elec-
t r o d e i n a m i x t u r e of methylene chloride-methyl a l c o h o l
w i t h a s u p p o r t i n g e l e c t r o l y t e of 0.1M tetraethylammonium
bromide. The halfwave p o t e n t i a l of oxazepam i n t h i s system
i s about -1.02V and t h e d i f f u s i o n c u r r e n t i s l i n e a r w i t h
c o n c e n t r a t i o n . T h i s method w a s a p p l i e d t o c a p s u l e s of
oxazepam.

O e l s c h l a g e r and coworkers36 a l s o found a l i n e a r

451
 CHARLES M. SHEARER AND CAESAR R . PILLA

r e l a t i o n between c o n c e n t r a t i o n and d i f f u s i o n c u r r e n t i n an
a c e t a t e b u f f e r system c o n t a i n i n g 20% dimethylformamide.
T h i s procedure w a s used t o a s s a y t a b l e t s c o n t a i n i n g oxaze-
pam. They a l s o c o r r e l a t e d half-wave p o t e n t i a l v e r s u s a
s a t u r a t e d calomel e l e c t r o d e , w i t h pH as shown below. The
pH w a s determined i n a Britton-Robinson b u f f e r c o n t a i n i n g
20% dimethylformamide.
PH 2.3 3.4 5.1 6.0 7.2 8.2 9.2
E % ( V ) -,695 -.825 -.955 -1.005 -1.08 -1.125 -1.185

Oeschlager and coworkers37 f u r t h e r i n v e s t i g a t e d t h e

e l e c t r o c h e m i s t r y of oxazepam and found t h a t i n a c i d b u f f e r s
i t i s reduced w i t h t h e u p t a k e of 4 e l e c t r o n s t o form 7-
chloro-5-phenyl-1,3,4,5-tetrahydro-2~-1,4-benzodiazepin-
2-one a t t h e dropping mercury e l e c t r o d e . However, i n
a l k a l i n e b u f f e r s t h e u n s t a b l e 4 , 5 dihydro d e r i v a t i v e which
i s formed f i r s t by consuming 2 e l e c t r o n , reacts f u r t h e r
forming a c y c l i c aldehyde-ammonia adduct which undergoes
r e d u c t i o n t o form t h e same p r o d u c t as is formed i n a c i d i c
solution.

6.8 Chromatographic A n a l y s i s

Many of t h e more common chromatographic technique6

have been a p p l i e d t o oxazepam.

6.81 Paper Chromatography

Oxazepam h a s been chromatographed on Whatman

%1 paper w i t h e i t h e r b u t a n o l , e t h a n o l , w a t e r (17:3:20)
upper s h a s e o r b u t a n o l , p y r i d i n e , w a t e r (6:4:3) a s t h e
e l u a n t 2.

6.82 Thin Layer Chromatography

The v a r i o u s e l u a n t systems used f o r t h i n l a y e r

chromatography on s i l i c a g e l p l a t e s f o r oxazepam are given
i n Table V I I . Table V I I I g i v e s s p r a y r e a g e n t s used f o r
t h e d e t e c t i o n of oxazepam on t h i n l a y e r chromatographs.

6.83 Gas Chromatography

Gas chromatography has been used t o analyze

oxazepam. The n e c e s s a r y d a t a of t h e v a r i o u s methods are
given as Table IX.

458
 OXAZEPAM

Table V I I
Thin Layer Chromatography System for Oxazepam
!k Eluant Reference
0.00 chloroform:e t h a n o m l ) 27
0.03 cyc1ohexane:diethylamine:benzene 40
(75:20:15)
0.04 benzene:ethyl acetate (5:l) 41
0.08 benzene: ethanol:ammonium hydroxide 40
(95:15:5)
0.08 chloroform:to1uene:methanol (10:9:1) 42
0.13 heptane: chloroform:ethanol (10:10: 1) 43
0.13 ch1oroform:cyclohexane:diethylamine 44
(40:50:10)
0.14 to1uene:nitromethane:methanol (11:8:1) 42
0.16 carbon tetrach1oride:methanol (90:10) 44
0-21 benzene:methanol:ammonium hydroxide 13
(9O:lO:1)
0.23 to1uene:diethylamine (80:20) 44
0.29 benzene:acet0ne:diethylamine (70:20:10) 43
0.29 isopropanol:isopropyl ether (16:84) 45
0-35 ch1oroform:methanol (10:1) 42
0.37 ethanol :water ( 96:4) 45
0.46 methanol:acetone (12:88) 45
0.48 methanol :methyl acetate (18:82) 45
0.50 benzene:ethano1:diethylamine (5:1:0.5) 46
0.50 ch1oroform:ethanol: acetone (8:l: 1) 41
0.51 cyclohexane:diethylamine (85:17) 44
0.52 carbon tetrach1oride:methanol (75:25) 44
0.55 acetone 40
0.57 methanol :ammonium hydroxide (100:1,5) 40
0.58 chloroform:acetone:methanol (70:20:10) 44
0.59 heptane: ch1oroform:ethanol (5:5:2) 47
0.60 ethyl acetate:1,2 dichloroethane (80:20) 44
0.60 isopropanol:ammonium hydroxide (20: 1) 43
0.66 benzene:acetone:methanol (55:35:10) 44
0.68 isopropano1:methanol (30:70) 44
0.68 ch1oroform:methanol: acetic acid (88:10: 2) 44
0.73 isopropano1:ammonium hydroxide:water 44
(75:17:18)
0.82 chloroform:acet0ne:diethylamine (50:40:10) 44
0.84 ethyl acetate:methanol:acetic acid 44
(80:20:10)
0.88 chloroform:acetic acid:methanol (15:114) 41

459
 CHARLES M. SHEARER AND CAESAR R . PlLLA

Sadee and Van d e r Kleijn38 found t h a t oxazepam

r e a r r a n g e d t o t h e q u i n a z o l i n e carboxaldehyde ( F i g u r e 8,
S t r u c t u r e IV) during g a s chromatography.
6.84 Column Chromatography
scribe a high pressure
S c o t t and B ~ m m e rd~e ~
l i q u i d chromatography system which w a s used f o r s e v e r a l
benzodiazepines, i n c l u d i n g oxazepam. The column was 100
cm. x 1 mm. i.d. s t a i n l e s s s t e e l packed w i t h Waters Asso-
c i a t e s Durapak rrOPN", 36-75 p m. p a r t i c l e diameter. The
e l u a n t w a s a mixture of hexane and isopropanol. An u l t r a -
v i o l e t d e t e c t o r w a s used.
Table V I I I
TLC Spray Reagents f o r t h e D e t e c t i o n of Oxazepam
Reagent -
Color L i g h t * Reference
Source
Bromine water orange - vis.
I1
13
Reinecke s o l u t i o n rose 13
11
Iodine s o l u t i o n brown 13
Sulfuric acid yellow II
13
I1
Dragendorf f yellow 46
It
Chlorine-o-toluidine violet 42
Cerric sulfate-Dragendorff red-or ang e II
42
Diphenylcarbazone l i g h t purple II
48
II
Silver acetate blue-purple 48
Mercuric s u l f a t e lavender I1
48
Cinnamaldehyde blue uv 40
Furfural reagent green It
40
Zinc (11) c h l o r i d e -
hydrochloric acid beige II
44
70% p e r c h l o r i c a c i d yellow-orange v i s . 44
Cerium (IV) s u l f a t e yellow uv 44
Formaldehyde-hydrochloric
acid l i g h t blue 11
44
40% o-phosphoric a c i d 1i g h t ye1 low vis. 44
Antimony (111) c h l o r i d e - l i g h t blue uv 44
acetic acid yellow via 44
Vanillin-sulfuric acid blue uv 44
yellow vis. 44

*vis. = v i s i b l e UV = u l t r a v i o l e t

460
 Table I X

G a s Chromatographic System f o r Oxazepam

Column Packing Column Carrier G a s Column Temperature Detector Ref*

3% OV 1 on G a s 2 m, x 2 mm. Nitrogen @ 245QC. flame i o n i - 49
Chrom Q (60180) glass 22 ml./min. zation

3% OV 1 7 on Gas 4 f t . x 4 mm. Argon-me thane ( 90 : 230OC. electron 50

Chrom Q (60180) glass 10) @ 100 mlJmin, capture

1.5% OV 1 on 3 f t . x 118 i n . H e l i u m @ 20 200 - 280°C. pro- t o t a l ion 38

z
fi HP Chrom G glass .
m l Imin. grammed current

3% OV 1 7 on 6 f t . x 118 i n . Nitrogen @ 245°C. flame i o n i - 38

Gas Chrom W stainless steel 70 ml./min. zation

3% OV 1 7 on 6 it. x 118 in. Argon-me thane ( 20 : 245OC. electron 38

G a s Chrom W s t a i n l e s s steel 1 ) @ 70 ml./min. capture
 CHARLES M. SHEARER AND CAESAR R . PILLA

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462
 OXAZEPAM

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463
 CHARLES M. SHEARER AND CAESAR R. PILLA

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464
 PHENAZOPYRIDINE HYDROCHLORIDE

Kenneth W. Blessel, Bruce C. Rudy, and Bernard Z. Senkowski

 KENNETH W. BLESSEL, BRUCE C. RUDY, AND BERNARD 2 . SENKOWSKI

INDEX

Analytical Profile - Phenazopyridine Hydrochloride

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 U1 traviolet Spectrum
2.4 Fluorescence Spectrum
2.5 Mass Spectrum
2.6 Optical Rotation
2.7 Melting Range
2.8 Differential Scanning Calorimetry
2.9 Thermogravimetric Analysis
2.10 Solubilities
2 , I1 X-ray Crystal Properties

3. Synthesis

4. Stability Degradation

5. Drug Metabolic Products

6. Methods of Analysis
6.1 Elemental Analysis
6.2 Phase Solubility Analysis
6.3 Thin Layer Chromatographic Analysis
6.4 Direct Spectrophotometric Analysis
6.5 Coulometric Analysis
6.6 Titrimetric Analysis
7. Acknowledgments
8. References

466
 PHENAZOPYRIDINE HYDROCHLORIDE

1. D e s c r i p t i o n

1.1 N a m e , Formula, M o l e c u l a r Weight

P h e n a z o p y r i d i n e h y d r o c h l o r i d e is 2,6-diamino-3-
( p h e n y 1 a z o ) p y r i d i n e monohydrochloride.

Cl1HI1N5 *HC1 M o l e c u l a r Weight: 249.70

1.2 Appearance, C o l o r , Odor

Phenazopyridine h y d r o c h l o r id e i s a l i g h t t o d ar k
r e d , o d o r l e s s c r y s t a l l i n e powder.

2. Physical Properties

2.1 I n f r a r e d Spectrum
The i n f r a r e d s p e c t r u m o f a sample of r e f e r e n c e
s t a n d a r d p h e n a z o p y r i d i n e h y d r o c h l o r i d e is shown i n F i g u r e 1
(1). The i n s t r u m e n t used w a s a P e r k i n E l m e r Model 621
r e c o r d i n g s p e c t r o p h o t o m e t e r . The sample was d i s p e r s e d i n
m i n e r a l o i l i n o r d e r t o r e c o r d t h e s p e c t r u m . The f o l l o w i n g
a s s i g n m e n t s have b e e n made f o r t h e bands i n F i g u r e 1 (1).

Band Assignment
3329 and 3275 2 5 cm-1 N-H s t r e t c h
3065 2 5 cm-1 a r o m a t i c C-H s t r e t c h
1 6 0 1 and 1500 5 5 cm-l aromatic r i n g v i b r a t i o n s
1638 If: 5 cm-1 NH2 d e f o r m a t i o n s
815 2 5 cm-1 o u t of p l a n e d e f o r m a t i o n of
H on pyridine ring
713 25 cm'l o u t o f p l a n e b e n d i n g o f NH2
682 5 cm-l o u t of p l a n e d e f o r m a t i o n s of
H on benzene r i n g
2.2 N u l e a r Magnetic Resonance Spectrum (NMR)
The NMR spectrum of p h e n a z o p y r i d i n e h y d r o c h l o r i d e
is shown i n F i g u r e 2 ( 2 ) . The s o l v e n t used was DMSO-d6,
t h e i n t e r n a l r e f e r e n c e w a s t e t r a m e t h y l s i l a n e and t h e

467
a,
7)
.d
k
0
4
0
k
T
I
&
aJ
E
'd
7)
'rl
U
6
8
(D f
8
E
W
0
B
k
u
V
a,
a
v)
a
a,
k
(D
k
W
C
I4
33NVlllWSNVUl '10
468
 I
469
 KENNETH W. BLESSEL, BRUCE C. RUDY, AND BERNARD 2. SENKOWSKI

solution concentration was 30.9 mg in 0.5 ml of solvent.

The spectral assignments are given in Table I (2).
Table I

NMR Spectral Data for Phenazopyridine Hydrochloride

No. of Chemical Coup1ing

Proton Each Shift (ppm) Multiplicity Constant

Proton on C5 1 6.36 doublet J H ~ - H ~ =Hz

~O
Protons meta and
para to phenylazo
nitrogen 3 7.25-7.65 mu1tip1et
Protons ortho to
phenylazo nitrogen 2 7.85-8.02 mu1 tiplet
Proton on C4 1 8.29 doublet
Amino protons 4 -8.7 singlet
(broad)

2.3 Ultraviolet Spectrum

The ultraviolet spectrum of phenazopyridine
hydrochloride (0.5 mg/100 ml of acidified 3 A alcohol) in
the region of 210-450 nm exhibits two maxima and three mini-
m . The maxima occur at 238-240 nm ( E = 2.2 x lo4) and
390-392 nm (E = 2.4 x lo4), while the minima are at 220 nm,
272 nm and 296 nm respectively (3). The spectrum is shown
in Figure 3.

2.4 Fluorescence Spectrum

The excitation and emission spectra o f phena-
zopyridine hydrochloride (10 pg/ml in methanol) are shown
in Figure 4 (4). The instrument used was a Farrand MK-1
recording spectrofluorometer. An excitation wavelength of
341 nm produced an emission spectrum with a maximum at
380 nm.

2.5 Mass Spectrum

The low resolution mass spectrum of phenazo-
pyridine is shown in Figure 5 (5). The spectrum was ob-
tained using a CEC 21-110 spectrometer w i t h an ionizing
voltage of 70 eV, which was interfaced with a Varian data

470
 Figure 3

U l t r a v i o l e t Spectrum of P h e n a z o p y r i d i n e H y d r o c h l o r i d e

W
471

V
z
4
m
(r
0
m
m
4

NANOMETERS
 Figure 4

Fluorescence Spectra of Phenazopyridine Hydrochloride

t
t
412

u)
z
W
t
z

NANOMETERS
L 1 I I I I I I I I
3
1
0
(D
0
0 *0 0
el
AllSN31NI 3AllVl3LI
473
 KENNETH W. BLESSEL, BRUCE C. RUDY, AND BERNARD 2.SENKOWSKI

system 100 MS. The d a t a s y s t e m a c c e p t e d t h e o u t p u t of t h e

s p e c t r o m e t e r , c a l c u l a t e d t h e masses, compared t h e i n t e n s i -
ties t o t h e b a s e peak and p l o t t e d t h i s i n f o r m a t i o n as a
series of l i n e s whose h e i g h t s were p r o p o r t i o n a l t o t h e i n -
tensities.
The m o l e c u l a r i o n of t h e f r e e b a s e was measured
a t m / e 213. O t h e r c h a r a c t e r i s t i c masses were observed a t
m / e 214, c o r r e s p o n d i n g t o (MSH), m / e 1 8 4 , which c o r r e s p o n d s
t o t h e l o s s of HN2 from t h e m o l e c u l a r i o n , m / e 1 3 6 , t h e
l o s s of a phenyl r i n g from t h e p a r e n t mass, and m / e 108
which is t h e 2,6-diamino-pyridinium m o i e t y ( 5 ) . A h i g h
r e s o l u t i o n s c a n confirmed t h e r e s u l t s of t h e low r e s o l u t i o n
spectrum.

2.6 Optical Rotation

P h e n a z o p y r i d i n e h y d r o c h l o r i d e e x h i b i t s no o p t i c a l
activity .
2.7 M e l t i n g Range
P h e n a z o p y r i d i n e h y d r o c h l o r i d e m e l t s w i t h decom-
p o s i t i o n a t a p p r o x i m a t e l y 235OC when a class I a p r o c e d u r e
is used ( 6 ) .

2.8 D i f f e r e n t i a l Scanning C a l o r i m e t r y (DSC)

The DSC s c a n of p h e n a z o p y r i d i n e h y d r o c h l o r i d e is
shown i n F i g u r e 6 ( 7 ) . The c u r v e ' k a s o b t a i n e d w i t h a
P e r k i n E l m e r DSC-1B C a l o r i m e t e r . The t e m p e r a t u r e program
used w a s 10°C/min. i n an atmosphere of n i t r o g e n . Under
t h e s e c o n d i t i o n s , t h e exothermic d e c o m p o s i t i o n o f phenazo-
p y r i d i n e h y d r o c h l o r i d e o c c u r s a t a p p r o x i m a t e l y 239OC.

2.9 Thermogravimetric A n a l y s i s (TGA)

The TGA s c a n showed no loss of w e i g h t as t h e
t e m p e r a t u r e was r a i s e d from ambient t o 115OC a t a r a t e of
10°C/min. ( 7 ) .

2.10 Solubility
The s o l u b i l i t y d a t a o b t a i n e d f o r a sample o f
r e f e r e n c e s t a n d a r d p h e n a z o p y r i d i n e h y d r o c h l o r i d e a t 25OC is
shown in T a b l e I1 ( 8 ) . The e q u i l i b r a t i o n t i m e w a s 20 h o u r s
a t 25OC.

474
 Figure 6

*rl
a

rl

'd
DSC Curve €or Phenazopyridine Hydrochloride

3
a,

k
h
0
V

a,
k
a,

0
1
3
475

TEMPERATURE "C
 KENNETH W. BLESSEL, BRUCE C. RUDY, A N D BERNARD 2. SENKOWSKI

Table I1

Phenazopyridine Hydrochloride - Solubilities

Solvent S o l u b i l i t y (rnglml)

3 A alcohol 2.7
benzene 0.3
chloroform 0.4
95% e t h a n o l 3.5
diethyl ether 0.2
-
2 propanol 2.1
m e t hano 1 2.7
petroleum e t h e r (30°-600) 0.1
water 3.2
1N H C 1 0.3
a c i d i f i e d 3A alcohol 3.2

2.11 Crystal Properties

The x-ray powder d i f f r a c t i o n d a t a from phenazo-
p y r i d i n e h y d r o c h l o r i d e is p r e s e n t e d i n Table I11 ( 9 ) . The
o p e r a t i n g c o n d i t i o n s of t h e i n s t r u m e n t are g i v e n below.

Instrument Conditions

General Electric Model XRD-6 Spectrogoniometer

Generator: 50 KV, 12-112 MA

Tube t a r g e t : Copper
Radiation: Cu Ka = 1.542
optics: 0.10 D e t e c t o r s l i t
M.R. S o l l e r s l i t
3' Beam s l i t
0.0007" Ni f i l t e r
4' t a k e o f f a n g l e
Goniometer: Scan a t O.2O 28 p e r minute
Detector: Amplifier g a i n - 1 6 c o a r s e ,
8.7 f i n e
Sealed p r o p o r t i o n a l c o u n t e r
t u b e and DC v o l t a g e a t
plateau
P u l s e h e i g h t s e l e c t i o n EL-
5 volts;
Eu - Out

476
 PHENAZOPYRI DINE HYDROCHLORIDE

Rate meter T.C. 4

2000 C/S f u l l scale
Recorder: C h a r t Speed 1 i n c h p e r 5
minutes
Samples: P r e p a r e d by g r i n d i n g a t room
temperature

T a b l e I11

I n t e r p l a n a r Spacings i n Phenazopyridine Hydrochloride

from X-ray Powder D i f f r a c t i o n Data

-
28 -
d* uIo-** 28
- -
d* UIo3
8.68 10.20 6 27.88 3.20 85
9.12 9.70 30 29.28 3.05 18
10.22 8.66 84 30.64 2.92 23
12.88 6.87 10 31.32 2.86 18
13.68 6.47 8 32.84 2.73 10
14.46 6.13 100 34.38 2.61 6
18.10 4.90 20 36.06 2.49 9
19.44 4.57 32 36.58 2.46 5
20.40 4.35 19 37.58 2.39 6
21. 82 4.07 25 40.02 2.25 4
22.10 4.02 21 41.14 2.19 3
23.06 3.86 10 42.32 2.14 8
24.06 3.70 10 43.78 2.07 2
25.74 3.46 32 47.00 1.93 4
26.38 3.38 85 48.75 1.87 3
27.10 3.29 46
nA
*d = ( i n t e r p l a n a r s p a c i n g )
2 Sin 8
**I/I,= r e l a t i v e i n t e n s i t y ( b a s e d on h i g h e s t i n t e n s i t y
of 100)

3. Synthesis
P h e n a z o p y r i d i n e may b e p r e p a r e d by c o u p l i n g benzene
diazonium c h l o r i d e w i t h a,a-diamino p y r i d i n e ( 1 0 ) .

