SNT 2315-2009进出口动物源性食品中氨基糖苷类药物残留测定方法 放射受体分析法

中华人民共和国出入境检验检疫行业标准进出口动物源性食品中氨基糖苷类药物残留测定方法放射受体分析法2009-07-07发布2010-01-16实施国家质量监督检验检疫总局I本标准的附录A为资料性附录。1进出口动物源性食品中氨基糖苷类药物残留测定方法放射受体分析法3试样制备与保存从混合原始样品中取出部分代表性样品，剔除脂肪组织后将可食部分取有代表性样品约500g,用组织绞碎机绞碎混匀，均分成两份，分别装入洁净容器中，密封，并标明标记，于-18℃以下冷冻保存。制样过程中应防止样品受到污染或发生残留物含量的变化。4方法提要5.1组织中庆大霉素-新霉素族测试试剂盒1)1)组织中庆大霉素-新霉素族、链霉素族测试试剂盒与CharmⅡ7600分析仪是由Charm公司提供的产品。给出2存24h;1)阴性对照浓缩干粉：加入10mL水溶解，此溶液可在2℃~6℃保存48h;或将溶液保存于方法同5.2中1);3)MSU萃取缓冲液：加入1000mL水溶解，在2℃~6℃可贮存2个月4)M2缓冲液：加入50mL水溶解，在2℃~6℃可贮存2个月；6.5高速组织捣碎分散匀质器7.1.1.1阴性对照液的配制：取2.0mLAg萃取缓冲液加入6.0mL水稀释，混匀后取出4.0mL作为1)组织中庆大霉素-新莓素族、链霉素族测试试剂盒与CharmⅡ76003于80℃恒温加热器中温育30min,取出后放置冰水浴中冷却10min,于3300r/min离心10min,转移节pH值6.0,此样液待测定。7.1.3检测步骤7.2链霉素族残留测定(用于链霉素和双氢链霉素的测定)7.2.1阴性和阳性对照液的配制匀后取出2.0mL,用6.0mLMSU萃取缓冲液稀释，取出2.0mL作为阳性对照液，使用前配制。称取10.0g样品于50mL具塞离心管中，加入30mLMSU萃取缓冲液，均质1min,涡旋混匀3min,于80℃恒温加热器中温育30min,取出后放置冰水浴中冷却10min,于3300r/min离心10min,转移全部上清液于另一50mL离心管中，用M2缓冲液调节pH值7.5,此样液待测定。7.2.3检测步骤7.2.3.2向离心管中加入2.0mL样品提取液(7.2.2),涡旋混匀10s,35℃±1℃温育2min。7.2.3.3取出后将绿色链霉素氘标记物药片压入离心管，涡旋混匀10s,35℃士1℃温育2min。8结果判定8.1控制点的确定8.1.1庆大霉素-新霉素族残留控制点的确定称取20.0g同类空白样品，添加0.2mLMSU多抗生素标准品溶液，充分混匀，按7.1.2和7.1.3进行提取与测定，测定6个非重复加标样品的cpm计数值，取结果平均值，该平均值乘以系数1.3即为控48.2结果判定10方法检出限链霉素方法检出限为500pg/kg;双氢链霉素方法检出限为500pg/kg;庆大霉素方法检出限为50μg/kg;新霉素方法检出限为30kg/kg;卡那霉素方法检出限为1004g/kg;安普霉素方法检出限为5(资料性附录)试剂盒与仪器性能监测A.2仪器性能监测取空的专用试管放入液体闪烁计数仪计数，cpm值需小于50,否则需调零。如测得的cpm值偏大，可能由液体闪烁计数仪有大量静电存在所引起，可用湿布将试管外壁湿润后放入液体闪烁计数仪进行应小于0.1。6ThestandardwasdraftedbyHenanEntry-ExitInspectionandQuarantineBureau.ThestandardwasmainlydraftedbyWeiWei,YangJizhou,Yuanping,LiuYafeng,GuoJunfeng,ZhuWeixia.firsttime.7SN/T2315—2009Thestandardspecifiesthemethodforthesamplepreparationanddeterminationofstreptomycin,di-hydrostreptomycin,gentamicin,neomycin,kanamycin,apramycinandparomomycinaminoglycosidesresiduesbyRadio-receptormethodinfoodstuffsofanimalorigin.Thisstandardisapplicabletothedeterminationofstreptomycin,dihydrostreptomycin,gentamicin,neomycin,kanamycin,apramycinandparomomycinaminoglycosidesresiduesinanimalmusclesinclu-dingpork,cattle,chicken,fish.Thefollowingnormativedocumentscontainprovisionswhich,throughreferenceinthistext,consti-tuteprovisionsofthisnationalstandard.Fordatedreferences,subsequentamendmentsto,orrevi-sionsof,anyofthesepublicationsdonotapply.However,partiestoagreementsbasedinthisnation-alstandardareencouragedtoinvestigatethepossibilityofapplyingthemostrecenteditionsofthenormativedocumentsindicatedbelow.Forundatedreferences,thelatesteditionofthenormativedocumentreferredtoapplies.GB/T6682waterforanalyticallaboratoryuse—SpecificationandtestmethodsTaketherepresentativeportionsfromthewholeprimarysample.Aftertrimmedfattissues,500gedibleportionisgrindedandblendedbyatissueblender