DL-Methionine EUROPEAN PHARMACOPOEIA 11.

– mobile phase B : 23 g/L solution of phosphoric acid R, water acceptance criterion for other/unspecified impurities and/or
for chromatography R, acetonitrile R1 (12.5:40:47.5 V/V/V) ; by the general monograph Substances for pharmaceutical use
Time Mobile phase A Mobile phase B
(2034). It is therefore not necessary to identify these impurities
(min) (per cent V/V) (per cent V/V)
for demonstration of compliance. See also 5.10. Control of
impurities in substances for pharmaceutical use) : A, B, C, D, E.
0-6 100 0
6 - 50 100 → 0 0 → 100
50 - 60 0 100

Flow rate : 1.0 mL/min. A. (2S)-2-amino-4-[(RS)-methylsulfinyl]butanoic acid

Detection : spectrophotometer at 205 nm. (L-methionine sulfoxide),
Injection : 50 μL.
Relative retention with reference to methionine (retention
time = about 6 min) : impurity A = about 0.5.
System suitability : reference solution (b) :
– resolution : minimum 5.0 between the peaks due to B. (2S)-2-amino-4-(methylsulfonyl)butanoic acid,
impurity A and methionine.
Calculation of percentage contents :
– for each impurity, use the concentration of methionine in
reference solution (a).
Limits :
C. (2RS)-2-(acetylamino)-4-(methylsulfanyl)butanoic acid,
– unspecified impurities : for each impurity, maximum
0.10 per cent ;
– total : maximum 0.3 per cent ;
– reporting threshold : 0.05 per cent.
Chlorides : maximum 200 ppm.
Dissolve 0.5 g of the substance to be examined in 5 mL of
dilute nitric acid R and dilute to 10 mL with the same acid. D. (2R)-2-[[(2RS)-2-(acetylamino)-4-(methylsulfanyl)-
Add 10 mL of strong hydrogen peroxide solution R and heat butanoyl]amino]-4-(methylsulfanyl)butanoic acid,
on a water bath for 30 min. Cool and dilute to 50 mL with
water R. Add 1 mL of silver nitrate solution R2 and mix. Allow
to stand protected from light for 5 min. Any opalescence
in the solution is not more intense than that in a standard
prepared at the same time and in the same manner using 2 mL
of chloride standard solution (50 ppm Cl) R. Examine the tubes
laterally against a black background.
Sulfates (2.4.13) : maximum 300 ppm. E. (2S)-2-[[(2RS)-2-(acetylamino)-4-(methylsulfanyl)-
butanoyl]amino]-4-(methylsulfanyl)butanoic acid.
Dissolve 0.5 g in 3 mL of dilute hydrochloric acid R and dilute
to 15 mL with distilled water R. 01/2017:0624
Ammonium (2.4.1, Method B) : maximum 200 ppm, corrected 11.0
determined on 0.10 g.
Prepare the standard using 0.2 mL of ammonium standard
solution (100 ppm NH4) R.
Iron (2.4.9) : maximum 10 ppm.
In a separating funnel, dissolve 1.0 g in 10 mL of dilute DL-METHIONINE
hydrochloric acid R. Shake with 3 quantities, each of 10 mL, of
methyl isobutyl ketone R1, shaking for 3 min each time. To DL-Methioninum
the combined upper layers, add 10 mL of water R and shake
for 3 min. Use the lower layer.
Loss on drying (2.2.32) : maximum 0.5 per cent, determined
on 1.000 g by drying in an oven at 105 °C. C5H11NO2S Mr 149.2
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on [59-51-8]
1.0 g.
DEFINITION
ASSAY DL-Methionine contains not less than 99.0 per cent
Dissolve 0.125 g in 5 mL of anhydrous formic acid R. Add and not more than the equivalent of 101.0 per cent of
30 mL of anhydrous acetic acid R. Titrate with 0.1 M perchloric (2RS)-2-amino-4-(methylsulfanyl)butanoic acid, calculated
acid, determining the end-point potentiometrically (2.2.20). with reference to the dried substance.
1 mL of 0.1 M perchloric acid is equivalent to 14.92 mg of
CHARACTERS
C5H11NO2S.
Almost white, crystalline powder or small flakes, sparingly
STORAGE soluble in water, very slightly soluble in ethanol (96 per cent).
Protected from light. It dissolves in dilute acids and in dilute solutions of the alkali
hydroxides.
IMPURITIES It melts at about 270 °C (instantaneous method).
Other detectable impurities (the following substances would,
if present at a sufficient level, be detected by one or other of IDENTIFICATION
the tests in the monograph. They are limited by the general First identification : A, C.

3370 See the information section on general monographs (cover pages)

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EUROPEAN PHARMACOPOEIA 11.0 Methotrexate

