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
Isomalt EUROPEAN PHARMACOPOEIA 11.0

Test solution. Dissolve 50 mg of the substance to be – temperature : 80 ± 3 °C.

examined in water R and dilute to 10 mL with the same Mobile phase : degassed water for chromatography R.
solvent. Flow rate : 0.5 mL/min.
Reference solution. Dissolve 50 mg of isomalt CRS in Detection : differential refractometer maintained at a constant
water R and dilute to 10 mL with the same solvent. temperature (e.g. 40 °C).
Plate : TLC silica gel F254 plate R. Injection : 20 μL.
Mobile phase : acetic acid R, propionic acid R, water R, ethyl Run time : 2.5 times the retention time of 1,1-GPM.
acetate R, pyridine R (5:5:10:50:50 V/V/V/V/V).
Relative retention with reference to 1,1-GPM
Application : 1 μL ; thoroughly dry the points of application (retention time = about 14 min): 1,6-GPS = about 1.2 ;
in warm air. impurity B = about 1.6 ; impurity C = about 2.0.
Development : over 1/2 of the plate. System suitability : reference solution (a) :
Drying : in a current of warm air. – resolution : minimum 2.0 between the peaks due to
Detection : dip for 3 s in a 1 g/L solution of sodium 1,1-GPM and 1,6-GPS.
periodate R and dry in a current of hot air ; dip for 3 s Limits :
in a mixture of 1 volume of acetic acid R, 1 volume of
anisaldehyde R, 5 volumes of sulfuric acid R and 90 volumes – impurities B, C : for each impurity, not more than the area
of anhydrous ethanol R ; dry in a current of hot air until of the corresponding peak in the chromatogram obtained
coloured spots become visible ; the background colour may with reference solution (b) (0.5 per cent) ;
be brightened in warm steam ; examine in daylight. – unspecified impurities : for each impurity, not more than the
area of the peak due to impurity C in the chromatogram
Results : the chromatogram obtained with the reference
solution shows 2 blue-grey spots with RF values of about obtained with reference solution (b) (0.5 per cent) ;
0.13 (1,6-GPS) and 0.16 (1,1-GPM). The chromatogram – total : not more than 4 times the area of the peak due to
obtained with the test solution shows principal spots impurity C in the chromatogram obtained with reference
similar in position and colour to the principal spots in the solution (b) (2.0 per cent);
chromatogram obtained with the reference solution. – disregard limit : 0.2 times the area of the peak due to
C. To 3 mL of a freshly prepared 100 g/L solution of impurity C in the chromatogram obtained with reference
pyrocatechol R add 6 mL of sulfuric acid R while cooling in solution (b) (0.1 per cent).
iced water. To 3 mL of the cooled mixture add 0.3 mL of Water (2.5.12) : maximum 7.0 per cent, determined on 0.300 g.
a 100 g/L solution of the substance to be examined. Heat As solvent, use a mixture of 20 mL of anhydrous methanol R
gently over a naked flame for about 30 s. A pink colour and 20 mL of formamide R1 at 50 ± 5 °C.
develops.◊
ASSAY
TESTS Liquid chromatography (2.2.29) as described in the test for
Conductivity (2.2.38) : maximum 20 μS·cm− 1. related substances with the following modification.
Dissolve 20.0 g in carbon dioxide-free water R with gentle Injection : test solution and reference solution (a).
heating (40-50 °C) and dilute to 100.0 mL with the same Calculate the percentage content of isomalt (1,1-GPM
solvent. Measure the conductivity of the solution while gently and 1,6-GPS) taking into account the assigned contents of
stirring with a magnetic stirrer. 1,1-GPM and 1,6-GPS in isomalt CRS.
Reducing sugars : maximum 0.3 per cent, expressed as LABELLING
glucose equivalent.
The label states the percentage contents of 1,1-GPM and
Dissolve 3.3 g in 10 mL of water R with gentle heating. Cool 1,6-GPS.
and add 20 mL of cupri-citric solution R and a few glass beads.
Heat so that boiling begins after 4 min and maintain boiling IMPURITIES
for 3 min. Cool rapidly and add 100 mL of a 2.4 per cent V/V Specified impurities : B, C.
solution of glacial acetic acid R and 20.0 mL of 0.025 M
Other detectable impurities (the following substances would,
iodine. With continuous shaking, add 25 mL of a mixture of
if present at a sufficient level, be detected by one or other of
6 volumes of hydrochloric acid R and 94 volumes of water R.
the tests in the monograph. They are limited by the general
When the precipitate has dissolved, titrate the excess of iodine
acceptance criterion for other/unspecified impurities. It
with 0.05 M sodium thiosulfate using 1 mL of starch solution R
is therefore not necessary to identify these impurities for
as indicator, added towards the end of the titration. Not less
demonstration of compliance. See also 5.10. Control of
than 12.8 mL of 0.05 M sodium thiosulfate is required.
impurities in substances for pharmaceutical use) : A, D.
Related substances. Liquid chromatography (2.2.29).
Test solution. Dissolve 0.200 g of the substance to be examined
in 4 mL of water R and dilute to 10.0 mL with the same solvent.
Reference solution (a). Dissolve 0.200 g of isomalt CRS in 4 mL
of water R and dilute to 10.0 mL with the same solvent.
Reference solution (b). Dissolve 10.0 mg of sorbitol CRS
(impurity C) and 10.0 mg of mannitol CRS (impurity B) in
20 mL of water R and dilute to 100.0 mL with the same solvent.
Precolumn :
– size : l = 30 mm, Ø = 4.6 mm ; A. 6-O-α-D-glucopyranosyl-β-D-arabino-hex-2-ulofuranose
– stationary phase : strong cation-exchange resin (calcium (isomaltulose),
form) R (9 μm) ;
– temperature : 80 ± 3 °C.
Column :
– size : l = 0.3 m, Ø = 7.8 mm ;
– stationary phase : strong cation-exchange resin (calcium
form) R (9 μm) ; B. D-mannitol,

