fda三聚氰胺的检验方法

GC-MSScreenforthePresenceofMelamine

(AdaptedfromFDA/ORAForensicChemistryCenterSOPT015)

RevisedApril10,

PURPOSE:

Thisprocedureprovidesageneralguideforthesamplepreparationandanalysisofwheatglutenandpetfoodmatricesformelamineusinggaschromatography/massspectrometry.

ThisprocedureisdesignedtoscreentheTMSderivativeofamethanolextractofthesampleanditincorporatesinstrumentalparameterswhichpermit10.5minuteruntimes.

SCOPE:

Thisprocedureisapplicabletodrywheatgluten,drypetfood,andwet“cutsandgravy”typepetfood.

Thisprocedureinvolvesextractionsinmethanolandthereforepreselectscompoundswhicharesolubleorpartiallysolubleinmethanol.

Duetosolubilitylimitationsofmelamineinmethanol,thismethodisintendedtobeaqualitativescreenonly.

RESPONSIBILITY:

Itistheresponsibilityoftheanalysttonoteanymodificationstothisprocedureintheworksheet.

DEFINITIONSANDACRONYMS:

TMS–trimethylsilyl

SAFETYCONSIDERATIONS:

Acceptedsafetymeasuresshouldbeemployedwhenworkingwithchemicalsandpressurizedgases.

EQUIPMENT:

Agilent5975GC-MSsystemequippedwitha30mDB-5MScolumn(orequivalent)

Reacti-Vap–Evaporating/HeatingModule

REAGENTS:

Methanol:HPLCgrade

Pyridine:CertifiedA.C.S.

BSTFAwith1%TMCS:bis(trimethylsilyl)trifluoroacetamidewith1%

Trimethylchlorosilane(e.g.SylonBFT,Supelco)

QCELEMENTS:

Ablankshouldberunattheonsetofeachanalysisandthenrandomlythroughouttheanalysisifthereissuspicionofcarryover.

Afortifiedsample(seePartC)shouldbeanalyzedwitheachsetofsamplestodemonstrateeffectivesystemperformance.

PROCEDURES(PROCESSDETAILS):

ThisproceduremaybeusedwiththeGCoperatingineitherthesplitlessorsplit(1:20)mode.

Generally,thesplitlessmodeisadvisableforsampleswheremoresensitivityisrequired.

Thesplitmodeissuitablewheresampleavailabilityisnotlimitedandtherecommendedamountcanbeprepared(~0.5gsamplepreparations).

A.MethanolExtract(UnderivatizedExtract):

UnknownWheatGluten/PetFoodSamples:

Transferapproximately0.500gofthesampleintoa20mlscintillationvialandadd5mlmethanol.

Vortexbrieflytomix,thensonicatefor10minutes.

Centrifugeforapproximately6minutesat4500rpmandthenfiltertheliquidextractintoaGCvialusinga0.45µmnylonfilterdisc.

Ifsampleislimited,reduceextractionvolumesandsampleweightsproportionatelyandanalyzeinthesplitlessmode.

Duplicatepreparationsarerecommended.

B.Trimethylsilyl(TMS)derivatives:

Melamineisdetectedasthe3-TMSderivativewithanominalmolecularweightof342amu.

Theretentiontimeofthe3-TMSderivativeisapproximately7.1minutesintheGC-MSchromatogram(seeFigures1-4).

Blanks:Transfer40µLofmethanolusedtoextractthesampletoanautosamplervial.

EvaporatetodrynessunderastreamofdryairusingaReacti-Vapmodulesetatapproximately70°C.

Add200µlpyridineand200µlBSTFA,vortexbrieflytomix,andincubateatapproximately70°Cfor30minutes.

KnownSamples:Ingeneral,ifworkingwithaknownconcentrationofanalyte(e.g.standards),appropriatedilutionsshouldbemadewithpyridine/BSTFAtoachieveanoncolumnconcentrationnogreaterthan100µg/mL.

Aftercalculatingsuitabledilutions,transferanappropriateamountofthestandardsolutiontoanautosamplervial.

EvaporatetodrynessunderastreamofdryairusingaReacti-Vapmodulesetatapproximately70°C.

Add200µlpyridineand200µlBSTFA,vortexbrieflytomix,andincubateatapproximately70°Cfor30minutes.

UnknownSamples:Transfer40µlofthemethanolextractpreparedforanalysisinsectionAintoanautosamplervial.

EvaporatetodrynessunderastreamofdryairusingaReacti-Vapmodulesetatapproximately70°C.

Add200µlpyridineand200µlBSTFA,vortexbrieflytomix,andincubateatapproximately70°Cfor30minutes.

C.SampleFortification:

Thismethodwassuccessfullyappliedtodetectmelamineinwetanddrypetfoodcomposites(Figure2)anddrywheatglutens(Figure3).

BasedonHPLC-UVresults,thismethodwasabletodetectmelaminelevelsdownto0.01%(byweight)inofficialFDAsamplesusing~0.500gsampleswithTMSderivatizationandsplitlessinjection.

Uncontaminatedwheatglutenandpetfoodcompositeswerespikedwithvariouslevelsofmelamineandtakenthroughthemethodusing~0.500gsamplesandsplitlessinjection.

