Animal feeding stuffs - Determination of residues of organochlorine pesticides - Gas chromatographic method (ISO 14181:2000)

This International Standard specifies a gas chromatographic method for the determination of residues of
organochlorine pesticides in animal feeding stuffs.
The method is applicable to animal feeding stuffs containing residues of one or more of the following
organochlorine pesticides and some of their isomers and degradation products: aldrin, op'-DDE, pp'-DDE, op'-DDT,
pp'-DDT, dieldrin, endosulfan, endrin, HCB, _-HCH (_-BHC), _-HCH (_-BHC), _-HCH (_-BHC), _-HCH
(_-BHC), heptachlor, heptachlor epoxide, op'-TDE (op'-DDD), pp'-TDE (pp'-DDD) and methoxychlor.
The lower determination limit for these organochlorine pesticides is 0,005 _g/g. However, the lower determination
limit is 0,01 _g/g for op'-DDT and pp'-DDT, and 0,05 _g/g for methoxychlor.

Futtermittel - Bestimmung des Gehalts an Organochlorpestizid-Rückständen - Gaschromatographisches Verfahren (ISO 14181:2000)

Anwendungsbereich
Diese Internationale Norm legt ein gaschromatographisches Verfahren zur Bestimmung des Gehalts an
Rückständen von Organochlor-Pestiziden in Futtermitteln fest.
Das Verfahren ist anwendbar auf Futtermittel, die Rückstände von einem oder mehreren der folgenden
Organochlor-Pestizide und einigen ihrer Isomere und Abbauprodukte enthalten: Aldrin, op0 -DDE, pp0-DDE,
op0 -DDT, pp0 -DDT, Dieldrin, Endosulfan, Endrin, HCB, a-HCH (a-BHC), v-HCH (v-BHC), g-HCH (g-BHC),
d-HCH (d-BHC), Heptachlor, Heptachlor-epoxid, op0-TDE (op0 -DDD), pp0 -TDE (op0 -DDD) und Methoxychlor.
Die untere Nachweisgrenze für diese Organochlor-Pestizide beträgt 0,005 µg=g. Die untere Nachweis-
grenze ist jedoch für op 0 -DDT und pp 0 -DDT 0,01 µg=g und für Methoxychlor 0,05 µg=g.

Aliments des animaux - Détermination des résidus de pesticides organochlorés - Méthode par chromatographie en phase gazeuse (ISO 14181:2000)

Krma - Določevanje ostankov organoklornih pesticidov - Plinska kromatografska metoda (ISO 14181:2000)

General Information

Status
Published
Publication Date
31-Jan-2001
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
01-Feb-2001
Due Date
01-Feb-2001
Completion Date
01-Feb-2001

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2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.RPDWRJUDIVNDFuttermittel - Bestimmung des Gehalts an Organochlorpestizid-Rückständen - Gaschromatographisches Verfahren (ISO 14181:2000)Aliments des animaux - Détermination des résidus de pesticides organochlorés - Méthode par chromatographie en phase gazeuse (ISO 14181:2000)Animal feeding stuffs - Determination of residues of organochlorine pesticides - Gas chromatographic method (ISO 14181:2000)65.120KrmilaAnimal feeding stuffsICS:Ta slovenski standard je istoveten z:EN ISO 14181:2000SIST EN ISO 14181:2001en01-februar-2001SIST EN ISO 14181:2001SLOVENSKI
STANDARD



SIST EN ISO 14181:2001



EUROPEAN STANDARDNORME EUROPÉENNEEUROPÄISCHE NORMEN ISO 14181September 2000ICS 65.120English versionAnimal feeding stuffs - Determination of residues oforganochlorine pesticides - Gas chromatographic method (ISO14181:2000)Aliments des animaux - Détermination des résidus depesticides organochlorés - Méthode par chromatographieen phase gazeuse (ISO 14181:2000)Futtermittel - Bestimmung des Gehalts anOrganochlorpestizid-Rückständen -Gaschromatographisches Verfahren (ISO 14181:2000)This European Standard was approved by CEN on 15 September 2000.CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this EuropeanStandard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such nationalstandards may be obtained on application to the Management Centre or to any CEN member.This European Standard exists in three official versions (English, French, German). A version in any other language made by translationunder the responsibility of a CEN member into its own language and notified to the Management Centre has the same status as the officialversions.CEN members are the national standards bodies of Austria, Belgium, Czech Republic, Denmark, Finland, France, Germany, Greece,Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and United Kingdom.EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMITÉ EUROPÉEN DE NORMALISATIONEUROPÄISCHES KOMITEE FÜR NORMUNGManagement Centre: rue de Stassart, 36
B-1050 Brussels© 2000 CENAll rights of exploitation in any form and by any means reservedworldwide for CEN national Members.Ref. No. EN ISO 14181:2000 ESIST EN ISO 14181:2001



