SN-T 0603-2013出口植物源食品中四溴菊酯殘留量檢驗(yàn)方法 液相色譜-質(zhì)譜質(zhì)譜法

中華人民共和國出入境檢驗(yàn)檢疫行業(yè)標(biāo)準(zhǔn)出口植物源食品中四溴菊酯殘留量檢驗(yàn)方法液相色譜-質(zhì)譜/質(zhì)譜法2013-11-06發(fā)布2014-06-01實(shí)施國家質(zhì)量監(jiān)督檢驗(yàn)檢疫總局I本標(biāo)準(zhǔn)代替SN0603—1996《出口糧谷中路咪啉殘留量檢驗(yàn)方法》?！恍薷牧藰?biāo)準(zhǔn)名稱；擴(kuò)大了適用范圍；——取消了SN0603—1996中的抽樣方法；——改進(jìn)了樣品前處理技術(shù)線路。 取消了SN0603—1996中GC檢測法和GC-MS確證方法，采用LC-MS/MS方法對四溴菊酯(路咪啉，tralomethrin)殘留量進(jìn)行準(zhǔn)確定性和定量。本標(biāo)準(zhǔn)由國家認(rèn)證認(rèn)可監(jiān)督管理委員會(huì)提出并歸口。本標(biāo)準(zhǔn)起草單位：中華人民共和國廣西出入境檢驗(yàn)檢疫局。本標(biāo)準(zhǔn)所代替標(biāo)準(zhǔn)的歷次版本發(fā)布情況為：1出口植物源食品中四溴菊酯殘留量1范圍本標(biāo)準(zhǔn)規(guī)定了植物源食品中四溴菊酯殘留量的液相色譜-質(zhì)譜/質(zhì)譜法(LC-MS/MS)測定和確證殘留量的測定和確證。2規(guī)范性引用文件下列文件對于本文件的應(yīng)用是必不可少的。凡是注日期的引用文件，僅注日期的版本適用于本文件。凡是不注日期的引用文件，其最新版本(包括所有的修改單)適用于本文件。試樣中殘留的四溴菊酯經(jīng)乙酸乙酯(含油脂較多樣品用乙腈)提取，提取液在37℃以下濃縮后用弗羅里硅土柱(含油脂較多樣品用中性氧化鋁柱，含色素較多樣品加活性炭柱)凈化，用LC-MS/MS進(jìn)行定性和定量(外標(biāo)法)。4試劑和材料4.7正己烷-乙酸乙酯(95+5)混合溶液：取正已烷95mL,加入5mL乙酸乙酯，混勻。4.8正己烷-乙酸乙酯(60+40)混合溶液；取正己烷60mL,加入40mL乙酸乙酯，混勻。4.11標(biāo)準(zhǔn)物質(zhì)儲備溶液：準(zhǔn)確稱取適量的標(biāo)準(zhǔn)物質(zhì)，用少量丙酮溶解，再加入正己烷配成濃度為1mg/mL的標(biāo)準(zhǔn)儲備液，在一18℃以下避光保存，有效期為12個(gè)月。4.12標(biāo)準(zhǔn)物質(zhì)中間溶液：根據(jù)需要取上述標(biāo)準(zhǔn)儲備溶液用正已烷稀釋配制成濃度為10pg/mL~24.13標(biāo)準(zhǔn)物質(zhì)工作溶液：根據(jù)需要取上述標(biāo)準(zhǔn)物質(zhì)中間溶液用正己烷稀釋配制成適當(dāng)濃度的標(biāo)準(zhǔn)工5.1液相色譜-質(zhì)譜/質(zhì)譜儀(LC-MS/MS)。5.8離心機(jī)：轉(zhuǎn)速大于4000r/min。6試樣制備與保存6.1試樣制備取代表性樣品約500g,將其可食部分先切碎，經(jīng)搗碎機(jī)充分搗碎均勻，裝入潔凈容器內(nèi)密封，標(biāo)明粉碎或磨碎固態(tài)樣品于4℃以下保存；搗碎半固態(tài)新鮮組織樣品于一18℃以下保存。在制樣過程3(95+5)混合溶液預(yù)淋。用3mL正己烷-乙酸乙酯(95+5)混合溶液分兩次溶解洗滌上述殘留物并轉(zhuǎn)液相色譜條件如下：b)流動(dòng)相：甲醇一10mmol乙酸銨(含0.1%甲酸)(80+20,體積比);4SN/T0603—2013b)掃描模式：多反應(yīng)監(jiān)測(MRM),條件參見表A.1。7.3.3液相色譜-質(zhì)譜/質(zhì)譜檢測和確證根據(jù)試樣中被測物的含量情況，選擇響應(yīng)值相近的標(biāo)準(zhǔn)工作溶液一起進(jìn)行測定，標(biāo)準(zhǔn)工作溶液和待測樣液中四溴菊酯的響應(yīng)值均應(yīng)在儀器線性響應(yīng)范圍內(nèi)，如超出應(yīng)進(jìn)行稀釋。在上述色譜條件下四溴菊酯參考保留時(shí)間為6min～8min。四溴菊酯標(biāo)準(zhǔn)品LC-MS/MS色譜圖參見附錄B(譜圖中雙峰為四溴菊酯及其同分異構(gòu)體)。按照上述液相色譜串聯(lián)質(zhì)譜條件測定樣品和標(biāo)準(zhǔn)工作溶液，樣品中待測物質(zhì)的保留時(shí)間與標(biāo)準(zhǔn)溶液中待測物質(zhì)的保留時(shí)間偏差在±2.5%之內(nèi)，定性離子對的相對豐度與濃度相當(dāng)?shù)臉?biāo)準(zhǔn)工作溶液的相對豐度一致，相對豐度允許偏差不超過表1規(guī)定的范圍，則可判斷樣品中存在對應(yīng)表1定性確證時(shí)相對離子豐度的最大允許偏差相對離子豐度/%允許的相對偏差/%士508結(jié)果計(jì)算和表述用色譜數(shù)據(jù)處理機(jī)或按式(1)計(jì)算試樣中四溴菊酯的含量，計(jì)算結(jié)果需扣除空白值。 (1)X——試樣中四溴菊酯含量，單位為毫克每千克(mg/kg);A——試樣中四溴菊酯的峰面積；c,——標(biāo)準(zhǔn)工作溶液中四溴菊酯的濃度，單位為微克每升(μg/LV---樣液最終定容體積，單位為毫升(mL);A,——標(biāo)準(zhǔn)工作溶液中四溴菊酯的峰面積；m——最終樣液所代表的試樣質(zhì)量，單位為克(g)。9定量低限和回收率9.1定量低限試樣添加濃度及回收率實(shí)驗(yàn)數(shù)據(jù)見表2。