1、A Referencenumbe r ISO13493:1998 (E) INTERNATIONAL STANDARD ISO 13493 Firstedition 19980901 MeatandmeatproductsDetermination ofchloramphenicolcontentMethod usingliquidchromatography ViandeetproduitsbasedeviandeDosageduchloramphnicol MthodeparchromatographieenphaseliquideISO13493:1998(E) ISO1998 Allr
2、ightsreserved.Unlessotherwisespecified,nopartofthispublicationmaybereproduced orutilizedinanyformorbyanymeans,electronicormechanical,includingphotocopyingand microfilm,withoutpermissioninwritingfromthepublisher. InternationalOrganizationforStandardization Casepostale56 CH1211Genve20 Switzerland Inte
3、rnet isoiso.ch PrintedinSwitzerland ii Foreword ISO(theInternationalOrganizationforStandardization)isaworldwide federationofnationalstandardsbodies(ISOmemberbodies).Theworkof preparingInternationalStandardsisnormallycarriedoutthroughISO technicalcommittees.Eachmemberbodyinterestedinasubjectforwhich
4、atechnicalcommitteehasbeenestablishedhastherighttoberepresented onthatcommittee.Internationalorganizations,governmentalandnon governmental,inliaisonwithISO,alsotakepartinthework.ISO collaboratescloselywiththeInternationalElectrotechnicalCommission (IEC)onallmattersofelectrotechnicalstandardization.
5、DraftInternationalStandardsadoptedbythetechnicalcommitteesare circulatedtothememberbodiesforvoting.PublicationasanInternational Standardrequiresapprovalbyatleast75%ofthememberbodiescasting avote. InternationalStandardISO13493waspreparedbyTechnicalCommittee ISO/TC34, Agriculturalfoodproducts ,Subcomm
6、itteeSC6, Meatandmeat products. AnnexAofthisInternationalStandardisforinformationonly.INTERNATIONALSTANDARD ISO ISO13493:1998(E) 1 MeatandmeatproductsDeterminationofchloramphenicol contentMethodusingliquidchromatography 1Scope ThisInternationalStandardspecifiesaliquidchromatographicmethodforthedeter
7、minationofthechloramphenicol contentofthemuscletissueofmeat,includingpoultry. Themethodissuitableforthedeterminationofchloramphenicolcontentsgreaterthan6,5 m g/kg. Testsampleswhichhavedeterioratedcannotbeanalysedwiththismethod. NOTEThisInternationalStandardmaybeapplicableforthedeterminationofthechlo
8、ramphenicolcontentofallkindsofmeat andmeatproducts.However,materialsotherthanmuscletissuewerenotincludedinthecollaborativetestingofthemethod. 2Normativereference Thefollowingstandardcontainsprovisionswhich,throughreferenceinthistext,constituteprovisionsofthis InternationalStandard.Atthetimeofpublica
9、tion,theeditionindicatedwasvalid.Allstandardsaresubjectto revision,andpartiestoagreementsbasedonthisInternationalStandardareencouragedtoinvestigatethe possibilityofapplyingthemostrecenteditionofthestandardindicatedbelow.MembersofIECandISOmaintain registersofcurrentlyvalidInternationalStandards. ISO3
10、696:1987, WaterforanalyticallaboratoryuseSpecificationandtestmethods. 3Definition ForthepurposesofthisInternationalStandard,thefollowingdefinitionapplies. 3.1 chloramphenicolcontentofmeatandmeatproducts massfractionofchloramphenicolresiduedeterminedaccordingtotheprocedurespecifiedinthisInternational
11、 Standard. NOTEThechloramphenicolcontentisexpressedinmicrogramsperkilogram.ISO13493:1998(E) ISO 2 4Principle Atestportionisextractedwithwater.Filtrationandsolidphaseextractionareusedtoisolatethelipophilic componentsfromtheaqueoussolution.Thechloramphenicoliselutedfromthecartridgewithdichloromethane.