2EN ISO 14181:2000 (E)CORRECTED
2001-08-30ForewordThe text of the International Standard ISO 14181:2000 has been prepared by TechnicalCommittee ISO/TC 34 "Agricultural food products" in collaboration with Technical CommitteeCEN/TC 327 "Animal feeding stuffs - Methods of sampling and analysis", the secretariat ofwhich is held by NEN.This European Standard shall be given the status of a national standard, either by publicationof an identical text or by endorsement, at the latest by March 2001, and conflicting nationalstandards shall be withdrawn at the latest by March 2001.According to the CEN/CENELEC Internal Regulations, the national standards organizations ofthe following countries are bound to implement this European Standard: Austria, Belgium,Czech Republic, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Italy,Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and the UnitedKingdom.Endorsement noticeThe text of the International Standard ISO 14181:2000 was approved by CEN as a EuropeanStandard without any modification.NOTE: Normative references to International Standards are listed in annex ZA (normative).SIST EN ISO 14181:2001



3EN ISO 14181:2000 (E)Annex ZA (normative)Normative references to international publicationswith their relevant European publicationsThis European Standard incorporates by dated or undated reference, provisions from otherpublications. These normative references are cited at the appropriate places in the text and thepublications are listed hereafter. For dated references, subsequent amendments to or revisionsof any of these publications apply to this European Standard only when incorporated in it byamendment or revision. For undated references the latest edition of the publication referred toapplies (including amendments).NOTE Where an International Publication has been modified by common modifications,indicated by (mod.), the relevant EN/HD applies.PublicationYearTitleENYearISO 36961987Water for analytical laboratory use -Specification and test methodsEN ISO 36961995SIST EN ISO 14181:2001



SIST EN ISO 14181:2001



ReferencenumberISO14181:2000(E)©ISO2000INTERNATIONALSTANDARDISO14181Firstedition2000-09-15Animalfeedingstuffs—Determinationofresiduesoforganochlorinepesticides—GaschromatographicmethodAlimentsdesanimaux—Déterminationdesrésidusdepesticidesorganochlorés—MéthodeparchromatographieenphasegazeuseSIST EN ISO 14181:2001



ISO14181:2000(E)PDFdisclaimerThisPDFfilemaycontainembeddedtypefaces.InaccordancewithAdobe'slicensingpolicy,thisfilemaybeprintedorviewedbutshallnotbeeditedunlessthetypefaceswhichareembeddedarelicensedtoandinstalledonthecomputerperformingtheediting.Indownloadingthisfile,partiesacceptthereintheresponsibilityofnotinfringingAdobe'slicensingpolicy.TheISOCentralSecretariatacceptsnoliabilityinthisarea.AdobeisatrademarkofAdobeSystemsIncorporated.DetailsofthesoftwareproductsusedtocreatethisPDFfilecanbefoundintheGeneralInforelativetothefile;thePDF-creationparameterswereoptimizedforprinting.EverycarehasbeentakentoensurethatthefileissuitableforusebyISOmemberbodies.Intheunlikelyeventthataproblemrelatingtoitisfound,pleaseinformtheCentralSecretariatattheaddressgivenbelow.©ISO2000Allrightsreserved.Unlessotherwisespecified,nopartofthispublicationmaybereproducedorutilizedinanyformorbyanymeans,electronicormechanical,includingphotocopyingandmicrofilm,withoutpermissioninwritingfromeitherISOattheaddressbeloworISO'smemberbodyinthecountryoftherequester.ISOcopyrightofficeCasepostale56CH-1211Geneva20Tel.+41227490111Fax+41227490947E-mailcopyright@iso.chWebwww.iso.chPrintedinSwitzerlandii©ISO2000–AllrightsreservedSIST EN ISO 14181:2001



