1、BRITISH STANDARD BS EN 13585:2002 Foodstuffs Determination of fumonisins B1 and B2 in maize HPLC method with solid phase extraction clean-up The European Standard EN 13585:2001 has the status of a British Standard ICS 67.060 NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBS EN
2、 13585:2002 This British Standard, having been prepared under the direction of the Consumer Products and Services Sector Policy and Strategy Committee, was published under the authority of the Standards Policy and Strategy Committee on 04 March 2002 BSI 04 March 2002 ISBN 0 580 39204 X National fore
3、word This British Standard is the official English language version of EN 13585:2001. The UK participation in its preparation was entrusted to Technical Committee AW/-/3, Food analysis Horizontal methods, which has the responsibility to: A list of organizations represented on this committee can be o
4、btained on request to its secretary. Cross-references The British Standards which implement international or European publications referred to in this document may be found in the BSI Standards Catalogue under the section entitled “International Standards Correspondence Index”, or by using the “Find
5、” facility of the BSI Standards Electronic Catalogue. A British Standard does not purport to include all the necessary provisions of a contract. Users of British Standards are responsible for their correct application. Compliance with a British Standard does not of itself confer immunity from legal
6、obligations. aid enquirers to understand the text; present to the responsible European committee any enquiries on the interpretation, or proposals for change, and keep the UK interests informed; monitor related international and European developments and promulgate them in the UK. Summary of pages T
7、his document comprises a front cover, an inside front cover, the EN title page, pages 2 to 14, and an inside back cover and a back cover. The BSI copyright date displayed in this document indicates when the document was last issued. Amendments issued since publication Amd. No. Date CommentsEUROPEANS
8、TANDARD NORMEEUROPENNE EUROPISCHENORM EN13585 December2001 ICS67.060 Englishversion FoodstuffsDeterminationoffumonisinsB1andB2inmaize HPLCmethodwithsolidphaseextractioncleanup ProduitsalimentairesDosagedesfumonisinesB1etB2 danslemasMthodeCLHPavecpurificationpar extractionenphasesolide LebensmittelBe
9、stimmungvonFumonisinB1undB2in MaisHPLCVerfahrenmitReinigungdurch Festphasenextraktion ThisEuropeanStandardwasapprovedbyCENon2November2001. CENmembersareboundtocomplywiththeCEN/CENELECInternalRegulationswhichstipulatetheconditionsforgivingthisEurope an Standardthestatusofanationalstandardwithoutanyal
10、teration.Uptodatelistsandbibliographicalreferencesconcernings uchnational standardsmaybeobtainedonapplicationtotheManagementCentreortoanyCENmember. ThisEuropeanStandardexistsinthreeofficialversions(English,French,German).Aversioninanyotherlanguagemadebytra nslation undertheresponsibilityofaCENmember
11、intoitsownlanguageandnotifiedtotheManagementCentrehasthesamestatusasthe official versions. CENmembersarethenationalstandardsbodiesofAustria,Belgium,CzechRepublic,Denmark,Finland,France,Germany,Greece, Iceland,Ireland,Italy,Luxembourg,Netherlands,Norway,Portugal,Spain,Sweden,SwitzerlandandUnitedKingd
