1、DRAFT FOR DEVELOPMENT DD CEN/TS 14537:2003 Foodstuffs Determination of neohesperidin- dihydrochalcon ICS 67.050 DD CEN/TS 14537:2003 This Draft for Development was published under the authority of the Standards Policy and Strategy Committee on 28 May 2003 BSI 28 May 2003 ISBN 0 580 41874 X National
2、foreword This Draft for Development is the English language version of CEN/TS 14537:2003. 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 of the need to gain experience in the
3、use of the method across a wider range of food products. It should be applied on this provisional basis, so that information 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
4、 the European organization responsible for its conversion to a European standard. A review of this publication will be initiated 2 years after its publication by the European organization so that a decision can be taken on its status at the end of its 3-year life. Notification of the start of the re
5、view period will be made in an announcement in the appropriate issue of Update Standards. According to the replies received by the end of the review period, the responsible BSI Committee will decide whether to support the conversion into a European standard, to extend the life of the Technical Speci
6、fication or to withdraw it. Comments should be sent in writing to the Secretary of BSI Technical Committee AW/-/3, Horizontal analysis, at British Standards House, 389 Chiswick High Road, London W4 4AL, giving the document reference and clause number and proposing, where possible, an appropriate rev
7、ision 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 or European publications referred to in this document may be found in the BSI Catalogue under the section entitle
8、d “International Standards Correspondence Index”, or by using the “Search” facility of the BSI Electronic Catalogue or of British Standards Online. Summary of pages This document comprises a front cover, an inside front cover, the CEN/TS title page, pages 2 to 13 and a back cover. The BSI copyright
9、date displayed in this document indicates when the document was last issued. Amendments issued since publication Amd. No. Date CommentsTECHNICALSPECIFICATION SPCIFICATIONTECHNIQUE TECHNISCHESPEZIFIKATION CEN/TS14537 April2003 ICS67.050 Englishversion FoodstuffsDeterminationofneohesperidindihydrochal
10、con LebensmittelBestimmungvonNeohesperidin Dihydrochalconmit Hochleistungsflssigkeitschromatographie(HPLC) ThisTechnicalSpecification(CEN/TS)wasapprovedbyCENon5January2003forprovisionalapplication. TheperiodofvalidityofthisCEN/TSislimitedinitiallytothreeyears.AftertwoyearsthemembersofCENwillbereques
11、ted tosubmittheir comments,particularlyonthequestionwhethertheCEN/TScanbeconvertedintoaEuropeanStandard. CENmembersarerequiredtoannouncetheexistenceofthisCEN/TSinthesamewayasforanENandtomaketheCEN/TSavailable .It ispermissibletokeepconflictingnationalstandardsinforce(inparalleltotheCEN/TS)untilthefi
12、naldecisionaboutthep ossible conversionoftheCEN/TSintoanENisreached. CENmembersarethenationalstandardsbodiesofAustria,Belgium,CzechRepublic,Denmark,Finland,France,Germany,Greece, Hungary,Iceland,Ireland,Italy,Luxembourg,Malta,Netherlands,Norway,Portugal,Slovakia,Spain,Sweden,SwitzerlandandUn ited Ki