ISO14181:2000(E)©ISO2000–AllrightsreservediiiContentsPageForeword.iv1Scope.12Normativereferences.13Principle.14Reagentsandmaterials.15Apparatus.35.1Grinder.56Sampling.57Preparationoftestsample.58Procedure.58.1General.58.2Testportion.58.3Extraction.58.4Columnclean-up.68.5Gaschromatography.69Expressionofresults.79.1Calculation.79.2Recovery.810Confirmationofidentity.811Precision.811.1Interlaboratorytest.811.2Repeatability.811.3Reproducibility.812Testreport.9AnnexA(informative)ExamplesofGCoperatingconditionsfororganochlorinepesticides.10AnnexB(informative)Resultsofinterlaboratorytest.11Bibliography.20SIST EN ISO 14181:2001



ISO14181:2000(E)iv©ISO2000–AllrightsreservedForewordISO(theInternationalOrganizationforStandardization)isaworldwidefederationofnationalstandardsbodies(ISOmemberbodies).TheworkofpreparingInternationalStandardsisnormallycarriedoutthroughISOtechnicalcommittees.Eachmemberbodyinterestedinasubjectforwhichatechnicalcommitteehasbeenestablishedhastherighttoberepresentedonthatcommittee.Internationalorganizations,governmentalandnon-governmental,inliaisonwithISO,alsotakepartinthework.ISOcollaboratescloselywiththeInternationalElectrotechnicalCommission(IEC)onallmattersofelectrotechnicalstandardization.InternationalStandardsaredraftedinaccordancewiththerulesgivenintheISO/IECDirectives,Part3.DraftInternationalStandardsadoptedbythetechnicalcommitteesarecirculatedtothememberbodiesforvoting.PublicationasanInternationalStandardrequiresapprovalbyatleast75%ofthememberbodiescastingavote.AttentionisdrawntothepossibilitythatsomeoftheelementsofthisInternationalStandardmaybethesubjectofpatentrights.ISOshallnotbeheldresponsibleforidentifyinganyorallsuchpatentrights.InternationalStandardISO14181waspreparedbyTechnicalCommitteeISO/TC34,Foodproducts,SubcommitteeSC10,Animalfeedingstuffs.AnnexesAandBofthisInternationalStandardareforinformationonly.SIST EN ISO 14181:2001



INTERNATIONALSTANDARDISO14181:2000(E)©ISO2000–Allrightsreserved1Animalfeedingstuffs—Determinationofresiduesoforganochlorinepesticides—Gaschromatographicmethod1ScopeThisInternationalStandardspecifiesagaschromatographicmethodforthedeterminationofresiduesoforganochlorinepesticidesinanimalfeedingstuffs.Themethodisapplicabletoanimalfeedingstuffscontainingresiduesofoneormoreofthefollowingorganochlorinepesticidesandsomeoftheirisomersanddegradationproducts:aldrin,op'-DDE,pp'-DDE,op'-DDT,pp'-DDT,dieldrin,endosulfan,endrin,HCB,-HCH(-BHC),-HCH(-BHC),-HCH(-BHC),-HCH(-BHC),heptachlor,heptachlorepoxide,op'-TDE(op'-DDD),pp'-TDE(pp'-DDD)andmethoxychlor.Thelowerdeterminationlimitfortheseorganochlorinepesticidesis0,005g/g.However,thelowerdeterminationlimitis0,01g/gforop'-DDTandpp'-DDT,and0,05g/gformethoxychlor.2NormativereferencesThefollowingnormativedocumentscontainprovisionswhich,throughreferenceinthistext,constituteprovisionsofthisInternationalStandard.Fordatedreferences,subsequentamendmentsto,orrevisionsof,anyofthesepublicationsdonotapply.However,partiestoagreementsbasedonthisInternationalStandardareencouragedtoinvestigatethepossibilityofapplyingthemostrecenteditionofthenormativedocumentindicatedbelow.Forundatedreferences,thelatesteditionofthenormativedocumentsreferredtoapplies.MembersofISOandIECmaintainregistersofcurrentlyvalidInternationalStandards.ISO3696:1987,Waterforanalyticallaboratoryuse—Specificationandtestmethods.ISO6498,Animalfeedingstuffs—Preparationoftestsamples.3PrincipleAtestportionisextractedwithacetone.Thefilteredextractisdilutedwithwaterandsaturatedsodiumchloridesolution.Thepesticidesarepartitionedintodichloromethane.Theconcentratedextractispurifiedonachromatographiccolumnof10%water-deactivatedsilicagel.Thepesticideresiduesaredeterminedbygaschromatographyusinganelectroncapturedetectororamassselectivedetector.4ReagentsandmaterialsUseonlyreagentsofrecognizedanalyticalgradeandwithapuritysuitableforpesticideresidueanalysis.Checkthepurityofthereagentsbyperformingablanktestunderthesameconditionsasusedinthemethod.Thechromatogramshouldnotshowanyinterferingimpurity.WARNING—Someoftheorganicsolventsaresuspectedcarcinogens.Usewithcare.4.1Water,complyingwithatleastgrade3inaccordancewithISO3696.SIST EN ISO 14181:2001



