EUROPEAN PHARMACOPOEIA 10.

0 Glutathione

D. To 2.0 mL of solution S (see Tests) add 0.1 mL of ASSAY

phenolphthalein solution R and 3.0 mL to 3.5 mL of 1 M Dissolve 0.130 g in 50 mL of carbon dioxide-free water R with
sodium hydroxide to change the colour of the indicator to gentle heating. Cool. Using 0.1 mL of bromothymol blue
red. Add a mixture of 3 mL of formaldehyde solution R, solution R1 as indicator, titrate with 0.1 M sodium hydroxide
3 mL of carbon dioxide-free water R and 0.1 mL of until the colour changes from yellow to blue.
phenolphthalein solution R, to which sufficient 1 M sodium 1 mL of 0.1 M sodium hydroxide is equivalent to 14.71 mg
hydroxide has been added to produce a pink colour. The of C5H9NO4.
solution is decolourised. Add 1 M sodium hydroxide until
a red colour is produced. The total volume of 1 M sodium STORAGE
hydroxide used is 4.0 mL to 4.7 mL. Protected from light.

TESTS 01/2017:1670
Solution S. Dissolve 5.00 g in 1 M hydrochloric acid with
gentle heating, and dilute to 50.0 mL with the same acid.
Appearance of solution. Solution S is clear (2.2.1) and
colourless (2.2.2, Method II). GLUTATHIONE
Specific optical rotation (2.2.7) : + 30.5 to + 32.5, determined
on solution S and calculated with reference to the dried Glutathionum
substance.
Ninhydrin-positive substances. Examine by thin-layer
chromatography (2.2.27), using a TLC silica gel plate R.
Test solution (a). Dissolve 0.10 g of the substance to be
examined in 5 mL of dilute ammonia R2 and dilute to 10 mL
with water R. C10H17N3O6S Mr 307.3
[70-18-8]
Test solution (b). Dilute 1 mL of test solution (a) to 50 mL
with water R. DEFINITION
L-γ-Glutamyl-L-cysteinylglycine.
Reference solution (a). Dissolve 10 mg of glutamic acid CRS in
water R and dilute to 50 mL with the same solvent. Fermentation product.
Content : 98.0 per cent to 101.0 per cent (dried substance).
Reference solution (b). Dilute 5 mL of test solution (b) to
20 mL with water R. CHARACTERS
Appearance : white or almost white, crystalline powder or
Reference solution (c). Dissolve 10 mg of glutamic acid CRS colourless crystals.
and 10 mg of aspartic acid CRS in water R and dilute to 25 mL
with the same solvent. Solubility : freely soluble in water, very slightly soluble in
ethanol (96 per cent) and in methylene chloride.
Apply to the plate 5 μL of each solution. Dry the plate in a
current of air for 15 min. Develop over a path of 15 cm using IDENTIFICATION
a mixture of 20 volumes of glacial acetic acid R, 20 volumes of A. Specific optical rotation (see Tests).
water R and 60 volumes of butanol R. Allow the plate to dry B. Infrared absorption spectrophotometry (2.2.24).
in air, spray with ninhydrin solution R and heat at 100-105 °C Comparison : glutathione CRS.
for 15 min. Any spot in the chromatogram obtained with test
solution (a), apart from the principal spot, is not more intense TESTS
than the spot in the chromatogram obtained with reference Solution S. Dissolve 5.0 g in distilled water R and dilute to
solution (b) (0.5 per cent). The test is not valid unless the 50 mL with the same solvent.
chromatogram obtained with reference solution (c) shows 2
clearly separated spots. Appearance of solution. Solution S is clear (2.2.1) and
colourless (2.2.2, Method II).
Chlorides (2.4.4). Dissolve 0.25 g in 3 mL of dilute nitric
acid R and dilute to 15 mL with water R. The solution, to Specific optical rotation (2.2.7) : − 17.5 to − 15.5 (dried
which 1 mL of water R is added instead of dilute nitric acid R, substance).
complies with the limit test for chlorides (200 ppm). Dissolve 1.0 g in water R and dilute to 25.0 mL with the same
solvent.
Sulfates (2.4.13). Dilute 5 mL of solution S to 15 mL with
distilled water R. The solution complies with the limit test for Related substances. Capillary electrophoresis (2.2.47).
sulfates (300 ppm). Prepare the solutions immediately before use.
Internal standard solution (a). Dissolve 0.100 g of
Ammonium (2.4.1). 50 mg complies with limit test B for
phenylalanine R in the electrolyte solution and dilute to
ammonium (200 ppm). Prepare the standard using 0.1 mL of
50.0 mL with the same solution.
ammonium standard solution (100 ppm NH4) R.
Internal standard solution (b). Dilute 10.0 mL of internal
Iron (2.4.9). In a separating funnel, dissolve 1.0 g in 10 mL of standard solution (a) to 100.0 mL with the electrolyte solution.
dilute hydrochloric acid R. Shake with 3 quantities, each of Test solution (a). Dissolve 0.200 g of the substance to be
10 mL, of methyl isobutyl ketone R1, shaking for 3 min each examined in the electrolyte solution and dilute to 10.0 mL
time. To the combined organic layers add 10 mL of water R with the same solution.
and shake for 3 min. The aqueous layer complies with the
limit test for iron (10 ppm). Test solution (b). Dissolve 0.200 g of the substance to be
examined in internal standard solution (b) and dilute to
Loss on drying (2.2.32). Not more than 0.5 per cent, 10.0 mL with the same solution.
determined on 1.000 g by drying in an oven at 105 °C. Reference solution (a). Dissolve 20.0 mg of the substance to
Sulfated ash (2.4.14). Not more than 0.1 per cent, determined be examined in internal standard solution (a) and dilute to
on 1.0 g. 10.0 mL with the same solution.

