SN-T3843-2014出口食品中红曲色素的测定

中华人民共和国出入境检验检疫行业标准出口食品中红曲色素的测定2014-01-13发布国家质量监督检验检疫总局I本标准按照GB/T1.1—2009给出的规则起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出口食品中红曲色素的测定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实验方法——标准物质：红曲红胺(Fmoc-8-AOC-OH,CASNO.126631-93-4,C₂₃H,NO₄)、红曲红素(monascusred,CASN0.874807-57-5,C₂aHzO₅)、红曲素(monascin,CASNO.21516-68-7,C₂H₂₆O₅)、红曲黄素(ankaflavin,CASNO.50980-32-0,C₂₃H₃₀O₅)纯度均大于或等于97%;1mL甲酸溶解于1000mL水中。23.2.4甲醇+0.1%甲酸水溶液(1+1,体积比)的配制500mL甲醇与500mL0.1%甲酸水溶液混合。使用前配制。准确称取适量的标准品，用甲醇配制成1.0mg/mL的标准贮备液，在0℃~4℃冰箱中保存。根据需要用甲醇+0.1%甲酸水溶液稀释配制适当浓度的标准工作液，现用现配， ——超声波发生器； 离心机：最大转速4000r/min以上 本方法所用的器具如下 3.4样品制备将所取原始样品1kg在瓷混样桶内充分混匀，气泡酒先超声脱气，再将混匀样品分装置洁净容器取有代表性样品1kg,将样品按四分法缩至200g,在玻璃研钵中研碎，再将混匀样品分装置洁净取有代表性样品1kg,将样品按四分法缩至500g,所取样品经组织捣碎机捣碎，再将混匀样品分3试样于0℃~4℃保存。在抽样及制样的操作过程中，应防止样品受到污染或发生含量的变化。称取试样约5g(精确至0.01g)置于50mL离心管中，加入30mL水，混匀后，于超声波清洗器中超声20min后，于4000r/min离心5min,过滤并用水定容至50mL容量瓶中，待净化。取10mL3.5.1中的待净化液，使该样液以小于1.0mL/min的流速通过C₈固相萃取柱。样液全部流出后，用5mL水淋洗，取10mL甲醇洗脱。整个固相萃取过程流速不超过1mL/min。洗脱液于45℃下用氮气吹干，残留物(相当于1g样品)用1mL流动相定容，涡旋混合1min,过微孔滤膜后，供时间/min流速/(mL/min)224根据试样中被测样液中被测物的含量情况，选取响应值相近的标准工作液进行色谱分析。标准工作液和样液中待测物的响应值均应在仪器线性响应范围内。在上述色谱条件下红曲红胺、红曲红素、红曲素、红曲黄素的参考保留时间见表2,红曲红胺、红曲红素、红曲素、红曲黄素标准品多反应监测色谱图参见附录B中图B.1,外标法定量。表2参考保留时间被测物名称保留时间/min红曲红胺红曲红素8.90红曲素红曲黄素在相同试验条件下，待测样品溶液中被测物的保留时间与标准工作溶液中被测物的保留时间的比值，偏差在±2.5%之内。根据定性选择离子对的种类及其相对丰度比对其进行阳性确证。定性时应当与浓度相当标准溶液的相对丰度一致，相对丰度允许偏差不超过表3规定的范围，则可判定样品中存在对应的被测物。表3定性确证时相对离子丰度的最大允许偏差相对离子丰度>20%～50%>10%～20%允许的相对偏差除不加试样外，均按上述步骤进行。3.6结果计算和表述用色谱数据处理机或用标准曲线按式(1)计算试样中红曲红胺、红曲红素、红曲素、红曲黄素残留式中：X;——试样中被测组分残留量，单位为毫克每千克(mg/kg);c;——从标准曲线读出的被测组分溶液浓度，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——样液所代表的最终试样的质量，单位为克(g)。5本方法的测定低限均为1.0mg/kg。回收率的实验数据(在不同添加浓度范围内)参见附录C中表C.1。4高效液相色谱法 标准物质；红曲红胺(Fmoc-8-AOC-OH,CASNO.126631-93-4,CaH,NO₄)、红曲红素(monascusred,CASN0.874807-57-5,C₃H₂₆O₅)、红曲素(monascin,CASNO.21516-68-7,C₂₁H₂₆O₅)、红曲黄素(ankaflavin,CASNO.50980-32-0,C₂₃H₃₀Os)纯度均大于或等 1mL甲酸溶解于1000mL水中，4.2.4甲醇+0.1%甲酸水溶液(1+1,体积比)的配制500mL甲醇与500mL0.1%甲酸水溶液混合。使用前配制。准确称取适量的标准品，用甲醇配制成1.0mg/mL的标准贮备液，在0℃~4℃冰箱中保存。根据需要用甲醇+0.1%甲酸水溶液稀释配制适当浓度的标准工作液，现用现配。6 将所取原始样品1kg在瓷混样桶内充分混匀，气泡酒先超声脱气，再将混匀样品分装置洁净容器取有代表性样品1kg,将样品按四分法缩至200g,在玻璃研钵中研碎，再将混匀样品分装置洁净取有代表性样品1kg,将样品按四分法缩至500g,所取样品经组织捣碎机捣碎，再将混匀样品分4.5测定步骤称取试样约5g(精确至0.01g)置于50mL离心管中，加入30mL水，混匀后，于超声波清洗器中超声20min后，于4000r/min离心5min,过滤并用水定容至50mL容量瓶中，待净化。7SN/T3843—2014准确移取5mL试样于50mL取10mL4.5.1中的待净化液，使该样液以小于1.0mL/min的流速通过C₈固相萃取柱。样液全45℃下用氮气吹干，残留物(相当于1g样品)用1mL流动相定容，涡旋混合1min,过微孔滤膜后，供时间/min流速/(mL/min)流动相流动相/%2230.1的响应值均应在仪器检测的线性范围内。标准溶液和样液等体积参差进样测定。以峰面积为纵坐标标准溶液对应的浓度为横坐标，绘制标准工作曲线。用保留时间定性，外标法定量。在上述色谱条件 (2)8本方法的测定低限均为5.0mg/kg。回收率的实验数据(在不同添加浓度范围内)参见附录C中表C.2。