BS ISO 22104-2021 水質(zhì).微囊藻毒素的測定.液相色譜法和串聯(lián)質(zhì)譜法（LC-MSMS）

BSISO22104:2021

BSIStandardsPublication

Waterquality—Determinationofmicrocystins—Methodusingliquidchromatographyand

tandemmassspectrometry(LC-MS/MS)

bsi.

BSISO22104:2021BRITISHSTANDARD

Nationalforeword

ThisBritishStandardistheUKimplementationofISO22104:2021.

TheUKparticipationinitspreparationwasentrustedtoTechnicalCommitteeEH/3/2,Physicalchemicalandbiochemicalmethods.

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@TheBritishStandardsInstitution2021

PublishedbyBSIStandardsLimited2021

ISBN9780580523854

ICS13.060.50

CompliancewithaBritishStandardcannotconferimmunityfromlegalobligations.

ThisBritishStandardwaspublishedundertheauthorityofthe

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Amendments/corrigendaissuedsincepublication

DateTextaffected

BSISO22104:2021

INTERNATIONALSTANDARD

ISO22104

Firstedition2021-07-01

Waterquality—Determinationof

microcystins—Methodusingliquidchromatographyandtandemmassspectrometry(LC-MS/MS)

QualitédeI'eau—Dosagedesmicrocystines—Méthodepar

chromatographieenphaseliquidecoupléeàlaspectrometriedemasseentandem(CL-SM/SM)

ReferencenumberISO22104:2021(E)

◎ISO2021

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ISO22104:2021(E)

COPYRIGHTPROTECTEDDOCUMENT

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ContentsPage

Foreword V

1Scope 1

2Normativereferences 1

3Termsanddefinitions 1

4Principle 2

5Interferences 2

5.1Biases 3

5.2Limitations 3

6Reagentsandstandards 3

6.1General 3

6.1.7Concentratedphosphate-freedetergen 4

6.2Preparationofsolutions 4

6.2.3Stocksolutionofinternalstandardsubstances 4

6.2.4Internalstandardsolution(IS1) 5

6.2.5Internalstandardsolution(IS2) 5

6.2.6MCYSTmixsolution(S1) 5

6.2.7MCYSTmixsolution(S2) 5

6.2.8MCYSTmixsolution(S3) 5

6.2.9MCYSTmixAsolution 5

6.2.10MCYSTmixBsolution 5

6.2.11MCYSTmixCsolution 5

6.2.12MCYSTmixDsolution 6

6.2.13Instrumentcheckmix(high)solution 6

6.2.14Calibrationcontrolstandard(CS1) 6

6.2.15Calibrationcontrolstandard(CS2) 6

7Apparatus 6

7.3Microsyringes 6

7.13Ultrasonicbath 7

7.15Liquidchromatograph(LC) 7

7.17Massspectrometer(MS) 7

8Sampling 7

9Procedure 8

9.1Preparationofsamples 8

9.1.1Genera 8

9.1.2Preparationofmethodblanksample 8

9.1.3Preparationoflaboratorycontrolspikesample 8

9.1.4Preparationofcalibrationcontrolsample 8

9.1.5Preparationofcalibrationstandardsolutions 8

9.1.6Preparationofdrinkingwaterandfreshwatersample 9

9.1.7Samplepreparationprocedurewithfreeze/thawcycles 9

9.2InstrumentalanalysisbyLC-MS/MSprocedure 10

9.2.1Instrumentset-upparameters 10

9.3Runprocessingandqualityassurance 13

9.3.1Runsequence 13

9.3.2Runcontroloperations/limits 13

10Calibration 14

11Evaluationandcalculationofresults 15

11.1Identificationandcalculations 15

11.2Calibrationcurveequationdetermination 15

11.3Internalstandardcalculation 15

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11.4Internalstandardrecoverycalculation 16

