EUROPEANSTANDARDNORMEEUROPEENNEEUROP?ISCHENORM

EN17250

January2020

ICS67.140.30;67.220.10

EnglishVersion

Foodstuffs-DeterminationofochratoxinAinspices,

liquorice,cocoaandcocoaproductsbyIACclean-upand

HPLC-FLD

Produitsalimentaires-DosagedeI'ochratoxineAdans

lesépices,laréglisse,lesproduitsàbasederéglisse,le

cacaoetlesproduitsàbasedecacaoparpurification

surcolonned'immuno-affinitéetCLHP-DFL

Lebensmittel-BestimmungvonOchratoxinAin

Gewürzen,Sü?holz,KakaoundKakaoerzeugnissen

nachIAC-ReinigungmitHPLC-FLD

ThisEuropeanStandardwasapprovedbyCENon18November2019.

CENmembersareboundtocomplywiththeCEN/CENELECInternalRegulationswhichstipulatetheconditionsforgivingthisEuropeanStandardthestatusofanationalstandardwithoutanyalteration.Up-to-datelistsandbibliographicalreferences

concerningsuchnationalstandardsmaybeobtainedonapplicationtotheCEN-CENELECManagementCentreortoanyCENmember

ThisEuropeanStandardexistsinthreefficialversions(English,French,German).AversioninanyotherlanguagemadebytranslationundertheresponsibilityofaCENmemberintoitsownlanguageandnotifiedtotheCEN-CENELECManagementCentrehasthesamestatusastheofficialversions.

CENmembersarethenationalstandardsbodiesofAustria,Belgium,Bulgaria,Croatia,Cyprus,CzechRepublic,Denmark,Estonia,Finland,France,Germany,Greece,Hungary,Iceland,Ireland,ltaly,Latvia,Lithuania,Luxembourg,Malta,Netherlands,Norway,

Poland,Portugal,RepublicofNorthMacedonia,Romania,Serbia,Slovakia,Slovenia,Spain,Sweden,Switzerland,TurkeyandUnitedKingdom.

EUROPEANCOMMITTEEFORSTANDARDIZATION

COMITEEUROPéENDENORMALISATION

EUROP?ISCHESKOMITEEFURNORMUNG

CEN-CENELECManagementCentre:RuedelaScience23,B-1040Brussels

◎2020CENAllrightsofexploitationinanyformandbyanymeansreservedRef.No.EN17250:2020EworldwideforCENnationalMembers.

2

ContentsPage

Europeanforeword 3

Introduction 4

1Scope 5

2Normativereferences 5

3Termsanddefinitions 5

4Principle 5

5Reagents 5

6Apparatusandequipment 9

7Procedure 10

8HPLCanalysis 11

9Calculation 13

10Precision 14

11Testreport 16

AnnexA(informative)Typicalchromatograms 17

AnnexB(informative)Precisiondata 21

Bibliography 27

3

Europeanforeword

Thisdocument(EN17250:2020)hasbeenpreparedbyTechnicalCommitteeCEN/TC275"Foodanalysis-Horizontalmethods",thesecretariatofwhichisheldbyDIN.

ThisEuropeanStandardshallbegiventhestatusofanationalstandard,eitherbypublicationofanidenticaltextorbyendorsement,atthelatestbyJuly2020,andconflictingnationalstandardsshallbewithdrawnatthelatestbyJuly2020.

Attentionisdrawntothepossibilitythatsomeoftheelementsofthisdocumentmaybethesubjectofpatentrights.CENshallnotbeheldresponsibleforidentifyinganyorallsuchpatentrights.

ThisdocumenthasbeenpreparedunderamandategiventoCENbytheEuropeanCommissionandtheEuropeanFreeTradeAssociation.

AccordingtotheCEN-CENELECInternalRegulations,thenationalstandardsorganisationsofthefollowingcountriesareboundtoimplementthisEuropeanStandard:Austria,Belgium,Bulgaria,Croatia,Cyprus,CzechRepublic,Denmark,Estonia,Finland,France,Germany,Greece,Hungary,Iceland,

Ireland,Italy,Latvia,Lithuania,Luxembourg,Malta,Netherlands,Norway,Poland,Portugal,Republicof

NorthMacedonia,Romania,Serbia,Slovakia,Slovenia,Spain,Sweden,Switzerland,TurkeyandtheUnitedKingdom.

