EN 17362-2020 动物饲料 取样和分析方法 用LC-MSMS测定饲料和复合饲料中五氯酚（PCP）

EUROPEANSTANDARDNORMEEUROPEENNEEUROPÄISCHENORM

EN17362

April2020

ICS65.120

EnglishVersion

Animalfeedingstuffs:Methodsofsamplingandanalysis-

Determinationofpentachlorophenol(PCP)infeed

materialsandcompoundfeedbyLC-MS/MS

Alimentspouranimaux:Méthodesd'échantillonnage

etd'analyse-Déterminationdelateneuren

pentachlorophénol(PCP)danslesmatièrespremières

pouralimentsdesanimauxetlesalimentscomposés

pouranimauxparCL-SM/SM

Futtermittel:Probenahme-und

Untersuchungsverfahren-Bestimmungvon

Pentachlorphenol(PCP)inFuttermittelundMischfuttermittelmittelsLC-MS/MS

ThisEuropeanStandardwasapprovedbyCENon7March2020.

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

EUROPAISCHESKOMITEEFÜRNORMUNG

CEN-CENELECManagementCentre:RuedelaScience23,B-1040Brussels

◎2020CENAllrightsofexploitationinanyformandbyanymeansreservedRef.No.EN17362:2020EworldwideforCENnationalMembers.

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EN17362:2020(E)

ContentsPage

Europeanforeword 3

1Scope 4

2Normativereferences 4

3Termsanddefinitions 4

4Principle 7

5Reagentsandmaterials 7

5.1Genera 7

5.2Chemicals 7

6Apparatus 10

6.1Analyticalbalance,accuracyto0,1mg 10

6.2Centrifuge 10

6.3Waterbath 10

6.4Liquidchromatographconnectedtoatriple-quadrupolemassspectrometer 10

7Sampling 11

8Preparationofthetestsample 12

9Procedure 12

9.1Genera 12

9.2Testportionsandextraction 12

9.3LC-MS/MS 13

10Calculationandexpressionofresults 13

10.1General 13

10.2Calibrationcriteria 14

10.3Identificationandconfirmation 14

10.4Calculation 14

10.5Recovery 15

11Precision 15

11.1Collaborativetest 15

11.2Repeatabilitylimit 15

11.3Reproducibilitylimit 15

12Testreport 16

AnnexA(informative)Collaborativetrial 17

Bibliography 19

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EN17362:2020(E)

Europeanforeword

Thisdocument(EN17362:2020)hasbeenpreparedbyTechnicalCommitteeCEN/TC327“Animalfeedingstuffs:Methodsofsamplingandanalysis",thesecretariatofwhichisheldbyNEN.

Thisdocumentshallbegiventhestatusofanationalstandard,eitherbypublicationofanidenticaltextorbyendorsement,atthelatestbyOctober2020,andconflictingnationalstandardsshallbewithdrawnatthelatestbyOctober2020.

Attentionisdrawntothepossibilitythatsomeoftheelementsofthisdocumentmaybethesubjectofpatentrights.CENshallnotbeheldresponsibleforidentifyinganyorallsuchpatentrights.

ThisdocumenthasbeenpreparedunderastandardizationrequestgiventoCENbytheEuropeanCommissionandtheEuropeanFreeTradeAssociation.

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

Latvia,Lithuania,Luxembourg,Malta,Netherlands,Norway,Poland,Portugal,RepublicofNorth

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

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EN17362:2020(E)

1Scope

Thisdocumentspecifiesaliquidchromatographicmethodwithtriple-quadrupolemassspectrometry(MS/MS)detectionforthedeterminationofpentachlorophenol(PCP)infeedmaterialsandcompoundfeed.

Thelimitofquantitation(LOQ)forthePCPdeterminationinguargum,fattyaciddistillates(FAD)andcompoundfeedis10μg/kg.Individuallaboratoriesareresponsibleforensuringthattheequipmentthattheyusewillachievethislimitofquantification.