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General Notices (1) apply to all monographs and other texts 2763
Glutathione EUROPEAN PHARMACOPOEIA 10.0

Reference solution (b). Dilute 5.0 mL of reference solution (a) – impurity C : not more than 1.5 times the ratio of the area
to 50.0 mL with the electrolyte solution. of the peak due to glutathione to the area of the peak due
Reference solution (c). Dissolve 0.200 g of the substance to be to the internal standard in the electropherogram obtained
examined in 5 mL of the electrolyte solution. Add 1.0 mL of with reference solution (b) (1.5 per cent) ;
internal standard solution (a), 0.5 mL of a 2 mg/mL solution – impurity D : not more than the ratio of the area of the
of L-cysteine R (impurity B) in the electrolyte solution, 0.5 mL peak due to glutathione to the area of the peak due to the
of a 2 mg/mL solution of oxidised L-glutathione R (impurity C) internal standard in the electropherogram obtained with
in the electrolyte solution and 0.5 mL of a 2 mg/mL solution reference solution (b) (1.0 per cent) ;
of L-γ-glutamyl-L-cysteine R (impurity D) in the electrolyte – impurities A, B, E : for each impurity, not more than
solution. Dilute to 10.0 mL with the electrolyte solution. 0.5 times the ratio of the area of the peak due to glutathione
Capillary : to the area of the peak due to the internal standard in the
electropherogram obtained with reference solution (b)
– material : uncoated fused silica ; (0.5 per cent) ;
– size : length to the detector cell = 0.5 m ; total length = 0.6 m ; – any other impurity : for each impurity, not more than
Ø = 75 μm. 0.2 times the ratio of the area of the peak due to glutathione
Temperature : 25 °C. to the area of the peak due to the internal standard in the
electropherogram obtained with reference solution (b)
Electrolyte solution. Dissolve 1.50 g of anhydrous sodium (0.2 per cent) ;
dihydrogen phosphate R in 230 mL of water R and adjust to
pH 1.80 with phosphoric acid R. Dilute to 250.0 mL with – total : not more than 2.5 times the ratio of the area of the
water R. Check the pH and, if necessary, adjust with phosphoric peak due to glutathione to the area of the peak due to the
acid R or dilute sodium hydroxide solution R. internal standard in the electropherogram obtained with
reference solution (b) (2.5 per cent) ;
Detection : spectrophotometer at 200 nm. – disregard limit : 0.05 times the ratio of the area of the
Preconditioning of a new capillary : rinse the new capillary peak due to glutathione to the area of the peak due to the
before the first injection with 0.1 M hydrochloric acid at internal standard in the electropherogram obtained with
138 kPa for 20 min and with water R at 138 kPa for 10 min ; reference solution (b) (0.05 per cent).
for complete equilibration, condition the capillary with the Chlorides : maximum 200 ppm.
electrolyte solution at 350 kPa for 40 min, and subsequently at Dissolve 0.5 g in 5 mL of dilute nitric acid R and dilute to
a voltage of 20 kV for 60 min. 10 mL with the same solvent. Add 10 mL of strong hydrogen
Preconditioning of the capillary : rinse the capillary with the peroxide solution R and heat on a water-bath for 30 min. Cool
electrolyte solution at 138 kPa for 40 min. and dilute to 50 mL with water R. Add 1 mL of silver nitrate
Between-run rinsing : rinse the capillary with water R at solution R2 and mix. Allow to stand for 5 min protected from
