SNT 2444-2010進(jìn)出口動物源食品中甲芐喹啉癸氧喹酯殘留量的測定液相色譜-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)進(jìn)出口動物源食品中甲芐喹啉和癸氧喹酯殘留量的測定液相色譜-質(zhì)譜/質(zhì)譜法2010-01-10發(fā)布2010-07-16實(shí)施國家質(zhì)量監(jiān)督檢驗(yàn)檢疫總局ISN/T2444—20101進(jìn)出口動物源食品中甲芐喹啉和癸氧喹酯殘留量的測定液相色譜-質(zhì)譜/質(zhì)譜法2規(guī)范性引用文件下列文件中的條款通過木標(biāo)準(zhǔn)的引用而成為禾標(biāo)準(zhǔn)的條款。凡是注日期的引用文件，其隨后所有的修改單(不包括勘誤的內(nèi)容)或修訂版均不適用于本標(biāo)準(zhǔn)，然而，鼓勵根據(jù)本標(biāo)準(zhǔn)達(dá)成協(xié)議的各方研究是否可使用這些文件的最新版本。凡是不注明日期的引用文件，其最新版本適用于本標(biāo)準(zhǔn)。4.7氯化鈉。4.100.005mol/L乙酸銨水溶液(含0.05%甲酸):準(zhǔn)確稱取0.3854g乙酸銨(4.6),用水溶解并轉(zhuǎn)移于99%。4.12標(biāo)準(zhǔn)儲備溶液：準(zhǔn)確稱取適量的甲芐喹啉標(biāo)準(zhǔn)物質(zhì)(4.11),用N,N-二甲基甲酰胺(4.3)配制成濃度為100mg/L的標(biāo)準(zhǔn)儲備溶液；準(zhǔn)確稱取適量的癸氧喹酯標(biāo)準(zhǔn)物質(zhì)(4.11),用三氯甲烷(4.4)配制25.2固相萃取裝置。5.7分析天平：感量0.1mg和0.01g。6.1試樣制備在制樣的操作過程中，應(yīng)防止樣品污染。7.1待測樣品溶液的制備稱取樣品5g(精確至0.01g)于50mL具塞離心管中，加入15mL乙腈(4.1),旋渦混勻1min,在往復(fù)式振蕩器上振蕩提取5min,3000r/min離心5min,將上清液轉(zhuǎn)移至另一50mL離心管中。在殘?jiān)屑尤?0mL乙腈(4.1),重復(fù)提取一次，5000r/min離心5min,合并乙腈層。在乙腈提取液中加入過量氯化鈉(4.7),旋渦混勻1min,5000r/min離心3min。取上層乙腈層5mL于15mL離心管中，45℃水浴下吹氮濃縮至3mL,加水定容至10mL,待凈化。將7.1.1中所得溶液轉(zhuǎn)人經(jīng)過預(yù)處理的OasisHLB固相萃取柱(4.14)中，以約3mL/min的流速浴下吹氮濃縮至近干，用樣品定容溶液(4.9)溶解并定容至1.0mL,旋渦混勻后過0.22μm濾膜稱取5份陰性樣品于5個50mL具塞離心管中，每份樣品5g(精確至0.01g),分別加入適量甲芐3喹啉和癸氧喹酯混合中間標(biāo)準(zhǔn)溶液(4.13),使甲芐喹啉和癸氧喹酯最終測定濃度分別為0.005mg/L、0.010mg/L、0.050mg/L、0.100mg/L、0.250mg/L。按7.1.1中“加入15mL乙腈……”和7.1.27.3陰性樣品基質(zhì)空白溶液的制備7.1.2操作。7.4.1液相色譜條件d)流動相、流速及梯度洗脫條件見表1。時間/min流速/(mL/min)0.005mol/L乙酸銨水溶液(含0.05%甲酸)/%乙腈/%00.300.300.300.300.307.4.2質(zhì)譜條件e)定性離子對：甲芐喹啉366.3/334.1,366.3/91.0;癸氧喹酯418.1/372.2,418.1/204.0;f)定量離子對：甲芐喹啉366.3/334.1其他參考質(zhì)譜條件參見附錄A。7.4.3液相色譜-質(zhì)譜/質(zhì)譜測定每種被測組分選擇1個母離子，2個以上子離子，在相同實(shí)驗(yàn)條件下，樣品中待測物質(zhì)的保留時間，與基質(zhì)標(biāo)準(zhǔn)溶液的保留時間偏差在±2.5%之內(nèi)；且樣品中各組分定性離子的相對豐度與濃度接近的基質(zhì)混合標(biāo)準(zhǔn)工作溶液中對應(yīng)的定性離子的相對豐度進(jìn)行比較，偏差不超過表2規(guī)定的范圍，則可判定為樣品中存在對應(yīng)的待測物。表2定性確證時相對離子豐度的最大允許偏差相對離子豐度/%>20～50>10～204定。甲芐喹啉和癸氧喹酯標(biāo)準(zhǔn)溶液的多反應(yīng)監(jiān)測(MRM)色譜圖參見附錄B中的圖B.1. (1) 表3雞肉、雞肝、雞腎、雞蛋(牛肝和牛奶中甲芐喹啉和癸氧喹酯不同添加水平回收率數(shù)據(jù)化合物名稱添加水平/(mg/kg)雞肉雞肝雞腎雞蛋牛肝牛奶甲芐喹啉0.01096.4～10585.0～10591:7～10889.0～10489.1～10193.7～1070.10098.905i91.0～10884.5～91.8101～10998.4～10792.4～99.00.20096.5～10491.796.787.2～97.889.7～101—2.0079.0～88.085.0~90.087.0～95.092.0～102——癸氧喹酯0.010.3～10581.8～11092.0～10985.0～107101～1100.10096.4m10288.6～10989.3.<10887.3～10183.2～10289.1～93.50.20078.0～95.796.1～10780.88.996.7～10291.~10289.4～105—2.0077.0～84.590.5～98.591.5一98.085.0～91.0 5(資料性附錄)參考質(zhì)譜條件)質(zhì)譜條件分析物參考保留時間/min母離子子離子采集時間/s錐孔電壓/V碰撞能量/eV甲芐喹啉91.0癸氧喹酯a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。