Second identification : B, C, D. STORAGE

A. Examine by infrared absorption spectrophotometry Store protected from light.
(2.2.24), comparing with the spectrum obtained with
DL-methionine CRS. Dry the substances at 105 °C. 01/2017:0560
B. Examine the chromatograms obtained in the test for related
substances. The principal spot in the chromatogram
obtained with test solution (b) is similar in position,
colour and size to the principal spot in the chromatogram
obtained with reference solution (a). METHOTREXATE
C. Dissolve 2.50 g in 1 M hydrochloric acid and dilute to
50.0 mL with the same acid. The angle of optical rotation Methotrexatum
(2.2.7) is − 0.05° to + 0.05°.
D. Dissolve 0.1 g of the substance to be examined and 0.1 g of
glycine R in 4.5 mL of dilute sodium hydroxide solution R.
Add 1 mL of a 25 g/L solution of sodium nitroprusside R.
Heat to 40 °C for 10 min. Allow to cool and add 2 mL of a
mixture of 1 volume of phosphoric acid R and 9 volumes of
hydrochloric acid R. A deep-red colour develops.
C20H22N8O5 Mr 454.4
TESTS [59-05-2]
Solution S. Dissolve 1.0 g in carbon dioxide-free water R and DEFINITION
dilute to 50 mL with the same solvent.
(2S)-2-[[4-[[(2,4-Diaminopteridin-6-yl)methyl]methyl-
Appearance of solution. Solution S is clear (2.2.1) and amino]benzoyl]amino]pentanedioic acid.
colourless (2.2.2, Method II).
Content : 97.0 per cent to 102.0 per cent (anhydrous substance).
pH (2.2.3). The pH of solution S is 5.4 to 6.1.
CHARACTERS
Related substances. Examine by thin-layer chromatography
(2.2.27), using silica gel G R as the coating substance. Appearance : yellow or orange, crystalline, hygroscopic
powder.
Test solution (a). Dissolve 0.2 g in water R and dilute to 10 mL
with the same solvent. Solubility : practically insoluble in water, in ethanol (96 per
cent) and in methylene chloride. It dissolves in dilute
Test solution (b). Dilute 1 mL of test solution (a) to 50 mL mineral acids and in dilute solutions of alkali hydroxides and
with water R. carbonates.
Reference solution (a). Dissolve 20 mg of DL-methionine CRS
in water R and dilute to 50 mL with the same solvent. IDENTIFICATION
Infrared absorption spectrophotometry (2.2.24).
Reference solution (b). Dilute 1 mL of reference solution (a)
to 10 mL with water R. Comparison : methotrexate CRS.
Apply separately to the plate 5 μL of each solution. Develop TESTS
over a path of 10 cm using a mixture of 20 volumes of Related substances. Liquid chromatography (2.2.29).
glacial acetic acid R, 20 volumes of water R and 60 volumes
of butanol R. Allow the plate to dry in air and spray with Test solution (a). Dissolve 40.0 mg of the substance to be
ninhydrin solution R. Heat the plate at 100 °C to 105 °C for examined in a mixture of 0.5 mL of dilute ammonia R1 and
15 min. Any spot in the chromatogram obtained with test 5 mL of mobile phase A and dilute to 100.0 mL with mobile
solution (a), apart from the principal spot, is not more intense phase A.
than the spot in the chromatogram obtained with reference Test solution (b). Dissolve 25.0 mg of the substance to be
solution (b) (0.2 per cent). examined in a mixture of 0.5 mL of dilute ammonia R1 and
5 mL of mobile phase A and dilute to 50.0 mL with mobile
Chlorides. Dissolve 0.25 g in 35 mL of water R. Add 5 mL phase A. Dilute 5.0 mL of this solution to 50.0 mL with mobile
of dilute nitric acid R and 10 mL of silver nitrate solution R2. phase A.
Allow to stand protected from light for 5 min. Any opalescence Reference solution (a). Dissolve 25.0 mg of methotrexate CRS
in the solution is not more intense than that in a standard in a mixture of 0.5 mL of dilute ammonia R1 and 5 mL of
prepared at the same time in the same manner using a mixture mobile phase A and dilute to 50.0 mL with mobile phase A.
of 10 mL of chloride standard solution (5 ppm Cl) R and 25 mL Dilute 5.0 mL of this solution to 50.0 mL with mobile phase A.
of water R (200 ppm). Examine the tubes laterally against a
black background. Reference solution (b). Dilute 5.0 mL of test solution (a) to
100.0 mL with mobile phase A. Dilute 5.0 mL of this solution
Sulfates (2.4.13). Dissolve 1.0 g in 20 mL of distilled water R, to 50.0 mL with mobile phase A.
heating to 60 °C. Cool to 10 °C and filter. 15 mL of the solution Reference solution (c). Dilute 5.0 mL of reference solution (b)
complies with the limit test for sulfates (200 ppm). to 25.0 mL with mobile phase A.
Loss on drying (2.2.32). Not more than 0.5 per cent, Reference solution (d). Dissolve 5 mg of the substance to
determined on 1.000 g by drying in an oven at 105 °C. be examined, 5 mg of 4-aminofolic acid R (impurity B),
Sulfated ash (2.4.14). Not more than 0.1 per cent, determined 5 mg of methotrexate impurity C CRS, 5 mg of methotrexate
on 1.0 g. impurity D CRS and 5 mg of methotrexate impurity E CRS in a
mixture of 0.5 mL of dilute ammonia R1 and 5 mL of mobile
ASSAY phase A and dilute to 100 mL with mobile phase A.
Dissolve 0.140 g in 3 mL of anhydrous formic acid R. Reference solution (e). Dissolve 8 mg of methotrexate for
Add 30 mL of anhydrous acetic acid R. Immediately after peak identification CRS (containing impurities H and I) in a
dissolution, titrate with 0.1 M perchloric acid, determining the mixture of 0.1 mL of dilute ammonia R1 and 1 mL of mobile
end-point potentiometrically (2.2.20). phase A and dilute to 20 mL with mobile phase A.
1 mL of 0.1 M perchloric acid is equivalent to 14.92 mg of Column :
C5H11NO2S. – size : l = 0.25 m, Ø = 4.0 mm ;

General Notices (1) apply to all monographs and other texts 3371
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