12Expressingofresults 16

13Testrepor 16

AnnexA(informative)Useofhighresolutionmassspectrometrydetectors(HRMS) 17

AnnexB(informative)Useofonlinesolidphaseextractioncoupledtoliquid

chromatographyfortheautomatedanalysisofmicrocystins 19

AnnexC(informative)Useofmanualsolidphaseextractionpriortoinstrumentalanalysis

forimprovedmethoddetectionlimits 25

Bibliography 32

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ISO22104:2021

Foreword

ISO(theInternationalOrganizationforStandardization)isaworldwidefederationofnationalstandardsbodies(ISOmemberbodies).TheworkofpreparingInternationalStandardsisnormallycarriedoutthroughISOtechnicalcommittees.Eachmemberbodyinterestedinasubjectforwhichatechnicalcommitteehasbeenestablishedhastherighttoberepresentedonthatcommittee.Internationalorganizations,governmentalandnon-governmental,inliaisonwithISO,alsotakepartinthework.ISOcollaboratescloselywiththeInternationalElectrotechnicalCommission(IEC)onallmattersofelectrotechnicalstandardization.

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ThisdocumentwaspreparedbyTechnicalCommitteeISO/TC147,Waterquality,SubcommitteeSC2,Physical,chemicalandbiochemicalmethods.

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BSISO22104:2021

INTERNATIONALSTANDARDISO22104:2021

Waterquality—Determinationofmicrocystins—

Methodusingliquidchromatographyandtandemmassspectrometry(LC-MS/MS)

WARNING—Personsusingthisdocumentshouldbefamiliarwithnormallaboratorypractice.Thisdocumentdoesnotpurporttoaddressallofthesafetyproblems,ifany,associatedwithitsuse.Itistheresponsibilityoftheusertoestablishappropriatesafetyandhealthpractices.

IMPORTANT—Itisabsolutelyessentialthattestsconductedinaccordancewiththisdocumentbecarriedoutbysuitablyqualifiedstaff.

1Scope

Thisdocumentspecifiesamethodforthequantificationoftwelvemicrocystinvariants(microcystin-LR,-LA,-YR,-RR,-LY,-WR,-HtyR,-HilR,-LW,-LF,[Dha?]-microcystin-LR,and[Dha7]-microcystin-RR)indrinkingwaterandfreshwatersamplesbetween0,05μg/lto1,6μg/1.Themethodcanbeusedtodeterminefurthermicrocystins,providedthatanalyticalconditionsforchromatographyandmassspectrometricdetectionhasbeentestedandvalidatedforeachmicrocystin.SamplesareanalysedbyLC-MS/MSusinginternalstandardcalibration.

Thismethodisperformancebased.Thelaboratoryispermittedtomodifythemethod,e.g.increasingdirectflowinjectionvolumeforlowinterferencesamplesordilutingthesamplestoincreasetheupperrworkingrangelimit,providedthatallperformancecriteriainthismethodaremet.

Detectionofmicrocystinsbyhighresolutionmassspectrometry(HRMS)asanalternativefortandemmassspectrometry(MS/MS)isdescribedinAnnexA.

Analternativeautomatedsamplepreparationmethodbasedonon-linesolidphaseextractioncoupledtoliquidchromatographyisdescribedinAnnexB.

Wheninstrumentalsensitivityisnotsufficienttoreachthemethoddetectionlimitsbydirectflowinjection,asolidphaseextractionclean-upandconcentrationstepisdescribedinAnnexC.

2Normativereferences

Thefollowingdocumentsarereferredtointhetextinsuchawaythatsomeoralloftheircontentconstitutesrequirementsofthisdocument.Fordatedreferences,onlytheeditioncitedapplies.Forundatedreferences,thelatesteditionofthereferenceddocument(includinganyamendments)applies.

ISO3696,Waterforanalyticallaboratoryuse—Specificationandtestmethods

3Termsanddefinitions

Notermsanddefinitionsarelistedinthisdocument.

