1、BRITISH STANDARD BS EN 14148:2003 Foodstuffs Determination of vitamin K1 by HPLC The European Standard EN 14148:2003 has the status of a British Standard ICS 67.050 BS EN 14148:2003 This British Standard, was published under the authority of the Standards Policy and Strategy Committee on 25 July 200
2、3 BSI 25 July 2003 ISBN 0 580 42317 4 National foreword This British Standard is the official English language version of EN 14148:2003. The UK participation in its preparation was entrusted to Technical Committee AW/-/3, Horizontal analysis, which has the responsibility to: A list of organizations
3、represented on this committee can be obtained 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 Catalogue under the section entitled “International Standards Correspondence I
4、ndex”, or by using the “Search” facility of the BSI Electronic Catalogue or of British Standards Online. This publication does not purport to include all the necessary provisions of a contract. Users are responsible for its correct application. Compliance with a British Standard does not of itself c
5、onfer immunity from legal obligations. aid enquirers to understand the text; present to the responsible international/European committee any enquiries on the interpretation, or proposals for change, and keep the UK interests informed; monitor related international and European developments and promu
6、lgate them in the UK. Summary of pages This document comprises a front cover, an inside front cover, the EN title page, pages 2 to 15 and a back cover. The BSI copyright notice displayed in this document indicates when the document was last issued. Amendments issued since publication Amd. No. Date C
7、ommentsEUROPEANSTANDARD NORMEEUROPENNE EUROPISCHENORM EN14148 July2003 ICS67.050 Englishversion FoodstuffsDeterminationofvitaminK1byHPLC ProduitsalimentairesDosagedelavitamineK1parCLHP LebensmittelBestimmungvonVitaminK1mitHPLC ThisEuropeanStandardwasapprovedbyCENon2May2003. CENmembersareboundtocompl
8、ywiththeCEN/CENELECInternalRegulationswhichstipulatetheconditionsforgivingthisEurope an Standardthestatusofanationalstandardwithoutanyalteration.Uptodatelistsandbibliographicalreferencesconcernings uchnational standardsmaybeobtainedonapplicationtotheManagementCentreortoanyCENmember. ThisEuropeanStan
9、dardexistsinthreeofficialversions(English,French,German).Aversioninanyotherlanguagemadebytra nslation undertheresponsibilityofaCENmemberintoitsownlanguageandnotifiedtotheManagementCentrehasthesamestatusasthe official versions. CENmembersarethenationalstandardsbodiesofAustria,Belgium,CzechRepublic,De
10、nmark,Finland,France,Germany,Greece, Hungary,Iceland,Ireland,Italy,Luxembourg,Malta,Netherlands,Norway,Portugal,Slovakia,Spain,Sweden,SwitzerlandandUn ited Kingdom. EUROPEANCOMMITTEEFORSTANDARDIZATION COMITEUROPENDENORMALISATION EUROPISCHESKOMITEEFRNORMUNG ManagementCentre:ruedeStassart,36B1050Bruss
11、els 2003CEN Allrightsofexploitationinanyformandbyanymeansreserved worldwideforCENnationalMembers. Ref.No.EN14148:2003EEN14148:2003(E) 2 Contents page Foreword3 1 Scope 4 2 Normativereferences 4 3 Principle4 4 Reagents.4 5 Apparatus .6 6 Procedure .7 7 Calculation8 8 Precision.9 9 Testreport . .10 An
12、nexA (informative) Figures .11 AnnexB (informative) Precisiondata 13 AnnexC (informative) AlternativeHPLCSystems.14 Bibliography 15EN14148:2003(E) 3 Foreword Thisdocument(EN14148:2003)hasbeenpreparedbyTechnicalCommitteeCEN/TC275“Foodanalysis Horizontalmethods”,thesecretariatofwhichisheldbyDIN. ThisE
13、uropeanStandardshallbegiventhestatusofanationalstandard,eitherbypublicationofanidenticaltextor byendorsement,atthelatestbyJanuary2004,andconflictingnationalstandardsshallbewithdrawnatthelatest byJanuary2004. AnnexesA,BandCareinformative. WARNINGTheuseofthisstandardcaninvolvehazardousmaterials,operat
14、ionsandequipment.This standarddoesnotpurporttoaddressallthesafetyproblemsassociatedwithitsuse.Itistheresponsibility oftheuserofthisstandardtoestablishappropriatesafetyandhealthpracticesanddeterminethe applicabilityofregulatorylimitationspriortouse. AccordingtotheCEN/CENELECInternalRegulations,thenat
15、ionalstandardsorganizationsofthefollowing countriesareboundtoimplementthisEuropeanStandard:Austria,Belgium,CzechRepublic,Denmark,Finland, France,Germany,Greece,Hungary,Iceland,Ireland,Italy,Luxembourg,Malta,Netherlands,Norway,Portugal, Slovakia,Spain,Sweden,SwitzerlandandtheUnitedKingdom.EN14148:200
16、3(E) 4 1Scope ThisEuropeanStandardspecifiesamethodforthedeterminationofvitaminK 1 infoodstuffsbyhighperformance liquidchromatography(HPLC).ThedeterminationofVitaminK 1 contentiscarriedoutbymeasurementofreduced phylloquinone.Themethodhasbeenvalidatedformilkandinfantformula,howeverlaboratoryexperience
17、sexist whichshowthatthemethodisalsoapplicabletoothertypeoffoodstuffs10. 2 Normativereferences ThisEuropeanStandardincorporatesbydatedorundatedreference,provisionsfromotherpublications.These normativereferencesarecitedattheappropriateplacesinthetextandthepublicationsarelistedhereafter.For datedrefere
18、nces,subsequentamendmentstoorrevisionsofanyofthesepublicationsapplytothisEuropean Standardonlywhenincorporatedinitbyamendmentorrevision.Forundatedreferencesthelatesteditionofthe publicationreferredtoapplies(includingamendments). ENISO3696, WaterforanalyticallaboratoryuseSpecificationandtestmethods(I
19、SO3696:1987). 3Principle AfterenzymaticremovaloffatfromthesamplevitaminK 1 isdeterminedinanappropriatesamplesolutionbyhigh performanceliquidchromatographicseparationcoupledwithpostcolumnreductionandsubsequentfluorometric detection.VitaminK 1 isomersarequantifiedasasingleunresolvedpeakwithaC 18 colum
20、n1to4. 4Reagents 4.1General Duringtheanalysis,unlessotherwisestated,useonlyreagentsofrecognisedanalyticalgradeandwaterofatleast grade1accordingtoENISO3696orusedistilledwater. 4.2Chemicalsandsolutions 4.2.1Methanol, massfraction w(CH 3 OH) 99,8% 4.2.2Ethanol, volumefraction (C 2 H 5 OH) 99,8% 4.2.3 R
21、eagentalcohol, (C 2 H 5 OH)=95% Mix950mlofethanol(4.2.2)with50mlofmethanol(4.2.1). 4.2.4Dichloromethane, w(CH 2 Cl 2 ) 99,5% 4.2.5nHexane,w(C 6 H 14 ) % 4.2.6 Lightpetroleum, bp.35Cto60C,p.a. 4.2.7Potassium hydroxide, w(KOH) % 4.2.8 Potassiumhydroxidesolution, substanceconcentration c(KOH)=10mol/lEN
22、14148:2003(E) 5 4.2.9 Potassiumdihydrogenphosphate, w(KH 2 PO 4 ) 99,5% 4.2.10Potassiumcarbonate, w(K 2 CO 3 ) 99,9% 4.2.11 Sodiumacetate,anhydrous, w(CH 3 COONa) 99,5% 4.2.12Aceticacid, w(CH 3 COOH) 99,8% 4.2.13 Zincchloride, w(ZnCl 2 ) 98% 4.2.14 Zinc,powder,particlesize63 m m, w(Zn) % 4.2.15 Phos
