SN-T3855-2014出口食品中乙二胺四乙酸二钠的测定

中华人民共和国出入境检验检疫行业标准出口食品中乙二胺四乙酸二钠的测定2014-01-13发布I本标准按照GB/T1.1—2009给出的规则起草。本标准由国家认证认可监督管理委员会提出并归口。1出口食品中乙二胺四乙酸二钠的测定本标准第一法规定了出口食品中乙二胺四乙酸二钠的液相色谱测定方法。本标准第二法规定了出口食品中乙二胺四乙酸二钠的液相色谱-质谱/质谱测定方法.2规范性引用文件下列文件对于本文件的应用是必不可少的。凡是注日期的引用文件，仅注日期的版本适用于本文GB/T6682分析实验室用水规格和试验方法第一法液相色谱法4.9乙二胺四乙酸二钠标准品：CAS:6381-92-6,纯度大于等于99%。4.10标准储备溶液：准确称取适量的乙二胺四乙酸二钠标准品，用水溶解并定容至100mL棕色容量瓶中，得浓度为10mg/mL的标准储备溶液，此溶液转移至储液瓶中4℃下储存一个月。24.11标准中间溶液：准确吸取乙二胺四乙酸二钠储备溶液10mL于50mL棕色容量瓶中，用水稀释成浓度为2mg/mL的标准中间溶液，此溶液转移至储液瓶中4℃下储存一个月。5仪器和设备6试样制备与保存取有代表性样品约500g,用组织捣碎机将样品加工成浆状，装入洁净容器作为试样，密封并标明取有代表性样品约500g于1000mL烧杯中，在70℃水浴中边加热边搅拌，去除部分二氧化碳，称取试样5g(精确到0.01g)于50mL玻璃具塞离心3SN/T3855—2014氯甲烷(4.2),用均质器以10000r/min均质2min,4500r/min离心5min。将上清液转移至50mL7.2.1样品溶液的衍生化8结果计算和表达 (1)4A——样液中乙二胺四乙酸二钠衍生物的峰面积；A,——标准工作液中乙二胺四乙酸二钠衍生物的峰面积；9测定低限和回收率本方法的测定低限为20mg/kg。样品的添加浓度及回收率的数据参见附录B中表B.1。第二法液相色谱液-质谱/质谱法11.8三氯化铁溶液：0.02mol。称取0.5406g三氯化铁(11.7),溶于90mL水中，转移到100mL容11.910%甲醇水溶液(含5mmol三正丁胺，pH=3.5):移取100mL甲醇(11.1),溶于880mL水中加入1.17mL三正丁胺(11.6),用冰乙酸(11.4)调节pII至3.5,转移到1000mL容量瓶中，用水定容至11.1095%甲醇水溶液(含5mmol三正丁胺，pH=3.5):移取950mL甲醇，溶于30mL水中，加入11.1110%甲酸甲醇水溶液：移取10mL甲酸(11.3),溶于40mL水中，转移到100mL容量瓶中，用11.12二胺四乙酸二钠标准品：CAS:6381-92-6,纯度大于等于99%。11.13标准储备溶液：分别准确称取适量的乙二胺四乙酸二钠标准品，用水溶解并定容至100mL棕5成浓度为2mg/mL的标准中间溶液，此溶液转移至储液瓶中4℃下储存一个月。取有代表性样品约500g,用组积捣碎机将样品加工成浆状，装人洁净容器作为试样，密封并标明称取试样5g(精确到0.01g)于50mL玻璃具塞离心管中，加入40mL水，待衍生化6称取试样约5g(精确到0.01g)于50mL螺旋盖聚丙烯离心管中，加入15mL水，再加20mL三氯甲烷(11.2),用均质器以10000r/min均质2min,4500r/min离心5min。将上清液转移至50mL玻璃具塞离心管中。再用15mL水重复提取两次，合并提取液于同一50mL玻璃具塞离心管中，待衍生向上述提取溶液中加入1.0mL三氯化铁溶液(11.8),混合，于超声波中超声20min,冷却至室温准确吸取适当浓度的标准工作液于50mL玻璃具塞离心管中，加入40mL水和1.0mL三氯化铁准确移取5mL上述衍生化样品溶液于25mL坡璃具塞离心管中，加入5mL三氯甲烷，在旋涡混合器上混合2min,4000r/min离心5min。取上清液过0.22μm滤膜(11.17),供液相色谱-质谱/质谱准确移取5mL上述衍生化样品溶液转移到MAX阴离子交换柱(11.16)中，控制流速在表1梯度洗脱程序表时间/min10%甲醇水溶液(11.9)/%95%甲醇水溶液(11.10)/%4.007化合物母离子子离子驻留时间/S锥孔电压/V碰撞能量/乙二胺四乙酸二钠衍生物·为定量离子。根据样液中被测物的含量情况，选定浓度与样液相近的标准工作溶液。标准工作溶液和样液中乙二胺四乙酸二钠衍生物响应值均应在仪器检测线性范围内。对标准工作溶液和样液等体积参插进样测定。在上述液相色谱-质谱条件下，乙二胺四乙酸二钠衍生物的保留时间为1.53min,乙二胺四乙酸二钠衍生物标准品的液相色谱-质谱/质谱多反应监测色谱图参见附录C中图C.1。在上述液相色谱-质谱条件下，样品中待测物质保留时间与标准工作溶液中对应的保留时间的偏差在±2.5%,且样品中被测物质的相对离子丰度与浓度相当标准工作溶液的相对离子丰度进行比较，相对丰度允许相对偏差不超过表3规定的范围，则可确定样品中存在对应的被测物相对离子丰度允许的相对偏差士25%士30%士50%用LC色谱数据处理机或按式(2)计算试样中乙二胺四乙酸二钠含量，计算结果需扣除空白值8SN/T3855—2014X——试样中乙二胺四乙酸二钠含量，单位为毫克每千克(mg/kg);A——样液中乙二胺四乙酸二钠衍生物的峰面积；c--—标准工作液中乙二胺四乙酸二钠衍生物的浓度，单位为微克每毫升(μg/mL);V——样液最终定容体积，单位为毫升(mL)A,——标准工作液中乙二胺四乙酸二钠衍生物的峰面积；16测定低限和回收率16.1测定低限本方法的测定低限为20mg/kg。16.2回收率样品的添加浓度及回收率的数据参见附录D中表D.1。(资料性附录)乙二胺四乙酸二钠衍生物标准品色谱图g表B.1样品的添加浓度及回收率的数据样品添加浓度/回收率范围/%样品添加浓度/回收率范围/%啤酒86.54～103.34蓝莓果酱89.63～103.2078.69～101.2380.54～100.3778.30～101.6582.45～99.90果酒89.10～100.63鱼罐头89.67～104.6875.68～97.5181.07～102.3779.30～100.6581.85～112.90果汁饮料89.61～100.07沙拉酱91.77～98.8778.65～101.1474.25～103.3878.90～106.8082.70～103.20茶饮料91.05～100.14金针姑罐头83.65～103.0982.36～95.2183.44～106.7893.15～108.5588.20～110.15八宝粥87.91～103.14板栗罐头82.92～103.2775.49～102.5779.65～101.2887.95～108.3582.85～105.35番茄酱89.32～101.7085.39～101.7386.95～107.80相对丰度