EUReferenceLaboratoryforpesticidesrequiringSingleResidueMethods(EURL-SRM)

QuickMethodfortheAnalysisof

NumerousHighlyPolarPesticidesinFoodInvolvingExtractionwithAcidifiedMethanolandLC-MS/MSMeasurement

II.FoodofAnimalOrigin(QuPPe-AO-Method)

CheckforlatestversionofthisMethodunder

www.quppe.eu

;olderversions:

obsoleteversions

Version3.3(30.12.2024,

DocumentHistory

,seepage

25

)

Authors:M.Anastassiades;A.-K.Sch?fer;E.Eichhorn;D.I.Kolberg;A.Benkenstein;S.Zechmann;

D.Mack;A.Barth;C.Wildgrube;B.Sauer;I.Sigalov;S.Goerlich;D.D?rk;G.Cerchia

EUReferenceLaboratoryforpesticidesrequiringSingleResidueMethods(EURL-SRM)

Address:CVUAStuttgart,Schaflandstr.3/2,DE-70736Fellbach,GermanyWeb:

www.eurl-pesticides.eu,

E-Mail:

EURL@cvuas.bwl.de

ChangesfromV3.2toV3.3arehighlightedinyellow

ScopeandShortDescription 2

ApparatusandConsumables 2

Chemicals 4

Disclaimer 5

Procedure 6

Samplepreparation 6

Extraction/Centrifugation/Filtration 6

Blankextracts 11

Recoveryexperiments 11

Preparationofcalibrationstandards 11

LC-MS/MSMeasurementConditions 13

ExemplaryLC-MS/MSchromatograms(methodM1.3) 13

ExemplaryLC-MS/MSchromatograms(methodM1.6) 14

ExemplaryLC-MS/MSchromatograms(methodM1.7) 16

ExemplaryLC-MS/MSchromatograms(methodM4.2) 17

CalibrationandCalculations 18

ValidationData 19

References 24

EUReferenceLaboratoryforpesticidesrequiringSingleResidueMethods(EURL-SRM)

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ScopeandShortDescription

Amethodisdescribedfortheresidueanalysisofverypolar,non-QuEChERS-amenable,pesticidesinfoodofanimalorigin.FollowingwateradjustmentandadditionofacidifiedmethanolandEDTA,residuesareextractedfromthetestportionviashaking.Followingcentrifugation,analiquotoftherawextractiscleaned-upbysimultaneousdilutionwithacetonitrileanddSPEwithODSsorbent,whichleadstoaprecipitationoradsorptionofalargeportionofco-extractives.Thecleaned-upextractiscentrifugedandfilteredandthensubjectedtodeterminativeanalysisviaLC-MS/MS.VariousLC-MS/MSmethodsforthesimultaneousanalysisofdifferentcombinationsofpesticidesareprovided.Quantificationisinmostcasesperformedwiththehelpofisotopicallylabelledanaloguesofthetargetanalytes,whichareusedasinternalstandards(ISTDs).Sofaravailable,theseISTDsareaddeddirectlytothetestportionatthebeginningoftheproceduretocompensateforanyfactorshavinganinfluenceontherecovery-ratessuchasvolume-deviations,analytelossesduringextractionandclean-upaswellasmatrix-effectsduringLC-MS/MS.

Howtocite(proposal):

QuickMethodfortheAnalysisofHighlyPolarPesticidesinFoodInvolvingExtractionwithAcidifiedMethanolandLC-orIC-MS/MSMeasurement-I.FoodofAnimalOrigin(QuPPe-AO-Method)–Version3.3(publishedonEURL-SRMwebsiteonMarch31,2022);M.Anastassiades;A.-K.Sch?fer;E.Eichhorn;D.I.Kolberg;A.Benkenstein;S.Zechmann;D.Mack;A.Barth;C.Wild-grube;B.Sauer;I.Sigalov;S.Goerlich;D.D?rk;G.Cerchia.

URL:

https://www.quppe.eu/quppe_doc.asp

ApparatusandConsumables

Powerfulsampleprocessingequipment,

e.g.StephanUM5orRetschGrindomixGM300orVorwerk-ThermomixTM31.Forliquidsamples(e.g.milk,eggs):itisalsopossibletousealesspowerfulblender,e.g.BraunMR5550handblenderwithchopperattachment.

LC-Plastictub,

forfilling-inliquidnitrogentoimmergethesamplespriortomilling(

5.1

),seelatestversionofQuPPe-PO-Method.

50mLcentrifugetubeswithscrewcaps,

fortheextractionstep.seelatestversionofQuPPe-PO-Method.

10mLcentrifugetubeswithscrewcaps,

forthed-SPEstep,seelatestversionofQuPPe-PO-Method.

Automaticpipettes,

seelatestversionofQuPPe-PO-Method.

10mLsolvent-dispenser,

seelatestversionofQuPPe-PO-Method.

2.7.Mechanicalshaker,

SeelatestversionofQuPPe-PO-Method.

Mechanicalgrinding/shakingaids,

e.g.stainlesssteelgrindingballs(?7-10mm).

WaterBath,

SeelatestversionofQuPPe-PO-Method.

Centrifuge,

seelatestversionofQuPPe-PO-Method.

DisposableSyringes,

seelatestversionofQuPPe-PO-Method.

DisposableSyringefilters,

seelatestversionofQuPPe-PO-Method.

Ultrafiltrationfilters,

seelatestversionofQuPPe-PO-Method.

Autosamplervials,

seelatestversionofQuPPe-PO-Method.

Notes:

-Theuseofplasticvialsishighlyrecommendedasseveralofthecompoundscoveredbythismethod(e.g.Phospho-nate,Nicotine,Paraquat,Diquat,StreptomycinandGlyphosate)1tendtointeractwithglass-surfaces.Suchinteractionswithglasssurfacesaretypicallymorepronouncedinsolutionsconsistingofaproticsolvents(e.g.acetonitrile).Increas-ingwatercontentand/oraciditytypicallyreducessuchinteractions.Percentlossesduetosuchinteractionsaretypicallyhigheratlowconcentrations.

Volumetricflaskwithstoppers,

seelatestversionofQuPPe-PO-Method.Mindtouseplasticcontainers(seenoteunder

2.14

).

Screw-capstoragevessels,

seelatestversionofQuPPe-PO-Method.Mindtouseplasticcontainers(seenoteunder

2.14

).

LC-MS/MSinstrumentation,

seelatestversionofQuPPe-PO-Method.

IC-MS/MSinstrumentation,

seelatestversionofQuPPe-PO-Method.

1Thelistofcompoundsrequiringplasticvesselsisnotcomprehensive.

Chemicals

Unlessotherwisespecified,usereagentsofrecognizedanalyticalgrade.Takeeveryprecautiontoavoidpossiblecon-taminationofwater,solvents,sorbents,inorganicsalts,etc.

Water(deionized),

Forwateradditiontothesamples.

Water,ultrapure,

e.g.preparedbyalaboratorywaterpurificationsystem.CommerciallyavailableMS-qualitywatercanbeusedforLC-MS/MSmobilephasesandIC-qualitywaterforIC-MS/MSmobilephases.

Methanol(atleastHPLCquality),

ForthepreparationofmobilephasespreferablyuseMS-qualitymethanol.

Acetonitrile(LC-MSquality),

ForthepreparationofmobilephasespreferablyuseMS-qualityacetonitrile.

Formicacid(concentrated;≥98%),

forthepreparationofmobilephasespreferablyuseMS-qualityformicacid.

AceticAcid(concentrated;≥98%),

forthepreparationofmobilephasespreferablyuseMS-qualityaceticacid.

Acidifiedmethanol,

seelatestversionofQuPPe-PO-Method.

