SNT 3264-2012 出口食品中鱼藤酮和印楝素残留量的检测方法

SN/T3264—2012出口食品中鱼藤酮和印楝素残留量的检测方法液相色谱-质谱/质谱法国家质量监督检验检疫总局ISN/T3264—2012本标准按照GB/T1.1—2009给出的规则起草。请注意本文件的某些内容可能涉及专利。本文件的发布机构不承担识别这些专利的责任。本标准由国家认证认可监督管理委员会提出并归口。本标准起草单位：中华人民共和国福建出入境检SN/T3264—2012出口食品中鱼藤酮和印楝素残留量的检测方法液相色谱-质谱/质谱法二乙烯基苯-吡咯烷酮聚合物填料的固相萃取小柱净化，液相色谱-质谱/质谱仪检测及确证，外标法印楝素：英文名Azadirachtin,分子式C₃₅H₄₄O₁₆,CASNo.11141-17-6,分子量720.71):纯度≥98%4.11鱼藤酮和印楝素标准贮备液(100mg/L):准确称取0.0100g鱼藤酮和印楝素标准物质，用甲醇溶解并定容至100mL,该标准储备液于4℃避光保存不超过1个月。4.12鱼藤酮和印楝素标准工作液：根据需要取适量标准贮备液，以20%乙腈水溶液稀释成适当浓度25.3离心机：4500r/min,配有50mL的具塞塑料离心管。5.7超声波清洗器。6试样制备和保存密封并标识。6.2试样保存3SN/T3264—20125mL饱和碳酸氢钠溶液(4.8)振摇后避光浸泡10min,加入15mL乙腈(4.1),涡动30s后超声提取约3g氯化钠(4.5)盐析，4500r/min离心3min离心，取上清液，加入2mL经乙腈饱和后的正已烷(4.2),振摇1min,4500r/min离心3min离心，弃去正己烷层，乙腈层于45℃减压旋转蒸发至近干，酸氢钠和15mL乙腈，振摇均匀后超声提取10min,450010mL乙腈重复提取1次，合并提取掖，加入约3g氯化钠盐析，4500rmin离心3min,取上清液，于7.1.3蜂蜜：称取2g(精确至0.01g)试样置于50mL具塞塑料离心管中，加入5mL饱和碳酸氢钠溶于45℃减压旋转蒸发至近干，以5mL20%甲醇水溶解残渣，按7.2步骤净化。7.1.4牛奶、果汁及葡萄酒等：称取2g(精确至0.01g)试样置于50mL具塞塑料离心管中，加入约2g碳酸氢钠和15mL乙腈，涡动30s后超声提取10min,4500r/min离心3min,移出有机相，残渣再加入10mL乙腈重复提取1次，合并提取液，加入约3g氯化钠盐析，4500r/min离心3min,取上清碳酸氢钠溶液振摇，加入15mL乙腈，涡动30s后超声提取10min,4500r/min离心3min,移出有机相，残渣再加入10mL乙腈重复提取1次，合并是取液，加入约3g氯化钠盐析，4500r/min离心3min,取上清液，加入5mL正己烷，振摇1mirl,4.500E/min离心3min,弃去正己烷层，乙腈层于45℃减压旋转蒸发至近干，以5mL20%甲醇水溶解残渣，按7.步骤净化。1mL/min,收集洗脱液，于45℃氮吹至近干，以20%乙腈水容液定容1mL,过0.22μm滤膜(4.14),7.3.1.3流动相：乙腈-5mmol/L乙酸铵缓冲液(4.9)(35+65,体积分数)。4SN/T3264—20127.3.2.2扫描方式：多反应监测(MRM)。7.3.2.3其他参考质谱条件参见表A.1。7.3.3液相色谱-质谱/质谱测定根据试样中被测物的含量情况，选取响应值适宜的标准工作液进行色谱分析。标准工作液和待测样液中鱼藤酮和印楝素的响应值均应在仪器线性响应范围内。按式(1)进行结果计算。在本方法条件下，鱼藤酮和印楝素的保留时间约为5.6min和4.2min,鱼藤酮和印楝素标准品的多反应监测(MRM)色谱图参见附录B。7.3.4定性标准进行样品测定时，如果检出的色谱峰保留时间与标准样品一致，并且在扣除背景后的样品谱图中，各定性离子的相对丰度与浓度接近的同样条件下得到的标准溶液谱图相比，最大允许相对偏差不超过表1中规定的范围，则可判断样品中存在对应的被测物。表1定性确证时相对离子丰度的最大允许偏差>20～50>10～207.4空白实验除不加试样外，均按上述步骤进行。8结果计算和表述用数据处理软件或按式(1)计算试样中鱼藤酮和印楝素药物的残留量，计算结果需扣除空白值：………………(1)X——试样中鱼藤酮或印楝素残留量，单位为微克每千克(μg/kg);c——从标准工作曲线得到的鱼藤酮和印楝素溶液浓度，单位为微克每升(μg/L);V——样品溶液最终定容体积，单位为毫升(mL);m——最终样液所代表的试样质量，单位为克(g)。9测定低限、回收率9.1测定低限9.2回收率食品中鱼藤酮和印楝素残留的添加回收数据见表2。5SN/T3264—2012样品名称鱼藤酮印楝素添加浓度回收率范围%添加浓度回收率范围%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苹果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茶叶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猪肝75.0～98.0277.2～106.5178.4～103.2476.3～101.5576.8～100.181.8～100.7鱼肉79.0～105.0277.5～100.3179.6～101.2476.2～101.7580.7～103.080.9～98.4虾肉81.0～97.0277.2～102.8180.6～101.8479.2～105.3580.3～96.382.7～101.4鸡肉83.0～97.0278.6～104.5180.4～104.2479.3～93.8581.2～96.981.9～106.86(资料性附录)表A-₁—鱼藤酮和印楝素的主要参考质谱参数化合物监测离子驻留时间m)S电压V碰撞能量鱼藤酮印楝素注：表中黑体字显示的离子为定量离子；对于不同质谱仪器，仪器参数可能存在差异，测定前应将质谱参数优化到最佳。1)非商业性表明：附录A所列参考质谱条件是在Agilent6460型液质联用仪上完成的，此处列出试验用仪器型号仅为提供参考，并不涉及商业目的，鼓励标准使用者尝试不同厂家或型号的仪器。7SN/T3264—2012(资料性附录)鱼藤酮和印楝素标准溶液的多反应监测色谱图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:RotenonemolecularformulaC₁₅H₈C₁₂FNO,CASNo.124495-18-7,molecularweight308.14;AzadirachtinmolecularformulaC₃₅H₄₄O₁₆,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