4

Introduction

ThemycotoxinochratoxinAhasachemicalstructurecomprisingadihydrocoumarinmoietylinkedtoamoleculeofL-β-phenylalanineviaanamidebond.OchratoxinAisproducedbyseveralfungalspeciesinthePenicilliumandAspergillusgenera,primarilyPenicilliumverrucosum,AspergillusochraceusandAspergilliofthesectionNigri,especiallyA.carbonarius.Cerealssuchaswheatareespeciallyafected,aswellasadiverserangeofotherfoodstuffssuchasdriedfruit,spices,cocoa,coffee,wine,beer,liquoriceandproductsthereof.

WARNING1-Suitableprecautionandprotectionmeasuresneedtobetakenwhencarryingoutworkingstepswithharmfulchemicals.Thelatestversionofthehazardoussubstancesordinance(EU)1907/2006[3]shouldbetakenintoaccountaswellasappropriatenationalstatementse.g.suchasin[4].

WARNING2—Theuseofthisdocumentcaninvolvehazardousmaterials,operationsandequipment.Thisdocumentdoesnotpurporttoaddressallthesafetyproblemsassociatedwithitsuse.Itistheresponsibilityoftheuserofthisdocumenttoestablishappropriatesafetyandhealthpracticesanddeterminetheapplicabilityofregulatorylimitationspriortouse.

WARNING3-OchratoxinAisapotentnephrotoxicagent,acarcinogenandhasgenotoxicproperties.OchratoxinAhasbeenclassifiedbyIARCasGroup2B.

5

1Scope

ThisdocumentspecifiesaprocedureforthedeterminationofochratoxinA(OTA)inchilli,paprika,blackandwhitepepper,nutmeg,spicemix,liquorice(rootandextracts),cocoaandcocoaproductsbyhighperformanceliquidchromatography(HPLC)withimmunoaffinitycolumnclean-upandfluorescencedetection(FLD).

Thismethodhasbeenvalidatedininterlaboratorystudiesviatheanalysisofbothnaturallycontaminatedandspikedsamplesrangingfrom1,0μg/kgto84,9μg/kgforspices(paprikaandchili[5],blackandwhitepepper,nutmegandspicemix[6]),rangingfrom7,7μg/kgto96,8μg/kgforliquoriceandliquoriceproducts[7]andrangingfrom2,1μg/kgto26,3μg/kgforcocoaandcocoaproducts[6].

Forfurtherinformationonthevalidation,seeClause10andAnnexB.

2Normativereferences

Thefollowingdocumentsarereferredtointhetextinsuchawaythatsomeoralloftheircontentconstitutesrequirementsofthisdocument.Fordatedreferences,onlytheeditioncitedapplies.Forundatedreferences,thelatesteditionofthereferenceddocument(includinganyamendments)appliesENISO3696,Waterforanalyticallaboratoryuse-Specificationandtestmethods(ISO3696)

3Termsanddefinitions

Notermsanddefinitionsarelistedinthisdocument.

ISOandIECmaintainterminologicaldatabasesforuseinstandardizationatthefollowingaddresses:

·IECElectropedia:availableat

/

·ISOOnlinebrowsingplatform:availableat

/obp/ui

4Principle

Spicesorliquoriceandliquoriceproductsareextractedwithamixtureofmethanolandaqueoussodiumhydrogencarbonatesolution,whereascocoaandcocoaproductsareextractedwithaqueousmethanol.Theextractisfiltered,dilutedwithphosphatebufferedsaline(PBS),polysorbate20(exceptforliquoriceandliquoriceproducts),andappliedtoanimmunoaffinitycolumncontainingantibodiesspecifictoochratoxinA.TheochratoxinAisisolated,purifiedandconcentratedonthecolumnthenreleasedusingmethanol.Thepurifiedextractisquantifiedbyreversed-phasehighperformanceliquidchromatography(RP-HPLC)coupledwithfluorescencedetection(FLD).

