SN-T 0603-2013出口植物源食品中四溴菊酯殘留量檢驗方法 液相色譜-質譜質譜法

中華人民共和國出入境檢驗檢疫行業(yè)標準出口植物源食品中四溴菊酯殘留量檢驗方法液相色譜-質譜/質譜法2013-11-06發(fā)布2014-06-01實施國家質量監(jiān)督檢驗檢疫總局I本標準代替SN0603—1996《出口糧谷中路咪啉殘留量檢驗方法》?！恍薷牧藰藴拭Q；擴大了適用范圍；——取消了SN0603—1996中的抽樣方法；——改進了樣品前處理技術線路。 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)殘留量進行準確定性和定量。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：中華人民共和國廣西出入境檢驗檢疫局。本標準所代替標準的歷次版本發(fā)布情況為：1出口植物源食品中四溴菊酯殘留量1范圍本標準規(guī)定了植物源食品中四溴菊酯殘留量的液相色譜-質譜/質譜法(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進行定性和定量(外標法)。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標準物質儲備溶液：準確稱取適量的標準物質，用少量丙酮溶解，再加入正己烷配成濃度為1mg/mL的標準儲備液，在一18℃以下避光保存，有效期為12個月。4.12標準物質中間溶液：根據需要取上述標準儲備溶液用正已烷稀釋配制成濃度為10pg/mL~24.13標準物質工作溶液：根據需要取上述標準物質中間溶液用正己烷稀釋配制成適當濃度的標準工5.1液相色譜-質譜/質譜儀(LC-MS/MS)。5.8離心機：轉速大于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，經搗碎機充分搗碎均勻，裝入潔凈容器內密封，標明粉碎或磨碎固態(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液相色譜條件如下：b)流動相：甲醇一10mmol乙酸銨(含0.1%甲酸)(80+20,體積比);4SN/T0603—2013b)掃描模式：多反應監(jiān)測(MRM),條件參見表A.1。7.3.3液相色譜-質譜/質譜檢測和確證根據試樣中被測物的含量情況，選擇響應值相近的標準工作溶液一起進行測定，標準工作溶液和待測樣液中四溴菊酯的響應值均應在儀器線性響應范圍內，如超出應進行稀釋。在上述色譜條件下四溴菊酯參考保留時間為6min～8min。四溴菊酯標準品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體)。按照上述液相色譜串聯質譜條件測定樣品和標準工作溶液，樣品中待測物質的保留時間與標準溶液中待測物質的保留時間偏差在±2.5%之內，定性離子對的相對豐度與濃度相當的標準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表1定性確證時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結果計算和表述用色譜數據處理機或按式(1)計算試樣中四溴菊酯的含量，計算結果需扣除空白值。 (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,——標準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,——標準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驗數據見表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)標準溶液LC-MS/MS色譜圖和保留時間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