5表2試樣不同添加濃度水平回收率范圍樣品名稱添加濃度/(mg/kg)回收率范圍/%大米糙米玉米小麥茶葉濃縮芒果汁大豆蘋果枯子白菜上海青Agilent6410BLC-MS/MS系統(tǒng)電噴霧離子源參考條件)被測物質(zhì)名稱定性離子對m/z定量離子對m/z裂解電壓V碰撞能最四溴菊酯682.8/440.4682.8/440.4-682.8/41214G7(資料性附錄)標(biāo)準(zhǔn)溶液LC-MS/MS色譜圖和保留時(shí)間8ThisstandardisdraftedinaccordancewithGB/T1.1—2009.ThisstandardreplacesSN0603—1996.ComparedwithSN0603—1996,thisstandardisofmajormodificationsasfollows:一Modifiedthenameofthestandard;一Enlargedthescopeofapplication;一Deletedsamplingprocedure;一Improvedsamplepretreatments;methodtodetermineandconfirm.ThisstandardwasproposedbyandisunderthechargeoftheCertificationandAccrcditationAdmin-strationofthePeople'sRepublicofChina.ThisstandardwasdrafedbyPRCGuangxiEntry-ExitInspectionandQuarantineThisstandardwasmainlydraftedbyQinWenchang,ZhengLing,HuangDaxing,Tianjijun,WuYujieSunGaoying,LiuRuifang,KongXiangjun.Thisstandardreplacedthepreviousversionofthereleaseofthestandardasfollows—SN0603—1996.9ThisstandardspecifiesthemethodofdeterminationandconfirmationoftralomethrinresiduesinfoodofplantoriginforimportandexportbyLC-MS/MSThisstandardisapplicabletothedeterminationandconfirmationoftralomethrinresiduesinrice,brownrice,maize,wheat,tea,concentratedmangojuice,soybean,apple,orange,cabbage,brassicacampestrisL.Thefollowingnormativedocumentscontainprovisionswhich,throughreferenceinthistext,consti-tuteprovisionsofthisstandard.Allpartiesaresubjecttoagreementsbasedonthisstandardareen-couragedtoinvestigatethepossibilityofapplyingthemostrecenteditionsofthestandardsindicatedbelow.Fordatedreference,subsequentamendmentsto,orrevisionsof,anyofthesepublicationsdonotapply.Forundatedreferences,thelatesteditionofthenormativedocumentreferredtoapplies.GB/T6682WaterforLaboratoryUse—Specifications.Theresiduesintestsamplesareextractedwithethylacetate(forsamplesrichinfatwithacetoni-trile),concentratedattemperaturebelow37℃,cleanedupbyflorisilSPE(forsamplesrichinfatwithneutralaluminacolumnSPE,andsamplewithdeepcolourcarbonSPEadded),thendeterminedandconfirmedbyGC-MS/MS.Unlessspecified,allreagentsusedshouldbeofanalyticalgrade."water"shouldbeoflevel1gradewhichconformswiththerequirementsofGB/T6682.4.1Ethylacetate:Chromatographicallypure.4.2Acetonitrile:ChromatographicallypureSN/T0603—20134.3n-Hexane:Chromatographicallypure.4.4Acetone:Chromatographicallypure4.6Anhydroussodiumsulfate:lgniteat650℃for4hours,cooltoroomtcmperatureindesicatorandkeepinatightlycontainerforuse.n-Hexane,mixadequately.n-Hexane,mixadequately.4.9Mobilephase:Methanol-10mmolammoniumacetate(with0.1%formicacid)(80+20,V/V).4.10Standardsubstance:Tralomethrin(CASNo.66841-25-6),Purity≥92%.4.11Standardstocksolution:Accuratelyweighadequateamountoftralomethrin,dissolvewithasmallamountofacetone,dilutewithn-Hexanetopfepareasolutionof1mg/mLasstandardstocksolution.Storedbelow-18℃andawayfromlight,thissolutionisvalidfor12months.4.12Standardmiddlesolutior:Pipetteadequateamountofstandardstocksolution,dilutewithn-Hexanetoprepareasolutionof10μg/mL100