1、DRAFT FOR DEVELOPMENT DD ENV 14194:2002 Foodstuffs Determination of saxitoxin and dc-saxitoxin in mussels HPLC method using post column derivatisation ICS 67.120.30 NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWDD ENV 14194:2002 This Draft for Development, having been prepare
2、d 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 16 May 2002 BSI 16 May 2002 ISBN 0 580 39628 2 National foreword This Draft for Development is the English language ve
3、rsion of ENV 14194:2002. This publication is not to be regarded as a British Standard. It is being issued in the Draft for Development series of publications and is of a provisional nature because the method has not been validated. It should be applied on this provisional basis, so that information
4、and experience of its practical application may be obtained. Comments arising from the use of this Draft for Development are requested so that UK experience can be reported to the European organization responsible for its conversion into a European Standard. A review of this publication will be init
5、iated 2 years after its publication by the European organization so that a decision can be taken on its status at the end of its three-year life. The commencement of the review period will be notified by an announcement in Update Standards. According to the replies received by the end of the review
6、period, the responsible BSI Committee will decide whether to support the conversion into a European Standard, to extend the life of the prestandard or to withdraw it. Comments should be sent in writing to the Secretary of BSI Technical Committee AW/-/3, Horizontal analysis, at 389 Chiswick High Road
7、, London W4 4AL, giving the document reference and clause number and proposing, where possible, an appropriate revision of the text. A list of organizations represented on this committee can be obtained on request to its secretary. Cross-references The British Standards which implement international
8、 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” facility of the BSI Standards Electronic Catalogue. Summary of pages This document comprises a front co
9、ver, an inside front cover, the ENV title page, pages 2 to 11 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 CommentsEUROPEANPRESTANDARD PRNORMEEUROPENNE EUROPISCHEVORNORM ENV14194 Apr
10、il2002 ICS67.120.30 Englishversion FoodstuffsDeterminationofsaxitoxinanddcsaxitoxinin musselsHPLCmethodusingpostcolumnderivatisation ProduitsalimentairesDterminationdelateneuren saxitoxineetendcsaxitoxinedanslesmoulesMthode parchromatographieliquidehauteperformanceaprs drivationpostcolonne Lebensmit
11、telBestimmungvonSaxitoxinundDCSaxitoxin inMuschelnHPLCVerfahrenmit Nachsulenderivatisierung ThisEuropeanPrestandard(ENV)wasapprovedbyCENon14December2001asaprospectivestandardforprovisionalapplication. TheperiodofvalidityofthisENVislimitedinitiallytothreeyears.AftertwoyearsthemembersofCENwillbereques
12、tedto submittheir comments,particularlyonthequestionwhethertheENVcanbeconvertedintoaEuropeanStandard. CENmembersarerequiredtoannouncetheexistenceofthisENVinthesamewayasforanENandtomaketheENVavailablepromp tly atnationallevelinanappropriateform.Itispermissibletokeepconflictingnationalstandardsinforce