4. S t a b i l i t y Degradation
P h e n a z o p y r i d i n e h a s been found t o b e s t a b l e i n d i s -
t i l l e d water and 0.1N sodium h y d r o x i d e when r e f l u x e d f o r
one hour on a steam b a t h . Some d e g r a d a t i o n o c c u r s i n 0.1N

417
 KENNETH W. BLESSEL, BRUCE C. RUDY, AND BERNARD A. SENKOWSKI

hydrochloric a c i d s o l u t i o n a t r e f l u x temperature. A f t e r
one hour a t r e f l u x i t w a s found, by q u a n t i t a t i v e d e n s i t o -
metry o n a t h i n - l a y e r chromatographic p l a t e , t h a t 20-25%
of t h e i n i t i a l amount of m a t e r i a l had degraded. The de-
g r a d a t i o n p r o d u c t s were y e l l o w b u t no i d e n t i f i c a t i o n w a s
attempted.

5. Drug M e t a b o l i c P r o d u c t s
The m e t a b o l i c f a t e of p h e n a z o p y r i d i n e h y d r o c h l o r i d e
h a s been s t u d i e d i n t h e r a b b i t and i n man (11). When a n
o r a l d o s e of 600 mg w a s g i v e n t o humans, a b o u t 80%w a s
e l i m i n a t e d i n t h e u r i n e w i t h i n 24 h o u r s . Of t h i s amount,
7.6% appeared as a n i l i n e , 19.9% as N-acetyl-p-aminophenol,
27.1% as p-aminophenol, 45.4% as t h e unchanged d r u g as w e l l
as a t r a c e amount of o-aminophenol. Triaminopyridine a l s o
w a s d e t e c t e d b u t n o t measured.

6. Methods of A n a l y s i s

6.1 Elemental Analysis

The r e s u l t s of a n e l e m e n t a l a n a l y s i s of a sample
of r e f e r e n c e s t a n d a r d p h e n a z o p y r i d i n e h y d r o c h l o r i d e a r e
p r e s e n t e d i n T a b l e I V (12).

Table I V

Elemental A n a l y s i s of P h e n a z o p y r i d i n e H y d r o c h l o r i d e

Element Theoretical % Found %

C 52.91 52.89
H 4.84 4.76
N 28.05 28.00
c1 14.20 14.23

6.2 Phase S o l u b i l i t y A n a l y s i s
Phase s o l u b i l i t y a n a l y s e s have been c a r r i e d o u t
f o r phenazopyridine hydrochloride. An example i s shown i n
F i g u r e 7 ( 8 ) . The s o l v e n t used w a s methanol w i t h a n
e q u i l i b r a t i o n t i m e of 20 hours a t 25OC.

6.3 Thin Layer Chromatographic A n a l y s i s

P h e n a z o p y r i d i n e c a n b e d e t e c t e d i n AZO GANTRISIN
t a b l e t s u s i n g t h e f o l l o w i n g TLC p r o c e d u r e . The t a b l e t s

478
 Figure 7

5 1 1 l ] l l l l ~ l l l l ~ l ~ l ~

I-
2
2
w 4- -
2 3
t o
m m
g 0 3' " .. -
" n
-
A

I'
s! 0 PHASE SOLUBILITY ANALYSIS

-
z g 2- Sample : Phenazopyridine Hydrochloride
o m Solvent : Methanol
k l L
Slope: 0.0%
3 0 Equilibration: 20 hrs at 25OC -
0 0 l -
' " E Extrapolated Solubility : 2.99 m g / g

0 I l r l l l r l l l r l l l l l l l l
 KENNETH W. BLESSEL, BRUCE C. RUDY, A N D BERNARD 2.SENKOWSKI

are ground t o a powder and a p o r t i o n d i s s o l v e d i n a c e t o n e .

This i s a p p l i e d t o t h e p l a t e ( s i l i c a g e l GF) and developed
f o r a t l e a s t 1 0 cm w i t h t h e s o l v e n t m i x t u r e chloroform:
heptane:3A a l c o h o l (45:45:10) i n a p a p e r - l i n e d , pre-
s a t u r a t e d tank. Phenazopyridine can be d e t e c t e d v i s u a l l y
as a y e l l o w s p o t a t a n Rf of 0 . 4 (13).

6.4 Direct S p e c t r o p h o t o m e t r i c A n a l y s i s
The 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 o f phena-
z o p y r i d i n e h y d r o c h l o r i d e i n 1.ON H C l i s , a c c o r d i n g t o t h e
N a t i o n a l Formulary, t h e method of c h o i c e f o r t h e a s s a y of
t h e b u l k d r u g (14). The sample i s d i s s o l v e d i n 1 . O N H C 1
and t h e absorbance d e t e r m i n e d a t t h e maximum a t a b o u t
480 nm. The q u a n t i t y of p h e n a z o p y r i d i n e h y d r o c h l o r i d e is
c a l c u l a t e d by comparison w i t h a sample o f r e f e r e n c e s t a n -
dard m a t e r i a l p r e p a r e d and measured i n a s i m i l a r way.
The above method is s u b j e c t t o t h e d i s a d v a n t a g e
of t h e low s o l u b i l i t y of p h e n a z o p y r i d i n e h y d r o c h l o r i d e i n
1.ON HC1. The s u b s t i t u t i o n of a c i d i f i e d 3A a l c o h o l as t h e
s o l v e n t would g i v e a p p r o x i m a t e l y a t e n - f o l d i n c r e a s e i n
t h e s o l u b i l i t y and eliminate t h e n e c e s s i t y of h e a t i n g t h e
s o l u t i o n i n o r d e r t o d i s s o l v e t h e r e q u i r e d amount of phena-
z o p y r i d i n e h y d r o c h l o r i d e . The maximum i n t h i s s o l v e n t
o c c u r s a b o u t 390 nm.

6.5 Coulometric A n a l y s i s
P h e n a z o p y r i d i n e h y d r o c h l o r i d e may b e d e t e r m i n e d
coulometrically i n a n a c i d i f i e d water-acetone s o l u t i o n ,
u t i l i z i n g a mercury p o o l e l e c t r o d e . The sample is reduced
a t a p o t e n t i a l of -0.40 v o l t s u n t i l t h e observed c e l l
c u r r e n t d r o p s t o 1/1000,of i t s i n i t i a l v a l u e . A b l a n k de-
t e r m i n a t i o n i s c a r r i e d o u t and any c o r r e c t i o n s made. Each
coulomb of e l e c t r i c i t y i s e q u i v a l e n t t o 646.9 mcg of phena-
zopyridine hydrochloride (6).

6.6 Titrimetric Analysis

P h e n a z o p y r i d i n e h y d r o c h l o r i d e may b e a s s a y e d by
a p o t e n t i o m e t r i c t i t r a t i o n w i t h HClO4. The sample i s d i s -
s o l v e d i n water which i s made b a s i c w i t h 10% NaOH and t h e
l i b e r a t e d base is extracted i n t o c h l o r o f orm. I t
i s t h e n 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 y w i t h 0.01N HClO4, i n
dioxane, u s i n g a g l a s s - c a l o m e l ( s l e e v e t y p e ) e l e c t r o d e
combination. Each ml of 0.01N HClO4 i s e q u i v a l e n t t o
2.497 mg of p h e n a z o p y r i d i n e h y d r o c h l o r i d e (15).

480
 PHENAZOPYRI DI NE HYDROCHLORIDE

7. Acknowledgments
The a u t h o r s wish t o acknowledge t h e S c i e n t i f i c
L i t e r a t u r e Department and t h e Research Records Off i c e of
Hoffmann-La Roche Inc. f o r 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
search f o r t h i s Analytical P r o f i l e .
 KENNETH W. BLESSEL, BRUCE C. RUDY, A N D B E R N A R D 2. SENKOWSKI

8. References

1. Hawrylyshyn, M., Hoffmann-La Roche Inc., Personal

Communication.
2. Johnson, J. H., Hoffmann-La Roche Inc., Personal
Communication.
3 . Rubia, L. B., Hoffmann-La Roche Inc., Personal
Communication.
4 . Boatman, J. , Hoffmann-La Roche Inc., Personal
Communication.
5 . Benz, W., Hoffmann-La Roche Inc., Personal
Communication.
6 . The National Formulary XIII, pp. 538-540 (1970).
7. Moros, S . , Hoffmann-La Roche Inc., Personal
Communication.
8. MacMullan, E. , Hoffmann-La Roche Inc., Personal
Communication.
9 . Hagel, R. , Hoffmann-La Roche Inc. , Personal
Communication.
10. Chichibabin, Zeide, J. Russ. Phys. Chern. SOC., 46,
1216 ( 1 9 1 4 ) .
11. Johnson, J. W. and Burba, J., Federation Proc.. g,
734 ( 1 9 6 6 ) .
12. Scheidl, F. , Hoffmann-La Roche Inc. , Personal
Communication.
13. Sokoloff, H., Hoffmann-La Roche Inc., Unpublished
Results .
1 4 . The National Formulary XIII, First Supplement,
p. 24 (1970).
1 5 . Senkowski, B., Hoffmann-La Roche Inc., Unpublished
Results.

482
 PHENY LEPHRINE HYDROCHLORIDE

Charles A . Gaglia, Jr.

Reviewed by E. L. Pratt and L. Chafetz

 CHARLES A. GAGLIA. JR.

CONTENTS

Analytical Profile - Phenylephrine Hydrochloride

1. Description
1 . 1 Name, F o r m u l a , Molecular W e i g h t
1 . 2 Appearance, C o l o r , Odor, Taste
2. Physical Properties
2 . 0 1 M e l t i n g Range
2.02 S o l u b i l i t y
2 . 0 3 pK
2.04 Optical Rotation
2.05 U l t r a v i o l e t S p e c t r u m
2.06 I n f r a r e d Spectrum
2 . 0 7 N u c l e a r M a g n e t i c Resonance S p e c t r u m
2 . 0 8 Mass S p e c t r u m
2.09 D i f f e r e n t i a1 Thermal A n a l y s i s
2 . 1 0 Thermal G r a v i m e t r i c A n a l y s i s
3. Synthesis
4. S t a b i l i t y - Degradation
5. Drug M e t a b o l i c Products
6. Methods of A n a l y s i s
6 . 1 Direct S p e c t r o p h o t o m e t r i c A n a l y s i s
6 . 2 Co 1 o r i me t ri c Ana l y s i s
6 . 2 1 I n d o p h e n o l Dye
6.22 Coupling w i t h p - N i t r o a n i l i n e
6.23 Coupling with 4-Aminoantipyrine
6.24 Complexation
6 . 2 5 C o u p l i n g w i t h Nitrous Acid
6.26 I d e n t i f i c a t i o n
6 . 2 7 O t h e r Methods
6 . 3 Chromatographic Methods o f A n a l y s i s
6.31 Paper Chromatography
6.32 T h i n L a y e r Chromatography
6 . 3 3 L i q u i d - L i q u i d Chromatography
6 . 3 4 Gas C h r o m a t o g r a p h y
6 . 3 5 Ion Exchange Chromatography
6 . 4 S p e c t ro f 1 u o r ome t r i c a n d P h o s p h or i met r i c
Analysis
6 . 5 Other Methods o f A n a l y s i s
7. References

484
 PHENY LEPHRINE HYDROCHLORIDE

1. Description
1.1 Name, F o r m u l a , M o l e c u l a r Weight
P h e n y l e p h r i n e H y d r o c h l o r i d e i s l-rn-Hy-
d r o xy -a-[ ( me t h y 1 am i n o ) me t h y 1 ] be n zy 1 a 1 co h o 1 h y -
d r o c h l o r i d e ( 1 ) . I t i s a l s o known a s l - a - h y d r o x y
- B -me t hyl a m i no- 3 -hyd roxy -1 -e t h y 1 benzene h y d r o -
c h l o r i d e ; m-methylaminoethanolphenol hydrochlo-
r i d e ; Neo S y n e p h r i n e h y d r o c h l o r i d e ; Meta-Syn-
e p h r i n e h y d r o c h l o r i d e ; A d r i a n o l ; rn-Sympatol ;
Meta-Sympatol; Neophryn; I s o p h r i n H y d r o c h l o r i d e ;
Oftal frine; Lexatol ( 2 ) .

OH
CsHl4ClN02 Mol. W t . 203.67
1.2Appearance, C o l o r , Odor, T a s t e
White o r n e a r l y w h i t e , o d o r l e s s c r y s t a l s
having a b i t t e r t a s t e .
2. Physical Properties
2.01 M e l t i n g Range
P he n -y 1 e .p h r i n e H C1 140 - 145°C (1)*
139 - 143°C (3)
P heny 1 eph r i n e b a s e 170 - 177°C (1)"
170 - 171OC (3)
*USP Speci f i c a t i ons
2.02 Solubility
F r e e l y s o l u b l e i n water and i n a l c o h o l

2.03 pK
pK, = 8 . 7 7 ( 4 )
pK2 = 9.84
2.04 Optical Rotation
[a];' = - 4 6 . 2 t o - 4 7 . 2 " (c=l) (2)

485
Fig. 1 Phenylephrine hydrochloride - IR spectrum o f 1 3 mm. K B r p e l l e t
from 1 mg. drug dispersed i n 200 mg. KBr - Instrument: Perkin-Elmer 621
 PHENYLEPHRINE HYDROCHLORIDE

2.05 U l t r a v i o l e t Spectrum
Solution X max. n m . x 10-3
0 . 0 5 N HC1 216 5.91
274 1.81
279s 1.65
0.05 N NaOH 2 39 8.95
292.5 3.04
Isosbestic 222.5 4.28
Points 260 6.66
278.5 1.67
2.06 I n f r a r e d Spectrum ( F i g u r e 1 )
The s p e c t r u m i n F i g u r e 1 was o b t a i n e d
using a Perkin-Elmer 621, I n f r a r e d Spectrophoto-
meter, A 1 3 mm. KBr p e l l e t c o n t a i n i n g 1 mg.
p h e n y l e p h r i n e HC1 a n d 2 0 0 mg. KBr w a s u s e d . Char-
a c t e r i s t i c band a s s i g n m e n t s a r e l i s t e d below.
B,and cm” A s s i gnmen t

3420, 3450 -OH

2 400-2800 N H*
1590 aromatic
1270 C-0 s t r e t c h a r o m a t i c
1080 C-0 s t r e t c h s e c o n d a r y a l c o h o l
900 a r o m a t i c o u t o f p l a n e bend s i n g l e
CH
780 a r o m a t i c o u t o f p l a n e b e n d meta
disubstituted
690 a r o m a t i c o u t o f p l a n e bend meta
di substi t u t e d
2.07 Nuclear Magnetic Resonance Spectrum
S o l v e n t : DMSO
I n s t r u m e n t : V a r i an A60
C o n c e n t r a t i o n : A p p r o x i m a t e l y 8%

48 I
 CHARLES A. GAGLIA. JR.

Phenylephrine Hydrochloride (Figure 2 )

ppm. ( f r o m TMS) Integral I d e n t i f ic a t i on
2.6 ( S ) 3 N-CH 3
2.8 - 3.2 (M) 2 CH 2 - N
27 -[!I*
6.7 7.5 (M)
1
1
4
CH
CHOH
aromatic
9 - 9.8 (B)* 3 phenolic OH,
NH':

P h e n y l e p h r i n e Base ( F i g u r e 3 )
ppm. ( f r o m TMS) Integral Identi fi cation
2.4 (S) 3 N-CH 3
2.55 ( D ) 2 CH 2 - N
4.5 ( T ) 1 CH
4 . 8 - 5 . 8 (B)* 3 OH ( 2 ) , NH
6.5 - 7.5 (M) 4 aromatic
* D i s a p p e a r on D20 e x c h a n g e
2.08 Mass S p e c t r u m
The low r e s o l u t i o n mass spectrum was
d e t e r m i n e d on a n MS 902 mass s p e c t r o m e t e r . The
s a m p l e was i n t r o d u c e d by a h e a t e d d i r e c t i n s e r -
t i o n p r o b e a t a t e m p e r a t u r e o f 150°C f o r t h e h y -
d r o c h l o r i d e and o f 1 0 0 ° C f o r the base.
Mass No. I n t e n s i t y HC1 I n t e n s i t y Base
167 10 38
148 2 6
133 1 4
121 5 lo
107 2 10
95 4 20
77 8 15
65 4 30
44 100 100
38 8 ---
36 26

488
Fig. 2 Time averaged NMR spectrum o f phenylephrine hydrochloride, 8 % i n
DMSO - Instrument: Varian A-60
 o
l-0
v)
W
F i g . 3 Time a v e r a g e d N M R s p e c t r u m o f p h e n y l e p h r i n e b a s e , a b o u t 8% i n

n
DMSO - I n s t r u m e n t : Varian A - 6 0
 PHENYLEPHRINE HYDROCHLORIDE

2.09 D i f f e r e n t i a1 T h e r m a l A n a l y s i s
O n s e t o f Me1 t i n g Peak E n d o t h e r m
P h e n y l e p h r i ne HC1 142°C 144°C
P h e n y l e p h r i ne Base 172°C 174°C

2.10 Thermal G r a v i m e t r i c A n a l y s i s
No s i g n i f i c a n t w e i g h t l o s s u n t i l decom-
p o s i t i o n a t 230°C f o r p h e n y l e p h r i n e HC1.

3. Synthesis

L e g e r l o t z was t h e f i r s t t o p r e p a r e p h e n y l -
e p h r i n e h y d r o c h l o r i d e by t h e h y d r o g e n a t i o n o f m-
hydroxy-w-methylamino-acetophenone i n t h e p r e s -
e n c e o f c o l l o i d a l p a l l a d i u m ( 5 ) . Bergmann a n d
Sul z b a c h e r ( 6 ) s y n t h e s i z e d p h e n y l e p h r i n e by
t r e a t i n g 5-(3'-benzyloxyphenyl)-3-methyl-2-oxa-
z o l i d o n e w i t h 40% h y d r o c h l o r i c a c i d s o l u t i o n .
R u s s e l l and C h i l d r e s s ( 7 ) a c h i e v e d t h e same e n d
b y r e f 1 u x i n g 3 - b e n z y l o x y - w - m e t h y l mandel ami de w i t h
L i A1H4 i n t e t r a h y d r o f u r a n (THF) t o p r o d u c e 3 - b e n -
z y 1o x y - a-me t h y 1 ami n o-me t h y 1 b e n z y 1 a1 c o h o l h y d r o -
chloride. T h i s compound was t h e n h y d r o g e n a t e d i n
t h e p r e s e n c e o f 5% p a l l a d i u m - C c a t a l y s t u n t i l one
e q u i v a l e n t o f H i s consumed. The h y d r o g e n a t i o n
o f 3-benzyloxy-a-methylamino-methyl b e n z y l a l c o h o l
was a l s o u s e d b y B r i t t e n ( 8 ) a n d , m o s t r e c e n t l y ,
R i z z i ( 9 ) as t h e l a s t s t e p s o f t h e i r s y n t h e s e s o f
phenylephrine.

4. Stability - Degradation

P h e n y l e p h r i n e h y d r o c h l o r i de i s s t a b l e as a
solid. The d e g r a d a t i o n o f aqueous s o l u t i o n s h a s
b e e n s t u d i e d b y E l - S h i b i n i e t aZ. ( 1 0 , l l ) . The
compound i s s t a b l e b e l o w pH 7 . Above pH 7 , d e g -
r a d a t i o n o c c u r s and a p p a r e n t l y i n v o l v e s t h e s i d e
c h a i n w i t h l o s s o f t h e s e c o n d a r y amine f u n c t i o n .
The p h e n o l i c g r o u p r e m a i n s i n t a c t . The decompo-
s i t i o n p r o d u c t s h a v e n o t been i d e n t i f i e d b u t 5 -
h y d r o x y - N - m e t h y l i n d o x y l has been p r o p o s e d . The
p r e s e n c e o f h e a v y m e t a l s , p a r t i c u l a r l y c o p p e r was
found t o c a t a l y z e t h e decomposition.