138 kPa for 1 min, with 0.1 M sodium hydroxide at 138 kPa light. Any opalescence in the solution is not more intense than
for 2 min, with water R at 138 kPa for 1 min, with 0.1 M that in a standard prepared at the same time and in the same
hydrochloric acid at 138 kPa for 3 min and with the electrolyte manner using 2 mL of chloride standard solution (50 ppm
solution at 138 kPa for 10 min. Cl) R. Examine the tubes laterally against a black background.
Injection : test solutions (a) and (b), reference solutions (b) Sulfates (2.4.13): maximum 300 ppm.
and (c) and the electrolyte solution (blank) : under pressure Dilute 5 mL of solution S to 15 mL with distilled water R.
(3.45 kPa) for 5 s. Ammonium (2.4.1, Method B): maximum 200 ppm,
Migration : apply a voltage of 20 kV. determined on 50 mg.
Run time : 45 min. Prepare the standard using 0.1 mL of ammonium standard
solution (100 ppm NH4) R.
Relative migration with reference to the internal
standard (about 14 min) : impurity A = about 0.77 ; Iron (2.4.9) : maximum 10 ppm.
impurity B = about 1.04 ; impurity E = about 1.2 ; In a separating funnel, dissolve 1.0 g in 10 mL of dilute
impurity C = about 1.26 ; impurity D = about 1.3. hydrochloric acid R. Shake with 3 quantities, each of 10 mL, of
methyl isobutyl ketone R1, shaking for 3 min each time. To the
System suitability : combined organic layers, add 10 mL of water R and shake for
– resolution : minimum 1.5 between the peaks due to the 3 min. The aqueous layer complies with the test.
internal standard and impurity B in the electropherogram Loss on drying (2.2.32) : maximum 0.5 per cent, determined
obtained with reference solution (c) ; if necessary, increase on 1.000 g by drying in an oven at 105 °C for 3 h.
the pH with dilute sodium hydroxide solution R ;
Sulfated ash (2.4.14): maximum 0.1 per cent, determined on
– peak-to-valley ratio : minimum 2.5, where Hp = height above 1.0 g.
the baseline of the peak due to impurity D and Hv = height
above the baseline of the lowest point of the curve ASSAY
separating this peak from the peak due to glutathione in In a ground-glass-stoppered flask, dissolve 0.500 g of the
the electropherogram obtained with reference solution (c); substance to be examined and 2 g of potassium iodide R in
if necessary, lower the pH with phosphoric acid R ; 50 mL of water R. Cool the solution in iced water and add
– check that in the electropherogram obtained with test 10 mL of hydrochloric acid R1 and 20.0 mL of 0.05 M iodine.
solution (a) there is no peak with the same migration time Stopper the flask and allow to stand in the dark for 15 min.
as the internal standard (in such case correct the area of the Titrate with 0.1 M sodium thiosulfate using 1 mL of starch
phenylalanine peak). solution R, added towards the end of the titration, as indicator.
Limits : test solution (b) : Carry out a blank titration.
1 mL of 0.05 M iodine is equivalent to 30.73 mg of C10H17N3O6S.
– corrected areas : divide all the peak areas by the
corresponding migration times ; STORAGE
– correction factors : for the calculation of content, multiply Protected from light.
the ratio of time-corrected peak areas of impurity and the
internal standard by the corresponding correction factor : IMPURITIES
impurity B = 3.0 ; impurity D = 1.4 ; Specified impurities : A, B, C, D, E.

