虾和蟹组织中CAP检测方法.docx

虾和蟹组织中CAP检测方法.docx

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虾和蟹组织中CAP检测方法

DeterminationofChloramphenicolResiduesinShrimpandCrabTissuesbyElectrosprayTripleQuadrupoleLC/MS/MS

JoeStorey,AlPfenning,SherriTurnipseed,GeneNandrea,RebeccaLee,CathyBurns,MarkMadson

DenverDistrictLaboratoryand

AnimalDrugsResearchCenter

FoodandDrugAdministration

P.O.Box25087,DenverCO80225-0087

Abstract:

ThisLIBdescribesasolidphaseextractionandcleanupprocedureforthedeterminationofchloramphenicolresiduesinshrimpandcrab.Chloramphenicolisdetected,afterchromatographicseparation,usingaThermoElectronTSQQuantumtriplequadrupolemassspectrometerwithanESIsourceoperatinginnegativeionmode.Aninternalstandardisusedtocorrectforvariabilityofextractionandinstrumentresponse.Fourproductionsofchloramphenicolandoneproductionoftheinternalstandardaremonitoredusingselectedreactionmonitoring（SRM）.Chloramphenicolcanbequantitatedandconfirmedatthe0.1ppblevel.

TheLaboratoryInformationBulletinisatoolfortherapiddisseminationoflaboratorymethods（orinformation）whichappeartowork.Itmaynotreportcompletedscientificwork.Theusermustassurehim/herselfbyappropriatevalidationproceduresthatLIBmethodsandtechniquesarereliableandaccurateforhis/herintendeduse.Referencetoanycommercialmaterials,equipment,orprocessdoesnotinanywayconstituteapproval,endorsement,orrecommendationbytheFoodandDrugAdministration.

Introduction

Recentlyithasbeenreportedthattheantibioticchloramphenicol（CAP）hasbeenfoundinseveralimportedfoodstuffsfromAsia,includingshrimp,crabandcrayfish.Initially,severalconfirmatoryanalyticalLC/MS/MSmethodsforchloramphenicolusingiontrapdetectionweredevelopedbytheDenverDistrictAnimalDrugsResearchCenter（ADRC）ofFDAtoaddressthisproblem.Specifically,LIBs4284,4287,4294,4281（1-4）describemethodsdevelopedtoqualitativelyconfirmCAPinshrimp,crayfish,crab,andhoney,respectively,at1ppborhigher.Usingthesemethodsoverthelastyear,severalFDAlaboratorieshavepositivelyconfirmedthepresenceofCAPinmanyimportsamplesoftheaforementionedfoods.Thesemethodsemploynegativeionmodewithelectrosprayionization.InthesemethodsfullscanMS2spectrawereobtainednotonlyfromtheparentCAPion（m/z=321）,butalsofromthecorrespondingm/z=323ionM+2（35Cl,37Cl）isotope.Theiontrapfullscandatagaveexcellentpositiveidentificationbutwasnotquiteassuccessfulforprecisequantitationorsensitiveenoughtodetectsub-ppbCAPlevels.ItwasrequestedthatamoresensitiveconfirmatorymethodforCAPbedevelopedforuseinthefield.

Togainthisaddedsensitivity,typicallyatriplequadrupoleLC/MS/MSoperatinginSelectedReactionMonitoringorSRMmodeisused.InSRM,insteadofmonitoringtheentiremassspectrumoftheproductionsofaparention,onlyafewselectedproductionsaremonitored.SeattleDistrictLaboratoryhasrecentlydevelopedatriplequadrupoleFinniganTSQ7000LC/MS/MSmethod（5）fortheanalysisofCAPinshrimp（LIB4290）whichseemstoworkwellwithaLOQof0.3ppbandaLODof0.08ppb.However,ournewermodeltriplequadrupoleinstrument（aTSQQuantum,alsousingnegativeionmodeESILC/MS/MS）gaveappreciabledownwarddriftinresponsetochloramphenicolaftermanyinjectionsduringarunsequence,possiblyduetoionsuppression.AnotherFDAlaboratory（verbalcommunication）hasalsoexperiencedasimilareffectoperatinginnegativeionmode.ForthisreasonwewouldnotbeabletousetheSeattlemethodwithoutaninternalstandard.

ThisLIBdescribesanadditionalmethodfortheLC/MS/MSdetectionofchloramphenicolinshrimpandcrab.DifferencesbetweenourmethodandtheSeattlemethodinclude:

1）Aninternalstandard（IS）,meta-Chloramphenicolorm-CAP,isaddedatthebeginningoftheextractioninourmethod.Theuseofaninternalstandardself-correctsforanyextractionvariabilityfromsampletosampleandshouldalsoself-correctforanydriftindetectorresponseduringarun.WiththeuseofanIS,0.1ppbCAPlevelsintissuecanbereliablyquantitated.

2）Theextractionemployedinourmethoddoesnotuseseparatoryfunnelsbutusesdisposableglasswaretominimizethepossibilityofcross-contamination.

3）AmobilephaseofonlyacetonitrileandwaterwithoutanysaltbuffersisusedinourmethodwhichshouldhelpminimizeMSmaintenance.