ISO14181:2000(E)2©ISO2000–Allrightsreserved4.2Hexane.4.3Acetone.4.4Dichloromethane.4.5Silicagel60,withamassfractionofwaterof10%.Activatesilicagel60,particlesize63mto200m,at130°Covernight,thencoolinadesiccator.Aftercoolingtoroomtemperature,pourthesilicagelintoanairtightglasscontainerandaddsufficientdistilledwatertobringthefinalmassfractionofwaterto10%.Shakethecontainermechanicallyorbyhandvigorouslyfor30sandallowtostandfor30minwithoccasionalshaking.After30minthesilicagelisreadyforuse.Itshouldnotbestoredformorethan6h.4.6Elutingsolvent:dichloromethaneinhexane,20%(byvolume).Mix1volumeofdichloromethanewith4volumesofhexane.4.7Inertgas,forexamplenitrogen.4.8Sodiumsulfate,anhydrous.4.9Sodiumchloride,saturatedsolution.4.10Pesticidereferencestandards,asfollows:aldrin[(1R,4S,4aS,5S,8R,8aR)-1,2,3,4,10,10-hexachloro-1,4,4a,5,8,8a-hexahydro-1,4:5,8-dimethano-naphthalene];op'-DDE[o,p'-(1,1-dichloro-2,2-bis(4-chlorophenyl)ethylene)];pp'-DDE[p,p'-(1,1-dichloro-2,2-bis(4-chlorophenyl)ethylene)];op'-DDT[o,p'-(1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane)];pp'-DDT[p,p'-(1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane)];dieldrin[(1R,4S,4aS,5R,6R,7S,8S,8aR)-1,2,3,4,10,10-hexachloro-1,4,4a,5,6,7,8,8a-octahydro-6,7epoxy-1,4:5,8-dimethanonaphthalene];endosulfan(6,7,8,9,10,10-hexachloro-1,5,5a,6,9,9a-hexahydro-6,9-methano-2,4,3-benzodioxathiepin3-oxide);endrin[(1R,4S,4aS,5S,6S,7R,8R,8aR)-1,2,3,4,10,10-hexachloro-1,4,4a,5,6,7,8,8a-octahydro-6,7-epoxy-1,4:5,8-dimethanonaphthalene];HCB(hexachlorobenzene);-HCH(-BHC)(-1,2,3,4,5,6-hexachlorocyclohexane);-HCH(-BHC)(-1,2,3,4,5,6-hexachlorocyclohexane);-HCH(-BHC;lindane)(-1,2,3,4,5,6-hexachlorocyclohexane);-HCH(-BHC)(-1,2,3,4,5,6-hexachlorocyclohexane);heptachlor(1,4,5,6,7,8,8-heptachloro-3a,4,7,7a-tetrahydro-4,7-methanoindene);heptachlorepoxide(heptachloroepoxy-tetrahydromethanoindene);SIST EN ISO 14181:2001



