ep8.0甲氨蝶呤

EUROPEAN PHARMACOPOEIA 8.0MethotrexateRelated substances .Examine by thin-layer chromatography (2.2.27),using silica gel G R as the coating substance.Test solution (a).Dissolve 0.2g in water R and dilute to 10mL with the same solvent.Test solution (b).Dilute 1mL of test solution (a)to 50mL with water R .Reference solution (a).Dissolve 20mg of DL -methionine CRS in water R and dilute to 50mL with the same solvent.Reference solution (b).Dilute 1mL of reference solution (a)to 10mL with water R .Apply separately to the plate 5μL of each solution.Develop over a path of 10cm using a mixture of 20volumes of glacial acetic acid R ,20volumes of water R and 60volumes of butanol R .Allow the plate to dry in air and spray with ninhydrin solution R .Heat the plate at 100°C to 105°C for 15min.Any spot in the chromatogram obtained with test solution (a),apart from the principal spot,is not more intense than the spot in the chromatogram obtained with reference solution (b)(0.2per cent).Chlorides .Dissolve 0.25g in 35mL of water R .Add 5mL of dilute nitric acid R and 10mL of silver nitrate solution R2.Allow to stand protected from light for 5min.Any opalescence in the solution is not more intense than that in a standard prepared at the same time in the same manner using a mixture of 10mL of chloride standard solution (5ppm Cl)R and 25mL of water R (200ppm).Examine the tubes laterally against a black background.Sulfates (2.4.13).Dissolve 1.0g in 20mL of distilled water R ,heating to 60°C.Cool to 10°C and filter.15mL of the solution complies with the limit test for sulfates (200ppm).Heavy metals (2.4.8).1.0g complies with test D for heavy metals (20ppm).Prepare the reference solution using 2mL of lead standard solution (10ppm Pb)R .Loss on drying (2.2.32).Not more than 0.5per cent,determined on 1.000g by drying in an oven at 105°C.Sulfated ash (2.4.14).Not more than 0.1per cent,determined on 1.0g.ASSAY Dissolve 0.140g in 3mL of anhydrous formic acid R .Add 30mL of anhydrous acetic acid R .Immediately afterdissolution,titrate with 0.1M perchloric acid ,determining the end-point potentiometrically (2.2.20).1mL of 0.1M perchloric acid is equivalent to 14.92mg of C 5H 11NO 2S.STORAGEStore protected from light.01/2009:0560corrected 7.0METHOTREXATEMethotrexatum C 20H 22N 8O 5M r 454.4[59-05-2]DEFINITION(2S )-2-[[4-[[(2,4-Diaminopteridin-6-yl)methyl]methyl-amino]benzoyl]amino]pentanedioic acid.Content :97.0per cent to 102.0per cent (anhydrous substance).CHARACTERSAppearance :yellow or orange,crystalline,hygroscopic powder.Solubility :practically insoluble in water,in ethanol (96per cent)and in methylene chloride.It dissolves in dilutemineral acids and in dilute solutions of alkali hydroxides and carbonates.IDENTIFICATIONInfrared absorption spectrophotometry (2.2.24).Comparison :methotrexate CRS .TESTSRelated substances .Liquid chromatography (2.2.29).Test solution (a).Dissolve 40.0mg of the substance to be examined in a mixture of 0.5mL of dilute ammonia R1and 5mL of mobile phase A and dilute to 100.0mL with mobile phase A.Test solution (b).Dissolve 25.0mg of the substance to be examined in a mixture of 0.5mL of dilute ammonia R1and 5mL of mobile phase A and dilute to 50.0mL with mobile phase A.Dilute 5.0mL of this solution to 50.0mL with mobile phase A.Reference solution (a).Dissolve 25.0mg of methotrexate CRS in a mixture of 0.5mL of dilute ammonia R1and 5mL of mobile phase A and dilute to 50.0mL with mobile phase A.Dilute 5.0mL of this solution to 50.0mL with mobile phase A.Reference solution (b).Dilute 5.0mL of test solution (a)to 100.0mL with mobile phase A.Dilute 5.0mL of this solution to 50.0mL with mobile phase A.Reference solution (c).Dilute 5.0mL of reference solution (b)to 25.0mL with mobile phase A.Reference solution (d).Dissolve 5mg of the substance to be examined,5mg of 4-aminofolic acid R (impurity B),5mg of methotrexate impurity C CRS ,5mg of methotrexate impurity D CRS and 5mg of methotrexate impurity E CRS in a mixture of 0.5mL of dilute ammonia R1and 5mL of mobile phase A and dilute to 100mL with mobile phase A.Reference solution (e).Dissolve 8mg of methotrexate forpeak identification CRS (containing impurities H and I)in amixture of 0.1mL of dilute ammonia R1and 1mL of mobile phase A and dilute to 20mL with mobile phase A.Column :–size :l =0.25m,Ø=4.0mm;–stationary phase :spherical end-capped octadecylsilyl silica gel for chromatography R (5μm).Mobile phase :–mobile phase A :mix 5volumes of acetonitrile for chromatography R and 95volumes of a 3.4g/L solution