SNT 2539-2010動物源食品中乙氧喹啉殘留量的測定方法 高效液相色譜法

ISN/T2539—20101SN/T2539—2010動物源食品中乙氧喹啉殘留量的測定方法高效液相色譜法高效液相色譜-質(zhì)譜確證方法。2方法提要試樣加入碳酸鈉溶液和丙酮振蕩提取后，用正己烷進(jìn)行液-液萃取分配，提取液經(jīng)濃縮定容后，用配熒光檢測器的高效液相色譜儀測定，外標(biāo)法定量，高效液相色譜-串聯(lián)質(zhì)譜儀確證。3.6碳酸鈉溶液(100g/L):準(zhǔn)確稱取100g碳酸鈉(3.4)于燒杯中加入適量水溶解，轉(zhuǎn)移至1L容量3.7乙酸銨溶液(20mmol/L):稱取1.54g乙酸銨(3.5),溶解于1L水中。3.8乙氧喹啉標(biāo)準(zhǔn)物質(zhì)(ethoxyquin,CAS編號：91-53-2,C?HgNO):3.10乙氧喹啉標(biāo)準(zhǔn)工作液：根據(jù)檢測需要移取一定體積的標(biāo)準(zhǔn)中間溶液用乙腈逐級稀釋成適當(dāng)濃度的標(biāo)準(zhǔn)工作溶液。標(biāo)準(zhǔn)工作溶液需現(xiàn)配現(xiàn)用。4儀器和設(shè)備4.1.高效液相色譜儀；配有熒光檢測器(FLD)。4.2高效液相色譜-串聯(lián)質(zhì)譜儀：配有電噴霧離子化源(ESI)。4.5渦旋混勻器。4.7分析天平：感量0.0001g和0.01g。25.1試樣制備5.1.1肉及動物內(nèi)臟5.2試樣保存稱取2g均勻試樣(精確至0.01g)于50mL具塞離心試管中，加入5mL碳酸鈉溶液(3.6)置于渦旋混勻器上混勻2min,加入5mL丙酮(3.1)置于渦旋混勻器上振蕩2min,加入15mL正己烷(3.2)正己烷再重復(fù)提取兩次，合并正己烷層于雞心瓶中，將提取液于30℃水浴下旋轉(zhuǎn)濃縮至約1mL,用平6.2.1高效液相色譜測定條件a)色譜柱：Ca,150mm×4/6mm(內(nèi)徑)/5b)色譜柱溫度：25℃;子乙腈=(7+3)(V/V);6.2.2.1高效液相色譜條件表1高效液相色譜-串聯(lián)質(zhì)譜儀梯度洗脫程序時間/min流速/(mL/min)5mmol/L乙酸銨水溶液/%乙腈/%036936.2.2.2質(zhì)譜條件參見附錄A。6.2.3測定6.2.3.1高效液相色譜測定根據(jù)樣液中乙氧喹啉的含量情況，選定濃度相近的乙氧喹啉標(biāo)準(zhǔn)工作溶液。標(biāo)準(zhǔn)工作溶液和樣液中乙氧喹啉的響應(yīng)值均應(yīng)在儀器檢測的線性范圍內(nèi)。對標(biāo)準(zhǔn)工作液和樣液等體積交替進(jìn)樣測定，以保留時間定性，測量樣液與標(biāo)準(zhǔn)工作液的峰面積比較定量。在上述高效液相色譜條件下，乙氧喹啉標(biāo)準(zhǔn)品色譜圖參見附錄B中圖B.1。6.2.3.2高效液相色譜-串聯(lián)質(zhì)譜確證樣品在高效液相色譜儀測定時，如果檢出色譜峰的保留時間與標(biāo)準(zhǔn)樣品相一致，則需對其進(jìn)行質(zhì)譜確證。用高效液相色譜-串聯(lián)質(zhì)譜進(jìn)行樣品確證時，在扣除背景后的樣品譜圖中，各定性離子的相對豐度與濃度接近的同樣條件下得到的標(biāo)準(zhǔn)溶液譜圖相比，在相同的實(shí)驗(yàn)條件下，樣液中被測物的質(zhì)量色譜峰保留時間與標(biāo)準(zhǔn)工作液相同，所選擇的定性離子均出現(xiàn)，而且所選擇的離子豐度比與標(biāo)準(zhǔn)樣品的離子豐度比相一致(允許偏差范圍見表2),則可判斷樣品中存在這種農(nóng)藥或相關(guān)化學(xué)品。如果不能確證，應(yīng)重新進(jìn)樣，以掃描方式(有足夠靈敏度)或采用增加其他確證離子的方式或用其他靈敏度更高的分析儀器來確證。在上述高效液相色譜-串聯(lián)質(zhì)譜條件下，乙氧喹啉標(biāo)準(zhǔn)溶液的選擇性離子流圖參見附錄C。表2定性確證時相對離子豐度的最大允許偏差相對離子豐度/%20=x50>10～20相對離子豐度最大允許偏差/%6.4結(jié)果計算用色譜工作站或按下式(1b計算試樣中乙氧喹啉的含量。X;——試樣中乙氧喹啉的殘留量，單位為毫克每千克(mg/kg);A——樣液中乙氧喹啉的峰面積；c?——標(biāo)準(zhǔn)工作液中乙氧喹啉的濃度，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)工作液中乙氧喹啉的峰面積；m——最終樣液所代表的試樣量，單位為克(g)。7測定低限和回收率0.01mg/kg。4表3樣品中乙氧喹啉添加濃度和回收率實(shí)驗(yàn)數(shù)據(jù)樣品名稱添加濃度/(mg/kg)樣品名稱添加濃度/(mg/kg)豬肉89.5～108.0蜂蜜89.2～100.384.2～102.488.8～101.286.8~98.284.1～90.5豬肝92.7～109.0雞蛋90.3～101.493.2～109.293.7～107.590.6～98.496.6～101.690.2～98.093.4～99.5豬腎85.8～102.0魚肉94.7～102.191.3～105.687.7～108.092.0～105.692.0～97.790.7～98.685.1～95.7雞肉84.8～101.1牛奶91.4～103.185.8～102.683.9～95.286.1～99.587.4～89.85(資料性附錄)質(zhì)譜條件)電噴霧離子化電離源(ESI),正離子監(jiān)測掃描方式’選擇離子監(jiān)測(SRM)氣簾氣(CUR)霧化氣(GS1)輔助加熱氣(GS2)碰撞氣(CAD)電噴霧電壓(IS)離子源溫度(TEM)表A.2選擇離子監(jiān)測條件待測物m/zm/z去簇電壓DP/V碰撞電壓CE/V碰撞室入室電壓EP/V碰撞室出室電壓CXP/V乙氧喹啉69a定量離子對。