Themethodisvalidatedinaninternationalcollaborativetrialforpentachlorophenolincompoundfeed,guargumandfattyaciddistillateintherangebetween9μg/kgand22μg/kg.

Theresultsofthecollaborativetrial,inwhich16laboratoriesparticipated,haveshownthatthemethodisapplicableforthedeterminationofPCPincompoundfeed,guargumandFADatthedesiredlimitof10μg/kg.Satisfactoryresultswereobtainedforonecompoundfeedsample,guargumandthetwoFADsamples(HorRat<2),whileforthesecondcompoundfeedsampleaHorRatvalueof2,2wasobtained.

2Normativereferences

Thefollowingdocumentsarereferredtointhetextinsuchawaythatsomeoralloftheircontentconstitutesrequirementsofthisdocument.Fordatedreferences,onlytheeditioncitedapplies.Forundatedreferences,thelatesteditionofthereferenceddocument(includinganyamendments)applies.ENISO6498,Animalfeedingstufs-Guidelinesforsamplepreparation(ISO6498)

3Termsanddefinitions

Forthepurposesofthisdocument,thefollowingtermsanddefinitionsapply.

ISOandIECmaintainterminologicaldatabasesforuseinstandardizationatthefollowingaddresses:

一IECElectropedia:availableat

/

一ISOOnlinebrowsingplatform:availableat

/obp

3.1

calibration

completesetofoperationswhichestimatesunderspecifiedconditionsthecalibrationfunctionfromobservationsoftheresponsevariableobtainedonreferencestates

[SOURCE:ISO3534-2:2006,3.5.13[1]]

3.2

collaborativetrial

interlaboratorycomparisons

organization,performanceandevaluationofmeasurementsortestsonthesameorsimilaritemsbytwoormorelaboratoriesinaccordancewithpredeterminedconditions

[SOURCE:ENISO/IEC17043:2010,3.4[2]]

EN17362:2020(E)

5

3.3

feedmaterial

productsofvegetableoranimalorigin,whoseprincipalpurposeistomeetanimals'nutritionalneeds,intheirnaturalstate,freshorpreserved,andproductsderivedfromtheindustrialprocessingthereof,andorganicorinorganicsubstances,whetherornotcontainingfeedadditives,whichareintendedforuseinoralanimal-feedingeitherdirectlyassuch,orafterprocessing,orinthepreparationofcompoundfeed,orascarrierofpremixtures

[SOURCE:Regulation(EC)No767/2009(Article3(2)(g))[3]]

3.4

HorRat

ratioofthereproducibilityrelativestandarddeviationtothatcalculatedfromtheHorwitzequationNote1toentry:PredictedrelativestandarddeviationPRSDR=2C-0,15

HorRatr=RSDR/PRSDR(1)

HorRatr=RSDr/PRSDr(2)

Note2toentry:Ifappliedtowithin-laboratorystudies,thenormalrangeofHorRat(r)is0,30to1,30.

Note3toentry:TocheckpropercalculationofPRSDr,aCof10-6shouldgiveaPRSDrof16%.Cisconcentrationexpressedasamassfraction(bothnumeratoranddenominatorexpressedinthesameunits).TheHorRatisindicativeofmethodperformanceforalargemajorityofmethodsinchemistry.Normalvaluesliebetween0,50and2,00.

[SOURCE:ISO16577:2016,3.75[4]]

3.5

limitofquantitation

measuredquantityvalue,obtainedbyagivenmeasurementprocedure,whichisthelowestconcentrationofameasurandthatcanbedeterminedwithanacceptablelevelofrepeatabilityprecisionandtrueness

Note1toentry:The"limitofquantitation"isnotaconceptdefinedinISO/IECGuide99:2007[5]buthasbeendefinedasdescribedabove.

Note2toentry:TheabbreviationLOQissometimesused.

