1、BRITISH STANDARD BS EN 1785:2003 Foodstuffs Detection of irradiated food containing fat Gas chromatographic/ mass spectrometric analysis of 2-alkylcyclobutanones The European Standard EN 1785:2003 has the status of a British Standard ICS 67.050 BS EN 1785:2003 This British Standard, was published un
2、der the authority of the Standards Policy and Strategy Committee on 12 August 2003 BSI 12 August 2003 ISBN 0 580 42454 5 National foreword This British Standard is the official English language version of EN 1785:2003. It supersedes BS EN 1785:1997 which is withdrawn. The UK participation in its pre
3、paration was entrusted to Technical Committee AW/-/3, Horizontal analysis, which has the responsibility to: 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
4、 referred to in this document may be found in the BSI Catalogue under the section entitled “International Standards Correspondence Index”, 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
5、provisions of a contract. Users are responsible for its correct application. Compliance with a British Standard does not of itself confer immunity from legal obligations. aid enquirers to understand the text; present to the responsible international/European committee any enquiries on the interpreta
6、tion, 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 This document comprises a front cover, an inside front cover, the EN title page, pages 2 to 16, an inside back cover and a back c
7、over. The BSI copyright notice displayed in this document indicates when the document was last issued. Amendments issued since publication Amd. No. Date CommentsEUROPEANSTANDARD NORMEEUROPENNE EUROPISCHENORM EN1785 August2003 ICS67.050 SupersedesEN1785:1996 Englishversion FoodstuffsDetectionofirradi
8、atedfoodcontainingfatGas chromatographic/massspectrometricanalysisof2 alkylcyclobutanones ProduitsalimentairesDtectiondalimentsioniss contenantdeslipidesAnalyseparchromatographieen phasegazeuse/Spectromtriedemassedes2 alkylcyclobutanones LebensmittelNachweisvonbestrahltenfetthaltigen LebensmittelnGa
9、schromatographisch/ massenspektrometrischeUntersuchungauf2 Alkylcyclobutanone ThisEuropeanStandardwasapprovedbyCENon20June2003. CENmembersareboundtocomplywiththeCEN/CENELECInternalRegulationswhichstipulatetheconditionsforgivingthisEurope an Standardthestatusofanationalstandardwithoutanyalteration.Up
10、todatelistsandbibliographicalreferencesconcernings uchnational standardsmaybeobtainedonapplicationtotheManagementCentreortoanyCENmember. ThisEuropeanStandardexistsinthreeofficialversions(English,French,German).Aversioninanyotherlanguagemadebytra nslation undertheresponsibilityofaCENmemberintoitsownl
11、anguageandnotifiedtotheManagementCentrehasthesamestatusasthe official versions. CENmembersarethenationalstandardsbodiesofAustria,Belgium,CzechRepublic,Denmark,Finland,France,Germany,Greece, Hungary,Iceland,Ireland,Italy,Luxembourg,Malta,Netherlands,Norway,Portugal,Slovakia,Spain,Sweden,Switzerlandan
12、dUn ited Kingdom. EUROPEANCOMMITTEEFORSTANDARDIZATION COMITEUROPENDENORMALISATION EUROPISCHESKOMITEEFRNORMUNG ManagementCentre:ruedeStassart,36B1050Brussels 2003CEN Allrightsofexploitationinanyformandbyanymeansreserved worldwideforCENnationalMembers. Ref.No.EN1785:2003EEN1785:2003(E) 2 Contents Page