1)附錄A所列參數(shù)是在API4006(資料性附錄)乙氧喹啉標(biāo)準(zhǔn)品液相色譜圖78AnnexA,BandCofthisstanThisstandardwasdraftedbyHunanEntry-ExitInspectionandQuarantineBureauofthePeople'sRepublicofChina.FuShanliang,LiYongjun,WangMeilingThisstandardisaprofessionalstandardpromulgatedforthefirsttime9Thisstandardspecifiesthehighperformanceliquidchromatographymethodofdeterminationandconfirmationofethoxyquinresiduesbyinpigmuscle,pigliver,pigkidney,chickenmuscle,fishmuscle,chickenegg,beehoneyandmilk.Thisstandardisapplicableforthedeterminationandconfirmationofdimethipinresiduesbyhighper-formanceliquidchromatographyinpigmuscle,pigliver,pigkidney,chickenmuscle,fishmuscle,chick-.enegg,beehoneyandmilk.Extractthepesticidesresiduesfromthesampleusingwithsodiumcarbonatesolutionandacetone.Theextractwasliquid-liquidpartitionedwithn-hexane.Performdeterminationandconfirmationbyhighperformanceliquidchromatographyequippedwithfluorescencedetection(FLD)andligiudchro-matography-tandemmassspectrometry,usingexternalstandardmethod.Unlessotherwisespecified,thereagentsshouldbeanalyticallypure,"Water"isredistilledwater.3.2n-Hexane:HPLCgrade.3.4Sodiumcarbonate.3.5Ammoniumacetate:HPLCgrade.3.6Sodiumcarbonatesolution(100g/L):Weigh100gsodiumcarbonate,dissolvedin1Lvolumewater.SN/T2539—20103.7Ammoniumacetatesolution(20mmol/L):Weigh1.54gammoniumacetate,dissolvedin1Lvolumewater.3.8Ethoxyquinstandard(C?H?NO,CASNo:91-53-2):Purity≥98%3.9Ethoxyquinstandardstoragesolution:Accuratelyweightanadequateamountofstandards,dis-solveinasmallvolumeofacetonitrile.Dilutewithacetonitriletoformastandardstocksolutionof100μg/mLinconcentration.Thestandardsolutionshouldbestoredbelow0℃~4℃andkeepindarkplace.3.10Ethoxyquinstandardworkingsolution:Weighamountofstandardstoragesolution,thendilutewithacetonitriletotherequiredcohcentrationasthestandardworkingsolution.Thestandardwork-ingsolutionshouldbemadewhileit-is-using.4.1Highperformanceliquidchromatography,equippedwithfluorescencedetection(FLD).4.2Liquidchromotograph-massspectrometry,oquipped.with.electrasprayionization(ESI).4.4Centrifuge:6000r/min.4.6Rotaryvacuumevaporator.4.7Analyticalbalance;0.0001g,0:01g4.8Nitrogenconcentrator.Therepresentativesamplesisreducedtoca500