49 1
 CHARLES A. GAGLIA, JR

T r o u p a n d Mitchner ( 1 2 ) c h a r a c t e r i z e d t h e
a c e t y l a t i o n of p h e n y l e p h r i n e i n t h e p r e s e n c e o f
a s p i r i n . The r e a c t i o n i s a p p a r e n t l y a c c e l e r a t e d
by t h e p r e s e n c e of p h e n y l e p h r i n e base a n d t h e
a v a i l a b i l i t y of a c e t a t e . The amine g r o u p a c e t y -
l a t e s p r e f e r e n t i a l l y . The hydroxyl g r o u p s become
a c e t y l a t e d a f t e r prolonged e x p o s u r e t o a s p i r i n .
Luduena e t aZ. ( 1 3 ) s t u d i e d t h e e f f e c t o f
u l t r a v i o l e t i r r a d i a t i o n on p h e n y l e p h r i n e s o l u -
t i o n s . E p i n e p h r i n e was i d e n t i f i e d a s t h e product.
Luduena p o s t u l a t e d t h e e p i n e p h r i n e could f u r t h e r
r e a c t t o produce o t h e r compounds. The f i n d i n g s
of West a n d W h i t t e t ( 1 4 ) s u p p o r t t h i s p o s t u l a t e .
S c h r i f t m a n ( 1 5 ) found from 1 2 t o 28% decompo-
s i t i o n of unbuffered phenylephrine s o l u t i o n s i n
one week a t v a r i o u s t e m p e r a t u r e s . He a l s o found
u p t o f i v e d e c o m p o s i t i o n p r o d u c t s . The s e c o n d a r y
amine f u n c t i o n was a b s e n t in a t l e a s t one of t h e
products.
Broadly a n d Roberts ( 1 6 ) found a second com-
p o u n d present i n s t r o n g acid (10 N hydrochloric
a c i d ) s o l u t i o n s of p h e n y l e p h r i n e . The compound
was n o t i d e n t i f i e d .
Misgen ( 1 7 ) d e t e r m i n e d t h e p h y s i c a l compati-
b i l i t y of p h e n y l e p h r i n e w i t h twenty-seven common
i n t r a v e n o u s a d m i x t u r e s . He found on adding a
s o l u t i o n of phenylephrine t o a s o l u t i o n of d i l a n -
t i n sodium a p r e c i p i t a t e formed w i t h i n two h o u r s .
F a g a r d ( 1 8 ) found p h e n y l e p h r i n e s o l u t i o n s t o
be s t a b l e i n brown g l a s s b o t t l e s , g i v e 1 % decom-
p o s i t i o n a f t e r 11 days i n low d e n s i t y p o l y e t h y l e n e
b o t t l e s a n d decompose t o 81% o f i n i t i a l when
s t o r e d f o r 130 days i n nylon b o t t l e s .
P e t r a g l i a a n d Dick ( 1 9 ) r e p o r t e d the s t a b i l i -
z a t i o n of p h e n y l e p h r i n e s o l u t i o n s t o s u n l i g h t by
a d d ’ i n g 0 . 2 % sodium m e t a b i s u l f i t e a n d 0.1% t a r t r a -
zine.
E l - S h i b i n i e t aZ. ( 1 1 ) i n d i c a t e d the stabi-

492
 PHENY LEPHRINE HYDROCHLORIDE

l i z i n g e f f e c t o f E D T A on b a s i c s o l u t i o n s o f
p h e n y l e p h r i ne.
Kisbye and Bols ( 2 0 ) f o u n d t h a t n o r a c e m i z a -
t i o n o f p h e n y l e p h r i n e o c c u r s a s a f u n c t i o n of pH.
P r a t t ( 2 1 ) found phenylephrine o p t i c a l l y s t a b l e
i n s o l u t i o n s a t pH 3 . 0 and 6 . 0 when r e f l u x e d f o r
3 hours.
Fourneau e t aZ. ( 2 2 ) r e p o r t e d t h e r e a c t i o n o f
phenylephrine w i t h aldehydes under "physiological
c o n d i t i o n s " t o p r o d u c e a m i x t u r e o f 4 , 6 - and 4 , 8 -
d i hydroxy-2-methyl-1 y 2, 3 , 4 - t e t r a h y d r o i soquino-
1ines.
5. Drug Metabolic Products
R e l a t i v e l y l i t t l e work h a s been r e p o r t e d on
t h e m e t a b o l i s m o f p h e n y l e p h r i n e . Bruce ( 2 3 ) r e -
ported phenylephrine i s excreted i n urine almost
e n t i r e l y a s t h e s u l f a t e c o n j u g a t e . Bruce r e -
ported 82% average t o t a l r e c o v e r y o f phenyl-
e p h r i n e from a t a b l e t f o r m u l a t i o n i n 24 h o u r s .
T y p i c a l u r i n e s a m p l e c o n t a i n e d from 86 t o 98% o f
t h e recovered p h e n y l e p h r i n e a s t h e s u l f a t e con-
j ugate .
Bogner and Walsh ( 2 4 ) , and C a v a l l i t o e t aZ.
( 2 5 ) showed b l o o d l e v e l and u r i n e e x c r e t i o n p a t -
t e r n s f o r p h e n y l e p h r i n e h y d r o c h l o r i d e and p h e n y l -
e p h r i n e t a n n a t e . T h e i r work i n v o l v e d t r i t i u m
l a b e l e d d r u g . M e t a b o l i c p r o d u c t s were n o t i d e n -
t i f ied.
R u b i n and K n o t t ( 2 6 ) r e p o r t e d a f l u o r o m e t r i c
procedure f o r determining phenylephrine i n serum.
Dombrowski and P r a t t ( 2 7 ) r e p o r t a p r o c e d u r e
f o r determining unmetabolized phenylephrlne i n
plasma.
6. Methods o f A n a l y s i s
6.1 D i rect Spectrophotometri c Analysis
The u l t r a v i o l e t a b s o r p t i o n band a t 2 7 2

493
 CHARLES A . GAGLIA, JR.

nm. i s due t o t h e p h e n o l i c s t r u c t u r e . The a b -

s o r b a n c e c a L be u s e d t o q u a n t i t a t e p h e n y l e p h r i n e
d i r e c t l y (28,29,30) o r a f t e r e x t r a c t i o n ( 3 1 ) .
S h i f t i n g t h e maxima t o 290 nm. i n b a s i c s o l u t i o n
has a l s o b e e n u s e d a s a d i r e c t a s s a y as w e l l a s a
d i f f e r e n t i a l technique t o quantitate phenyl-
e p h r i n e (32,33). O x i d a t i on o f p h e n y l e p h r i n e t o
m-hydroxy benzaldehyde and measuring absorbance
a t 2 5 7 nm. a n d / g r 315 nm. i n a c i d i c o r n e u t r a l
s o l u t i o n s o r a t 2 3 7 , 267 a n d 357 nm. i n b a s i c
s o l u t i o n h a s a l s o b e e n c a r r i e d o u t ( 3 4 ) . The
oxidation also offers increased s e n s i t i v i t y over
d i r e c t U.V. U . V . i s a common d e t e c t i o n t e c h n i q u e
f o r t h i n l a y e r , paper and column chromatographic
techniques.
6.2 Col o r i r n e t r i c A n a l y s i s
P h e n v l e D h r i n e h a s b e e n id e n t i f i e d a n d
q u a n t i t a t e d b y a’ v a r i e t y o f c o l o r i m e t r i c t e c h -
niques.
6.21 I n d o p h e n o l Dye
I n d o p h e n o l dye i s f o r m e d b y t h e
r e a c t i o n o f p-Me2NC6H4NH2C1 a n d K 3 F e ( C N ) 6 w i t h
p a r a u n s u b s t i t u t e d p h e n o l s i n a1 k a l i n e medi a ( 3 5
36,37). The d y e r e s u l t i n g f r o m t h e r e a c t i o n w i t h
p h e n y l e p h r i n e has an a b s o r b t i on maximum a b o u t
6 2 0 nm.

6.22 Coupling with p-Nitroaniline

Phenylephrine may be c o u p l e d w i t h
diazotized p-nitroaniline i n a c i d s o l u t i o n (38,
39). The r e s u l t i n g compound i s made b a s i c a n d
d e t e r m i n e d a t 495 nm.
6.23 Coup1 i n g w i t h 4-Ami n o a n t ip y r i n e
P-aminoantipyrine i s a selective
coupling agent f o r phenols w i t h t h e para p o s i t i o n
free. The r e a c t i o n i s c a r r i e d o u t i n a l k a l i n e
b u f f e r s o l u t i o n pH = 9 i n t h e p r e s e n c e o f K 3 F e -
(CN),. The r e s u l t i n g a b s o r b t i o n maximum a t 460
nm, i s q u a n t i t a t i v e f o r p h e n y l e p h r i n e (40,41 , 4 2 ) .
6.24 Complexation
Phenylephrine forms complexes w i t h

494
 PHENY LEPHRINE HYDROCHLORIDE

v a r i o u s s u l f o p h t h a l e i n dyes i n n e u t r a l t o s l i g h t -
l y b a s i c s o l u t i o n . The r e s u l t i n g complexes a r e
then extracted i n t o a polar organic solvent and
t h e c o l o r d e t e r m i n e d s p e c t r o p h o t omet ri c a l l y ( 4 3 ,
44)
6.25 C o u p l i n g w i t h N i t r o u s Acid
When s o l u t i o n s o f P h e n"v l e.P h r i n e
a r e h e a t e d w i t h mercury s a l t s then c o u p l e d w i t h
n i t r o u s a c i d , a red c o l o r d e v e l o p s . The peak a t
495 n m . h a s been used t o q u a n t i t a t e p h e n y l e p h r i n e
(45,46). Detailed s t u d i e s o f t h e reaction condi-
t i ons have been c o n d u c t e d ( 4 7 , 4 8 ) .
6.26 Identification
P h e n y l e p h r i n e u n d e r g o e s many c o l or
r e a c t i o n s . S e v e r a l schemes f o r i d e n t i f y i n g
p h e n y l e p h r i n e a l o n e and i n t h e p r e s e n c e o f o t h e r
d r u g s have been d e v e l o p e d ( 4 9 , 5 0 , 5 1 , 3 ) .
6.27 Other Methods
Many o t h e r q u a n t i t a t i v e c o l o r re-
a c t i o n s have been k e p o r t e d ' i n t h e l i t e r a t u r e .
The r e a c t i o n p r o d u c t w i t h i o d i c a c i d i s d e t e r -
m i n e d a t 420 n m . ( 5 0 ) . P h e n y l e p h r i n e r e a c t s w i t h
2 , 6 - d i c h l o r o q u i n o n e i n n e u t r a l s o l u t i o n and i s
d e t e r m i n e d a t 625 nm. ( 5 2 ) . The o x i d a t i o n o f
p h e n y l e p h r i n e t o an a l d e h y d e f o l l o w e d by r e a c t i o n
w i t h t h i o b a r b i t u r i c a c i d (53) o r 3-methylbento-
thiazolin-2-one (54) i s also quantitative. Nin-
hydrin r e a c t s w i t h phenylephrine t o produce a
p i n k c o l o r w i t h a m a x i m u m a b s o r b a n c e a t 440 n m .
(55)
6.3 C h r o m a t o g r a p h i c Methods o f A n a l y s i s
6.31 P a p e r Chromatography
P a p e r chromatoqraDh.y has been u s e d
t o i s o l a t e phenylephrine from-its decomposition
p r o d u c t s ( 1 5 , 5 6 ) and from o t h e r sympathicomi -
metics (57,58,59,60,37). T a b l e 6.31 -1 summarizes
t h e 1i t e r a t u r e f o r p a p e r chromatographic s e p a r a -
t i o n o f phenylephrine.

495
 CHARLES A. GAGLIA, JR.

T A B L E 6.31 -1

Sol vent Method o f

Sys tern V i s u a l i z a t i on R f x 100 Reference
A n i nhydri n 57 57
B d i a z o t i zed p- 63 15
sul f a n i l i c
dragendorff
U.V. deni tometry

C n i nhydri n 67 56
D n o t a v a i 1a b l e -- 60
E indophenol 10 37

A = n-butanol /aceti c aci d/water 4:5:1

B = n-butanol /aceti c aci d/water 5:l :3
C = p h e n o l c o n t a i n i n g 15% v / v 0 , I N H C l
D = butanol /to1 uene/aceti c aci d/water
1 0 0 : 1 0 0 : 50:50
E = benzyl a l c o h o l / a c e t i c a c i d / w a t e r 5:2:5

496
 PHENYLEPHRINE HYDROCHLORIDE

TABLE 6 . 3 2 - 1

Method o f
System R f x 100 D e t e c t io n Reference
I 5 s p r a y 1% I 2 i n 61
methanol and/or
dragendorff's
reagent

I1 21 I1 61

I11 33 II
61

IV 60 II
61

V 45 I1
61

VI -- p o t a s s i um 62
f e r r i cyanide
0.6% w/v i n
0 . 5 % w / v NaOH
quan. U V . den-
si t o m e t r y
VII 50 n i nhydrin 63

S i l i c a Gel P l a t e s
Coated w i t h D e v e l o p i n g Sol v e n t

I 0.1 NaOH c y c l ohexane/benzene/

diethylamine
75:15:10 ( v / v )

I1 0.1 M NaOH methanol

111 0 . 1 M NaOH acetone

IV 0 . 1 M KHSOI, methanol

V 0 . 1 M KHSOI, 95% ethanol

497
 CHARLES A. GAGLIA. JR.

TABLE 6.32-1 (continued)

S i l i c a Gel P l a t e s
Coated with Developing Sol vent
VI c e l l u l o s e 2 5 0 1-1 n-butanol/aceti c
acid/water 4 : l : 5
v / v organic phase as
the developing s o l -
vent
VII s i l i c a gel G n-butanol l a c e t i c
aci d/water 1 2 :1 :7
v / v organic phase as
t h e developing sol-
vent

498
 PHENYLEPHRINE HYDROCHLORIDE

6.32 T h i n L a y e r Chromatography
The t h i n l a y e r c h r o m a t o g r a p h i c R f
v a l u e s f o r p h e n y l e p h r i n e i n a number o f s o l v e n t
s y s t e m s a r e g i v e n i n T a b l e 6.32-1
6.33 L i q u i d - L i q u i d Chromatography
P h e n y l e p h r i ne l e n d s i t s e l f r e a d i l y
t o l i q u i d - l i q u i d chromatography, The d i f f i c u l t y
i n e x t r a c t i n g p h e n y l e p h r i n e f r o m aqueous s o l u -
t i o n s has been u s e d t o a d v a n t a g e t o remove o t h e r
compounds f r o m phen l e p h r i n e . L e v i n e and D o y l e
( 6 4 , 6 5 ) and Cox ( 6 6 7 p r e s e n t e d t h e t h e o r e t i c a l
aspects o f l i q u i d - l i q u i d p a r t i t i o n systems.
T h e i r w o r k i n c l u d e s t h e p a r t i t i on c o e f f i c i e n t s o f
many d r u g s and i s p r e s e n t e d s o t h a t o p t i m u m c o n -
d i t i o n s f o r p a r t i c u l a r s e p a r a t i o n s may be s e l e c t -
ed. T a b l e 6.33-1 summarizes t h e p r a c t i c a l appl i-
c a t i ons o f 1 iqu id - 1 iq u i d 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 o f phenylephrine.

6.34 Gas C h r o m a t o g r a p h y
Gas c h r o m a t o g r a p h y has been u s e d
t o s e p a r a t e , i d e n t i f y and q u a n t i t a t e p h e n y l -
ephrine. A summary o f t h e gas c h r o m a t o g r a p h i c
d a t a i s p r e s e n t e d i n T a b l e 6.34-1.
6.35 I o n Exchange C h r o m a t o g r a p h y
Table 6.35-1 summarizes t h e l i t e r -
a t u r e on i o n e x c h a n g e s e p a r a t i o n o f p h e n y l e p h r i n e .

6.4 S p e c t r o f l u o r o m e t r i c and P h o s p h o r i m e t r i c
Analysis
P h e n y l e p h r i n e has n a t i v e f l u o r e s c e n t
p r o p e r t i e s . U d e n f r i e n d et Q Z . ( 8 0 ) r e p o r t e d 270
nm. as t h e w a v e l e n g t h o f e x c i t a t i o n w i t h 305 nm.
being the wavelength o f emission. The f l u o r e s -
c e n c e o c c u r s i n aqueous a c i d s o l u t i o n w i t h a r e -
p o r t e d s e n s i t i v i t y o f 0.04 p g . / m l . Rubin and
K n o t t ( 2 6 ) used a f l u o r o m e t r i c p r o c e d u r e t o d e t e r -
m i n e p h e n y l e p h r i n e i n serum. Winefordner (81)
d e t e r m i n e d p h e n y l e p h r i ne b y i t s p h o s p h o r e s c e n t
properties a t l i q u i d nitrogen temperatures i n
e t h a n o l ic s o l u t i o n s . The w a v e l e n g t h s o f e x c i t a -
t i o n a r e 2 9 0 a n d 2 4 0 nm. w i t h p h o s p h o r e s c e n c e

499
 CHARLES A. GAGLIA, JR.

TABLE 6.32-1

Method o f
System R f x 100 Detect i o n Reference
I 5 spray 1% I2 i n 58
methanol and/
o r dragendorff I s
reagent

I1 21 II
58
I11 33 I1
58
IV 60 II
58
V 45 II
58
VI -- p o t a s s ium 59
ferricyanide
0 . 6 % w/v i n
0 . 5 % w/v NaOH
quan. U V . d e n -
s itometry
VII 50 n inh y d r i n 60

S i l i c a Gel P l a t e s
Coated w i t h Developing Sol v e n t
I 0 . 1 M NaOH Cyc 1 o hex a n e / b e n z e n e /
diethylamine
7 5 : 1 5 : 1 0 (v/v)
I1 0 . 1 M NaOH methanol
I11 0 . 1 M NaOH acetone
IV 0.1 M KHSOk met hano l
V 0 . 1 M KHSOk 95% e t h a n o l

500
 PHENY LEPHRINE HYDROCHLORIDE

T A B L E 6.32-1 (continued)

S i l i c a Gel P l a t e s
Coated with Developing Solvent
VI c e l l u l o s e 250 1.1 n-butanol lacetic
a c i d l w a t e r 4 : 1 : 5 v/v
organic phase as the
developing solvent
VII s i l i c a gel G n-butanollacetic
acid/water 12:1:7
v/v organic phase as
the developing s o l -
vent

501
 TABLE 6 . 3 3 - 1
Other Compounds
Col umn Stationary Present Not
Support Phase Mobile Phase Interfering Reference
c e l i t e 545 acetic acid chl oroform codeine 67
NaCL ( s a t ' d ) wash t h e n d e x t rometh o r p h a n
ether el u t ion phenyl p r o p a n o l amine
HC1
chlorpheniramine
p h e n i ramine
pyrilamine
doxy1 amine s u c c i n a t e
t r i p e 1 e n n a m i n e ci t r a t e
p h e n y l t o 1 oxamine
d i hydrogen c i t r a t e
aspirin
c e l i t e 545 pH 5 . 8 b u f f e r chloroform p h e n y l p r o p a n o l ami ne 68
pH 5.1 b u f f e r wash t h e n e l u t e HC1
w i t h 2.4% v/v d e x t rome t h o r p han H B r
DEHP i n c h l o r o - glyceryl guai acol a t e
form acetaminophen
chlorpheni ramine
p h e n y l t o l o x a m i ne c i t r a t e
aspirin
phenolphthalein
p y r i 1 amine ma1 e a t e
g u l f i i;oxgzol e
romp en1 r a m i n e ma1 e a t e
 T A B L E 6.33-1 ( c o n t i n u e d )
Other ComDounds
Column Stationary P r e s e n t Not
Support Phase Mobile Phase Interfering Ref e rence
c e l i t e 545 sodi u m chloroform wash codeine s u l f a t e 69
a c i d washed borate then a c e t y l a t e me t h a p y r i 1 en e H C1
and e l u t e a c e t y - p y r i l a m i n e maleate
1 a t e d phenyl- d-amphetamine s u l f a t e
e p h r i ne w i t h
chloroform
saponify
ul
0
c e l i t e 545 s o d i um same a s above codeine phosphate 70
w aci d washed borate chl orpheni ramine
ma1 e a t e
c e l i t e 545 various c h 7 o rof orm wash chlorpheniramine 71
aci d washed acids a n d elute w i t h ma1 e a t e
bases ethanol p y r i l a m i n e maleate
codeine phosphate
phenyl p r o panol ami ne
HC1