9a)气帘气(CUR):18Psi;b)雾化气(GS1):25Psi;c)辅助气(GS2):45Psi;d)电喷雾电压(IS):5500.00V;e)碰撞气(CAD):7.0Psi;f)离子源温度(TEM):500℃;名称定量离子对定性离子对碰撞气能量去簇电压V红曲红胺382.2>179.0382.2>179.0382.2>160.0红曲红素383.2>365.1383.2>365.1383.2>224.3红曲素359.2>215.0359.2>215.0359.2>261.0红曲黄素387.2>261.2387.2>261.2387.2>215.2标准品的色谱图XICof+MRM(21pairs).382.2/179.0DaID:HQHAfromSample2(1.0)ofC+DTD1102.wittTurboSpray)Max.1.6e6cps382.2>179.0XICof+MRM(21pairs):359.2/215.0DaIDHQSfromSample2(1.0)ofC+DTD1102.wift(TurboSpray)Max.1.3e6eps.00e6-359.2>215,(一XIC000of+MRM(2lpairs):387.2/261.2Da7.81ID:HQHVANG-10fromSample21.0)ofC+DTD1102.wifftTurboSpray)Max1520.0eps387.2>261.2.8.3611.2113.5114.14、33.364d00:3.552.56.2.87.4..897.23Time,minXICof+MRM(21pairs):383.2/365.0DaIDXHQHVNG-10fromSample2(1.0)ofC+DTD1102.wiffTurboSpray)9.6015.0017,317.7119.5120.3422.6223.0724.1025.4227.27Max.1380.0eps29.2330.2330.g33.28,33.651.3802.503.394.685,3529.78.92.9.20383.2>365.84_10.7212.5314.1me,min(资料性附录)红曲色素添加水平/(mg/kg)配制酒冰淇淋饼干火腿肠腐乳红曲红胺90.0～91.089.0～97.089.0～99.089.0～96.088.0～100.087.0～101.687.2～100.689.5～99.690.2~98.488.7～98.491.3～98.790.3～97.490.3～97.491.3～97.492.4～97.4红曲红素90.0～97.090.0～97.089.0～95.088.0～97.090.0～95.091.2～99.692.8～98.289.6～98.490.2～97.891.8--99.291.7～97.392.7～98.891.8～98.891.8～98.890.0～96.0红曲素90.0～97.090.0～97.088.0～99.088.0～97.090.0～95.087.0～99.687.2～99.489.8～98.090.2～98.491.8～98.492.4～96.892.3～96.490.4～97.390.4～97.790.4～97.8红曲黄素89.0～98.090.0～96.089.0～96.088.0～97.090.0～96.091.2～99.693.2～98.492.6～99.290.2~97.891.8～98.892.4～98.892.8～96.892.4～96.892.9～96.892.0～96.1红曲色素添加水平回收率范围/%配制洒冰淇淋饼干火腿肠腐乳红曲红胺红曲素Thisstandardwasdraftedaccotakeontheresponsibilitytoidentifythesepatents.sstandardwasproporCertificationandAccreditation.publicofChina.Themaindraftersofthisstandardare:Mashumin,Ronghui,Zhangxun,Mujun,Zhangdaihuchuan,Hutingting,KangmingqinNote:ThisEnglishversion,atranslationfromtheChinesetext,iThisstandardisapplicabletothedeterminationandconfirmatred,monascin,ankaflavinresidueinpreparationwine,icecream,biscuits,sausage,fermentedbeancurdforexport.umentsdated,whichisdoesn'tdatedreferencedocuments,thelatestversion(includesallofamendments)apdocument.Liquidsamplewaterulcation,sampleliquiduponelutionvolume,forliquidchromatograspectrometrydeterminatio—FormicAcid:HPLCgrade;—Standardmaterial:monascorubramine(Fmoc-8-AOC-OH,CASNO.126631-93-4,CaH,NO4),Accordingtoneedto—Centrifuge:4000r/min;—Centrifugaltube:50firstultrasounddegassing,againwillblendingsamplepointainersealing,assamples,andindicatethemark.process,shouldpreventsamplescontaminatedortheoccurrenceofthechangmatographyweredetermined.a)LCcolumn:ZorbaxCarbohydrateb)Columntemperatured)Mobilephase:A:water,B:acetonitrile,gradientelutionseetable1;f)lonsource:electricsprayionsource(ESI)g)Scanningmode:positiveionsscanningh)Detectingway:morereactionmonitoring(MRM)I)OthermassspectrumparametersseTimeminVelocitymL/minMobilephaseA%MobilephaseB%22Accordingtothesamplewasmeasuredthesampleliquidanalyteincontentssimilarstandardworkingliquidchromatographicanalysis.Workingstandardsolutionandsamplesolu-tionanalyteresponsevalueshouconditionsofmonascorubramine,monascusred,monascin,ankafiavinreferenceretentiontimeisshownintable2,standardmultiplereactionchromatogramofappendixBinstandardmethodforquantitative.ObjectnameRetentiontime/minmonascorubraminemonascusred8.90monascinankafiavinAtthesameexperimentalstatus,whenthedeviationbetwecntheretentiontimeofthetestsampleandthetimeofthestandardworkingsolutioniswithin±2.5%,andtheallowancebetweentherelativeabundanceofthequalificationionsofthecomponentsandtherelativeabundanceofthestandardworkingsolutionisnotovertherangedescribedintable3,ajudgmentthatthepesticidalresidueex-istsinthesampleisconfirmed.Table3—MaximumpermittedtolerancesforrelativeionintensitieswhileconfirmationRelativeionicabundanceAllowedrelativedeviationInadditiontonotaddsamples,allaccordingtotheabovestepsCalculatethecontentofmonascorubramine,monascusred,monascin,ankaflavinresidueinthetestsamplebydataprocessororaccordingtothefollowedformula(1)…………(1)X,—Theresiduecontentofanalyteinthetesc;—Readfromstandardcurvemeasuredcomponentssolutionconcentration,unitismicrogrampermilliliter(μg/mL);V—Thefinalvolumeofthesamplesolution,unitismilliliter(mL);m—Massofthetestsample,unitisTherecoveryofexperimentaldata(indifferentaddingconcentrationrange)inAppendixCtableC.1.Liquidsamplewaterultrasonicextraction,centrifugationanddeterminedcapacity,solidandsemisolidsamplesusingwatersolubilityconstantvolume,extractionandsolidphaseextractioncolumnpurifi-cation,sampleliquiduponelutionvolume,forliquidchromatography,quantifiedbyexternalstandardAllthereagentsusedshouldbeofHPLC.gradeunlessspecified,"water"isthefirs—Methanol:HPLCgrade;—FormicAcid:HPLCgrade;—Standardmaterial:monascorubramine(Fmoc-8-AOC-OH,CASNO.126631-93-4,C₂₃H₂,NO₄)monascusred(CASN0.874807-57-5,Cz₃H—Solidphaseextractioncolumn:using:Ca,1.0g,6mL,orequivalent.Beforeusing5mLinmethanol,—TFMicro-porefilterfilm:0.20μm.4.2.3Preparationof0.1%formicacidDissolve1.0mLformicacid1000mLwat4.2.4Preparationofmethanol+0.1%formicacidsolution(1+1,thevolumeratio)Accuratelyweighappropriateamountofstandards,withmethanolmadeinto1.0mg/mLstandardstocksolution,at0℃~4℃keptintherefrigeratorAccordingtoneedtousethemethanol+0.1%formicacidsolutiondilutionoftheconcentrationofthestandardworkproperlypr4.3Apparatusandequipment—Highlyeffectiveliquidphasecolorspectrometer:equippedwithphotodiodearraydetector(DAD)—Ultrasonicgenerator;—Centrifuge:4000r/min;—