ISOandIECmaintainterminologicaldatabasesforuseinstandardizationatthefollowingaddresses:

—ISOOnlinebrowsingplatform:availableat

/obp

一IECElectropedia:availableat

/

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4Principle

Thismethodisdesignedtoidentifyandquantifytotal(free+intracellular)microcystinsinwaterbydirectflowinjectionliquidchromatographyandtandemmassspectrometry(LC-MS/MS)withelectrosprayionizationl1J,[21.12microcystins(Table1)aredeterminedquantitativelybymulti-pointcalibrationusingnodularinasinternalstandard.

Table1—Microcystinvariantsincludedinthemethod

Microcystinvariant

CAS-RNa

Molecularformular

Microcystin-LR

101043-37-2

C?9H?74N10012

Microcystin-RR

111755-374

C49H75N13012

Microcystin-LA

96180-79-9

C46H67N?O12

Microcystin-YR

101064-48-6

C52H?2N10013

Microcystin-LY

123304-10-9

C52H71N?013

Microcystin-WR

138234-58-9

C54H73N11012

Microcystin-HtyR

913178-65-1

C52H?2N10013

Microcystin-HilR

N/A

CsoH76N10012

Microcystin-LW

157622-02-1

C54H?2N?O12

Microcystin-LF

154037-70-4

C52H71N?012

[Dha7]-Microcystin-LR(dmLR)

120011-66-7

C48H?2N10012

[Dha7]-Microcystin-RR(dmRR)

131022-02-1

C48H73N13012

aCAS-RN:ChemicalAbstractsSystemRegistrationNumber.

Nodularincanbenaturallyoccurringinbrackishwatersamples.Blanklevelsshouldbecheckedbeforeanalysisforthesesamples.Alternatively,15N-labelledmicrocystinsurrogatesshouldbeusedifavailable.

NOTESomemicrocystins(e.g.demethylatedRRvariants)havethesameexactmassandasimilarchromatographicbehaviour.Whilesomecanbedistinguishedbytheirfragmentation(e.g.[Asp3,Mdha?]MC-RRand[MeAsp3,Dha?]]MC-RR),othersevenshowthesamefragmentation[e.g.Asp3,Mdha?]MC-RRand[Asp3,Dhb]MC-RR).

Watersamplesarehomogenizedtodispersecellaggregates.A5mlaliquotistransferredtoa15mlcentrifugetube,internalstandardisadded,andcellsarelysedbythreecyclesoffreeze/thaw.Solidparticlesandcelldebrisarecentrifugedandsyringefiltereddirectlyintoanautosamplervial.QuantificationofmicrocystinsisdonebyaninternalstandardmethodusingLC-MS/MSorHRMS(AnnexA).

Alternatively,lysedandfilteredsamplescanbeinjectedusinganon-lineSPEinstrumentalconfiguration(AnnexB)ormanualSPE(AnnexC)foranincreasedsensitivity.

5Interferences

Thisanalysiswasdevelopedusingliquidchromatography(LC)tandemmassspectrometry(MS/MS)withelectrosprayionization(ESI),onatriplequadrupolemassspectrometer.Acquisitionmodewasbasedonmultiplereactionmonitoring(MRM).Isobaricinterferencesthatarenotresolvedbychromatographyortheunitmassresolutionofthetandemquadrupolesmaybepresentinsomesamples.Thesesamplesmayrequireadditionalselectivityviaadditionalsampleclean-upand/orhigh-resolutionmassspectrometry(HRMS).SomemicrocystinswiththesameexactmassmightnotbeabletobedistinguishedbyHRMS,butbytheirdifferentfragmentationpatterns,afewcongenerscannotbedistinguishedbyeitheroftheseapproaches.

Variableinstrumentresponseand/orinconsistentretentiontimesmaybeobservedinthefirstgradientrunsoftheday.Thecolumnrequiresconditioningbyrunningatleastonegradientprogrampriortothefirstsampleinjectionoftheday.