ISO14181:2000(E)©ISO2000–Allrightsreserved3op'-TDE(op-DDD)[o,p'-1,1-dichloro-2,2-bis(4-chlorophenyl)ethane];pp'-TDE(pp'-DDD)[p,p'-1,1-dichloro-2,2-bis(4-chlorophenyl)ethane];methoxychlor[1,1,1-trichloro-2,2-bis(4-methoxyphenyl)ethane].4.11Internalstandard:mirex(1,3,5-tribromobenzene)orpentachloronitrobenzene.4.12Pesticidestandardandinternalstandardsolutions.4.12.1Stocksolutions,ofconcentration1000g/ml.Prepareastocksolutionofeachpesticidereferencestandard(4.10)andoftheinternalstandard(4.11)asfollows.Weigh,tothenearest0,1mg,amassofapesticidereferencestandard(4.10)ortheinternalstandard(4.11)whichwillresultinasolutionwithacontentofreferencestandardorinternalstandardof1000g/ml.Whileweighing,observethecleannessofthestandardmatrial.Transfertheweighedmassintoavolumetricflask,dissolveinhexaneorinanothersolventsuchastolueneorisooctane.However,dissolve-HCHinacetone.Dilutetovolumewiththesamesolventandmixwell.Thesesolutionsarestablefor6monthswhenstoredat4Cinthedark.4.12.2Intermediatesolutions,ofconcentration10g/ml.Pipette1mlofeachstocksolution(4.12.1)intoindividual100mlvolumetricflasks.Dilutetovolumewithhexane.Thesolutionsarestableforatleast3monthswhenstoredat4°Cinthedark.NOTEThestabilityofproperlystoredpesticidestandardsisverywidelyknown.Investigationshaveshownthatallneatpesticidestandardstestedarestablefor15yearswhenstoredat–18°Candthatstocksolutionsofpesticidestandardsintolueneof1mg/mlarestableforatleast3yearswhenstoredat–18°C.Thefollowingrecommendedpracticemaybeusedforlongerstorage.TransferportionsofthepreparedstandardsolutionstoambervialswithPTFE-linedscrewcaps.Weighthevialsandstoreat–20°C.Whenneeded,removeavialfromthefreezer,bringtoroomtemperatureandweigh.Iftheaccumulatedlossinmassduetoevaporationis10%ormoreoftheprefrozennetmass,discardthevial.Weighandrefreezestockstandardsandintermediatesolutionsthatareinuseformorethan3months(usuallyin25mlvials).Otherwisethepreparedstandardsolutions(usuallyin2mlvials)maybestoredat4°Candshallbediscardedafter3months.4.12.3Workingsolution,ofconcentration0,05g/ml.Pipettea0,5mlofeachintermediatesolution(4.12.2)intoa100mlvolumetricflaskanddilutetovolumewithhexane.Thesolutionisstablefor1monthwhenstoredat4°Cinthedark(see4.12.2).5ApparatusBeforeuse,washallglasswarethoroughlywithdetergentfreeofinterferingsubstances.Rinsewithwater,thenwithacetoneanddry.Avoidtheuseofplasticscontainersanddonotlubricatethestopcockswithgrease,otherwiseimpuritiescouldbeintroducedintothesolvents.Usuallaboratoryequipmentand,inparticular,thefollowing.5.1Separatingfunnels,of500mlcapacitywithpolytetrafluoroethylene(PTFE)stopcocksandstoppers.5.2Filteringflasks,of500mlcapacity.SIST EN ISO 14181:2001



ISO14181:2000(E)4©ISO2000–Allrightsreserved5.3Büchnerfunnel,ofporcelain,withinternaldiameter90mm.5.4Graduatedtubes,of10mlcapacity,withpolytetrafluoroethylene(PTFE)stoppers.5.5Glasschromatographictube,about300mminlength,8mmto10mminternaldiameter,withcoarsefrittedplateofporositygradeP100(poresizeindex40mto100m[2])orglasswoolplug.5.6Rotaryvacuumevaporator,providedwithround-bottomflasksof100mland500mlcapacitiesandawaterbathat40C2°C.5.7Mechanicalshakerorhigh-speedblender.5.8Gaschromatographicsystemconsistingof:splitlessoron-columninjectionsystem;column;electroncapturedetectorormassselectivedetector;electrometer;mVrecorderorintegrator;datahandlingsoftwareandcomputersystem.Eachinjectionport,columnovenanddetectorshallbeprovidedwithanindependentheatingdevicecontrolledtothenearest0,1C.Thechromatographicsystemshallbeadjustedandtheparametersoptimizedaccordingtothecharacteristicsoftheinstrumentused.5.8.1InjectiondeviceAnautosampleroranyotheradequateinjectiondevicemaybeused.Formanualinjections,useamicrosyringeof1lto5lcapacitywithaneedlelengthsuitableforthemodeofinjection(splitlessoron-column).Beforeinjectingthesolutionintothegaschromatograph,rinsethesyringe10timeswithpuresolvent,then5timeswithsolution.Afterinjection,rinsethesyringe5timeswithpuresolvent.5.8.2ColumnTheuseofcapillarycolumnscoatedwithmid-rangepolaritystationaryphases(e.g.SE-30,SE-54,OV-17,orequivalent)isrecommended.Standardglasscolumns,oflength2mto4mandofinternaldiameter2mmto4mm,packedwithamixtureof2,5%QF1+1%OV11+0,5%XE60onChromosorbWHP0,125mmto0,15mmparticlesize,oranyotherstationaryphasesandinertsupportsrecommendedfororganochlorineresidueanalysescouldbeusedasanalternative.Thetemperatureprogrammeforthecolumnshallbechosentoseparatethepesticidesmixturespecifiedinclause1intoindividualcomponents(seeannexA).Afterinstallinganewcolumn,itshallbeconditionedforatleast24hatatemperatureslightlyabovethemaximumproposedoperatingtemperature,withcarriergasflowingthroughitandtheendofthecolumndisconnectedfromthedetector.SIST EN ISO 14181:2001



ISO14181:2000(E)©ISO2000–Allrightsreserved55.8.3DetectorUseanelectroncapturedetector(ECD)operatingeitherinconstantcurrentmodeorinconstantfrequencymodeatthepolarizationvoltage,width,amplitudeorfrequencyofthepulsesatwhichamaximumof0,05ngheptachlorepoxidecauses40%to50%full-scaledeflection.5.8.4Carriergasandmake-upgasPurenitrogen(oxygen-free),pureheliumorpurehydrogen,oramixtureofargonandmethane[90+10,byvolumeor(95+5)%byvolume].5.9Grinder6SamplingItisimportantthatthelaboratoryreceiveasamplewhichistrulyrepresentativeandhasnotbeendamagedorchangedduringtransportorstorage.SamplingisnotpartofthemethodspecifiedinthisInternationalStandard.ArecommendedsamplingmethodisgiveninISO6497[1].7PreparationoftestsamplePreparethetestsampleinaccordancewithISO6498.Grindaportionofthewell-mixedlaboratorysample(dryorlowmoistureproductssuchascerealsandcerealproducts,oilseedsandoilseedmeals,mixedfeeds,hay,etc.)sothatitpassescompletelythroughasievewith1mmapertures.Mixthoroughly.Chopupthehighmoistureproducts(e.g.grasses,silages,etc.)andmixthoroughlytoobtainhomogeneoussamples.8Procedure8.1GeneralCarryoutsteps8.2to8.4usingboththepreparedtestsample(clause7)andablanksample.Theblanksampleshallbefreeofresiduesatorabovethelimitsofdetectionfoundfrompreviousdeterminations.Theblankextractisusedtopreparethematrix-matchedstandardsolution(see8.5.3).8.2TestportionWeigh,tothenearest0,1g,50gofthepreparedtestsample(clause7)fordryorlowmoistureproducts,or100gforhighmoistureproducts,intoa1000mlconicalflask.8.3ExtractionAddsufficientwatertothetestportionsothatatotalwateramountofabout100gisobtained.Allowthesampletosoakforabout5min.Add200mlofacetone.Closetheflasktightlyandshakefor2honamechanicalshakerorhomogenizefor2mininahigh-speedblender.FilterthehomogenatewithsuctionthroughaBüchnerfunnel(5.3)fittedwithfilterpaperofmediumporosity,intoa500mlfilteringflask(5.2).Washtheconicalflaskortheblendercupandtheresidueonthefilterpaperwithtwo25mlportionsofacetone,collectingthewashingsinthesamefilteringflask.SIST EN ISO 14181:2001