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2764 See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 10.0 Glycerol

TESTS
Solution S. Dilute 100.0 g to 200.0 mL with carbon dioxide-free
water R.
Appearance of solution. Solution S is clear (2.2.1). Dilute
A. L-cysteinylglycine, 10 mL of solution S to 25 mL with water R. The solution is
colourless (2.2.2, Method II).
Acidity or alkalinity. To 50 mL of solution S add 0.5 mL of
phenolphthalein solution R. The solution is colourless. Not
more than 0.2 mL of 0.1 M sodium hydroxide is required to
B. (2R)-2-amino-3-sulfanylpropanoic acid (cysteine), change the colour of the indicator to pink.
Refractive index (2.2.6) : 1.470 to 1.475.
Aldehydes : maximum 10 ppm.
Place 7.5 mL of solution S in a ground-glass-stoppered
flask and add 7.5 mL of water R and 1.0 mL of decolorised
pararosaniline solution R. Close the flask and allow to stand
for 1 h at a temperature of 25 ± 1 °C. The absorbance (2.2.25)
of the solution measured at 552 nm is not greater than that
of a standard prepared at the same time and in the same
manner using 7.5 mL of formaldehyde standard solution
C. bis(L-γ-glutamyl-L-cysteinylglycine) disulfide (5 ppm CH2O) R and 7.5 mL of water R. The test is not valid
(L-glutathione oxidised), unless the standard is pink.
Esters. Add 10.0 mL of 0.1 M sodium hydroxide to the final
solution obtained in the test for acidity or alkalinity. Boil
under a reflux condenser for 5 min. Cool. Add 0.5 mL of
phenolphthalein solution R and titrate with 0.1 M hydrochloric
acid. Not less than 8.0 mL of 0.1 M hydrochloric acid is
required to change the colour of the indicator.
D. L-γ-glutamyl-L-cysteine,
Impurity A and related substances. Gas chromatography
E. unknown structure (product of degradation). (2.2.28).
Test solution. Dilute 10.0 mL of solution S to 100.0 mL with
01/2019:0496 water R.
corrected 10.0 Reference solution (a). Dilute 10.0 g of glycerol R1 to 20.0 mL
with water R. Dilute 10.0 mL of the solution to 100.0 mL with
water R.
Reference solution (b). Dissolve 1.000 g of diethylene glycol R
in water R and dilute to 100.0 mL with the same solvent.
GLYCEROL Reference solution (c). Dilute 1.0 mL of reference solution (b)
to 10.0 mL with reference solution (a). Dilute 1.0 mL of this
Glycerolum solution to 20.0 mL with reference solution (a).
Reference solution (d). Mix 1.0 mL of the test solution and
5.0 mL of reference solution (b) and dilute to 100.0 mL with
water R. Dilute 1.0 mL of this solution to 10.0 mL with water R.
C 3H 8O 3 Mr 92.1 Reference solution (e). Dilute 5.0 mL of reference solution (b)
[56-81-5] to 100.0 mL with water R.
Column :
DEFINITION – size : l = 30 m, Ø = 0.53 mm ;
Propane-1,2,3-triol. – stationary phase : cyanopropyl(3)phenyl(3)methyl(94)pol-
Content : 98.0 per cent m/m to 101.0 per cent m/m (anhydrous ysiloxane R.
substance). Carrier gas : helium for chromatography R.
CHARACTERS Split ratio : 1:10.
Aspect : syrupy liquid, unctuous to the touch, colourless or Linear velocity : 38 cm/s.
almost colourless, clear, very hygroscopic. Temperature :
Solubility : miscible with water and with ethanol (96 per cent), Time Temperature
slightly soluble in acetone, practically insoluble in fatty oils (min) (°C)
and in essential oils. Column 0 100
0 - 16 100 → 220
IDENTIFICATION
16 - 20 220
First identification : A, B. Injection port 220
Second identification : A, C.
Detector 250
A. Refractive index (see Tests).
B. Infrared absorption spectrophotometry (2.2.24). Detection : flame ionisation.
Preparation : to 5 mL add 1 mL of water R and mix carefully. Injection : 0.5 μL.
Comparison : Ph. Eur. reference spectrum of glycerol (85 per Elution order : impurity A, glycerol.
cent). System suitability : reference solution (d) :
C. Relative density (2.2.5) : 1.258 to 1.268. – resolution : minimum 7.0 between the peaks due to
impurity A and glycerol.

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