SN-T3855-2014出口食品中乙二胺四乙酸二鈉的測定

中華人民共和國出入境檢驗檢疫行業(yè)標(biāo)準(zhǔn)出口食品中乙二胺四乙酸二鈉的測定2014-01-13發(fā)布I本標(biāo)準(zhǔn)按照GB/T1.1—2009給出的規(guī)則起草。本標(biāo)準(zhǔn)由國家認(rèn)證認(rèn)可監(jiān)督管理委員會提出并歸口。1出口食品中乙二胺四乙酸二鈉的測定本標(biāo)準(zhǔn)第一法規(guī)定了出口食品中乙二胺四乙酸二鈉的液相色譜測定方法。本標(biāo)準(zhǔn)第二法規(guī)定了出口食品中乙二胺四乙酸二鈉的液相色譜-質(zhì)譜/質(zhì)譜測定方法.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GB/T6682分析實驗室用水規(guī)格和試驗方法第一法液相色譜法4.9乙二胺四乙酸二鈉標(biāo)準(zhǔn)品：CAS:6381-92-6,純度大于等于99%。4.10標(biāo)準(zhǔn)儲備溶液：準(zhǔn)確稱取適量的乙二胺四乙酸二鈉標(biāo)準(zhǔn)品，用水溶解并定容至100mL棕色容量瓶中，得濃度為10mg/mL的標(biāo)準(zhǔn)儲備溶液，此溶液轉(zhuǎn)移至儲液瓶中4℃下儲存一個月。24.11標(biāo)準(zhǔn)中間溶液：準(zhǔn)確吸取乙二胺四乙酸二鈉儲備溶液10mL于50mL棕色容量瓶中，用水稀釋成濃度為2mg/mL的標(biāo)準(zhǔn)中間溶液，此溶液轉(zhuǎn)移至儲液瓶中4℃下儲存一個月。5儀器和設(shè)備6試樣制備與保存取有代表性樣品約500g,用組織搗碎機將樣品加工成漿狀，裝入潔凈容器作為試樣，密封并標(biāo)明取有代表性樣品約500g于1000mL燒杯中，在70℃水浴中邊加熱邊攪拌，去除部分二氧化碳，稱取試樣5g(精確到0.01g)于50mL玻璃具塞離心3SN/T3855—2014氯甲烷(4.2),用均質(zhì)器以10000r/min均質(zhì)2min,4500r/min離心5min。將上清液轉(zhuǎn)移至50mL7.2.1樣品溶液的衍生化8結(jié)果計算和表達 (1)4A——樣液中乙二胺四乙酸二鈉衍生物的峰面積；A,——標(biāo)準(zhǔn)工作液中乙二胺四乙酸二鈉衍生物的峰面積；9測定低限和回收率本方法的測定低限為20mg/kg。樣品的添加濃度及回收率的數(shù)據(jù)參見附錄B中表B.1。第二法液相色譜液-質(zhì)譜/質(zhì)譜法11.8三氯化鐵溶液：0.02mol。稱取0.5406g三氯化鐵(11.7),溶于90mL水中，轉(zhuǎn)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)調(diào)節(jié)pII至3.5,轉(zhuǎn)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，轉(zhuǎn)移到100mL容量瓶中，用11.12二胺四乙酸二鈉標(biāo)準(zhǔn)品：CAS:6381-92-6,純度大于等于99%。11.13標(biāo)準(zhǔn)儲備溶液：分別準(zhǔn)確稱取適量的乙二胺四乙酸二鈉標(biāo)準(zhǔn)品，用水溶解并定容至100mL棕5成濃度為2mg/mL的標(biāo)準(zhǔn)中間溶液，此溶液轉(zhuǎn)移至儲液瓶中4℃下儲存一個月。取有代表性樣品約500g,用組積搗碎機將樣品加工成漿狀，裝人潔凈容器作為試樣，密封并標(biāo)明稱取試樣5g(精確到0.01g)于50mL玻璃具塞離心管中，加入40mL水，待衍生化6稱取試樣約5g(精確到0.01g)于50mL螺旋蓋聚丙烯離心管中，加入15mL水，再加20mL三氯甲烷(11.2),用均質(zhì)器以10000r/min均質(zhì)2min,4500r/min離心5min。將上清液轉(zhuǎn)移至50mL玻璃具塞離心管中。再用15mL水重復(fù)提取兩次，合并提取液于同一50mL玻璃具塞離心管中，待衍生向上述提取溶液中加入1.0mL三氯化鐵溶液(11.8),混合，于超聲波中超聲20min,冷卻至室溫準(zhǔn)確吸取適當(dāng)濃度的標(biāo)準(zhǔn)工作液于50mL玻璃具塞離心管中，加入40mL水和1.0mL三氯化鐵準(zhǔn)確移取5mL上述衍生化樣品溶液于25mL坡璃具塞離心管中，加入5mL三氯甲烷，在旋渦混合器上混合2min,4000r/min離心5min。