3138 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 11.0 Isoniazid

Impurity E. Liquid chromatography (2.2.29). Use freshly

prepared solutions.
Solvent mixture : acetonitrile R, water R (50:50 V/V).
Solution A. To 2.0 mL of benzaldehyde R add acetonitrile R
and dilute to 100.0 mL with the same solvent. Use the solution
C. D-glucitol (D-sorbitol), within 4 h.
Test solution. Dissolve 50.0 mg of the substance to be
examined in 1.0 mL of water R, add 5.0 mL of solution A and
shake well. Wait for 45 min for completion of derivatisation
and dilute to 10.0 mL with the solvent mixture.
Reference solution. Dissolve 20.0 mg of hydrazine sulfate R
(equivalent to 4.925 mg of impurity E) in water R and dilute to
50.0 mL with the same solvent. Dilute 2.5 mL of the solution
to 100.0 mL with water R. To 1.0 mL of this solution add
2.5 mL of solution A and shake well. Wait for 45 min for
D. 1-O-α-D-glucopyranosyl-D-arabino-hex-2-ulofuranose completion of derivatisation and dilute to 25.0 mL with the
(trehalulose). solvent mixture. Dilute 7.5 mL of this solution to 10.0 mL
with the solvent mixture.
Column :
07/2018:0146 – size : l = 0.25 m, Ø = 4.6 mm ;
– stationary phase : end-capped octadecylsilyl silica gel for
chromatography R (5 μm).
Mobile phase : water for chromatography R, acetonitrile R
(40:60 V/V).
ISONIAZID Flow rate : 1.0 mL/min.
Detection : spectrophotometer at 300 nm.
Isoniazidum Injection : 10 μL.
Run time : 1.5 times the retention time of benzaldehyde azine.
Retention time : benzaldehyde azine = about 13 min.
System suitability : reference solution:
– repeatability : maximum relative standard deviation of
5.0 per cent determined on 6 injections.
C 6H 7N3O Mr 137.1
[54-85-3] Calculation of percentage content :
– for impurity E, use the concentration of hydrazine sulfate
DEFINITION in the reference solution.
Pyridine-4-carbohydrazide. Limit :
Content : 99.0 per cent to 101.0 per cent (dried substance). – impurity E : maximum 15 ppm.
CHARACTERS Related substances. Liquid chromatography (2.2.29). Use
freshly prepared solutions.
Appearance : white or almost white, crystalline powder or
colourless crystals. Test solution. Dissolve 25.0 mg of the substance to be
examined in mobile phase A and dilute to 25.0 mL with
Solubility : freely soluble in water, sparingly soluble in ethanol mobile phase A.
(96 per cent).
Reference solution (a). Dilute 1.0 mL of the test solution to
IDENTIFICATION 100.0 mL with mobile phase A. Dilute 1.0 mL of this solution
to 10.0 mL with mobile phase A.
First identification : A, B.
Reference solution (b). Dissolve 5 mg of isonicotinic acid R
Second identification : A, C. (impurity A), 5 mg of isonicotinamide R (impurity B) and
A. Melting point (2.2.14) : 170 °C to 174 °C. 5 mg of nicotinoyl hydrazide R (impurity D) in mobile phase A
B. Infrared absorption spectrophotometry (2.2.24). and dilute to 50.0 mL with mobile phase A. Dilute 1.0 mL of
the solution to 10.0 mL with mobile phase A. Dilute 1.0 mL of
Comparison : isoniazid CRS.
this solution to 10.0 mL with the test solution.
C. Dissolve 0.1 g in 2 mL of water R and add 10 mL of a warm Column :
10 g/L solution of vanillin R. Allow to stand and scratch the
wall of the test tube with a glass rod. A yellow precipitate is – size : l = 0.25 m, Ø = 4.6 mm ;
formed, which, after recrystallisation from 5 mL of ethanol – stationary phase : base-deactivated end-capped octadecylsilyl
(70 per cent V/V) R and drying at 100-105 °C, melts (2.2.14) silica gel for chromatography R (5 μm).
at 226 °C to 231 °C. Mobile phase :
TESTS – mobile phase A : mix 3 volumes of methanol R and
97 volumes of a buffer solution prepared as follows :
Solution S. Dissolve 2.5 g in carbon dioxide-free water R and dissolve 13.6 g of potassium dihydrogen phosphate R in
dilute to 50 mL with the same solvent. 950 mL of water for chromatography R, adjust to pH 6.9
Appearance of solution. Solution S is clear (2.2.1) and not with strong sodium hydroxide solution R, add 30 mg of
more intensely coloured than reference solution BY7 (2.2.2, triethanolamine R and dilute to 1000 mL with water for
Method II). chromatography R ;
pH (2.2.3): 6.0 to 8.0 for solution S. – mobile phase B : methanol R ;

General Notices (1) apply to all monographs and other texts 3139