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5.1Biases

Alllabwarethatcontactsmicrocystinsshouldhaverelativelyinertsurfaces;otherwise,compoundlossesmayoccurbyadsorptionontotheglass.Unscratchedborosilicateglasswareorpolyethyleneisrecommended.Tofurtherminimizethiseffect,samplepreparationshouldbecarriedoutinatimelymannerandquantificationbymatrixmatchedcalibrationstandardsispreferred.

Analyticalresults(methodprecisionandaccuracy)arecalculatedbyinternalstandardquantitationmethodsandmaybeaffectedbydifferencesintherecoveryoftheinternalstandardrelativetothatofthetargetcompounds.Whenavailable,15N-labelledmicrocystinsshouldbeusedforthispurpose.

Theconcentrationofon-sitesampleswillvarygreatlydependingonthedensityofalgaeateachsamplingpoint,andtheconcentrationdifferencewillalsobelargeforeachmicrocystins.Giventhis,themulti-pointcalibrationcurvesforthemicrocystins,usingafixedamountofinternalstandard,arenon-linear.Quantificationisdonebyasecondorder(quadratic)curve-fittingprocedure.

5.2Limitations

Thesamplepreparationmethodisrestrictedtowatersamples.Applicabilityofthemethodtosampleswithveryhighorganiccontent,suchaswatercontaininghighconcentrationsofhumicmaterials,isunknown.

Theworkingrangeofthismethodis0,05μg/lto1,6μg/l.Ifsampleswithahighermicrocystinconcentrationthan1,5μg/larefoundorpredicted,asmalleraliquotofsampleshouldbetaken,andadilutionfactorappliedtothefinalresult.Surfacewaterscontainingthickcyanobacterialbloomsmayinterferewiththeinstrumentalanalysis.Inthesecases,asmalleramountofsamplecanbediluted,andvolumeshouldberecordedforthefinalcalculationofmicrocystinsconcentration.

Standardsofspecificmicrocystinvariantsarenotalwaysavailableonacontinuousbasis.Foreignsuppliersaresometimesrestrictedbylawandarenotalwaysabletoexportalgaltoxinstandardstodifferentcountries.Beforebeingused,newlypreparedstandardsshallbecomparedtostandardsincurrentuse.Purityofthedifferentlotsofstandardsshouldbecheckedagainstreferencematerialswhenavailable.Alternatively,puritycanalsobeconfirmedusinguniversaldetectorlikeHPLC-UV(ISO20179).

6Reagentsandstandards

6.1General

Ifavailable,reagentsofpuritygrade"foranalysis"or“forresidueanalysis”areused.Theamountofimpuritiescontributingtotheblankvalueorcausingsignalinterferencesshallbenegligible.Thisshallbecheckedregularly(seesectionforblankvaluemeasurements).

Solvent,waterandreagentsintendedforuseaselutionagentsshallbecompatiblewithHPLCandmassspectrometry.

Microcystinsarepotenthepatotoxins.Laboratorysafetymeasuresshouldbestrictlyfollowedthroughoutthesamplepreparation(includinglabgloves,labcoat,safetyglasses)topreventhumanexposuretothesetoxins.

NOTE1Highpuritygradesofsolventapplicableforuseareavailablecommercially.

NOTE2Reagentslistedas“prepareasrequired”haveanexpirydateofoneyearfromthemomenttheywereprepared.

NOTE3Preparedstandardsolutionsarestoredat(5±3)°℃,withanexpirydateofoneyearfromthemomenttheywereprepared.

Stockandintermediatestandardsolutionsshouldbeusedasareference,otherstockandintermediateconcentrationsareacceptabletopreparethefinalworkingsolutions.

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6.1.1Water,conformingwiththerequirementsofISO3696,grade1orequivalentandwithoutanyinterferingblankvalues.