/%(资料性附录)时间/min时间/min表D.1样品的添加浓度及回收率的数据样品添加浓度/回收率范围/%样品添加浓度/回收率范围/%啤酒蓝莓果酱果酒鱼罐头果汁饮料8T.08～100.32沙拉酱茶饮料金针菇罐头八宝粥板栗罐头番茄酱ThisstandardisdraftedinaccordaThisfileisnotresponsibletoidentifythese.ThisstandardwasproposedbyandisunderthechargeofNationalRegulatoryCommissionforCerti-ThisstandardwasdraftedbyHeilongjiangEntry-ExitInspectionandQuarantineBureaPeople'sRepublicofChina.ThemaindraftersofthisstandardareYangChangzhi,WangChuansong,WeiDongxu,WangWei,HanGuangyuan,WuYan,ChengYang,TianFen.ThefirstmethodinthisstandardspecifiesthemethodofdeterminationofEDTAdisodiuminfood-ThesecondmethodinthisstandardspecifiesthemethodofdeterminationofEDTAdisodiuminfoodstuffsforexportbyLC-MS/MSThefirstmethodinthisstandardisapplicabletothedeterminationofEDTAdisodiuminbeer,fruitwine,fruitdrink,teadrink,eightingredientporridge,tomatoketchup,blueberryjam,cannedfish,saladjam,cannedgoldenneedlemushroom,cannedchestnut.ThesecondmethodinthisstandardisapplicabletothedeterminationandconfirmationofEDTAdis-odiuminbeer,fruitwine,fruitdrink,teadrink,eightingredientporridge,tomatoketchup,blueberryjam,cannedfish,saladjam,cannedgoldenneedlemushroom,cannedchestnutThefollowingnormativedocumentscontainprovisionswhich,throughreferenceinthistext,con.stitueprovisionsofthisProfessionalStandard.Fordatedreferences,subsequentamendmentsto,orrevisionsof,anyofthesepublicationsdonotapply.However,partiestoagreementsbasedinthisProfesssionalStandarareencouragedtoinvestigatethepossibilityofapplyingthemostrecentedi-tionsofthenormativedocumentsindicatedbelow.Forundatedreferences,thelasesteditionofthenormativedocumentreferredtoappliesGB/T6682Waterforanalyticallaboratoryuse—SpecificationandtestmethodTheEDTAdisodiuminthetestsamplesareextractedwithwater.TheextractedisderivedfromFeClyandcleanedupbychloroform.ThepurifiedsolutionisdeterminedbyLC,usingexternalstandardmethod.Unlessotherwisespecified,allregentsareanalyticallypure,"water"isthefirstgradewaterprescribedbyGB/T66824.1Methanol:LCgrade.4.2Chloroform:LCgrade.4.5Phosphate.4.6Ferricchloride(FeCl₃·6H₂O).4.7Ferricchloridesolution,0.02mol/L:Weigh0.5406gFerricchloride(4.6),dissolvewith90mldeionizedwater.Transferdissolvedsolutioninto100mLvolumetricflask,addapproximately0.03mLofhydrochloricacid(4.4)anddilutetofinedvolumeof100mL4.8Dipotassiumhydrogenphosphatesolution,0.075mol/L(pH2.3):Weigh10.207gdipotassiumhydrogenphosphate(4.3),dissolvewith900mLdeionizedwater.AdjustpHto2.3withphosphateanddilutetofinedvolumeof1000mL.4.9Ethylenediaminetetraaceticaciddisodiumstandard:EDTAdisodium,CAS:6381-92-6,purity4.10EDTAdisodiumstockstandardsolution:AccuratelyweighanadequateamountofEDTAdiso-diumstandardintoa100mLbrownvolumetricflask,dissolveanddilutetovolumewithdeionizedwaterprepareasolutionof10mg/mLasthestandardstocksolution.bepreservedbyavoidinglightintherefrigeratorat4℃foronemonth.4.11Intermediatestandardsolution:Accuratelypitionintoa50mLbrownvolumetricflask,dissolveanddilutetovolumewithdeionizedwaterprepareasolutionof2mg/mLasintermediatestandardsolution.Themixtureintermediatestandardsolutioncanbepreservedbyavoidinglightintherefrigeratorat4Coronemonth.4.12Standardworkingsolution:Accordingtotherequirement,diluteastandardworkingsolutionofappropriateconcentrationsolutionjustbeforeuse4.13Watermembranefilter:0.45μm.5Apparatusandequipment5.3Tissuehomogenizer5.4Centrifuge:5000r/min5.6Highspeedhomogenizer:12000r/min5.8Polypropylenecentrifugetubes-withscrew-cap:50mL.5.9Glasscentrifugetubeswithgroundstopper:25mL,50mL.6Preparationandstorageqfsample6.1.1Eightingredientporridge,cannedfish,cannedgoldenneedlemushroomandcannedchestnutTakeapproximately500gofrepresentativesampleinto1000mLbeakerwithperiodicstirringbyaglasssticktodischargeCO₂at70℃.Thesampleisplacedincleancontainersasthetestsampwhichisscaledandlabeledaftercooldownsampleroomtemperature.6.2StorageoftestsampleThetestsamplesofbeer,fruitwine,fruitdrink,teadrinkshouldbestoredatThetestsamplesofeightingredientporridge,tomatoketchup,blueberryjam,cannedfish,saladjarm,cannedgoldenneedlemushroomandcannedchestnutshouldbestoredbelow-18℃.Inthecourseofsamplingandsamplepreparation,precautionsshallbetakentoavoidcontaminationoranyfactorswhichmaycausethechangeofresiduecontent.Weighca.5g(accurateto0.01g)ofthetestsampleintoa50mLglasscentrifugetubeswithgroundstopper,add40mLofdeionizedwaterforderivatization.7.1.2Eightingredientporridge,tomatoketchup,blneedlemushroomandcannedchestnutWeighca.5g(accurateto0.01g)ofthetestsampleintoa50mLpolypropylenecentrifugetubeswithscrew-cap.Add15mLofdeionizedwaterand20mLofchloroform(4.2).Homogenizethetubeat10000r/minfor2minonhighspeedhomogenizer,andthencentrifugeatTransferthesupernatantlayerintoa50mLglasscentrifugetubeswithgroundstopper.Theresiduesisextractedtwicemorewith15mLofdeionizedwater.Combinethesupernatantlayerintothesame50mLglasscentrifugetubeswithgroundstopperforderivatizationandcleaningupAdd1mLofferricchloridesolution(4.7)intoaboveextractsandmixwellinultrasonicwavema-chinefor20min.Aftercooldownextractsroomtemperature,transferextractsinto50mLbrownvolumetricflaskanddilutetovolumewithdeionizedwater.Accuratelypipetthestandardworkingsolutionofappropriateconcentrationinto50mLglasscentri-fugetubeswithgroundstopper,add40mLofdeionizedwaterand1mLofferricchloridesolution.Thefollowingoperatingwith7.2.1.Accuratelypipet5mLofthesampleextractedsolutionofderivatizationaboveinto25mLglasscen-trifugetubeswithgroundstopper.Add5mLofchloroformandmixathighspeedonavortexmixerfor2min,andcentrifugeat4000r/minfor5min.ThesupernatantlayerisfilteredthroughtheMobilephase:Methanol-0.075mol/L(pH2.3)dipotassiumhydrogenphosphatesolution(10:90,V/V).AccordingtotheestimatedapproximateconcentrationofEDTAdisodiuminthesamplesolution,selectthestandardworkingsolutionofsimilarconcentrationtothatofsamplesolution.TheresponsesofEDTAdisodiuminthestandardworkingsolutionandthesamplesolutionshouldbeinthelinearrangeoftheinstrumentaldetection.Thestandardworkingsolutionshouldbeinjectedrandomlyin-be-tweentheinjectionsofthesamplesolutionofequalvolume.Undertheabovechromatographiccondi-tion,theretentiontimeofdiaveridineisca.7.8min.ForthechromatogramofEDTAdisodiumstand.ard,seeFigure.A.1inAnnexA.Theoperationoftheblanktestisthesameasthatdescribedinthemethodofdetermination,butwithoutadditionofsample.CalculatethecontentofEDTAdisodiumresidueinthetestsamplebyHPLCdataprocessothefollowedFormula(1).TheblankvalueshouldbesubtractedfromtheaboveresultofcalculationWhere;X-theresiduecontentofEDTAdisodiuminthetestsamples,mg/kg;A—thepeakareaofEDTAdisodiumderivativeinthetestsamplesolution;c—theconcentrationofEDTAdisodiumderivativeinthestandardworkingsolution,ug/mL;V—thefinalvolumeofsamplesolution,mL;As—thepeakareaofEDTAdisodiumderivativeinthestandardworkingsolution;m—thecorrespondingmassoftestsampleinthefinalsamplesolution,g.Thelimitofdeterminationofthismethodis20mg/kg.TherecoveryandthefortifyingconcentrationofEDTAdisodiumindifferentmatrixisshownbyTa-ThesecondLC-MS/MSmethodTheEDTAdisodiuminthetestsamplesareextractedwithwater.TheextractedisderivedfromFe-Cl₃,cleanedupbychloroformandMAXanionexchangecolumn.ThepurifiedsolutionisdeterminedbyLC-MS/MS,usingexternalUnlessotherwisespecified,allregentsareanalyticallypure,“water”isthefirstgradewaterpre-scribedbyGB/T668211.16MAXanionexchangecolumn;150mg,6mLorequivalent.Washcolumnwith5mLmethanol,11.17Watermembranefilter:0.22μm.12.1Liquidchromatography-massspectrometryequippedwithelectrosprayionsourceandtriqua-12.2Electronicbalance:accurateto0.01mgand0.01g.12.3Tissuehomogenizer.12.6Highspeedhomogenizer:12000r/min.12.9Polypropylenecentrifugefubeswithscrew-cap;50mlThesampleisplacedincleancontainersasthetestsample,whichissealedandlabeled.Takeapproximately500gofrepresontativesampleinto1000mLbeakerwithperiodicstiringbyaglasssticktodischargeCOzat70℃.Thesampleisplacedincleawhichissealedandlabeledaftercooldownsampleroomtemperature.Thetestsamplesofeightingredientporridge,tomatoketchup,blueberryjam,cannedfish,saladjam,cannedgoldenneedlemushroomandcannedchestnutshouldbestoredbelow-18℃.Inthecourseofsamplingandsamplepreparation,precautionsshouldbetakentoavoidcontaminationoranyfac-torswhichmaycausethechangeofresiduecontent.Weighca.5g(accurateto0.01g)ofthetestsampleintoa50mLglasscentrifugetubeswithgroundstopper,add40mLofdeionizedwaterforderivatization14.1.2Eightingredientporridge,tomatoketchup,blueberryjam,cannedfish,saladjam,cannedgoldenWeighca.5g(accurateto0.01g)ofthetestsampleintoa50mLpolypropylenecentrifugetubeswithscrew-cap.Add15mLofdeionizedwaterand20mLofchloroform(11.2).Homogenizethetubeat10000r/minfor2minonhighspeedhomogenizer,andthTransferthesupernatantlayerintoa50mLglasscentrifugetubeswithgroundstopper.Theresiduesisextractedtwicemorewith15mLofdeionizedwater.Combinethesupernatantlayerintothesame50mLglasscentrifugetubeswithgroundstopperforderivatizationandcleaningup.Add1mLofferricchloridesolution(11.8)intoahoveextractsandmixwellinultrasonicwavema-chinefor20min.Aftercooldownextractsroomtemperature,transferextractsinto50mLbrownAccuratelypipetthestandardworkingsolutionofappropriateconcentrationinto50mLglasscentri-fugetubeswithgroundstopper,add40mLofdeionizedwaterand1mLofferricchloridesolution.14.3.1Beer,fruitwine,fruitdrink,teadrink,blueberryjamandcannedchestnutAccuratelypipet5mLofthesampleextractedsolutionofderivatizationaboveinto25mLglasscen-trifugetubeswithgroundstopper.Add5mLofchloroformandmixathighspeedonavortexmixerfor2min,andcentrifugeat4000r/minfor5min.Thesupernatantlayerisfilteredthroughthe0.22μmmembranefilter(11.17)andreadyforLC-MS/MSdetermination.14.3.2Eightingredientporridge,tomatoketchup,cannedfish,saladjamandcannedgoldenneedlemushroomAccuratelypipet5mLofthesampleextractedsolutionofderivatizationaboveintoMAXanionexchangecolumn(11.14)atarateof1mL～2mL,discardtheefflucnt.Washthecolumnwithwith5mLofdeionizedwater,5mLofmethanol,3mLofformicacid-methanol-aqueoussolution(11.16)atarateof1mL~2mL,discardtheeffluent.Elutewith6mLofformicacid-methanol-aque-oussolution(11.11),collectalleluatesandblowtodrynessinnitrogenevaporatorbelow80℃.ThesolutionisreadyforLC-MS/MSdetermination.Time/min5mmol/L(pH3.5)tributylaminemethanolaqueoussolution(11.9)/%5mmol/L(pH3.5)tributylaminemethanolaqueoussolution(11.10)/%0.002.002.503.004.00SN/T3855—2014Scanmode:MultipDesolvationgasflow:550L/h,GascellPiranipressure:3.30×10~“kPa,Ar,purity≥99.999%,Table2MainmassreferenceparametersCompoundParent(Q1)Daughter(Q3)Dwelltime/SConevoltage/VCollisionenergy/EDTAdisodium344.02255.97300.03°athequantitativeionAccordingtotheestimatedapproximateconcentrationofEDTAdisodiuminthesamplesolution,selectthestandardworkingsolutionofsimilarconcentrationtothatofsamplesolution.TheresponsesofEDTAdisodiuminthestandardworkingsolutionandthesamplesolutionshouldbeinthelinearrangeoftheinstrumentaldetection.Thestandardworkingsolutionshouldbeinjectedrandomlyin-be-tweentheinjectionsofthesamplesolutionofequalvolume.UndertheaboveLC-MS/MSoperatingconditions,theretentiontimeofEDTAdisodiumisabout1.53min.LC-MS/MSmultiplereactionmo.nitoringchromatogramofEDTAdisodiumstandardareshownrespectivelybyFigureC.1inAnnexC.UndertheaboveLC-MS/MSoperatingconditions,theratioofthechromatographicretentiontimeolSN/T3855—2014analyteshallcorrespondtothatofthecalibrationsolutionatatoleranceof±2.5%.Therelativeinten-sitiesofthedetectedionsofeachanalyteshallcorrespondtothoseofthecalibrationstandardsolu-tionatcomparableconcentrations,theallowedrelativedeviationofrelativeintensityislessthanwithintheTable3tolerance,thesamecompoundinsamplemustbeconfirmed.Table3MaximumpermittedtolerancesforrelativeionintensitieswhileconfirmationRelativeionintensities>50%>20%～50%>10%～20%permittedrelativetolerancesTheoperationoftheblanktestisthesameasthatdescribedinthemethodofdetermination,butwithoutadditionofsample.15CalculationandexpressionofrfollowedFormula(2).Theblankvalueshouldbes