欧洲药典胰蛋白酶

Trypsin EUROPEANPHARMACOPOEIA7.0 Statichead-spaceconditionswhichmaybeused: —equilibrationtemperature:80°C, —equilibrationtime:45min, —transferlinetemperature:110°C, —pressurisationtime:2min, —injectiontime:12s. Temperature: Time (min) Temperature ( ° C) Column 0 -5 40 5 - 18 40 → 200 Injection port 150 Detector 250 Detection:flameionisation. Injection:1.0mL. Thepeakduetoethyleneoxideisidentifiedbyinjecting solutionsofethyleneoxideofincreasingconcentration. Determinethecontentofethyleneoxide(ppm)inthesubstance tobeexaminedusingthefollowingexpression: A1 = areaofthepeakduetoethyleneoxideinthe chromatogramobtainedwiththetestsolution, A2 = areaofthepeakduetoethyleneoxideinthe chromatogramobtainedwiththereferencesolution, m1 = massofethyleneoxideinthereferencesolution,in micrograms, m2 = massofthesubstancetobeexaminedinthetest solution,ingrams, m3 = massofthesubstancetobeexaminedinthe referencesolution,ingrams. Limit: —ethyleneoxide:maximum1ppm. Heavy metals (2.4.8):maximum20ppm. 1.0gcomplieswithtestF.Preparethereferencesolutionusing2mLofleadstandardsolution(10ppmPb)R. Loss on drying (2.2.32):maximum5.0percent,determinedon1.000gbydryinginanovenat105°Cfor4h. Sulfated ash (2.4.14):maximum0.4percent,determinedon1.0g. ASSAY Dissolve0.200gin100.0mLofwaterR.Dilute10.0mLof thissolutionto100.0 mLwithwaterR.Dilute10.0mLto100.0mLwithwaterR.Measuretheabsorbance(2.2.25)atthe absorptionmaximumat 350nm. CalculatethepercentagecontentofC33H42O19 takingthespecificabsorbancetobe250. STORAGE Inanairtightcontainer,protectedfromlight. 01/2011:0694 TRYPSIN Trypsinum [9002-07-7] DEFINITION Trypsinisaproteolyticenzymeobtainedbytheactivation oftrypsinogenextractedfromthepancreasofmammals.Ithasanactivityofnotlessthan0.5microkatalpermilligram, calculatedwithreferencetothedriedsubstance.Insolution,ithasmaximumenzymicactivityatpH8;theactivityisreversiblyinhibitedatpH3,thepHatwhichitismoststable. PRODUCTION Theanimalsfromwhichtrypsinisderivedmustfulfilthe requirementsforthehealthofanimalssuitableforhuman consumption. Themethodofmanufactureisvalidatedtodemonstratethattheproduct,iftested,wouldcomplywiththefollowingtest. Histamine (2.6.10):notmorethan1μgofhistaminebaseper0.2microkataloftrypsinactivity. Usea10g/Lsolutionofthe substancetobeexaminedin0.0015MboratebuffersolutionpH8.0Rinactivatedbyheatingonawater-bathfor30min.Carryoutdilutionswitha9g/LsolutionofsodiumchlorideR. CHARACTERS Appearance:whiteoralmostwhite,crystallineoramorphouspowder,hygroscopicifamorphous. Solubility:sparinglysolubleinwater. IDENTIFICATION A.Dilute1mLofsolutionS(seeTests)to100mLwith waterR.Inadepressioninawhitespot-plate,mix0.1mL ofthissolutionwith0.2mLoftosylargininemethylesterhydrochloridesolutionR.Areddish-violetcolourdevelops within3min. B.Dilute0.5mLofsolutionSto5mLwithwaterR.Add0.1mLofa20g/Lsolutionoftosyl-lysyl-chloromethanehydrochlorideR.AdjusttopH7.0,shakefor2handdiluteto50mLwithwaterR.Inoneofthedepressionsofa whitespot-plate,mix0.1mLofthissolutionwith0.2mLoftosylargininemethylesterhydrochloridesolutionR.No reddish-violetcolourdevelopswithin3min. TESTS Solution S.Dissolve0.10gincarbondioxide-freewaterRanddiluteto10.0mLwiththesamesolvent. Appearanceof solution.SolutionSisnotmoreopalescentthan referencesuspensionIII(2.2.1). pH (2.2.3):3.0to6.0forsolutionS. Specific absorbance(2.2.25):13.5to16.5,determinedatthe absorptionmaximumat280nm;maximum7.0,determinedat theabsorptionminimumat250nm. Dissolve30.0mgin0.001Mhydrochloricacidanddiluteto100.0mLwiththesameacid. Chymotrypsin . Testsolution.To1.8mLofbuffersolutionpH8.0Radd7.4mL ofwaterRand0.5mLof0.2MacetyltyrosineethylesterR.Whileshakingthesolution,add0.3mLofsolutionSandstart atimer.Afterexactly5min,measurethepH(2.2.3). Referencesolution.Prepareinthesamemannerasthetest solution,replacingsolutionSby0.3mLofa0.5g/Lsolutionof chymotrypsinBRP,andmeasurethepH(2.2.3)exactly5min afteraddingthechymotrypsin. ThepHofthetestsolutionishigherthanthatofthereference solution. Losson drying (2.2.32):notmore than5.0percent,determined on0.500gbydryingat60°Catapressurenotexceeding0.67kPafor2h. Microbial contamination TAMC:acceptancecriterion104 CFU/g(2.6.12). TYMC:acceptancecriterion102 CFU/g(2.6.12). 