13、(inparalleltothe ENV)untilthefinal decisionaboutthepossibleconversionoftheENVintoanENisreached. CENmembersarethenationalstandardsbodiesofAustria,Belgium,CzechRepublic,Denmark,Finland,France,Germany,Greece, Iceland,Ireland,Italy,Luxembourg,Malta,Netherlands,Norway,Portugal,Spain,Sweden,Switzerlandand
14、UnitedKingdom. EUROPEANCOMMITTEEFORSTANDARDIZATION COMITEUROPENDENORMALISATION EUROPISCHESKOMITEEFRNORMUNG ManagementCentre:ruedeStassart,36B1050Brussels 2002CEN Allrightsofexploitationinanyformandbyanymeansreserved worldwideforCENnationalMembers. Ref.No.ENV14194:2002EENV14194:2002(E) 2 Foreword Thi
15、sdocument(ENV14194:2002)hasbeenpreparedbyTechnicalCommitteeCEN/TC275,“Foodanalysis Horizontalmethods“,thesecretariatofwhichisheldbyDIN. AccordingtotheCEN/CENELECInternalRegulations,thenationalstandardsorganizationsofthefollowing countriesareboundtoannouncethisEuropeanPrestandard:Austria,Belgium,Czec
16、hRepublic,Denmark,Finland, France,Germany,Greece,Iceland,Ireland,Italy,Luxembourg,Malta,Netherlands,Norway,Portugal,Spain, Sweden,SwitzerlandandtheUnitedKingdom. 1Scope ThisEuropeanPrestandardspecifiesamethodforthequantitativedeterminationofsaxitoxin(STX),dcsaxitoxin (dcSTX)andthequalitativedetermin
17、ationofneosaxitoxin,andthegonyautoxinsGTX2andGTX3inmussels. ThemethodcanalsobeusedtoidentifytheNsulfocarbamoyltoxinsC1,C2,GTX5andGTX6afterhydrolysis and,ifthesetoxinsarepresent,toexcludefalsepositiveresultsforGTX2,GTX3,neosaxitoxinandsaxitoxin.For musselthelimitofquantificationisforsaxitoxin0,04mg/k
18、gmusselmeatandfordcsaxitoxin0,03mg/kgmussel meat(signal/noise=10). ThelimitsofdetectionforC1,C2,GTX2,GTX3,GTX5,GTX6andneosaxitoxinhavenotbeendetermined. 2 Normativereferences ThisEuropeanPrestandardincorporatesbydatedorundatedreference,provisionsfromotherpublications.These normativereferencesarecite
19、dattheappropriateplacesinthetextandthepublicationsarelistedhereafter.For datedreferences,subsequentamendmentstoorrevisionsofanyofthesepublicationsapplytothisEuropean Prestandardonlywhenincorporatedinitbyamendmentorrevision.Forundatedreferencesthelatesteditionofthe publicationreferredtoapplies(includ
20、ingamendments). ENISO3696:1995, WaterforanalyticallaboratoryuseSpecificationandtestmethods(ISO3696:1987) 3Principle PSP(ParalyticShellfishPoisoning)toxinsareextractedfrommusselshomogenatewithanacidicaqueoussolution. Aftercentrifugationthesupernatantispurifiedbysolidphaseextraction(SPE)overaC18cleanu
21、pcartridge.Part oftheextractisdirectlyinjectedontheHPLCfortoxindetermination.Aftertheseparationontheanalyticalcolumn postcolumnderivatisationisused,withoxidationwithperiodicacidandafteracidificationthederivatizedtoxinsare detectedusingfluorimetricdetection.HPLCanalysisisseparatedintwoparts:asystemto
22、separatethesaxitoxin grouptoxins(saxitoxin,dcsaxitoxinandneosaxitoxin)andasystemtoseparatetheGTXgrouptoxins(GTX2and GTX3).IdentificationafterhydrolysisisbasedonthetransformationoftheNsulfocarbamoyltoxinstotheir correspondingcarbamoyltoxins. WARNINGPSPtoxinsarestrongneurotoxins.Glovesandsafetyglasses
