SNT 0351-2009进出口食品中丙线磷残留量检测方法

中华人民共和国出入境检验检疫行业标准代替SN0351—1995进出口食品中丙线磷残留量检测方法2009-07-07发布2010-01-16实施国家质量监督检验检疫总局I——扩大了使用范围；1进出口食品中丙线磷残留量检测方法本标准规定了进出口食品中丙线磷残留量检测的气相色谱测定和液相色谱-质谱/质谱确证方法。 线磷残留量的测定和确证。2方法提要样品经乙腈或乙酸乙酯提取后，通过固相萃取小柱净化，采用气相色谱(火焰光度检测器)测定，外标法定量。液相色谱-质谱/质谱确证。3试剂和材料3.6无水硫酸钠：650℃灼烧4h,在干燥器内冷却至室温，储于密闭干燥器中备用。3.8正已烷-丙酮(2+1,体积比).取正己烷1006L,加入50mL丙酮，混匀。3.10丙线磷标准储备液：准确称取适量两线磷，用乙酸乙酯配制成浓度为1.00mg/mL的标准储备3.11丙线磷标准中间溶液；准确吸取适量标准储备液，用乙酸乙酯稀释至浓度为100.0mg/mL的标3.12丙线磷标准工作液：使用前根据需要将标准中间溶液用乙酸乙酯稀释成适当浓度的标准工作液，3.14氟罗里硅土固相萃取柱：1000mg,3mL,或相当者。3.16CHROMABONDXTR固相萃取柱：3000mg,15mL,或相当者。4仪器和设备2称取5g试样(精确至0.01g)于50mL离心管中，加2g无水硫酸钠(3.6),加15mL乙酸乙酯(3.2),匀质提取1min,振荡提取20min,4000r/min离心5min,移取上清液于试管中，再分别用10mL称取10g试样(精确至0.01g)于50mL离心管中，加10g无水硫酸钠，加15mL乙酸乙酯，匀质提取1min,4000r/min离心5min,移取上清液于25mL容量瓶中，再用10mL乙酸乙酯洗涤残渣一次，涡旋振荡1min,4000r/min离心5min,合并提取液于容量瓶中，用乙酸乙酯定容至刻度。取12.5ml3称取1.0g试样(精确至0.01g)于10mL离心管中，加3mL水，浸泡20min,加0.5g无水硫酸钠，振荡混匀，再分别用2mL乙酸乙酯提取3次，旋涡振荡提取1min,4000r/min离心3min,合并提取6.1.5蜂蜜称取2g试样(精确至0.01g)于50mL离心管中，加3mL水，0.5g氯化钠(3.7),振荡混匀，过固集于50mL浓缩瓶中，在45℃以下旋转浓缩至约1mL石墨化碳黑固相萃取柱(3.13)用2×2mL乙酸乙酯活化，弃去流出液。将待净化溶液过石墨化碳黑固相萃取柱，再用2×2mL乙酸乙酯洗脱，流速控制为1mL/min,收集全部流出液于试管中，在45℃以下氮吹至近干，用乙酸乙酯定容至1.0mL,待测。2mL;再过石墨化碳黑固相萃取柱，在柱上填装0.5g无水硫酸钠，用2×2mL乙腈活化。将浓缩液过石墨45℃以下氮吹至约0.5mL,用乙酸乙酯定容至2.0mL,过0.45μm滤膜(3.17)后，待测。在石墨化碳黑固相萃取柱上填装0.5g无水硫酸钠，用2×2mL乙酸乙酯活化，弃去流出液。将待净化液过柱，用2×2mL.乙酸乙酯洗脱，流速控制为1mL/min,收集全部过柱溶液，在45℃下吹氮浓缩至约1mL。氨基柱(3.15)先用2×2mL正己烷-丙酮活化，将浓缩液过氨基柱后，3×1mL正己烷-丙酮洗脱，流速控制为1mL/min,收集全部流出液于试管中，在45℃以下氮吹至约0.5mL,用乙酸乙酯定容至1.0mL,待测。6.2.4蜂蜜6.3测定6.3.1气相色谱条件c)进样口温度：220℃;d)检测器温度：245℃;e)载气：氮气(纯度99.999%),流量7.0mL/min;46.3.2气相色谱测定6.4.1.1液相色谱参考条件表1.流动相梯度表时间/min流速/(mL:/min)水/%6.4.1.2质谱参考条件被测物名称定性离子对(m/z)采集时间去簇电压碰撞能量丙线磷6.4.2液相色谱-质谱/质谱法定性确证5的相对丰度进行比较，偏差不超过表3规定的范围，被确证的样品可判定为丙线磷阳性检出。丙线磷标准品的子离子全扫描质谱图和多反应监测(MRM)色谱图参见附录B中图B.1、图B.2。表3定性确证时相对离子丰度的最大允许偏差>20～50>10～206.5空白试验7结果计算和表述A——样液中丙线磷的峰面积；8测定低限和回收率8.1测定低限8.2回收率丙线磷添加浓度及同收率的数据见表4。样品名称添加浓度/(μg/kg)回收率范围/%大米绿豆菠菜荷兰豆6表4(续)样品名称添加浓度/(μg/kg)回收率范围/%柑橘葡萄猪肉鸡肉罗非鱼茶叶5板栗猪肝蜂蜜7(资料性附录)丙线磷的标准物质气相色谱图图A.1丙线磷的标准物质的气相色谱图(GC-FPD)8ntensity/epsntensity/eps(资料性附录)丙线磷标准物质LC-MS/MS质谱图和色谱图a)图B.2丙线磷标准物质多反应监测(MRM)色谱图(5μg/L)9ThisstandardsubstitutedSN0351—1995《methodfordeterminationofethoprophosresiduesincere.ThedifferencesbetweenthisstandardandSN0351—1995areasfollows:一Enlargedscopeaboutapplyed;一AddedmethodofconfirmationbyGaschromatographyandliquid,massspectrometry;—Removed“2SamplingandSamplePreparation”inSN0351-1995,added“Preparationandstorageoftestsample";一Improvedontechnicallineaboutextractionofsample.AnnexAandBofthisstandardareinformativeannexs.ThisstandardwasproposedbyandisunderthechangedoftheCertificationandAccreditationAd.ministrationofthePeople'sRepublicofChina.ThisstandardwasdraftedbyGuangdongEntry/ExitInspectionandOlarantineBureauofthePeople'sRepublicofChina,ZhejangEntry-ExitInspectjonandQuarantineBureauofthePeople'sRepublicofChina,BeijingEntry-ExitInspectionandQuarfntineBureauofhhePedple'sRepublicofChinaandHe.beiEntry-ExitInspectionandOuarantineBurThemaindreftersofthisstandaldareChenJie,XieJiahiun,WangLan,WuYingxuan,LinHaidan,Zhengziqiang,XuChaoyiandWangFengchiThisstandardispublishedforthefirsttimein1995.Thestandardismodifiedforthefirsttime.DeterminationofethoprophosresiduesinfoodsThestandardspecifiesthedeterminationandconfirmationofethoprophosresiduesinfoodsbyGaschromatographyandliquidmassspectrometry.Thisstandardisapplicabletothedeterminationandconfirmationofethoprophosresiduesinrice,mungbean,spinage,vegetablepea,orange,grape,chestnut,tea,pork,chicken,porkliver,tilapiaandhoney.Theresiduesintestsamplesareextractedwithacetonitrileorethylacetate.TheextractsarecleanedupbyneutralaluminaSPEcartridgeorflorisilSPEcartridgeoractivecarbonSPEcartridge.Determi-nationandconfirmationmadebyGC(FPD)andLC-MS/MSusingexternalstandardmethod.Unlessotherwisespecified,allreagentsusedshouldbeanalyticalgrade,“water”isdistilledwater3.2Ethylacetate.3.4Acetonitrile:Chromatogramgrade.3.6Anhydroussodiumsulfate:Igniteat650℃for4h,andkeepinadesicatoraftercooling3.7Sodiumchloride.3.8n-Hexane-acetone(2+1,V/V)3.9Ethoprophos:(C₈HgO₂PS₂,CASNo:13194-48-4):Purity≥99.0%,3.10Standardstocksolution:Accuratelyweighadequateamountofethoprophosstandards,dis-solvewithethylacetateandprepareasolutionof1.00mg/mLasstandardstocksolution.Standard3.11Standardmiddlesolution:pipetteadequateamountofstandardstocksolution,dilutewitheth-ylacetatetoprepareasolutionof100.0μg/mLasstandardworkingsolution.Storedinarefrigerator3.12Standardworkingsolutions:pipetteadequateamountofstandardmiddlesolution,dilutewithethylacetatetoprepareappropriateconcentrationstandardworkingsolutions.Storedinarefrigera-3.13ActivecarbonSPEtubes:250mg,3mL,ENVI-Carb,orequivalent.3.14FlorisilSPEcartridge:1000mg,3mL,orequivalent3.15NH₂SPEcartridge:200mg,3mL,orequivalent.3.16CHROMABONDXTRSPEcartridge;3000mg,15mL,orequivalent.3.17Syringedrirevenfilter:0.45μm(FH).4.1Gaschromatograph,equippedwithflamephotometricdetector4.3Analyticalbalance:0.1mgand0.01g4.5HighSpeedHomogenizer.4.7Rotaryvacuumevaporator.4.8Nitrogenevaporator.SN/T0351—20094