12、om. EUROPEANCOMMITTEEFORSTANDARDIZATION COMITEUROPENDENORMALISATION EUROPISCHESKOMITEEFRNORMUNG ManagementCentre:ruedeStassart,36B1050Brussels 2001CEN Allrightsofexploitationinanyformandbyanymeansreserved worldwideforCENnationalMembers. Ref.No.EN13585:2001EEN13585:2001(E) 2 Contents page Foreword2 1
13、 Scope 3 2 Normativereferences 3 3 Principle3 4 Reagentsandmaterials .3 5 Apparatus .4 6 Sampling.5 7 Preparationofthetestsample .5 8 Procedure .6 9 Calculation6 10 Precision.7 11 Testreport 8 AnnexA (informative) Typicalchromatogram10 AnnexB (informative) Recoveryandrelativestandarddeviation11 Anne
14、xC (informative) Precisiondata. .12 Bibliography 15 Foreword ThisEuropeanStandardhasbeenpreparedbyTechnicalCommitteeCEN/TC275,“FoodanalysisHorizontal methods“,thesecretariatofwhichisheldbyDIN. ThisEuropeanStandardshallbegiventhestatusofanationalstandard,eitherbypublicationofanidenticaltextor byendor
15、sement,atthelatestbyJune2002,andconflictingnationalstandardsshallbewithdrawnatthelatestby June2002. TheannexesA,BandCareinformative. AccordingtotheCEN/CENELECInternalRegulations,thenationalstandardsorganizationsofthefollowing countriesareboundtoimplementthisEuropeanStandard:Austria,Belgium,CzechRepu
16、blic,Denmark,Finland, France,Germany,Greece,Iceland,Ireland,Italy,Luxembourg,Netherlands,Norway,Portugal,Spain,Sweden, SwitzerlandandtheUnitedKingdom.EN13585:2001(E) 3 1Scope ThisEuropeanStandardspecifiesamethodforthedeterminationoffumonisinB 1 (FB 1 )andfumonisinB 2 (FB 2 )in maizeusinghighperforma
17、nceliquidchromatography(HPLC). ThemethodhasbeensuccessfullyvalidatedinaninterlaboratorystudyaccordingtoAOACGuidelinesfor CollaborativeStudies1onmaizecontaining405g/kgto6732 g/kgfumonisinB 1 and152 g/kgto2619 g/kg fumonisinB 2 .Themethodworkswellwithmaizeorminimallyprocessedmaize(e.g.fresh,driedandmi
18、lledmaize), butdoesnotprovidereliableresultswithmostmaizebasedprocessedproducts. 2 Normativereferences ThisEuropeanStandardincorporatesbydatedorundatedreference,provisionsfromotherpublications.These normativereferencesarecitedattheappropriateplacesinthetext,andthepublicationsarelistedhereafter.For d
19、atedreferences,subsequentamendmentstoorrevisionsofanyofthesepublicationsapplytothisEuropean Standardonlywhenincorporatedinitbyamendmentorrevision.Forundatedreferencesthelatesteditionofthe publicationreferredtoapplies(includingamendments). ENISO3696 WaterforanalyticallaboratoryuseSpecificationandtest
20、methods(ISO3696:1987) . 3Principle Fumonisinsareextractedfromthesampleofmaizewithamixtureofmethanolandwater.Thefilteredextractis purifiedonastronganionexchange(SAX)solidphaseextraction(SPE)cartridge,andthefumonisinsareeluted withamixtureofaceticacidandmethanol.Theextractisevaporatedandtheresidueisre
21、dissolvedinmethanol andophthaldialdehyde/2mercaptoethanol(OPA/MCE)isaddedtoformfluorescentfumonisinderivatives.The derivativesareanalysedbyreversephasehighperformanceliquidchromatography(HPLC)withfluorescence detection. WARNINGFumonisinsarehepatoxic,nephrotoxicandcarcinogenictoratsandmice.Effectsonh
22、umans arenotfullyknown.Observeappropriatesafetyprecautionsforhandlingfumonisins.Anylaboratoryspills shouldbewashedwitha5%solutionofsodiumhypochlorite. 4 Reagentsandmaterials 4.1General Duringtheanalysis,unlessotherwisestated,useonlyreagentsofrecognizedanalyticalgradeandonlydistilled waterorwaterofgr