23、shouldbewornatalltimes,and allstandardandsamplepreparationstagesshouldbecarriedoutinafumecupboard.ENV14194:2002(E) 3 4Reagents 4.1General Duringtheanalysis,unlessotherwisestated,useonlywateraccordingtograde1ofENISO3696:1995. Allchemicalsshallbeofproanalysis(p.a)quality,unlessotherwiseindicated. Refe
24、rencematerials(calibrantsofthetoxins)originatingfromothersourcesthanindicatedmayalsobeusedif wellcharacterisedandwithawelldefinedmassconcentration. 4.2Methanol 4.3Acetonitrile, HPLCquality 4.4 Hydrochloricacid, volumeconcentration (HCl) 25%,(acidimetric) 4.4.1 Hydrochloricacidsolution, substanceconc
25、entration c=1,0mol/l Dilute130mlofhydrochloricacidsolution(4.4)with870mlofwater. 4.4.2 Hydrochloricacidsolution, c=0,1mol/l Dilute100mlofhydrochloricacidsolution(4.4.1)with900mlofwater. 4.5 Octanesulfonicacidsolution, c=0,5mol/l Dissolve2,9gofoctanesulfonicacidsodiumsaltmonohydratein25,0mlofwater. 4
26、.6Phosphoric acid, themassfractionis85% 4.6.1Phosphoric acid solution, c=0,5mol/l Take6,7mlofphosphoricacid(4.6)anddiluteto200,0mlwithwater. 4.7 Ammoniasolution, =25%and =1% Forthe1%solution,dilute40mlammoniasolution =25%with960mlofwater. 4.8 Periodicacidsolution, c=0,01mol/l Dissolve1,14gofperiodic
27、acidinwateranddiluteupto500mlwithwater. 4.9 Aceticacid, 100% 4.9.1 Aceticacidsolution1, c=1mol/l Take57,2mlofaceticacidsolution(4.9)anddiluteto1,0lwithwater. 4.9.2 Aceticacidsolution2, c=0,2mol/l Dilute200mlofaceticacidsolution1(4.9.1)with800mlofwater. 4.9.3 Aceticacidsolution3, c=0,03mol/l Dilute15
28、0mlofaceticacidsolution2(4.9.2)with850mlofwater.ENV14194:2002(E) 4 4.10Helium 4.11 Saxitoxinstandardsolutions 4.11.1 Saxitoxinstocksolution, massconcentration =1,0g/ml Prepareacalibrationsolutioncontaining1,0g/mlofsaxitoxininaceticacidsolution3(4.9.3).Storethecalibration solutioncool(atapproximately
29、+4C)andinthedark.Thissolutionisstableforatleast6months. NOTE1 Anampoulecontainingapproximately0,2mlofsaxitoxincalibrationsolution,massconcentrationofsaxitoxinof 0,14mg/mlisforexampleavailablefromtheNationalResearchCouncilCanada,Halifax,Canada(setofcalibrantsPSP1).In ordertoobtainacalibrationsolution
30、of1,0 m g/ml,thecontentoftheampoulecanbequantitativelytransferredto27,8mlacetic acid3(4.9.3)(totalvolume28,0ml).Thetotalvolumecanbecheckedandadjustedbyweighing(5.9),usingdensityofwaterf or calculations.Forthispurposeweightheampoulebeforeopening,takecarethatallglassispreservedafteropeningandweigh aga
31、inafterthecalibrantsolutionistransferred. NOTE2 AcalibrationsolutioncontainingGTX1andGTX4isalsoavailableandmaybeusedforqualitativelydetermination ofthesetoxins.ThishasnotbeentestedinthecertificationstudyoftheSt andardMeasurements&TestingProgramme,designed tocertifythemassfractionsofsaxitoxinanddcsax
32、itoxininmusselreferencematerials,see6. 4.11.2 Saxitoxincalibrationsolutions, =0,05g/mlto0,30g/ml Prepareforthedeterminationofsaxitoxinacalibrationseriesofincreasingconcentrationinthemassconcentration rangeof0,05g/mlto0,30g/mlfromsaxitoxinstocksolution(4.11.1)inaceticacidsolution3(4.9.3).Prepare fres
33、heverydayofanalysis. 4.12 Dcsaxitoxinstandardsubstance Storecoolandinthedark. 4.13 Dcsaxitoxinstocksolution, =1,0g/ml Preparefromdcsaxitoxinstandardsubstance(4.12)adcsaxitoxincalibrationsolution1,0g/mlinaceticacid solution3(4.9.3)byweighing(5.9),usingdensityofwaterforcalculations.Thissolutionisstabl