3156 Seetheinformationsectionongeneralmonographs(coverpages) EUROPEANPHARMACOPOEIA7.0 Tryptophan AbsenceofEscherichiacoli(2.6.13). AbsenceofSalmonella(2.6.13). ASSAY Theactivityoftrypsinisdeterminedbycomparingthe rateatwhichithydrolysesbenzoylarginineethylesterhydrochlorideRwiththerateatwhichtrypsinBRPhydrolysesthesamesubstrateinthesameconditions. Apparatus.Useareactionvesselofabout30mLcapacityprovidedwith: —adevicethatwillmaintainatemperatureof25.0±0.1°C; —astirringdevice(forexample,amagneticstirrer); —alidwithholesfortheinsertionofelectrodes,thetipof aburette,atubefortheadmissionofnitrogenandtheintroductionofreagents. Anautomaticormanualtitrationdevicemaybeused.Forthelatter,theburetteisgraduatedin0.005mLandthepH meterisprovidedwithawide-rangescaleandglass-calomelorglass-silver-silverchlorideelectrodes. Testsolution.Dissolvesufficientofthesubstancetobe examinedin0.001Mhydrochloricacidanddiluteto25.0mL withthesameacidinordertoobtainasolutioncontaining approximately700nanokatalspermillilitre. Referencesolution.Dissolve25.0mgoftrypsinBRPin0.001Mhydrochloricacidanddiluteto25.0mLwiththesameacid. Storethesolutionsat0-5°C.Warm1mLofeachsolutiontoabout25°Cover15minanduse50μLofeachsolutionforeachtitration.Carryoutthetitrationinanatmosphereof nitrogen.Transfer10.0mLof0.0015MboratebuffersolutionpH8.0Rtothereactionvesseland,whilestirring,add1.0mL ofafreshlyprepared6.86g/Lsolutionofbenzoylarginine ethylesterhydrochlorideR.Whenthetemperatureissteady at25.0±0.1°C(afterabout5min)adjusttopH8.0exactly with0.1Msodiumhydroxide.Add50μLofthetestsolution andstartatimer.MaintainatpH8.0bytheadditionof0.1M sodiumhydroxide,thetipofthemicroburettebeingimmersedinthesolution;notethevolumeaddedevery30s.Followthe reactionfor8min.Calculatethevolumeof0.1Msodiumhydroxideusedpersecond.Carryoutatitrationinthesame mannerusingthereferencesolutionandcalculatethevolume of0.1Msodiumhydroxideusedpersecond. Calculatetheactivityinmicrokatalspermilligramusingthefollowingexpression: m = massofthesubstancetobeexamined,inmilligrams; m′ = massoftrypsinBRP,inmilligrams; V = volumeof0.1Msodiumhydroxideusedpersecondbythetestsolution; V′ = volumeof0.1Msodiumhydroxideusedpersecondbythereferencesolution; A = activityoftrypsinBRP,inmicrokatalspermilligram. STORAGE Inanairtightcontainer,protectedfromlight,atatemperature of2°Cto8°C. LABELLING Thelabelstates: —theactivityinmicrokatalspermilligram; —fortheamorphoussubstance,thatitishygroscopic. 01/2009:1272 corrected 7.0 TRYPTOPHAN Tryptophanum C11H12N2O2 Mr 204.2[73-22-3] DEFINITION (S)-2-Amino-3-(1H-indol-3-yl)propanoicacid. Content:98.5percentto101.0percent(driedsubstance). CHARACTERS Appearance:whiteoralmostwhite,crystallineoramorphouspowder. Solubility:sparinglysolubleinwater,slightlysolubleinethanol(96percent).Itdissolvesindilutesolutionsofmineralacids andalkalihydroxides. IDENTIFICATION Firstidentification:A,B. Secondidentification:A,C,D. A.Specificopticalrotation(seeTests). B.Infraredabsorptionspectrophotometry(2.2.24). Preparation:discs. Comparison:tryptophanCRS. C.Examinethechromatogramsobtainedinthetestfor ninhydrin-positivesubstances. Results:theprincipalspotinthechromatogramobtained withtestsolution(b)issimilarinposition,colourandsize totheprincipalspotinthechromatogramobtainedwith referencesolution(a). D.Dissolveabout20mgin10mLofwaterR.Add5mLofdimethylaminobenzaldehydesolutionR6and2mLofhydrochloricacidR1.Heatonawater-bath.Apurple-blue colourdevelops. TESTS Appearanceof solution. Thesolutionisclear(2.2.1)andnot moreintenselycolouredthanreferencesolutionBY6 (2.2.2,MethodII). Dissolve0.1gin1Mhydrochloricacidanddiluteto10mL withthesameacid. Specific optical rotation (2.2.7):-30.0to-33.0(dried substance). Dissolve0.25ginwaterR,heatingonawater-bathifnecessary, anddiluteto25.0mLwiththesamesolvent. Ninhydrin-positive substances.Thin-layerchromatography(2.2.27). Solventmixture:glacialaceticacidR,waterR(50:50V/V). Testsolution(a).Dissolve0.10gofthesubstancetobe examinedinthesolventmixtureanddiluteto10 mLwiththe solventmixture. Testsolution(b).Dilute1mLoftestsolution(a)to50mLwiththesolventmixture. Referencesolution(a).Dissolve10mgoftryptophanCRSinthesolventmixtureanddiluteto50mLwiththesolvent mixture. Referencesolution(b).Dilute5mLoftestsolution(b)to20mL withthesolventmixture. GeneralNotices(1)applytoallmonographsandothertexts 3157