23、ade1asdefinedinENISO3696.SolventshallbeofqualityforHPLCanalysis. 4.2Methanol, abs. 4.3Methanolsolution, volumefraction (CH 3 OH)=75% Mix75partspervolumeofmethanol(4.2)with25partspervolumeofwater. 4.4Acetonitrilesolution, (CH 3 CN)=50% Mix50partspervolumeofacetonitrilewith50partspervolumeofwater. 4.5
24、ophosphoricacid, (H 3 PO 4 )=85% 4.6Aceticacidmethanolsolution, (CH 3 COOH)=1%forelutingtheSPEcolumn. Mix1partpervolumeofglacialaceticacidwith99partspervolumeofmethanol(4.2).EN13585:2001(E) 4 4.7ophthaldialdehyde(OPA) 4.82mercaptoethanol(MCE) 4.9Sodiumdihydrogenphosphatesolution, substanceconcentrat
25、ion c(NaH 2 PO 4 2H 2 O)=0,1mol/l Dissolve15,6gofNaH 2 PO 4 2H 2 Oin1lofwater. 4.10 Disodiumtetraboratesolution, c(Na 2 B 4 O 7 10H 2 O)=0,1mol/l Dissolve3,8gofNa 2 B 4 O 7 10H 2 Oin100mlofwater. 4.11 Sodiumhydroxidesolution, c(NaOH)=0,1mol/l Dissolve0,4gofNaOHin100mlofwater. 4.12 Mobilephase Mix77v
26、olumepartsofmethanol(4.2)with23volumepartsofsodiumdihydrogenphosphatesolution(4.9).Adjust topH3,35withophosphoricacid(4.5).Filterthesolutionthrougha0,45 mmembrane(5.7)filter. ThemobilephasecompositionmayhavetobeadjustedtoconformwithindividualHPLCcolumncharacteristics. 4.13Derivatizationreagent Disso
27、lve40mgofOPA(4.7)in1mlofmethanol(4.2)anddilutewith5mlofdisodiumtetraboratesolution(4.10). Add50 lofMCE(4.8)andmix.Thereagentsolutionisstableforuptooneweekatroomtemperatureinadark andcappedambervial. 4.14FB 1 andFB 2 standardsolution PrepareindividualstocksolutionsofFB 1 andFB 2 atmassconcentrationso
28、f250g/mlinacetonitrilesolution(4.4). Commerciallyavailablestandardsolutionsmaybeused.Transfer100 laliquotsofeachstocksolutiontoaglass vialandadd300 lofacetonitrilesolutiontoyield500 lofastandardsolutioncontainingbothfumonisinsat individualmassconcentrationof50 g/ml.Ifacalibrationcurveismade,mixdiffe
29、rentaliquotsofstandardsolutions (i.e.20 l,50 l,100 land200 l)ofindividualfumonisinsanddiluteto500 lwithacetonitrilesolutiontoobtain therelevantcalibrationsolutions. Fumonisinstandardsolutionsarestableuptoatleastsixmonthswhenstoredatapproximately4 C. 5Apparatus Usuallaboratoryequipmentand,inparticula
30、r,thefollowing: 5.1HPLCapparatus, comprisingthefollowing 5.1.1 Highperformanceliquidchromatograph, isocraticpumpsettodelivere.g.1ml/minconstantflowrate, equippedwithaninjectionsystemcapabletodelivere.g.10 l. 5.1.2 Analyticalreversephaseseparatingcolumn, e.g.octyldecylsilane(ODS),whichensuresabaselin
31、e resolutionofthefumonisinpeaksfromallotherpeaks,withthefollowingcharacteristics: stainless steel;EN13585:2001(E) 5 alengthof150mm; aninnerdiameterof4,6mm; astationaryphasewithparticlesizeof5 m; asuitablecorrespondingreversephaseguardcolumn. Columnsofotherdimensionscanalsobeused. 5.1.3Fluorescence d
32、etector withthecapabilityofusingexcitationwavelengthof =335nmandemission wavelengthof =440nm. 5.1.4 Datasystem 5.2Blender,homogenizer,ormixer. 5.3Flutedfilterpaper 5.4Stronganionexchangingsolidphaseextractioncolumn, e.g.BondElut 1 ) SAXcartridges, containing500mgofsorbent,orsimilarhasbeenfoundtobesu
33、itable. 5.5SPEextractionmanifold 5.6Solventevaporator, withheatingmodule,orsimilar. 5.7Membranefilter, foraqueoussolutions,withaporesizeof0,45 m. 6Sampling Itisimportantthatthelaboratoryreceivesasamplewhichistrulyrepresentativeandhasnotbeendamagedor changedduringtransportorstorage. 7 Preparationofth