6(資料性附錄)標(biāo)準(zhǔn)的多反應(yīng)監(jiān)測(MRM)色譜圖366.3>334.418.1>372.2366.3>91b)癸氧喹酯418.1>2047AnnexAandBofthisstandardisaninformativeannexThisstandardwasproposedbyandisunderthechargeoftheNationalRegulatoryCommissionforCertificationandAccreditationofthePeople'sRepublicofChina.ThestandardwasdraftedbyShenzhenAcademyofInspectionandQuarantine,ShenzhenEntry-ExitInspectionandQuarantineBureauofthePeople'sRepublicofChinaThemaindraftersofthisstandardareKangHaining,XiaoChengui,XieLigi,HanRuiyang,ShenJin-can,JinBaohui,Linli,ZhangJianyingThisstandardisaprofessionalstandardforentry-exitinspectionandquarantinepromulgatedforthefirsttime.8Thisstandardspecifiesthemethodsofdeterminationbyliquidchromatography-electrospraytandemmassspectrometryofnequinateanddecoquinateinfoodstuffsofanimalorigin.Thisstandardisapplicabletothequalitativeandquantitativedeterminationquinateinchicken,chickenliver,chickenkidney,egg,bovineliverandmilk.tion.Forthedatedquoteddocuments,noneoftherevised-contents(whichdoesntentforcorrectingerrors)ortherevisededitionsafterthisstandardisapplicabletHowever,everybodywhocomest6anagreementthroughthisstandardisencouragedtostudydocumentswithoutdate,theirlastededitionsareapplicabletothisstandard.Theresiduesofnequinateanddecoquinateinthetestsamplearesingexternalstandardmethod.Unlessotherwisespecified,allreagentsshouldbeofanalyticalgrade;wat9N,N-dimethylformamide:HPLCgrade;Trichloromethane:HPLCgrade;Ammoniumacetate:HPLCgrade;30%acetonitrileaqueoussolution:Mix30mLactonitrile(4.1)with70mLwater.4.9Sampledilutesolution:Mix50mLactonitrile(4.1)with50mLwater,add0.1mLformicacid(4.5),mixed.4.100.005mol/Lammoniumacetateaqueoussolution(contain)0.05%formicacid):Accuratelyweigh0.3854gammoniumacetate(4.6),dissolvewithwater,add0.5mLformicacid(4.5),dilute4.11Standards:Nequinate(CasNo-13997-19-8),decoquinate(CasNo.:18507-89-6),puritymorethan99%.4.12Standardstocksoltion{Accuratelywelghadequatenequinate(4.11),dissolveinN,N-dime.estandardstocksolutionof100mg/Linconcentration.Accuratelythylformamide(4.3)tomakeweighadequatedecoquinate(4)11),dissolve'intrichloromethéne(4.4)tomakeastandardstocksolu.4.13Intermediatestandardsmixedsolution:accuratelydrawanadequatevolumestandardstosolution(4.12),dilutewithacetonitrile(4.1)tomakeanintermediatestan10.0mg/Linconcentration.Thesolutioncanbepreservedinthetemperatureat4.15Syringefilter:0.22μm.5.1Liquidchromatographytandemmassspectrometry,equippedwithelectrosprayionsource.5.4Homogenizer.5.5Vortexmixer.5.6Centrifuge:Rotatespeedmorethan5000r/min.5.11Reciprocatingtypeoscillator.6SamplepreparationandstorageTakeapproximately500gofrepresentativesample,smashthoroughlybyahomogenizer,mixthoChooseabout500grepresentativesample,devidedintotwopart,andputintosamplecontainer,Incourseofsampling,itshouldbetakentoavoidcontaminaWeigh5gsample(accurateto0.01g)ina50mLcentrifugetube,add15mLacetonitrile(4.1