蟹肉中CAP检测方法Word文件下载.docx

蟹肉中CAP检测方法Word文件下载.docx

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extractionofthelipidsintotheheptane,followedbyextractionoftheaqueousphasewithEtOAc;

dissolutionintomethanol-water;

filtration;

andseparation/detection/confirmationusingLC/MS/MS.Crabmeatwasfortifiedat0.10（1st）,0.10（2nd）,0.25,0.50,and1.0ng/g（ppb）chloramphenicol.Averageabsoluterecoverieswere53,51,67,84,and86%respectively,withRSD’salllessthan1%.Fourdaughterions（m/z152,176,194and257）weremonitoredoffofthem/z321precursorion.Determinationwasbasedonastandardcurveusingthepeakareasofthem/z152daughterion（thebasepeak）,forstandardsolutionsequivalentto0.10,0.20,0.50,and1.0ppbintissue（madewithcontrolcrabextract）.Asetofsixmatrixcontrols（unfortifiedcrabmeat）werealsoanalyzed,inwhichnochloramphenicolwasdetected.Foridentificationpurposes,theionratios（ofeachdaughterionversusthebasedaughterion）ofthefortifiedcrabversusthoseofthechloramphenicolstandards,agreedwithin10%（relative）atchloramphenicolconcentrationsof0.25-1.0ppb,butincreasedtoupto21%forthem/z257ioninonesetofthe0.10ppbfortifiedcrab.

Note:

ThisLaboratoryInformationBulletin（LIB）isatoolfortherapiddisseminationoflaboratorymethodswhichappeartowork.Itdoesnotnecessarilyreportcompletedscientificwork.UsersmustassurethemselvesbyappropriatevalidationproceduresthatLIBmethodsandtechniquesarereliableandaccuratefortheirintendeduse.Referencetoanycommercialmaterials,equipment,orprocessesdoesnotinanywayconstituteapproval,endorsement,orrecommendationbytheFoodandDrugAdministration.

Introduction

Chloramphenicol（CAP）isabroadspectrumantibioticthatwasdevelopedaround1950andithasveryeffectiveantibacterialproperties.Severalyearsofclinicalusehasproducedasignificantamountofevidencerelativetothetoxiceffectsofchloramphenicolonhumans.Thereisapotentialformisuseindomesticandinternationalmarketsbecausechloramphenicoliseffectiveinanimaltherapy,includingaquaculturespecies.Chloramphenicolresiduemaybepresentinthetissueoftreatedfoodproducinganimalstherebyposingahealthrisktosomeconsumers.Consequently,FDAdoesnotpermititsuseinfoodproducinganimals.Antibioticresiduesinfoodareaglobalpublichealthconcernbecausetheyhelptopromotetheevolutionofbacteriatobecomeresistanttomanyofourantibiotics.

AnalyticalmethodsforCAPinshrimphavebeenavailableforanumberofyearsforGC/MS.（3,4）Recently,theFDAhasdevelopedLC/MSmethodsforCAPinshrimp1andCAPincrab.（5）Althoughthelattercrabmethodisspecificforouranalyte,wefoundthattheformershrimpmethodiseasiertoperform,givesslightlybetterrecoveries,andmakesuseoftriplequadrupoletechnologywhichallowsfordaughterionidentificationandismorerobustthaniontrapmethodologies.Wehavefurtherrefinedtheshrimpmethodtomakeitmorestreamlinedandsaferbyusingmultiplecentrifugetubesinsteadofseparatoryfunnels,andbyusingheptaneinsteadofhexane（tominimizeexposuretoneurotoxicsolvents）.

ReagentsandChemicals

EthylAcetate,N-Heptane,Methanol,andAcetonitrile（HighPurity,HPLC,ResidueGrade）.

GlacialAceticAcid,AmmoniumAcetate,andSodiumChloride,ReagentGrade.

Chloramphenicol,USPReferenceStandard（LotN）.

De-ionizedWater（needstobefilteredwhenusedforHPLC）.

Diluent:

1:

1Methanol:

Watermadebymixingequalvolumesofeachsolvent.

MobilePhaseA:

10mMAmmoniumAcetateand0.1%AceticAcidinHPLCgradewater.Two

（2）litersofthissolutionismadebyplacing1.547gofammoniumacetateand2.00mLofglacialaceticacidintoa2000mLvolumetricflaskanddilutingtothemarkwithHPLCgradewater.

MobilePhaseB:

95:

5Acetonitrile:

MobilePhaseA.One

（1）literofthissolutionismadebyadding50mLofMobilePhaseAtoa1000mLvolumetricflaskanddilutingtothemarkwithacetonitrile.

4%SodiumChloride:

（4%NaCl）tomakeone

（1）literofthissolutionweigh40.0gofsodiumchloride（NaCl）intoa1000mLvolumetricflaskanddilutetothemarkwithlaboratoryde-ionizedwater.

Apparatus

Instrument:

FinniganTSQwithSurveyorHPLC.LiquidChromatograph/MassSpectrometer（LC/MS/MS）[SeeInstrumentParameters]

ChromatographicColumn:

PhenomenexLUNA5µ

mC18150x2mm

FoodProcessor:

Robot-CoupemodelR10,orequivalent.

Centrifuge:

Mustbecapableofholding50mLcentrifugetubesand3000revolutionsperminute（rpm）.