Acidifiedmethanolwith30%water,

forfatextraction,preparedbypipetting10mLofformicacid(

3.5

)intoa1000mLvolumetricflask,followedby300mLwater(

3.1

)andfillinguptovolumewithmethanol(

3.3

).

C18-sorbent(ODSsorbent),

seelatestversionofQuPPe-PO-Method.

Ammoniumformate(p.a.)

Ethylenediaminetetraaceticacidtetrasodium

seelatestversionofQuPPe-PO-Method.

10%aqueousEDTAsolution,

seelatestversionofQuPPe-PO-Method.

Dryice,

seelatestversionofQuPPe-PO-Method.

PesticideStandards,

ofknownpurity.

Pesticidestocksolutions,

seelatestversionofQuPPe-PO-Method.Mindtouseplasticcontainers(seenoteunder

2.14

).

Pesticideworkingsolutions/mixtures,

seelatestversionofQuPPe-PO-Method.Mindtouseplasticcontainers(seenoteunder

2.14

).

InternalStandards(ISs),

ofknownpurity.

ISStocksolutions,

seelatestversionofQuPPe-PO-Method.Mindtouseplasticcontainers(seenoteunder

2.14

).

IS-workingsolutionI(IS-WSln1)forspikingsamplespriortoextraction,

seelatestversionofQuPPe-PO-Method.Mindtouseplasticcontainers(seenoteunder

2.14

).

IS-workingsolutionII(IS-WSln2)forpreparationofcalibrationstandards,

seelatestversionofQuPPe-PO-Method.Mindtouseplasticcontainers(seenoteunder

2.14

).

Disclaimer

Thismethodreferstoseveraltradenamesofproductsandinstruments,whicharecommerciallyavailableandsuit-ableforthedescribedprocedure.ThisinformationisgivenfortheconvenienceoftheusersofthismethodanddoesnotconstituteanendorsementbytheEURLoftheproductsnamed.Theapplicationofthismethodmayinvolvehazardousmaterials,operationsandequipment.Itistheresponsibilityoftheusersofthismethodtoestablishap-propriatesafetyandhealthpracticespriortouse.Anyconsumablesandchemicalsusedintheprocedureshouldbeperiodicallychecked,e.g.throughreagentblanktests,foranyrelevantlevelsoftheanalytesofinterest.

Procedure

Samplepreparation

Toobtainrepresentativetest-portionsfromthelaboratorysample,proceedasrequiredbytherespectiveregulationsandguidelines.

Eggsaredeshelledandhomogenizedbyahand-blender(

2.1

)untilafreeflowingmixtureisobtained.Proceedsimi-larlywithnon-homogenizedmilk(e.g.iffathasseparated).Homogenizedmilkcanbeusedassuch.

Animaltissues(muscle,kidneyandliver)arepreferablymilledcryogenically(e.g.usingdryice).Thisisdonetoreduceanalytedegradationandparticlesizes,withthelatterresultinginimprovedhomogeneityandresidueaccessibility.Onepossibilityforcryogenicmillingistocutlargeunitscoarselytoca3x3cmpieces,freezethemandthenmillthemforca.1-2minuteswithapowerfulmill.Thenadddryice(ca.150-200gper500gsample)andcontinuemillinguntilbarelyanycarbondioxidefumesareobserved.Alternativelyfillaplasticorpolystyrenecontainerwithaca.5-15cmthicklayerofliquidnitrogenandimmersethesamplepiecesintotheliquidnitrogen.Whencompletelyfrozentrans-ferthematerialintoapowerfulknifemillandgrindathighspeeduntilitgetsasnow-likeconsistency.Ifnecessary,crushlargeunitswithahammerbeforemilling.Ifthematerialstartsdefrostingduringmilling,addsomemoreliquidnitrogenordryiceandcontinuemillingasdescribedabove.Placethehomogenateimmediatelyinthefreezer.

