虾中CAP检测方法Word文档格式.docx

虾中CAP检测方法Word文档格式.docx

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Introduction

Chloramphenicol（CAP）wasfirstisolatedfromStreptomycesvenezuelaeinthe1940’s（1,2）,isawiderangeantibioticwhichinterfereswithproteinsynthesisofmanygram-negativeandgram-positivebacteria（3）,andhastoxiceffectsonhumans（4）.Miscellaneoustoxiceffectsareduetothedichloridecarbonalphatothecarbonylgroup;

thiscarbonreadilyundergoessubstitutionwithnucleophilessuchasthosefoundonproteins（5）.Seethefigurebelow.

Themainpotentialhumantoxicityisdepressionofredbloodcellproductioninbonemarrowleadingtoaplasticanemia（3,6）.Inspiteofitspotentialtoxicity,CAPissometimesusedattherapeuticdosesfortreatmentofseriousinfectionsinhumans;

however,ithasnotbeenpossibletoidentifyasafelevelofhumanexposuretoCAP.Becauseoftheunpredictableeffectsofdoseondifferentpatientpopulations,Federalregulationsprohibititsuseinfoodproducinganimalsandanimalfeedproducts（7）.

EventhoughuseofCAPinmeatproducinganimalsandaquacultureisbannedintheEuropeanUnion（EU）,CanadaandUnitedStates,illegaluseofCAPtotreatseafoodproductsremainsapossibilityduetoitsbroadspectrumactivity,readyavailabilityandlowcost（8）.BoththeEUandCanadahaverecentlydetectedlowlevelsofCAPinimportedshrimpfromChina,ThailandandVietnam（7,9,10）.Canada’sandEU’spresentmethodologyallowdetectionofCAPat2.5and0.3ppbrespectively（7,11）.TheofficialmethodusedbytheU.S.detectsCAPinshrimpatthe5ppblevel（7）;

however,modificationsandnewmethodsshouldlowerourdetectionlimittoatorbelow1ppb（7）.

AsofSeptember2002,therewereover24,000referencestoCAPinthescientificliteraturewith4000ofthesedealingwithdetection;

however,onlyafewofthese

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referencesdealtwithdetectionofCAPinaquaculture.AcommonapproachtotheanalysisofCAPinseafoodtissuesisfirstcleanuputilizingliquid/liquidextractionandsolidphaseextraction（SPE）followedbyderivatizationtoformvolatilederivatives,andanalysisbyGC/ElectronCaptureDetection（GC-ECD）（8,12）.MunnsGC-ECDmethodgaveadetectionlimitof1ppbinshrimp,andthelowestspikelevelusedbytheconfirminglaboratorieswas5ppb（12）.LiquidChromatography（LC）usingUVdetectionwasalsocommonlyused（13–19）whichgavedetectionandquantitationlimitsat5and10ppbrespectivelyinaquaculturetissue（13）.GCusingmassspectrometry（MS）detectionwasusedinseveralpublications（20-22）withdetectionlimitsatabout1ppb.ThereafewpapersinvolvingLC-MSseparationandsemi-quantitativedetectionofCAPinaquaculture（23–25）withadetectionlimitinshrimpat0.5ppb（24）.LC-MSmethodsforseparationanddetectionofCAPappeartohavethebestpotentialforbothlowdetectionlevelsandstrongconformationalinformation.SeveraladditionalCAPLC-MSproceduresformatricesotherthanseafoodtissuearereported（26-31）.Atleasttwoenzymelinkedimmunosorbentassay（ELISA）kitshaverecentlybeendeveloped（VeratoxandRidascreen）whichclaimtobeabletodetectCAPinseafoodtissueintheppttolowppbregion（32,33）;

however,resultsusingthesekitshavenotbeenpublished.AnadditionalaspectofshrimpsamplepreparationusingdryicewaspresentedinapublicationbyBunch,et.al.（34）.

TheLC/MS/MSmethoddescribedhereallowsforquantitativedetectionofCAPinshrimpatthesub-ppblevel,allowsforqualitativeconfirmationofthepresenceofCAPinshrimp,anddoesnotrequiretheuseofaninternalstandard.