Themethodsuccessfullydetectedmelamine(asthe3TMSderivative)at14micrograms/ginthewheatglutenandat7micrograms/ginthepetfoodcomposite.

Atlowspikelevels,extractedionchromatogramswereusedtoverifythepresenceoffourmajorionsofthemelamine-TMSmassspectrum(m/z=342,327,171,and99).

Samplefortificationexperimentstodemonstratesuccessfuldetectionofmelamineinthespecificmatrixofinterestshouldbeperformedbytheindividuallaboratories.

D.StandardPreparation:

Melamineissparinglysolubleinmethanolandslightlymoresolubleinmethanol/watermixtures.

Upto~1.5mg/mLofmelaminestandardcanbedissolvedina50/50mixtureofmethanol/waterwithsonicationfor30minutes.

Ifmoreconcentratedsolutionsofmelaminearedesired,dimethylsulfoxide(DMSO)maybeusedasaprimarysolventfollowedbymethanoloracetonitrileforserialdilutions.

Note:Duetothepresenceofwaterinthestandardsolution,drydowntimesforthe

melaminestandardontheReacti-Vapwillbeincreased.

E.InstrumentParameters:

INSTRUMENT:

5975AgilentGC6890NSerieswith7683BSeriesautosampler

DETECTOR:

AgilentMassSelectiveDetector(MSD)model5975i

SOFTWARE:

EnhancedChemstationversionD.02.00.275;Wiley7thEditionandNIST02MSLibrary

COLUMN:

J&WScientificDB-5MS5%PhenylMethylSiloxanewith10MDuraguard

Part#19091S-433,Serial#US4937937H

ID:0.25mm

FilmThickness:0.25microns

Length:30meters(nominal)

CARRIERGAS:

Helium

FlowRate:1.3ml/min

Avg.LinearVelocity:35cm/sec

Pressure:17.5psi(initial)

Mode:constantflow

INJECTION:

InjectionType:

Splitless;orSplit(1:20)

InjectionVolume:1µl

InjectorTemperature:250ºC

SplitlessInjectionPurgeFlow:

10.0ml/minat2.0minutes

GCPARAMETERS:

StartTemperature:75ºC;Hold:1.0min

RampRate:30ºC/min

FinalTemperature:300ºC;Hold:2.0min

TransferLineTemperature280ºC

RunTime:

10.5minutes

MSPARAMETERS:

MassRange:40-450amu

ScanMode:Full(ElectronIonization)

FilamentDelay:

4.2min(splitless);3.8min(split)

Threshold:100

MSQuad:150ºC

MSSource:230ºC

F.PeakIdentification:

Themassspectrumofthe3-TMSderivativeofmelamine(Figure1)exhibitsthemolecularionatm/z=342,amethylgrouplossatm/z=327,andsignificantionsatm/z=171,99,and73.

Thestandardofmelamineshouldbeprepared(andTMS-derivatized)andanalyzedalongwiththesampleprepsonthesamedaytoconfirmtheidentification.

Figures2aand2bareexamplesofaGC-MSchromatogramofmelaminecontaminatedpetfood.

AdditionalpeakspresentintheTICareconsistentwithTMSderivativesofphosphateandvarioussugars.

Figure3isanexampleofaGC-MSchromatogramofmelaminecontaminatedwheatgluten.

Figure1

Figure2a

Figure2b

Figure3

Webpageupdatedby

mdt

-April11,,10:26AMET

UpdatedFCCDevelopmentalMelamineQuantitation(HPLC-UV)

April2,

SamplePreparation/Extraction:

Wheatgluten:GroundinaRetschZM100centrifugalrotormill(ringsieve0.5mm).

Moistpetfood:Moistchunksandgravywereblendedtotheconsistencyofagrittypuddingpriortosampling.

Samplesweighedintoglassscintillationvials.Extractusing50:50acetonitrile:water.

Procedure:Addindicatedvolumeofextractionsolvent(seenotebelow)1.Capandvortexthoroughly,getaggressivewiththisstep(criticalduetoslowdissolutionrateofmelamine).Sonicate30minutes.Filterportionofextractthrough2-stageGMF-nylon(0.45mm)filters.Dilutefilteredextract250

lextract+750

lsolvent2tomaintainsolubilityofmatrixcomponents.

1Wheatgluten:Extractinproportion0.1gto10ml.Formelaminecontentsabove2%w/w,extractinproportion0.05gto15ml.

1Moistpetfood:Extractinproportion2.0–2.5gto10ml.

2Solventforfinaldilutionmaybewateror0.1NHCl.Finaldilutionisnecessaryforcompatibilitywithion-pairingchromatography.Wehaveobservedsomedifferencesinbehaviorbetweenthewheatglutenandpetfoodsampleswithrespecttomaintainingsolubilityduringfinaldilution(thesearemostlikelymatrixcomponentswhichfallout).0.1NHClseemstohelpmaintainsolubilityforfinaldilutionofthewheatglutens.

HPLC-UVOperatingParameters:

Column:ZorbaxRxC8(retentionistoohighonC18column)