3.6

precision

closenessofagreementbetweenindicationsormeasuredquantityvaluesobtainedbyreplicate

measurementsonthesameorsimilarobjectsunderspecifiedconditions

Note1toentry:Measurementprecisionisusuallyexpressednumericallybymeasuresofimprecision,suchasstandarddeviation,variance,orcoefficientofvariationunderthespecifiedconditionsofmeasurement.

Note2toentry:The'specifiedconditions'canbe,forexample,repeatabilityconditionsofmeasurement,intermediateprecisionconditionsofmeasurement,orreproducibilityconditionsofmeasurement(seeISO5725-1[6]).

Note3toentry:Measurementprecisionisusedtodefinemeasurementrepeatability,intermediatemeasurementprecision,andmeasurementreproducibility.

Note4toentry:Sometimes'measurementprecision'iserroneouslyusedtomean'measurementaccuracy!.

EN17362:2020(E)

6

[SOURCE:ISOIECGuide99:2007,2.15[5]]

3.7

repeatability

precisionunderrepeatabilityconditions

Note1toentry:Repeatabilitycanbeexpressedquantitativelyintermsofthedispersioncharacteristicsoftheresults.

[SOURCE:ISO3534-2:2006,3.3.5[1]]

3.8

repeatabilitylimit

r

repeatabilitycriticaldifferenceforaspecifiedprobabilityof95%

[SOURCE:ISO3534-2:2006,3.3.9[1]]

3.9

repeatabilitystandarddeviation

standarddeviationoftestresultsormeasurementresultsobtainedunderrepeatabilityconditions

Note1toentry:Itisameasureofthedispersionofthedistributionoftestormeasurementresultsunderrepeatabilityconditions.

Note2toentry:Similarly,"repeatabilityvariance"and"repeatabilitycoefficientofvariation"canbedefinedandusedasmeasuresofthedispersionoftestormeasurementresultsunderrepeatabilityconditions.

[SOURCE:ISO3534-2:2006,3.3.7[1]]

3.10

reproducibility

precisionunderreproducibilityconditions

Note1toentry:Reproducibilitycanbeexpressedquantitativelyintermsofthedispersioncharacteristicsoftheresults.

Note2toentry:Resultsareusuallyunderstoodtobecorrectedresults.

[SOURCE:ISO3534-2:2006,3.3.10[1]]

3.11

reproducibilitylimit

R

reproducibilitycriticaldifferenceforaspecifiedprobabilityof95%

[SOURCE:ISO3534-2:2006,3.3.14[1]]

EN17362:2020(E)

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3.12

reproducibilitystandarddeviation

standarddeviationoftestresultsormeasurementresultsobtainedunderreproducibilityconditions

Note1toentry:Itisameasureofthedispersionofthedistributionoftestormeasurementresultsunderreproducibilityconditions.

Note2toentry:Similarly,"reproducibilityvariance"and"reproducibilitycoefficientofvariation"canbedefinedandusedasmeasuresofthedispersionoftestormeasurementresultsunderreproducibilityconditions.

[SOURCE:ISO3534-2:2006,3.3.12[1]]

4Principle

InordertocheckforthepresenceofPCP,atestportionofsamplematerialisfortifiedwithinternalstandards(13C-PCP).ThetestportionisextractedusingaQuEChERSapproachforcompoundfeedandFAD

A'reversed'QuEChERSapproachisusedforguargum.ForFADsamples,lipidsareremovedpriortoQuEChERSextractionbytheadditionofsulfuricacid.

Finalextractsfromallmatricesareanalysedbyliquidchromatographycoupledtotriplequadrupolemassspectrometry,operatedinnegativeelectrosprayionizationmode.

Identificationisdoneonthebasisofretentiontimeandmass-to-chargeratio.Quantificationisdoneusingtheinternalstandardmethod.

5Reagentsandmaterials

5.1General

Useonlyreagentsofrecognizedanalyticalgradeandwithapuritysuitablefororganochlorineresidueanalysis.Checkthepurityofthereagentsbyperformingablanktestunderthesameconditionsasusedinthemethod.Thechromatogramshouldnotshowanyinterferingimpurityattheretentiontimeofcompoundsofinterest.

5.2Chemicals

5.2.1Diethylamine(DEA)

5.2.2Acetonitrile(ACN)

5.2.3Methanol

5.2.4Acetone

5.2.5n-Hexane

5.2.6Sulphuricacid(concentrated,95%-98%)

5.2.7Deionizedwater

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EN17362:2020(E)

5.2.8Sodiumhydroxide

Sodiumhydroxidesolution(10M)

Weigh4gofsodiumhydroxide(5.2.8)andadd10mldeionizedwater(5.2.7)toachieveaconcentrationof10M.Storeatroomtemperatureinaclosedglassbottlewithaplasticcaporstopper.

5.2.9Magnesiumsulphate

5.2.10Sodiumchloride

5.2.11Formicacid

5.2.12Ammoniumformate

Ammoniumformatesolution(1M)

Weigh6,3gammoniumformate(5.2.12)andadd100mldeionizedwater(5.2.7)toachieveaconcentrationof1M.Storethesolutionatroomtemperature.Thesolutionisstableundertheseconditionsforatleast1month.

5.2.13MobilephaseA

Take1mlammoniumformatesolution(1M)(

)and

add999mldeionizedwater(5.2.7)and20μLformicacid(5.2.11).Mixwell.Storethesolutionatroomtemperature.Thesolutionisstableundertheseconditionsforatleast1month.

5.2.14MobilephaseB

Take1mlammoniumformatesolution(1M)(

)and

add50mldeionizedwater(5.2.7),949mlmethanol(5.2.3)and20μlformicacid(5.2.11)andmixwell.Storethesolutionatroomtemperature.Thesolutionisstableundertheseconditionsforatleast1month.

5.2.15Pentachlorophenol(PCPsodiumsalt,95%purity)

PCPStocksolution1(2000μg/ml)

Weigh25mg(±0,01mg)ofPCP(5.2.15)(takingimpuritiesintoconsideration)andadd12,5mldeionizedwater(5.2.7)toachieveaconcentrationof2000μg/ml.Storethesolutioninarefrigeratorat4℃(±3℃).Thesolutionisstableundertheseconditionsforatleast12months.

PCPStocksolution2(1ng/μl)

Dilute50μlofPCPStocksolution1(

)to

100mldeionizedwater(5.2.7)inavolumetricflask(5.2.20).Storethesolutioninarefrigeratorat4℃(±3℃).Thesolutionisstableundertheseconditionsforatleast12months.

PCPWorkingsolution1(0,1ng/μl)

Take100μLofPCPStocksolution2(

)and

add900μlacetonitrile(5.2.2).Preparethissolutiondaily.

PCPWorkingsolution2(0,01ng/μl)

Take50μlofPCPWorkingsolution1(

).Add450μlacetonitrile(5.2.2).Preparethissolutiondaily.

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EN17362:2020(E)

5.2.1613C₆-pentachlorophenolofcertifiedpurity(>99%,100ng/μlinnonane)asaninternalstandard

13C₆-PCPStocksolution(10ng/μl)

Take1mlof13C₆-PCPat100ng/μl(5.2.16)anddilutewithacetone(5.2.4)to10mlinavolumetricflask(5.2.20).Mixwell.Storethesolutioninarefrigeratorat4℃(±3℃).Thesolutionisstableundertheseconditionsforatleast12months.

13C₆-PCPWorkingsolution1(1ng/μl)

Take100μlof¹3C₆-PCPStocksolution(

)andadd900μlacetonitrile(5.2.2).Thesolutionisstable

undertheseconditionsforatleast12months.

13C6-PCPWorkingsolution2(0,1ng/μl)

Take50μlof13C₆-PCPWorkingsolution1(

)and

add450μlacetonitrile(5.2.2).Preparethissolutiondaily.

