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JapanPositiveListSystem

TestMethodforCephemsinAnimalProductsOasisHLB60mg/3cc,LC/MS

Cephazolin,Cephapirin,Cephalexin,Cephalonium,Cephoperazone&CephuroximeTestMethod(Meat,Fisheryproducts)

Substancestobeanalyzed

Agro-chemicalsubstance

Analysiscompound

Cephazolin

Cephazolin

Cephapirin

Cephapirin

Cephalexin

Cephalexin

Cephalonium

Cephalonium

Cephoperazone

Cephoperazone

Cephuroxime

Cephuroxime

Apparatus

Liquidchromatograph,massspectrometer(LC/MS)

Reagents

UsethereagentslistedinSection2oftheGeneralRulesexceptforthefollowing:Acetonitrile Usethetypemadeforliquidchromatograph

Water UsethetypemadeforliquidchromatographMethanol Usethetypemadeforliquidchromatograph

Divinylbenzene-N-vinylpyrrolidoneco-polymermini-column(60mg)

Fill60mgofDivinylbenzene-N-vinylpyrrolidoneco-polymerintopolyethylenecolumnofinternaldiameter12-13mm,orusesubstancewithequivalentseparationcharacteristics.

(OasisHLB60mg/3cc,P/N:WAT094226)

StandardCephazolinSodium Contains97%ormoreCephazolinSodiumStandardCephapirinSodium Contains90%ormoreCephapirinSodiumStandardCephalexin Contains99%ormoreCephalexinStandardCephalonium Contains98%ormoreCephalonium

StandardCephoperazoneSodiumContains95%ormoreCephoperazoneSodiumStandardCephuroximeSodium Contains99%ormoreCephuroximeSodium

Procedure

Extraction

Meat

Take5.00goftestmaterialandadd100mLofmethanol/0.2%metaphosphoricacid(3:7),thenfilterbysuction.Add20mlofmethanol/0.2%metaphosphoricacidsolution(3:7)totheresidueonthefilterpaper,andrepeattheextractionprocess.Combinethefilteredliquidsandconcentrateitto30mlinconditionbelow40°C.

Fat

Take5.00goftestmaterial.Add30mLn-hexaneand70ml0.2%metaphosphoricacid,thenhomogenize.Centrifugefor5minutesunder3,000rpmandcollectthe0.2%metaphosphoricacidsolutionlayer.

Liver,kidney,milk&otherpartsofthebody

Take5.00goftestmaterialandadd100mLmethanol/0.2%metaphosphoricacid(3:7),thenhomogenize.Add3gofdiatomiteandfilterbysuction.Add20mlofmethanol/0.2%metaphosphoricacid(3:7)totheremainingresidueonthefilterpaper,andrepeattheextractionprocess.Combinethefilteredliquidsandconcentrateitto30mlbelow40°C.

Clean-up

Add5mLmethanoland5mLwatertoconditionthedivinyl-benzene-N-vinyl-pyrrolidoneco-polymermini-column(60mg)(OasisHLB60mg/3cc,P/N:WAT094226).Discardtheeffluent.Afterloadingthesampleobtainedinextractionstep1),addwater5mL,anddiscardtheeffluent.Then,addmethanol5mLtothecolumnandcollecttheeluant.Evaporatetheeluantinconditionbelow40°C.Add1.0mLofwater/methanol(7:3)totheresiduetoreconstitute.Thiswillbecomethetestsolution.

Calibrationcurves

Dilute19mgofstandardCephaloniumin3mLwater,andaddmethanoltomakeupto100mL.Otherstandardmaterialsshouldbedilutedwith10mg/100mLmethanolsolution.Dilutewithwater/methanol(7:3)tomakeseveralconcentrationsofstandardsolutionswithin0.05-5mg/L.Then,injectintoLC/MS.Usepeaksintheresultingchromatogramstoprepareacalibrationcurveusingthepeakheightorpeakmethod.

Determination

InjecttestsolutionintoLC/MS,anddeterminethecontentsofeachanalyte,usingLC/MSorLC/MS/MSresultsandthecalibrationcurvespreparedintheabovestep5.

Confirmation

PerformLC/MSmeasurement.

Measuringconditions

LC/MS

Column:Octadecylsilanizedsilicagel(I.D.2.0-6.0mm,length100-250mm,particlesize2-5μm)Columntemperature:40°C

Mobilephase:Gradientofacetonitrileand0.01%formicacidmixture,concentration(1:19)to(1:1)over20minutes.

Majormonitoringion(m/z):

Cephazolin

atESI+

455

Cephapirin

atESI+

424

Cephalexin

atESI+

348

Cephalonium

atESI+

459

Cephoperazone

atESI+

646orESI- 644

Cephuroxime

atESI-

423

Expectedretentiontime:10min(Cephapirin)

Limitofquantitation

0.01mg/kgforeachanalysismaterial

Others

Testprocedureoutline

Extractcephazolin,cephapirin,cephalexin,cephalonium,cephoperazone&cephuroximefromsamplewithmethanol/0.2%metaphosphoricacidsolution,or0.2%metaphosphoricacid.Afterclean-upwithdivinylbenzene-N-vinyl-pyrrolidoneco-polymermini-column,analyseandcheckwithLC/MS.

Notes

ThevolumeofstandardsolutionandtestsolutiontobeinjectedinLC/MSshouldbe10μLforcolumnof3.0mminternaldiameter.Therearecaseswhenthevolumetobeinjectedisdifferentduetocolumnorapparatus.