SN1920-2007进出口动物源性食品中敌百虫、敌敌畏、蝇毒磷残留量的检测方法液相色谱-质谱质谱法

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敌百虫、敌敌畏、蝇毒磷残留量的检测方法液相色谱-质谱/质谱法Determinationoftrichlorfor,dichlorvosandcoumaphosresiduesinfoodstuffsofanimaloriginforimportandexport—LC-MS/MSmethod2007-05-23发布I本标准的附录A、附录B和附录C均为资料性附录。本标准由国家认证认可监督管理委员会提出并归口。本标准由中华人民共和国重庆出入境检验检疫局、中华人民共和国湖南出入境检验检疫局和中华人民共和国浙江出入境检验检疫局负责起草。本标准系首次发布的出入境检验检疫行业标准。1进出口动物源性食品中敌百虫、敌敌畏、蝇毒磷残留量的检测方法液相色谱-质谱/质谱法质谱/质谱检验方法。2试样的制备与保存分割肉：取样品中有代表性的约500g,用组织捣碎机捣碎，装入洁净容器作为试样，密封并做好标肠衣：取有代表性样品约100g,用剪刀剪碎至2mm以下，装入洁净容器作为试样，密封并做好标识，于-18℃冰箱内保存。蜂蜜：取有代表性样品约500g,搅拌均匀后装入洁净容器内密封并做好标识，于一18℃冰箱内保存。制样操作过程中应防止样品受到污染或发生残留物含量的变化。3方法提要4试剂和材料4.3乙酸乙酯。4.4二氯甲烷。4.5环己烷。4.6硫酸钠：650℃灼烧4h,在干燥器内冷却至室温，贮于密封瓶中备用。4.850mmol/L乙酸铵溶液：称取0.385g乙酸铵溶于1000mL水中。4.9敌百虫(Trichlorfor,CAS号：52-68-6,分子式：C₄H₈Cl₃O₄P)标准品：纯度大于或等于98.0%。4.10敌敌畏(Dichlorvos,CAS号：62-73-7,分子式：C₄H₇Cl₂O₄P)标准品：纯度大于或等于97.6%。4.11蝇毒磷(Coumaphos,CAS浓度的混合标准储备液。此溶液在0℃~4℃冰箱中避光保存，可使用3个月。4.13空白样品提取液：用不含敌百4.14标准工作溶液：根据需要用空白样品提取液将标准储备液稀释成50ng/mL、100ng/mL、2200ng/mL、500ng/mL的混合标准工作溶液。置于0℃~4℃冰箱中避光保存，可使用3d。5仪器和设备5.1高效液相色谱-质谱/质谱仪：三重四极杆质谱检测器，配电喷雾离子源(ESI)或相当者。5.2超声波清洗器。5.3旋涡混匀器。5.4旋转蒸发仪。5.6均质器。5.70.45μm微孔滤膜。6.1提取与净化6.1.1分割肉a)准确称取5g均匀试样(精确到0.01g)于50mL具塞试管中，加入5g无水硫酸钠混匀，再加入15mL二氯甲烷，用均质器(10000r/min)均质2min,4000r/min离心3min,将有机相转移至100mL梨形蒸馏瓶中，残渣再用2×10mL二氯甲烷均质提取两次。b)离心合并有机相，于40℃旋转蒸发至2mL,将样液转移至5mL刻度试管中，并用少量二氯甲烷洗涤梨形蒸馏瓶，合并洗涤液到刻度试管中，室温通氮浓缩至干。定量加入1.0mL乙腈溶解残渣，加入2mL环己烷旋涡混匀2min后，2500r/min离心3min,将下层乙腈相过6.1.2肠衣准确称取5g试样(精确到0.01g)于50mL具塞离心瓶中，加入2g无水硫酸钠混匀，再加入15mL二氯甲烷，盖上盖混匀，置于超声波清洗器中超声30min,冷却后将有机相过滤转移至100mL梨形蒸馏瓶中，残渣再用2×10mL用二氯甲烷混匀超声提取两次，离心合并有机相。接下来按照6.1.1中b)的步骤进行。称取5g试样(精确到0.01g)置于50mL具塞离心管中，加10mL水，加25mL乙酸乙酯，于旋涡混合器上混匀1min,以3000r/min离心5min,将上层乙酸乙酯提取液收集于浓缩瓶中，残渣再加入20mL乙酸乙酯，重复上述操作，合并乙酸乙酯提取溶液。在50℃以下减压浓缩至约3mL后转移至离心管，用5mL乙酸乙酯分两次洗涤浓缩瓶，合并洗涤液于离心管中，用氮气吹干干。加1.0mL乙腈溶6.2.1液相色谱-质谱/质谱条件a)色谱柱：柱填料为十八烷基硅烷键合相的色谱柱，4.6×150mm,5μm或相当者；i)质谱/质谱参考条件参见附录B。36.2.2定性、定量测定按照6.2.1液相色谱-质谱/质谱条件测定样液和标准工作溶液，外标标准曲线法测定样液中的敌百虫、敌敌畏、蝇毒磷残留量。样品中待测物残留量应在标准曲线范围之内，如果残留量超出标准曲线范围，应用空白样品提取液(4.13)进行适当稀释。在上述色谱条件下，敌百虫、敌敌畏、蝇毒磷的质量色谱峰保留时间约为13.2min,14.1min,15.9min。标准品的多反应监测色谱图参见附录C中的在相同的实验条件下，样品与标准工作液中待测物质的质量色谱峰相对保留时间在2.5%以内，并且在扣除背景后的样品质量色谱图中，所选择的离子对均出现，同时与标准品的相对丰度允许偏差不超过表1规定的范围，则可判断样品中存在对应的被测物。表1使用定性液相色谱-质谱/质谱时相对离子丰度最大容许误差相对丰度(基峰)/(%)液相色谱-质谱/质谱时相对离子丰度最大允许误差/(%)大于20至小于等于50大于10至小于等于20小于等于107结果计算和表述用数据处理软件中的外标法，或绘制标准曲线，按照式(1)计算样品中敌百虫、敌敌畏、蝇毒磷残留量。……式中：X——试样中敌百虫、敌敌畏、蝇毒磷的残留量，单位为毫克每千克(mg/kg);c——由标准曲线而得的样液中敌百虫、敌敌畏、蝇毒磷的浓度，单位为纳克每毫升(ng/mL);V——样液最终定容体积，单位为毫升(mL);m——最终样液所代表的试样质量，单位为克(g)。8测定低限、回收率8.1测定低限本方法敌百虫、敌敌畏、蝇毒磷的测定低限均为0.010mg/kg。