Vortexmixer;一Centrifugaltube:50mL,polypropylene,aplug;—Pipette:1000μL,100μL.firstultrasounddegassing,againwillblendingsamplepointsdevicecleancontainersealing,asasaple,indicatethemark.researchingrindbroken,thenblendingsamplepointsdeviceclTake1kgrepresentatitakenfromsamplebyorganizationbeattainersealing,assamples,andindicatethemarkprocess,shouldpreventsamplescontaminatedortheoccurrenceofthechangeofthecontent.inultrasoniccleaningimplementinultrasonic20min,in4000r/mincentrifugal5min,filterandwaterthroughtheCigsolidphaseextractioncolumn.Liquidsampleafterallout,with5mLwatercolumnmobilephasethecapacity,vortexmixed1min,amicroporousmembranefilter,forliquidchromaHPLCconditionsusedinthismethodareasfolb)Columntemperature:30℃;d)Mobilephase:A:water,B;acetonitrile,gradientelutionseetable4;f)Detectedwavelength:230nmTimeminVelocitymL/minMobilephaseA%MobilephaseB%22234.5.3.2ChromatographicAccordingtothesampleinliquid,monascuspigmentconcentration,selectedpeakareaofasimilarueofallshouldintheinstrumentoflinearrangedetection.Standardsolutionandsamplessuchasfluidofsamplesizevariesdetermination.Topeakareafory-coordinate,standardsolutionofthecor-respondingtotheconcentrationoftheabscissadenotes,drawstandardworkcurve.Keeptimewithqualitative,quantitativeexternalstandardmethod.Intheabovechromatographicconditions,monascuspigmentofretentiontimeformonascorubramine6.81min,monascin11.71min,chromatoInadditiontonotaddsamples,allaccordingtotheabovestepsCalculatethecontentofmonascorubramine,monascin,residueinthetestsamplebydataprocessororaccordingtothefollowedformula(2)X,—Theresiduecontentofanalyteinthetestsample,unitismic₁—Readfromstandardcurvemeasuredcomponentssolutionconcentration,unitismicrogrampermilliliter(μg/mL);V—Thefinalvolumeofthesamplesolution,unitismilliliter(mL);m—Massofthetestsample,unitisgram4.7Detectionlimi4.7.1LimitofdeterminationThelimitofdeterminationofthemethodis5.0mg/kg.Therecoveryofexperimentaldata(indifferentaddingconcentrationrange)inAppendixCtableC.2(Informative)b)Nebulizer(GS1):25Psi;c)Auxiliarygas(GS2):45Psi;d)lonsprayvoltage(IS):5500.00Ve)Collisiongas(CAD):7.0Psif)Probetemperature(TEM):500℃;g)SeetableA.1forquantitativeion,qualitativeion,declusteringpotentialvoltage,collisiongasenergyDeclusteringVmonascorubraminemonascin1)Non-commercialstatement:thereferenceparamatersinAnnexAisaccomplishedbyAP14000LC/MS/MS,theequipmentanditsmodelinvolvedinthestandardmethodareonlyforreferenceandnotrelatedtoanycommer.cialaim,andtheanalystsareencoueagedtouseequipmentsofdifferentcorporationordifferentmodel.AnnexBXICof+MRM(21pairs):382.2/179.0DaID:HQHAfromSample2(1.0)ofC+DTD1102.wifl(TurboSpray)Max1.6e6eps3.890.0l1me,minXICof+MRM(21pairs):359.2/2