5Reagents

Useonlyreagentsofrecognizedanalyticalgradeandwatercomplyingwithgrade1ofENISO3696,unlessotherwisespecified.Commerciallyavailablesolutionswithequivalentpropertiestothoselistedmaybeused.

5.1Nitrogen,minimum99,95%purity.

5.2Methanol,technicalgrade.

5.3Methanol,HPLCgrade.

5.4Acetonitrile,HPLCgrade.

6

5.5Glacialaceticacid,99%purity.

5.6Toluene,UVgrade.

5.7Sodiumhydrogencarbonate,minimum99,5%purity.

5.8Sodiumchloride(NaCl),minimum99%purity.

5.9Disodiumhydrogenphosphatedodecahydrate(Na2HPO?·12H?O),minimum99%purity.

5.10Potassiumdihydrogenphosphate(KH?PO4),minimum99%purity.

5.11Potassiumchloride(KCl),minimum99%purity

5.12Sodiumhydroxide(NaOH),minimum99%purity.

5.13Hydrochloricacidsolution,volumefractionφ(HCl)=37%(acidimetric).

5.14Hydrochloricacidsolution,substanceconcentrationc(HCl)=0,1mol/L.

Dilute8,28mlofhydrochloricacidsolution(5.13)to11withwater.

5.15Sodiumhydroxidesolution,c(NaOH)=0,2mol/L.

Dissolve8gofsodiumhydroxide(5.12)in1lofwater.

5.16Aceticacidsolution,massconcentrationp(CH?COOH)=10g/L.

Dilute9,5mlofglacialaceticacid(5.5)to1lwithwater.

5.17Polysorbate20

5.18Polysorbate20solution,p(TweenR201)=20g/L.

Dissolve20gofPolysorbate20(5.17)in1000mlofwater.

5.19Phosphatebufferedsalinesolution(PBS),pH=7,4.

Dissolve8gofsodiumchloride(5.8),2,9gofdisodiumhydrogenphosphate(5.9),0,2gofpotassiumdihydrogenphosphate(5.10)and0,2gofpotassiumchloride(5.11)in900mlofwater.Afterdissolution,adjustthepHto7,4withhydrochloricacidsolution(5.14)orsodiumhydroxidesolution(5.15)asappropriate,thendiluteto1lwithwater.

Alternatively,aPBSsolutionwithequivalentpropertiescanbepreparedfromcommerciallyavailablePBSmaterial.

5.20Sodiumhydrogencarbonatesolution,p(NaHCO3)=30g/l.

Dilute30gofsodiumhydrogencarbonate(5.7)in1000mlofwater.

1Tween@20isatradenameofapolysorbate20-typenonionicsurfactantavailablefromdifferentsuppliers.ThisinformationisgivenfortheconvenienceofusersofthisEuropeanstandardanddoesnotconstituteanendorsementbyCENofthisproduct.Equivalentproductsmaybeusediftheycanbeshowntoleadtothesameresults.

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5.21ExtractionsolutionA(forspices,liquoriceandliquoriceproducts).

Mixmethanol(5.2)withsodiumhydrogencarbonatesolution(5.20)(50+50,v+v).Mixwell.

5.22ExtractionsolutionB(forcocoaandcocoaproducts).

Mixmethanol(5.2)withwater(80+20,v+v).Mixwell.

5.23MobilephaseA(forpaprikaandchilli).

Mixmethanol(5.3)withacetonitrile(5.4),waterandglacialaceticacid(5.5)(35+35+29+1,v+v+v+v).

5.24MobilephaseB(forliquoriceandliquoriceproducts).

Mixmethanol(5.3)withwaterandglacialaceticacid(5.5)(70+30+1,v+v+v).

5.25MobilephaseC(forblackandwhitepepper,nutmeg,spicemix,cocoaandcocoaproducts).

Mixmethanol(5.3),acetonitrile(5.4),waterandglacialaceticacid(5.5)(28+28+39+1,v+V+v+v).