Overviewofmethod

TheextractionusedinthemethoddescribedinthisLIBisasimplifiedversionofthatdescribedinLIB4284.Tengramsoftissuecomposite（withaddedm-CAPinternalstandard）isextracted（3x）inammoniatedethylacetate.Thecombinedextractsareevaporatedandre-dissolvedinwater.Ahexanewashremovesfats.Theextractisthenappliedtoareverse-phaseSPEcolumnandtheCAPiselutedwithmethanol.Themethanolisevaporatedtodrynessandtheextractisreconstitutedinwater,filteredandinjectedintotheLC/MSforanalysis.Them/z321[M-H]ionforbothCAPandtheinternalstandardm-CAPisisolatedinQ1,withfiveproductionsisolatedinQ3.ConfirmationofCAPismadebycomparisonofionchromatogrampeakswiththoseofaCAPstandard.QuantitationofCAPismadebytakingtheratiooftheCAPbasepeakarea（m/z152）totheinternalstandardm-CAPbasepeakarea（m/z207）.ThisMSapproachisanadaptation（asperamanuscriptkindlyprovidedtoDenverLaboratorybyFDA’sCenterforVeterinaryMedicine）ofaCanadianmethod（6）.TheCanadianmethod’sHPLCcolumnwithamodifiedmobilephaseisusedinthisLIB.Alsowefoundthatbyaddingtheinternalstandardm-CAPatthebeginningoftheextractioninsteadofattheendincreasedthereproducibilityofthemethod.Finally,wealsousedahigherlevelofinternalstandard（0.3ppb）togiveamoreintenseresponse.

InternalStandardpreparation:

（fortificationsolutionpreparationisinitalics）.

Theinternalstandardusedism-chloramphenicol（m-CAP）,wascustomsynthesizedfortheFDA.

1.A100µg/mLstockstandardm-CAPsolutioninacetonitrile（acn）isprepared.

2.A1:

100dilutioninacnofthestockstandardgivesanintermediatestandardconcentrationof1µg/mLor1000ng/mLm-CAP.

3.ThreemLoftheintermediatestandardisdilutedto500mLwithwatertogiveam-CAPconcentrationof6ng/mLdiluentsolution（ThisdiluentsolutionisusedtopreparetheLC/MSstandards）.

4.FivemLoftheintermediatestandardisdilutedto250mLwithwatertogivethem-CAPfortificationsolutionof20ng/mL.Every10.0gsampleisfortifiedwith150µLofm-CAPfortificationsolutionfora0.3ppbIS（internalstandard）concentration—thisisequivalenttoa6ng/mLfinalvialconcentrationofm-CAP.

Chloramphenicolstdpreparation:

（preparationoffortificationsolutionsinitalics）.

ChloramphenicolstandardusedisthecurrentUSPlot.

1.A100µg/mLstockstandardCAPsolutioninacetonitrile（acn）isprepared.

2.A1:

100dilutionwithm-CAPdiluentofthestockstandardgivesanintermediatestandardconcentrationof1µg/mLor1000ng/mLCAP.

3.A1:

10dilutionoftheintermediateCAPstandardwithm-CAPdiluentgivesa100ng/mLCAPconcentrationstandardequivalenttoa5ppbCAPstandard.

4.A6:

10and4:

10dilutionofthe5ppbCAPstandardwithm-capdiluentgives3ppband2ppbstandardsrespectively.A5.00mLto25.00mLdilutionwithm-capdiluentgivesa1ppbCAPstandard.

5.A6.00,3.00,1.00,and0.00mLdilutionofthe1ppbCAPstandardto10.00mLwithm-CAPdiluentgives0.6,0.3,0.1,and0ppbCAPstandardsrespectively.The1.0,0.6,0.3and0.1ppbCAPstandardswillhaveCAPconcentrationsof20,12,6,and2ng/mLrespectively.The0.3ppbstandardcanbeusedastheCCVsolution.Asimilar,butpreparedwithadifferentCAPstocksolution,0.1ppbICVCAPstandard（butusingthesamediluentsolution）isalsoprepared.

6.PreparationofCAPfortificationsolutions.（Thesesolutionsdonothavem-capdiluentinthembutaredilutedwithwater）.Usingthe100ug/mLCAPstocksolutionabovea1:

100dilutionismadewithwater;1.00mLofthissolutionisdilutedto10mLwithwatertogiveCAPfortificationsolution#1of100ng/mLconcentration.A2:

10dilutionoffortificationsolution#1givesfortificationsolution#2of20ngCAP/mLinwater.For10.0gtissue,preparea0.1ppbspikebyadding50µLoffortificationsolution#2.Preparea0.3ppbspikebyadding30uLoffortificationsolution#1.Preparea0.6ppbspikebyadding60µLoffortificationsolution#1andsoon.

Samplepreparation

1.Placepeeled（ifapplicable）tissueinblender,andblendwithdryiceusingrobotcoupemixerwithpulsedactionuntilcontentsareuniform（7）.

2.Accuratelyweigh（tothenearesttenthofagram）10.0gofblendedmeatcompositeintoa50mLpolypropylene（P/P）centrifugetube.Fortifyasdescribedinthestandardpreparationsection.

3.Add20mLofextractionsolution（EtOAC:

NH4OH,98:

2）homogenizewithvortexeruntiltheentiremassisbrokenup（about30sec）.MixonpulsedMultitubeVortexerfor10minutes.

4.Centrifugefor7min@4000rpm,decantintoanother50mLpolypropylenecentrifugetubeandplaceinN-Evapsetat50oCtobeginevaporationundernitrogen.

5.Repeatextractionsteps3and4twomoretimes,combiningtheextracts.

6.Finishevaporatingtodrynessundernitrogen.

7.Add30mLof0.05%aceticacidinH2Otodriedextract,vortexca.1mintoloosenresiduethencontinuetovortexaminimumof5min.

8.Add10mLofhexanetothe50mLtube,andcap.Inverttubegentlyseveraltimessothatemulsionsdonotformbutensureanysolidsaredissolved,centrifuge3minat4000rpm.Aspiratewithdisposablepipetteanddiscardhexanelayer.Repeathexanede-fattingsteptwicemorewithtwoadditional5mLportionsofhexanea