ISO14181:2000(E)6©ISO2000–AllrightsreservedMeasurethevolumeofthefiltrate(V1)andtransferone-fifthofthesolution(V2)toa500mlseparatingfunnel.Add250mlofwater,about50mlofsaturatedsodiumchloridesolution(4.9)and100mlofdichloromethane(4.4)totheseparatingfunnel.Stopperandshakefor2min.Allowthephasestoseparateanddrawoffthelowerphase(dichloromethane)intoasecond500mlseparatingfunnel(5.1).Repeattheextractiontwicewith50mlofdichloromethanethencombinetheextractsinthesame500mlseparatingfunnel.Washthedichloromethaneextractwithtwo100mlportionsofwater,discardingthewashings.Filterthedichloromethaneextractthroughafilterpapercontainingabout20gofsodiumsulfate(4.8)intoa500mlflaskofavacuumevaporator.Rinsetheseparatingfunnelandthesodiumsulfatewithtwo10mlportionsofdichloromethaneandaddtotheflask.Concentratethesolutiontoabout2mlundervacuumatatemperaturenotexceeding40C.Transferthesolutiontoa10mlgraduatedtubeusing1mlto2mlofhexaneandconcentrateundernitrogentoabout1ml.Donotallowthesolutiontodrycompletelyorlossesofpesticidesmayoccurbecauseofvolatilityorpoorsolubility.8.4Columnclean-up8.4.1PreparationofthecolumnTransfer5gofsilicagel(4.5)toaglasschromatographictube(5.5).Add5gofanhydroussodiumsulfate(4.8)onthetopofthesilicagel.Washthepreparedcolumnwith20mlofhexane.NOTEPrepackedsilicaorflorisilcartridges(e.g.Millipore-SEPPAK)maybeusedinsteadofasilicagelcolumn,aftercheckingforefficiencyandabsenceofinterferences.8.4.2PurificationTransferquantitativelytheconcentratedextract(8.3)onthetopofthecolumn(8.4.1)using1mlto2mlportionsofhexane.Elutetheorganochlorinepesticideswith50mlofelutingsolvent(4.6)andcollecttheeluateinthe100mlflaskoftherotaryvacuumevaporator(5.6).Concentratetheeluatetoabout2mlundervacuumatatemperaturenotexceeding40°C.Addabout10mlofhexaneandconcentrateoncemoretoabout1ml.Repeattwotimes,leavingabout1mlofhexaneatthefinalevaporation.Transfertoa10mlgraduatedtubeusing1mlto2mlofhexane.Dilutethesampleextractto10mlwithhexanefordeterminationbychromatography.Whenaninternalstandardmethodisused,add1mloftheintermediatesolution(4.12.2)oftheinternalstandard(4.11)tothefinalextractbeforedilutingto10mlwithhexane.Keeptheblankextracttopreparethematrix-matchedstandardsolution(see8.5).8.5Gaschromatography8.5.1PreparationofthesystemEquilibratethegaschromatographicsystemundertherecommendedoperatingconditions(5.8).Ifthecarriergasvolumeflowratethroughthecolumnisbelow25ml/min,introduceasupplementalcontributionofgasattheoutletofthecolumninordertoensureasufficientflowofgasthroughtheelectroncapturedetector(make-upgas).Drythecarriergasbypassingitthrough0,5nmmolecularsievetraps,previouslyactivatedat350Cfor4hto8h,installedinthecarriergasline.SIST EN ISO 14181:2001



ISO14181:2000(E)©ISO2000–Allrightsreserved7Reactivatethemolecularsieveseachtimeanewgascylinderisassembledandasoftenasneeded.8.5.2CheckingthelinearityofthesystemCheckthelinearityofthesystemfrom0,05ngto0,5ngofheptachlorepoxide.Prepareworkingsolutionswithhetachlorepoxidecontentsrangingfrom0,01g/mlto0,1g/ml.Inject1l.Plottheresponsefactor(area/mass,innanograms,ofheptachlorepoxideinjected)versusthemass,innanogramsofheptachlorepoxideinjected.Thegraphshallbestraightlineparalleltothex-axis.Ifnot,establishtherangeofconcentrationswithinwhichthedetectorresponseislinear.8.5.3DeterminationInject1lto2lofworkingstandardsolution(4.12.3),thenthesamevolumeofthesampleextract(8.4.2).Dilutethesampleextractifnecessary.Identifytheindividualpesticidepeaksonthebasisofretentiontimes.Determinetheamountofpesticidesbycomparingthesizeofthesamplepeakswiththoseoftheknownamountofthecorrespondingpesticidepeakintheworkingstandardsolution.Whentheresultsbasedonsolventstandardsindicateresiduelevelsatorabove50%oftheappropriateMRL,amatrix-matchedsolutionshouldbeused,preparedbyaddingtotheblankextract(8.4.2)appropriateamountsofintermediatesolutions(4.12.2)ofthosepesticidesidentifiedinthesamplesolution,sothatthesizeofthepeaksofthisreferencesolutioniswithin25%ofthesizeofthepeaksinthesamplesolution.Bringto10mlwithhexane.Injectintothegaschromatographthesamevolumeasforthesamplesolution.Determinetheamountofpesticidesbycomparingthesizeofthesamplepeakswiththoseoftheknownamountofthecorrespondingpesticidepeakinthematrix-matchedstandardsolution.9Expressionofresults9.1CalculationCalculatetheresidueofeachindividualpesticideinthetestsamplebytheequation:sis23si14AAmVVwAAmVVwherewisthecontentofanindividualpesticideresidue,inmicrogramspergram,inthetestsample;Aisthearea(orheight)ofthepesticidepeakinthesamplesolution;Asisthearea(orheight)ofthecorrespondingstandardpesticidepeakintheworkingstandardsolutionorinthematrix-matchedstandardsolution;Aiisthearea(orheight)oftheinternalstandardpeakinthesamplesolution;Asiisthearea(orheight)oftheinternalstandardpeakintheworkingstandardsolutionorinthematrix-matchedstandardsolution;msisthemass,innanograms,ofpesticideinthevolumeinjectedintothegaschromatograph;SIST EN ISO 14181:2001



