SNT 3264-2012 出口食品中魚藤酮和印楝素殘留量的檢測方法

SN/T3264—2012出口食品中魚藤酮和印楝素殘留量的檢測方法液相色譜-質(zhì)譜/質(zhì)譜法國家質(zhì)量監(jiān)督檢驗檢疫總局ISN/T3264—2012本標準按照GB/T1.1—2009給出的規(guī)則起草。請注意本文件的某些內(nèi)容可能涉及專利。本文件的發(fā)布機構(gòu)不承擔識別這些專利的責任。本標準由國家認證認可監(jiān)督管理委員會提出并歸口。本標準起草單位：中華人民共和國福建出入境檢SN/T3264—2012出口食品中魚藤酮和印楝素殘留量的檢測方法液相色譜-質(zhì)譜/質(zhì)譜法二乙烯基苯-吡咯烷酮聚合物填料的固相萃取小柱凈化，液相色譜-質(zhì)譜/質(zhì)譜儀檢測及確證，外標法印楝素：英文名Azadirachtin,分子式C??H??O??,CASNo.11141-17-6,分子量720.71):純度≥98%4.11魚藤酮和印楝素標準貯備液(100mg/L):準確稱取0.0100g魚藤酮和印楝素標準物質(zhì)，用甲醇溶解并定容至100mL,該標準儲備液于4℃避光保存不超過1個月。4.12魚藤酮和印楝素標準工作液：根據(jù)需要取適量標準貯備液，以20%乙腈水溶液稀釋成適當濃度25.3離心機：4500r/min,配有50mL的具塞塑料離心管。5.7超聲波清洗器。6試樣制備和保存密封并標識。6.2試樣保存3SN/T3264—20125mL飽和碳酸氫鈉溶液(4.8)振搖后避光浸泡10min,加入15mL乙腈(4.1),渦動30s后超聲提取約3g氯化鈉(4.5)鹽析，4500r/min離心3min離心，取上清液，加入2mL經(jīng)乙腈飽和后的正已烷(4.2),振搖1min,4500r/min離心3min離心，棄去正己烷層，乙腈層于45℃減壓旋轉(zhuǎn)蒸發(fā)至近干，酸氫鈉和15mL乙腈，振搖均勻后超聲提取10min,450010mL乙腈重復(fù)提取1次，合并提取掖，加入約3g氯化鈉鹽析，4500rmin離心3min,取上清液，于7.1.3蜂蜜：稱取2g(精確至0.01g)試樣置于50mL具塞塑料離心管中，加入5mL飽和碳酸氫鈉溶于45℃減壓旋轉(zhuǎn)蒸發(fā)至近干，以5mL20%甲醇水溶解殘渣，按7.2步驟凈化。7.1.4牛奶、果汁及葡萄酒等：稱取2g(精確至0.01g)試樣置于50mL具塞塑料離心管中，加入約2g碳酸氫鈉和15mL乙腈，渦動30s后超聲提取10min,4500r/min離心3min,移出有機相，殘渣再加入10mL乙腈重復(fù)提取1次，合并提取液，加入約3g氯化鈉鹽析，4500r/min離心3min,取上清碳酸氫鈉溶液振搖，加入15mL乙腈，渦動30s后超聲提取10min,4500r/min離心3min,移出有機相，殘渣再加入10mL乙腈重復(fù)提取1次，合并是取液，加入約3g氯化鈉鹽析，4500r/min離心3min,取上清液，加入5mL正己烷，振搖1mirl,4.500E/min離心3min,棄去正己烷層，乙腈層于45℃減壓旋轉(zhuǎn)蒸發(fā)至近干，以5mL20%甲醇水溶解殘渣，按7.步驟凈化。1mL/min,收集洗脫液，于45℃氮吹至近干，以20%乙腈水容液定容1mL,過0.22μm濾膜(4.14),7.3.1.3流動相：乙腈-5mmol/L乙酸銨緩沖液(4.9)(35+65,體積分數(shù))。4SN/T3264—20127.3.2.2掃描方式：多反應(yīng)監(jiān)測(MRM)。7.3.2.3其他參考質(zhì)譜條件參見表A.1。7.3.3液相色譜-質(zhì)譜/質(zhì)譜測定根據(jù)試樣中被測物的含量情況，選取響應(yīng)值適宜的標準工作液進行色譜分析。標準工作液和待測樣液中魚藤酮和印楝素的響應(yīng)值均應(yīng)在儀器線性響應(yīng)范圍內(nèi)。按式(1)進行結(jié)果計算。在本方法條件下，魚藤酮和印楝素的保留時間約為5.6min和4.2min,魚藤酮和印楝素標準品的多反應(yīng)監(jiān)測(MRM)色譜圖參見附錄B。7.3.4定性標準進行樣品測定時，如果檢出的色譜峰保留時間與標準樣品一致，并且在扣除背景后的樣品譜圖中，各定性離子的相對豐度與濃度接近的同樣條件下得到的標準溶液譜圖相比，最大允許相對偏差不超過表1中規(guī)定的范圍，則可判斷樣品中存在對應(yīng)的被測物。表1定性確證時相對離子豐度的最大允許偏差>20～50>10～207.4空白實驗除不加試樣外，均按上述步驟進行。8結(jié)果計算和表述用數(shù)據(jù)處理軟件或按式(1)計算試樣中魚藤酮和印楝素藥物的殘留量，計算結(jié)果需扣除空白值：………………(1)X——試樣中魚藤酮或印楝素殘留量，單位為微克每千克(μg/kg);c——從標準工作曲線得到的魚藤酮和印楝素溶液濃度，單位為微克每升(μg/L);V——樣品溶液最終定容體積，單位為毫升(mL);m——最終樣液所代表的試樣質(zhì)量，單位為克(g)。9測定低限、回收率9.1測定低限9.2回收率食品中魚藤酮和印楝素殘留的添加回收數(shù)據(jù)見表2。