13、 Foreword3 1 Scope 3 2 Normativereferences 3 3 Principle3 4 Reagents.4 5 Apparatus .5 6 Samplingtechnique .6 7 Procedure .6 8 Evaluation.7 9 Limitations8 10 Validation8 11 Testreport 9 AnnexA (informative) Figures . 11 Bibliography 15EN1785:2003(E) 3 Foreword Thisdocument(EN1785:2003)hasbeenprepared
14、byTechnicalCommitteeCEN/TC 275,“Foodanalysis Horizontalmethods“,thesecretariatofwhichisheldbyDIN. ThisEuropeanStandardshallbegiventhestatusofanationalstandard,eitherbypublicationofanidenticaltextor byendorsement,atthelatestbyFebruary2004,andconflictingnationalstandardsshallbewithdrawnatthelatest byF
15、ebruary2004. ThisdocumentsupersedesEN1785:1996. ThisEuropeanStandardwaselaboratedonthebasisofaprotocolduringaconcertedactionoftheEuropean Commission(DG XII C.5).ExpertsandlaboratoriesfromE.U.andEFTAcountries,contributedjointlytothe developmentofthisprotocol. Thepredecessorofthepresentstandard(EN1785
16、1996)hasbeenelaboratedfollowingamandateofthe EuropeanCommission. AnnexAisinformative. AccordingtotheCEN/CENELECInternalRegulations,thenationalstandardsorganizationsofthefollowing countriesareboundtoimplementthisEuropeanStandard:Austria,Belgium,CzechRepublic,Denmark,Finland, France,Germany,Greece,Hu
17、ngary,Iceland,Ireland,Italy,Luxembourg,Malta,Netherlands,Norway,Portugal, Slovakia,Spain,Sweden,SwitzerlandandtheUnitedKingdom. 1Scope ThisEuropeanStandardspecifiesamethodfortheidentificationofirradiationtreatmentoffoodcontainingfat.Itis basedonthemassspectrometric(MS)detectionofradiationinduced2alk
18、ylcyclobutanonesaftergas chromatographic(GC)separation1to3. Themethodhasbeensuccessfullytestedininterlaboratorytrialsonrawchicken,pork,liquidwholeegg,salmon andCamembert4to8. Otherstudiesdemonstratethatthemethodisapplicabletoawiderangeoffoodstuffs9to21. 2 Normativereferences ThisEuropeanStandardinco
19、rporatesbydatedorundatedreferences,provisionsfromotherpublications.These normativereferencesarecitedattheappropriateplacesinthetextandthepublicationsarelistedhereafter.For datedreferences,subsequentamendmentstoorrevisionsofanyofthesepublicationsapplytothisEuropean Standardonlywhenincorporatedinitbya
20、mendmentorrevision.Forundatedreferencesthelatesteditionofthe publicationreferredtoapplies(includingamendments). ENISO3696:1995, WaterforanalyticallaboratoryuseSpecificationandtestmethods(ISO3696:1987). 3Principle Duringirradiation,theacyloxygenbondintriglyceridesiscleavedandthisreactionresultsinthef
21、ormationof 2alkylcyclobutanonescontainingthesamenumberofcarbonatomsastheparentfattyacidandthealkylgroupis locatedinringposition2.Thus,ifthefattyacidcompositionisknown,the2alkylcyclobutanonesformedcanbe predicted. The2alkylcyclobutanoneswhichwereanalysedininterlaboratorystudieswere2dodecylcyclobutano
22、ne(DCB) and2tetradecylcyclobutanone(TCB)whichareformedfrompalmiticandstearicacid ,respectively,during irradiation.Todate,thereisnoevidencethatthe2alkylcyclobutanonescanbedetectedinunirradiatedfoods4,EN1785:2003(E) 4 9to21.The2alkylcyclobutanonesareextractedusingnhexaneornpentanealongwiththefat.Theex
