SNT4318-2015出口食品中香豆素、6-甲基香豆素、二氢

中华人民共和国出入境检验检疫行业标准SN/T4318—2015出口食品中香豆素、6-甲基香豆素、二氢香豆素、7-甲氧基香豆素、醋硝香豆素、7-乙氧基-4-甲基香豆素含量的测定液相色谱-质谱/质谱法2016-04-01实施国家质量监督检验检疫总局I本标准按照GB/T1.1—2009、GB/T20001.4—2001和SN/T0001—1995给出的规则起草。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。本标准起草单位：中华人民共和国湖北出入境检验检疫局。1出口食品中香豆素、6-甲基香豆素、二氢香豆素、7-甲氧基香豆素、醋硝香豆素、7-乙氧基-4-甲基香豆素含量的测定液相色谱-质谱/质谱法本标准规定了出口食品中香豆素、6-甲基香豆素、二氢香豆素、7-甲氧基香豆素、醋硝香豆素和7-乙氧基-4-甲基香豆素的液相色谱-质谱/质谱测定力法。本标准适用于出口饼干、饮料(包括含酒精饮料)冰淇淋和口香糖中香豆素、6-甲基香豆素、二氢香2方法提要饼干、饮料(包括含酒精饮料)、冰湛淋样品中的香豆素、6-甲基香豆素、二氢香豆素、7-甲氧基香豆素、醋硝香豆素和7-乙氧基-4-甲基香豆素用无水乙醇提取、□香楠样品单的香豆素、6-甲基香豆素、一氢香豆素、7-甲氧基香豆素、醋硝香豆素和7-乙氧基-4-甲基膏豆索用甲醇和水提取；固相萃取小柱净化后，用液相色谱串联质谱法进行测定，外标法定量。3试剂和材料除另有规定外，所有试剂均为分断纯，水为超结水。3.2无水乙醇。3.4无水硫酸钠。3.5甲醇-水(1+9,体积比):10mL甲醇，用水定容至100mL3.6甲醇-水(1+1,体积比):50mL甲醇，用水定容至100mL。3.70.005mol/L醋酸铵溶液：称取0.3854g醋酸铵，以水溶解定容至1000mL。3.80.1%甲酸的0.005moL/L醋酸铵溶液：取0.1mL甲酸，以0.005moL/L醋酸铵溶液定容3.9香豆素、6-甲基香豆素、二氢香豆素、7-甲氧基香豆素、醋硝香豆素和7-乙氧基-4-甲基香豆素标准物质：纯度大于或等于95%,其他信息参见附录A。3.10标准储备液：准确称取适量标准品(精确至0.0001g),用甲醇溶解配制浓度为1.0mg/mL的标准储备液，保存于一18℃冰箱内。3.11中间标准溶液：移取1.0mL标准储备液于10mL容量瓶中，用甲醇定容，配成100.0pg/mL,保存于4℃冰箱内。3.12基质标准工作液：临用前根据需要，移取适量的中间标准溶液，用空白基质溶液稀释至适当浓度。3.13HLB固相萃取柱(3mL,60mg)或相当者：使用前用5mL甲醇、5mL水淋洗活化。2SN/T4318—20154仪器和设备4.1高效液相色谱-质谱/质谱仪，配有电喷雾离子源(ESI)。4.2振荡器。4.4快速溶剂浓缩仪。4.6涡旋混合器。5试样制备和保存取有代表性样品50g,混合混匀，分成2份，分别装入洁净容器内，密封并标识，将试样存放于4℃在制样过程中，应防止样品受到污染或发生残留物含量的变化。6测定步骤准确称取样品1.00g于50mL离心管内，加入5g无水硫酸钠和10mL无水乙醇，振荡10min,在8000r/min下离心5min,取上层于另一50mL离心管中，残渣再分别加入10mL无水乙醇重复提取2次，合并上清液，45℃快速溶剂浓缩仪减压浓缩近干，用1mL甲醇溶解残渣，再加8mL水涡旋混匀，8000r/min离心5min,上清液用水定容至10mL,待净化。准确称取样品0.5g(精确至0.01g)于15mL离心管中，加入2mL甲醇提取，涡旋提取2min,10000r/min离心5min,上清液转移至另一15mL离心管中，残渣分别用2mL甲醇-水(1+1,体积比)重复提取2次，合并提取液。提取液经氮气吹干浓缩近2mL,加入7mL水，涡旋混匀，8000r/min离心5min,上清液用水定容至25mL,待净化。将上述溶液取5mL过预淋洗好的HLB固相萃取柱，用5mL甲醇-水(1+9,体积比)淋洗柱子。待淋洗液全部流出后，减压抽干3min。用8mL甲醇洗脱，接取全部洗脱液。在40℃下，洗脱液用氮液相色谱条件如下，b)流动相1[适用于饼干、饮料(包括含酒精饮料)、冰淇淋中香豆素类化合物测定]:含0.1%甲酸3SN/T4318—2015的0.005moL/L醋酸铵溶液-乙腈-甲醇(20+10+70,体积比);流动相2(适用于口香糖中香豆素类化合物测定):含0.1%甲酸的0.005mol/L醋酸铵溶液-甲醇-乙腈(48+50+2,体积比);d)进样量：5μL;e)柱温：35℃。6.3.2质谱条件质谱条件如下：a)离子源：电喷雾离子源；b)扫描方式：正离子扫描；c)检测方式：多反应监测(MRM);d)其他参考质谱条件参见附录B。6.3.3定量测定在仪器最佳工作条件下，对基质标准溶液进样。用基质标准工作曲线按外标法定量，基质标准工作溶液和样品溶液中被测物的响应值均应在仪器检测线性响应范围内。根据试样中被测样液的含量情况，选取响应值相近的基质标准工作液进行色谱分析。对基质标准溶液和样液等体积参插进样测定。在上述色谱条件下香豆素标准溶液多反应监测(MRM)色谱图参见附录C。6.3.4定性测定在相同实验条件下，样品中待测物质的保留时间，与基质标准溶液的保留时间偏差在±2.5%之内；每种化合物的质谱定性离子至少应包括一个母离子和两个子离子，且样品中各组分定性离子的相对丰度与浓度接近的基质混合标准工作溶液中对应的定性离子的相对丰度进行比较，偏差不超过表1规定的范围，则可判定为样品中存在对应的待测物。表1定性测定时相对离子丰度的最大允许偏差6.4空白试验除不加试样外，均按上述操作步骤进行。7结果计算和表述用色谱数据处理软件或按式(1)计算试样中被测物含量，计算结果需将空白值扣除：…………X;——试样中被测物含量，单位为微克每千克(μg/kg);A;——样液中被测物的峰面积；c;——标准工作溶液中被测物的浓度，单位为纳克每毫升(ng/mL);4A,——标准工作溶液中被测物的峰面积；m:——最终样液所代表的试样量，单位为克(g)。8.1测定低限素、醋硝香豆素和7-乙氧基-4-甲基香豆素的测定低限均为50.0μg/kg。口香素、二氢香豆素、7-甲氧基香豆表硝香豆素和7乙氧4甲基有豆素的测定低限均为250.0μg/kg。回收率数据参见附录D。5SN/T4318—2015(资料性附录)标准物质基本信息表A.1标准物质基本信息英文名称中文名称分子式相对分子质量结构式coumarin香豆素91-64-5C.H,O₂6-methylcoumarin6-甲基香豆素92-48-8H.Odihydrocoumarin二氢香豆素7-methoxycoumarin7-甲氧基香豆素531-59-9CH,O₃acenocoumarol醋硝香豆素152-72-7C₀H₁₅NO35332OO7-ethoxy-4-methylcoumarin7-乙氧基-4甲基香豆素CTH₂O₃204.226(资料性附录)参考质谱条件!参考质谱条件：a)电喷雾电压(IS):5500V;b)雾化气压力(GS1,kPa):414(氮气);c)气帘气压力(CUR,kPa):172(氮气);d)辅助气流速(GS2,kPa):414(氮气);e)离子源温度(TEM):550℃;f)碰撞电压(CE)、去簇电压(DP)、碰撞室入口电压(EP)和碰撞室出口电压(CXP)见表B.1。表B.1主要参考质谱参数化合物母离子m/z子离子m/z去簇电压V碰撞电压V碰撞室入口电压V碰撞室出口电压V香豆素6-甲基香豆素二氢香豆素7-甲氧基香豆素醋硝香豆素354.3296.1*7-乙氧基-4-甲基香豆素表示定量离子对。7时间/min图C.1流动相1条件下香豆素标准溶液的多反应监测(MRM)色谱图时间/min图C.2流动相2条件下香豆素标准溶液的多反应监测(MRM)色谱图85时间/min9强度/cps图C.7流动相1条件下7-甲氧基香豆素标准溶液的多反应监测(MRM)色谱图时间/min强度/cps强强度/cps强度/cps图C.10流动相2条件下醋硝豆素标准溶液的多反应监测(MRM)色谱图时间/min图C.11流动相1条件下7-乙氧基-4-甲基香豆素标准溶液的多反应监测(MRM)色谱图(资料性附录)回收率数据表D.1不同基质中回收率数据基质化合物添加浓度回收率%相对标准偏差饼干香豆素70.6,85.0,79.5,83.1,6-甲基香豆素102.6,94.3,102.6,10二氢香豆素88.4,91.4,80.5,109.8,7-甲氧基香豆素82.8,101.0,108.6,87.0,99.7,94.1醋硝香豆素99.0,85.2,107.2,80.8,105.7,104.5,87.2,84.57-乙氧基-4-甲基香豆素101.4,107.5,92.6,,82.4饮料香豆素81.2,80.5,86.4,109.7,16-甲基香豆素89.6,81.1,108.5,71.2,799.8,108.6,82.4,83.3,二氢香豆素80.6,84.8,72.3,109.1,186.8,85.1,109.5,107.4,1095.8,105.4,106.7,81.8,1087-甲氧基香豆素88.2,92.4,108.3,108.0,80基质化合物添加浓度回收率%相对标准偏差饮料7-甲氧基香豆素醋硝香豆素90.0,91.5,106.6,80.1,7-乙氧基-4-甲基香豆素85.6,94.1,93.4,71.2,887.2,82.5,107.4,105.2,冰淇淋香豆素88.2,73.4,99.5,73.0,99.5,92.3,83.8,85.8,16-甲基香豆素97.4,78.8,85.4,95.5,82.6,109.2,88.2,100.4,95.0,87.7,103.6,83.1,108二氢香豆素94.3,74.4,80.6,106.8,7-甲氧基香豆素80.8,,109.8,109醋硝香豆素81.4,103.5,86.6,81.1,181.1,104.0,94.4,94.7,17-乙氧基-4-甲基香豆素93.6,95.6,105.3,81.5,182.6,108.0,107.3,91.5,10含酒精饮料香豆素98.7,98.5,85.1,109,7,6-甲基香豆素80.0,109.6,102.8,109.8,992.1,84.4,101.6,107.3,96.6,87.2,95.4,90.1,二氢香豆素表D.1(续)基质化合物添加浓度回收率%相对标准偏差含酒精饮料二氢香豆素7-甲氧基香豆素92.4,102.8,80.1,105.2,95醋硝香豆素80.6,107.7,89.3,109.2,1085.8,89.8,93.8,101.4,7-乙氧基-4-甲基香豆素83.4,96.0,90.3,87.5,8口香糖香豆素88.5,106.5,100.3,80.4,82.3,84.4,100.5,104.6,6-甲基香豆素83.6,87.0,84.6,100.1,10584.2,92.1,93.3,94.0,二氢香豆素98.3,94.8,108.6,88.3,195.5,98.8,92.0,81.5,1090.5,102.3,91.9,102.0,1067-甲氧基香豆素84.8,103.3,108.0,82.7,81.3,88.4,80.2,96.6,10醋硝香豆素85.0,108.3,96.2,83.4,195.1,88.7,109.8,96.3,87-乙氧基-4-甲基香豆素95.5,90.2,106.5,83.3,187.6,87.5,101.0,91.4,9SN/T4318—2015ThisstandardwasdraftedaccordingtoGB/T1.1—2009,GB/T20001.4—2001andSN/T0001-1995.Pleasenotethatsomeoftheelementsofthisdocumentmaybeinvolvedinpatent,issuedthedocu-mentdoesnotassumetheresponsibilityidentificationofthesepatents.ThisstandardwasproposedbyandwasundertheohergeofNationaltificationandAccreditation.ThestandardwasdraftedbyHuRepublicofChina.ThisstandardwasmainlydraftedoyZhaoXiaoye.FuXiaofang.ZhangJianfeng,WangPeng,LiJing,YeCheng,ShangYinzhu,HuXiaozhong.SN/T4318—2015Determinationofcoumarin,6-methylcoumarin,dihydrocoumarin,7-methoxycoumarin,acenocoumarol,7-ethoxy-4-methylcoumarinresiduesThisstandardspecifiesthemethodofdeterminationofccumarn.6metiyicoumarin.dihydrocoumarin,7-methoxycoumarin,acenocoumarol,7-ethoxy-4-methylcoumarinresiduesinfoodforexport—LC/MS/MSmeth-Thisstandardisapplicabletothequafiutveandqualtatvetetemnatanofcoumarin.6methylcoumarin,dihydrocoumarin,7-methoxycoumarinacenocoumarol,7-ethoxy-4-methyicoumarinresiduesinbiscuits,drinkandgumforexport.Thecontentsofcoumarin.6-metfylcoumarin,dinydrocoumarin7-methoxycoumarin,acenocoumarol,7-ethoxy-4-methylcoumarininbiscuits,drink(ineludingalcoholicdrink)oricecreamisextractedwithabsolutealcohol.Thecontentsofcoumarin.6-methylcoumarin,dihydrocoumarin,7-methoxycoumarin,acenocoumarol,7-ethoxy-4-methyicoumanningumisextraotedwithmethnolandwater.ThereafterbeingcleanedupbytheHLBSPEcolumn,thecontentsaredeterminedbyLC/MS/MS,quantifiedbyexternalstandardmethod.Unlessspecified,allreagentsusedshouldbeofanalyticalgrade;"water"isultrapurewater.3.1Methanol:Chromatographypure.3.2Absolutealcohol.3.3Acetonitrile:Chromatographypure.3.4Anhydroussodiumsulfate.SN/T4318—20153.5Methnol-water(1+9,V/V):pipette10mLmethnolto100mLmarkanddilutewithmethanol.3.6Methnol-water(1+1,V/V):pipette50mLmethnolto100mLmarkanddilutewithmethanol.3.70.005mol/Lammoniumacetatesolution:Weighamount0.3854gammoniumacetate,dissolved3.80.1%formicacid0.005mol/Lammoniumacetatesolution:pipette0.1mLformicaciddiluteto100mLwith0.005mol/Lammoniumacetatesolution.3.9Coumarin,6-methylcoumarin,dihydrocoumarin,7-methoxycoumarin,acenocoumarol,7-ethoxy-4-meth-ylcoumarinstandardch3.10Standardstocksolution:Accuratelyweighappropriatestandard,dissolveandquantitativelywithmethnol.Theconcentrationofthesolutionis1.0mg/mL.Thestocksolutionshouldbestoredat-18℃refrigeratory.3.11Standardworkingsolution:dilutestandardstocksolutionwithmethanoltoobtain100.0μg/mLconcentrationofthestandardsolution.Thestocksolutionshouldbestoredat4℃refrigeratory.3.12Matrixstandardworkingsolution:accordingtotherequirement,accuratelymeasurestandardworkingsolutiontoa10mLvolumetricflask,dilutewithblanksampleextract.3.13HLBSPECartridge:60mg,3mL.orequivalent.ConditionHLBSPEand5mLwaterbeforeusing.3.14Membrane:organic,0.22μm.4.1Liquidchromatographycoupledwithelectrosprayionizationtriplequadrupolemassspectrome-try.4.3Centrifuge:8000r/min.4.4Sampleconcentrator.4.5Analyticalbalance:0.1mgand0.01g.SN/T4318—2015Takeapproximately50gofrepresentativesample,mixthoroughly,dividedinto2portionsandplacedintoacleancontainer,sealedandlabeled.Thesampleshouldbestored4℃.Inthecourseofsamplingandsamplepreparation,precautionsmustbetakentoavoidthecontamina-tionoranyfactorswhichmaycausethechangeofresiduecontent.6.1.1Biscuits,drink(includingalcoholicdrink)aAccuratelyweight1gofthetestsample(accurateto0.01g)intoa50mLcentrifugetube,add5gan.8000r/min.Thesupernatantistakenintoother50mLcentrifugetube.Themixturewasexttwicewith10mLabsolutealcohol.Combinethesupernatantintothesamecentrifugetube.Theresidue,add8mLwaterandmixthem,thencentrisupernatantto10mLmarkwithwater.Bereadyforcleanup.Accuratelyweight0.5gofthetestsample(accurateto0.01g)intoa15mLcentrifugetube,add2mLamethnol,intenselymother15mLcentrifugetube.Themixturewasextractedtwicewith2mLmethnol-water(1+1,V/V).Combinethesupernatantintothesamecentrifugetube.Theextractsolutionisevaporatedtonearpernatantto25mLmarkwithwater.BereadyforcleanuDrawtheabovesolution5mLthroughaper-conditionedHLBSP5mLmethnol-water(then2.5mLmethanolisaddedtoreconstitutetheresidue.AfterbeingreadyforanalysisbyLC-MS/MS.SN/T4318—20156.3.1LCoperatingconditionsLCoperatingconditionswereasfollows:a)LCcolumn:C₁g(3.5μm,2.1mmi.d.×150mm);orequivalent.b)Mobilephase1(biscuits,drink,alcoholicdrinkandicecream):0.1%formicacid0.005mol/Lam-moniumacetatesolution-acetonitrile-methnol(20+10+70,V/V/V).Mobilephase2(gum):0.1%for-c)Flowrate:0.2mL/min.d)Injectionvolume:5μL.e)Columntemperature:35℃.MS/MSoperatingconditionswereasfolows!a)lonsource:ESI.b)Scanningmodel:positiveion.c)Monitoringmodel:Multiplereactionmonitor(MFM).d)ReferencedconditionsseeAnnexBrAccordingtotheapproximateconcentrationoftargetcompoundinsamplesolution,selectthematrixstandardworkingsolutionwithsimilarresponsestothatofsamplesolution.Theresponsesoftargetcompoundinthematrixstandardworkingsolutionandthesamplesolutionshouldbewithinthelinearrangeoftheinstrumentdetection.Reconstituted(MRM)chromatogramofstandardworkingsolutionUndertheLC-MS/MSoperatingconditions,thematrixstandardworkingsolutionandsamplesolutionisinjected.Iftheretentiontimesofsamplechromatogrampecksareconsistentwiththatofstandardsolution,calibrationcurvemethodisusedforquantitativemeasurement.Therelativeintensitiesofsampletransiyionsshallcorrespondtothoseofstandardsolutiontransitionsforconfirmation.Theconcentrationofstandardsolutionshouldbesamewiththoseofsamplesolution.Thepermittedtol-SN/T4318—2015relativeintensity/%permittedtolerances/%Theoperationoftheblanktestisthesameasthatdescribedinthemethodofdetermination,butwithomissionofsampleaddition,CalculatethecontentofmethiocarbresidueinthetestsamplebyLC-MS/MSdataprocessororac-cordingcordingtothefollowedformula(1), (1)Where:X,—theresiduecontentofinthetestsample,ug/kg.A,—thepeakareaoftargetcompoundinthesarplesolution.c,-theconcentrationoftargetcompoundinfhematrigitandardworkingsolution.ng/mL.V—thefinalvolumeofthesamplesolution,mL.A。-thepeakareaoftargetcompoundinthematrixstangardworkngsolution.m,—thecorrespondingmassofthetestsampleiithefiralsamglesolution,g.Thelimitofdetermination(LOQ)forcoumarin.6-methylcoumarin,dihydrocoumarin,7-methoxycouma-rin,acenocoumarol,7-ethoxy-4-methylcoumarininbiscuits,drink,alcoholicdrinkandicecreamis50.0μg/kg.Thelimitofdetermination(LOQ)forcoumarin.6-methylcoumarin,dihydrocoumarin,7-me-thoxycoumarin,acenocoumarol,7-ethoxy-4-methylcoumariningumis250.0μg/kg.SN/T4318—2015TableA.1—TheinformationofthestandardMolecularFormulaMolecularWeight7-ethoxy-4-methylcou-AnnexBAPI4000QLC/MS/MSreferencedconditions"Referencedconditions:c)CUR:172kPa.d)GS2:414kPa.e)TEM:550℃f)Precursorion,Production,Collisionenergy(CE),Declusteringpotential(DP),Entrancepotential(EP)andCollisioncellexitpotential(CXP)seeTableB.1.CompoundPrecursorion[M+H]'m/zProductionm/zDPVVEPVCXPVcoumarin6-methylcoumarindihydrocoumarin7-methoxycoumarinacenocoumarol354.3296.1*7-ethoxy-4-methylcoumarinAnnotation:thesymbol"*"representsthequantitativetransition.1)Non-commercialstatement:theequipmentsandtheirtypeinvolvedinthestandardmethodarenotrelatedtocommercialaims,anditisencouragedtouseequipmentsofdifferentcorporationordifferenttype.SN/TAnnexC(Informative)MRMchromatogramsofthestandard5Time/min5FigC.2MRMchromatogramsofcoumarinwithmobilephase2SN/TTime/minTime/minFigC.6MRMchromatogramsofdihydTime/minTime/minFigC.7MRMchromatogramsof7-methoxycoumarinwithmoTime/minTime/minTime/minTime/minSN/TSN/T4318—2015%%83.8,75.2,98.0,72.5,189.7,107.6,82.4,102.5,8188.4,91.4,80.5,109.8,82.8,101.0,108.6,87.0,90.3,81.1,106.2,89.0,899.0,85.2,107.2,80.8,81.1,109.3,84.0,83.8,194.2,92.8,105.2,95.7,