Isolatedandpre-homogenizedanimalfat,suchascommercialbutterfatorrenderedlardmaybeusedassuch.Trimmedadiposetissueshouldbehomogenized.Thiscanbedoneeitheratroomtemperatureusingahighspeedknifemillorcryogenicallybycuttingthefatinsmallpieces(e.g.2x2cm)freezingitoutandhomogenizingitwithapowerfulknifemill.Forthispurpose,placethefrozenfatpiecesintothemill,adddryice(ca.4:1ratio)andmilluntilafree-flowingpowderisobtained.Alternatively,immersethefatpiecesintoliquidnitrogenandmillwithaknifemilltoobtainafreeflowingpowder.Fillthemilledmaterialintoasuitablevesselorbagandfreezeimmediately.

Extraction/Centrifugation/Filtration

Thegeneralanalyticalprocedureataglanceisshownin

Figure1

forliver,milk,kidney,muscleandeggandin

Figure

2

foranimalfat.

Weighingofanalyticalportions

Weigharepresentativeanalyticalportion(ma)ofthesamplehomogenate(

5.1

)intoa50mLcentrifugetube(

2.2

).Incaseofanimaltissues(e.g.liver,muscle,kidney)aswellasmilkandeggweigh10g0.1gofthehomogenizedsample.Incaseofanimalfatweigh5g0.05g.

Adjustmentofwatercontent

Addwater(

3.1

)totheanalyticalportion(

5.2.1

),toreachatotalwatercontentofca.10gperportion.

Theamountofwatertobeaddedtotheanalyticalportionisshownin

Table1

.Noextrawaterisaddedinthecaseofanimalfat.

Notes:

-WherenoISsareusedorwheretheyareaddedafterextractaliquotation,wateradjustmentto10gisessentialforminimizingthevolumetricerrortoacceptablelevels.WheretheappropriateISsareemployedbeforeanyaliquotation,wateradjustmentislesscriticalandmaybeskippedforcommoditiescontaining>80%naturalmoisture,orforcom-moditiescontaining>70%naturalmoistureiftheanalyticalprocedureinvolvedtheadditionof1mLaqueousEDTAsolution(seebelow).ThewatercontainedintheaqueoussolutionEDTAsolutionaddedduringtheextractionstep

(5.2.3

)isalsoconsideredintheoverallwatercontent.Keepinmindthatthewatervolumeadjustmentsin

Table1

areapproximate.

Table1:Adjustmentofwatercontentforvariousmatrixesofanimaloriginaccordingtotheirnaturalwatercontent.

Commodity

Sampleweight

Typicalnatural

watercontenting/100g

Water

tobeadded

Volume

EDTAsolu-tion

Wateradd.

maybeskipped*

IS-WSln1

addede.g.

Extra

Formicacid

ExtractionSolu-tion

Milk(wholefat)

10g

85

0.5mL

1mL

Yes

100μL

100μL

10mLMeOH+1%FA

(3.7)

Milk(1.5%fat)

10g

90

-

1mL

Yes

100μL

100μL

Egg

10g

75

1.5mL

1mL

No

100μL

100μL

Liver

10g

70

2mL

1mL

No

100μL

100μL

Kidney

10g

80

1mL

1mL

Yes

100μL

100μL

Muscle

10g

80

1,5mL

1mL

Yes

100μL

100μL

Animalfat

5g

-

-

-

Notapplic.