12、The organicphaseisevaporatedandpurifiedbyliquidliquidextractionwithwaterandtoluene.Thechloramphenicolis measuredwithreversephasechromatographybyultraviolet(UV)detection. 5Reagents Useonlyreagentsofrecognizedanalyticalgrade,unlessotherwisespecified. 5.1 Water,complyingwithatleastgrade3inaccordancewit
13、hISO3696.Thewatershallbefreeoforganic compounds. 5.2 Nitrogen,suitableforevaporatingsolvents. 5.3 Dichloromethane. 5.4 Toluene. 5.5 Acetatebuffer, c(CH 3 CO 2 Na)=0,01mol/l,pH=4,3. Dissolve0,82gofanhydroussodiumacetateinabout970mlofwater.AdjustthepHto4,3with50%( m/m)dilute aceticacid(CH 3 CO 2 H)usi
14、ngthepHmeter(6.1).Transferthesolutiontoa1000mlonemarkvolumetricflask. Dilutetothemarkwithwaterandmix. 5.6Acetonitrile, suitableforUVspectroscopy. 5.7Mobilephase. Add750mlofacetatebuffer(5.5)to250mlofacetonitrile(5.6)andmixthoroughly. Beforeuse,filtertheeluentthrougha0,22 mmfilter(6.2)anddegas. 5.8Ch
15、loramphenicolstocksolution, 100 mg/ml. Weigh,tothenearest0,1mg,10mgofchloramphenicolandtransferittoa100mlonemarkvolumetricflask.Dilute tothemarkwithmethanolandmix. Thisstocksolutionisstablefor1monthwhenstoredinthedark. 5.9 Chloramphenicolstandardsolutions. Pipette5,0mlofthestocksolution(5.8)intoa100
16、mlonemarkvolumetricflask.Dilutetothemarkwithwaterand mix. Preparefourstandardsolutionsbydiluting1,0ml,2,0ml,5,0mland15,0mlofthissolutionto100mlwithwaterto obtainsolutionswithachloramphenicolcontentof0,05 mg/ml,0,10 mg/ml,0,25 mg/mland0,75 mg/mlrespectively. Thesestandardsolutionsarestablefor1weekwhe
17、nstoredinthedark. 6Apparatus Usuallaboratoryapparatusand,inparticular,thefollowing.ISO ISO13493:1998(E) 3 6.1pHmeter. 6.2Membranefilter, oflowdeadvolumeandporesize0,22 mm. 6.3Mechanicalorelectricalequipment capableofhomogenizingthelaboratorysample. Thisincludesahighspeedrotationalcutter,oramincerfit
18、tedwithaplatewithaperturesnotexceeding4,0mmin diameter. 6.4Laboratoryblender (e.g.Stomacherblender 1) orvortextype). 6.5Filterpaper, quantitative,fastfiltrationrate,ofdiameterabout15cm. NOTE Forexample,Whatman41provedtobesuitable 1) . 6.6Extractioncartridges, ofcapacity20ml,containingdiatomaceousear
19、ththatextractslipophiliccomponents fromaqueoussolutions. NOTE Extrelut,manufacturedbyMerck,Darmstadt,Germany(No.11737),provedtobesuitable 1) . 6.7Waterbath orheatingblock,capableofbeingmaintainedat(401)C,withequipmentfordryingwith nitrogen(5.2);or rotaryvacuumevaporator. 6.8Centrifugetubes, ofcapaci
20、ty25ml. 6.9Vortexmixer, operatingatarotationfrequencyofabout700min -1 . 6.10Centrifuge, operatingataradialaccelerationofabout1000 g. 6.11Micropipettes, ofcapacity300 ml. 6.12Liquidchromatograph, equippedwith: aconstantflowpump; aninjector; areversephaseC 8 orC 18 columnwithaninternaldiameterof3mm,le
21、ngthof20cm,andparticlesizeof 5 m m,oracolumnofequivalentquality; aUV/VISdetectorsuitableformeasurementsatawavelengthof285nm;ifavailable,adiodearraydetector (forconfirmationpurposes); arecorderwithvariablemeasuringrangeoranintegrator. 7Sampling SamplingisnotpartofthemethodspecifiedinthisInternational