5.2.17Calibrationstandards

PreparecalibrationmixturesaccordingtoTable1inafinalvolumeof1,0mlofacetonitrile.Therangeofconcentrationscanbeadapteddependingontheexpectedconcentrationsinthesample,aslongastherangeislinear.Storethecalibrationmixturesat4℃(±3℃).Preparethesesolutionsdaily.

Table1-Calibrationmixtures

Level

Concentration

ng/ml

PCP

0,01ng/μl()

PCP

0,1ng/μl()

PCP

1ng/μl()

Internal

standard0,1ng/μl()

ACN

(5.2.2)

1

0

20μl

980μl

2

0,1

10μl

20μl

970μl

3

0,5

50μl

20μl

930μl

4

1,0

10μ

20μl

970μl

5

5,0

50μl

20μl

930μl

6

10

100μl

20μl

880μl

7

40

40μl

20μl

940μl

5.2.18Polypropylene(PP)screwcapcentrifugetube,50ml

5.2.19Glasstubewithscrewcap(polytetrafluoroethylene(PTFE)inlay),12ml

5.2.20Volumetricflask,10ml

5.2.21Volumetricflask,100ml

5.2.22LC-MSvial,2ml

5.2.23pHpaper,range0-14,oranothersuitabletestingrange

EN17362:2020(E)

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6Apparatus

6.1Analyticalbalance,accuracyto0,1mg

6.2Centrifuge

6.3Waterbath

6.4Liquidchromatographconnectedtoatriple-quadrupolemassspectrometer

6.4.1LCconditions

TheliquidchromatographisequippedwithaSymmetryC18column(5μm,3,0mm×150mm)orwithacomparablecolumn.Theflowofthemobilephaseiskeptconstantat500μl/min,whilethecolumniskeptat40℃.Theinjectionvolumeis20μl.

ThegradientprogramofmobilephasesAandB(5.2.13and5.2.14,respectively)ismentionedinTable2.

Table2-MobilephasegradientforLC-MS/MSanalysis

Time

[min]

MobilephaseA

[%]

MobilephaseB

[%]

Flow

[μl/min]

0

55

45

500

1

55

45

500

4

8

92

500

9

8

92

500

9,5

55

45

500

16

55

45

500

6.4.2MSconditions

Themass-spectrometershallbecapableofMultipleReactionMonitoring(MRM)andshouldbetunedaccordingtothemanufacturer'sdescription.InMS/MSmode,nofragmentationofPCPcanbeobservedatthehighestcollisionenergysetting.Therefore,aMRMtransitionchannelmaybeusedwithminimalcollisionenergyusingthesamemassesforprecursorandproductionselectioninordertoreducebackgroundnoise.TheMSparametersaregiveninTable3.

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Table3-MSparameters

Source

ESI

negative

Capillary(kV)

3,50

Cone(V)

30

Sourcetemperature(℃)

120

Desolvationtemperature(℃)

350

Conegasflow(l/h)

200

Desolvationgasflow(l/h)

600

Analyser

LM1resolution

12,5

HM1resolution

12,5

Ionenergy1

1,0

LM2resolution

12,5

HM2resolution

12,5

Ionenergy2

1

Multiplier

750

NOTEItisadvisedtooptimizethesesettingsfortheusedinstrument.Theshownvaluesareindicative.

TheselectedmassfragmentsareshowninTable4.Atleastonequantifiertransitionandonequalifiertransitionshallbemeasured.

NOTETheMRMtransitionqualifierandquantifiercanbeoptimizedfortheusedinstrument.

Table4—MRMtransitionsforPCP

Analyte

MRM

Transition

Quantifier

MRM

Transition

Qualifier1

MRM

Transition

Qualifier2

Dwell

time

[s]

Cone

[V]a

Collision

energy

[eV]a

PCP

262,8>262,8

264,8>264,8

266,8>266,8

0,2

30

7

13C6-PCP

272,8>272,8

274,8>274,8

276,8>276,8

0,2

30

7

aConevoltagesandcollisionenergiesshouldbeoptimizedfortheusedinstrument.Theshownvaluesareindicative.