5.26MobilephaseD(HPLCcolumnwashingsolutionforliquoriceandliquoriceproducts).

100%methanol(5.3).

5.27Immunoaffinitycolumn

TheimmunoaffinitycolumncontainsantibodiesraisedagainstochratoxinA.Thecolumnshallhaveacapacityofnotlessthan100ngofochratoxinAandshallgivearecoveryofnotlessthan85%whenappliedasastandardsolutionofochratoxinAinamixtureof15partspervolumeofmethanol(5.2)and85partspervolumeofPBSsolution(5.19)containing3ngofochratoxinA.

5.28OchratoxinA,incrystalformorasafilminampoulesorasacertifiedstandardsolution.

5.29OchratoxinAstocksolution,p(ochratoxinA)=10μg/ml.

PrepareastocksolutionofochratoxinA(5.28)inamixtureoftoluene(5.6)andglacialaceticacid(5.5)inratio99+1(v+v)withanominalconcentrationof10μg/ml.

Todeterminetheexactconcentration,recordtheabsorptioncurvebetweenawavelengthof300nmand370nmin5nmstepsin1cmquartzcellsinaspectrometerwiththesolventmixture(toluene+glacialaceticacid,99+1,v+v)asreference.IdentifythewavelengthformaximumabsorptionandcalculatethemassconcentrationofochratoxinA,p,inμg/ml,usingFormula(1):

(1)

where

Amaxisthemaximumabsorbancevaluedeterminedfromtheabsorptioncurve(here:at333nm);

MisthemolarmassofochratoxinA,ing/mol(here:M=403,8g/mol);

εisthemolarabsorptioncoeficientofochratoxinAinthesolventmixture(toluene+glacial

aceticacid:99+1,v+v),inm2/mol(here:544m2/mol);bisthepathlengthofthequartzcell,incm.

Thissolutioncanbeusedfor6monthsifstoredatapproximately-18℃.Allowtoreachroomtemperaturebeforeopening.Confirmthemassconcentrationofthesolutionifitisolderthan6months.

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Thisstepmaybeomittedwhenusingacertifiedstandardsolution,providedthattheproductcomeswithacertificate,givingsufficientevidenceonthecorrectnessofthestatedmassfraction.Thecertifiedstandardsolutionthenservesasstocksolution.

TheexactmassconcentrationsofochratoxinAinthestandard,spikingandcalibrationsolutionsarecalculatedfromtheinitialconcentrationofthisstocksolutionandthesubsequentvolumesused.

5.30OchratoxinAstandardsolution,p(ochratoxinA)=1μg/ml.

Pipette(6.4)100μlofochratoxinAstocksolution(5.29)intoa1mlvolumetricflask(6.10),drybyagentleflowofnitrogen(5.1),thendiluteto1ml(uptothemark)withtheappropriatemobilephase(5.23,5.24or5.25)andshakeitvigorously.Thisgivesastandardsolutioncontaining1μg/mlofochratoxinA.Thissolutioncanbeusedfor6monthsifstoredatapproximately-18℃.Allowtoreachroomtemperaturebeforeopening.Confirmthemassconcentrationofthesolutionifitisolderthan6months.

5.31OchratoxinAspikingsolution,p(ochratoxinA)=400ng/ml.

Pipette1mlofochratoxinAstocksolution(5.29)intoa25mlvolumetricflask(6.10)anddilutetothemarkwithamixtureofacetonitrile(5.4)andglacialaceticacid(5.5)inaratioof99+1,v+v,andshake.Thisgivesaspikingsolutioncontaining400ng/mlofochratoxinA.

Thissolutioncanbeusedfor6monthsifstoredatapproximately-18℃.Allowtoreachroomtemperaturebeforeopening.Confirmthemassconcentrationofthesolutionifitisolderthan6months.