22、Standard.Arecommendedsamplingmethodis giveninISO310011. Itisimportantthatthelaboratoryreceiveasamplewhichistrulyrepresentativeandhasnotbeendamagedor changedduringtransportorstorage. Proceedfromarepresentativesampleofatleast200g.Storethesampleinsuchawaythatdeteriorationand changeincompositionarepreve
23、nted. 1)Theseareexamplesofsuitableproductsavailablecommercially.Thisinformationisgivenfortheconvenienceofusersof thisInternationalStandardanddoesnotconstituteanendorsementbyISOoftheseproducts.ISO13493:1998(E) ISO 4 8Preparationoftestsample Allowthesampletoreachroomtemperature.Removeexcessoffatandine
24、dibleparts. Homogenizethelaboratorysamplewiththeappropriateequipment(6.3).Takecarethatthetemperatureofthe samplematerialdoesnotriseabove25C.Ifamincerisused,passthesampleatleasttwicethroughthe equipment. Fillasuitableairtightcontainerwiththepreparedsample.Closethecontainerandstoreinsuchawaythat deter
25、iorationandchangeincompositionareprevented. Storethesample,ifnecessary,atatemperaturebelow -18C. Analysethesampleassoonaspracticable,butalwayswithin24hafterhomogenization. 9Procedure NOTEIfitisrequiredtocheckwhethertherepeatabilitylimit(see11.2)ismet,carryouttwosingledeterminationsin accordancewith9
26、.1to9.6. 9.1General Inconjunctionwiththeanalysisofthetestsolution(oraseriesoftestsolutions),analyseaspikedblanksamplewith achloramphenicolcontentof10 mg/kgandablanksample. 9.2Testportion Weigh10g( m)ofthepreparedtestsample(seeclause8)tothenearest0,1gina100mlconicalflask. 9.3Preparationofextract Add4
27、0,0mlofwaterandmixvigorouslyfor3minwiththelaboratoryblender(6.4). Thevolume( V 1 )ofthewaterphaseobtainedis40,0mlplusthevolumeofwaterinthetestportion(normallyabout 7,5mlwaterin10gofsample). Filterthesamplethroughafilterpaper(6.5). 9.4Solidphaseextraction Transfer20,0ml( V 2 )ofthefiltratetoanextract
28、ioncartridge(6.6). After(150,2)min,elutethechloramphenicolwith70mlofdichloromethane(5.3).Evaporatetheorganicphaseto avolumeofabout1mlunderagentlestreamofnitrogen(5.2)inthewaterbath(6.7)orusingtherotaryvacuum evaporator(6.7). Transfertheresiduetoacentrifugetube(6.8)withabout10mlofdichloromethane(5.3)
29、. Evaporatecarefullytoabsolutedryness.ISO ISO13493:1998(E) 5 9.5Liquidliquidextraction Add400 ml( V 3 )ofwaterand2,0mloftoluene(5.4)totheresidueandmixgentlyfor1minatarotationfrequencyof about700min -1 onthevortexmixer(6.9). Centrifugefor5minataradialaccelerationof1000 ginthecentrifuge(6.10). Removea
30、smuchaspossibleoftheorganicphasewithapipetteanddiscardit. Add1,5mloftolueneandmixgentlyfor1minatarotationfrequencyofabout700min -1 onthevortexmixer(6.9). Centrifugefor5minataradialaccelerationof1000 ginthecentrifuge(6.10). Removeasmuchaspossibleoftheorganicphasewithapipetteanddiscardit. Transfer300
31、mloftheaqueousphasetoasuitablecontainerusingamicropipette(6.11). 9.6Chromatographicanalysis 9.6.1Chromatographicconditions Parameter Setting Wavelength 285nm Detectorrange absorbanceof0,005to0,01atfullscaleofrecorder Recorderrange 10mV Paperspeed 1,0cm/min Mobilephase(5.7)volumeflowrate 0,6ml/min In
32、jectionvolume 100 ml NOTE Theinjectionvolumeandthevolumeflowratedependonthecolumndimensions. 9.6.2Chromatographicprocedure Waituntiltheliquidchromatograph(6.12)systemisstab ilized.Injecttheblanksample,thespikedblanksample,the fourchloramphenicolstandardsolutions(5.9),thetestsolutionobtainedin9.5,and