7Sampling

Inordertobetrulyrepresentative,thesampleshallnothavebeendamagedorchangedduringtransportorstorage.Samplingisnotpartofthemethodspecifiedinthisdocument.ArecommendedsamplingmethodisgiveninENISO6497[7].

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EN17362:2020(E)

8Preparationofthetestsample

PreparethetestsampleinaccordancewithENISO6498.

Dryorlowmoistureproductssuchascerealsandcerealproducts,compoundfeedsandhayshouldbegroundcarefullysothatthetestsamplepassescompletelythroughasievewith1mmapertures.Mixthoroughly.

9Procedure

9.1General

Analyseineachseriesthefollowingsamples:

1)Proceduralblank;

2)Blankmatrix(n=1);

3)Blankmatrixspiked(n=2);

4)Allsamples.

NOTEAmatrixcouldbeguargum,fattyaciddistillateand/orcompoundfeed.Anyblankmatrixsampleproventobeblankinapreviousruncanbeusedforqualitycontrol.

9.2Testportionsandextraction

9.2.1Guargum

Weigh1,0g(±0,05g)ofthepreparedtestsampleintoa50mlPPscrewcapcentrifugetube(5.2.18).Add10mlacetonitrile(5.2.2)andshakeimmediatelyfor10s.Spikethesamplewith20μl13C6-PCPworkingsolution1(1ng/μl)(

).Add

10mldeionizedwater(5.2.7)andshakefor1min.Addthepre-weighedsalts[4gmagnesiumsulphate(5.2.9)and1gsodiumchloride(5.2.10)].Shakeimmediatelyfor1min.Centrifugefor5minat2000g.Transfer1mlacetonitrilephaseintoaLC-MSvial(5.2.22).

9.2.2Compoundfeed

Forawetcompoundfeedweigh10,0g(±0,10g)ofthepreparedtestsampleintoa50mlPPscrewcapcentrifugetube(5.2.18),andspikewith20μl13C₆-PCPWorkingsolution1(1ng/μl)(

)and

letitrestforatleast1h.

Foradrycompoundfeedweigh2,5g(±0,10g)ofthepreparedtestsampleintoa50mlPPscrewcapcentrifugetube(5.2.18)andspikewith20μl13C₆-PCPWorkingsolution1(1ng/μl)(

),add

7,5mldeionizedwater(5.2.7),andletitrestforatleast1h.

Add10mlacetonitrile(5.2.2)andshakefor1min.Addthepre-weighedsalts[4gmagnesiumsulphate(5.2.9)and1gsodiumchloride(5.2.10)],immediatelyshakeintenselyfor1min.Centrifugefor5minat2000g.Transfer1mlacetonitrilephaseintoaLC-MSvial(5.2.22).

9.2.3Fattyaciddistillate

Weigh1,0g(±0,05g)ofthepreparedtestsampleintoa12mlglasstubewithscrewcap(polytetrafluoroethylene(PTFE)inlay)(5.2.19).Spikethesamplewith20μl13C₆-PCPWorkingsolution1(1ng/μl)(

).Add

5mlofhexane(5.2.5)andmixuntilthesampleisdissolved.Add1mlconcentratedsulfuricacid(5.2.6).Placethetubesduring30mininawaterbath(6.3)at60℃.Mixcarefullyevery10min.

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EN17362:2020(E)

Leavethesamplestocooltoroomtemperatureandcentrifugefor5minat700g.Transfertheorganiclayertoacleanglasstube(5.2.19).

Repeattheextractionofthesulphuricacidinthefirsttube,usingasecondportionof5mlhexane(5.2.5).Combinethehexanephasesinthesecondglasstube.Add1mlconcentratedsulphuricacid(5.2.6)tothecombinedhexanephases.Placethetubesduring30mininawaterbath(6.3)at60℃.Mixcarefullyevery10min.Leavethesamplestocooltoroomtemperatureandcentrifugefor5minat700g.Transferthehexanetoa50mlPPscrewcapcentrifugetube(5.2.18).