23、phatebufferpH7,9to8,0 Dissolve54,0gofpotassiumdihydrogenphosphate(4.2.9)inapproximately350mlofwater,adjustthepHto7,9 to8,0withpotassiumhydroxidesolution(4.2.8)anddiluteto500mlwithwater. 4.2.16 Zincchlorideacetatesolution Weigh13,7gofzincchloride(4.2.13),4,1gofanhydroussodiumacetate(4.2.11)and3,0gofa
24、ceticacid(4.2.12) ina50mlvolumetricflask,dissolveinmethanol(4.2.1)anddiluteto50mlwithmethanol. 4.2.17 LipasetypeVII e.g.from Candidarugosa,activityca.1000U/mgorsuitablealternative 1) ;otherenzymesourcesfrom Pseudomonasand Rhizopusspeciescanalsobeusedconsideringthedifferentactivityprofile. 4.2.18 HPL
25、CMobilephase Mix100mlofdichloromethane(4.2.4),900mlofmethanol(4.2.1)and5mlofzincchlorideacetatesolution (4.2.16).Filterthrougha0,45 m mfilter. 4.3VitaminK 1 standardsubstance(Phyllochinone,3Phythylmenadione), w(C 31 H 46 O 2 ) 99% VitaminK 1 canbeobtainedfromvarioussuppliers.Thepurityofthephylloquin
26、onestandardmayvary.Itis thereforenecessarytodeterminetheconcentrationofthecalibrationsolutionbyUVspectrometry(see concentrationtest4.4.4) 4.4Stocksolutions 4.4.1Precautions VitaminK 1 isverysensitivetolight.Measureshavetobetakentoprotectthestandardandthecorresponding solutionsduringthewholeprocedure
27、e.g.byusinggenerallybrownglassware. 4.4.2Vitamin K 1 stocksolutionI, massconcentration r (C 31 H 46 O 2 ) 1,0mg/ml Weighaccuratelyapproximately100mgofvitaminK 1 standardsubstance(4.3)intoa100mlvolumetricflask dissolveinmethanol(4.2.1)anddiluteto100ml.Thissolutioncanbestoredundernitrogenfor3monthsat2
28、0C inthedark. NOTE TheamountofvitaminK1canbedifficulttodissolveinmethanol. 1) e.g.L1754;Sigma ChemicalCo,P.O.14508,St.Louis,MO63178USA.Thisproductwasusedintheinterlaboratory study.ThisinformationisgivenfortheconvenienceofusersofthisstandardanddoesnotconstituteanendorsementbyCENof theproductnamed.Equ
29、ivalentproductsmaybeusediftheycanbeshowntoleadtothesameresults.EN14148:2003(E) 6 4.4.3Vitamin K 1 stocksolutionII, r (C 31 H 46 O 2 ) 50,0 m g/ml Pipette5,0mlofvitaminK 1 stocksolutionI(4.4.2),intoa100mlvolumetricflaskanddilutetovolumewith methanol(4.2.1).Thissolutioncanbestoredundernitrogenfor1mont
30、hat20Cinthedark. 4.4.4 Concentrationtest Evaporate5,0mlofvitaminK 1 stocksolutionII(4.4.3)bymeansofarotaryevaporatorunderpartialvacuumor underastreamofnitrogen.Redissolvetheresiduein25,0mlofnhexane(4.2.5)orlightpetroleum(4.2.6). Measuretheabsorbanceofthissolutionina1cmcellagainstnhexaneorlightpetrol
31、eumasreferenceatthe maximumwavelengthofabout248nmwithaspectrometer(5.1).CalculatethevitaminK 1 massconcentration r ,in microgramspermillilitre,ofthevitaminK 1 stocksolutionII(4.4.3)accordingtoequation(1): 419 5 10 A 4 248 = r (1) where A 248 istheabsorptionvalueofthesolutionatthemaximumwavelengthofa
32、bout248nm; 419 isthe 1% 1cm A valueofvitaminK 1 innhexane(4.2.5)orlightpetroleum(4.2.6)at248nm5; 10 4 istheconversionof 1% 1cm A tomicrogrammepermilliliter; 5 isthedilutionstepduringsolventchangefrommethanoltonhexane. 4.5Standardsolutions 4.5.1 Intermediatestandardsolution,vitaminK 1 , r (C 31 H 46
