SNT 2315-2009進(jìn)出口動(dòng)物源性食品中氨基糖苷類(lèi)藥物殘留測(cè)定方法 放射受體分析法

中華人民共和國(guó)出入境檢驗(yàn)檢疫行業(yè)標(biāo)準(zhǔn)進(jìn)出口動(dòng)物源性食品中氨基糖苷類(lèi)藥物殘留測(cè)定方法放射受體分析法2009-07-07發(fā)布2010-01-16實(shí)施國(guó)家質(zhì)量監(jiān)督檢驗(yàn)檢疫總局I本標(biāo)準(zhǔn)的附錄A為資料性附錄。1進(jìn)出口動(dòng)物源性食品中氨基糖苷類(lèi)藥物殘留測(cè)定方法放射受體分析法3試樣制備與保存從混合原始樣品中取出部分代表性樣品，剔除脂肪組織后將可食部分取有代表性樣品約500g,用組織絞碎機(jī)絞碎混勻，均分成兩份，分別裝入潔凈容器中，密封，并標(biāo)明標(biāo)記，于-18℃以下冷凍保存。制樣過(guò)程中應(yīng)防止樣品受到污染或發(fā)生殘留物含量的變化。4方法提要5.1組織中慶大霉素-新霉素族測(cè)試試劑盒1)1)組織中慶大霉素-新霉素族、鏈霉素族測(cè)試試劑盒與CharmⅡ7600分析儀是由Charm公司提供的產(chǎn)品。給出2存24h;1)陰性對(duì)照濃縮干粉：加入10mL水溶解，此溶液可在2℃~6℃保存48h;或?qū)⑷芤罕４嬗诜椒ㄍ?.2中1);3)MSU萃取緩沖液：加入1000mL水溶解，在2℃~6℃可貯存2個(gè)月4)M2緩沖液：加入50mL水溶解，在2℃~6℃可貯存2個(gè)月；6.5高速組織搗碎分散勻質(zhì)器7.1.1.1陰性對(duì)照液的配制：取2.0mLAg萃取緩沖液加入6.0mL水稀釋?zhuān)靹蚝笕〕?.0mL作為1)組織中慶大霉素-新莓素族、鏈霉素族測(cè)試試劑盒與CharmⅡ76003于80℃恒溫加熱器中溫育30min,取出后放置冰水浴中冷卻10min,于3300r/min離心10min,轉(zhuǎn)移節(jié)pH值6.0,此樣液待測(cè)定。7.1.3檢測(cè)步驟7.2鏈霉素族殘留測(cè)定(用于鏈霉素和雙氫鏈霉素的測(cè)定)7.2.1陰性和陽(yáng)性對(duì)照液的配制勻后取出2.0mL,用6.0mLMSU萃取緩沖液稀釋?zhuān)〕?.0mL作為陽(yáng)性對(duì)照液，使用前配制。稱(chēng)取10.0g樣品于50mL具塞離心管中，加入30mLMSU萃取緩沖液，均質(zhì)1min,渦旋混勻3min,于80℃恒溫加熱器中溫育30min,取出后放置冰水浴中冷卻10min,于3300r/min離心10min,轉(zhuǎn)移全部上清液于另一50mL離心管中，用M2緩沖液調(diào)節(jié)pH值7.5,此樣液待測(cè)定。7.2.3檢測(cè)步驟7.2.3.2向離心管中加入2.0mL樣品提取液(7.2.2),渦旋混勻10s,35℃±1℃溫育2min。7.2.3.3取出后將綠色鏈霉素氘標(biāo)記物藥片壓入離心管，渦旋混勻10s,35℃士1℃溫育2min。8結(jié)果判定8.1控制點(diǎn)的確定8.1.1慶大霉素-新霉素族殘留控制點(diǎn)的確定稱(chēng)取20.0g同類(lèi)空白樣品，添加0.2mLMSU多抗生素標(biāo)準(zhǔn)品溶液，充分混勻，按7.1.2和7.1.3進(jìn)行提取與測(cè)定，測(cè)定6個(gè)非重復(fù)加標(biāo)樣品的cpm計(jì)數(shù)值，取結(jié)果平均值，該平均值乘以系數(shù)1.3即為控48.2結(jié)果判定10方法檢出限鏈霉素方法檢出限為500pg/kg;雙氫鏈霉素方法檢出限為500pg/kg;慶大霉素方法檢出限為50μg/kg;新霉素方法檢出限為30kg/kg;卡那霉素方法檢出限為1004g/kg;安普霉素方法檢出限為5(資料性附錄)試劑盒與儀器性能監(jiān)測(cè)A.2儀器性能監(jiān)測(cè)取空的專(zhuān)用試管放入液體閃爍計(jì)數(shù)儀計(jì)數(shù)，cpm值需小于50,否則需調(diào)零。如測(cè)得的cpm值偏大，可能由液體閃爍計(jì)數(shù)儀有大量靜電存在所引起，可用濕布將試管外壁濕潤(rùn)后放入液體閃爍計(jì)數(shù)儀進(jìn)行應(yīng)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