.Thehomogenizedsampleisdividedequallyintotwoandputinasuitablecleancontainer.Afterbeingsealedandlabeled,thesampleshouldbestoredatbelow-18℃.Inthecourseofsamplingandsamplepreparation,precautionsmustbetakentoavoidcontaminationoranyfactorswhichmaycausethechangeofresiduecontent.8SN/T2315—2009Radio-receptormethodbasesics.Sothespecificityofthereceptorcanrecompetitionwithanexemptamountof[3H]labeledaminoglycosidesforthespecificityofthtorsites.Afterreactioninthetemperatureitwascentrifuged,andtheunboticsthatwerediscarded.ThenitwUnlessotherwisespecified,allreagentsshouldbeofanalyticaTgrade;wateristhefirstgradewater5.1Tissuegentamicinandneomycin-typetestkit¹)followingparts.heldfor48hat2℃~6℃;orfrozenat.below-15C.Defrostthereconstitutedconcentratforeusing,itmaybestoredfor24hat2℃~6℃;shouldbeusedbeforetheexpiration1)Thisinformationisgivenfortheconvenienceofthestandarduser,butthisdoesnotimprovethattheproductisapproved.Ifothereuqalproductshavethesameeffect,theyareencouragedtouse9Itmustbeperformancetestedofeachtestkitbeforeusing,seeAnnexA.Itismainlycompothefollowingparts.constitutebuffermaybeheldfor482)MSUMulti-AntimicrobialConcentrateStandard:Reconstitutebufferdissolvein10mLwaterbe-foreusing.Theconcentrationisequalto1000pg/Lstreptomycinstandard.Thestorageofsolu-3)MSUextractionbuffer:ReconstitutebufferdisSolvein1000mLwaterandthesolutionisstoredfortwomonthsat2℃~6℃;4)M2Buffer:Dissolvein50mLwater.Itisheldfortwomonthsat2℃~6℃;5)Streptomycin-typebindingrengenttablet:Whitecolour,itisstoredatbelow-15℃andshouldbeusedbeforetheexpirationdatefromthepackagingdate;6)streptomycin-typedeuteratetabletrGreencolour,itisstoredatbelow-15Candshouldbeusedbeforetheexpirationdatefromthepgckagingdate.5.4Hydrochloricacid.water.5.60.1mol/Lhydrochloricacidsolution:Taken9mLhydrochloricacidisslowlyaddto400mL~600mLwater.Aftermixed,thesolutionisdilutedto1000mLwithwater.proved.Ifothereugalproductshavethesameeffect,theyareencouragedtouse.6.3pHmeters:accurateto±0.1.6.4Temperatureconstantincubator:80℃±2℃,35℃±1℃,6.5Homogenizer.6.8Pressedstickfortablet.6.9Borosilicateglasstesttubewithcap:8mL7.1Theanalysisofgentamicinandneomycin-type(forthedeterminationofgentamicin,neomycin,7.1.1Negativecontrolandpositivecontrolsolutionpreparation7.1.1.1Negativecontrolsolutionpreparation:Dilute2.0mLAgextractionbufferwith6.0mLofwatertomakethenegativecontrolsolutions.Afetermixing,use4.0mLasnegativecontrolsolution.Weigh20.0gofsampletoa50mLcentrifugetubewithacap.Add20mLAgextractionbuffertothecentrifugetube.Homogenizefor1minandvortexfor3min.Theso3300r/min,andthenthesupertantwastransferredtoanother50mLcentrifugetubeandadjustedtopH6.0with0.1mol/Lsodiumhydroxide(5.5)or0.1mol/Lhydrochloricacid(5.6).Thesolutionisreadyfordetermination.7.1.3.2Add4.0mLsampleextractsolution(7.1.2)tothecentrifugetube.Usingthepressstickoftablets,pressyellowtabletthroughintothetube.Vortex-mixingfor15sandincubateat35℃±7.1.3.3Aftertakenout,thesolutioniscentrifugedfor5minat3300r/min,thesupernatantswere7.1.3.4Add0.3mLwater,Vortex-mixingfor10stodisslovethesolidinthetube.Add3mLscin-tillationfluid(5.7)tothetesttube.Aftervortex-mixed,thesolutionwastakenintocountin7600analyzerandreadcpmvalueon3Hchannelfor60s7.2Theanalysisofstreptomycin-type(forthedeterminationofdihydrostrepmycinandstreptomy-7.2.1Negativecontrolandpositivecontrolsolutionpreparation7.2.1.1Negativecontrolsolutionpreparation;Dilute2.0mLnegativeconcentratesolutionwith6.0mLMSUextractionbuffer.Use2.0mLsolutionasnegativecontrolandthesolutibeforeusing.sing.Weigh10.0gofhomogenizedsampletoa50mLcentrifugetubewithacap.Add30mLMSUextrac-tionbuffertothecentrifugetube.Homogenizefor1minandvortexfor3min.Thesolutionwasincu-takenup,centrifugedfor10minat3300r/min,thesupernatantsweretransferredtoanother50mLcentrifugetubeandadjustedtopH7.5withM2buffer.Thesolutionisreadyfordetermination.7.2.3.1Usingthebluntendofpressstickoftablets,presswhitetabletintoanemptycentrifuge7.2.3.2Add2.0mLdilutedsampleextracttothecentrifugetube.Aftervortex-mixedfor10s,theSN/T2315—20097.2.3.3Aftertakenout,greentabletswerepressedintothecentrifugetubeandvortex-mixedfor7.2.3.4Afterincubated,centrifugefor5minat3300r/min,thesupernatantswerediscardedim-7.2.3.5Add0.3mLwater,mixthotesttube.Aftervortex-mixed,thesolutionwastakenintocountin7600analyzerandreadcpmvalueon3Hchannelfor60s.8.1Determination20.0gblanksamplespiked0.PmLMSUMulti-Antimicrobialconcentratestandard.Aftermixedthoroughly,extractionanddeterminationprocedureisthesameastheprocedu(7.1.2and7.1.3).Sixspikesampleswerepreparedandthen6cpmvalueswereobtained.Finally,theresultswereaveraged.Thecontrolpointinclude:averagenumbers×(1+30%).8.1.2Thecontrolpointofstreptomycin-type10.0gblanksamplespiked0.5mLMSUMulti-Antimicrobialconcentratestandard.Aftermixedthoroughly,extractionanddeterminationprocedure/isthesameastheproceduredescribedabove(7.2.2and7.2.3).SixspikeSampleswerepreparedandthén6cpmvalueswereobtained.Finally,theresultswereaveraged.Thecontrolpointinclude:averagenumbers×(1+30%).lfthesamplecpmvalueisgreaterthanthecontrolpoint,thesampleisnegative.Ifthesamplecpmstandard(AnnexA.1)forgentamicinandneomycin-type,negativecontrol(7.1.1.2)shouldberetested.Forstreptomycin-type,negativecontrol(7.1.1.1)andcontrol(7.2.1.1)andcontrol(7.2.1.2)shouldbersampleretestedcpmvalueisgreaterthanthecontrolpoint,thesampleisnegative.Ifthesultislessthancontrolpoint9Verificationtesttestusingotherdetectionmethod.Thelimitofdeterm