13、ngdom. EUROPEANCOMMITTEEFORSTANDARDIZATION COMITEUROPENDENORMALISATION EUROPISCHESKOMITEEFRNORMUNG ManagementCentre:ruedeStassart,36B1050Brussels 2003CEN Allrightsofexploitationinanyformandbyanymeansreserved worldwideforCENnationalMembers. Ref.No.CEN/TS14537:2003ECEN/TS14537:2003(E) 2 Contents 1 Sco
14、pe 4 2 Normativereferences 4 3 Principle4 4 Reagents.4 5 Apparatus .5 6Procedure.5 7 Calculation7 8 Precision.7 9 Testreport . .8 AnnexA (informative) Precisiondata 9 AnnexB (informative) AlternativeHPLCSystems.10 AnnexC (informative) Exampleforachromatogram 11 Bibliography 13CEN/TS14537:2003(E) 3 F
15、oreword Thisdocument(CEN/TS14537:2003)hasbeenpreparedbyTechnicalCommitteeCEN/TC275“Foodanalysis Horizontalmethods”,thesecretariatofwhichisheldbyDIN. AccordingtotheCEN/CENELECInternalRegulations,thenationalstandardsorganizationsofthefollowing countriesareboundtoannouncethisTechnicalSpecification:Aust
16、ria,Belgium,CzechRepublic,Denmark, Finland,France,Germany,Greece,HungaryIceland,Ireland,Italy,Luxembourg,Malta,Netherlands,Norway, Portugal,Slovakia,Spain,Sweden,SwitzerlandandtheUnitedKingdom. AnnexesA,BandCareinformative.CEN/TS14537:2003(E) 4 1Scope ThisTechnicalSpecificationspecifiesanHPLCmethodf
17、orthedeterminationofneohesperidindihydrochalcon (NHDC)infoodstuffs. Ithasbeenvalidatedinacollaborativetestwithcherryyoghurtcontaining42,7mg/kgandonamultivitamindrink containing35,6mg/lNHDC1. Themethodhasbeensuccessfullyappliedtoarangeofotherfoodsincludingmarzipan,bakeryproducts,cream, custardpowder,
18、chocolate,icecream. 2 Normativereferences ThisTechnicalSpecificationincorporatesbydatedorundatedreference,provisionsfromotherpublications.These normativereferencesarecitedattheappropriateplacesinthetext,andthepublicationsarelistedhereafter.For datedreferences,subsequentamendmentstoorrevisionsofanyof
19、thesepublicationsapplytothisTechnical Specificationonlywhenincorporatedinitbyamendmentorrevision.Forundatedreferencesthelatesteditionof thepublicationreferredtoapplies(includingamendments). ENISO3696, WaterforanalyticallaboratoryuseSpecificationandtestmethods(ISO3696:1987). 3Principle Thesampleisdil
20、utedwithmethanolorextractedwithamethanol/watermixtureandpossiblyfiltered.NHDCis separatedbyHPLConareversedphase,detectedspectrometricallyanddeterminedbytheexternalstandard method1. 4Reagents 4.1General Duringtheanalysis,unlessotherwisestated,useonlyreagentsofrecognisedanalyticalgradeandwaterofatleas
21、t grade1accordingtoENISO 3696orusedistilledwater. 4.2tetranbutylammoniumhydrogensulfatesolution(mobilePhaseA) forHPLCrecommended Substanceconcentration c=0,01mol/l 4.3Methanol(mobilePhaseB) 4.4HPLCmobilephase Forthemobilephase,tetra nbutylammoniumhydrogensulfatesolution(4.2)andmethanol(4.3)areapplie
22、din appropriateratios(gradient,e.g.asgivenin6.3.1).Filterthroughamembranefilter(5.2)beforeuse. 4.5Standardsubstance TheNHDCstandardsubstanceshallfulfiltherequirementsaccordingtoEUdirective95/31/EU2. CommerciallyavailableNHDCcancontain4molofwater.Ifusingthiscommerciallyavailablesubstance,takeits wate