Method of Q u a n t i t a t i o n - U.V.
 T A B L E 6.34.1
Column I ns trumen t a1
Condi t i ons Conditions Derivative Reference
8 f t . , 3 mm. I . D . , S E - c o l . temp. 135"C, flow base 72
30 1 .15% on gas chrom- r a t e 30 m l . / m i n . i n l e t acetone
P 100-140 mesh p r e s . 31 p s i butanone
6 f t . , 3 mm. I.D., c o l . temp. 135"C, i n l e t a c e t o n e 72
QFI-0065 (Dow Corni n g ) pres. 30 psi B - i o n i -
2.8% on chromsorb 60- z a t i on d e t e c t o r
80 mesh
VI
0
P 2 M g l a s s , 4 mm. I . D . , i n j . 300"C, d e t c . 260°C t r i f l u o r o - 73 a
0.1% s i l i c o n e o i l ( D C - flame i o n i z a t i o n d e t c . acetic acid
710) on 60-80 mesh he1 i urn/hydrogen/ai r
dimethyl-dichlorosilane flow r a t e 80/80/450 m l J
t r e a t e d g l a s s beads m i n . , resp. program col.
100°C t o 200°C a t l O " C /
min.
6 f t . , 4 mm. I.D., 10% d e t c . 300°C f l a m e i o n i - HMDS ( h e x a - 74
F-60 (methyl p o l y s i l o - z a t i on t e m p . program methyldi s i 1 -
xane) on gas chrom-P 100"C-2OO0C 8 1.5"C/ azane)
80-100 mesh m i n . n i t r o g e n 12 psi acetone
a i r 40 p s i , hydrogen cycl obutanone
20 psi
a = assayed t a b l e t s and s y r u p s
 TABLE 6 . 3 5 - 1
Compounds
Method o f Present Not
Res in Type M o b i l e Phase Quan. Interfering Reference
Amberl it e weakly 75% e t h a n o l tit r a t i o n codeine phos- 75
IR-45 basic phate
p o t a s s i um
guaiacol-
sul fonate
camphor
menthol
Dowex sulfonic w a t e r ash azo APAP
50-X-1 aci d then e ute coup1 in g t heny 1 d iami ne 39
Dowex w i t h 0.5 N Millon's HC1
50-X-2 H+ f o r m HCI reagent caffein
Dowex
50-X-8
Dowex
50-X-12
Dowex
50-X-16
Dowex
50-W-X-1
Amberl it e
I R 120
 T A B L E 6.35-1 (continued)
Compounds
Method of P r e s e n t Not
Resin Type Mobile Phase Quan. Interfering Re f e ren ce
Type A P01Y - g r a d i e n t pH U.Y. met anep h ri ne 76
styrene 10-12 c r 0 . 1 5 p -synephri ne
sulfonic M t o 0.37 M + 13 o t h e r com-
acid N H t NHbOH pounds
form
AG 5OW-X-4 strong 1 N HC1 i n 50% U.Y. codeine phosphate 77
cation met ha no1 chl orpheni rami ne 78
maleate
prome t h a z i ne HC1
methapyrilene HC1
d e x t romet h o r p h a n H B r
A1 g i n i c cation 0 . 0 1 N HC1 U.Y. p y r i l ami ne ma1 e a t e 79
Acid codeine phosphate
acetaminophen
 PHENY LEPHRINE HYDROCHLORIDE

o c c u r r i n g a t 390 n m .
6.5 Other Methods o f A n a l y s i s
A non-aqueous t i t r a t i o n o f p h e n . y l e p h r i n e
t o a crystal violet end point u s i n g perchloric
a c i d i n d i o x a n e - a c e t i c a c i d medium h a s been r e -
ported ( 8 2 ) .
Bromi n a t i on has been used t o d e t e r m i n e phenyl-
e p h r i ne u s i n g bromine w a t e r ( 8 3 ) and coul ometri -
c a l l y g e n e r a t e d bromine ( 8 4 ) .
T h e i n c r e a s e i n b l o o d pressure o f b o t h r a t s
and g u i n e a p i g s i s t h e b a s i s o f a b i o l o g i c a l
assay o f phenylephrine ( 8 5 ) . Salicylamide, N-
a c e t y l -p-aminophenol and c h l o r p h e n i r a m i n e m a l e a t e
d o n o t i n t e r f e r e . Sample s i z e s o f 1 . 5 t o 80 u g .
have been d e t e r m i n e d .
I n t e r f e r e n c e r e f r a c t o m e t r y ( 8 6 ) has been u s e d
as a q u a n t i t a t i v e micro method of phenylephrine
analysis.
7. References
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71. D. J . S m i t h , J . A s s o c . Offic. A n a l . Chem.
4 9 ( 3 ) 9 536-41 ( 1 9 6 6 ) .
72. E. Brochmann-Hanssen and A. B . Svendsen,
J . Pharm. S c i . , 5 1 ( l o ) , 9 3 8 - 4 1 ( 1 9 6 2 ) .
73. C. H i s h t a a n d R. G . L a u b a c k , J . Pharm.
Sci., 58(6) 745-46 ( 1 9 6 9 ) .
74. P . C a p e l l a and E . C . H o r n i n g , AnaZ. Chem.
3 8 ( 2 ) , 31 6 - 2 1 ( 1 9 6 6 ) .
75. M . C . V i n c e n t , E . K r u p s k i a n d L. F i s c h e r ,
J . A m . Pharm. A s s o c . , S c i . E d . , 46, 85,
( 1957).
76. T . A . Wheaton a n d I . S t e w a r t , A n a l . B i o -
chem. 12, 5 8 5 - 5 9 2 ( 1 9 6 5 ) .
77. K . 0. M o n t g o m e r y , P . V . J e n n i n g s and M.
H . W e i n s w i g , J . Pharm. S c i . , 56(1>, 1 4 1 -
43 ( 1 9 6 7 ) .
78. Idem. i b i d . , 3 9 3 - 9 5 ( 1 9 6 7 ) .
79. F. De F a b r i z i o , J . Pharm. S c i . , 5 7 ( 4 ) ,
644-45 ( 1968).
80. S. U d e n f r i e n d , D. E . Duggan, B. M. V a s t a
a n d B . B . B r o d i e , J . Pharmacol. E x p .
T h e r . 120, 2 6 - 3 2 ( 1 9 5 7 ) .
81. J . D. W i n e f o r d n e r and M. T i n , A n a l . Chim.
A c t a , 31, 2 3 9 - 4 5 ( 1 9 6 4 ) .
82. R. P o h l o u d e k - F a b i n i a n d K. K o n i g ,
Pharmazie, 13, 7 5 2 - 5 6 ( 1 9 5 9 ) .
83. W . Awe a n d H . S t o h l m a n n , P h a r m a z i e , 12,
647-51 ( 19 5 7 ) .
84. G. P a t r i a r c h e , J . Pharm. B e Z g . , 1 9 ( 7 - 8 1 ,
299-317 ( 1 9 6 4 ) , CA 63:16125e.
85. J . S . N a r a v a n e a n d S. S. S a m a r t h ,
I n d i a n J . Pharm., 2 8 ( 4 ) , 99-102 ( 1 9 6 6 ) .
86. N. H . C h o u l i s , J . Pharm. Sci., 5 6 ( 9 ) ,
1206, ( 1 9 6 7 ) .
( L i t e r a t u r e s e a r c h c o v e r s t h r o u g h Decem-
b e r 1971).

511
 CHARLES A. GAGLIA, JR.

Acknowledgment
The a u t h o r w i s h e s t o a c k n o w l e d g e t h e
a s s i s t a n c e o f Dr. R. C . G r e e n o u g h i n p r e p a r i n g
and i n t e r p r e t i n g s p e c t r a l d a t a i n t h i s p r o -
file.

512
 TOLBUTAMIDE

William F. Beyer and Erik H. Jensen

 WILLIAM F. BEYER AND ERIK H. JENSEN

CONTENTS

1. Description
1.1. Name, Formula, Molecular Weight
1.2. Appearance, Color, Taste, Odor
2. Physical Properties
2.1. Solubility
2.2. Melting Range
2.3. Crystal Properties
2.31. Crystal Morphology
2.311. System and Class
2.312. Axial Ratio
2.313. Interfacial Angles
2.314. Habit
2.32. Optical Properties
2.321. Refractive Indicies
2.322. Molecular Refraction
2.323. Optic Axial Angle
2.324. Dispersion
2.325. Optic Orientation
2.326. Common Crystal Orientation
2.327. Optic Sign
2.33. Fusion Properties
2.34. X-Ray Diffraction
2.4. Infrared Spectrum
2.5. Nuclear Magnetic Resonance Spectrum
2.6. Mass Spectrum
2.7. Ultraviolet Spectrum
2.8. pKa
2.9. Differential Scanning Calorimetry
3. Synthesis
4 . Stability
5. Drug Metabolites
6 . Methods of Analysis
6.1. Elemental Analysis
6.2. Phase Solubility
6.3. Titr imetric
6.4. Ultraviolet Spectrophotometric
6.5. Colorimetric

5 14
 TOLBUTAMIOE

6.6. Gas Chromatographic

6.7. Liquid Chromatographic
6.8. Paper Chromatographic
6.9, Thin Layer Chromatographic
6.10. Coulometric
7. Pharmacokinetics and Toxicity
8. Identification
9. References Cited

515
 WILLIAM F. BEYER AND ERIK H. JENSEN

1. Description
1.1. Name, Formula, Molecular Weight
- -
To1butamide is 1-Buty1 3-(p to1y 1sul fony1)urea
It is also referred to as: N-(4-methyl-benzenesulphonyl)-
'.
N'-n-butyl-urea2, to1 lsulfonylbutylurea~,3-(p-tolyl-4-
3
sulfony1)-1-butylurea , N-(sulfonyl-p-methylbenzene)-N'-n-
butylurea3. The most commonly used trade marks are Orinase
and Rastinon; 14 additional are listed in the Merck Index
8th Edition3. Tolbutamide is a sulfonamide but it is not
a sulfanilamide derivative,

0 - - S O 2 - $" - 4C - N -
CH3 I c4H9

12H 1BN 2' '3 Mol. Wt. 270.35

1.2. Appearance, Color, Taste, and Odor

Tolbutamide is a white, or practically white,
crystalline powder, It has a slightly bitter taste and is
practically odorless1 .
2. Physical Properties

2.1. Solubility
Practically insoluble in water but forms water-
soluble salts with alkalies. It is soluble in alcohol and
in chloroform1. It is soluble to the extent of 7.8 mg/ml
in toluene and 4.4 mg/ml in ethyl acetate:heptane (1:3)4.

516
 TOLBUTAMIDE

2.2.M e l t i n g Range
The m e l t i n g range of tolbutamide has been r e -
ported a s 126-132'l and 128.5-129.5'3.

2.3. Crystal Properties

2.31. C r y s t a l Morphology5

2.311. System and C l a s s

Orthorhombic, rhombic pyramidal,
MM2
2.312. Axial Ratio
a:b:c = 0.4504: 1:0.3864

2,313. I n t e r f a c i a l Angles
(101) A (ioi) = 810; (011) A
(oil) = 1380; ( i 2 0 ) A (iio) = 960

2,314.
Habit
Tabular {OlO) w i t h {lOl), {Oll),
[i20) . Supplementary twinning is common, but u s u a l l y
w i t h t h e composition p l a n e and r e e n t r a n t a n g l e s v i s i b l e .

2.32. Optical Properties5

2,321.
R e f r a c t i v e I n d i c i e s (5893A)
Nx = 1.544; Ny = 1.550; Nz =
1.604; geometric mean = 1.562

2,322. Molecular R e f r a c t i o n
Observed = 69.4; c a l c u l a t e d = 70.4

2,323. O p t i c A x i a l Angle (58938)

2V = 38' c a l c u l a t e d from
r e f r a c t i v e i n d i c i e s ; 42' u s i n g M a l l a r d ' s c o n s t a n t .

2.324. Dispersion
1: > v , s t r o n g .

517
 WILLIAM F. BEYER AND E R I K H. JENSEN

2.325. Optic Orientation

a=Y; b = X

2.326.
Common C r y s t a l O r i e n t a t i o n
(010) showing c e n t e r e d o b t u s e
bisectrix interference figure.

2.327. O p t i c Sign
Positive

2.33. Fusion P r o p e r t i e s
When tolbutamide i s melted and slowly
cooled, an u n s t a b l e form c r y s t a l l i z e s which, upon r e h e a t i n g ,
slowly undergoes a s o l i d - s o l i d phase t r a n s f o r m a t i o n t o t h e
s t a b l e form5.

2.34. X-ray D i f f r a c t i o n
P r e c e s s i o n and Weissenberg photographs of
t h e X-ray d i f f r a c t i o n p a t t e r n of s i n g l e c r y s t a l s of USP
Tolbutamide were made i n a study t o update t h e powder d i f -
f r a c t i o n f i l e 6 . The c r y s t a l s were found t o be orthorhombic,
and s y s t e m a t i c absences uniquely determined t h e space group
a s P nma. The u n i t c e l l parameters measured were i n good
agreement w i t h t h e o r i g i n a l d e t e r m i n a t i o n of Q h e l l 5 . The
l a b e l i n g of t h e u n i t c e l l axes given by S h e l l has, how-
e v e r , been permuted t o a g r e e w i t h t h e I n t e r n a t i o n a l Tables
-
f o r X-ray C r y s t a l l o g r a p h y convention f g r space grgup P nma.
The new c e l l dimensions a r e : a = 20.14A; b = 9 . 0 7 ~ ; and
c = 7.781.

The DIFMAX computer program was used t o a r r i v e a t t h e

indexed r e f l e c t i o n s , 2 8 v a l u e s , and d s p a c i n g s of Table I.
I n space group P nma, t h e f o l l m i n g c o n d i t i o n s l i m i t
possible reflections:

0, k, l : k + 1 m u s t be even

h , k, o:h m u s t be even

Accordingly, d e l e t i o n s of s s t e m a t i c a l l y a b s e n t r e f l e c -
t i o n s were made i n Table I. g

518
 TOLBUTAMIDE

TABLE I
Possible X-ray Diffraction Maxima for Tolbutamide

H K L 2 Theta D

2 0 0 8.78 10.0608
1 0 1 12.19 7.2507
2 1 0 13.13 6.7332
2 0 1 14.38 6.1510
0 1 1 15.00 5.8998
1 1 1 15.63 5.6614
2 1 1 17.40 5.0893
3 0 1 17.44 5.0780
4 0 0 17.61 5.0304
0 2 0 19.57 4.5308
3 1 1 20.02 4.4299
4 1 0 20.17 4.3981
4 0 1 21.01 4.2231
2 2 0 21.48 4.1312
0 0 2 22.86 3.8864
1 2 1 23.12 3.8423
4 1 1 23.21 3.8278
1 0 2 23.28 3.8159
2 2 1 24.37 3.6480
2 0 2 24.53 3.6253
5 0 1 24.89 3.5737
1 1 2 25.30 3.5168
3 2 1 26.33 3.3807
4 2 0 26.45 3.3666
2 1 2 26.45 3.3659
3 0 2 26.48 3.3627
6 0 0 26.55 3.3536
5 1 1 26.79 3.3245
3 1 2 28.28 3.1526
6 1 0 28.35 3.1451
4 2 1 28.87 3.0893
6 0 1 28.96 3.0792
4 0 2 29.00 3.0755
0 2 2 30.26 2.9499
1 2 2 30.60 2.9187
6 1 1 30.63 2.9155

519
 WILLIAM F. BEYER AND ERIK H . JENSEN

TABLE I
(Continued)
H K L 2 Theta D
4 1 2 30.66 2.9123
2 3 0 30.87 2.8930
2 2 2 31.57 2.8307
0 3 1 31.75 2.8154
5 2 1 31.86 2.8059
5 0 2 31.98 2.7956
1 3 1 32.06 2.7883
2 3 1 33.00 2.7113
3 2 2 33.14 2,7002
7 0 1 33.19 2.6960
6 2 0 33.20 2.6955
5 1 2 33.51 2.6713
3 3 1 34.51 2.5960
4 3 0 34.60 2.5896
7 1 1 34.68 2.5841
I 0 3 34.88 2.5697
6 2 1 35.20 2.5467
4 2 2 35.23 2.5446
6 0 2 35.31 2.5390
8 0 0 35.66 2.5152
2 0 3 35.75 2.5091
0 1 3 36.01 2.4911
1 1 3 36.30 2.4722
4 3 1 36.53 2.4568
6 1 2 36.72 2.4448
8 1 0 37.05 2.4235
2 1 3 37.14 2.4181
3 0 3 37.16 2.4169
8 0 1 37.54 2.3930
5 2 2 37.77 2.3791
1 3 2 37.95 2.3683
3 1 3 38.51 2.3352
2 3 2 38.76 2.3206
7 2 1 38.83 2.3169
8 1 1 38.88 2.3137
7 0 2 38.93 2.3110
5 3 1 39.00 2.3069
4 0 3 39.06 2.3034
0 4 0 39.75 2.2654

520
 TOLBUTAMIDE

Figure 1 gives the X-ray powder diffraction pat-

tern for USP Tolbutamide obtained with a General Electric
XRD-5 Diffractometer using C u K d 1, 50 KVP, 16 MA, and
tray mount6,

2.4. Infrared Spectrum

Tolbutamide can be identified bv means of its
infrared spectrum (Figure 2). The spectrLm of USP Tolbut-
amide7 was obtained from a Nujol mull using a Perkin-Elmer
Model 421 spectrophotometer. The principal peaks are
assigned as follows: 3320, 3190 (urea, NH stretch), 2920,
2850 (alkane, CW stretch including Nujol), 1700, 1660
(urea, C=O stretch), 1600, 1500 (aromatic, C=C stretch),
1555 (urea, amide 11), 1460, 1375 (alkane, CH deformation
including Nujol), 1335, 1160 (sulfonamide, S=O stretch),
and 815 cm-1 (aromatic, CH deformation for para substi-
tution). Other peaks occur at 1320, 1310, 1250, 1220, 1190,
1120, 1095, 900, 740, 725, and 660 cm-l.

2.5. Nuclear Magnetic Resonance Spectrum

The nuclear magnetic resonance (NMR) spectrum of
tolbutamide was obtained using a Varian instrument Model
A-60 D. Figure 3 gives the spectrum8. The tolbutamide
was dissolved in deutero chloroform with tetramethylsilam
as the internal reference. The NMR proton spectral assign-
ments are given in Table I18.

2.6. Mass Spectrum

Tolbutamide mass-spectral data are given in
Table III9. The data were obtained using an Atlas CH4
instrument. The l o s s of 64 mass units from the molecular
ion is attributed to the loss of S02, a unique loss of
these elements from the middle of the tolbutamide molecule.
The molecular ion was observed at mfe 270. At 70 ev the
most intense peak was observed at m/e 91 (15.4% of total
ionization). This peak can be represented as a C7H7+ion
which yields CgHg+(m/e 65; metastable at 46.4) upon
expulsion of an acetylene molecule after decomposition.

521
522
 0
0
m
0
0
b
a,
a
W
0
i
0 0 0 0 0 0 0 0 0 6
o m a b u , u , a n ~ -
33NWlllHISNWMl %
523
 a
\
I
\-

Figure 3. Nuclear magnetic resonance spectrum of tolbutamide

 TOLBUTAMIDE

TABLE I1
NMR Spectral Assignments for Tolbutamide
Chemical
Shape Shift J (Hz)
Distorted Triplet 0.88 6.5

Broad Multiplet

CH3-CgHq-
- Singlet 2.43

CH3-CH2-CH2-CH2- Quartet 3.23 6.0 (av.)

-
-N_H-CH2- Triplet 6.57 6.0 (av.)

H
Apparent Doublet 7.33 8.0 (av.)
CH3 QS

CH3 9
H
H
S Apparent Doublet 7.83 8.0 (av.)

-02S-NK-CO- Broad Singlet 9.7

2.7. Ultraviolet Spectrum

The UV spectrum of USP Tolbutamide is shown in
Figure 4 . The spectrum was obtained with a Cary 15
spectrophotometer using a 15 mcg/ml solution of tolbut-
amide in anhydrous ethanol. The spectrum, obtained with
a 1-cm cell, shows A max at 228 nm.

525
 WILLIAM F. BEYER AND E R I K H. JENSEN

0.3

WAVELENGTH (nm)

Figure 4 . Ultraviolet spectrum of tolbutamide

526
 TOLBUTAMIDE

TABLE 111

Mass Spectral Data for Tolbutamide

% total ionization
-
ml e 70 ev -
19 ev
270 2.2 7.5
255 0.2
241 0.3
227 1.1
215 0.4
206 6.5 35.7
184 0.2
171 0.6 0.5
163 0.3
155 7.1 0.4
139 0.6
115 2.0 1.5
ioa 14.0 19.4
107 5.3 11.5
99 3.2 3.3
91 15.4 0.1
73 3.2 3.1
72 2.3 0.5
65 4.6
30 12.6 5.7

2.8. p ~ a
The pKa' of tolbutamide by two separate pro-
cedures was 5.43 at 25OC and 5.32 at 37.5OC10.

2.9. Differential Scanning Calorimetry

The absolute purity of tolbutamide can be deter-
mined using differential scanning calorimetry. The purity
of Tolbutamide USP Reference Standard with this technique
was 99 12. A Perkin-Elmer Differential Scanning
Calorimeter Model 1 - B was used at a scan speed of 1.25'1
min. at a sensitivity of 2 m callsec f u l l scale.

