SN-T3848-2014出口食品中茶多酚的检测方法高效液相色谱法

中华人民共和国出入境检验检疫行业标准出口食品中茶多酚的检测方法高效液相色谱法Determinationmethodofteapolyphenolresiduesinfoodsforexport—HPLCmethod2014-04-09发布国家质量监督检验检疫总局I本标准按照GB/T1.1—2009给出的规则起草。本标准由国家认证认可监督管理委员会提出并归口。本标准由中华人民共和国东莞出入境检验检疫局检验检疫综合技术中心、中华人民共和国北京出入境检验检疫局负责起草。1出口食品中茶多酚的检测方法高效液相色谱法本标准规定了出口食品中茶多酚的高效液相色谱检测方法。2规范性引用文件3方法提要4试剂和材料除另有规定外，所有试剂均为分析纯，水为GB/T6682规定的一级水。4.4表没食子儿茶素没食子酸酯标准品(EGCG);CAS号989-51-5,分子式C₂H₁O,相对分子质量458.37,纯度大于等于95%。4.5表没食子儿茶素标准品(EGC):CAS号970-74-1,分子式C₁sH₄O₇,相对分子质量30于等于95%,4.6表儿茶素没食子酸酯标准品(ECG):CAS号1257-08-5,分子式CsH₁gO₁,相对分子质量442.37,纯度大于等于92%,4.7表儿茶素标准品(EC):CAS号490-46-0,分子式C₃H₄O₆,相对分子质量290.27,纯度大于等于97%。25.4旋涡混合器。6.1试样制备与保存明标记，于0℃~4℃保存。取有代表性样品1kg,将样品按四分法缩至200g,在粉碎机中粉碎，再将混匀样品分装至洁净容取有代表性样品1kg,将样品按四分法缩至200g,所取样品经组织捣碎机捣碎，再将混匀样品分在制样的操作过程中，应防止样品受到污染。称取2g试样(精确到0.001g)于10mL带刻度具塞离心管中，加入甲醇定容至10mL,混匀，涡旋振荡2min,超声20min,在一18℃冰柜中冷冻10min,以6000r/min离心5min,上清液经0.45μm称取2g试样(精确到0.001g)于250mL具塞锥形瓶内，加入50mL正已烷振荡提取，用滤纸过滤提取液(必要时40℃水浴中超声溶解),移入10mL带刻度离心管中，用少量甲醇冲洗圆底烧瓶3次3SN/T3848—2014b)流动相梯度洗脱程序见表1;表1梯度洗脱程序时间/min甲醇/%0.5%甲酸水溶液/%054556.3.2定量测定取待测溶液20μL注入高效液相色谱仪，根据峰面积从标准曲线上查出待测液中茶多酚各组分的的标准色谱图参见附录A中的图A.1。6.3.3定性测定在相同测试条件下，样品中待检测物质与相应的标准品的保留时间应保持一致，偏差在±2.5%之内。二极管阵列检测器所获得的紫外光谱图中，待测物与标准品有相同的最大吸收波长，且两者偏差在6.4空白试验除不加试样外，其余均按上述步骤(6.2～6.3)同时完成试验，7结果计算结果按式(1)计算，计算结果需扣除空白值 (1)式中：X.试样中茶多酚组分的含量，单位为毫克每千克(mg/kg)V样液最终定容体积，单位为毫升(mL)平行测定结果用算术平均值表示，计算结果保留三位有效数字。48定量限(LOQ)和回收率本方法的添加回收率范围参见附录B中表B.1。5茶多酚组分(4种儿茶素)液相色谱图6(资料性附录)食品中茶多酚组分(4种儿茶素)的添加回收率范围茶多酚组分(4种儿茶素)在不同基质中的添加回收率范围(n=10)儿茶素回收率范围/%(添加浓度：10mg/kg～200mg/kg)植物油69.1～98.0方便米面70.1～97.6糕点74.7～99.6油炸食品73.6～95.6肉灌肠73.9～92.0(添加浓度：2mg/kg～20mg/kg)植物油74.7～104.5方便米面73.2～103.2糕点79.4～108.6油炸食品77.3～103.1肉灌肠76.4～96.3(添加浓度：4mg/kg~40mg/kg)植物油76.2～99.6方便米面72.9～107.1糕点74.4～102.0油炸食品73.8～93.3肉灌肠70.3～95.9(添加浓度：2mg/kg～20mg/kg)植物油71.6～98.6方便米面73.5～102.0糕点77.2～96.9油炸食品76.1～100.肉灌肠72.6～105.37ThisstandardwasdraftedinaccordancewiththeruleofGB/T1.1—2009.ThisstandardwasproposedbyandwasunderthechargedofNationalRegulatoryCommissionforCertificationandAccreditation.ThisstandardwasdraftedbyIntegratedTechnologyCentrEntry-ExitInspectionandQuarantineBureauofthePeople'sRepublicofChina,BeijingEntry-ExitIn-spectionandQuarantineBureauofthePeople'sRepublicofChina.ThisstandardwasmainlydraftedbyFuDengzhou,LiXiaolin,LiJu,HuangWei,YangXuejiao,WenJianchang,WangWei,ZhangJiangfeng.8Thisstandardspecifiesthedeterminationmethodofteapolyphenolresiduesinfoodsforexportconvenientrice,friedfoodandmeatenema,itcannotbeappliedtoteaandteaproducts.Thefollowingdocumentsareindispensablefortheapplicationofthisdocument.Fordatedreferences,onlythedatedversioncanbereferredinthisdocument.Thecitateddocumentswithoutdate,latestversioncanbereferredinthisdocument.3Methodsummarywereextractedwithn-hexane,extractcanbefrozenandcentrifugated,HPLCmethedtests,Unlessotherwisespecified,allreagentsaretheanalyticalreagentARandthefirstdegreedwaterre.quiredbyGB/T6682.4.1Acetonitrile;chromatographicgrade.4.2Methanol;chromatographicgrade.4.3Formicacid;chromatographicgrade94.4EGCG:CAS989-51-5,C₂H₁₈O,formulaweight458.37,purityquotient≥95%,4.5EGC:CAS970-74-1,CsH₄O₇,formulaweight306.27,purityquotient≥95%.4.6ECG:CAS1257-08-5,CsHO,formulaweight442.37,purityquotient≥92%.4.7EC:CAS490-46-0,C₁₆H₄O₆,formulaweight290.27,purityquotient≥97%.4.8Teapolyphenolcomponentstandardstocksolution:weighingaccuratelytheamountofEGCG、EGC、ECG、EC(accurateto0.0001g),dissolvedinmethanolandconstantedvolumeto10mL,thenwecangetthestandardstocksolutionofEGCG、EGC、ECG、EC(theconcentrationof0.5mg/mL),savingat0℃~4℃,validingforaperiodoftwoweeks.4.9Teapolyphenolcomponentsofmixedstandardsolution:takingappropriatetheabove-mentionedteapolyphenolcomponentstandardstocksolutionaccordingtotheneed,dilutingcompo-nentsofmixedstandardsolutionindebitaspissitudinebywater,makinginpaceofusing.4.10Microporousfilteringfilm:0.45μm,organicphase.5.1Highperformanceliquidchromatograph:withUVdetector/DADdetector.5.2Balance:accurateto0.0001gand0.01g.5.3Centrifuge:thespecdofnotlessthan6000r/min.5.5Centrifugetube:PTEF,10mLMixingtheoriginalsamplesof1kginthesamplebarrel,andputtingthedividedsampletothecleancontainertoseal,signedassample,savingat0℃~4℃.6.1.2Cakes,convenientriceandnoodle,friedfood0℃~4℃.Takingarepresentativesampleof1kg,pickiingat0℃~4℃.Chromatographyconditionsareasfollowsa)Chromatographycoc)Currentspeed:1.0mL/min;d)Columntemperature:40℃;e)Wavelengthdetection:278nm;f)Samplevolume:20μLtime/minmethanol/%0.5%formicacidaqueoussolution/%05455matogramofstandardscanbereferredfromFigureA.1ofAnnexA.Inthesametestcondition,theretentiontimeoftestmaterialandthecorrespondingstandardsam-plesshouldbeconsistent,deviationin±2.5%,Uitravioletspectraobtainedbydiodearraydetector,theobjecttobemeasuredandthestandardhasthemaximumabsorptionwavelengthofthesame,andthedeviationin±2nmInadditiontoaddnospecimen,theothersarecompletedatthesametimetestaccordingtotheabovesteps(6.2～6.3).ResultsarecalculatedaccordingtoFormula(1),thecalculationresultsneedtobedeductedfromtheblankvalue.X,—consistenceofteapolyphenolinthesample,mg/kg;fthe