6.1.2Methanol,CH?OH,LC-MSgrade.

6.1.3Acetonitrile,CH?CN,LC-MSgrade.

6.1.4Formicacid,CHCOOH,LC-MSgrade,massfraction≥98%.

6.1.5Electrospraytuningmixture,inaccordancewiththespecificationoftheinstrumentmanufacturer.

6.1.6Sodiumthiosulfatepentahydrate,Na2S?O3·5H?0,99%purity.

6.1.7Concentratedphosphate-freedetergent.

6.1.8InternalstandardsubstanceslikeNodularin,(CASno118399-22-7,≥95%puritydeterminedbyHPLC)orisotopelabelledcompoundsofreferencesubstances.

6.1.9ReferenceSubstancesaslistedinTable1,withknownmassfractionorpurity≥95%determinedbyHPLC.

6.1.10Microcystin-LR,10ng/μlcertifiedreferencestandard.

6.2Preparationofsolutions

6.2.1Tapwater,quenchedwithsodiumthiosulfateat150mg/l(forcalibrationstandardsolutions,QCsamplesandsampledilutions).

Themethodblank,calibrationstandardsolutions,QCsamplesandsampledilutions(ifnecessary)aremadewithquenchedlaboratorytapwater.Thisquenchedwaterismadebytaking1loftapwaterandadding1,5mlofsodiumthiosulfatepreservativesolution(i.e.150mgsodiumthiosulfate)(6.2.2).Capthebottleandshakevigorouslytomix.Thiswaterispreparedasrequiredbeforesamplepreparationinordertoquenchanyresidualchlorineinthetapwaterwhichwouldoxidizethemicrocystins.Storethereagentwateratroomtemperature.Quenchingisnotnecessaryifitcanbeensuredthattheusedtapwaterisprovidedwithoutchlorination.

NOTEDependingontheapplication,methodblank,calibrationstandardsolutions,QCsamplesandsampledilutionscanbepreparedwithothermatricessuchasmineralwater.

6.2.2Sodiumthiosulfatepreservativesolution,Na?S?O3,100mg/ml.

Intoa11volumetricflaskput157gofNa?S?O?·5H?O(6.1.6),correspondingto100gofanhydrousNa?S?O3.Dissolveinwater(6.1.1),andmakeupto11withpurewater.Prepareasrequired.Storethepreservativeatroomtemperature.

6.2.3Stocksolutionofinternalstandardsubstances

Preparesolutionswithamassconcentrationof,forexample,200ng/μl.

Forthisuse,forexample,transfer10mgofaninternalstandard(6.1.8)toaseparate50mlvolumetricflaskanddissolveitinmethanol(6.1.2).Filluptothe50mlmarkwithmethanol(6.1.2).Theconcentrationofthissolutionis200ng/μl.

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6.2.4Internalstandardsolution(IS1)

Prepareaworkingsolutionwithinternalstandardmassconcentrationsof,forexample,8,0ng/μleach.

Forthisuse,forexample,transfer1,0mlofeachinternalstandardstocksolution(6.2.3)toa25mlflaskandfilluptothemarkwithmethanol(6.1.2).

6.2.5Internalstandardsolution(IS2)

Prepareaworkingsolutionwithinternalstandardmassconcentrationsof,forexample,80pg/μleach.

Forthisuse,forexample,transfer250μlofeachinternalstandardstocksolution(6.2.4)toa25mlflaskandfilluptothemarkwithmethanol(6.1.2).

6.2.6MCYSTmixsolution(S1)

Prepareasolutionwithmicrocystinmassconcentrationsof,forexample,4ng/μl.