521
 WILLIAM F. BEYER AND ERIK H. JENSEN

W. F. Beyer and E. H. Jensen

3. Synthesis
The patent procedure for tolbutamide2 gives the
following example for its preparation: 50 gms of n-butyl
isocyanate are stirred at RT into a suspension of 96 gms
of sodium 4-methyl-benzenesulfamide in 120 ml of dry
nitrobenzene, and the mixture is then heated for 7 hours
at 100°C. After being cooled, the reaction mixture, which
is a thick magma, is diluted with methylene chloride or
ethyl acetate, and the sodium salt of the sulfonylurea
formed is separated by centrifuging. The centrifuged
crystalline residue freed from organic solvents is dis-
solved in 500-600 ml of water heated at 5OoC and decolor-
ized with charcoal. The precipitate obtained by acidi-
fication with dilute hydrochloric acid is dissolved in an
equivalent quantity of dilute ammonia solution (about
1:20), again treated with charcoal and reprecipitated with
dilute hydrochloric acid. In this manner tolbutamide is
obtained in analytically pure form in a yield of 70-80 per-
cent of theory. Menzer et all3 list p-tolylsulfonamide
and p-tolylsulfonylurea as the primary impurities to be
expected in the synthesis of tolbutamide.

4. Stability
Because of the absence of p-amino groups, which are
common to antibacterial sulfonamides, tolbutamide cannot
be acetylated. Its p-methyl group, however, renders tol-
butamide susceptible to oxidation, occurring chiefly in
biological systems.
Thermal decomposition of tolbutamide has been reported
by Menzer et al13, with reformation of p-tolylsulfonamide
and synthesis of butylisocyanate. The latter then reacts
with unconverted butylamine and ammonia to form N,N-di-
butylurea and N-butylurea. The authors also isolated four
additional by-products of tolbutamide, one identified as
p-tolylsulfonylbiuret.
The hydrolysis of tolbutamide in an acid environment
was investigated by Vogtl4 and in alkaline solution by
Haussler and Hajduls. A quantitative dissociation of tol-
butamide to p-toluenesulfonamide and n-butyl isocyanate
was reported by Ulrich and Sayighl6 to take place in
inert solvent at 160 to 180°C. A significant degradation

528
 TOLBUTAMIDE

occurred in some o/w creams when the drug wa dissolved in

the oil phase of the emulsion at 70 to 80 C 17. No such
loss occurred when the sulfonylurea was incorporated in
the base at room temperature. The authors concluded that
the instability of tolbutamide in the oil phase of the
emulsion was due to components containing hydroxyl groups.
The investigations were expanded to study the dissociation
of tolbutamide at 80OC in twelve primary alcohols and in
polyethylene glycol 40018. Tolbutamide was shown to
dissociate to give butylamine and p-toluenesulfonyl
isocyanate. N-(p-toluenesulfonyl) carbamate, formed by
the reaction of the sulfonylisocyanate with the alcohols,
was present in the equilibrium mixture. Bottari et all9,
in studies investigating the reaction products of tolbut-
amide and other N-substituted sulfonylureas in alcohols,
water, and amines, concluded that dissociation, rather
than solvolysis, was the most likely mechanism by which
sulfonylureas undergo breakdown at rela ively low temper-
atures. A report by Chubb and SimmonsZS indicates that
tolbutamide reacts with refluxing methanol to form the
butylamine salt of methyl p-tolylsulfonylcarbamate. They
subscribe to a mechanism of methanolysis for the reaction
rather than one of pyrolysis.

5. Drug Metabolites
Oxidation of tolbutamide through its p-methyl group
appears to be the principal manner of degradation of
tolbutamide in man. The p-methyl group is oxidized to
form a carboxyl group, converting tolbutamide into its
principal metabolite, 1-butyl-3-p-carboxyphensulfony-
lurea (carboxytolbutamide)21s22.

The tolbutamide metabolite is highly soluble over the

critical acid range of urinary pH values, and its solu-
bility increases with an increase in pH. The measured
solubility at pH 5 is 2.8 mg/ml, increasing to 20 mg/ml
at pH 5.5; atpH6.0 by extrapolation, the solubility
becomes 300 mg/m123. At 37.5OC and at various pH ranges
the following carboxyto1butamide:tolbutamide solubility
ratios were determined: pH 5.0, 13:l; pH 5.5, 50:l;
pH 6.0, 350:11°. A pKa1 of 3.54 at 37.5OC has been deter-
mined for the metabolitelo.

529
 WILLIAM F. BEYER A N D E R I K H. JENSEN

The amount of m e t a b o l i t e i n u r i n e c a n be d e t e r m i n e d by
measuring t h e c o l o r found when amyl a c e t a t e - e x t r a c t e d
u r i n e i s added t o 0.1% d i n i t r o f l u o r o b e n z e n e and h e a t e d a t
150'24.

6. Methods o f A n a l y s i s

6.1. E l e m e n t a l A n a l y s i s of t o l b u t a m i d e 4 ,

E 1e men t % Theory % Reported

Carbon 53.31 53.43

Hydrogen 6.71 6.99
Nitrogen 10.36 10.29
Sulfur 11.86 11.88

6.2. Phase S o l u b i l i t y A n a l y s i s
Phase s o l u b i l i t y p r o f i l e s o f t o l b u t a m i d e were
o b t a i n e d w i t h s o l v e n t s y s t e m s of t o l u e n e and e t h y l a c e t a t e :
h e p t a n e ( 1 : 3 ) , g i v i n g s o l u b i l i t i e s of 7.79 k0.15 mg/gm and
4 . 4 1 k0.08 mg/gm r e s p e c t i v e l y 4 . The c a l c u l a t e d p u r i t y
of t h e t o l b u t a m i d e sample based on i t s s o l u b i l i t y p r o f i l e
i n t o l u e n e was 100.1 k0.70% and i n e t h y l a c e t a t e : h e p t a n e
(1:3) i t was 99.6 k0.41%.

6.3. Titrimetric
The p r o c e d u r e of F r a n c h i 2 5 depends on t i t r a t i o n
o f t o l b u t a m i d e w i t h sodium methoxide i n anhydrous p y r i -
dine-chloroform-methanol.

I n a r e p o r t e d t i t r i m e t r i c method of a s s a y f o r t o l -
butamide i n non-aqueous mediaz6, 50 t o 150 mg i s d i s s o l v e d
i n 10 m l of anhydrous a c e t o n e o r p y r i d i n e and t i t r a t e d
w i t h 0 . 1 1 sodium methoxide t o a p h e n o l p h t h a l e i n e n d p o i n t ,
I n a m i x t u r e of benzene and methanol ( 2 : 1 ) , thymol b l u e
c a n be used a s i n d i c a t o r . A s i m i l a r p r o c e d u r e was r e p o r t e d
by Dave and P a t e 1 2 7 .

S i m i o n o v i c i and ConuZ8 developed a d i r e c t

t i t r a t i o n method whereby a p p r o x i m a t e l y 300 mg o f

530
 TOLBUTAMIDE

t o l b u t a m i d e i s d i s s o l v e d i n 25 m l of a c e t o n e p r e v i o u s l y
n e u t r a l i z e d t o c r e s o l r e d and t i t r a t e d w i t h 0.1; sodium
hydroxide t o a r o s e - v i o l e t c o l o r .

I n a p r o c e d u r e depending on h y d r o l y s i s , t h e previous
a u t h o r s 2 8 t r e a t e d a p p r o x i m a t e l y 300 mg of t o l b u t a m i d e w i t h
10 m l o f e t h a n e d i o l and 2 m l of conc. h y d r o c h l o r i c a c i d ,
and a p p l i e d 120-122'C h e a t f o r 30 min. The m i x t u r e was
then d i l u t e d with water, t r e a t e d i n a Kjeldahl f l a s k with
1 5 g sodium h y d r o x i d e and t h e amine was d i s t i l l e d o f f i n t o
25 m l o f 0.1N h y d r o c h l o r i c a c i d , t h e e x c e s s d e t e r m i n e d by
t i t r a t i o n . Assay v a r i a t i o n o f +1.0% was r e p o r t e d .

P a r i k h and Mukherji2' developed a t i t r a t i o n pro-

c e d u r e f o r t o l b u t a r n i d e i n which t o l b u t a m i d e was c o n v e r t e d
i n t o i t s sodium s a l t , combining i t q u a n t i t a t i v e l y w i t h
s i l v e r n i t r a t e t o form a n i n s o l u b l e s i l v e r s a l t . F e r r i c
ammonium s u l p h a t e was used a s t h e i n d i c a t o r and 0.05;
ammonium t h i o c y a n a t e a s t h e t i t r a n t .

Beyer and Houtman3' d e s c r i b e d automated t i t r i -

metric procedures f o r the analysis of tolbutamide t a b l e t s
u s i n g a modified F i s c h e r T i t r a l y z e r . 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 f o r t h e p r o c e d u r e was a p p r o x i m a t e l y 1%.

The USP a s s a y p r o c e d u r e 1 f o r t o l b u t a m i d e depends

upon t h e t i t r a t i o n o f t h e d r u g i n n e u t r a l i z e d aqueous
a l c o h o l w i t h sodium h y d r o x i d e a s t h e t i t r a n t , and phenol-
p h t h a l e i n as i n d i c a t o r .

6.4. U l t r a v i o l e t Spectrophotometric
S p i n g l e r and K a i s e r J L d e t e r m i n e d t o l b u t a m i d e i n
serum a f t e r l y o p h i l i z a t i o n , e x t r a c t i o n w i t h a c i d i f i e d
e t h y l a c e t a t e , r e d u c t i o n t o d r y n e s s , and f i n a l l y d i s s o l -
u t i o n i n methanol. Absorbances a t 228 nm and 280 nm were
used i n q u a n t i t a t i n g tolbutamide.

F o r i s t e t a132 d e v e l o p e d an a n a l y t i c a l p r o c e d u r e
f o r t h e d e t e r m i n a t i o n o f t o l b u t a m i d e i n plasma. The

531
 WILLIAM f. BEYER A N D ERIK H . JENSEN

procedure depends on c h l o r o f o r m e x t r a c t i o n o f weakly

a c i d i f i e d plasma, c o n c e n t r a t i o n o f t h e e x t r a c t i o n t o d r y -
n e s s , d i s s o l u t i o n of t h e d r y r e s i d u e i n e t h a n o l , t r e a t -
ment o f t h e s o l u t i o n w i t h c h a r c o a l , and measurement of
t h e a b s o r b a n c e of a n a l c o h o l i c s o l u t i o n a t 228 nm.
Experiments w i t h human and dog plasma gave r e c o v e r i e s o f
added t o l b u t a m i d e o f a b o u t 98-99%. R e f i n ments i n t h e
and P a l a z z o l i 34 .
method were r e o r t e d by Bladh and Norden3', and D e l a v i l l e

A p r o c e d u r e f o r t h e automated a n a l y s i s of
t o l b u t a m i d e t a b l e t s h a s been r e p o r t e d u s i n g Technicon
C o r p o r a t i o n ' s AutoAnalyzer equipment35. The a n a l y s i s was
c a r r i e d o u t a t a w a v e l e n g t h of 263 nm a t a sampling r a t e
of 201hour. A c o e f f i c i e n t o f v a r i a t i o n o f a p p r o x i m a t e l y
1%was o b t a i n e d .

The USP X V I I I l p r o c e d u r e f o r t o l b u t a m i d e t a b l e t s
depends upon t h e UV a b s o r b a n c e of e x t r a c t e d t a b l e t s i n
c h l o r o f o r m a t a wavelength o f 263 nm. A UV d i s s o l u t i o n
r a t e t e s t f o r t o l b u t a m i d e t a b l e t s i s d e s c r i b e d i n t h e 1st
supplement t o t h e USP X V I I I u s i n g t r i s ( h y d r o x y m e t h y 1 )
aminomethane b u f f e r a t pH 7.6 and a s t i r r i n g r a t e o f
150 rpm. F i l t e r e d samples a r e r e a d a t a w a v e l e n g t h o f
226 nm. The t e s t s p e c i f i e s t h a t t h e t i m e r e q u i r e d f o r
50% of t h e l a b e l e d amount of t o l b u t a m i d e i n t a b l e t s t o
d i s s o l v e i s n o t more t h a n 45 m i n u t e s ,

6.5. Colorimetric
McDonald and S a ~ i n s k di e~v e~l o p e d a c o l o r i -
m e t r i c method i n v o l v i n g t h e r e a c t i o n between t o l b u t a m i d e ,
2 - n a p h t h o l , sodium n i t r i t e , and c o n c e n t r a t e d s u l f u r i c
a c i d forming a red c o l o r . The method i s r e p o r t e d t o be
a p p l i c a b l e over t h e c o n c e n t r a t i o n range of approximately
50 mcg t o 1 0 mg o f t o l b u t a m i d e p e r m l o f s o l u t i o n .

C h ~ l s k di e~s c~r i b e d a method f o r t h e d e t e r -

m i n a t i o n o f t o l b u t a m i d e i n serum by e x t r a c t i n g a c i d i f i e d
serum w i t h c h l o r o f o r m . A f t e r r e d u c i n g t h e c h l o r o f o r m
e x t r a c t t o d r y n e s s , a n a l c h o l i c s o l u t i o n o f p-N-dimethyl-
aminobenzaldehyde i s added and t h e s o l u t i o n reduced t o
d r y n e s s . The d r y r e s i d u e i s h e a t e d €or 2-112 h o u r s a t

532
 TOLBUTAMIDE

7 O o C and a l c o h o l i s added. Absorbance o f t h e s o l u t i o n s

i s measured a t 395 nm. The a v e r a g e r e c o v e r y o f t o l b u t a m i d e
from serum was r e p o r t e d t o be n e a r 100% w i t h a s t a n d a r d
d e v i a t i o n o f a p p r o x i m a t e l y 5%.

The c o l o r i m e t r i c t e r m i n a t i o n o f serum t o l b u t -
amide d e v e l o p e d by S p i n g l e r " depends on t h e r e a c t i o n o f
t o l b u t a m i d e and d i n i t r o f l u o r o b e n z e n e a t 15OoC f o l l o w i n g
e x t r a c t i o n o f a c i d i f i e d serum w i t h amyl a c e t a t e . The
a b s o r b a n c e i s d e t e r m i n e d a t a b o u t 380 nm. A f t e r a s t u d y
o f t h e method o f S ~ i n g l e r 3 ~P i, g n a r d 3 g s u g g e s t e d improve-
ments t h a t were r e p o r t e d t o i n c r e a s e s p e c i f i c i t y and
r a n g e of u s e f u l n e s s . Among t h o s e s u g g e s t e d were p u r i f i -
c a t i o n of r e a g e n t s and l e n g t h e n i n g t h e h e a t i n g t i m e from
5 t o 30 m i n u t e s a t a t e m p e r a t u r e of 100°C i n s t e a d o f
15OoC.

D ~ r f m ~ l l ree rp o~r t~e d t h e r e a c t i o n o f t o l b u t -

amide i n a l k a l i n e media w i t h diacetylmonoxime and N-phenyl-
a n t h r a n i l i c a c i d , f o l l o w e d by a c i d i f i c a t i o n and h e a t and
t h e a d d i t i o n o f sodium p e r s u l f a t e and sodium a c e t a t e t o
form a b l u e c o l o r .

Mesnard and C r o c k e t t 4 1 e x t e n d e d t h e work o f

R i c h t e r 4 * , which i n v o l v e d t h e d e t e r m i n a t i o n of a l i p h a t i c
amino a c i d s , t o t h e a n a l y s i s of t o l b u t a m i d e i n b i o l o g i c a l
f l u i d s . The method i s based on t h e s t r o n g y e l l o w c o l o r a -
t i o n o f s u b s t i t u t e d ammonium p i c r a t e s i n s e l e c t e d anhy-
d r o u s s o l v e n t s , i n which p i c r i c a c i d i s e s s e n t i a l l y c o l o r -
l e s s . Of t h e two a b s o r b a n c e maxima observed (355 nm and
412 nm) t h e maximum a t 412 nm w a s used t o a v o i d a b s o r p t i v e
m a t e r i a l s i n blood and u r i n e t h a t c o u l d i n t e r f e r e a t t h e
355 nm wavelength. The a u t h o r s a l s o reviewed o t h e r
methods f o r t h e d e t e r m i n a t i o n of t o l b u t a m i d e and o t h e r
non-amino hypoglycemic su I f ~ n a m i d e9 s4 4~ . ~

Kern45 r e p o r t e d a p r o c e d u r e f o r t h e d e t e r -
m i n a t i o n of t o l b u t a m i d e i n blood f o l l o w i n g e x t r a c t i o n
w i t h e t h y l e n e chloride a t pH 5. A f t e r n i t r a t i o n , d i a z o -
t i z a t i o n and c o u p l i n g w i t h N(1-naphtyl) e t h y l e n e d i a m i n e ,
a n a z o dye i s produced t h a t i s measured a t 547 nm.

533
 WILLIAM F. BEYER AND ERIK H. JENSEN

A number o f o t h e r i n v e s t i g a t o r s have d e s c r i b e d
co l o r ime t r i c p r o c e d u r e s f o r t o 1b u t amid e46-49 .
6.6. Gas Chromatographic
A g a s - l i q u i d c h r o m a t o g r a p h i c method f o r t h e
d e t e r m i n a t i o n of t o l b u t a m i d e i n b l o o d , u r i n e , and t a b l e t s
was r e p o r t e d by S a b i h and SabihSO. A method f o r b l o o d and
u r i n e i n v o l v e d e x t r a c t i o n o f t o l b u t a m i d e from a c i d i f i e d
plasma o r u r i n e and c o n v e r s i o n t o t h e m e t h y l d e r i v a t i v e
with dimethylsulfate i n t h e presence of base. A g a s
chromatograph (F & M 5755B) w i t h a f l a m e i o n i z a t i o n
d e t e c t o r and f i t t e d w i t h a s t a i n l e s s s t e e l column was u s e d .
The column was packed w i t h d i a t o m a c e o u s e a r t h (Gas chrom Q)
and c o a t e d w i t h 5% DC-200. T e m p e r a t u r e s o f 205-210' f o r
t h e column, 320' f o r t h e d e t e c t o r , and 330' f o r t h e
i n j e c t i o n p o r t were u s e d . A d d i t i o n a l GLC methods f o r t o l -
butamide i n blood have b e e n r e p o r t e d by P r e s c o t t and Red-
man51 and Simmons e t a152 a l s o i n v o l v i n g m e t h y l a t i o n w i t h
dimethyl s u l f a t e .

6.7. L i q u i d Chromatographic
A l i q u i d chromatographic a s s a y procedure f o r
t o l b u t a m i d e i n t a b l e t s ( a l s o a p p l i c a b l e t o b u l k d r u g ) was
d e s c r i b e d by B e ~ e r ~ A~ duPont . Model 820 L i q u i d Chromato-
g r a p h w i t h a n HCP column ( s t a i n l e s s s t e e l ) 1 M l o n g x 2.1
mm I D and a mobile p h a s e of pH 4.4 monobasic sodium
c i t r a t e b u f f e r i n 15% methanol a t a f l o w r a t e of 0 . 3 6 m l /
min were used f o r t h e a n a l y s i s . 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 w a s less t h a n 2% and r e c o v e r y was q u a n t i t a t i v e ,

6.8. Paper Chromatographic

Chakrabarti54 r e p o r t e d a paper chromatographic
a n a l y s i s o f t o l b u t a m i d e i n v a r i o u s s o l v e n t s y s t e m s . The
developed p a p e r i s r e a c t e d w i t h p h e n y l h y d r a z i n e and
s p r a y e d w i t h a s o l u t i o n of ammoniacal Ni2+ g i v i n g p i n k t o
v i o l e t s p o t s , d e p e n d i n g on t h e c o n c e n t r a t i o n of t o l b u t -
amide s o l u t i o n s .

Hentrichfj5 d e s c r i b e d a paper chromatographic

s e p a r a t i o n o f t o l b u t a m i d e by c h r o m a t o g r a p h i n g t h e b u t y l
amine produced when t o l b u t a m i d e i s r e a c t e d w i t h a m o d i f i e d
F o l i n r e a g e n t . A f t e r h e a t i n g t h e p a p e r a t 180-200', a
brown s p o t i s produced.

534
 TOLBUTAMIDE

Abdel-Wahab and E l - A l l a ~ yr~e p~o r t e d a procedure

u t i l i z i n g paper chromatography f o r r a d i o i s o t o p e s and t h e
d e t e r m i n a t i o n of t o l b u t a m i d e . The a u t h o r s employed
v a r i o u s d e v e l o p e r s and c o l o r i n g r e a g e n t s such a s n i n h y d r i n .
R a d i o - a c t i v a t i o n of d r i e d , developed chromatograms, u s i n g
1131was a p p l i e d t o determine Rf v a l u e s . I n v e s t i g a t i o n s of
S35 l a b e l e d t o l b u t a m i d e showed t h a t paper chromatography,
accompanied by r a d i o s c a n n i n g o r a u t o r a d i o g r a p h y could be
used f o r t h e s e p a r a t i o n , d e t e c t i o n , and d e t e r m i n a t i o n of
tolbutamide.