100μL

none

10mLMeOH:Water

(7:3)+1%FA

(3.8)

*ifsuitableISisusedbeforealiquotation

Extraction

Liver,Kidney,Muscle,MilkandEgg:

Add10mLacidifiedmethanol(

3.7

)andanappropriatesmallvolume(e.g.100μl)oftheIS-WSln-1(

3.19

)containingisotopicallylabelledanaloguesoftheanalytesofinterest(addedISmass=mISsample).Addanextraamountof100μLformicacid(

3.5

).Closethetubeandshakeforafewsecondstodistributetheacidandallowproteinstocoagulate.Add10%aqueousEDTAsolution(

3.12

)andshakeeitherfor1minbyhandorfor2-15minbyanautomaticshaker.Notes:

WherenoISsareusedtheaimshouldbetoreachatotalvolumeoftheliquidphaseascloseaspossibleto20mL,which

correspondsto0.5g/0.25gsamplepermLextractif10g/5gsampleareused.Thisvolumewillmainlyconsistofthewaternaturallycontainedinthesample,thewateraddedduringtheprocedure(includingthatoftheEDTAsolution),theextractionsolventadded,theISsolutionaddedaswellastheextravolumeofformicacid.VolumecontractionisalsotakingplacetoacertaindegreeanditispartlycomplementedbytheadditionofISandformicacid.Furtheralter-nativestoavoiderrorsduetovolumetricdeviationsarecalibrationsthatcompensateforrecovery,suchastheapproachofstandardadditionstosampleportionsandtheproceduralcalibrationsapproachusingasuitableblankmatrix.

ForscreeningpurposestheIScanbealternativelyaddedtoanaliquotofthesampleextract(e.g.the1mLtransferredtotheautosamplervial,seebelow),assumingthat1mLextractentailsexactly0.25gsampleequivalents.ThiswaytheaddedamountofISpersamplecanbedrasticallyreduced(e.g.40-foldifaddedto1mLextract).TheISaddedatthisstepwillcompensateformatrixeffectsincludingretention-timeshiftsbutnotforrecoveryandvolumedeviations.Thequantitativeresultshouldthereforebeconsideredtentative.Formoreaccuracysamplesshouldbere-extractedwiththeISbeingaddedtotheanalyticalportionbeforealiquotation.

Animalfat(isolatedfatoradiposetissuehomogenate):

Add10mLacidif.methanolwith30%water(

3.7

)andanappropriatesmallvolume(e.g.100μl)ofIS-WSln-1(

3.19

)containingisotope-labelledanaloguesofanalytes(addedISmass=mISsample).Addmechanicalaids,e.g.3to5grindingballs(?7-10mm)(

2.8

),closethetubeandshakevigorouslyfor5to20min(byapowerfulmechanicalshaker).

Ifnogrindingaidsareathand,alternatively,closethetube,shakewellforafewsecondsandplaceitinawaterbathof80°Cfor3-4minutesuntilthefathascompletelymelted.Whilestillhot,shakeintensivelyfor1minutebyhandorfor2-15minbyanautomaticshaker,toensuredistributionofthepolarpesticidesintotheaqueousphase.

Notes:

Duetothepoormiscibilityoftheaqueousmethanolwiththefat,thefinalextractvolumecanbeconsideredasbeing10mL,whichcorrespondsto0.5gsamplepermL.

ForscreeningpurposestheIScanbealternativelyaddedtoanaliquotofthesampleextract(e.g.the1mLaliquottransferredtotheautosamplervial,seebelow),assumingthat1mLextractentailsexactly0.5gsampleequivalents.Thisway,theaddedamountofISpersamplecanbedrasticallyreduced(e.g.10-foldifaddedto1mLextract).Seefurthercommendsunder

5.2.3.

Althoughmeltingpointsofanimalfatusuallyarebetween30and50°Citismoresuitabletoheatupthesampletoatleast60°Ctoensurethatthefatmeltsquicklyandstaysliquidduringshaking.

Freeze-OutandCentrifugation

Dependingontheavailablecentrifugationequipmenttherearevariousoptions,e.g.:

(1)Ambientcentrifugation:Centrifugetheextractsfrom

5.2.3

for5minat≥3,000g(thehigherthecentrifu-gationforcethebetter).ThisprocedureisNOTrecommendedforextractsofcommoditieswithhighfatcontent(e.g.liver,wholefatmilk,eggs).Forsuchcommoditiesbetterusethefollowingoptions

(2)

or

(3)

.