8.2回收率8.2.1肠衣中敌百虫添加浓度及其回收率数据：添加浓度在0.010mg/kg时，回收率在73.0%～94.0%之间；添加浓度在0.020mg/kg时，回收率在78.0%～95.0%之间；添加浓度在0.040mg/kg时，回收率在78.0%～95.0%之间。8.2.2肠衣中敌敌畏添加浓度及其回收率数据：添加浓度在0.010mg/kg时，回收率在76.0%～98.0%之间；添加浓度在0.020mg/kg时，回收率在78.0%～98.0%之间；添加浓度在0.040mg/kg时，回收率在86.0%～95.0%之间。8.2.3肠衣中蝇毒磷添加浓度及其回收率数据：添加浓度在0.010mg/kg时，回收率在75.0%～95.0%之间；添加浓度在0.020mg/kg时，回收率在80.0%～96.0%之间；4添加浓度在0.040mg/kg时，回收率在77.0%～92.0%之间。8.2.4分割肉中敌百虫添加浓度及其回收率数据：添加浓度在0.010mg/kg时，回收率在92.0%～115%之间；添加浓度在0.020mg/kg时，回收率在86.5%～113%之间；添加浓度在0.040mg/kg时，回收率在80.8%～101%之间。8.2.5分割肉中敌敌畏添加浓度及其回收率数据：添加浓度在0.010mg/kg时，回收率在93.0%～110%之间；添加浓度在0.020mg/kg时，回收率在88.5%～108%之间；添加浓度在0.040mg/kg时，回收率在84.3%～110%之间。8.2.6分割肉中蝇毒磷添加浓度及其回收率数据：添加浓度在0.010mg/kg时，回收率在91.0%～112%之间；添加浓度在0.020mg/kg时，回收率在79.0%～102%之间；添加浓度在0.040mg/kg时，回收率在73.9%～95.3%之间。8.2.7蜂蜜中敌百虫添加浓度及其回收率数据：添加浓度在0.010mg/kg时，回收率在70.0%～110%之间；添加浓度在0.020mg/kg时，回收率在79.0%～104%之间；添加浓度在0.040mg/kg时，回收率在80.0%～107%之间。8.2.8蜂蜜中敌敌畏添加浓度及其回收率数据：添加浓度在0.010mg/kg时，回收率在70.0%～92.0%之间；添加浓度在0.020mg/kg时，回收率在71.0%～91.0%之间；添加浓度在0.040mg/kg时，回收率在70.0%～96.0%之间。8.2.9蜂蜜中蝇毒磷添加浓度及其回收率数据：添加浓度在0.010添加浓度在0.020添加浓度在0.040mg/kgmg/kgmg/kg时，回收率在79.0%～110%之间；时，回收率在74.0%～105%之间；时，回收率在71.0%～104%之间。5(资料性附录)梯度洗脱程序表A.1流动相梯度洗脱程序时间/min甲醇/(%)50mmol/L乙酸铵/(%)090.090.095.095.090.025.0090.0(资料性附录)API4000LC-MS/MS系统电喷雾离子源参考条件：a)窗帘气(CUR):15.00Pa;b)雾化气(GS1):40.00Pa;c)辅助加热气(GS2):45.00L/min;d)碰撞气(CAD):7.00Pa;e)离子源喷雾器电压(IS):5000.00V;g)定性离子对、定量离子对、去簇电压、碰撞能量、碰撞室出口电压见表B.1。表B.1待测物定性离子对、定量离子对、去簇电压、碰撞能量和碰撞室出口电压待测物m/zm/z去簇电压/V碰撞能量/V碰撞室出口电压/V敌百虫Trichlorfon259.0°63.026.5256.9221.263.0敌敌畏Dichlorvos221.3°63.025.5223.363.025.0蝇毒磷Coumaphos363.3“307.096.025.020.0363.3227.096.0a为定量离子对。6(资料性附录)标准品多反应监测色谱图3000001ARM|30=0.252011022aruhonSmon00B1-u)150A₅₂11.四叠加离子色谱图Kax.63a*.敌百虫259.0→109.220XEalIKRM|7D*0.221211039amuhonFanOamD0I-00100XCal1MRM130*0.262319032amu""iomFandelad1Hom0s¹200811-2a*Iuo)2敌敌畏221.3→109.27Kax.4,Fm*0蝇毒磷363.3→307.02图C.1敌百虫、敌敌畏、蝇毒磷标准溶液多反应监测色谱图7AnnexA,BandCofthisstandardareinformativeannexes.tificationandAccreditationofthePeoplesRepublicofChina.ThestandardwasdraftedbyChongqingEntry-ExitInspectionandQuarantineBureauofthePeople'sna,andZhejiangEntry-ExitInspectionandQuarantineBureauofthePeople'sRepublicofChina.ThisstandardwasmainlydraftedbyWangGuoming,DaiHua,XieWen,ZhuGe,WangMeiling,LiXianliang,LiYingguo,ZhangLei,ZhouQimingandWangXiangxian.Thisstandardisaprofessionalstandardforentry-exitinspectionandquarantinepromulgatedforthefirsttime.Note:ThisEnglishversion,atranslationfromtheChinesetext,isonlyforreference.8byliquidchromatography-massspectrometryoftrichloffor,dichlorvosandcoumaphosresiduesinanimalmuscle,saltedcasingandhoney.Thisstandardisapplicabletothedeterminationoftrichloffor,dichlorvosandcoumaphosresiduesinanimalmuscle,saltedcasingandhoney.Muscle:About500grepresentativesamplesshouldbetakenfromallsamples,thengrindedandblen-dedbyatissueblendertoproducehomogenoussamples,putinsuitablecleancontainer.Afterbeingsealedandlabeled,thesamplesshouldbestoredatbelow-18Casing:About100grepresentativesamplesshouldbetakenfromallsamples,thengrindedandblen-dedbyaforfex,putinsuitablecleancontainer.Afterbeingsealedandlabeled,thesamplesshouldbeHoney:About500grepresentativesamplesshouldbetakenfromallsamples,thenmixroundtopducehomogenoussamples,putinsuitablecleancontainer.Afterbeingsealedandlabeled,thesamplesshouldbestoredatbelow-18℃.Inthecourseofsamplingandsamplepreparation,precautionshouldbetakentoavoidcontaminationoranyfactorthatmaycausethechangeofresiduecontent.EthylAcetateordichloromethane.Afterbeingconcentratedandreconstituted,theresiduesaredeter-minedbyliquidchromatography-massspectrometry,quantifiedbyexternalstandardmethod.Unlessspecified,allreagentsshouldbeofanalyticalgrade;“Water”isthedistilledwater.94.2Methanol:HPLCgrade.4.4Dichloromethane.4.5Cyclohexane.4.6Sodiumsulfate:lgniteat650℃for4h,cooltoroomtemperatureinadesiccatorandstoreinsealedcontainer.4.7Ammoniumacetate:GR.4.850mmol/Lammoniumacetate:Weigh0.385gammoniumacetate,dissolvedin1000mLwa-ter.4.9Trichlorfor(CASNO:52-68-6formula:C₄H₈Cl₃4.11Coumaphos(CASNO:56-72-4,formula:C₄Hi₆CIO₅PS),purity≥99.9%.4.12Standardstocksolution:Accuratelyweightrichlorfor,dichlorvoswithmethanol,andmixtohomogeneity.Theconcentrationofthesolutionis100g/mL,canbepre-servedatthetemperature0℃~4℃forthreemonths.chlorvosandcoumaphos.4.14Standardworkingsolution:accordingtotherequirement,dilutemixedstandardsolutionwithblankmatrixextractsolution.Theconcentrationofthesolutionis50ng/mL,100ng/mL,200ng/mLand500ng/mL.Itcanbepreservedatthetemperature0℃~4℃forthreedays.5Apparantusandequipme5.1Liquidchromatography-massspectrometry,equippedwithelectrosprayionsourceandtriqua-5.2UItrasonicextractor.5.3Mechanicalshaker.SN/T1920—20075.5Centrifuge:5000r/min.5.6Homogenizer.5.7Membranefilter:0.45μm.a)Accuratelyweigh5gofthetestsample(accurateto0.01g)intoa50mLpolypropylenecentri-fugetubewithcap,add5ganhydroussodiumsulfateand15mLofdichloromethane,homogenizefor2min(10000r/min),thencentrifugeat4000r/minfor3min.Transferthesupematanttoa100mLevaporatedflask,theresiduewashomogenizedandextractedwith2×10mLdichlo-romethane.b)Aftercentrifuging,combinethesupernatanttotheevaporatedflask.Evaporatethedichlo-romethanetoabout2mLwithrotaryevaporatorbelow40℃,removetheresiduestoa5mLcentrifugetube,washevaporatedflaskwithlittledichloromethane,combinethedichloromethanesolutiontograduatedtesttube,evaporatetheextracttodrynessonN-Evapatroomtempera-ture.Theresidueisdissolvedwith1.0mLofacetonitrileand2mLofcyclohexane.Votexandcentrifugeat2500r/minfor3min.Filterthenetherlayerthrougha0.45μmmembraneintoHPLCvials.ThesolutionisreadyforLC-MS/MSdetermination.Accuratelyweigh5gofthetestsample(accurateto0.01g)intoa50mLpolypropylenecentrifugetubewithcap,add5ganhydroussodiumsulfate,add15mLofdichloromethaneandvortex,thenex-tractinultrasonicextractorfor30min.Aftercooling,transferthesupematanttoa100mLevaporat-edflask,theresiduewasvortexedandextractedwith2×10mLdichloromethane.Aftercentrifuging,combinethesupernatanttotheevaporatedflask.Then,dealwiththesampleaccordingtothestepof6.1.1b).Accuratelyweigh5gofthetestsample(accurateto0.01g)intoa50mLpolypropylenecentrifugetubewithcap,add10mLwaterand25mLethylacetate,shakefor1minonamechanicalshakerat2000r/min,thencentrifugeat3000r/minfor5min.Transferthesupernatanttoaevaporatedflask,theresidueswasre-extractedwith20mLethylacetateastheaboveprocedure,combinethesuper-natant,evaporatedtoabout3mLwithrotaryevaporatorinawater-bathbelow50℃,removetheres-iduetoanewcentrifugetube,washtheevaporatedflasktwiceuse5mLethylacetate,combinetheeluate,evaporatetheeluatetodrynessonN-Evap,dissolvedby1.0mLofmethanol,FilterthroughaSN/T1920—20070.45μmmembraneintoHPLCvials.ThesolutionisreadyforLC-MS/MSdetermination.6.2.1LC-MS/MSoperationconditiona)LCcolumn:Ca,4.6×150mm,5μm(orotherconformablecolumn);b)Mobilephase:methanol:50mmol/Lammoniumacetate,theelutiongradientislistedinAnnexA;d)Columntemperature:40℃;e)Injectorvolume:10μL;f)lonsource:ESl;g)Scanningmodel:positiveion;h)Monitoringmodel:multiplereactionmonitoring(MRM);i)ReferencedconditionsseenAnnexB.6.2.2QualificationandquantificationtestAccordingtothemethodof6.2.1,detecttheresiduesoftrichlorfor,dichlorvosandcoumaphosinthetestsamplesolution,thestandardworkingsolution.Theresponseoftrichlorfor,dichlorvosandcoumaphosshouldbeinthelinearrangeoftheinstrumentaldetection.Iftheresponseisoutofthelinearrange,thesampleshouldbedilutedwiththeblankmatrixextractsolution(4.13)tosuitableconcertration.Undertheabovechromatographconditions,thereferenceretentiontimeoftrichlorfor,dichlorvosandcoumaphosisabout13.2min,14.1min,15.9min.ReconstitutedionchromatogramofstandardworkingsolutionislistedinAnnexC.Underthesameconditionsofexperiment,theretentiontimeoftheunknownsampleisthesameasthestandardworkingsolution