of anhydrous sodium dihydrogen phosphate R previouslyadjusted to pH 6.0with a 42g/L solution of sodiumhydroxide R ;–mobile phase B :mix 50volumes of acetonitrile forchromatography R and 50volumes of a 3.4g/L solutionof anhydrous sodium dihydrogen phosphate R previouslyadjusted to pH 6.0with a 42g/L solution of sodiumhydroxide R ;Time (min)Mobile phase A (per cent V/V )Mobile phase B (per cent V/V )0-1010010-20100→950→520-2895→505→5028-375050Flow rate :1.5mL/min.Detection :spectrophotometer at 280nm.Injection :20μL of test solution (a)and reference solutions (b),(c),(d)and (e).General Notices (1)apply to all monographs and other texts2735Methotrexate EUROPEAN PHARMACOPOEIA8.0Identification of impurities:use the chromatogram supplied with methotrexate for peak identification CRS and the chromatogram obtained with reference solution(e)to identify the peaks due to impurities H and I.Relative retention with reference to methotrexate (retention time=about18min):impurity B=about0.3; impurity C=about0.4;impurity E=about1.4;impurity I=about1.5;impurity H=about1.6.System suitability:–resolution:minimum2.0between the peaks due to impurities B and C and minimum1.5between the peaks due to impurity D and methotrexate,in the chromatogram obtained with reference solution(d);minimum1.5between the peaks due to impurities I and H in the chromatogram obtained with reference solution(e);if the resolutionbetween impurity D and methotrexate does not comply, increase theflow rate to meet the requirement.Limits:–correction factors:for the calculation of content, multiply the peak areas of the following impurities bythe corresponding correction factor:impurity E=0.8;impurity I=1.4;–impurity C:not more than the area of the principal peak in the chromatogram obtained with reference solution(b)(0.5per cent);–impurities B,E:for each impurity,not more than0.6times the area of the principal peak in the chromatogramobtained with reference solution(b)(0.3per cent);–impurities H,I:for each impurity,not more than twice the area of the principal peak in the chromatogram obtained with reference solution(c)(0.2per cent);–unspecified impurities:for each impurity,not more than0.5times the area of the principal peak in the chromatogramobtained with reference solution(c)(0.05per cent);–sum of impurities other than B,C and E:not more than the area of the principal peak in the chromatogram obtained with reference solution(b)(0.5per cent);–disregard limit:0.3times the area of the principal peak in the chromatogram obtained with reference solution(c)(0.03per cent).Enantiomeric purity.Liquid chromatography(2.2.29). Test solution.Dissolve20.0mg of the substance to be examined in the mobile phase and dilute to100.0mL with the mobile phase.Reference solution(a).Dilute1.0mL of the test solution to 100.0mL with the mobile phase.Reference solution(b).Dissolve4.0mg of(RS)-methotrexate R in the mobile phase and dilute to100.0mL with the mobile phase.Column:–size:l=0.15m,Ø=4.0mm;–stationary phase:bovine albumin R bound to silica gel for chromatography R(7μm)with a pore size of30nm. Mobile phase:add500mL of a7.1g/L solution of anhydrous disodium hydrogen phosphate R to600mL of a6.9g/L solution of sodium dihydrogen phosphate monohydrate R,mix,and adjust to pH6.9with dilute sodium hydroxide solution R;to 920mL of this mixture add80mL of propanol R.Flow rate:1.5mL/min.Detection:spectrophotometer at302nm.Injection:20μL.System suitability:reference solution(b):–resolution:minimum2.0between the peaks due to methotrexate and impurity F.Limit:–impurity F:not more than3times the area of the principal peak in the chromatogram obtained with referencesolution(a)(3.0per cent).Heavy metals(2.4.8):maximum50ppm.1.0g complies with test C.Prepare the reference solution using 5mL of lead standard solution(10ppm Pb)R.Water(2.5.12):maximum13.0per cent,determined on0.10g. Sulfated ash(2.4.14):maximum0.1per cent,determined on 1.0g.ASSAYLiquid chromatography(2.2.29)as described in the test for related substances with the following modification. Injection:test solution(b)and reference solution(a).Calculate the percentage content of C20H22N8O5from the declared content of methotrexate CRS.STORAGEIn an airtight container,protected from light. IMPURITIESSpecified impurities:B,C,E,F,H,I.Other detectable impurities(the following substances would, if present at a sufficient level,be detected by one or other of the tests in the monograph.They are limited by the general acceptance criterion for other/unspecified impurities and/or by the general monograph Substances for pharmaceutical use(2034).It is therefore not necessary to identify these impurities for demonstration of compliance.See also5.10. Control of impurities in substances for pharmaceutical use):A, D,G,J,K,L.A.