(2)Ambientcentrifugationfollowingfreeze-out:Placetheextractsfrom

5.2.3

intoafreezer(e.g.atca.-80°C

for30minoratca.-20°Cfor>120min)andcentrifugewhilestillcoldfor5minat≥3,000g.Highercentrif-

ugationforces(e.g.≥10,000g)andcoldcentrifugationarepreferred.Thisprocedureissuitablefortheextractsofallsamples.

Refrigeratedhigh-speedcentrifugation:Centrifugetheextractsfrom

5.2.3

for>20minathighcentrifuga-tionspeed(e.g.>10,000g)andlowtemperatures(e.g.lowerthan-5°C).Centrifugationtimemaybere-ducedto5miniftheextractispre-frozen.Thisprocedureissuitableforextractsofallsamples.

Notes:

Solidmetalrackssuitableforfalcontubes(e.g.VWR?ModularBlocksforConical-Bottom50mLCentrifugeTubes)maybeusedtospeedupfreeze-out.

Lowtemperaturesreducethesolubilityofinterferingmatrixcomponentsresultinginincreasedprecipitation,whichconsiderablyfacilitatesthefiltrationstepaswellasthesubsequentLC-MS/MSanalysisbyreducingmatrixeffectsandincreasingthelifespanofcolumns.Itisrecommendedtoproceedimmediatelywiththenextstepstoavoidre-dissolu-tionofmatrixcomponents.Otherwisetransferanaliquotofthecoldsupernatantintoasealablecontainerforlateruse.

dSPEanddilutionwithACNforremovaloflipidsandproteinprecipitation

Liver,Kidney,Muscle,MilkandEgg:

Transfera2mLaliquotofthesupernatantfrom5.2.4intoa10mLcentrifugetubewithscrewcap(

2.4

),alreadycontaining2mLofacetonitrile(

3.4

)and100mgofC18-sorbent(

3.9

)andshakefor1min.Centrifugefor5minat

>3,000g(see

2.10

).

Animalfat:

Wherethesupernatantwasisolatedwhilestillverycold,thisstepmaybeskipped.Otherwise,transfera4mLaliquotofthesupernatantfrom5.2.4intoa10mLcentrifugetubewithscrewcap(

2.4

),whichalreadycontains200mgofC18-sorbent(

3.9

)andshakefor1min.Thencentrifugefor5minat>3,000g(see

2.10

).

Filtration

Bysyringefilter:

Withdrawanaliquot(e.g.2-3mL)ofthesupernatantfrom

5.2.4

or

5.2.5

usingasyringe(

2.11

)andfilteritthroughasyringefilter(

2.12

)eitherdirectlyintoanauto-samplervial(

2.14

)orintoasealablestoragevessel

(2.16

).

Byultrafiltration(optional):

Transfera3mLaliquotofthesupernatantfrom

5.2.5

intoanultrafiltrationunit(

2.13

)andcentrifugeat3,000guntilenoughfiltrateiscollected(e.g.5-10min).Transferanaliquotofthefiltrateintoanautosamplervial.

Notes:

Thecleaned-upextractwillcontainca.0.5gsampleequivalentspermLextractinthecaseofanimalfatandforallothercommodities0.25gsampleequivalentspermLwhere10gsample(e.g.milk,liver)areemployed.

InsteadofaddingtheISatthebeginningoftheprocedureitcanbeaddedtoanaliquot(e.g.1mL)ofthefinalsampleextract.Thisway,theaddedamountofISpersamplecanbedrasticallyreduced(e.g.40-fold2ifaddedto1mLextract).TheISaddedatthisstepwillcompensateformatrixeffectsincludingretention-timeshifts.Quantitativeresultshouldhoweverbeconsideredastentative.Formoreaccuracysamplesshouldbere-analyzedwithISbeingaddedinstep

5.2.3.