34、eforatleast 5months(seeBibliography,3). 4.14 Dcsaxitoxincalibrationsolution, =0,05 m g/mlto0,30g/ml Prepareforthedeterminationofdcsaxitoxinacalibrationseriesofincreasingconcentrationinthemass concentrationrangeof0,05 m g/mlto0,30g/mlfromDcsaxitoxinstocksolutioninaceticacidsolution3(4.9.3). Preparefr
35、esheverydayofanalysis. NOTE Forthequantitativedeterminationofsaxitoxinanddcsaxitoxinacombinedcalibrationsolutionforthese2toxins canbemadeifthevalleybetweenthe2toxinpeaksintheHPLCchromatogramis 10%ofthesumofthepeakheights.Ifthis conditionisfulfilledintheHPLCseparation,onecombinedcalibrationsolutionco
36、ntainingsaxitoxinanddcsaxitoxincanbe preparedinthemassconcentrationrangeof0,05g/mlto0,30g/ml(forbothtoxins)inaceticacidsolution3(4.9.3). 4.15 NeoSaxitoxinstandardsolutions 4.15.1 Neosaxitoxincalibrationsolution, =2,0g/ml Prepareacalibrationsolutioncontaining2,0g/mlofneosaxitoxininaceticacidsolution3
37、(4.9.3).Storethe calibrationsolutioncool(atapproximately+4C)andinthedark.Thissolutionisstableforatleast6months. NOTE1 Anampoulecontaining0,2mlneosaxitoxincalibrationsolution,massconcentration0,14mg/mlisforexample availablefromtheNationalResearchCouncilCanada,Halifax,Canada(setofcalibrantsPSP1).Inord
38、ertoobtainacalibratio n solutionof2,0 m g/ml,thecontentoftheampoulecanbequantitativelytransferredto13,8mlaceticacidsolution3(4.9.3)(total volume14,0ml).Thetotalvolumecanbecheckedandadjustedbyweighing(5.9),usingdensityofwaterforcalculations.ENV14194:2002(E) 5 NOTE2 AcalibrationsolutioncontainingGTX1a
39、ndGTX4isalsoavailableandmaybeusedforqualitativelydetermination ofthesetoxins.ThishasnotbeentestedinthecertificationstudyoftheSt andardMeasurements&TestingProgramme,designed tocertifythemassfractionsofsaxitoxinanddcsaxitoxininmusselreferencematerials,see6. 4.15.2 Neosaxitoxincalibrationsolutionforqua
40、litativeanalyses, =1,0g/ml Add50lofNeosaxitoxincalibrationsolution(4.15.1)to50lofaceticacidsolution3(4.9.3).Preparefreshevery dayofanalysis. 4.16 GTX2/GTX3standardsolutions 4.16.1 GTX2/GTX3calibrationsolution, =2,0g/mland0,5g/mlrespectively Prepareacalibrationsolutioncontaining2,0g/mlofGTX2and0,5g/m
41、lofGTX3inaceticacidsolution3 (4.9.3).Storethecalibrationsolutioncool(atapproximately+4C)andinthedark.Thissolutionisstableforat least6months. NOTE1 Anampoulecontainingapproximately0,2mlGTX2(0,12mg/ml)andGTX3(0,029mg/ml)standardsubstanceis forexampleavailablefromtheNationalResearchCouncilCanada,Halifa
42、x,Canada(setofcalibrantsPSP1).Inordertoobtain acalibrationsolutionof2,0 m g/mlofGTX2andof0,5 m g/mlofGTX3,thecontentoftheampoulecanbequantitatively transferredto11,8mlaceticacidsolution3(4.9.3)(totalvolume12,0ml).Thetotalvolumecanbecheckedandadjustedby weighing(5.9),usingdensityofwaterforcalculation
43、s.Forthispurposeweightheampoulebeforeopening,takecarethatall glassispreservedafteropeningandweighagainafterthecalibrantsolutionistransferred. NOTE2 AcalibrationsolutioncontainingGTX1andGTX4isalsoavailableandmaybeusedforqualitativelydetermination ofthesetoxins.ThishasnotbeentestedinthecertificationstudyoftheSt andardMeasurements&TestingProgramme,designed tocertifythemassfractionsofsaxitoxinanddcsaxitoxininmusselreferencematerials,see6. 4.16.2 GTX2/GTX3calibrationsolutionforqual