(2,4-diaminopteridin-6-yl)methanol,B.R1=NH2,R2=R3=R4=H:(2S)-2-[[4-[[(2,4-diamino-pteridin-6-yl)methyl]amino]benzoyl]amino]pentanedioic acid(4-aminofolic acid,aminopterin),H.R1=NH2,R2=R4=CH3,R3=H:(2S)-2-[[4-[[(2,4-di-aminopteridin-6-yl)methyl]methylamino]benzoyl]ami-no]-5-methoxy-5-oxopentanoic acid(methotrexate5-methyl ester),I.R1=NH2,R2=R3=CH3,R4=H:(4S)-4-[[4-[[(2,4-diaminopteridin-6-yl)methyl]methylamino]benzoyl]-amino]-5-methoxy-5-oxopentanoic acid(methotrexate1-methyl ester),J.R1=NH2,R2=R3=R4=CH3:dimethyl(2S)-2-[[4-[[(2,4-diaminopteridin-6-yl)methyl]methylamino]benzoyl]-amino]pentanedioate(methotrexate dimethylester),C.(2S)-2-[[4-[[(2-amino-4-oxo-1,4-dihydropteridin-6-yl)-methyl]methylamino]benzoyl]amino]pentanedioic acid(N-methylfolic acid,methopterin),2736See the information section on general monographs(cover pages)EUROPEAN PHARMACOPOEIA 8.0MethylnicotinateD.4-[[(2-amino-4-oxo-1,4-dihydropteridin-6-yl)methyl]-methylamino]benzoic acid (N 10-methylpteroicacid),E.4-[[(2,4-diaminopteridin-6-yl)methyl]methylamino]-benzoic acid (4-amino-N 10-methylpteroic acid,APA),F.(2R )-2-[[4-[[(2,4-diaminopteridin-6-yl)methyl]methyl-amino]benzoyl]amino]pentanedioic acid ((R)-methotrexate),G.(2S )-2-[[4-[[4-[[(2,4-diaminopteridin-6-yl)methyl]-methylamino]benzoyl]methylamino]benzoyl]amino]-pentanedioicacid,K.R =H:(2S )-2-[(4-aminobenzoyl)amino]pentanedioic acid,L.R =CH 3:(2S )-2-[[4-(methylamino)benzoyl]amino]-pentanedioic acid.01/2008:2129METHYL NICOTINATEMethylisnicotinasC 7H 7NO 2M r 137.1[93-60-7]DEFINITIONMethyl pyridine-3-carboxylate.Content :99.0per cent to 101.0per cent (anhydrous substance).CHARACTERSAppearance :white or almost white powder.Solubility :very soluble in water,in ethanol (96per cent)and in methylene chloride.IDENTIFICATION First identification:B.Second identification:A,C,D.A.Melting point (2.2.14):40°C to 42°C.B.Infrared absorption spectrophotometry (2.2.24).Comparison :methyl nicotinate CRS .C.Thin-layer chromatography (2.2.27).Test solution .Dissolve 10mg of the substance to beexamined in methanol R and dilute to 2mL with the same solvent.Reference solution .Dissolve 10mg of methyl nicotinate CRS in methanol R and dilute to 2mL with the same solvent.Plate :TLC silica gel F 254plate R .Mobile phase :methanol R ,toluene R (10:90V/V ).Application :2μL.Development :over 2/3of the plate.Drying :in air.Detection :examine in ultraviolet light at 254nm.Results :the principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with the reference solution.D.To 0.5g add 0.1g of citric acid R and 0.2mL of acetic anhydride R .Heat cautiously for 1min.A yellow colour is produced which turns first to orange,then to red and then to violet.TESTSRelated substances .Liquid chromatography (2.2.29).Test solution .Dissolve 25mg of the substance to be examined in the mobile phase and dilute to 25.0mL with the mobile phase.Reference solution (a).Dissolve 25mg of nicotinic acid R in the mobile phase and dilute to 25.0mL with the mobile phase.To 0.5mL of this solution add 0.5mL of the test solution and dilute to 100mL with the mobile phase.Reference solution (b).Dilute 1.0mL of the test solution to 100.0mL with the mobile phase.Dilute 1.0mL of this solution to 10.0mL with the mobile phase.Column :–size :l =0.25m,Ø=4mm,–stationary phase :octadecylsilyl silica gel for chromatography R (5μm).Mobile phase :acetic acid R ,water R ,acetonitrile R (1:29:70V/V/V ).Flow rate :1mL/min.Detection :spectrophotometer at 261nm.Injection :20μL.Run time :3times the retention time of methyl nicotinate.Retention time :methyl nicotinate =about 3.3min.System suitability :reference solution (a):–resolution :minimum 2between the peaks due to impurity A and methyl nicotinate.Limits :–impurity A :not more than the area of the principal peak in the chromatogram obtained with reference solution (b)(0.1per cent),–any other impurity :for each impurity,not more than the area of the principal peak in the chromatogram obtained with reference solution (b)(0.1per cent),–total :not more than 5times the area of the principal peak in the chromatogram obtained with reference solution (b)(0.5per cent),–disregard limit :0.5times the area of the principal peak in the chromatogram obtained with reference solution (b)(0.05per cent).General Notices (1)apply to all monographs and other texts2737。