210-foldinthecaseofanimalfat

QuPPe-AO-Methodataglance

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ProcedureforLiver,Kidney,Muscle,MilkandEgg

Weighsamplehomogenateintoa50mLcentrifugetube

Milk,Egg,Kidney,MuscleandLiver:10g±0,1g

Adjustwatercontentofsampleto10mL(notmandatoryifIL-ISisused)

Liver:+2mL;Muscle+1.5mL;Kidney+1mL,Egg+1.5mL,Wholefatmilk:+0.5mL;skimmedmilk:noaddition

Add100μLisotopically-labelledinternalstandard(IL-IS)mix

Liver,Milk,Kidney,Muscle,Egg:

Add10mLMeOHcontaining1%formicacid+extra100μLformicacid,closetubeandshake

Add1mL10%aqueousEDTAsolution

Shakethoroughlyfor5-15minbyamechanicalshaker

Option1

Freeze-outsampletillcompletelyfrozen

e.g.30minat-80°Cor>90minat-20°C

ImmediatelyCentrifuge

>3,000gfor5min(>10,000gpreferred)(refrigeratedcentrifugationpreferred)

Option2

RefrigeratedHigh-SpeedCentrifugation

e.g.>10,000gat-10°C

for≥20min

dSPEanddilutionwithACNforremovaloflipidsandproteinprecipitationTransfer2mLofrawextractintoatubecontaining100mgC18-sorbentand2mLACN,Shakefor1minandcentrifugeat>3,000gfor5min

Filteraliquotofsupernatant

Centrifugationassistedultrafiltrationthrougha10kDacut-offfilter(e.g.polyethersulfonemembrane)

LC-MS/MSanalysis

Figure1:Methodataglanceliver,milk,kidney,muscleandegg.

ProcedureforAnimalFat

Weighsamplehomogenateintoa50mLcentrifugetube

Fat:5g±0,05g

Add100μLisotopically-labelledinternalstandard(IL-IS)mix

Add10mLMeOHcontaining30%waterand1%formicacid

Heatuptubeuntilthefatismeltedcompletely

(e.g.placeinan80°Cwaterbath,don‘theatlongerthanneeded)

Addmechanicalaids,e.g.3to5stainlesssteelgrindingballs

(?7-10mm)

Immediatelyshakethoroughly(for1minbyhandorfor2-15minbyautomaticshaker)

Closethetubeandshakevigorouslyfor5to20min

(byamechanicalshaker)

Centrifugationoption1or2

Option1

Freeze-outsampletillcompletelyfrozen

e.g.30minat-80°Cor>90minat-20°C

ImmediatelyCentrifuge

>3,000gfor5min(>10,000gpreferred)(refrigeratedcentrifugationpreferred)

Option2

RefrigeratedHigh-SpeedCentrifugation

e.g.>10,000gat-10°C,for≥20min

dSPEforremovaloflipids

Transfer4mLaliquotofrawextractintotubecontaining200mgC18-sorbentpermLextract,Shakefor1min

Centrifugeat3,000gfor5min

Filtersupernatantintoaplasticvial

Syringefilterwith0.2μmporesize(e.g.H-PTFEorregeneratedcellulose)

(plasticvialsarerecommendedassomecompoundstendtointeractwithglass)

LC-MS/MSanalysis

Figure2:Methodataglancefat

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Blankextracts

Usinghomogenatesofsuitableblankcommodities(notcontaininganydetectableresiduesoftheanalytesofinter-est),proceedsamplepreparationexactlyasdescribedunder

5.2

butSKIPTHEADDITIONOFISTDs.

