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