Forthisuse,forexample,transfer100μg[Dha?]microcystin-LR(dmLR),100μg[Dha?]microcystin-RR(dmRR),100μgmicrocystin-LF,100μgmicrocystin-LW,100μgmicrocystin-WR,100μgmicrocystin-LY,100μgmicrocystin-HtyR,and100μgmicrocystin-HilRtoa25mlvolumetricflaskanddissolveitinmethanol(6.1.2).Makeupto25mlwithmethanol(6.1.2).Theconcentrationofeachmicrocystinis4ng/μl.

6.2.7MCYSTmixsolution(S2)

Prepareasolutionwithmicrocystinmassconcentrationsof,forexample,400pg/μl.

Forthisuse,forexample,transfer2500μlofsupplementalmicrocystinmixsolutionsolution(6.2.6)toa25mlflaskandfilluptothemarkwithmethanol(6.1.2)

6.2.8MCYSTmixsolution(S3)

Prepareasolutionwithmicrocystinmassconcentrationsof,forexample,40pg/μl.

Forthisuse,forexample,transfer250μlofsupplementalmicrocystinmixsolution(6.2.6)toa25mlflaskandfilluptothemarkwithmethanol(6.1.2).

6.2.9MCYSTmixAsolution

Prepareasolutionwithmicrocystinmassconcentrationsof,forexample,20ng/μl

Forthisuse,forexample,transfer500μgofmicrocystin-LR,500μgofmicrocystin-RR,500μgofmicrocystin-YRand500μgofmicrocystin-LAtoa25mlvolumetricflaskanddissolveitinmethanol(6.1.2).Makeupto25mlwithmethanol(6.1.2).Theconcentrationofeachmicrocystinis20ng/μl.

6.2.10MCYSTmixBsolution

Prepareasolutionwithmicrocystinmassconcentrationsof,forexample,2,0ng/μl.

Forthisuse,forexample,dilute2,5mlofMCYSTmixAsolution(6.2.9)to25mlwithmethanolina25mlvolumetricflask.Theconcentrationofeachmicrocystinis2,0ng/μl

6.2.11MCYSTmixCsolution

Prepareasolutionwithmicrocystinmassconcentrationsof,forexample,200pg/μl.

Forthisuse,forexample,dilute250μlofMCYSTmixsolutionA(6.2.9)to25mlwithmethanolina25mlvolumetricflask.Theconcentrationofeachmicrocystinis200pg/μl.

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6.2.12MCYSTmixDsolution

Prepareasolutionwithmicrocystinmassconcentrationsof,forexample,20pg/μl.

Forthisuse,forexample,dilute25μlofMCYSTmixsolutionA(6.2.9)to25mlwithmethanolina25mlvolumetricflask.Theconcentrationofeachmicrocystinis20pg/μl.

6.2.13Instrumentcheckmix(high)solution

Intoa25mlvolumetricflaskput25μlofMCYSTmixsolutionA(6.2.9)and250μlofsupplementalmicrocystinmixsolution(6.2.6).Makeupto25mlwithpurewater(6.1.1).Theconcentrationsofmicrocystins-LR(6.2.10),-RR,-LA,-YRare20pg/μl.Theremainingsupplementalmicrocystinsareataconcentrationof40pg/μl.

6.2.14Calibrationcontrolstandard(CS1)

Thecalibrationcontrolstandardisareferencesubstancesolutionproducedindependentlyoftheotherstocksolutions(6.2.5to6.2.13),e.g.asolutionfromanalternativebatchormanufacturer.

Forthisuse,forexample,amicrocystin-LR,10ng/μlcertifiedconcentrationstandardcanbepurchasedorprepared.

Othermicrocystinswithcertifiedconcentrationshouldalsobeusedtovalidatestandardmixtureconcentrationwhenavailable.

6.2.15Calibrationcontrolstandard(CS2)

Prepareasolutionwithmicrocystinmassconcentrationsof,forexample,100pg/μl.

Forthisuse,forexample,dilute100μlofcalibrationcontrolstandardCS1(6.2.14)to10mlwithmethanolina10mlvolumetricflask.Theconcentrationofea