Recoveryexperiments

-SeelatestversionofQuPPe-PO-Method.Inthecaseoffatsamplesincurredresidueswillbebettersimulatedifthefatportionstobeanalyzedarefirstmelted(waterbath),thencooleddownabitandspikedwhilethefatisstillliquid(e.g.at45°C)andpreferablyusingstandardsdilutedinmethanolratherthanwatertoensurebettermiscibility.Followinggentlestirringtodistributetheresiduesthespikedfatportionsarelefttosolidify(e.g.placedinthefridgeorfreezer)beforebeingextractedasshownabove.Afat-incorporatedresiduewillrepresentaworst-casesituationwheretheanalytesarepresentinthefatinteriorandthusnotreadilyaccessibletotheextractingsolvent.

Preparationofcalibrationstandards

Solvent-basedcalibrationstandards

Anexemplarypipettingschemeforthepreparationofsolvent-basedcalibrationstandardsisshownin

Table2

.Thecalculationofthemass-fractionWRofthepesticideinthesample,whenISTDisused,isshownin

5.7.1

.

Note:Wheresolvent-basedcalibrationsareusedtheuseofIL-ISTDsforquantificationisessentialastheISTDcompensatesforanymatrix-relatedsignalsuppressions/enhancements.

Thoughmatrix-matchedcalibrationisconsideredthebestoption,solvent-basedcalibrationscanalsoproduceaccurateresultsasIL-ISscancompensateforerrorsirrespectiveonwhetherthecalibrationissolvent-based,matrix-basedormatrix-matched.Nevertheless,insomecasestheuseofmatrix-basedcalibrationsaretobepreferredoversolvent-basedcalibrationsasthematrixpresentcandecreaseunwantedinteractionswithsurfaces(e.g.intheinjectorarea)thusleadingtopeakshapesandretentiontimesthatareclosertothoseobservedfromsampleextracts.

Matrixbasedandmatrixmatchedcalibrationstandards

Transfersuitablealiquotsoftheblankextract(

5.3

)toauto-samplervialsandproceedasshownin

Table2

.

Thecalculationofthemass-fractionWRofthepesticideinthesampleusingmatrix-matchedcalibrationstandards,withandwithouttheuseofIL-ISs,isshownin

5.7.1

and

respectively.

Table2:Exemplarypipettingschemeforthepreparationofcalibrationstandards

Calibrationstandards

Solventbased(

5.5.1)

Matrix-matched(

5.5.2

)

usingIL-IS4

withoutIL-IS5

usingIL-IS4

Calibr.levelsinμgpesticide/mLORinμgpesticide/“IL-IS-portion”1

0.056

0.1

0.25

0.05

0.1

0.25

0.05

0.1

0.25

Blankextract(

5.3

)

-

-

-

875μL

875μL

875μL

825μL

825μL

825μL

1:1(v/v)mixofwater(

3.1

)and

acidifiedmethanol(

3.7

)

925μL

900μL

825μL

100μL

75μL

-

100μL

75μL

-

Pesticide working

solutions(

3.16)

2

0.5μg/mL

25μL

50μL

125μL

25μL

50μl

125μL

25μL

50μL

125μL

IS-WSln-2(

3.20)

1,3

50μL

50μL

50μL

-

-

-

50μL

50μL

50μL

Totalvolume

1000μL

1000μL

1000μL

1000μL

1000μL

1000μL

1000μL

1000μL

1000μL

1OneIL-ISportionwouldcorrespondtotheIL-ISmasscontainedin50μLIS-WSln-2(whichintheparticularexampleisaddedtoeachcalibrationstandard).

2Theconcentrationofthepesticideworkingsolution(s)shouldbesufficientlyhightoavoidexcessivedilutionoftheblankextract,whichwouldresultinmatrixeffectdeviations.

3Forcalibrationstandardsof1mLitishighlyrecommendedtopreparetheIS-WSln-2

(3.20

)bydilutingIS-WSln-1

(3.19

)40-fold.Thesamevolumeandpipetteasin

5.2.3

canbeusedforpreparingthecalibrationstandards.

4WhenemployingIL-ISs,matrix-matchinga