5.32Calibrationsolutions

Preparesixcalibrationsolutionsfromthestandardsolution(5.30)asfollows:

Withappropriatepipettes(6.4)transfere.g.thevolumesoftheochratoxinAstandardsolution(5.30)separatelyeachintovolumetricflasksasspecifiedinTable1.Filleachvolumetricflaskuptothemarkwiththeappropriatemobilephase(5.23,5.24or5.25),closeandshakemanually.ThisresultsinsixochratoxinAcalibrationsolutionswithapproximatelytheconcentrationsaslistedinTable1.

Thesesixcalibrationsolutionscoverarangefromapproximately1,2μg/kgtoapproximately100μg/kgforochratoxinAforallspices,cocoaandcocoaproductsandfromapproximately2,4μg/kgtoapproximately200μg/kgforliquoriceandliquoriceproducts.

Protectcalibrationsolutionsfromlight.Thesesolutionscanbeusedfor1monthifstoredatapproximately-18℃.

Table1-Preparationofcalibrationsolutions

Calibration

solution

Standard

solution(5.30)

μl

Final

volume

ml

Massconcentrationofcalibrationsolution

ng/ml

1

15

50

0,3

2

15

25

0,6

3

25

25

1

4

50

10

5

5

150

10

15

6

250

10

25

TransferthesecalibrationsolutionsintoLCvials(6.9)beforeinjection.

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6Apparatusandequipment

Usuallaboratoryglasswareandequipment,inparticular,thefollowing:

6.1Laboratorybalance,accuracy:0,01g

6.2Analyticalbalance,accuracy:0,1mg

6.3Laboratoryshaker,andshakerforcentrifugetubes

6.4Pipettes,e.g.100μlto2000μland4mlvolumetricpipettes,suitablefororganicsolvents.

6.5Disposablesyringereservoir,of100mlcapacity,andattachmentstofittoimmunoaffinitycolumns.

6.6Glassmicrofibrefilterpaper,1,6μmretentionsize,150mmdiameter,orequivalent.Asanalternative,filterpaper(6.7)maybeusedwhentheyhavebeenproventogiveequivalentresults.

6.7Cellulosefilterpaper,11μmporesize,150mmdiameter.

6.8SPEvacuummanifold/elutionstation.

6.9LCvials,approximately2mlcapacity,or2mlLCvialwithinsert,withcrimpcapsorequivalent.

6.10Volumetricflasks,ofvariouscapacities(e.g.1ml,2ml,5ml,10ml,25ml,50ml).

6.11Conicalflasks,100mlor500mlwithscrewcap,orsimilarrecipient.

6.12HPLCsystem,comprisingthefollowing:

6.12.1HPLCpump,gradient,capableofmaintainingavolumeflowrateof0,8ml/minpulsefreeand1,0ml/minpulsefree.

6.12.2Injectionsystem.

6.12.3Pre-column,ofsuitabledimensions,withstationaryphasematerialthesameorsimilartotheanalyticalcolumn.

6.12.4Reversed-phaseHPLCcolumn.

AsuitablecolumnandappropriateHPLCconditions(isocraticorgradientprogramme)witharetentionfactorofatleasttwothatensuresbaselineseparationtodistinguishpeaksofochratoxinAfromallothersignals

SomeexamplesofcolumnswhichhavebeenfoundtobesuitablearegiveninAnnexA.

6.12.5Degasser,optional,fordegassingmobilephases(5.23;5.24;5.25;5.26).

6.12.6Columnoven,capableofmaintainingaconstanttemperature.

6.12.7Fluorescencedetector.

6.12.8Dataevaluationsystem.

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7Procedure

7.1Preparationofthetestsample

Mixorstirthelaboratorysamplethoroughlybeforeremovingthetestportion.Weighthetestportionintoaconicalflaskorsimilarrecipientof500ml(6.11).

7.2Extraction

Forspices,cocoaandcocoaproductsweighatestportionof12,5g(m)tothenearest0,1g,andforliquoriceandliquoriceproductsweighatestportionof10g(m)tothenearest0,1g.

Thetestportionsolventratioshallbe1to8exceptforliquoriceandliquoriceproductswherethesolventratioshallbe1to20.