Equipment:

Wrist-ActionMechanicalShaker,VortexMixer,andNitrogenEvaporatorwithheatedwaterbath.

AspirationDevice:

Fitaboredstopperinatrapflask.Connectarmofflasktovacuumsourcewithvacuumhosing.SnuglyinsertalengthofTeflonorflexibleplastictubingintotheboreholeofthestopper.Attachadisposablepipettortiptothe“workingend”ofthetubing.Thisendisthesnoutthatisusedtosuctionoffthehexanefromtheaqueouslayer,andthetipcanbechangedbetweeneachsample.Thisdeviceallowsfordeftaspirationofthetoplayerofsolventifthesnouttipisplacedagainstthetubewallslightlyabovetheliquidsurface,foraspirationoftheverythinsolidlayerthatsometimesformsbetweenaliphaticandaqueouslayers.

CentrifugeTubes:

Fiftymilliliter（50mL）polypropylene,conical,withscrew-caps.

Syringes:

1mLpolypropyleneforfilteringextract.

SyringeFilters:

13mmx0.2µ

mPVDF（polyvinylidenefluoride）membranefilters.

VolumetricGlassware:

VariousclassApipettesandflasks.

InstrumentParameters

Minutes

Mobile

PhaseA

PhaseB

100%

0%

15

20%

80%

15.5

20.5

Chromatography

Gradient:

（seetableatright）

ApproximateRetentionTimeofCAP:

~12minutes

FlowRate:

200µ

L/minute

ColumnOven:

40°

C

AutosamplerConditions

InjectionVolume:

10µ

L

Syringeflushandwashvolume:

6mL

SampleTrayTemperature:

10°

MassSpectroscopy

Ionization:

NegativeIonElectrospray

SprayVoltage:

1.5kV

SheathGas:

N2@80psi

CapillaryTemperature:

350°

SourceOffsetVoltage:

5V

PrecursorIon（Q1）:

m/z321

CollisionGas（Q2）:

Argon@2.5milliTorr

CollisionVoltage:

26V

ProductIons（Q3）:

m/z257,m/z194,m/z176,m/z152

ElectronMultiplierVoltage:

1.27kV

SamplePreparation

Onehundredgrams（100g）ofcookedcrabmeatandtwohundredgrams（200g）ofdryicewereplacedintheRobot-Coupefoodprocessor.Thismixturewasthenprocessedtoafinepowderconsistency.Thismixtureofpowderedcrabanddryicewasthende-gassedovernightinafreezerbeforeproceeding.（Thereareacoupleofsafetyremindershere:

Themixtureshouldnotbestoredinsealedcontainers,astheevolvinggaswillbuilduppressurepresentingapossibleburstinghazard.Thesecondpointisthatdependingonthetotalamountofdryiceinvolved,anasphyxiationhazardcoulddevelopinawalk-infreezer.）Thisdry-icetechniqueisbasedontheworkofBunch,et.al.

（2）

SampleExtractionandClean-up

[SeeFigure1.LabAid,forvisualflowchartofthemethod.]Tengrams（10g）ofdegassed,frozencrabpowderwereweighedintothefirst（ofthree）fiftymilliliter（50mL）plasticcentrifugetubes.Twentymilliliters（20mL）ofethylacetate（EtOAc）wereaddedtothecentrifugetubeandcappedtightly.Thetubewasshakenvigorouslyfortenminutes,usingawrist-actionmechanicalshaker.Thetubewasthencentrifugedforfiveminutesat3000RPM,andthesupernatantwasdecantedintothesecondcentrifugetube.Another20mLportionofEtOAcwasaddedtothesample;

thetubewascappedandvigorouslyshakenforabout5minutes.Thesampletubewascentrifugedagainforfiveminutesat3000RPMandthesupernatantwasalsodecantedintothesecondcentrifugetube.Thefirstsampletubewiththecrabpelletwasthendiscarded（itisadvisabletoallowthetubetodryinafumehoodbeforeplacinginthetrash）.

Theextractinthesecondsampletubewasreducedtodrynesswithastreamofnitrogeninawaterbathat45-50°

C（oruntilalltheEtOAcisgoneandjustadropofnon-evaporatingliquidremains）.Atthistimetwomillilitersofmethanolwasaddedtothesecondsampletube,cappedandspunonthevortexmixerforabouta30seconds.Twentyfivemilliliters（25mL）of4%NaClsolutionandtwentymilliliters（20mL）ofheptanewereaddedtothesecondsampletube.Thismixturewasthenvigorouslyshaken（byhand）foraboutthirtyseconds,andthenallowedtoseparateforseveralminutes（oruntilanyemulsionbreaksup）.Thetoplayerofheptanewasremovedbyaspirationanddiscarded.Thede-fattingextractionwasthenrepeatedwithanother20mLaliquotofheptane,andthistoowasremovedanddiscarded.

Thechloramphenicolwasthenextractedfromtheaqueousphaseremaininginthesecondcentrifugetube,byaddingfifteenmilliliters（15mL）ofEtOAc,cappingtightlyandshakingvigorouslybyhandforaboutone

（1）minute.Themixturewasthenallowedtostandforseveralminutes,oruntiltheupperorganicphasewasclear.Itisimportantthatallemulsionbebrokenbefo