ISO14181:2000(E)8©ISO2000–AllrightsreservedV1isthetotalvolumeoffiltrate,inmillilitres,obtainedintheextractionstep;V2isthevolumeofthefiltrate,inmillilitres,usedinthepurificationstep;V3isthefinalvolume,inmillilitres,ofthetestsolution,takingintoaccountanyfurtherdilutionthatisnecessary;V4isthevolume,inmicrolitres,ofthesamplesolutioninjectedintothegaschromatograph;misthemass,ingrams,ofthetestportion.9.2RecoveryVerifytheperformanceofthemethodbyrecoveryexperimentsmadeonfortifiedblanksamplesatthe0,05g/glevel.Addtothetestportionofablanksampleaknownamountofpesticidesolution.Letitstandfor30minthenanalysethefortifiedtestportiontogetherwiththeoriginalblanksamplewithnoaddedpesticides.Therecovery(%)iscalculatedforeachpesticideas100[(analysedamountinthefortifiedsampleminustheoriginalamountinthesample)/(addedamount)]Therecoverycoefficientforeachindividualpesticideshallbebetween70%and110%.Whenaresidueexceedingamaximumresiduelimit(MLR)istobeconfirmed,theconcurrentrecoverylevelshouldbeapproximatelysimilartothatofthesample.10ConfirmationofidentityAnalysesforconfirmationoftheidentityandquantityofpesticidesshallbeperformed,particularlywhentheresultsobtainedcorrespondtoorexceedthemaximumresiduelimits(MRLs),eitherbychromatographyonasecondcolumnofsignificantlydifferentpolarityor,wheretheapparatusisavailable,byusingGC-MStoquantifyandconfirmtheidentityofthepesticide.11Precision11.1InterlaboratorytestDetailsofaninterlaboratorytestontheprecisionofthemethodaregiveninannexB.Thevaluesderivedfromthistestmaynotbeapplicabletoconcentrationrangesandmatricesotherthanthosegiven.11.2RepeatabilityTheabsolutedifferencebetweentwoindependentsingletestresults,obtainedusingthesamemethodonidenticaltestmaterialinthesamelaboratorybythesameoperatorusingthesameequipmentwithinashortintervaloftime,willinnotmorethan5%ofcasesexceedtherepeatabilitylimitrderivedfromTablesB.1toB.17.11.3ReproducibilityTheabsolutedifferencebetweentwosingletestresults,obtainedusingthesamemethodonidenticaltestmaterialindifferentlaboratoriesbydifferentoperatorsusingdifferentequipment,willinnotmorethan5%ofcasesexceedthereproducibilitylimitRderivedfromTablesB.1toB.17.SIST EN ISO 14181:2001



ISO14181:2000(E)©ISO2000–Allrightsreserved912TestreportThetestreportshallspecify:allinformationnecessaryforthecompleteidentificationofthesample;thesamplingmethodused,ifknown;thetestmethodused,withreferencetothisInternationalStandard;alloperatingdetailsnotspecifiedinthisInternationalStandard,orregardedasoptional,togetherwithdetailsofanyincidentswhichmayhaveinfluencedthetestresult(s);thetestresultobtained,orthetwotestresultsobtainediftherepeatabilityhasbeenchecked.SIST EN ISO 14181:2001



ISO14181:2000(
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