An i n f r a r e d i d e n t i f i c a t i o n o f t o l b u t a m i d e i n
human serum employing paper chromatography was r e p o r t e d
by K r i v i s and F o r i s t 5 7 . The procedure depends upon ex-
t r a c t i o n of serum w i t h chloroform, e v e n t u a l r e d u c t i o n t o
d r y n e s s , d i s s o l u t i o n i n methylene c h l o r i d e , and a p p l i c a t i o n
t o prewashed Whatman No. 1 paper. A f t e r development i n a
butanol-water-piperidine (81: 17: 2) system by d e s c e n d i n g
chromatography, t h e t o l b u t a m i d e zone was e l u t e d w i t h w a t e r .
A potassium bromide m i c r o p e l l e t prepared from a methylene
c h l o r i d e e x t r a c t i o n of t h e aqueous e l u a t e was t h e n
examined by i n f r a r e d spectrophotometry.

6.9, Thin Layer Chromatographic

S t r i c k l a n d b 8 d e s c r i b e d TLC procedures f o r t h e
s e p a r a t i o n and d e t e c t i o n o f microgram amounts of t o l b u t -
amide i n t h e presence of acetohexamide, chlorpropamide,
and phenformin HC1. Various s o l v e n t systems and s p r a y r e -
a g e n t s were used t o determine r e l a t i v e Rf v a l u e s . A
s o l v e n t system c o n s i s t i n g of acetone-benzene-water
(65:30:5) s e p a r a t e d t o l b u t a m i d e and t h e o t h e r t h r e e a n t i -
d i a b e t i c a g e n t s . The l i m i t of d e t e c t i o n f o r t o l b u t a m i d e
u s i n g UV was about 1 mcg.

A TLC procedure f o r t h e d e t e c t i o n of t o l b u t a m i d e
i n blood and u r i n e was r e p o r t e d by Baumler and R i p p s t e i n 5 9 .
The d r u g i s e x t r a c t e d w i t h e t h e r and chromatographed
u s i n g K i e s e l g e l - c e l l u l o s e ( 1 : l ) a s t h e TLC s u p p o r t and
developed w i t h benzene-methanol ( 4 : 1). Tolbutamide
a p p e a r s a s a v i o l e t s p o t when t h e developed p l a t e i s
sprayed w i t h n i n h y d r i n - s t a n n o u s c h l o r i d e and h e a t e d , t h e n
sprayed w i t h a c i d i f i e d n i n h y d r i n and h e a t e d a g a i n .

535
 WILLIAM F. BEYER AND E R I K H. JENSEN

Guven e t a160 i d e n t i f i e d t o l b u t a m i d e amongst

o t h e r a n t i - d i a b e t i c d r u g s by TLC on s i l i c a g e l u s i n g a
s o l v e n t system composed of b u t a n o l - a c e t i c acid-water
(10:2:1). A s o l u t i o n of copper s u l f a t e (10%) ammonia (2%)
( 5 : l ) was used t o d e t e c t t h e s p o t s , w i t h t o l b u t a m i d e
e x h i b i t i n g a green color.

A r e p o r t by Hutzul and Wright61 f o r t h e

d e t e c t i o n of s m a l l amounts of i m p u r i t i e s ( f o r example,
0.05% p - t o l u e n e s u l f o n y l u r e a ) i n t o l b u t a m i d e , makes use
of a "Moscow" method of TLC developed by Mistryukov62.
The p l a t e , 5x8x1/8" f r o s t e d window pane, i s covered w i t h
adsorbent and maintained h o r i z o n t a l l y w h i l e t h e d e v e l o p i n g
s o l u t i o n i s fed t o one edge by c a p i l l a r y a c t i o n .
E-Toluenesulfonylurea is s e p a r a t e d from t o l b u t a m i d e u s i n g
Davison s i l i c a g e l a s a d s o r b e n t , benzene-acetone-
m e t h a n o l - a c e t i c a c i d (70: 20: 9: 1) a s d e v e l o p i n g s o l v e n t ,
and v i s u a l i z e d u s i n g mists of hypochlorous a c i d , e t h a n o l ,
and an aqueous s o l u t i o n of a c e t i c a c i d w i t h i o d i d e and
s a t u r a t e d w i t h b e n z i d i n e . For t h e d e t e c t i o n of 1-
toluenesulfonamide, an a d s o r b e n t of aluminum oxide and
a developing s o l v e n t of benzene-acetone-methanol
(70:20:10) was used. Agents f o r v i s u a l i z a t i o n were t h e
same a s t h o s e f o r p t o l u e n e s u l f o n y l u r e a .

Menzer e t a l l 3 u s e d a developing system com-

p r i s e d of c h 1 o r o f o r m : g l a c i a l a c e t i c a c i d (99:l) and
s i l i c a g e l H p l a t e s , 0.25 mm t h i c k t o s e p a r a t e t o l b u t -
amide from i t s by-products. E v a l u a t i o n s were made under
UV l i g h t a f t e r spraying with xanthydrol s o l u t i o n . Their
work a l s o d i s c l o s e d a new by-product i n t h e s y n t h e s i s of
tolbutamide: p t o l y l s u l f o n y l b i u r e t . Table I V summarizes
their results.

536
 TOLBUTAMIDE

TABLE IV

Relative TLC Rf Values for Tolbutamide and By-products

Using a Ch1oroform:Glacial Acetic Acid (99:l)
Developing Solution

Compound -
Rf
Tolbutamide 0.85
N,N'-Dibutylurea 0.56
P-Toly lsu 1fonamide 0.47
P-Toly lsu1fony lurea 0.26
P-Tolylsulfonylbiuret 0.13
N-Butylurea 0.12

Reisch et a163 reported five TLC developing

systems using n-butanol in combination with two to three
other organic solvents. Spots were detected on silica gel
G plates when sprayed with visualizing agents. TLC was
applied by Neidlein et a164 and Glogner et d5 in
separating tolbutamide from other sulfonamides.

6.10. Coulometric
A Mercurocoulometric method for the determin-
ation of tolbutamide has been reported by Kalinowski and
Korzybski66 and V O ~ C U ~ ~ .

7. Pharmacokinetics and Toxicity

The half-life in human subjects for 1 gm of tolbut-
amide given as a single dose following an overnight fast
is reported by McMahon et a168 to be 5.7 hours. The LD50
f o r tolbutamide administered orally to rats is 2,344 mg/
kgm and in mice injected IP is 1,232 mg/kgm68.

Inactivation of tolbutamide to carboxytolbutamide occurs

in man and is rapidly excreted in urine as the principal
metabolite.

537
 WILLIAM F. BEYER AND ERIK H. JENSEN

8. Identification
I d e n t i f i c a t i o n t e s t s f o r tolbutamide a r e given i n
U.S.P. X V I I I 2 . The t e s t s depend upon: a ) t h e i n f r a r e d
a d s o r p t i o n spectrum of a m i n e r a l o i l d i s p e r s i o n of t h e
drug i n t h e range o f 2 t o 1 2 p; b) f o r m a t i o n of an
orange-red c o l o r a f t e r t h e d r u g i s r e f l u x e d w i t h d i l u t e
s u l f u r i c a c i d , steam d i s t i l l e d i n d i l u t e h y d r o c h l o r i c
a c i d a f t e r b e i n g made s t r o n g l y a l k a l i n e , made a l k a l i n e
w i t h a c e t a t e and b o r a t e b u f f e r , and r e a c t e d i n an i c e b a t h
w i t h p - n i t r o a n i l i n e and sodium hydroxide; and c ) pro-
d u c t i o n of p-toluenesulfonamide ( m e l t i n g between 136' and
141') by r e f l u x i n g i n d i l u t e s u l f u r i c a c i d , c o o l i n g t h e
s o l u t i o n , c o l l e c t i n g and p u r i f y i n g t h e c r y s t a l s .

538
 TOLBUTAMI DE

9. References

1. "United States Pharmacopeia," 18th Ed., Mack

Printing Co., Easton, Pa.

2. Ruschig, H., W. Aumiiller, G. Korger, H. Wagner,

J. Scholz, and A . BPnder, U.S. Patent 2,968,158,
January 17, 1961 (assigned to The Upjohn Co.).

3. The Merck Index, 8th Edition, Merck and Co., Inc.

Rahway, N.J. (1968).

4. Humphrey, L. M., The Upjohn Co., unpublished data.

5. Shell, J. W., Anal. Chem., 30, 1577 (1958).

6. Zipple, K. G., C. G. Chidester, C. G. Waber, and

D. J. Duchamp, The Upjohn Co., unpublished data.

7. Muelman, P. A. and M. L. Knuth, The Upjohn Co.,

unpublished data.

8. Slomp, G., The Upjohn Co., unpublished data.

9. Grostic, M. F., R. J. Wnuk, and F. A. MacKellar,

J. Am. Chem. SOC., g,(1966).

10. Forist, A . A , , T. Chulski, Metabolism, 3 , 807

(1956).

11. Bowman, P. B., The Upjohn Co., unpublished data.

12. Perkin-Elmer Trade Publication, Thermal Analysis

News Letter No. 5.

13. Menzer, M., J. Presewowski, and U. Haug, Pharmazie

-
26, 738 (.i97i).

14. Vogt, H., Pharm. Zentralh, 98, 651 (1959).

15. Haussler, A . and P. Hajdu, Arch, Pharm., 295, 471

(1962).

539
 WILLIAM F. BEYER A N D ERIK H. JENSEN

16. U l r i c h , H. and A.A.R. Sayigh, Angew. Chem. I n t .

E d . Engl. 2, 724 (1966).

17. B o t t a r i , F., M. F. S a e t t o n e , B. G i a n n a c c i n i , and

M. E. LoBrutto, Rass. Dermatol, S i f i l o g r . , 20,
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18. B o t t a r i , F., M. M a n n e l l i , and M. F. S a e t t o n e , J.

Pharm. S c i . , 2,1663 (1970).

19. B o t t a r i , F., B. G i a n n a c c i n i , E. N a n n i p i e r i , and

M. F. S a e t t o n e , J. Pharm. S c i . , 61, 602 (1972).

20. Chubb, F. L. and D. L. Simmons, Can. J. Pharm.

S c i . , 1,28 (1972).

21. L o u i s , L. H . , S. S , F a j a n s , J . W . Conn, W , A.
S t r u c k , J. B. Wright, and J. L. Johnson, J. Am.
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22. P h y s i c i a n s Desk R e f e r e n c e , 26 E d . , Medical

Economics, I n c . , 1972.

23. Tucker, H. A., "Oral A n t i d i a b e t i c Therapy 1956

1965: w i t h P a r t i c u l a r R e f e r e n c e t o Tolbutamide
( O r i n a s e ) , " C. C. Thomas, P u b l i s h e r , 1965.

24. Nelson, E . , I. O ' R e i l l y , and T. C h u l s k i , C l i n .

Chim. Acta, 3 , 774 (1960).

25. Franchi, G., A t t i . Accad. F i s i o c r i t . Siena 6,61

(1956-57).

26. Kracmarova, J . , J. Kracmar, C e s k o s l . Farm., 1,

566 (1958).

27. Dave, J. B., and J. L. P a t e l , I n d i a n . J . o f

Pharm. Z l , 226 (1959).

28. S i m i o n o v i c i , R., and I. Conu, Rev. Chim. Bucha-

rest, Lo, 107 (1959).

540
 TOLBUTAM I DE

29. P a r i k h , P. M., S. P. M ukhe r j i , I n d i a n J. o f

Pharrn. , 2l, 110 (1959).

30. Beyer, U. F. and R. L. Houtman, Ann. N.Y:Acad.

S c i . , 130, 539 (1965).

31. S p i n g l e r , H. and F. K a i s e r , A r z n e i r n i t t e l - F o r s c h ,
6 , 760 (1956).
I

32. F o r i s t , A.A., W. L. M i l l e r , J r . , J. Krake, W. A .

S t r u c k , Proc. SOC. E x p t l . B i o l . Med., 96, 180
(1957).

33. Bladh, E . and A. Norden, Ac t a . Pharrnacol. T o x i c o l .

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14, 188 (1958).

34. D e l a v i l l e , G . , and M. P a l a z z o l i , Ann. B i o l . C l i n .

( P a r i s ) , l6, 481 ( 1958) .

35. Beyer, W. F. and R. L. Houtman, Ann. N.Y. Acad.

S c i . , 130, 535 (1965).

36. McDonald, H. and V. Sawins k i , Texas Rep. B i o l .

Med., l6, 479 (1958).

37. C h u l s k i , T., J. Lab. and C l i n . Med. 53, 490

( 1959) .

38. S p i n g l e r , H . , K l i n . Wochschr, -
35, 533 (1957).

39. P i g n a r d , P., Ann. B i o l . C l i n . ( P a r i s ) , l6, 4 7 1

( 1958) .

40. D or f r nuller , T . , "Das A r t z l i c h e Laboratorium"

(1957).

41. Mesnard, P. and R. C r o c k e t t , Rev. Esp. F i s i o l ,

-
16, 163 (1960).

42. R i c h t e r , D., Biochem. J., 32, 2 (1938).

 WILLIAM F. BEYER A N D ERIK H . JENSEN

43. Mesnard, P. and R , Crockett., Chim. Anal., 9,

346 (1960).

44 Mesnard, P. and R. Crockett, m, 42, 381

(1960).

45. Kern, W., Anal. Chem. 35, 50 (1963).

46. Wermuth, C. G., and P. Morand, Trav. SOC. Pharm.

Montpellier, 20, 234 (1960).

47. Jung, L. M., C. G. Wermuth, and P. Morand,

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21, 170 (1961).
x,
48. Alessandro, A., R. Emer, and G. Abbondanza,
G, Med. Mil., 116,827 (1966).

49. Kaistha, K. K. and W. N. French, J. Pharm. Sci.,

-
57, 459 (1968).

50. Sabih, K. and K. Sabih, J. Pharm. Sci., 59, 782

(1970) .
51. Simmons, D. L., R. J. Ranz, and P. Picotte,
J. Chromatog.71, 421 (1972).

52 Prescott, L. F. and D. R. Redman, J. Pharm.

Pharmac., 24, 713 (1972).

53. Beyer, W. F., Anal. Chem., 44, 1312 (1972).

54. Chakrabarti, J. K., J. Chromatog., 8 , 414 (1962).

55. Hentrich, K., Pharmazie, l8, 405 (1963).

56. Abdel-Wahab, M. F. and R. M. El-Allawy, Isoto-

penpraxis, 4, 371 (1968).

57. Krivis, A. F. and A. A. Forist, Microchem, J.

-5,553 (1961).

542
 TOLBUTAMIDE

58. Strickland, R. D., J. Chromatog., 24, 455 (1966).

59. Baumler, J. and S . Rippstein, Dt. ApothZtg. 107,

1647 (1967).

60. Gcven, K. C., S. Gecgil, and 0. Pekin, Eczacilik

BElteni, 2, 158 (1966).

61. Hutzul, M. and G. Wright, Canadian J. Pharm. S t i . ,

-
3 , 4 (1968).

62. Mistryukov, E. A,, Collection Czech. Chem.

Commun. 26, 2072 (1961).

63. Reisch, J., H. Bornfleth, and G. L. Tittel,

Pharm. Ztz Apotheker Ztg, 109, 74 (1964).

64. Neidlein, R., G. KlGgel, and U. Lebert, Pharm.

Ztg. Ver. Apotheker Ztg, 110, 651 (1965).

65. Glogner, P., H. Lange, and R, Pfab, Med. Welt, 52,

2876 (1968).

66. Kalinowski, K. and R. Korzybski, Acta Polon.

Pharm. 2,221 (1963).

67. Voicu, A., Farrnacia l0, 399 (1962).

68. McMahon, F. G., H. L. Upjohn, 0. S. Carpenter,

J. B. Wright, H. Oster, and W. E. Dulin, Current
Therapeutic Research, 4 , 330 (1962).

This monograph is based to a great extent on a preliminary

compilation of analytical information on tolbutamide by
Dr. Arlington A. Forist. His background data facilitated
the preparation of the monograph. Mrs. Betty Breseman
deserves special recognition for preparation of the manu-
script in its final form. The authors also wish t o
acknowledge the valuable secretarial support of Mrs. Mari-
lynn K. Nelson.

543
 TRIMETHAPHAN CAMSYLATE

Kenneth W. Blessel, Bruce C. Rudy, and Bernard Z. Senkowski

 KENNETH W. BLESSEL, BRUCE C. R U D Y , A N D B E R N A R D 2 . SENKOWSKI

INDEX

1. Description
1.1 Name, Formula, Molecular Weight
1.2 Appearance, Color, Odor
1.3 Isomeric Forms

2. Physical Properties
2.1 Infrared Spectrum
2.2 Nuclear Magnetic Resonance Spectrum
2.3 Ultraviolet Spectrum
2.4 Fluorescence Spectrum
2.5 Mass Spectrum
2.6 Optical Rotation
2.7 Melting Range
2.8 Differential Scanning Calorimetry
2.9 Thermogravimetric Analysis
2.10 Solubility
2.11 X-ray Crystal Properties
3. Synthesis
4. Stability Degradation
5. Drug Metabolic Products
6. Methods of Analysis
6.1 Elemental Analysis
6.2 Phase Solubility Analysis
6.3 Thin Layer Chromatographic Analysis
6.4 Direct Spectrophotometric Analysis
6.5 Colorimetric Analysis
6.6 Non-Aqueous Titration
7. Acknowledgment
8. References

546
 TRIMETHAPHAN CAMSY LATE

1. Description

1.1 Name, Formula, Molecular Weight

Trimethaphan camsylate is (+)-1.3-dibenzyldeca-
hydro-2-oxoimidazo~4,5-c]thieno [ 1,2-a] -thiolium 2-bxo-10-
bornane sulfonate.

C32H40N205S2 Molecular Weight: 596.81.

1.2 Appearance, Color, Odor
Trimethaphan camsylate is a white crystalline
powder which is odorless or has a slight odor.

1.3 Isomeric Forms

Trimethaphan camsylate has four possible isomers,
grouped in two pairs of enantiomers.

2. Physical Properties

2.1 Infrared Spectrum

The infrared spectrum of a sample of reference
standard trimethaphan camsylate is shown in Figure 1 (1).
The spectrum was recorded using a 5% w/v solution in
chloroform, utilizing a Perkin Elmer 621 Spectrophotometer
equipped with 0.1 mm NaCl liquid cells. The following
assignments of some of the bands in the spectrum have been
made which are shown in Table I below (1).

Table I

-
Band Assignment

1735 c
m
'
l C=O stretch in the camphor-
sulfonic acid moiety
1700 cm-l C=O stretch of the trimetha-
phan moiety

547
 8
In
8
8-
8
3 -
I
f
0 0
e;: 0
8:
R W
8'
v)
(u
8
0
M
8
In
M
8
U
33NWlllWSNWUl Yo
548
 TRIMETHAPHAN CAMSY LATE

1447 cm-l CH2 and CH3 deformations

701 cm-1 out-of-plane deformations o f
t h e monosubs t i t u t e d benzene
rings.

2.2 Nuclear Magnetic Resonance Spectrum (NMR)

The NMR spectrum of a sample of r e f e r e n c e stan-
dard trimethaphan camsylate i s shown i n F i g u r e 2 ( 2 ) . The
s o l v e n t used was DM!SO-d6 and t h e c o n c e n t r a t i o n of t h e
trimethaphan camsylate was 54.8 mg/0.5 m l . Due t o t h e
complex n a t u r e of t h e spectrum, a c o n s i d e r a b l e amount of
spin-spin decoupling w a s n e c e s s a r y i n o r d e r t o o b t a i n t h e
assignments shown i n Table I1 ( 2 ) . An a r b i t r a r y set of
numbers was used on t h e s t r u c t u r a l formula, shown below,
f o r ease i n p r e s e n t i n g t h e assignments.

Table I1

NMR S p e c t r a l Assignments f o r Trimethaphan Camsylate

Total
No. of Chemical Coup1i n g
Proton Protons S h i f t (ppm) Multiplicity Constant

c18 p r o t o n s 3 0.73 Singlet

C19 protons 3 1.03 Singlet
C14 and C15 4 1.10-1.60 Multiplet
pro t o n s
c16 and C20 3 -2.3 Mu1 t i p l e t
protons
C12 protons 2 -3.0 Triplet
Cg and Cll 4 4.0-4.3 2 p a i r s of J(C.:i)-16 Hz
protons 4.6-5.0 Doublets

549
550
 TRIMETHAPHAN CAMSYLATE

c1,c2 ,c3 ,C4’ 11 2.0-3.8 Two sets of

C6,C7 and C8 4.5-5.9 Multiplets

p r o tons
aromatic 10 7.34 Singlet
protons

2.3 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 of r e f e r e n c e s t a n d a r d
trimethaphan camsylate (a) is shown i n F i g u r e 3 (3). The
c o n c e n t r a t i o n w a s 1.00 mg/ml i n chloroform. The c u r v e
shows a maximum a t 258 nm (E = 3.9 x lo2) having two
s h o u l d e r s on t h e r i s i n g p o r t i o n of t h e curve. Also shown
on t h e same f i g u r e is t h e b a s e l i n e s c a n (b) and t h e u l t r a -
v i o l e t spectrum of t h e bromocresol g r e e n complex ( c ) w i t h
trimethaphan camsylate which is used f o r c o l o r i m e t r i c
determination.

