Isoleucine EUROPEAN PHARMACOPOEIA 11.

Mobile phase : glacial acetic acid R, water R, butanol R

(20:20:60 V/V/V).
Application : 5 μL.
B. 2-(difluoromethoxy)-1,1,1-trifluoroethane, Development : over 2/3 of the plate.
Drying : in air.
Detection : spray with ninhydrin solution R and heat at
105 °C for 15 min.
Results : the principal spot in the chromatogram obtained
C. (2RS)-2-chloro-2-(chlorodifluoromethoxy)-1,1,1- with the test solution is similar in position, colour and size
trifluoroethane, to the principal spot in the chromatogram obtained with
the reference solution.
TESTS
Appearance of solution. The solution is clear (2.2.1) and not
D. 1,1-dichloro-1-(difluoromethoxy)-2,2,2-trifluoroethane,
more intensely coloured than reference solution BY6 (2.2.2,
Method II).
Dissolve 0.5 g in a 103 g/L solution of hydrochloric acid R and
dilute to 10 mL with the same solution.
E. 1,1-dichloro-1-(chlorodifluoromethoxy)-2,2,2- Specific optical rotation (2.2.7) : + 40.0 to + 43.0 (dried
trifluoroethane, substance).
Dissolve 1.00 g in hydrochloric acid R1 and dilute to 25.0 mL
with the same acid.
Ninhydrin-positive substances. Amino acid analysis
(2.2.56). For analysis, use Method 1.
F. propanone (acetone).
The concentrations of the test solution and the reference
solutions may be adapted according to the sensitivity of the
01/2017:0770 equipment used. The concentrations of all solutions are
adjusted so that the system suitability requirements described
in general chapter 2.2.46 are fulfilled, keeping the ratios of
concentrations between all solutions as described.
Solution A : dilute hydrochloric acid R1 or a sample preparation
ISOLEUCINE buffer suitable for the apparatus used.
Test solution. Dissolve 30.0 mg of the substance to be
Isoleucinum examined in solution A and dilute to 50.0 mL with solution A.
Reference solution (a). Dilute 1.0 mL of the test solution to
100.0 mL with solution A. Dilute 2.0 mL of this solution to
10.0 mL with solution A.
Reference solution (b). Dissolve 30.0 mg of valine R
(impurity A) in solution A and dilute to 100.0 mL with
C6H13NO2 Mr 131.2 solution A. Dilute 1.0 mL of the solution to 250.0 mL with
[73-32-5] solution A.
Reference solution (c). Dissolve 30.0 mg of proline R in
DEFINITION
solution A and dilute to 100.0 mL with solution A. Dilute
(2S,3S)-2-Amino-3-methylpentanoic acid. 1.0 mL of the solution to 250.0 mL with solution A.
Product of fermentation or of protein hydrolysis. Reference solution (d). Dissolve 30.0 mg of leucine R
Content : 98.5 per cent to 101.0 per cent (dried substance). (impurity C) in solution A and dilute to 100.0 mL with
solution A. Dilute 1.0 mL of the solution to 250.0 mL with
CHARACTERS solution A.
Appearance : white or almost white, crystalline powder or Reference solution (e). Dilute 6.0 mL of ammonium standard
flakes. solution (100 ppm NH4) R to 50.0 mL with solution A. Dilute
Solubility : sparingly soluble in water, slightly soluble in 1.0 mL of this solution to 100.0 mL with solution A.
ethanol (96 per cent). It dissolves in dilute mineral acids and Reference solution (f). Dissolve 30 mg of isoleucine R and
in dilute solutions of alkali hydroxides. 30 mg of leucine R (impurity C) in solution A and dilute to
IDENTIFICATION 50.0 mL with solution A. Dilute 1.0 mL of the solution to
200.0 mL with solution A.
First identification : A, B.
Blank solution : solution A.
Second identification : A, C.
Inject suitable, equal amounts of the test solution, blank
A. Specific optical rotation (see Tests). solution and reference solutions (a), (b), (c), (d) and (f) into
B. Infrared absorption spectrophotometry (2.2.24). the amino acid analyser. Run a program suitable for the
Comparison : isoleucine CRS. determination of physiological amino acids.
C. Thin-layer chromatography (2.2.27). System suitability : reference solution (f) :
Test solution. Dissolve 10 mg of the substance to be – resolution : minimum 1.5 between the peaks due to
examined in a 10.3 g/L solution of hydrochloric acid R and isoleucine and impurity C.
dilute to 50 mL with the same solution. Calculation of percentage contents :
Reference solution. Dissolve 10 mg of isoleucine CRS in a – for impurity A, use the concentration of impurity A in
10.3 g/L solution of hydrochloric acid R and dilute to 50 mL reference solution (b);
with the same solution. – for impurity C, use the concentration of impurity C in
Plate : TLC silica gel plate R. reference solution (d) ;