23、rcontentintoaccount.ThemolecularweightofdriedNHDCis612,6g/mol.CEN/TS14537:2003(E) 5 4.6Standardtestsolutions DissolveanappropriateamountofNHDCwithamixtureofmethanol/water(50+50,volumefraction)anddissolve thissolutionagainwithamixtureofmethanol/water(50+50,volumefraction)toobtainstandardsolutionswhic
24、h willgivepeakscomparabletothoseobtainedwiththesamplesolution. Thissolutionmaybestoredfor3monthsinarefrigeratorsetat+4C. 5Apparatus Useusuallaboratoryapparatusand,inparticular,thefollowing: 5.1Filterdevice, e.g.filtrationunitforvacuum,glass,consistingofglasssinterplateD2(diameter50mm), 250mlfunnelan
25、d1lconicalflask,eachwithgroundjoint. 5.2Membranefilter forfilteringthemobilephase,withporesizeof0,45 m m(orless). NOTE Filteringofthemobilephaseaswellasofthesamplesolutionthroughamembranefilterpriortouseorinjectionis supposedtoincreaselongevityofthecolumns. 5.3Ultrasonicbath 5.4Stomacher, optional,e
26、g.forextractionandpreparationofsolidsamples. 5.5HPLCsystem, consistingof a pump; asampleinjectingdevice; aUVdetectorwithvariablewavelengthsetting; anevaluatingsystem. 5.6HPLCcolumn Analyticalreversedphasecolumn,acidresistant,diameter4mm,length250mm,filledwithparticlesize5 m m,e.g. LiChrospher 60RPs
27、electB 1) ,oranotheracidresistantRPphase.Toprotecttheanalyticalcolumn,itis recommendedtouseaprecolumnofsimilarcharacteristics. ColumndimensionsotherthanthosespecifiedinthisTechnicalSpecificationmaybeused(e.g.ofdiameter 3,0mmto4,6mm,length100mmto250mm).Separationparameters(flow,injectionvolume)haveto
28、beadapted toguaranteeequivalentresults. 6Procedure 6.1Preparationofthetestsample Homogenisethetestsample.Shakeliquidsamples,homogeniseviscousandsemisolidsamplesbystirringand grindsolidsampleswithanappropriatemillandmixagain. 1) LiChrospher 60RPselectBisanexampleofasuitableproductavailablecommerciall
29、y.Thisinformationisgivenforthe convenienceofusersofthisTechnicalSpecificationanddoesnotconstituteanendorsementbyCENofthisproduct.CEN/TS14537:2003(E) 6 6.2Preparationofthesampletestsolution 6.2.1Liquid samples Dilute50mlofsampleina100mlvolumetricflaskwithmethanoltothemark,mixandplacetheflaskfor10mini
30、n anultrasonicbath.Filterthissuspensionthroughafoldedfilterandthanthoughamembranefilter(5.2). 6.2.2 Viscousandsemisolidsamples Weigh10gofthehomogenisedsampleintoa100mlvolumetricflaskandadd50mlofmethanol.Shakeandplace for10minintoanultrasonicbath.Lettheflaskstanduntilthecontentreachesroomtemperaturea
31、nddilutewith watertothemark.Placethissuspensionagaininanultrasonicbathfor10min,filterthissuspensionthrougha foldedfilterandthenthoughamembranefilter(5.2). NOTE Inthecollaborativetrialwithcherryyoghurt,itisunlikelythatthevolumeoftheinsolublematerialexceeded1%of thefinalsuspensionvolume.Insuchcircumst
32、ances,anycorrectionbycalculationforthereducedvolumeofextractcanbe consideredunnecessary. 6.2.3 Solidsamples Weigh10gofthehomogenisedsampleintoastomacherbag,add50mlofmethanol,add50mlofwaterlessthan thewaterwhichisalreadyincludedinthesample.Shake,placeinthestomacherandstomachfor10min.Filterthe suspens
33、ionthroughafoldedfilterandthanthroughamembranefilter(5.2). 6.3HPLCdetermination 6.3.1HPLC conditions Theseparationandthequantificationhaveproventobesatisfactoryiffollowingexperimentalconditionsare followed: StationaryPhase: accordingto5.6. Mobilephase: applyagradientasinTable1: Table1Gradientofmobil