Repeattheextractionofthesulphuricacidinthesecondglasstube,usingaportionof5mlhexane(5.2.5).Centrifugefor5minat2000g.Transferandcombinethehexaneinthe50mlPPscrewcapcentrifugetube.Add0,2ml10Msodiumhydroxide(5.2.8)and5mldeionizedwater(5.2.7)tothecombinedhexaneinthetube.Shakeduring1minandcentrifugefor5minat2000g.Transferthehexanetoasecond50mlPPscrewcapcentrifugetube(5.2.18).

Add0,1ml10Msodiumhydroxide(

)and

5mldeionizedwater(5.2.7)tothehexaneinthesecondtube.Shakeduring1minandcentrifugefor5minat2000g.Discardthehexanephasefromthesecondtube.Combinethewaterphasefrombothtubes,inthesecondtube.

Add10mlacetonitrile(5.2.2)tothecombinedwaterphasesandadjustthepHto<3byadding0,2mlconcentratedsulphuricacid(5.2.6).CheckwithapHpaper.Mixthetube,addthepre-weighedsalts[4gmagnesiumsulphate(5.2.9)and1gsodiumchloride(5.2.10)].Shakeimmediatelyfor1min.Centrifugefor5minat2000g.Transfer1mlacetonitrilephaseintoaLC-MSvial(5.2.22).

9.3LC-MS/MS

9.3.1Preparationofthesystem

EquilibratetheLCsystemundertherecommendedoperatingconditions(6.4).TuneandcalibratetheMS.

9.3.2Checkinginstrumentsettings

Injectcalibrationlevel5(5.2.17)andcheckpeakshapeandretentiontime.

9.3.3Determination

Injectasufficientaliquot(e.g.20μl)ofallthecalibrationstandardsolutions(5.2.17)andanequalvolumeofthesampleextracts.IdentifythePCPpeakonbasisofretentiontimeandtransitionionratio.

DeterminetheamountofPCPbycomparingthesizeofthesamplepeakswiththoseoftheknownamountinthecalibrationstandardsolutions(5.2.17).Calibrationisbasedoninternalstandardprinciple.

10Calculationandexpressionofresults

10.1General

Calculationsareperformedusingdataacquisitionsoftware.Settingsasbelow:

一Beforeprocessingalldata,theretentiontimeofPCPischeckedand,ifnecessary,modifiedintheprocessingmethod.

-Useaseven-pointinternalstandardcalibrationandcalculatethecorrelationcoefficientr².Itisadvisedtousea1/xweighting.

-Afterprocessingofthedata,everyresultismanuallycheckedforcorrectintegration.

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EN17362:2020(E)

10.2Calibrationcriteria

Criteriaforcorrelationcoefficientis>0,995.Theresultsshouldfitwithinthecalibrationcurve.Whenaresultexceedsthethresholdsofthecalibrationcurve,thesampleshouldbedilutedandreanalyseduntilitfitswithinthecalibrationcurve.

10.3Identificationandconfirmation

PCPisidentifiedbasedonitsretentiontimeandMRMtransitionsincludingtheratiooftheMRMtransitions.Therelativeintensitiesofthedetectedions,expressedasapercentageoftheintensityofthemostintenseion,shallcorrespondtothoseofthecalibrationstandard,eitherfromcalibrationstandardsolutionsorfromspikedsamples,atcomparableconcentrations,measuredunderthesameconditionswithinatoleranceof±30%.

ThedifferencebetweenthechromatographicretentiontimeofPCPandthatofthe13C₆-internalstandardshallbenomorethan0,1min.

10.4Calculation

Forallcalibrationlevelstherelativeresponsefactor(RRF)iscalculatedasfollows:

where

AxistheareaofthequantifierionofPCP;

Aisistheareaofthequantifierion