Forspicesadd100ml(V1)ofextractionsolutionA(5.21).Forcocoaandcocoaproducts,add100ml(V1)ofextractionsolutionB(5.22).Forliquoriceandliquoriceproducts,add200ml(V1)ofextractionsolutionA(5.21).Shakebyhandforafewsecondstoobtainahomogeneoussuspension,andthenshakefor40minwithalaboratoryshaker(6.3).

Filteratleast10mloftheextractthroughthe150mmglassfibre(orcellulose)filterpaper(6.6,6.7),conicallyfolded.Collectthefilteredextractinascrewcapconicalflask(6.11)forfurtheranalysis.Proceedimmediatelywiththeimmunoaffinitycolumnclean-upprocedure(7.3).

7.3Immunoaffinitycolumnclean-up

Connecttheimmunoaffinitycolumn(5.27)tothevacuummanifold(6.8)andattachasyringereservoir(6.5)totheimmunoaffinitycolumn.

Theimmunoaffinitycolumnshallbeallowedtoreachroomtemperaturepriortousing.

Forspices,cocoaandcocoaproductstransferinthereservoir(6.5)50mlofPBS(5.19)and1mlof2%Polysorbate20solution(5.18)andpipette(6.4)4ml[V?]ofthefilteredextractasobtainedin7.2andmix.

Forliquoriceandliquoriceproductstransferinthereservoir(6.5)15mlofPBS(5.19),andpipette0,5ml[V2]ofthefilteredextractasobtainedin7.2andmix.

Drawthemixture(extractandPBS)throughthecolumnbygravityatasteadyflowrate(theflowrateshallresultinadroppingspeedof1drop/s,whichisabout3ml/min)untilallextracthaspassedthecolumnandthelastsolventportionreachesthefritofthecolumn.

Ifnecessary,theprocessmaybeacceleratedbyapplyingslightpressuretotheimmunoaffinitycolumnbyasyringeorbyapplyinglittlevacuum(e.g.byusingthevacuumsystemdescribedin6.8).Inbothcases,attentionshallbepaidnottoexceedtheflowrateof3ml/min(=1drop/s).

CAUTION—Ifusingavacuummanifold,extracareisnecessarytoavoidincreasingtheflowratethroughthecolumnasrecoverycanadverselybeaffected.

7.4Preparationofsampletestsolutions

Forpaprikaandchilli,washthecolumnwith10mlofwaterataratenotexceeding3ml/min.Drythecolumnbypushing50mlairthroughitwithasyringe.Forspices,cocoaandcocoaproductsandliquoriceandliquoriceproductswashthecolumnwith1mlof2%Polysorbate20solution(5.18)followedby10mlofwaterataratenotexceeding3ml/min.Forliquoriceandliquoriceproductsrepeatthewashingsteps.Drythecolumnbypassingnitrogenorairthroughitforabout1sto2s,anddiscardtheeluatefromthisstageoftheclean-upprocedure.

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Forspices,cocoaandcocoaproductsplacea2mlvolumetricflask(6.10)underthecolumnandpass0,5mlofmethanol(5.3)throughthecolumn,collectingtheeluate.Afterthelastdropsofmethanolhavepassedthroughthecolumn,waitapproximately1min.Thenaddafurther0,5mlofmethanol(5.3)inthesamevolumetricflaskandcollecttheeluate.Finallyadd0,5mlofmethanol(5.3)andcontinuetocollecttheeluate.Carefullypassairthroughthecolumninordertocollectanyfinaldrops.Addaceticacidsolution(5.16)intotheflaskuptothemarkandshake.Thisresultsinafinalvolumeofexactly2,0ml[V?].TransferasufficientportionintoanLCvial(6.9)andcap

Forliquoriceandliquoriceproducts,placea2mlvial(6.9)underthecolumnandpass0,5mlofmethanol(5.3)throughthecolumn,collectingtheeluate.Afterthelastdropsofmethanolhavepassedthroughthecolumn,waitapproximately1min.Thenaddafurther0,5mlofmethanol(5.3)andcollecttheeluate.Finallyadd0,5mlofmethanol(5.3)andcontinuetocollecttheeluate.Carefullypassairthroughthecolumninordertocollectanyfinaldrops.