34、etestsample Grindthesampletopassthrougha1,0mmsieveandhomogenizethesample. 8Procedure 8.1Extraction Place50gofthetestsampleintoasuitableglassorplasticcontainer(e.g.a250mlplasticcentrifugebottle). Extractfor3minwith100mlofmethanolsolution(4.3)usingtheblender(5.2)atapproximately10000min 1 .The timeneed
35、edforcompleteextractioncanvarywiththetypeofequipmentused. Centrifugetheblendedextractfor10minat500 gandfilterthesupernatantthroughaflutedfilterpaper(5.3). CheckthepHoftheeluateandadjust,ifnecessary,withsodiumhydroxidesolution(4.11)tobetweenpH5,8and pH6,5. 1 BondElut isanexampleofasuitableproductavai
36、lablecommercially.Thisinformationisgivenfortheconvenienceofusers ofthisEuropeanStandardanddoesnotconstituteanendorsementbyCENofthisproduct.EN13585:2001(E) 6 8.2Cleanup AttachtheSPEcartridge(5.4)totheSPEmanifold(5.5)andconditionbywashingsuccessivelywith5mlof methanol(4.2)andthenwith5mlofmethanolsolut
37、ion(4.3).Whilemaintainingaflowrateofnomorethan 2ml/min,applya10mlaliquotofthefilteredsampleextracttotheSPEcartridge.WashtheSPEcartridgewith8ml ofmethanolwatersolution(4.3),followedby3mlofmethanol(4.2).Donotallowthecartridgetorundry.Discard thewashings.Elutethefumonisinswith10mlofmethanolicaceticacid
38、1%(4.6)ataflowratenotmorethan 1ml/min.Collecttheeluateinasuitablevial. Transfertheeluateinthecollectionvialtoasuitablevialandevaporatetheeluatetodrynessbyusingthesolvent evaporator(5.6)undernitrogenatapproximately60C.Washthecollectionvialwith1mlofmethanol(4.2)and addthewashingtothesuitablevial.Evapo
39、ratetheadditionalmethanoltodrynessundernitrogen,ensurethatall theaceticacidhasevapored,andcapthevial.Retainthedriedresidueatapproximately4CuntilHPLCanalysis, thatshouldbeperformedwithintwoweeks. 8.3Derivatizationanddetermination 8.3.1 Standardderivativesolution Transfer25 lofthefumonisinstandardsolu
40、tion(4.14)tothebaseofasmalltesttube.Add225 lderivatization reagent(4.13),mixthesolutionsvigorously,andinjectanaliquot(e.g.10 l=0,050gFB 1 andFB 2 )ofthe derivatizedsolutionontotheHPLCcolumn(5.1.2)atareproducibletimewithin3minafteradditionofthe derivatizationreagent.Ifasinglepointcalibrationisused,ad
41、justthesensitivityofthefluorescencedetector(5.1.3) toatleastan80%recorderresponse. 8.3.2 Maizetestsolution Redissolvethedriedresidue(see8.2)in200 lofmethanol(4.2).Acetonitrilesolution(4.4)mayalsobeused. Transfer25 lofthissolutiontothebaseofasmalltesttubeandadd225 lofthederivatizationreagent(4.13). M
42、ixthesolutionsandinjectanaliquot,e.g.10 lofthederivatizedsolutionontotheHPLCcolumn(5.1.2)ata reproducibletimewithin3minafteradditionofthederivatizationreagent(4.13).Iffumonisinchromatographic peaksexceedthoseoffumonisinstandardsolutionorexceedthehighestpointofthecalibrationcurve(4.14),make additiona
43、ldilutionsofthesampleextractswithmethanol(4.2)andrepeatderivatization. NOTE1 Itiscriticaltoadheretoreproducibletimesbetweentheadditionofthereagent(4.13)andtheinjectionontotheHPLC column,becauseofprogressivedecayinfluorescenceofthefumonisinderivativesthatoccursafterperiodsinexcessto4min. NOTE2 Limitsofdetectionandquantificationvaryconsiderablyaccordingtothesensitivityofthedetectorused.Limits