)intoeachcentrifugetube,vortexmixfor1min,thenshake5mininordertoextractthoroughly,centri-10mLacetonitrile(4.1)intotheleavings,repeattheprocessonceagain,centrifugedat5000r/minfor5min,combinetheupperliquidfornextprocess.Addsuperfluoussodiumchloride(4.7)intotheextraction,vortexmixfor1min,centrifugeat5000r/minfor3minsothattheaqueoussolutionwassaturatedandwoulddividefromtheacetonitrile.Accuratemeasure5mLupperacetonitrileextrac-tionintoa15mLcentrifugetube,concentratedwithwaterforfurthercleanup.Transfertheextraction(7.1.1)ontothepre-conditionedHLBcartridge(4.14)andkeeptheflowrateat3mL/min.discardtheeffluence.Rinsethecartridgewith3mL30%acetonitrileaqueoussolution(4.8)and3mLwater,discardtheeffluence.Drythecartridgebydrawingairthroughitfor15min.Fi-nallyelutetheobjectswith3mLacetonitrile(4.1)intoa15mLcentrifugetube,concentratetheelu-enttonearlydry,anddiluteto1.0mLwiththesampledilutesyringefilter(4.15)forLC-MS/MSanalysis.Weighfiveblanksampleinfive50mLcentrifugetubes,each5g(accurateto0.01intermediatestandardsmixedsolution(4.13),makethefinallydeterminatedconcentrationare0.005mg/L,0.010mg/L,0.050mg/L,0.100mg/L,0.250mg/L.Add15mLacetonitrile(4.1)intoeachcentrifugetube,thenextprocessfollowthe7.1.1“Add15mLacetoniWeigh5goftheblanksampa)Column:AcquityBEHCcolumn,50mm×2.1mm(i.d.),1.7μm,orequivalent;SN/T2444—2010c)InjectionvolumTime/minFlowrate/(mL/min)0.005mol/LAmmoniumacetatesolutionformicacid)/%'Acetonitrile/%0b)Scanmode:positiveme)Qualitative204.0:pairs:nequinate366.3/334.1,366.3/91.0,decoquinate418.1/372.2,418.1/f)Quantitativeionpairs:nequinate366.3/334.1,decoquinate418.1/372.2;Otheroperatingconditionsarelisteditentiontimeofanalytebetweentestsampleandstandardsolutioniswithin±2.5%underthesameexperimentconditions,andthedifferenceofrelativeionratioofanalytebetweentestsampleandlistedintable2),correspondinganalRelativeionratio/%>20～50>10～20Maxdeviationallowed/%Undertheoptimizedinstrumentworkingcondition,differentmixedmatrixcalibrationsolutionsareTheresponseofnequinateanddecoquinateinthestandardworkingsolutionaearrangeofstandardworkingsolution,thesolutionshouldbedilutedbeforetiplereactionmonitoring(MRM)chromatograms-of-nequinate-and-decoquinateareshownasfigureThecalculationofnequinateanddecoquinatecontentinthesampleisXWhere…………mX-theresiduecontentofanalytec—theconcentrationofanalytewhichisquantifiedbystandardcalibrationcuV—thefinalvolumeofsamplesolution,mL;m—thecorrespondingmassoftestsampleinthefinalsamplesolution,g.Recoverydatasforchicken,chickenlCompoundAddedlevel/(mg/kg)Recovery/%ChickenChickenChickenkidneyEggBovineMilkNequinate0.01096.4～10585.0～10591.7～10889.0～10489.1～10193.7～1070.10098.9～10591.0～10884.5～91.8101～10998.4～10792.4～99.00.200 —96.5～104101～10878.9～84.991.7～96.787.2～97.889.7～1