23、tractis thenfractionatedusingadsorptionchromatographypriortoseparationusinggaschromatographyanddetection withamassspectrometer.Other2alkycyclobutanones,e.g.2(tetradec5enyl)cyclobutanonederivedfromoleic acid,havealsobeenidentifiedinirradiatedfoodstuffs8,15to20. NOTE Asanalternativeprocedureforextract
24、ionandpurificationofthe2alkylcyclobutanones,supercriticalfluidextraction (SFE),hasbeensuccessfullyemployed18,21.Argentationchromatographyhasbeenusedeffectivelyforthedetectionof foodsirradiatedatverylowdosesorcontainingirradiatedingredients17.Liquidchromatography(LC)GCMScouplinghas beenusedsuccessful
25、lyasanalternativeprocedureforpurificationanddetection14.Itshould,however,benotedthatthese alternativeprocedureshavenotbeenvalidatedbyinterlaboratorytrials. 4Reagents 4.1General Allreagentsandmaterialsusedshallbeofrecognizedanalyticalgradethepurityofwhichhastobetested regularlybytheanalysisofblanksam
26、ples.Watershallbeofatleastgrade3accordingtoENISO3696:1995. 4.2nHexane 1) 4.3Sodiumsulfate, anhydrous 4.4Diethylether 4.5Stockstandardsolutions nhexaneorisooctanemaybeusedtopreparesolutionsof2cyclohexylcyclohexanone(5 g/ml),and2 dodecylcyclobutanone 2) and2tetradecylcyclobutanone 2) (100g/ml).Storeat
27、20C. 4.6Workingstandardsolutions nhexaneorisooctanemaybeusedtopreparesolutionsof2cyclohexylcyclohexanone(0,5 g/ml)(internal standard),2dodecylcyclobutanoneand2tetradecylcyclobutanone(10g/ml).Storeat20C. 4.7Florisil 3) , 150mto250m(60meshto100mesh),pesticideresidueanalysisgrade. Beforeuse,activatethe
28、adsorbentbyheatingat550Cforatleast5horovernight.Coolinadesiccator.Keepwell sealedaftercooling. PreparedeactivatedFlorisil byadding20partsofwaterto100partsofadsorbent(m/m).Approximately30gof activatedFlorisil isrequiredtopreparesufficientdeactivatedadsorbentforeachcolumn.Ensurethatthe deactivatedFlor
29、isil containsnolumpsandthatthepowderflowsfreely.Leavetoequilibrateovernight.Usewithin oneweek. 1) nHexanewasthesolventusedtovalidatethemethod.However,itisalsopossibletousenpentaneonhealth groundsprovideditcanbeshowntoleadtothesameresult. 2) ContacttheNationalStandardizationOrganizationsfortheavailab
30、ilityofreferencestandards 3) Florisil isanexampleforasuitableproductavailablecommercially.Thisinformationisgivenfortheconvenienceof usersofthisstandardanddoesnotconstituteanendorsementbyCENofthisproduct.EN1785:2003(E) 5 4.8Nitrogen, forconcentratingsolutions 4.9Helium, ascarriergas 5Apparatus 5.1Gen
31、eral Usuallaboratoryapparatusand,inparticular,thefollowing: 5.2Electricblender 5.3Soxhletapparatus, withsuitableflaskofe.g.250mlandextractorofe.g.100ml. 5.4Celluloseextractionthimbles, e.g.oflength80mmto100mm,withaninternaldiameterof30mm. Extractionwithnhexanepriortousemaybenecessary. 5.5Cottonwool,
32、 nonabsorbent,washedinnhexanepriortouse. 5.6Electricheatingmantle or waterbath 5.7Chromatographictube, madeofglass,havingalengthof300mmandwithaninternaldiameterof20mm, fittedwithafrit,apolytetrafluoroethylene(PTFE)stopcockandagroundglassjointatthetop. 5.8Separatingfunnel, or droppingfunnel , e.g.of2
33、50ml,withagroundglassjoint. 5.9Rotaryevaporator, withevaporationflaskandawaterbathcapableofbeingcontrolledat45C(low vacuum,approximately25kPa). 5.10 Apparatusforconcentrationofsolutions undernitrogen. 5.11 Gaschromatograph(GC)glassvials 5.12Gaschromatograph(GC) linkedtoa massspectrometer(MS). 5.13Ca