33、againthechloramphenicol standardsolutions(5.9). Checkforchloramphenicolsignalsinthesamplechromatogramsattheretentiontimeofchloramphenicol. 9.6.3Measurement Measurethechloramphenicolpeakheightsorpeakareasofthetestsolutionandthechloramphenicolstandard solutions. Theresponsesobtainedforthechloramphenic
34、olstandardsolutionsshallbelinearlyrelatedtothechloramphenicol contentsofthesesolutions. NOTEConfirmationcanbecarriedoutwithadiodearraydetectorforchloramphenicolcontentsexceeding10 mg/kg.ISO13493:1998(E) ISO 6 10Calculation Calculatethechloramphenicolcontentofthetestsampleusingtheequation: w hV V hmV
35、 = r 13 2 s where w isthechloramphenicolcontent,inmicrogramsperkilogram,ofthetestsample; h isthepeakheightorpeakarea,inlengthorareaunits,foundforthetestsolution; h s isthepeakheightorpeakarea,inlengthorareaunits,foundforoneofthestandardsolutions(5.9); r isthechloramphenicolcontent,inmicrogramspermil
36、lilitre,ofthestandardsolution; m isthemass,ingrams,ofthetestportion(9.2); V 1 isthevolume,inmillilitres,ofthewaterphaseobtainedaftermixingin9.3( V 1 =40ml+thevolumeofwaterin thetestportion); V 2 isthevolume,inmillilitres,offiltratetransferredin9.4totheextractioncartridge( V 2 =20ml); V 3 isthevolume
37、,inmicrolitres,ofwateraddedin9.5totheresidue( V 3 =400 ml). Reporttheresultroundedtothenearest0,1 mg/kg. Theresultshallnotbecorrectedforrecovery.Therecoveryshallbespecifiedinthetestreport(clause12). 11Precision 11.1Interlaboratorytests Theprecisionofthemethodwasestablishedbyaninterlaboratorytestcarr
38、iedoutinaccordancewithISO5725 2 2) . Theresultsofthisinterlaboratorytesthavebeenpublished(seereference5).Thevaluesderivedfromthistest maynotbeapplicabletoconcentrationrangesandmatricesotherthanthosegiven. Theresultsofanotherinterlaboratorytest,carriedoutinaccordancewithISO5725,showthatrecoveryformea
39、t, meatproductsandpoultryisreproducibleandapproximately55%. 11.2Repeatability Theabsolutedifferencebetweentwoindependentsingletestresults,obtainedusingthesamemethodonidentical testmaterialinthesamelaboratorybythesameoperatorusingthesameequipmentwithinashortintervaloftime, willinnotmorethan5%ofcasese
40、xceed2,1 mg/kgforachloramphenicolcontentof10 mg/kg. 2)ISO5725:1986(nowwithdrawn)wasusedtoobtaintheprecisionresults.ISO ISO13493:1998(E) 7 11.3Reproducibility Theabsolutedifferencebetweentwosingletestresults,obtainedusingthesamemethodonidenticaltestmaterial indifferentlaboratorieswithdifferentoperato
41、rsusingdifferentequipment,willinnotmorethan5%ofcasesexceed 4,9 mg/kgforachloramphenicolcontentof10 mg/kg. 12Testreport Thetestreportshallspecify: allinformationnecessaryforthecompleteidentificationofthesample; thesamplingmethodused,ifknown; thetestmethodused,withreferencetothisInternationalStandard;
42、 alloperatingdetailsnotspecifiedinthisInternationalStandard,orregardedasoptional,togetherwithdetailsof anyincidentsthatoccurredwhichmayhaveinfluencedthetestresult(s); thetestresultobtained,orthetwotestresultsobtainediftherepeatabilityhasbeenchecked; therecovery.ISO13493:1998(E) ISO 8 AnnexA (informative) Bibliography 1 ISO31001:1991, MeatandmeatproductsSamplingandpreparationoftestsamplesPart1:Sampling. 2 ISO5725
