1、BRITISH STANDARD BS EN 14177:2003 Foodstuffs Determination of patulin in clear and cloudy apple juice and puree HPLC method with liquid/liquid partition clean-up The European Standard EN 14177:2003 has the status of a British Standard ICS 67.080.10; 67.160.20 BS EN 14177:2003 This British Standard w
2、as published under the authority of the Standards Policy and Strategy Committee on 13 January 2004 BSI 13 January 2004 ISBN 0 580 43217 3 National foreword This British Standard is the official English language version of EN 14177:2003. The UK participation in its preparation was entrusted to Techni
3、cal 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 referred to in this document ma
4、y 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 provisions of a contract. Users
5、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 interpretation, or proposals for change, a
6、nd 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 15 and a back cover. The BSI copyright notice displayed in this docum
7、ent indicates when the document was last issued. Amendments issued since publication Amd. No. Date CommentsEUROPEANSTANDARD NORMEEUROPENNE EUROPISCHENORM EN14177 December2003 ICS67.080.10;67.160.20 Englishversion FoodstuffsDeterminationofpatulininclearandcloudyapple juiceandpureeHPLCmethodwithliquid
8、/liquidpartitionclean up ProduitsalimentairesDterminationdelateneuren patulinedanslejusdepommesetdanslacompotede pommes,limpidesettroublesMthodepar chromatographieenphaseliquidehauteperformance avecpurificationparpartageliquide/liquide LebensmittelBestimmungvonPatulininklaremund trbemApfelsaftundApf
9、elpreeHPLCVerfahrenmit ReinigungdurchFlssig/FlssigVerteilung ThisEuropeanStandardwasapprovedbyCENon3November2003. CENmembersareboundtocomplywiththeCEN/CENELECInternalRegulationswhichstipulatetheconditionsforgivingthisEurope an Standardthestatusofanationalstandardwithoutanyalteration.Uptodatelistsand
10、bibliographicalreferencesconcernings uchnational standardsmaybeobtainedonapplicationtotheManagementCentreortoanyCENmember. ThisEuropeanStandardexistsinthreeofficialversions(English,French,German).Aversioninanyotherlanguagemadebytra nslation undertheresponsibilityofaCENmemberintoitsownlanguageandnoti
11、fiedtotheManagementCentrehasthesamestatusasthe official versions. CENmembersarethenationalstandardsbodiesofAustria,Belgium,CzechRepublic,Denmark,Finland,France,Germany,Greece, Hungary,Iceland,Ireland,Italy,Luxembourg,Malta,Netherlands,Norway,Portugal,Slovakia,Spain,Sweden,SwitzerlandandUn ited Kingd
12、om. EUROPEANCOMMITTEEFORSTANDARDIZATION COMITEUROPENDENORMALISATION EUROPISCHESKOMITEEFRNORMUNG ManagementCentre:ruedeStassart,36B1050Brussels 2003CEN Allrightsofexploitationinanyformandbyanymeansreserved worldwideforCENnationalMembers. Ref.No.EN14177:2003EEN14177:2003(E) 2 Contents Page Foreword. 4
13、 1 Scope . 5 2 Normativereferences . 5 3 Principle. 5 4 Reagents 5 4.1 General. 5 4.2 Sodiumcarbonatesolution,massconcentration rr (Na 2 CO 3 ) 15g/l 5 4.3 Aceticacid,volumeconcentration jj (CH 3 COOH) 98% 5 4.4 Sodiumsulfateanhydrous . 5 4.5 pH4water 5 4.6 Absoluteethanol, jj (CH 3 CH 2 OH) 99,7%.
