SN-T3845-2014出口火锅底料中多种合成色素的测定

中华人民共和国出入境检验检疫行业标准出口火锅底料中多种合成色素的测定2014-01-13发布I请注意本文件的某些内容可能涉及专利。本文件的发布机构不承担识别这些专利的责任。本标准由国家认证认可监督管理委员会提出并归口1出口火锅底料中多种合成色素的测定本标准规定了出口火锅底料中多种合成色素的液相色谱检测方法。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件。凡是不注日期的引用文件，其最新版本(包括所有的修改单)适用于本文件。GB/T6682分析实验室用水规格和试验方法试样经60℃水浴融化，依次用甲醇-丙酮混合溶液和尿素甲醇溶液(含5%氨水溶液)提取试样中的合成色素，液液萃取分配后分别经聚酰胺树脂层析柱和凝胶渗透色谱净化，液相色谱测定，外标峰面积4.7氨水：浓度为28%。4.10甲醇-丙酮溶液(1+1,体积比);将甲醇(4.1)和丙酮(4.2)等体积混合均匀。4.11乙酸乙酯-环已烷溶液(1+1,体积比);将乙酸乙酯(4.3)和环己烷(4.4)等体积混合均匀。4.1280%甲醇溶液(含0.1%磷酸):取80mL甲醇(4.1),加入0.1mL磷酸(4.5),用水稀释至100mL。4.132mol/L尿素甲醇溶液(含5%氨水溶液):称取12g尿素(4.6)水(4.7),用甲醇(4.1)稀释至100mL。2水溶解并稀释至1000mL。纯度均≥90%,各物质信息参见附IV、苏丹橙G和苏丹红7B标准贮备液于100mL容量瓶中，用甲醇-丙酮溶液(4.10)稀释成10pg/mL的混合标准中间液，置于4℃保存。4.23聚酰胺树脂层析柱：在层析柱管底部塞入一薄层脱脂棉，称取1.0g聚酰胺树脂(4.22),用10mL5仪器和设备5.8离心机：转速不低于5000r/min。6试样制备与保存3SN/T3845—20147.2.3聚酰胺树脂层析柱净化将水相待净化液转移至聚酰胺树脂层析柱中，以2mL/min～3mL/min流经层析柱，同时用5mL4表1梯度洗脱程序时间min甲醇%0.01moL/L磷酸盐缓冲液%007.3.2液相色谱测定在仪器最佳工作条件下，对标准工作溶液进样，用标准工作曲线按外标法定量。标准工作溶液和样液中各种合成色素的响应值均应在仪器的线性范围内。在上述色谱条件下，各目标化合物的参考保留时间参见附录B中表B.1。标准溶液的液相色谱图参见附录C中图C.1,8空白试验除不加试样外，均按上述操作步骤进行。9结果计算和表述用色谱数据处理机或按式(1)计算试样中合成色素的含量，计算结果须扣除空白值： (1)x;——试样中待测物质的含量，单位为毫克每千克(mg/kg);c;——从标准曲线上得到的待测物质的溶液浓度，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);m——最终样液所代表的试样质量，单位为克(g)。10测定低限和回收率10.1测定低限本标准对于火锅底料中新红、苋菜红、胭脂红、诱惑红、赤藓红、日落黄、酸性红G、酸性大红GR、罗丹明B、对位红、苏丹红I、苏丹红Ⅱ、苏丹红Ⅲ、苏丹红IV、苏丹橙G和苏丹红7B的测定低限均为mg/kg.回收率本标准的添加水平与回收率范围参见附录D中表D.1。5表A.1标准物质信息化合物英文名称CAS号分子式新红Ci₈H₁₂N₃O₁S₃Na₃苋菜红C₂₀H₁N₂Na₃O₁₀S₃胭脂红诱惑红AlluraRedACCgH₁₄N₂Na₂O₈S₂赤藓红日落黄C₈H₁₃N₃Na₂O₈S₂C₂₂H₄N₄Na₂O₇S₂对位红C₁H₁N₃O₃苏丹红IC₁₆H₁₂N₂O苏丹红Ⅱ苏丹红ⅢC₂₂H₁₆N₄O苏丹红IVC₂₄H₂₀N₄OC₂H₀N₂O₂C₂₄H₂₁N₅6(资料性附录)参考保留时间表B.1十六种合成色素的参考保留时间化合物参考保留时间min化合物参考保留时间min新红苋菜红苏丹红I胭脂红对位红日落黄苏丹红Ⅱ酸性红G苏丹红Ⅲ诱惑红苏丹红IV赤藓红(资料性附录)标准溶液色谱图图C.1十六种合成色素标准溶液的液相色谱图(0.05mg/L8(资料性附录)不同添加水平十六种合成色素回收率数据化合物添加水平mg/kg回收率范围%新红60.5～71.565.7～72.668.4～76.0苋菜红69.7～81.866.4～80.665.5~79.7胭脂红69.5～75.573.6～86.870.5～82.3日落黄81.7～90.591.4～98.991.8～96.5赤藓红81.0～100.192.9～100.278.3～87.2诱惑红91.0～100.190.8～99.584.7～94.6酸性红G80.6～90.686.3～90.282.7～93.681.0～87.180.5～86.780.7～89.072.0～92.081.4～98.078.8～97.19化合物添加水平mg/kg回收率范围%对位红0.0172.0～87.00.0275.9～87.40.0579.4～88.80.0174.1～83.40.0275.9～85.20.0576.5～84.50.0176.7～97.10.0281.7～96.00.0578.0～92.1苏丹红Ⅱ0.0169.9～89.10.0277.5～92.00.0581.7～97.0苏丹红Ⅲ0.0175.0～97.30.0275.5～92.40.0579.5～93.70.0175.0～87.50.0274.1～87.70.0579.5～92.5苏丹红IV0.0169.8～80.70.0269.8～82.10.0582.5～82.4tificationandAccreditationofthePeople'sRepublicofChina.RepublicofChina.ThisstandardwasmainlydraftedbyXiCunxian,TangBobin,ZhangLei,LiXianliang,ZhengXiaoling,DeterminationofmultiplesyntheticdyesinbottommaterialofchafingdishforexportThisstandardspecifiesthemethodsofsamplepreparationandqualifiedandquantifieddeterminationdishforexport.Thisstandardisapplicabletothedeterminationofnewred,carmine,amaranth,allurared,erythrosine,sunsetThefollowingnormativedocumentscntainprovisionwhiprovisionofthispart.Fordaterefeirences,subsequentamgatethepossibilityofapplyingthemostrecenteditionsofthenormativedocumentsindicatedbamide-methanolsolution(contain5%ammoniumhydroxidesolution),respectively,afterliquid-GPC,respectively.TheanalytewasdeterminedbyHPLC,quantifiedSN/T3845—20144.4Cyclohexane.4.7Ammoniumhydroxide:28%4.8Sodiumdihydrogenphosp4.9Disodiumhydrogen4.10Methanol-acetonesolution(1+1,V/V):Equivalentvolumemixtureofmethanol(4.1)andacetone(4.2).4.11Ethylacetate-cyclohexanesolution(1+1,V/V):Equivalentvolumemixtureofethylacetate(4.3)andcyclohexane(4.4)4.1280%methanolsolution(contain0.1%phosphoricacid):Dilute80mLmethanol(4.phosphoricacid(4.5)withwatertothevolumeof100mL4.132mol/LCarbamide-methanolsolution(contain5%ammoniumhydroxidesolution):Dilute12.0gcarbamide(4.6)and5mLammoniumhydroxide(4.7)withmethanol(4.1)tothevolumeof4.1450%Phosphoricacidsolution:Equivalentvolumemixtureofphosphoric(4.5)andwater4.165%Ammoniumhydroxide-methanolsolution:Dilute5mLammoniumhydroxide(4.7)with4.170.01mol/Lphosphatebuffersolution:Accuratelyweight1.41gsodiumdihydrogenphosphateanhydrous(4.8)and1.56disodiumhydrogenphosphateanhydrous(4.9),dissolvedin1000mLwater,andmixtohomogeneity4.19Stockstandardsolutioamaranth,allurared,erythrosine,sunsetyellow,acidredG,acidscarletstockstandardsolutionwas3months.4.21Standardworkingsolution:Accordingtotherequirement,nol-acetonesolution(4.10)justbeforeuse.1.0gpolyamide(4.22)with0.1%Phosphoric4.24Mermbranefilter:0.22μm,Hydrophilicpolyethersu5.2GelpermeationchromatograpSN/T3845—20145.9Chromatographycolumn:1.5cm×10cmAbout500grepresentativfist,thengrindedandblendedbyablendeAccuratelyweigh5.00gofthetestsample(accurateto0.01g)intoa50mLcentrifugetubewithcap,add20mLmethanol-acetonesolution(4.10),heatedin60℃water-bathfor10min,vortexfor2min,thencentrifugeat5000r/minfor5min.Transferthesupernatantto150mLdistillationflask,theresiduewasre-extractedoncewith20mLmethanol-acetonesolution(4.10),andre-extractedwith15mL2mol/LCarbamide-methanolsolution(4.13)fortwice.Aftercentrifuging,combinethesupernatanttothedistillationflask;evaporatetheextractnearlytodrynessonrotaryevaporatorbe-Thedistillationflaskwaswashedwith5mLofethylacetate-cyclohexane(4.11)and5mLofwaterfortwice,respectivly,andthewashingsolutionwastransferredintoa50mLpolypropylenecentrifugetube,vortex,for2minandcentrifuged.Theorganicphasewastransferredtoa10mLcol-orimetrictube,concentratednearlytodrynesswithevaporatorat40℃,dissolvedresiduewith5mLethylacetale-cyclohexane(4.11),cleanupwithGPC.Theaqueousphasewasaddedphosphoricacidsolution(4.14),mixedtocleanupCleanuptheorganicphasesolution(7.2)withGPCaccordingthefollowingconditions:b)Mobilephase:ethylacetate-cyclohexane(4.11).nol(4.12),thendrythecolumnwithair.TheanalytesarCombinedGPCcleanupsolution(7.2.1)and9mLseveraltimes,combinethesolution.Evaporatetheelutesolutionnearlytodryat40flow.Dilutetheresiduetothevolumeof1.0mLofmethanol-acetone(4.10),vortexmixed,filteredthroughhydrophilicpolyethersulfonemembrane,andfinalsolua)LCcolumn:Cg,250mm×4.6(i.d.),5μm,(orotherconformablecolumn),c)Detectionwavelength:programvariablewavelength,0～13.7min,520nm;13.71min~15min.420nm;15.01min～30min,520nm.f)Injectorvolume:10uL.g)Equilibriumtime:5.0min.00Undertheoptimizedinstrumentsworkingcondition,differentconcentrationsolutionsareinjectionintotheinstrumentinseparatsamplesolutionshouldbewithinthelinearrangeoftheinstrumentaldetection.Undertheoptimizedinstrumentsworkingconditiontheret9CalculationandexpressionofresultCalculatingthecontentofanalytesconcentrationaccordingtotheformula(1):c,一theconcentrationofanalytewhichisquantifiedbystandardcalibrationcurV—thefinalvolumeofsamplesolution,mLm—thecorrespondingmassoftestsampleinthefinalsamplesolution,gThelimitofquantificationfornewred,carmine,amaranth,allurared,erythrosine,sunsetyellow,acidAccordingtotheexperimentalCompoundCASNo.MolecularformulaNewRed220658-76-4C₀H₁₂N₃O₁,S₃Na₃915-67-3C₂₀H₁₁N₂Na₃O₀S₃CarmineC₂₂H₂₀O₁₃AlluraRedAC25956-17-6CH₁₄N₂Na₂O₄SErythrosinB568-63-8SunsetYellow2783-94-0C₁₆H₀N₂Na₂O,S₂AcidRed13734-67-6CgH₁₃N₃Na₂OS₂BrilliantCrocein5413-75-2C₂H₁₄N₄Na₂O,S₂RhodamineB81-88-9C₂₈H₃₁CIN₂O₃ParaRed6410-10-2C₁gH₁₁N₃O₃842-07-9C₁H₁₂N₂O3118-97-6CH₁₆N₂O85-86-9C₂H₁₆N₄O85-83-6C₂₄H₂₀N₄OSudanOrangeG2051-85-6C₁₂H₀N₂O₂6368-72-5C₂₄H₂₁N₅(informative)Compound