2.4 Fluorescence Spectrum

TrimethaPhan camsylate (1 mg/ml i n methanol) has
extremely weak e x c i t a t i o n and e m i s s i o n s p e c t r a which are
shown i n F i g u r e 4 (4). The i n s t r u m e n t used w a s a Farrand
MK-1 r e c o r d i n g s p e c t r o f l u o r o m e t e r . E x c i t a t i o n a t 290 nm
produced an emission spectrum having a maximum a t 416 nm.

2.5 Mass Spectrum

The low r e s o l u t i o n mass spectrum of a sample of
r e f e r e n c e s t a n d a r d trimethaphan camsylate i s shown i n
F i g u r e 5 ( 5 ) . The spectrum was o b t a i n e d w i t h t h e a i d of a
CEC 21-110 mass s p e c t r o m e t e r a t an i o n i z i n g energy of
7 0 eV, i n t e r f a c e d w i t h a Varian d a t a system 100 MS. The
d a t a system a c c e p t e d t h e o u t p u t of t h e s p e c t r o m e t e r , c a l -
c u l a t e d t h e masses, compared t h e i n t e n s i t i e s t o t h a t o f
t h e b a s e peak and p l o t t e d t h e i n t e n s i t i e s as a series of
l i n e s whose h e i g h t s were p r o p o r t i o n a l t o t h e i n t e n s i t i e s .
Trimethaphan camsylate i s a thiophanium s a l t and
a s such h a s v e r y low v o l a t i l i t y , t h e r e f o r e , a h i g h l y
c h a r a c t e r i s t i c mass spectrum cannot b e expected. The
h i g h e s t mass observed by low r e s o l u t i o n was m / e 488. The
b a s e peak was observed a t m / e 9 1 , corresponding t o
C H5-CH20. A high r e s o l u t i o n s c a n showed masses up t o
m f e 578 which probably arises from t h e l o s s of H20 from
t h e molecular i o n . Other masses were observed a t m / e 548,
probably due t o t h e l o s s of SO from t h e p a r e n t mass and

551
 KENNETH W. BLESSEL, BRUCE C. RUDY, AND BERNARD Z.SENKOWSKI

Figure 3

Ultraviolet Spectra
(a) Trimethaphan Camsylate
(b) Solvent
(c) Trimethaphan Camsylate - Bromocresol Green Complex

I.c

8:
.i

C
7 I I I I I
!m x)o 350 400 450 500 5
WAVELENGTH, nm.

552
 0
0
rp
8
In
v)
a
W
+
o w
4
a
0
0
rn
0
0
N
553
t s
-I:
554
 TRIMETHAPHAN CAMSY LATE

m / e 545, due t o t h e l o s s of SH and H20 from t h e m o l e c u l a r

i o n . These peaks a r e r a t h e r p e c u l i a r i n t h a t t h e y a p p e a r
t o a r i s e from fragments c o n t a i n i n g b o t h t h e a c i d and b a s e
p o r t i o n s of t h e molecule ( 5 ) .

2.6 Optical Rotation

The v a l u e r e p o r t e d f o r t h e s p e c i f i c r o t a t i o n of
trimethaphan c a m s y l a t e i n t h e United S t a t e s Pharmacopeia
XVIII i s "not less t h a n +20° and n o t more t h a n +23' d e t e r -
mined i n a s o l u t i o n c o n t a i n i n g 400 mg i n each 10 m l " ( 6 ) .
A graph of s p e c i f i c r o t a t i o n as a f u n c t i o n of wavelength i s
a l s o p r e s e n t e d i n F i g u r e 6 ( 7 ) . The d a t a were o b t a i n e d by
c o n v e r t i n g r o t a t i o n v a l u e s from a J a s c o ORD-UV 5 i n s t r u -
ment t o s p e c i f i c r o t a t i o n . The s p e c i f i c r o t a t i o n w a s z e r o
a t 294 nm and 225 nm. It can b e s e e n t h a t t h e s p e c i f i c
r o t a t i o n changes r a p i d l y a t wavelengths below 300 nm.

2.7 M e l t i n g Range
Trimethaphan c a m s y l a t e melts w i t h decomposition
over a range of 230-235'C when a Class I a p r o c e d u r e is
used ( 6 ) .

2.8 D i f f e r e n t i a l Scanning Calorimetry (DSCl

The DSC c u r v e f o r a sample of r e f e r e n c e s t a n d a r d
trimethaphan c a m s y l a t e is shown i n F i g u r e 7 ( 8 ) . T h i s
curve was o b t a i n e d a t a s c a n r a t e of 10°C/min. i n a n i t r o -
gen atmosphere u t i l i z i n g a P e r k i n E l m e r DSC-1B. The ex-
t r a p o l a t e d o n s e t of t h e m e l t i n g endotherm, accompanied by
decomposition, i s 234.9 f. 0.loC and t h e peak is a t 238.4 2
0.4OC. A l l t e m p e r a t u r e s have been c o r r e c t e d . Because of
t h e decomposition d u r i n g t h e m e l t , t h e AHf cannot b e cal-
c u l a t e d w i t h much r e l i a b i l i t y , however, i t s v a l u e is
approximately 1 2 k c a l / m o l e ( 8 ) .

2.9 Thermogravimetric A n a l y s i s (TGA)

The TGA c u r v e f o r r e f e r e n c e s t a n d a r d t r i m e t h a p h a n
camsylate showed no weight l o s s from ambient t e m p e r a t u r e
t o 265OC a t a h e a t i n g r a t e of 10°C/min. A t about 265OC,
weight l o s s began which amounted t o approximately 70% o f
t h e weight a t 3 4 5 O C ( 8 ) .

555
 KENNETH W. BLESSEL, BRUCE C. RUDY, A N D BERNARD 2.SENKOWSKI

Figure 6

Specific Rotation vs Wavelength f o r Trlmethaphan Camsylate

+ 400 -

I -
7 1200-
-
1600
--
2000 --
-
2400
-
2800
-
-
3200 -
-
3600
-
-
4000 --
4400 --
4800
--

556
 TRIMETHAPHAN CAMSYLATE

Figure 7

DSC Curve of Trimethaphan Camsylate

I I I I I

Endothermic

\ 234.9'C

1
Exothermic

I I I I I
250 240 230 220 210
"C

557
 KENNETH W. BLESSEL. BRUCE C. RUDY, A N D BERNARD 2 . SENKOWSKI

2.10 Solubility
The s o l u b i l i t y d a t a shown i n T a b l e I11 was ob-
t a i n e d a t 25OC f o r r e f e r e n c e s t a n d a r d t r i m e t h a p h a n cam-
s y l a t e (9).

T a b l e I11

S o l u b i l i t y Data f o r Trimethaphan Camsylate

Solvent S o l u b i l i t y (rng/ml)

diethyl ether 0.01

p e t r o l e u m e t h e r (30-60') 0.05
-
2 propanol 20.15
3A a l c o h o l 89.06
chloroform >500.
95% e t h a n o l 175.80
benzene 2.59
methano 1 >500.
water >500.

2.11 Crystal Properties

The x-ray powder d i f f r a c t i o n d a t a € o r a sample
of r e f e r e n c e s t a n d a r d t r i m e t h a p h a n c a m s y l a t e is g i v e n i n
Table I V ( 1 0 ) . The o p e r a t i n g p a r a m e t e r s of t h e i n s t r u m e n t
are g i v e n below.

Instrumental Conditions

General E l e c t r i c Model XRD-6 S p e c t r o g o n i o m e t e r

Generator: 50 KV, 12-112 MA

Tube t a r g e t : Cu K, = 1 . 5 4 2 8
optics: 0.1' D e t e c t o r s l i t
M.R. S o l l e r s l i t
3' Beam s l i t
0.0007" N i f i l t e r
4' t a k e o f f a n g l e
Goniometer : Scan a t 0.2' 28 p e r m i n u t e
Detect o r : Amplifier g ain - 1 6 co u r s e,
8.7 f i n e
Sealed proportional counter
t u b e and DC v o l t a g e a t
p 1a t e a u

558
 TRIMETHAPHAN CAMSY LATE

P u l s e h e i g h t s e l e c t i o n EL-
5 volts
Eu - o u t
Rate meter T.C. 4
2000 CIS f u l l scale
Recorder: Chart Speed 1 i n c h p e r 5
mi n u t e s
Samples : P r e p a r e d by g r i n d i n g a t room
t e mp e r a t u r e .
Table I V
I n t e r p l a n a r S p a c i n g s i n Trimethaphan Camsylate from Powder
D i f f r a c t i o n Data
-
28 d (1)* I/1*** -
28 d (1)* ZL1,- **
7.04 12.5 100 26.94 3.31 16
10.94 8.09 14 28.06 3.18 18
12.00 7.38 13 28.86 3.09 14
12.26 7.22 19 29.92 2.99 25
12.52 7.07 53 30.42 2.94 2
12.76 6.94 23 31.12 2.87 4
14.14 6.26 52 31.66 2.83 4
14.84 5.97 7 32.54 2.75 8
16.22 5.46 96 34.54 2.60 10
17.54 5.06 81 35.10 2.56 6
18. 68 4.75 44 35.86 2.50 3
19.24 4.61 98 36.96 2.43 3
20.78 4.27 57 37.44 2.40 6
21.28 4. 1 8 48 37.84 2.38 4
21.94 4.05 20 38.96 2.31 4
22.34 3.98 21 39.56 2.28 3
22.82 3.90 11 40.40 2 .2 3 4
23.28 3.82 10 41.00 2.20 1
24.50 3.63 25 41.76 2.16 3
24.86 3.58 11 43.00 2.10 4
25.20 3.53 9
25.58 3.48 13
25.94 3.43 4
nX
*d = ( i n t e r p l a n a r d i s t a n c e )
2 Sin 8
**I/ = r e l a t i v e i n t e n s i t y ( b a s e d on h i g h e s t i n t e n s i t y
I0 of 1 0 0 )

559
 K E N N E T H W. BLESSEL, BRUCE C. RUDY, A N D B E R N A R D Z . SENKOWSKI

3. Synthesis
Trimethaphan c a m s y l a t e can b e o b t a i n e d as a by-product
i n t h e s y n t h e s i s of b i o t i n . The s y n t h e t i c r o u t e s t o b i o t i n
have been r e p o r t e d i n s e v e r a l p a t e n t s (11-13).

4. S t a b i l i t y Degradation
A study of t h e s t a b i l i t y of trimethaphan camsylate was
c a r r i e d o u t by h e a t i n g t h e material i n t h e d r y form f o r
d i f f e r e n t p e r i o d s of t i m e . The s o l i d w a s d i s s o l v e d i n
d o u b l e d i s t i l l e d water a t a c o n c e n t r a t i o n of 5% and a n
estimate of s t a b i l i t y was made by n o t i n g t h e d e g r e e o f
t u r b i d i t y c a u s e d by d e c o m p o s i t i o n p r o d u c t s . It w a s ob-
s e r v e d t h a t on h e a t i n g a t 100°C f o r p e r i o d s up t o 2 h o u r s ,
o n l y a s l i g h t t u r b i d i t y was n o t e d i n t h e s o l u t i o n ( 1 4 ) .
The d e c o m p o s i t i o n of t r i m e t h a p h a n c a m s y l a t e u n d e r e x t r e m e
c o n d i t i o n s w a s a l s o s t u d i e d . I t was found t h a t r e f l u x i n g
a 5% aqueous s o l u t i o n f o r 90 h o u r s c a u s e d a p p r o x i m a t e l y
60% d e c o m p o s i t i o n , c a l c u l a t e d from t h e amount of l i b e r a t e d
f r e e acid (15).

5. Drug M e t a b o l i c P r o d u c t s
S c u r r and Wyman ( 1 9 ) r e p o r t e d i n 1954 t h a t t h e meta-
b o l i c p r o d u c t s o f t r i m e t h a p h a n c a m s y l a t e were unknown. A
s e a r c h of t h e l i t e r a t u r e from t h a t p o i n t up t o t h e p r e s e n t
d i d n o t add t o t h e c u r r e n t knowledge p e r t a i n i n g t o meta-
b o l i s m of t h e d r u g . S i n c e t h e p h y s i o l o g i c a l a c t i o n of t h e
d r u g commences and c e a s e s v e r y r a p i d l y , t h e e f f e c t o r
n a t u r e of t h e m e t a b o l i t e s i s u n c e r t a i n .

6. Methods of A n a l y s i s

6.1 Elemental Analysis

The r e s u l t s of a n e l e m e n t a l a n a l y s i s of a s a m p l e
of reference standard trimethaphan camsylate are presented
i n Table V ( 1 6 ) .
Table V
E l e m e n t a l A n a l y s i s of Trimethaphan Camsylate
E l emen t % Theory % Found
C 64.40 64.39
H 6.76 6.81
N 4.69 4.76
S 10.74 10.78

560
 TRIMETHAPHAN CAMSY LATE

6.2 Phase S o l u b i l i t y Analysis

The r e s u l t s of a phase s o l u b i l i t y a n a l y s i s a s an
i n d i c a t i o n of p u r i t y i s shown i n F i g u r e 8 f o r a r e f e r e n c e
s t a n d a r d sample o f trimethaphan camsylate ( 9 ) . The s o l v e n t
used was a c e t o n e w i t h a n e q u i l i b r a t i o n t i m e of 20 hours a t
25OC. The remainder of t h e e x p e r i m e n t a l c o n d i t i o n s and
r e s u l t s a r e shown i n F i g u r e 8.

6.3 Thin Layer Chromatography

A t h i n l a y e r chromatographic system h a s been de-
veloped f o r t h e s e p a r a t i o n of h y d r o l y s i s p r o d u c t s of tri-
methaphan camsylate from t h e p a r e n t s u b s t a n c e (17). The type
of p l a t e used w a s s i l i c a g e l G, w h i l e t h e d e v e l o p i n g solvent
.
was methanol: 10% aq. H2SO4 (90 :10) A f t e r t h e sol- t k o n t h a
ascended f o r 1 5 cm t h e p l a t e i s a i r d r i e d and sprayed w i t h
modified Dragendorff r e a g e n t . The Rf v a l u e of trimethaphan
camsylate was 0.5 w h i l e t h a t of t h e major h y d r o l y s i s pro-
duct was 0 . 7 .

6.4 Direct Spectrophotometric Analysis

Trimethaphan c a m s y l a t e , I n i n j e c t a b l e s o l u t i o n ,
can b e assayed d i r e c t l y by a UV a b s o r p t i o n procedure. This
procedure i n v o l v e s t h e d i l u t i o n of 5 m l of ampul s o l u t i o n
(50 mg/cc) t o one l i t e r . The absorbance of t h i s s o l u t i o n
i s measured a t wavelengths from 254-259 nm. The maximum
a b s o r p t i o n i n t h i s range i s used t o c a l c u l a t e t h e amount
of trimethaphan camsylate p r e s e n t i n t h e ampul by compari-
son w i t h a sample of r e f e r e n c e s t a n d a r d m a t e r i a l prepared
and measured i n a similar way (18).

6.5 C o l o r i m e t r i c Analysis
Trimethaphan camsylate can be determined c o l o r i -
m e t r i c a l l y by f o r m a t i o n of t h e bromocresol green i o n p a i r
followed by e x t r a c t i o n as d e s c r i b e d i n t h e f o l l o w i n g pro-
cedure. A volume of s o l u t i o n e q u i v a l e n t t o a b o u t
100 m g of trimethaphan camsylate is d i l u t e d t o o n e
l i t e r . A 10-ml a l i q u o t of t h i s s o l u t i o n is b u f f e r e d a t a
pH of 5 . 3 by t h e a d d i t i o n of a phosphate b u f f e r . Then
5-ml of a bromocresol green s o l u t i o n i n t h e phosphate buf-
f e r i s added, a f t e r which t h e aqueous s o l u t i o n is e x t r a c t e d
w i t h two 25-ml p o r t i o n s of chloroform. The combined
chloroform e x t r a c t s a r e d i l u t e d t o 100 m l and t h e absorb-
ance determined a t about 420 nm. The q u a n t i t y of t r i m e t h a -
phan camsylate p r e s e n t i s c a l c u l a t e d by comparison w i t h a

561
 Figure 8

5 I l l 1 ~ l l l l ~ l l l l ~ l l l l

e
z
z?!lLI 4 - -
23 0 - s n n - n "
A

k $
v)

z: 3-
PHASE SOLUBILITY ANALYSIS
-
= u Sampte : Trimethophan Camsylate
zz ' -;I 2 -
Solvent : Acetone
Slope : 0.19 Ole -
-E:
O
+ Equilibration : 2 0 hrs at 25'C
-lo
3 L Extrapolated Solubility : 3.72 m g / q
-: -
Q I
 TRIMETHAPHAN CAMSY LATE

known c o n c e n t r a t i o n of r e f e r e n c e s t a n d a r d material similar-

l y prepared and measured (6).

6.6 Non-Aqueous T i t r a t i o n
The non-aqueous t i t r a t i o n d e s c r i b e d i n t h e USP
XVIII is t h e accepted method f o r t h e a n a l y s i s of t r i m e t h a -
phan camsylate i n t h e b u l k form ( 6 ) . The sample i s d i s -
solved i n a c e t i c anhydride and t i t r a t e d w i t h 0.1N H C l O 4
u t i l i z i n g a p o t e n t i o m e t r i c end-point. Each m l of 0.1N
HC104 is e q u i v a l e n t t o 59.68 mg of trimethaphan camsylate.

7. Acknowledgment
The a u t h o r s wish t o acknowledge t h e a s s i s t a n c e of t h e
Research Records O f f i c e of Hoffmann-La Roche I n c . f o r t h e i r
l i t e r a t u r e search.

563
 KENNETH W. BLESSEL, BRUCE C. RUDY, AND BERNARD 2 . SENKOWSKI

8. References

1. Hawrylyshyn, M., Hoffmann-La Roche Inc., Personal

Communication.
2 . Johnson, J . H., Hoffmann-La Roche Inc., Personal
Communication.
3. Rubia, L. B., Hoffmann-La Roche Inc., Personal
Communication.
4 . Boatman, J., Hoffmann-La Roche Inc., Personal
Communication.
5. Benz, W., Hoffmann-La Roche Inc., Personal
Communication.
6 . The United S t a t e s Phamacopeia XVIII, p p . 755-757
(1970).
7. Toome, V., Hoffmann-La Roche Inc., Personal
Communication.
8. Moros, S., Hoffmann-La Roche Inc., Personal
Communication.
9 . MacMullan, E., Hoffmann-La Roche Inc., Personal
Communication.
1 0 * Hagel, R., Hoffmann-La Roche Inc., Personal
Communication.
11. United States Patent 2 , 4 8 9 , 2 3 2 - 2 , November, 1 9 4 9 .
1 2 . United States Patent 2 , 4 8 9 , 2 3 8 , November, 1 9 4 9 .
13. United States Patent 2 , 5 1 9 , 7 2 0 , August, 1 9 7 0 .
1 4 . Rubin, S., Hoffmann-La Roche Inc., Unpublished Data.
1 5 . Sternbach, L., Hoffmann-La Roche Inc., Unpublished
Data.
16. Scheidl, F., Hoffmann-La Roche Inc., Personal
Communication.
1 7 . Sokoloff, H., Hoffmann-La Roche Inc., Unpublished
Results .
1 8 . Keller, C., Hoffmann-La Roche Inc., Personal
Communication.
1 9 . Scurr, C. F. and Wyman, J . P., Lancet, 266, 338
(1954) .

5 64
 TROPICAMIDE

Kenneth W. Blessel, Bruce C. Rudy, and Bernard Z. Senkowski

 KENNETH W. BLESSEL, BRUCE C. RUDY, A N D BERNARD Z . SENKOWSKI

INDEX

Analytical P r o f i l e - Tropicamide
1. Description
1.1 N a m e , Formula, Molecular Weight
1.2 Appearance, Color, Odor

2. Physical Properties
2.1 I n f r a r e d Spectrum
2.2 Nuclear Magnetic Resonance Spectrum
2.3 U l t r a v i o l e t Spectrum
2.4 F l u o r e s c e n c e Spectrum
2.5 Mass Spectrum
2.6 Optical Rotation
2.7 Melting Range
2.8 D i f f e r e n t i a l Scanning Calorimetry
2.9 Thermogravimetric A n a l y s i s
2.10 S o l u b i l i t i e s
2 . 1 1 X-ray C r y s t a l P r o p e r t i e s
2 . 1 2 D i s s o c i a t i o n Constant

3. Synthesis

4. S t a b i l i t y Degradation

5. Drug Metabolic P r o d u c t s

6. Methods of Analysis
6.1 Elemental A n a l y s i s
6.2 Phase S o l u b i l i t y Analysis
6.3 Thin Layer Chromatographic Analysis
6.4 Direct Spectrophotometric A n a l y s i s
6.5 Non-Aqueous T i t r a t i o n

7. Acknowledgments

8. References

5 66
 TROPICAM I DE

1. Description
1.1 Name, Formula, Molecular Weight
Tropicamide is N-ethyl-2-phenyl-N-(4-pyridyl-
methyl)-hydracrylamide. OH

C17H20N2N2 Molecular Weight: 284.36

1.2 Appearance, Color, Odor
Tropicamide is a white crystalline odorless
powder.

2. Physical Properties

2.1 Infrared Spectrum

The infrared spectrum of a sample of reference
standard tropicamide is shown in Figure 1 (1). The
spectrum was recorded on a KBr pellet containing 0.5 mg of
tropicamide and 300 mg of KBr, using a Perkin Elmer 6 2 1
Spectrophotometer. The following assignments have been
made of the bands in Figure 1 (I).