5SN/T3264—2012樣品名稱魚藤酮印楝素添加濃度回收率范圍%添加濃度回收率范圍%大米83.5～102.0275.2～104.5184.6～101.8479.6～105.2579.3～97.880.5～101.3花椰菜81.0～103.0284.8～106.5179.4～96.8480.1～102.4580.8～108.778.9～100.3蘋果81.0～96.0281.4～102.8179.6～98.4486.6～106.5578.4～100.390.1～102.4木耳81.0～99.0282.1～108.2179.0～99.8485.6～104.4581.4～104.581.4～104.8茶葉78.0～103.0274.6～107.6178.8～102.2476.2～100.3577.4～99.778.9～104.8蜂蜜84.0～101.0279.4～102.8180.2～97.6480.5～103.2580.5～99.780.5～100.5牛奶76.2～104.3278.6～109.6176.4～106.6476.2～95.5576.2～102.881.9～106.8豬肝75.0～98.0277.2～106.5178.4～103.2476.3～101.5576.8～100.181.8～100.7魚肉79.0～105.0277.5～100.3179.6～101.2476.2～101.7580.7～103.080.9～98.4蝦肉81.0～97.0277.2～102.8180.6～101.8479.2～105.3580.3～96.382.7～101.4雞肉83.0～97.0278.6～104.5180.4～104.2479.3～93.8581.2～96.981.9～106.86(資料性附錄)表A-?—魚藤酮和印楝素的主要參考質(zhì)譜參數(shù)化合物監(jiān)測離子駐留時間m)S電壓V碰撞能量魚藤酮印楝素注：表中黑體字顯示的離子為定量離子；對于不同質(zhì)譜儀器，儀器參數(shù)可能存在差異，測定前應(yīng)將質(zhì)譜參數(shù)優(yōu)化到最佳。1)非商業(yè)性表明：附錄A所列參考質(zhì)譜條件是在Agilent6460型液質(zhì)聯(lián)用儀上完成的，此處列出試驗用儀器型號僅為提供參考，并不涉及商業(yè)目的，鼓勵標準使用者嘗試不同廠家或型號的儀器。7SN/T3264—2012(資料性附錄)魚藤酮和印楝素標準溶液的多反應(yīng)監(jiān)測色譜圖8SN/T3264—2012Pleasenotethatsomeofthecontentofthisdocumentmayinvolvepatent,ThepublisherofthispublicofChina,ChineseAcademyInspectionanThemaindraftersofthisstandardareYangFang,ZhangFeng,TongYugui,L9SN/T3264—2012Thestandardspecifiesthemethodofdetefoodstuffsforexportbyliquidchromatography-tandemmassspmushroom,tea,honey,liver,fish,shrimpandchicken.ThisstandardrefertothefollowingstandardsandthelGB/T6682Waterforanalyticallaboratoryuse—SpecExtractionoftherotenoneandazadirachtinresiduewithacetonitrile.Aftersalting-outofacetonitriresiduesweredeterminedbyliquidchromatography-tandemm4.1Acetonitrile:HPLCgrade.SN/T3264—20124.6Ammoniumacetate.4.8Saturatedsodiumbicarbonatesolution:Weighappropriateamountofsodiumbicarbonate,dis-solveinwatertillsa4.95mmol/Lammoniumacetate:Weigh-0-38yammoniamacetatein800mLwater,piping2mL4.10Standardofrotenoneandazadirachtin:RotenonemolecularformulaC??H?C??FNO,CASNo.124495-18-7,molecularweight308.14;AzadirachtinmolecularformulaC??H??O??,CASNo.11141-17-6,molecularweight720.71,purity≥98%4.11Stocksolutionsofrotenoreandazadirachtin(100-mg/L):Accuratelyweighrotenoneandaza-dirachtinstandardmaterial,dissolvewithmethanoltoavolumeof100mL,andstoreatapproximately4℃in4.12CalibrationsolutionsofrotenoneandazadirachtinDiluteappropriatevolumeofssolutionstoaintendedconcentratiohwithacetonitrile-water(2+8,V/V),andpreparefreshly.4.13Polymersolidphaseextractioncatridge:60mg,3-mL4.14Filtermembrane:0.22μm,nylonsyringefilter5Apparatus5.1Liquidchromatography-tandemmassspectrometryEquippedwithelectrospray(ESI)LCinter.face.5.2Electronicbalance:Accuracy0.1mgand0.01g,respectively.5.5Tissueshomogenizer5.8Solidphaseextractionequipment.5.9Pressuredgasblowingconcentrator.6Samplepreparationandstorage6.1Samplepreparationter)andmixedwellwithatissurehomoge6.1.2Tea,grainsandnutsmogenizer,dividetheprepared6.2SamplestorageSamplessuchtea,wine,honeyandgrainsstoreatO℃~4℃,samplessuchasvegetables,fruits,takentoavoidcontaminationoranyfactors7.1.1Tea,grainsandnuts:Weigh1gofthepreparedtestsamplesintoa50mLstopperedplasticcentrifugetube,add5mLsaturatedsodiumbicarbonatesolution(4.9)andletstandindarknessforSN/T3264—2012thesupernatanttoanothercleantubeandrepeattheextracttrile.Collecttheentiresupernatants,addingca3gsodiumchlorium(4.6)forsalting-outandaftermethanol-water(2+8,V/V),thenclean-uptheextractsaccordingtoclean-upstep7.2.centrifugetube,add15mLacetonitrile(4.1),extractwithultersonicmachinefor10min,andthen,acetonitrile.Collecttheentiresupernatants,addingca3gsodiumchlorium(4.6)forsalting-outandaftercentrifugingat4500r/minfor3min,collec45℃,Dissolvetheresiduesin5mLofmethanol-water(2+8,V/V),thenclean-uptheextractsac-7.1.3Honey:Weigh2gofthepreparedtestsampletrile,extractwithultersonicmachinefor10min,andthenmethanol-water(2+8,V/V),thenclean-uptheextractsaccordingtoclean-upstep7.2.7.1.4Milk,Juice,andwine:weigh7.1.5Meatandmeatplasticcentrifugetube,adding5mLsaturatedsodiumbicarbonatesolution(4.9)andmixedwell,Thenadding15mLacetonitrile(4.1),extractwithultersonicmachinefor10min,andthen,transferthesupernatanttoanothercleantubeandrepeattheextracttrile.Collecttheentiresupernatants,addingca3gsodiumchlorium(4.6)forsalting-outandaftermethanol-water(2+8,V/V),thenclean-uptheextractsaccordingtoclean-upstep7.2.trile.CollecttheentireSN/T3264—20127.3.1.1Column:PhenomenexLunaCigcolumn,150mm×2.0mm(i.d.),3μm,orequivalent7.3.1.3Mobilephase:acetonitrile-5mmolammoniumacetatebuffer(4.9)(35+65,V/V).7.3.1.5Injectionvolume:10μL7.3.2.1lonsource:ESI,positiveionisationmode.7.3.2.2Scanmode:multiplereactionmonitoring(MRM)mode7.3.2.3OtherreferencemassoperatingconditionsarelistPreparestandardsolutionscontaining7.3.4ConfirmationtestThequalificationionsofthebetweenthetwodaughterionsfortRelativeintensity(ofbasepeak)/%>20～50>10～20MaximumpermittedtolerancesforrelativeSN/T3264—2012Theoperationoftheblanktestisthesameasthatdescribedinthemethodofdetermination,butwithomissionofsampleaddition.8CalculationandexpressionoftheresultCalculatetheconcentrationoftherotenoneandazadirachtinresidueinsampleaccordingtothefollowingequation:………………WhereV—thefinalvolumeofthesamplesolution,mL9.1LimitofdeterminationTherecoverydataofrotenoneandazadirachtinresiduesdeterminedbyHPLC-MS/MSisSN/T3264—2012MatrixRecovery%Recovery%rice83.2～104.5293.3～103.2186.2～92.9487.8～98.6581.8～93.082.3～112.386.6～106.5282.9～95.0190.1～102.44