14、6 4.7 Ethylacetate. . 6 4.8 Perchloricacid, jj (HClO 4 )=60%. 6 4.9 Acetonitrile 6 4.10 Endogalacturonaseenzymesolution,typicalactivity1400U/g . 6 4.11 HPLCmobilephase 6 4.12 Patulinstocksolution. 6 4.13 Patulinstandardsolution . 6 4.14 Patulinstandardsolutionforcalibration 7 5 Apparatus 7 5.1 Gener
15、al. 7 5.2 Displacementpipettesof5ml,1ml,200 m land50 m lcapacitywithappropriatepipette tips 7 5.3 Analyticalbalance . 7 5.4 UVspectrophotometer,doublebeamandrecordingsuitableformeasurementat250nm to350nm . 7 5.5 Quartzcells,ofopticalpathlength1cm. . 7 5.6 Centrifuge,capableofoperatingat4500 g . 7 5.
16、7 Centrifugetubesof50mlcapacitywithscrewcaplids . 7 5.8 Rotaryevaporator,withwaterbathsetat40C . . 7 5.9 Filterpaper,withporesize20mto25m . 7 5.10 Disposablesyringefilters,of0,2 m mporesize(optional) 7 5.11 HPLCapparatus,comprisingthefollowing . 7 5.12 Roundbottomflasks,of250mlcapacitywithgroundglas
17、sjoint. . 8 6 Procedure 8 6.1 Preparationoftestsamples. 8 6.2 Extractionofpatulin . 8 6.3 Removalofinterferingacidiccompounds . 8 6.4 Preparationofthesampletestsolution . 9 7 Spikingprocedure . . 9 7.1 Clearapplejuice. . 9 7.2 Cloudyapplejuice 9 7.3 Applepuree . 9 8 HPLCdetermination . 9 8.1 Calibra
18、tiongraph . . 9 8.2 HPLCoperatingconditions. . 10 9 Calculation. 10EN14177:2003(E) 3 10 Precision.10 10.1 Interlaboratorytest. .10 10.2 Repeatability 11 10.3 Reproducibility.11 10.4 Testreport 12 AnnexA (informative) Precisiondata .13 Bibliography15EN14177:2003(E) 4 Foreword Thisdocument(EN14177:200
19、3)hasbeenpreparedbyTechnicalCommitteeCEN/TC275“Foodanalysis Horizontalmethods”,thesecretariatofwhichisheldbyDIN. ThisEuropeanStandardshallbegiventhestatusofanationalstandard,eitherbypublicationofanidentical textorbyendorsement,atthelatestbyJune2004,andconflictingnationalstandardsshallbewithdrawnat t
20、helatestbyJune2004. AnnexAisinformative. WARNINGTheuseofthisstandardcaninvolvehazardousmaterials,operationsandequipment. Thisstandarddoesnotpurporttoaddressallthesafetyproblemsassociatedwithitsuse.Itisthe responsibilityoftheuserofthisstandardtoestablishappropriatesafetyandhealthpracticesand determin
21、etheapplicabilityofregulatorylimitationspriortouse. AccordingtotheCEN/CENELECInternalRegulations,thenationalstandardsorganizationsofthefollowing countriesareboundtoimplementthisEuropeanStandard:Austria,Belgium,CzechRepublic,Denmark, Finland,France,Germany,Greece,Hungary,Iceland,Ireland,Italy,Luxembo
22、urg,Malta,Netherlands, Norway,Portugal,Slovakia,Spain,Sweden,SwitzerlandandtheUnitedKingdom.EN14177:2003(E) 5 1Scope ThisEuropeanStandardspecifiesamethodforthedeterminationofpatulininapplejuicesandapplepuree usinghighperformanceliquidchromatography(HPLC).Themethodhasbeenvalidatedforthedetermination
23、ofpatulinviatheanalysisofnaturallycontaminatedandspikedsamplesinclearapplejuiceatlevelsranging from26 m g/lupto128 m g/l,incloudyapplejuiceatlevelsrangingfrom26 m g/lupto106 m g/landinapple pureeatlevelsrangingfrom23 m g/kgupto121 m g/kg. 2 Normativereferences ThisEuropeanStandardincorporatesbydated