3136 See the information section on general monographs (cover pages)

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EUROPEAN PHARMACOPOEIA 11.0 Isomalt

– for any ninhydrin-positive substance detected at 570 nm,

use the concentration of isoleucine in reference solution (a) ;
– for any ninhydrin-positive substance detected at 440 nm,
use the concentration of proline in reference solution (c) ; if
a peak is above the reporting threshold at both wavelengths, A. (2S)-2-amino-3-methylbutanoic acid (valine),
use the result obtained at 570 nm for quantification.
Limits :
– impurities A and C at 570 nm : for each impurity, maximum
0.3 per cent ; B. (2R)-2-amino-3-sulfanylpropanoic acid (cysteine),
– any ninhydrin-positive substance : for each impurity,
maximum 0.2 per cent ;
– total : maximum 1.0 per cent ;
– reporting threshold : 0.05 per cent. C. (2S)-2-amino-4-methylpentanoic acid (leucine),
The thresholds indicated under Related Substances
(Table 2034.-1) in the general monograph Substances for
pharmaceutical use (2034) do not apply.
Chlorides (2.4.4): maximum 200 ppm. D. (2S)-2-amino-4-(methylsulfanyl)butanoic acid
Dissolve 0.25 g in water R and dilute to 15 mL with the same (methionine).
solvent.
Sulfates (2.4.13) : maximum 300 ppm. 07/2019:1531
Dissolve 0.5 g in 3 mL of dilute hydrochloric acid R and dilute
to 15 mL with distilled water R.
Ammonium. Amino acid analysis (2.2.56) as described in
the test for ninhydrin-positive substances with the following
modifications. ISOMALT(2)
Injection : test solution, reference solution (e) and blank
solution.
Isomaltum
Limit :
– ammonium at 570 nm : not more than the area of the
corresponding peak in the chromatogram obtained with
reference solution (e) (0.02 per cent), taking into account
the peak due to ammonium in the chromatogram obtained
with the blank solution.
Iron (2.4.9) : maximum 10 ppm.
In a separating funnel, dissolve 1.0 g in 10 mL of dilute
hydrochloric acid R. Shake with 3 quantities, each of 10 mL, of
methyl isobutyl ketone R1, shaking for 3 min each time. To the
combined organic layers add 10 mL of water R and shake for C12H24O11 Mr 344.3
3 min. Use the aqueous layer.
Loss on drying (2.2.32) : maximum 0.5 per cent, determined C12H24O11,2H2O Mr 380.3
on 1.000 g by drying in an oven at 105 °C. Anhydrous isomalt : [64519-82-0]
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on DEFINITION
1.0 g. Mixture of 6-O-α-D-glucopyranosyl-D-glucitol
(6-O-α-D-glucopyranosyl-D-sorbitol ; 1,6-GPS) and
ASSAY 1-O-α-D-glucopyranosyl-D-mannitol (1,1-GPM).
Dissolve 0.100 g in 3 mL of anhydrous formic acid R. Add Content : 98.0 per cent to 102.0 per cent for the mixture of
30 mL of anhydrous acetic acid R. Titrate with 0.1 M perchloric 1,6-GPS and 1,1-GPM and neither of the 2 components is less
acid, determining the end-point potentiometrically (2.2.20). than 3.0 per cent (anhydrous substance).
1 mL of 0.1 M perchloric acid is equivalent to 13.12 mg of
C6H13NO2. ♦CHARACTERS
Appearance : white or almost white powder or granules.
STORAGE Solubility : freely soluble in water, practically insoluble in
Protected from light. anhydrous ethanol.♦

IMPURITIES IDENTIFICATION
First identification : A.
Specified impurities : A, C.
◊Second identification : B, C.◊
Other detectable impurities (the following substances would,
if present at a sufficient level, be detected by one or other of A. Examine the chromatograms obtained in the assay.
the tests in the monograph. They are limited by the general Results : the 2 principal peaks in the chromatogram
acceptance criterion for other/unspecified impurities. It obtained with the test solution are similar in retention time
is therefore not necessary to identify these impurities for to the 2 principal peaks in the chromatogram obtained
demonstration of compliance. See also 5.10. Control of with reference solution (a).
impurities in substances for pharmaceutical use) : B, D. ◊B. Thin-layer chromatography (2.2.27).
(2) This monograph has undergone pharmacopoeial harmonisation. See chapter 5.8. Pharmacopoeial harmonisation.

General Notices (1) apply to all monographs and other texts 3137
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