34、pillarycolumn, withsuitableperformancecharacteristics,seeAnnexA.EN1785:2003(E) 6 5.14Laboratoryoven,capableofbeingmaintainedat100C 6 Samplingtechnique Whentakingsamples,givepreferencetothosepartsofthefoodwhichhaveahighfatcontent(e.g.chickenskin). Keepthesampleinasealableglassvesselorinafatfreemetalf
35、oil. 7Procedure 7.1Samplepreparation Coarselychopthesamplesoffoodandthen,withtheexceptionofCamembert,homogenizeinanelectricblender (5.2).Camembertshouldbecutintosmallcubespriortoextraction.Forliquidwholeegg,ensurethatthesampleis thoroughlymixedpriortosampling. 7.2Fatextraction Weigh20gofanhydroussod
36、iumsulfate(4.3)and20gofwellmixedhomogenizedsampleintoanextraction thimble(5.4),mixandplugwithcottonwool(5.5).Extrasodiumsulfatemaybeusedifnecessary.Forfoodstuffs havingalowfatcontent,itmaybenecessarytoincreasethesizeofthetestsampleandquantityofanhydrous sodiumsulfateaccordingly.Itisrecommendedthatli
37、quideggisdriedat100Cfor12hpriortoextraction.Athin filmofeggpartiallydried(2hat100C)hasgivencomparableresults.Alternativedryingprocedures,e.g.freeze drying,maybeusedprovidedrecoveryof2alkylcyclobutanonesischecked(see7.6). Pour100mlofnhexane(4.2)intoasuitableflask(5.3)andplaceextractorontop.Placeextra
38、ctionthimbleinthe extractorandadd40mlofnhexane.Placetheflaskontheheatingmantle(5.6)andcondenserontopofthe extractor.Refluxandextractgentlyfor6h.Thesolventshouldsiphonoverfourtimesinapproximately1h. Removetheflaskfromtheheatanddisposeofthethimbleandthenhexaneintheextractor.Transferthelipid extractfro
39、mtheflasktoa100mlstopperedglassmeasuringcylinderandadjustthevolumeto100mlwithmore solvent.Add5gto10gofanhydroussodiumsulfate,stopper,mixandleaveovernight. Alternativefatextractionproceduresmaybeusediftheycanbeshowntoleadtothesameresults. 7.3Preparationofthelipidextract 7.3.1 Determinationoflipidcont
40、entmethodI Dryduplicateflasksforatleast4horovernightat100C.Coolandweigh.Pipetteanaliquotoflipidextract(7.2) intoeachflaskandrotaryevaporate(5.9)todryness.Dryforatleast4horovernightat100Candreweigh. Alternatively,toprovideamorerapidmeasurementoflipidcontent,pipetteanaliquotoflipidextract(7.2)intoa pr
41、eweighedglassvial.Evaporatethesolventunderastreamofnitrogen(5.10)toconstantweight.Calculatethe volumeofextractrequiredtoprovideapproximately200mgoflipid. 7.3.2 DeterminationoflipidconcentmethodII Alternativelyto7.3.1,concentratethewholelipidextract(7.2)toafewmillilitres(2mlto3ml)usingarotary evapora
42、tor(5.9).Transfertheconcentratedlipidextracttoasmall(e.g.10ml)sealablepreweighedglassvial.Dry thesampleunderastreamofnitrogen(5.10)toconstantweight. 7.4Florisil columnchromatography PrepareaFlorisil column(20cmto21cm)usingachromatographictube(5.7),deactivatedFlorisil (4.7)andn hexane(4.2).Allowthenh
43、exaneleveltodroptojustabovethetopoftheFlorisil . Takeavolumeoftheextract(7.3.1)whichprovidesapproximately200mgoflipidandconcentrateifnecessary. Thefinalvolumeshouldnotexceed5ml.Alternatively,dissolve200mgofthelipidobtainedin7.3.2innotmore than5mlofnhexane. Applythelipidextractquantitativelytothecolumn(recordexactweightoflipidapplied)andallowthenhexane l