24、orundatedreference,provisionfromotherpublications.These normativereferencesarecitedattheappropriateplacesinthetextandthepublicationsarelistedhereafter. Fordatedreferences,subsequentamendmentstoorrevisionsofanyofthesepublicationsapplytothis EuropeanStandardonlywhenincorporatedinitbyamendmentorrevisio
25、n.Forundatedreferencesthelatest editionofthepublicationreferredtoapplies(includingamendments). ENISO3696, WaterforanalyticallaboratoryuseSpecificationandtestmethods(ISO3696:1987) 3Principle Cloudyapplejuiceandapplepureearetreatedwithapectinaseenzyme.Patulinisextractedfromapplejuice orenzymetreatedpu
26、reewithethylacetate.Thesolventextractiscleanedupbyliquidliquidextractionwith aqueoussodiumcarbonatesolution.Theethylacetateextractisdriedwithanhydroussodiumsulfate.After evaporationofethylacetate,patulinisquantitativelydeterminedbyhighperformanceliquidchromatography (HPLC)withultraviolet(UV)detectio
27、n. 4Reagents 4.1General Duringtheanalysis,unlessotherwisestated,useonlyreagentsofrecognizedanalyticalgradeandonly distilledwaterorwaterofgrade1asdefinedinENISO3696.SolventsshallbeofqualityforHPLCanalysis. 4.2 Sodiumcarbonatesolution,massconcentration rr (Na 2 CO 3 ) 15g/l Dissolve1,5gofsodiumcarbona
28、tein100mlofwater. 4.3 Aceticacid,volumeconcentration jj (CH 3 COOH) 98% 4.4 Sodiumsulfateanhydrous 4.5 pH4water AdjustwatertopH4withaceticacid.EN14177:2003(E) 6 4.6 Absoluteethanol, jj (CH 3 CH 2 OH) 99,7% 4.7Ethyl acetate 4.8 Perchloricacid, jj (HClO 4 )=60% 4.9Acetonitrile 4.10 Endogalacturonaseen
29、zymesolution,typicalactivity1400U/g Unitdefinitiontheamountofenzymewhichcatalysesthedecreaseinviscosityofa1%pectinsolutionby 20%in5minatapH3,4andat25 C. NOTE Packagevolumesof100mlto200mlarepractical. 4.11 HPLCmobilephase Mix95partspervolumeofwaterwith5partspervolumeofacetonitrile(4.9)and0,095partspe
30、rvolumeof perchloricacid(4.8),anddegas. 4.12 Patulinstocksolution WARNINGPatulinisasuspectmutagenandhasbeenreportedtohaveimmunotoxicandneurotoxic properties. Glovesandsafety glassesshouldbewornatalltimesandallstandardandsample preparationstagesshouldbecarriedoutinafumecupboard. Dissolve5mgofpatulino
31、rthecontentsof1ampoule(ifpatulinhasbeenobtainedasafilm)inethylacetate (4.7).Transferthesolutiontoa25mlvolumetricflaskanddilutevolumewithethylacetatetoproducea solutioncontainingapproximately200 m g/mlofpatulin.Storethestocksolutioninafreezeratlessthan0 C. 4.13 Patulinstandardsolution Evaporate1000 m
32、 lofthestocksolution(4.12)todrynessundernitrogenandthenimmediatelydissolveitin 20mlofethanol(4.6)toobtainamassconcentrationofapproximately10 m g/mlofpatulin. Todeterminetheexactconcentration,recordtheabsorptioncurvebetween250nmand350nmina1cm quartzcellwithethanolasreference.Identifythewavelengthform
33、aximumabsorption.Calculatethemass concentrationofpatulin, r pat ,inmicrogramspermillilitreusingequation1: d e r = 100 max M A pat (1) where: A max istheabsorbancedeterminedatthemaximumoftheabsorptioncurve(here:atapproximately 276nm); M istherelativemolecularmassofpatulin(M=154,12g/mol); e istherelat