.11Solidphaseextractionvacu4.13Centrifugetube,polytetrafluoroethyle4.14Heart-shapedflask,25mL.5.1.1Spinage,vegetablepea,orange,grape5.1.3Pork,chicken,porkliver,tilapiaroomtemperature.Inthecourseofsamplemelting,precautionsmustbetakentoavoidevaporationofwaterfromthesample.Placeinacleancontainer,whichislabeledandsealed.Tea,honey,grainandnutshouldbestoredat0℃~4℃.VegetableandfruitshouldbestoredInthecourseofsamplingandsamplepreparation,precautionmustbetakentoavolidcontamination6.1.1Rice,mungbean,chestnutWeigh5g(accurateto0.01g)oftestsampleintoa50mLcentrifugetube,add15mLofethylacetateand2gofanhydroussodiumsulfate.Homogenize-thesamplefor1min,thencentrifugationfor5minat4000r/min.Thesupernatantlayerwastransferedintoglasstube.Residuewasrinsedtwicewith10mLofethylacetateandcombinedtheorganicsolvent.Evaporatetheorganicsolventto2mLwithnitrogenevaporatorat45℃forcleanup.6.1.2Spinage,vegetablepea,orange,grapeWeighaccurately10g(accurateg60.01g)oftestsampleintoa.50mLcentrifugetube,add10gofanhydroussodiumsulfateand15mLofethylacetate,thenmixed1nin,thencentrifugationfor5minat4000r/min.Thesupernatantlayerwastransferedintoglasstube.Residuewasrinsedtwicewith10mLofethylacetateandcomblnedtheorganicsolvent.inthe-25mLcolorimetrictube.Mixwellandtransfer12.5mLofextractsolutionforcleaningup.6.1.3Pork,chicken,porkliver,tllapiaandchestnutWeigh5goftestsampleinto50mLcentrifudetube,add10gofanhydroussodiumsulfate,15mLoacetonitrile.Homogenizethesamplefor1min,thenusing10imLofacetonitrilewashthetoolholdebitsandtransferittotheformercentrifugetube.Aftercentrifugationfor5minat6000r/min,trans.fer10mLofthesupernatantlaVerintoa20mLtubeforgdleaningup.Weigh1.0g(accurateto0.01g)oftestsampleintoa10mLcentrifugetube,add3.0mLwaterinitandimmersefor20min.Add0.5gofsodiumchloride,vortextomixandadd3×2mLofethylacetatetoextract,vortexfor2mineachtime,centrifugeat4000r/minfor3min,andcombinedthe3timessupernatantsforcleaningupWeigh2g(accurateto0.01g)oftestsampleintoa50mLcentrifugetube,add3mLwater,0.5gofsodiumchloride,vortextomix.ThedilutedsolutionwaspassedthroughaCHROMABONDRTXSPEcar-tridge(3.16)underwhichconnectwithacartridgefilledin1ganhydroussodiumsulfate,keepfor5min,thenrinsethecartridgewith35mLofethylacetate,ataflowrateof1.0mL/min.Collecttheelu50mLheart-shapedflask.Evaporatenearlyto1mL6.2.1Rice,mungbeathroughtheNH₂cartridge(3.15).Rinsethecolumnwith3×a)GCcolumn:HP-5capillarycolumn,30m×0.32mm(i.d.)