Evaporatethemethanoliceluatetodrynessapplyingagentlestreamofnitrogen(5.1)atabout30℃to35℃.Re-dissolvethepurifiedsampleresiduesin200μl[V?]ofmobilephaseB(5.24),capvialandshakeonavortexmixer(6.3)foratleast15s,makingsurethelowerpartofthevialisthoroughlyrinsedbythesolvent.TransferthetestsolutionintoanLCvial(6.9)andanalyse.

CAUTION—Sincealltheimmunoaffinitycolumneluateisusedforthequantitativeanalysisitisveryimportanttodrytheimmunoaffinitycolumneffectivelybyairafterthewashingstepandaftertheelutionbymethanol.Shakingthevialsbeforeinjectionisalsocriticaltoensurehomogeneityofthesolutionpriorinjection.

7.5Spikingprocedure(optional,ifnocertifiedreferencematerial(CRM)isused)

Fromtheblanksamplesofspices,cocoaandcocoaproductsweighatestportionof12,5gtothenearest0,1gandfromtheblanksamplesofliquoriceandliquoriceproductsweigha10gtestportiontothenearest0,1gintoaconicalflaskorsimilarrecipientof500ml(6.11).Pipette40μlofochratoxinAspikingsolution(5.31)ontotheblankmatrix.Afteradditionofthespikingsolution,letthesolventevaporateinafumecupboardforatleast2hpriortoextraction.Proceedasin7.2startingwiththeadditionoftheextractionsolution.

8HPLCanalysis

8.1HPLCoperatingconditions

8.1.1General

InjectequalvolumesofthesampletestsolutionandofeachcalibrationsolutionintotheHPLC-FLD-system.

8.1.2Paprikaandchilli(mobilephaseA)

WhenusinganRP-C18typecolumnasspecifiedunder6.12.4andthemobilephaseAasspecifiedin5.23,thefollowingsettingshavebeenshowntobeapplicable:

Flow:0,8ml/min

Injectionvolume:20μl

Columnoventemperature(includingtheguardcolumn):22℃±1℃

Autosampler(optional)temperature:15℃to20℃

Excitationwavelength:332nm

Emissionwavelength:476nm

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OchratoxinAhasaretentiontimeofapproximately6,6min.Othercolumndimensionsmaybeused,providedthattherequiredresolutionisachieved.Thisshallbedemonstrated(maximumoverlapofochratoxinAwithanyotherpeaksifpresentshallbelessthan10%).Theflowratemaybeadjustedaccordingtothecolumndimension.AtypicalchromatogramforpaprikaisshowninAnnexA,FigureA.1.

8.1.3Liquoriceandliquoriceproducts(mobilephaseB)

WhenusinganRP-C18typecolumnasspecifiedunder6.12.4andthemobilephaseBspecifiedin5.24,thefollowingsettingshavebeenshowntobeapplicable:

Flow:

1,0ml/min

Injectionvolume:

Columnoventemperature(includingtheguardcolumn):

Autosampler(optional)temperature:

Excitationwavelength:

Emissionwavelength:

20μl

22℃±1℃

15℃to20℃

332nm

476nm

OchratoxinAhasaretentiontimeofapproximately10min.AtypicalchromatogramforliquoriceextractpowderisshowninAnnexA,FigureA.2.

Afterimmunoaffinitycolumnclean-upthepurifiedextractmaystillcontainsubstancesthatcanelutelaterorinsubsequentchromatographicrunsappearingasbroadpeaks.Topreventthis,washthecolumnfor10minwithmobilephaseDaswashingsolution(5.26)followedby10minre-equilibrationwithmobilephaseB(5.24)priortothenextrun.Thenecessityofthewashingstepvarieswithdifferentsampletypesandmaybeskippedifnointerferencesarenotedfromlateelutionpeaks.

13

8.1.4Spices,cocoaandcocoaproducts(mobilephaseC)

Whenusingaphenyl-hexylcolumnasspecifiedunder6.12.4andthemobilephaseCspecifiedin5.25,thefollowingsett