-
Band Assignment

3396 cm-’ OH stretch

1620 cm-1 CEO stretch
1595 and 1493 cm-l Aromatic Ring Vibrations
810 cm-1 Monosubstituted Pyridine
Ring
760 and 707 cm-l Monosubstituted Benzene Ring

2.2 Nuclear Magnetic Resonance Spectrum (NMR)

The NMR spectrum of a sample of reference stan-
dard tropicamide is shown in Figure 2 (2). The sample
solution contained 62.5 mg of tropicamide per 0.5 ml o f
C D C l 3 . Due to the complex spectrum observed, extensive
spin decoupling experiments were carried out in order to
evaluate the coupling constants and chemical shifts. Con-
secutive irradiations were performed at 58.8 Hz, 6 5 . 4 Hz,
196 Hz, and 200 Hz which simplified the spectrum to the

561
5 68
569
 KENNETH W. BLESSEL, BRUCE C. RUDY, AND BERNARD Z. SENKOWSKI

e x t e n t t h a t t h e f o l l o w i n g assignments could b e made ( 2 ) .

Table I

NMR S p e c t r a l Data f o r Tropicamide*

No. o f Chemical
Each Shift Multiplicity

C5 p r o t o n s 3 0.98,1.09 (2) J [ CH3-CH2-N] =

t r i p 1e ts 7Hz
C protons 2 3.27 octet J[N-CHZ-CH~] =
4
7Hz
OH-pro ton 1 3.60 s i n g l e t (broad)
c(5 p r o t o n s 2 3.75 multiplet
C 1 and C2 p r o t o n s 3 Q3.8-4.9 multiplet
cg and Cl1 protons 2 7.05 triplet J Q - H ~= 5 ~ z
ar0rnati.c p r o t o n s
on t r o p i c 5 7.35
a c i d moiety
Cg and Cl0 p r o t o n s 2 8.50 doublet J H ~ -=H5Hz
~
*A r b i t r a r y numbers were a s s i g n e d t o t h e atoms i n t h e
s t r u c t u r e f o r ease i n p r e s e n t a t i o n of d a t a .

2.3 U l t r a v i o l e t Spectrum
The u l t r a v i o l e t spegtrum of tropicamide i n t h e
r e g i o n of 200-400 nm is shown i n F i g u r e 3- ( 3 ) . The
spectrum shows a maximum a t 254 nm ( E = 5.1 x 103) and a
minimum a t 235-237 nm. The s Q l u t i o n c o n c e n t r a t i o n was
0.025 mg/ml i n 0.1N H C 1 .
 TROPICAM I DE

Figure 3

Ultraviolet Spectrum of Tropicamide

210 250 300 350

NANOMETERS

571
 KENNETH W. BLESSEL, BRUCE C. RUDY, AND BERNARD 2 . SENKOWSKI

2.4 Fluorescence Spectrum

An e x c i t a t i o n axid emission s c a n were c a r r i e d o u t
with a methanol s o l u t i o n of r e f e r e n c e s t a n d a r d t r o p i c a m i d e .
There w a s , however, no f l u o r e s c e n c e observed (4).

2.5 Mass Spectrum

The l o w - r e s o l u t i o n mass spectrum of t r o p i c a m i d e
is shown i n F i g u r e 4 ( 5 ) . The spectrum w a s o b t a i n e d u s i n g
a CEC 21-110 s p e c t r o m e t e r w i t h a n i o n i z i n g v o l t a g e of
70 eV, which was i n t e r f a c e d w i t h a V a r i a n d a t a system 100
MS. The d a t a system a c c e p t e d t h e o u t p u t of t h e s p e c t r o -
meter, c a l c u l a t e d t h e masses, compared t h e i r i n t e n s i t i e s t o
t h e b a s e peak and p l o t t e d t h i s i n f o r m a t i o n as a series of
l i n e s whose h e i g h t s were p r o p o r t i o n a l t o t h e i n t e n s i t i e s .
The molecular i o n was measured a t m / e 284. The
b a s e peak a t m / e 254 a r i s e s due t o t h e l o s s of
CH2 = 0 from t h e molecular i o n by a McLafferty r e a r r a n g e -
ment. The i o n a t m / e 225 probably arises v i a a s k e l e t a l
rearrangement of m / e 254 l e a d i n g t o t h e loss of HCO.
Cleavage between t h e benzyl carbon and t h e carbonyl group
g i v e s r i s e t o t h e i o n a t m / e 163. The i o n a t m / e 92 is
t h e n i t r o g e n - c o n t a i n i n g tropylium c a t i o n , C6H614+ (5).

2.6 Optical Rotation

Tropicamide does n o t e x h i b i t o p t i c a l a c t i v i t y .

2.7 Melting Range

The m e l t i n g r a n g e r e p o r t e d i n t h e United S t a t e s
Pharmacopeia X V I I I f o r t r o p i c a m i d e i s 96-1OO0C when a
Class I procedure is used ( 6 ) .

2.8 D i f f e r e n t i a l Scanning C a l o r i m e t r y (DSC)

The DSC s c a n of a sample of r e f e r e n c e s t a n d a r d
t r o p i c a m i d e is shown i n F i g u r e 5 ( 7 ) . The t e m p e r a t u r e w a s
r a i s e d a t a r a t e of 10°C/min. i n an atomosphere of f l o w i n g
n i t r o g e n . A s i n g l e endotherm w a s o b s e r v e d , t h e e x t r a p -
o l a t e d o n s e t of which w a s 95.5 & 0.2OC. The peak of t h e
m e l t i n g endotherm w a s observed a t 98.6 5 0.2OC. All t e m p e r
a t u r e s are c o r r e c t e d . The v a l u e of AHf f o r t h e m e l t i n g
endotherm was c a l c u l a t e d t o b e 8.8 kcal/mole.

2.9 Thermogravimetric Analysis (TGA)

The r e s u l t s of a TGA s c a n of t r o p i c a m i d e i n d i -
c a t e d t h a t no weight loss o c c u r r e d from ambient t e m p e r a t u r e

572
 Figure 4

Mass Spectrum of Tropicamide

KENNETH W. BLESSEL, BRUCE C. RUDY,AND BERNARD 2. SENKOWSKI

Figure 5

DSC Curve f o r Tropicamide

1 I I I
AHf = 8.8 kcal / mole

I I I I
80 90 too 110 I :0
OC

574
 TROPICAM I DE

t o 15OoC. A s i n g l e w e i g h t l o s s w a s o b s e r v e d b e g i n n i n g a t
15OoC and c o n t i n u i n g t o 334OC a t which p o i n t 100% of t h e
sample w e i g h t had been l o s t ( 7 ) .

2.10 Solubility
The s o l u b i l i t y d a t a shown i n T a b l e IIwas o b t a i n e d
f o r a sample of r e f e r e n c e s t a n d a r d t r o p i c a m i d e a t a temper-
a t u r e of 25OC ( 8 ) .

T a b l e I1

S o l u b i l i t i e s of Tropicamide

Solvent S o l u b i l i t y (mg/ml)

3A a l c o h o l 235.0
benzene 25.9
chloroform >500.
95% e t h a n o l 321.5
diethyl ether 3.9
2 - propanol 112.4
methanol >500.
p e t r o l e u m e t h e r (30-60') 0.2
water 5.7

2.11 Crystal Properties

T a b l e I11 g i v e s i n t e r p l a n a r s p a c i n g s from x-ray
powder d i f f r a c t i o n d a t a f o r t r o p i c a m i d e ( 9 ) . The o p e r a t i n g
p a r a m e t e r s of t h e i n s t r u m e n t are g i v e n below.

Instrumental Conditions:

G e n e r a l E l e c t r i c Model XRD-6 S p e c t r o g o n i o m e t e r

Generator: 50 KV, 12-112 MA

Tube t a r g e t : Copper
Radiation: Cu Ka = 1.542 8
optics: 0.1' D e t e c t o r s l i t
3' B e a m s l i t
0.0007" N i f i l t e r
4O t a k e o f f a n g l e
Goniometer: Scan a t 0.2' 28 p e r m i n u t e

575
 KENNETH W. BLESSEL. BRUCE C. RUDY, AND BERNARD 2 . SENKOWSKI

Detector: Amplifier g a i n - 1 6 course,

8.7 f i n e
Sealed p r o p o r t i o n a l counter
t u b e and DC v o l t a g e a t
plateau
-
P u l s e h e i g h t s e l e c t i o n EL
5 volts
Eu - out
Rate meter T.C. 4
2000 c/s f u l l scale
Recorder: Chart speed 1 inch p e r 5
minutes
Samples : P r e p a r e d by g r i n d i n g a t room
temper a t u r e
T a b l e 111
I n t e r p l a n a r Spacings from Powder D i f f r a c t i o n Data
- 28 - - **
d* I/Io- 28
- d* - I/TV**

10.54 8.39 9 29.74 3 .OO 11

13.76 6.44 75 30.06 2.97 12
14.60 6.07 63 31.42 2.85 4
18.14 4.89 27 32.00 2.80 8
18.94 4.69 47 33.06 2.71 5
19.84 4.47 12 34.46 2.60 9
20.40 4.35 67 35.18 2.55 8
20.88 4.25 72 35.52 2.53 15
21.88 4.06 100 37.04 2.43 4
22.60 3.93 28 37.54 2.40 4
23.22 3.83 13 39.00 2.31 7
24.30 3.66 19 39.82 2.26 4
25.38 3.51 12 40.22 2.24 8
26.10 3.41 4 40.62 2.22 4
26.46 3.37 4 41.66 2.17 2
27.54 3.24 30 42.24 2.14 4
28.00 3.19 5 42.70 2.12 3
28.94 3.09 41 45 * 54 1.99 6

* d = ( i n t e r p l a n a r spacing) nX
2 Sin 8

** I/Io = r e l a t i v e i n t e n s i t y ( b a s e d o n h i g h e s t
i n t e n s i t y of 100)

576
 TROPICAMIDE

2.12 Dissociation Constant

The pKa of t r o p i c a m i d e was d e t e r m i n e d by s p e c t r o -
p h o t o m e t r i c a n a l y s i s and by a p o t e n t i o m e t r i c t i t r a t i o n .
The v a l u e o b s e r v e d w a s 5 . 2 by s p e c t r o p h o t o m e t r y and 5 . 3
by p o t e n t i o m e t r y ( 1 0 ) .

3. Synthesis
Tropicamide may b e p r e p a r e d by t h e c o n d e n s a t i o n o f
e t h y l - ( y - p i c o l y l ) -amine w i t h t r o p i c a c i d c h l o r i d e , i n the
p r e s e n c e o f b a s e , c a r r i e d o u t i n anhydrous c h l o r o f o r m (11).

4. S t a b i l i t y Degradation
A s t u d y h a s b e e n c a r r i e d o u t i n which t h e s t a b i l i t y o f
t r o p i c a m i d e i n o p t h a l m i c s o l u t i o n was d e t e r m i n e d ( 1 2 ) . A
3% s o l u t i o n of t r o p i c a m i d e i n o p t h a l m i c s o l u t i o n w a s main-
t a i n e d a t t e m p e r a t u r e s r a n g i n g from O°C t o 45OC f o r p e r i o d s
of t i m e up t o 1 2 weeks. I n order t o gain information about
p o s s i b l e breakdown p r o d u c t s of t r o p i c a m i d e , pH measurements,
t u r b i d i t y d a t a and a d i r e c t s p e c t r o p h o t o m e t r i c a s s a y was
performed a t t h e s t a r t and a f t e r 3 , 6 and 1 2 weeks. No
e v i d e n c e o f d e c o m p o s i t i o n w a s found a € t e r p e r i o d s o f up t o
1 2 weeks a t each of t h e above t e m p e r a t u r e s ( 1 2 ) .

5. Drug M e t a b o l i c P r o d u c t s
Tropicamide i s used e x c l u s i v e l y € o r o p t h a l m i c s o l u t i o n s
i n t h i s c o u n t r y , and is a p p l i e d t o p i c a l l y . F o r t h i s r e a s o n
no m e t a b o l i c s t u d i e s h a v e b e e n p u r s u e d .

6. Methods of A n a l y s i s

6.1 Elemental Analysis

The r e s u l t s o f a n elemental a n a l y s i s of a sample
of r e f e r e n c e s t a n d a r d t r o p i c a m i d e are p r e s e n t e d i n T a b l e I V
(13).

E l emen t % Theory % Found

C 71.81 71.87
H 7.09 7.13
N 9.85 9.93

6.2 Phase S o l u b i l i t y Analysis

Phase s o l u b i l i t y a n a l y s e s have been c a r r i e d o u t
f o r t r o p i c a m i d e t o e s t i m a t e t h e - p u r i t y of t h e sample. An
 KENNETH W. BLESSEL, BRUCE C. RUDY, AND BERNARD 2 . SENKOWSKI

example is shown i n F i g u r e 6 ( 8 ) where t h e s o l v e n t u s e d w a s

t o l u e n e and t h e e q u i l i b r a t i o n t i m e was 20 h o u r s a t 25%.

6.3 Thin Layer Chromatographic A n a l y s i s

A TLC s y s t e m h a s been developed which h a s proved
t o b e u s e f u l f o r a n a l y s i s of t r o p i c a m i d e . The a d s o r b a n t
f o r t h e system is s i l i c a g e l and t h e d e v e l o p i n g s o l v e n t is
ch1oroform:methanol:concentrated ammonium h y d r o x i d e
(90:10:2). The s o l v e n t f r o n t is allowed t o t r a v e l f o r
about 1 5 cm i n a p r e - s a t u r a t e d t a n k . The p l a t e is a i r dried
and t h e n s p r a y e d w i t h iodine-modified Dragendorff r e a g e n t .
The approximate Rf of t r o p i c a m i d e i n t h i s s y s t e m is 0 . 6 5
(14).

6.4 Direct S p e c t r o p h o t o m e t r i c A n a l y s i s
Tropicamide may b e a s s a y e d s p e c t r o p h o t o m e t r i c a l l y
i n opthalmic s o l u t i o n a f t e r an e x t r a c t i o n i n t o chloroform
and a back e x t r a c t i o n i n t o d i l u t e s u l f u r i c a c i d . The ab-
s o r b a n c e of t h i s s o l u t i o n is measured a t t h e wavelength of
maximum a b s o r b a n c e a t a b o u t 253 nm. The amount o f t r o p i c -
a m i d e i n t h e o p t h a l m i c s o l u t i o n i s c a l c u l a t e d by comparison
w i t h a r e f e r e n c e s t a n d a r d sample of t r o p i c a m i d e measured
i n a similar way ( 6 ) .

6.5 Non-Aqueous T i t r a t i o n
The non-aqueous t i t r a t i o n d e s c r i b e d i n t h e USP
X V I Z I i s t h e p r e f e r r e d method f o r t h e a n a l y s i s of t r o p i c a -
mide i n t h e b u l k form ( 6 ) . The sample is t i t r a t e d i n
g l a c i a l a c e t i c acid with O.1N HClO4 i n acetic acid, using
c r y s t a l v i o l e t as t h e i n d i c a t o r . One m l of 0.1N HClO4 is
e q u i v a l e n t t o 28.44 mg o f t r o p i c a m i d e .

7. Acknowledgments
The a u t h o r s wish t o acknowledge t h e S c i e n t i f i c Litera-
t u r e Department and t h e Research Records O f f i c e of
Hoffmann-La Roche I n c . f o r t h e i r a s s i s t a n c e i n t h e l i t e r a -
ture search f o r t h i s analytical profile,

578
 1 I I I I 1 1 1 1 1 I 1 I '

-
-
- n n
-
n n
#
.

-
-
PHASE SOLUBILITY ANALYSIS -
Sample : Tropicomide -
Solvent : Toluene
Slope : .150/0 -
Equilibrotian : 2 0 hrs at 25OC -
Extrapolated Solubility : 12.35 mq/g
-
-
-
-
-
-
1 1 1 1 1 1 1 1 , ) 1 1 1 1 l l l l d
0 25
 KENNETH W. BLESSEL, BRUCE C. R U D Y , A N D B E R N A R D 2.SENKOWSKI

8. References

1. Hawrylyshyn, M., Hoffmann-La Roche Inc., Personal

Communication.
2. Johnson, J. H., Hoffmann-La Roche Inc., Personal
Communication.
3. Rubia, L. B., Hoffmann-La Roche Inc., Personal
Communication.
4. Boatman, J., Hoffmann-La Roche Inc. , Personal
Communication.
5. Benz, W., Hoffmann-La Roche Inc., Personal
Communication.
6. The United States Pharmacopeia XVIII, pp. 762-763
(1970) .
7. Moros, S., Hoffmann-La Roche Inc., Personal
Communication.
8. MacMullan, E., Hoffmann-La Roche Inc., Personal
Communication.
9. Hagel, R., Hoffmann-La Roche Inc., Personal
Communication.
10. Heveran, J., Hoffmann-La Roche Inc., Unpublished
Results.
11. Hoffmann-La Roche Inc., United States Patent
2,726,245 (1955) .
12. Bollinger, A., Hoffmann-La Roche Inc., Unpublished
Data.
13. Scheidl, F., Hoffmann-La Roche Inc., Personal
Communication.
14. Sokoloff , H. , Hoffmann-La Roche Inc. , Unpublished
Results.

580
 CUMULATIVE INDEX
Italic numerals refer to Volume numbers.

Acetaminophen, 3, 1 Lavatesenol Bitartrate, 1, 149; 2, 573

Acetohexamide, I, 1; 2,573 Meperidine Hydrochloride, 1. 175
Alpha-Tocopheryl Acetate, 3, 111 Meprobamate, I, 209
Amitriptyline Hydrochloride, 3, 127 Methadone Hydrochloride, 3, 365
Ampicillin, 2. 1 Methyprylon, 2, 363
Chlorprothixene, 2, 63 Nortriptyline Hydrochloride, 1, 233; 2, 573
Chloral Hydrate, 2, 85 Oxazepam, 3, 441
Chlordiazepoxide, 1, 15 Phenazopyridine Hydrochloride, 3,465
Chlordiazepoxide Hydrochloride, 1, 39 Phenelzine Sulfate, 2, 383
Clidinium Bromide, 2, 145 Phenylephrine Hydrochloride, 3, 483
Cycloserine, I, 5 3 Potassium Phenoxymethyl Penicillin, 1, 249
Cyclothiazide, 1, 66 Primidone, 2, 409
Dexamethasone, 2, 163 Propiomazine Hydrochloride, 2, 439
Diazepam, 1. 79 Propoxyphene Hydrochloride, I , 301
Digitoxin, 3, 149 Sodium Cephalothin, 1, 319
Dioctyl Sodium Sulfosuccinate, 2, 199 Sodium Secobarbital, I, 343
Diphenhydramine Hydrochloride, 3, 173 Sulfamethoxazole, 2, 467
Echothiophate Iodide, 3, 233 Sulfisoxazole, 2, 487
Erythromycin Estolate, I, 101; 2, 573 Tolbutamide, 3, 513
Ethynodiol Diacetate, 3, 253 Triamcinolone, 1, 367; 2, 571
Fludrocortisone Acetate, 3. 281 Triamcinolone Acetonide, I, 397; 2, 571
Fluorouracil, 2, 221 Triamcinolone Diacetate, I, 423
Fluphenazine Enanthate, 2, 245 Triclobisonium Chloride, 2, 507
Fluphenazine Hydrochloride, 2, 263 Triflupromazine Hydrochloride, 2, 523
Nurazepam Hydrochloride, 3, 307 Trimethaphan Camsylate, 3, 545
Halothane, I, 119; 2, 573 Trimethobenzamide Hydrochloride, 2, 55 1
Iodipnmide, 3, 333 Tropicamide, 3, 565
Isocarboxazid, 2, 295 Vinblastine Sulfate, I, 443
Isopropamide, 2, 3 15 Vincristine Sulfate, 1, 463
Levallorphan Tartrate, 2, 339

58 1