取上清液過0.22μm濾膜(11.17),供液相色譜-質(zhì)譜/質(zhì)譜準(zhǔn)確移取5mL上述衍生化樣品溶液轉(zhuǎn)移到MAX陰離子交換柱(11.16)中，控制流速在表1梯度洗脫程序表時間/min10%甲醇水溶液(11.9)/%95%甲醇水溶液(11.10)/%4.007化合物母離子子離子駐留時間/S錐孔電壓/V碰撞能量/乙二胺四乙酸二鈉衍生物·為定量離子。根據(jù)樣液中被測物的含量情況，選定濃度與樣液相近的標(biāo)準(zhǔn)工作溶液。標(biāo)準(zhǔn)工作溶液和樣液中乙二胺四乙酸二鈉衍生物響應(yīng)值均應(yīng)在儀器檢測線性范圍內(nèi)。對標(biāo)準(zhǔn)工作溶液和樣液等體積參插進樣測定。在上述液相色譜-質(zhì)譜條件下，乙二胺四乙酸二鈉衍生物的保留時間為1.53min,乙二胺四乙酸二鈉衍生物標(biāo)準(zhǔn)品的液相色譜-質(zhì)譜/質(zhì)譜多反應(yīng)監(jiān)測色譜圖參見附錄C中圖C.1。在上述液相色譜-質(zhì)譜條件下，樣品中待測物質(zhì)保留時間與標(biāo)準(zhǔn)工作溶液中對應(yīng)的保留時間的偏差在±2.5%,且樣品中被測物質(zhì)的相對離子豐度與濃度相當(dāng)標(biāo)準(zhǔn)工作溶液的相對離子豐度進行比較，相對豐度允許相對偏差不超過表3規(guī)定的范圍，則可確定樣品中存在對應(yīng)的被測物相對離子豐度允許的相對偏差士25%士30%士50%用LC色譜數(shù)據(jù)處理機或按式(2)計算試樣中乙二胺四乙酸二鈉含量，計算結(jié)果需扣除空白值8SN/T3855—2014X——試樣中乙二胺四乙酸二鈉含量，單位為毫克每千克(mg/kg);A——樣液中乙二胺四乙酸二鈉衍生物的峰面積；c--—標(biāo)準(zhǔn)工作液中乙二胺四乙酸二鈉衍生物的濃度，單位為微克每毫升(μg/mL);V——樣液最終定容體積，單位為毫升(mL)A,——標(biāo)準(zhǔn)工作液中乙二胺四乙酸二鈉衍生物的峰面積；16測定低限和回收率16.1測定低限本方法的測定低限為20mg/kg。16.2回收率樣品的添加濃度及回收率的數(shù)據(jù)參見附錄D中表D.1。(資料性附錄)乙二胺四乙酸二鈉衍生物標(biāo)準(zhǔn)品色譜圖g表B.1樣品的添加濃度及回收率的數(shù)據(jù)樣品添加濃度/回收率范圍/%樣品添加濃度/回收率范圍/%啤酒86.54～103.34藍莓果醬89.63～103.2078.69～101.2380.54～100.3778.30～101.6582.45～99.90果酒89.10～100.63魚罐頭89.67～104.6875.68～97.5181.07～102.3779.30～100.6581.85～112.90果汁飲料89.61～100.07沙拉醬91.77～98.8778.65～101.1474.25～103.3878.90～106.8082.70～103.20茶飲料91.05～100.14金針姑罐頭83.65～103.0982.36～95.2183.44～106.7893.15～108.5588.20～110.15八寶粥87.91～103.14板栗罐頭82.92～103.2775.49～102.5779.65～101.2887.95～108.3582.85～105.35番茄醬89.32～101.7085.39～101.7386.95～107.80相對豐度/%(資料性附錄)時間/min時間/min表D.1樣品的添加濃度及回收率的數(shù)據(jù)樣品添加濃度/回收率范圍/%樣品添加濃度/回收率范圍/%啤酒藍莓果醬果酒魚罐頭果汁飲料8T.08～100.32沙拉醬茶飲料金針菇罐頭八寶粥板栗罐頭番茄醬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(FeCl?·6H?O).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,cannedgoldenneedlemushroomandcannedchestnutTakeapproximately500gofrepresentativesampleinto1000mLbeakerwithperiodicstirringbyaglasssticktodischargeCO?at70℃.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-Cl?,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