;thequalificationionsforeverycompoundmustbefound.Forthesameanalysisbatchandthesamecompound,thevariationrangeoftheionratiobetweenthetwodaughterionsfortheunknownsampleandthestandardworkingsolutionatthesimilarconcentrationcannotbeoutofrangeoftable1.Table1—MaximumpermittedtolerancesforrelativeionintensitieswhileconfirmationRelativeintensity/(%)Permittedtolerances/(%)7CalculationandexpressionofresultCalculatingthecontentoftrichlorfor,dichlorvosandcoumaphosresidueconcentrationinthesampleiscarriedoutbyLC/MS/MSdataprocessororaccordingtotheformula(1): (1)c—theconcentrationoftrichlorfor,dichlorvosandcoumaphosinthetestsamplecalculatedbycali-brationcurve,ng/mL;m—thecorrespondingmassoftestsampleinthefinalsamplesolution,g;V—thefinalvolumeofsamplesolution,mL.8.1LimitofquantificationThelimitofquantificationfortrichlorfor,dichlorvosandcoumaphosis0.010mg/kg8.2.1Accordingtotheexperimentaldate,thefortifyingconcentrationoftrichlorforincasinganditscorrespondingrecoveriesare:0.010mg/kg,therecoveryis73.0%～94.0%;0.020mg/kg,therecoveryis78.0%～95.0%;0.040mg/kg,therecoveryis78.0%～95.0%.8.2.2Accordingtotheexperimentaldate,thefortifyingconcentrationofdichlorvosincasinganditscorrespondingrecoveriesare:0.010mg/kg,therecoveryis76.0%～98.0%;0.020mg/kg,therecoveryis78.0%～98.0%;0.040mg/kg,therecoveryis86.0%～95.0%.8.2.3Accordingtotheexperimentaldate,thefortifyingconcentrationofcoumaphosincasinganditscorrespondingrecoveriesare:0.010mg/kg,therecoveryis75.0%～95.0%;0.020mg/kg,therecoveryis80.0%～96.0%;0.040mg/kg,therecoveryis77.0%～92.0%.8.2.4Accordingtotheexperimentaldate,thefortifyingconcentrationoftrichlorforinmusleanditscorrespondingrecoveriesare:0.010mg/kg,therecoveryis92.0%～115%;0.020mg/kg,therecoveryis86.5%～113%;0.040mg/kg,therecoveryis80.8%～101%.SN/T1920—20078.2.5Accordingtotheexperimentaldate,thefortifyingconcentrationofdichlorvosinitscorrespondingrecoverie0.010mg/kg,therecoveryis93.0%～110%;0.020mg/kg,therecoveryis80.040mg/kg,therecoveryis84.3%～110%.8.2.6Accordingtotheexperimentaldate,thefortifyingconcentrationofcoumaphosinitscorrespondingrecoverie0.010mg/kg,therecoveryis91.0%～112%;0.020mg/kg,therecoveryis79.0%～102%;0.040mg/kg,therecoveryis73.9%～95.3%.8.2.7Accordingtotheexperimentaldate,thefortifyingconcentrationoftrichlorforinitscorrespondingrecoverie0.010mg/kg,therecoveryis70.0%～110%;0.020mg/kg,therecoveryis79.0%～104%;0.040mg/kg,therecoveryis80.0%～107%.8.2.8Accordingtotheexperimentaldate,thefortifyingconcentrationofdichlorvosinitscorrespondingrecoverie0.010mg/kg,therecoveryis70.020mg/kg,therecoveryis70.040mg/kg,therecover