34、ivemolarabsorptioncoefficientofpatulininethanol,(here:1460m 2 /mol,asgivenin AOACOfficialMethods,1995,NaturalToxins,Patulin,49.6.01.C(d); d isthepathlengthofthequartzcellincentimetres.EN14177:2003(E) 7 Storethestandardsolutioninafreezeratlessthan0 C.Solutionsstoredthiswayarestablefor2months. Ensuret
35、hestandardsolutionisallowedtoreachroomtemperaturebeforeusetoavoidincorporationofwater bycondensation. 4.14 Patulinstandardsolutionforcalibration Evaporatetodryness500lofthestandardsolution(4.13)oranaliquotwhichisequivalenttoanabsolute amountof5gofpatulin,dissolvein5mlofpH4water(4.5)toobtainamassconc
36、entrationof1 m g/mlof patulin. Thissolutioncanbestoredinarefrigeratorat4 C.Asolutionstoredinthiswayisstableforatleast8weeks. 5Apparatus 5.1 General Usuallaboratoryequipmentand,inparticular,thefollowing: 5.2 Displacementpipettesof5ml,1ml,200 m land50 m lcapacitywithappropriatepipette tips 5.3 Analyti
37、calbalance 5.4 UVspectrophotometer,doublebeamandrecordingsuitableformeasurementat250nm to350nm 5.5 Quartzcells,ofopticalpathlength1cm 5.6 Centrifuge,capableofoperatingat4500 g 5.7 Centrifugetubesof50mlcapacitywithscrewcaplids 5.8 Rotaryevaporator,withwaterbathsetat40C 5.9 Filterpaper,withporesize20m
38、to25m 5.10 Disposablesyringefilters,of0,2 m mporesize(optional) Testeachbatchbeforeusetoensurethatpatulinisnotadsorbedontothefilter. 5.11 HPLCapparatus,comprisingthefollowing 5.11.1 Injectionsystem,avalveinjectionsystemwitha50 m linjectionloop 5.11.2 Pump,isocratic,pulsefree,capableofmaintainingavol
39、umeflowrateof1ml/min 5.11.3 UVdetector,fittedwithananalyticalflowcellandsetat276nm 5.11.4 AnalyticalreversephaseHPLCcolumn,forexampleC 18 octyldecylsilane(ODS)(fullyorsuperend cappedwithacarbonloadingof12%to17%hasbeenfoundsuitable)whichensuresresolutionofpatulin fromallotherpeaks.Themaximumheightofo
40、verlappingpeakshouldersshallbelessthan10%ofthe maximumpeakheight(itcouldbenecessarytoadjustthemobilephaseforsufficientbaselineresolution).A suitableprecolumnshouldbeused.EN14177:2003(E) 8 5.11.5 Datasystem 5.11.6 SwitchingvalveorsecondHPLCpump,optional,toallowwashingofanalyticalcolumnbetween injecti
41、ons 5.12 Roundbottomflasks,of250mlcapacitywithgroundglassjoint 6Procedure 6.1 Preparationoftestsamples 6.1.1 Clearapplejuice Nopreparationisrequired.Proceedasdescribedin6.2to6.4. 6.1.2Cloudy apple juice Transfer20ml+0,1mlofcloudyjuicetoacentrifugetube(5.7)andadd150 m lofenzymesolution(4.10). Coverwi
42、ththescrewcapandshakewelltomix.Leaveovernightatroomtemperatureorfor2hat40C,after whichcentrifugeat4500 gfor5min. 6.1.3Apple puree Weigh,tothenearest0,1g,a10gtestportionofthepureeintoacentrifugetube(5.7),add150 m lofenzyme solution(4.10)followedby10mlofwater.Coverwiththescrewcapandshakewell,withhomog
43、enizer dependingonthesample,tomix.Leaveovernightatroomtemperatureorfor2hat40C,afterwhich centrifugeat4500 gfor5min. 6.2 Extractionofpatulin Pipette10mlofclearjuiceorcloudyjuiceaspreparedin6.1.2intoa100mlseparatingfunnel.Forapple pureepipette10mlofapplepureeaspreparedin6.1.3intoa100mlseparatingfunnel.Thisisequivalentto 5gapplepuree.Add20mlofethylacetate(4.7)andshakefor1min.Allowthelayerstoseparatethendrain themintotwoseparateconicalflasks.Transfertheaqueousla