33、O 2 ) 2,5 m g/ml Pipette5,0mlofvitaminK 1 stocksolutionII(4.4.3),intoa100mlvolumetricflaskanddilutetovolumewith methanol(4.2.1). 4.5.2 StandardtestsolutionforHPLC,vitaminK 1 , r (C 31 H 46 O 2 ) 25,0ng/ml Pipetteappropriatevolumese.g.1mlofthevitaminK 1 intermediatestandardsolution(4.5.1)intobrownvol
34、umetric flaskse.g.100mlandaddmethanol(4.2.1)todilutetovolume.Preparethissolutionfresheveryday. 5Apparatus Uselaboratoryapparatusand,inparticular,thefollowing: 5.1UVSpectrometer UVspectrometercapableofmeasuringabsorptionsatdefinedwavelengths,withappropriatecells,e.g.of1cm length. 5.2HPLCsystem HPLCsy
35、stem,consistingofapump,asampleinjectingdevice,afluorescencedetectorwithanexcitation wavelengthsetate.g.243nmandanemissionwavelengthsetate.g.430nmandanevaluatingsystemsuchas anintegrator.EN14148:2003(E) 7 5.3HPLCcolumn Analyticalreversedphasecolumn,e.g.ofdiameter3,0mmto4,6mm,length100mmto250mm,filled
36、with particlesize3 m mto10 m m. ParticlesizesandcolumndimensionsotherthanthosespecifiedinthisEuropeanStandardmaybeused. Separationparametershavetobeadaptedtosuchmaterialstoguaranteeequivalentresults. Othersystems(seeannexC)canbeusedprovidingthatasatisfactoryseparationofphylloquinonefromotherco extra
37、ctivesisachieved. 5.4Postcolumnreductor Astainlesssteelorglasscolumnplacedbetweenanalyticalcolumnandfluorescencedetector,e.g.ofdiameter 2,0mmto6,0mm,length10mmto150mm,filledwithzincpowder(4.2.14). 5.5Filterdevice Membranefilterwithporesizeof,e.g.0,45 m mareappropriate. NOTE Filteringofthemobilephase
38、aswellasofthesamplesolutionthroughamembranefilterpriortouseorinjectionis supposedtoincreaselongevityofthecolumns. 6Procedure 6.1Precautions VitaminK 1 isverysensitivetolight.Measureshavetobetakentoprotectthesampleandthecorresponding solutionsduringthewholeproceduree.g.byusinggenerallybrownglassware.
39、 6.2Preparationofthetestsample Homogenisethetestsample.Grindcoarsematerialwithanappropriatemillandmixagain.Measuressuchaspre coolinghavetobetakentoavoidexposingtohightemperatureforlongperiodsoftime. 6.3Preparationofthesamplesolution 6.3.1 Sampleextraction Weighanappropriateamountofthesampletothenear
40、estmg,e.g.1gpowderyor10gliquidmaterialina closabletesttubeoraconicalflask.Vortexpowderysamplewith15mlofwaterof40C,supplementliquid sampleswith5mlofwaterof40C.Runablankjustwiththereagentsbyomittingthesample(see6.5). 6.3.2 Enzymetreatment Add5mlofphosphatebufferpH7,9to8,0(4.2.15)andmix.Add1,0glipase(4
41、.2.17),vortex,stopperandshaketo disperseforapproximately2minto3min.Incubatethemixtureatatemperatureof37C 2Cfor2h.Atregular intervals,e.g.20min,shakemanuallythemixturevigorously. 6.3.3Extraction Cooltoroomtemperature,add10mlofreagentalcohol(4.2.3)and1,0gofpotassiumcarbonate(4.2.10)andmix well.Addadef
42、inedvolume V E ofnhexane(4.2.5),e.g.30mlandshakevigorously.Allowseparationofthephases byleavingtostandinthedarkorbycentrifuginge.g.at2000 gfor10min.Thenhexaneextractcanbestored overnightifkeptat4Cundernitrogeninthedark.EN14148:2003(E) 8 6.3.4 Phasetransferanddilution PipetteanaliquotV A ofthenhexane
43、phase(6.3.3)e.g.0,5mlforfortifiedsamplesor5,0mlfornonfortified samplesintoavial.Removethesolventundernitrogenandredissolvetheresidueinadefinedvolume Vof methanol(4.2.1),e.g.1,0ml.ThisisthefinalsampletestsolutionfortheHPLCanalysis. 6.4Identification IdentifythevitaminK 1 bycomparisonoftheretentiontimeofthepeakinthechrom