×0.25mmorequivalent;c)Injectionporttemperature:220℃;d)Detectiontemperature:245℃;e)Carriergas:Nitrogen,purity≥99.999%;f)Injectionmode:Splitless,purgeonafteg)Injectionvolumn:1.0μL.Accordingtoapproximateconcentrationofanalyte,selectthestandardworkingsolutionwithsimilarresponsetothatofsamplesolution.Theresponsesofethoprophosinthestandardworkingsolutionandsamplesolutionshouldbeinthelinearrangeoftheinstrumentaldetection.Thestandardworkingsolutionshouldbeinjectedrandomlyinbetweentheinjectionsofsamplesolutionofequalvolume.AttheaboveGCconditions,theretentiontimeis6.8min,andtheGCchromatogramoftheethoprophosstandardseeannexA.6.4.1LC-MS/MSoperatingconditionsa)Column:DiscoveryCig,5μm,150mm×2.1mm(i.d.)orequivalent;b)Columntemperature:40℃;c)Mobilephase:Methanol-water(50+50,V/V);SN/T0351—2009f)Gradient:seetable1.Time/minFlowrate/(mL/min)Water/%Methanol/%0.30-a)lonsource;ESI;b)Scanmode:negativemode;d)lonsprayvoltage(IS):4500V;e)Nebulizergas,curtaingas,heatergasandfcollision,gasarehighpuritynitrogenorequivalent,optimizetheflowrateofeachgasforeachtherequirementofthesensitivityofmassspectrometer;g)Quelityions,quantityions,declusteringpofential(DP)andcollisionenergy(CE)areshownintaNote:Non-commercialstatement;theequipmentsandtheirtypesAPI3000involvedinthestandard-methodarenotrelatedtocommercialaims,andtheanalystsareencouragedtouseequipmentsofdifferentcorporationordifferenttype.Transfer0.5mLtestsamplesolutionwithGCanalysisevaporateundernitrogentodrynessat45℃.DissolvetheresidueanddilutewithmethanolforLC-MS/MSanalysis.LC-MS/MSshouldbeappliedforconfirmationthequalityoftheethoprophoswhenthequantityofethoprophosinsampleishigherthanthedetectionlimit.Thestandardsolutionandsamplesolutionwereanalyzedaccordingtotheoperatingconditionassignedin6.4.1.Thequalitativeionsforeachanalystincludeoneprecursorionandtwoproductionsatleast.Underthesamedeterminationconditions,theretentiontimeofthean-alyteinthesampleshallmatchthatofthecalibrationstandardwithinthetolerances2.5%.Therelativeintensitiesofthedetected-ions-of-each-analystrshall-correspondtothoseofthecalibrationstandardatcomparableconcentrations,withinthetolerancesshawnintable3,thenthecorrespond.inganalytemustbepresentinthesample.ForLC-MS/MSchromatogram(MRM)ofethoprophosstandards,seefigureB.1andfigureB.2-in-annexB.Table3—Maximumpermittedtolerancesforrelativeionintensitieswhileconfirmatior>10～20Theoperationoftheblanktestisthesameasthedescribedinthemethodofdetermingtion.butwiththeomissionofsampleaddition.CalculationthecontentofethoprophosresiduesinthetestsamplebyGCdataprocessororaccordingtotheformula(1).Theblankva