34、ephases Time,min EluentA,% EluentB,% 0 4 10 25 30 40 85 85 70 40 5 5 15 15 30 60 95 95 Flowrate: 1,2ml/min Injectionvolume: 20l Detection: UV,at285nm 6.3.2Identification Injectthesameappropriatevolumes,e.g.20 m lofthestandardtestsolution(4.6)aswellasofthesampletest solution(6.2)intotheHPLCsystem(5.5
35、).CEN/TS14537:2003(E) 7 IdentifyNHDCbycomparisonoftheretentiontimeofthepeakinthechromatogramsobtainedwiththesampletest solutionandwiththestandardsolution.Peakidentificationcanalsobeperformedbyaddingsmallamountsofthe appropriatestandardsolutionstothesampletestsolutionandbycheckingthepeakpuritybyUVVIS
36、spectrum, e.g.usingadiodearrayspectrometer. 6.3.3Determination Tocarryoutthedeterminationbyexternalcalibration,integratethepeakareasordeterminethepeakheightsofthe sampleandcomparetheresultswiththecorrespondingvaluesforthestandardsubstanceoruseacalibration graph.Checkthelinearityofthecalibrationgraph
37、 7Calculation Calculatethemassconcentration, ,ofNHDCinmilligramperlitreorthemassfraction,w,inmilligramper kilogramofthesampleusingequation(1): 000 1 or 0 2 2 1 1 1 = m V A m V A w r (1) where A 1 isthepeakareaforNHDCobtainedwiththesampletestsolution(6.2),inunitsofarea; A 2 isthepeakareaforNHDCobtai
38、nedwiththestandardtestsolution(4.6),inunitsofarea; V 1 isthetotalvolumeofsampletestsolution(6.2),inmillilitre; V 2 isthetotalvolumeofstandardtestsolution(4.6),inmillilitre; m 1 isthemassof(dried)NHDCcontainedin V 2 , inmilligram; m 0 isthesamplemass,inmillilitreorgram. Reporttheresultwithoutdecimalp
39、laces. 8Precision 8.1General TheprecisiondataforthedeterminationofNHDCwereestablishedin1999byacollaborativestudyoncherry yoghurtandonamultivitamindrinkasshowninannexA.Thedataderivedfromthiscollaborativestudymaynot beapplicabletoanalyteconcentrationrangesandsamplematricesotherthanthosegiveninannexA.
40、8.2Repeatability Theabsolutedifferencebetweentwosingletestresultsfoundonidenticaltestmaterialbyoneoperatorusingthe sameapparatuswithintheshortestfeasibletimeintervalwillexceedtherepeatabilitylimit r innotmorethan5%of thecases.Therepeatabilityisdependentontheconcentrationleveloftheanalyteinthesample.
41、 ThevaluesforNHDCare: cherryyoghurt x =42,7mg/kg r=2,6mg/kg multivitamindrink x =35,6mg/l r=1,8mg/lCEN/TS14537:2003(E) 8 8.3Reproducibility Theabsolutedifferencebetweentwosingletestresultsobtainedonidenticalmaterialreportedbytwolaboratories willexceedthereproducibilitylimit Rinnotmorethan5%ofthecase
42、s. ThevaluesforNHDCare: cherryyoghurt x =42,7mg/kg R=9,8mg/kg multivitamindrink x =35,6mg/l R=5,4mg/l 9 Testreport Thetestreportshallcontainatleastthefollowingdata: a) allinformationnecessaryforthecompleteidentificationofthesample; b) areferencetothisTechnicalSpecificationortothemethodused; c) theda
43、teandtypeofsampleprocedure(ifknown); d) thedateofreceipt; e) thedateoftest; f) theresultsandtheunitsinwhichtheresultshavebeenexpressed; g) anyparticularpointsobservedinthecourseofthetest; h) anyoperationsnotspecifiedinthemethodorregardedasoptionalwhichmighthaveaffectedtheresults.CEN/TS14537:2003(E) 9 Annex A (informative) Precisiondata ThefollowingprecisiondatahavebeendefinedinaninternationalcollaborativestudyaccordingtoISO57253 carriedoutbytheFederalGermanInstituteofConsumersHealthandVeterinaryM