μg/mLasstandardmiddlesolution.Storedbelow-18℃andawayfromlight,thsolutionisvalidfor3months.4.13Standardworkingsolution\Pipetteadequateamountofstandardmiddlesolution,dilutewithn.Hoxanotoprepareaappropriateconccntrationasstandardworkingsolutionwhenusing.4.15NeutralaluminacolumnSPE:1g/6mL.4.16CarbonSPE:500mg/6mL.4.17Syringedrivenfilter:0.22μm(FH).5.1LC-MS/MSspectrometry5.2Balance:Accuracy0.01gand0.0001g5.5Homogenizer.5.7Shaker.5.8Centrifuge:Rotatingspeed≥4000r/min.5.9Rotaryvacuumevaporator.5.11Solidphaseextractionvacuummanifold.6Preparationandstorageof/testsamples6.1Preparationoftestsamples6.1.1solidanddrysamplese.g,grain,teaandsoybean:Takeabout500gofrepresentativesampleofeach.Grindinapulverizerforgranulewithsizelessthan2.0mmrespectively.Keepthepreparedsampleinacleancontainer,sealedandlabeledTakeabout500gofrepresentativesampleofeach.Keeptheediblepartsandcutthemintosmalbeled.Grindedsolidanddrysamplesshouldbestoragedbelow4℃.Mashedtissuesamplesshouldbestor-7.1.1Grainandtea:Weigh2goftestsample(accurateto0.01g)intoa50mLcen3gofsodiumchlorideand10mLofwater.Fullyshakeandthenstandfor20min.Add10mLofethylacetate.Homogenizefor30sat10000r/min.Shakefor10min.Centrifugefor5minat4000r/min.Transferthesupernatantintoaconcentrationflaskof150mL.Repeattheextractioninthesamewaywith10mLofethylacetate.Combinethesupernatantsandevaporateittonearlydrynessbelow37℃forcleaningup7.1.2Concentratedmangojuice:Weigh2goftestsample(accurateto0.01g)intoa50mLcentri-fugetube.Add3gofsodiumchlorideand5mLofwater.Fullyshakeandthenadd10mLofethylace-tate.Thesubsequentoperationsarethesameas.1.3Fruitsandvegetables:Weigh5goftestsatube.Add1.5gofsodiumchlorideand10mLofethylacetate.Thesubsequentoperationsarethesameas.1.4Soybean:Weigh2goftestsample(accurateto0.01g)intoa50mLcentrifugetube.Add2gofsodiumchlorideand20mLofacetonitrile.Homogenizefor30sat10000r/min.Shakefor10min.Centrifugefor5minat4000r/min.Transferthesupernatantintoaconcentrationflaskof150mL.Repeattheextractioninthesamewaywith10mLofacetonitrile.Combinethesupernatantsandevaporateittonearlydryn7.2.1Forsampleswithlightcolore.g.brownrice,rice,wheat,maize,cabbageandapple:Add2cmhighofanhydroussodiumsulfateintoaflorisilSPEcartridge.ConditiontheflorisilSPEcartridgewithDissolvetheresiduesandwashtheflasktwotimeswith3mLofn-Hexane-Ethylacetatemixedsolu-tion(95+5,V/V).Passthewashesthroughthecartridgeandelutethecartridgewith15mLofn-Hexane-Ethylacetatemixedsolution(95+5,V/V).Collecttheeluentsintoa50mLtubeandevap.orateundernitrogentodrynessbelow37℃.Dissolveanddilutetheresiduesbyadding1mLofmo-bilcphasointothetube,andfilterthesolutionwith0.22μmmcmbrancforLC-MS/MSanalysis.7.2.2Forsampleswithdeepcoloure.g.tea,brassicacampestrisL.,orange,concentratedmangojuice:SetonecarbonSPEontoaflorisilSPEcartridgebyaconnector.Add2cmhighofanhydrousso-diumsulfateintothecarbonSPE.ConditionthecarbonandflorisilSPEcartridgewith10mLofn-Hex-ane-Ethylacetatemixedsolution(60+40,V/V)beforeuseanddiscardthesolvent.Dissolvetheresiduesandwashtheflasktwotimeswith3mLofn-Hexane-Ethylacetatemixedsolution(60+40,V/V).Passthewashesthroughthecartridgeandelutethecartridgewith30mLofn-Hexane-Ethylacetatemixedsolution(60+40,V/V).Collecttheeluentsintoaconcentrationflaskof150mL.Evap-oratetheeluentstonearlydrynesswithrotaryvacuumevaporatorbelow37℃,andthentodrynessundernitrogenfurther.Dissolveanddilutetheresiduesbyadding1mLofmobilephaseintotheflask,andfilterthesolutionwith0.22μmmembrancforLC-MS/MSanalysis.7.2.3Forsamplesrichinfate.g.soybean:Usene10mLofacetonitrilebeforeuse.Discardthesolvent.DissolvetheresiduesandtransferthesolutionintotheneutralaluminacolumnSPEwith2mLofacetonitrile.Washtheflasktwotimeswith10mLofacetonitrileandpassthewashesthroughthecartridgerespectively.Collecttheeluentsintoacon-centrationflaskof150mL.Evaporatetheeluentstonearlydrynesswithrotaryvacuumevaporatorbelow37℃,andthentodrynessundernitrogenfurther.Dissolveanddilutetheresiduesbyadding1mLofmobilophaseintotheflask,andfilterthesolutionwith0.22μmmembraneforLC-MS/MSanal-ysis.HPLCoperatingconditionisasfollowinga)Column:Ca,50mm×4.6mm(i.d.),1.8μmorequivalent.b)Mobilephase:Methanol-10mmolammoniumacetate(with0.1%formicacid):(80+20,V/V).c)Flowrate:0.5mL/min.d)Columntemperature:40℃e)Injectionvolume:10μL.MSoperatinglonditionisasfollowing:a)lonizationmode:ESl,Positive.annexA.Accordingtotheapproximateconcentrationofanalyteinsamplesolution,selectthestandardworkingsolutionwithsimilarresponsestothatofsamplesolution.TheresponsesoftheanalyteinSN/T0603—2013thestandardworkingsolutionandthesamplesolutionshouldbewithinthelinearrangeoftheinstru-mentdetection,otherwisediluted.Undertheaboveoperatingcondition,theretentiontimeoftralomethrinis6min~8min.ThechromatogramoftralomethrinseeannexA.Undertheabovedeter-minationcondition,thevariationrangeoftheretentiontimeforthepeakofanalyteinthesamplcandinthestandardworkingsolutioncannotbeoutofrangeof±2.5%,thevariationrangeoftheionra-tiobetweenthetwodaughterionsforthesampleandthestandardworkingsolutionatthesimilarconcentrationcannotbeoutofrangeoftable1,andthenthecorrespondinganalytemustbepresentinthesample.Relativeintensity/%>20～50>10～20Permittedtolerances/%Theoperationoftheblanktestisthesameasthedescribedinthemethodofdetermination,butwiththeomissionofsampleaddition.CalculationoftheresiduecontentofTralomethrininthetestsamplecanbecarriedoutbyLC-MS/MSdataprocessororaccordingtotheformula(1)/theblankvalueshouldbesubtractedfromtheresultofcalculation.Where:X—theresiduecontentofTralomethrininthetestsample,mg/kg;A—the