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Chemicals,Equipment&

Conditions

Reagents

a.EthylAcetate:

HighPurity;

VWRCatalogNo.BJ099-4

b.N-Hexane:

ChromARHPLCGrade;

VWRCatalogNo.MK516706

c.Methanol:

HPLCGrade;

JTBaker

d.Acetonitrile:

e.Water:

GeneratedfromMilliporeMilli-QPlus–UltraPureWaterSystem

f.GlacialAceticAcid:

SigmaACSReagentGrade;

CatalogNo.A-0808

g.AmmoniumAcetate:

SigmaUltraGrade;

CatalogNo.A-7330

h.SodiumChloride:

SpectrumChemicalMfg.Corp;

CatalogNo.S1240

i.SodiumSulfate:

AldrichACSReagentGrade;

CatalogNo.23,931-3

j.Chloramphenicol:

USPReferenceStandard;

LotN

k.Diluent:

1:

1Methanol:

UltrapureWater

Equipment

a.LC:

ThermoQuestSurveyorMSPumpandautosampler

b.MSDetector:

ThermoQuestFinniganTSQ7000withAPI2usedin

electrospraymodewithmetalneedleoption（API-2metalneedlekit;

partno.OPTON-53003fromThermoFinnigan）.

c.Instrumentsoftware:

XcaliburHomePage,Version1.2;

SurveyorAS,Version1.2SP1;

SurveyorMSpump,Version1.2;

TSQMS,Version1.1

d.LCColumn:

PhenomenexLUNA5mC18150x2mm

e.RobotCoupemodelR10

f.Centrifugecapableofholding50mLcentrifugetubesandspinning

at3000rpm

g.Mechanicalshaker:

BurrellWristActionShaker（Armsarerotatedsotubes

areshakeninahorizontalposition.）

h.Vortexmixer:

VortexJr.MixerfromAmericanScientificProducts,catalogno.S82251-1

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EquipmentContinued

i.N-EVAP:

OrganomationN-EVAPAnalyticalEvaporator

j.SampleTubes:

50mLpolypropyleneconicalcentrifugetubes（"

BLUE

MAX"

）,sterile,30x115mm,catalogno.21008-940fromVWR

k.Micropipettors:

WheatonCalibraDigitalMicropipettors;

10-100L（catalogno.851164）,100-1000L（catalogno.851168）.AcalibrationcheckwasperformedwithDiluentmassmeasurementsbeforeuse.

l.PipetTips:

10-100L,yellow,polypropylene,catalogno.851272fromWheaton;

100-1000L,blue,polypropylene,catalogno.10020fromSorensonBioScience

m.CottonTippedApplicators:

Puritancottontippedapplicatorswithwoodenhandles（Thewoodenendisusedtoloosenthecompositeaftercentrifuging.）

n.125mLGlassSeparatoryFunnels

o.8cmGlassFunnels

p.GlassWool:

CorningFiberglass8m（Thisiswashedwithacetone

andairdriedafterbeingreceivedintoourlab.）

q.SyringeFilters:

Whatman4mmPVDFsyringefilterswithtube

tips,0.2m,catalogno.28137-512fromVWR

r.1mLDisposableSyringes:

catalogno.BD309602fromVWR

s.Snap-CapSampleTubes:

0.5mLpolypropylenetubeswithattached

cap,catalogno.1605-0099fromUSA/Scientific

t.VolumetricPipets&

Flasks:

ClassA

Standards

Calibrationandspikestandardswerepreparedin50:

50Methanol:

UltraPureWater（Diluent）fromUSPCAP.Preparationofatypicalsetofstandardsisgivenbelow.

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StandardsContinued

-37.1mgCAPdissolvedin50.0mLmethanoltoyield742,000ng/mL（#1）

（note:

thefollowingstandardsweredilutedwithDiluent）

-3.00mL#1to100.0mLtoyield22,300ng/mL（#2）

-3.00mL#2to50.0mLtoyield1,340ng/mL（#3）

-3.00mL#3to100.0mLtoyield40.ng/mL

-The40.ng/mLstandardwasuseddirectlyanddilutedtoyieldsixstandards1.0

through40ng/mL.Eachofthesixwasagaindiluted1:

1withunspikedsamplematrixtoyieldCAPconcentrationsfrom0.50to20.ng/mL;

thesewereusedforthecalibrationcurves.Theconcentrationofshrimpinthefinaldilutionofeachstandardwas10g/mL.

SpikedSamples

SpikerecoverieswereperformedatCAPconcentrationsof0.10,0.25,0.50,and1.0ppbintheshrimp.Fiverecoveriesateachlevelwererunalongwithbothareagentandasampleblank.Thespikewasaddedtotheshrimpbeforeextractioninstep（b）oftheextraction-cleanupprocedure.

LCConditions

MobilePhase:

A--0.1%aceticacida