g,Theeatablepotionsarethoroughlygroundandho.SN/T2539—2010mogenizedinameatgrinder.Andthenareplacedinacleancontainerasthetestsample,whichisTherepresentativesamplesisreducedtoca500g,homogenizedinagrinder.Andthenareplacedinacleancontainerasthetestsample,whichissealedandlabeled.5.2StorageoftestsamplesThetestsamplesofbeehoneyshouldbestoredbelow4℃.Thetestsamplesofmilk,freshmeatandmeatproductsshouldbestoredbelow-18℃.Inthecourseofsamplingandsamplepreparation,pre-cautionshouldbetakentoavoidcontaminationoranyfactorswhichmaycausethechangeofresiduecontent.Weighca2g(accurateto0.01g)ofthetestsampleintoa50mLcentrifugetube.Add5mLsodiumcarbonatesolution(3.6),blendfor2mininvortexmxer.Add5.mLacetone(3.1),blendfor2mininvortexmixer.Thenhexane15mLwasaddedandshakenvigorouslyfor2min,centrifugefor3mintrationwasrepeatedtwicemorewith15|mLHexane(3.2),combinetheextracts.Concentratethegenpurge.Dissolvetheresidueanddiluteexactlyto1/0mLwithacetonitrile(3.3).Filtratedthrougha0.45μmmembranefilter,thefiltrateisreadyforHPLCandLC-MS/MSdetermination.6.2Determination6.2.1HPLCconditionsa)Octadecylsilica(ODS)oreqivalentcolumn(4.6×150mm,5μm),ortheequivalent;b)Columntemperature:25℃;c)Mobilephaseandgradientprogram;d)Wavelengh:360nm,435nm;f)Injectionvolume:10μL.SN/T2539—20106.2.2.1HPLCconditionsa)Octadecylsilica(ODS)(150mm×4.6mm.i.d.,5.0μm),ortheequivalent;b)Columntemperature:25℃;c)Flowrate:0.50mL/min;d)Mobilephaseandgradientprogramseenintable1;Table1—MobilephaseandTime/minFlowrate/(mL/min)5mmol/Lmmoniumacetatesolution/%Acetonitril/%0369e)Injectionvolume:5μL.SeeinannexA.Accordingtotheapproximateconcentrationofethoxyquininthetestsamplesolution,selectthestandardworkingsolutionwithsimilarpeakareatothatofsamplesolution.Thestandardworkingso-lutionshouldbeinjectedrandomlyinbetweentheinjectionsofsamplesolutionofequalvolume.Theresponsesofethoxyquinstandardworkingsolutioninandsamplesolutionshouldbeinthelinearrangeoftheinstrumentaldetector.UndertheaboveHPLCoperatingconditions,theHPLCchromatog-raphyofethoxyquinstandardseeinannexBfigureB.1.AccordingtotheHPLC-MS/MSoperatingconditionsdescribedin6.2.2,analyzethestandardsolutionandsamplesolution.Iftheretentiontimeofsamplechromatogrampeaksareconsistentwiththestandard,theconfirmationtestshouldbeconducted.Thetargetanalytecanbeconfirmedwhensub-tractedchromatogramfrombackgroundcompensation,selectedionsareallpresentandtherelativeSN/T2539—2010ionabundanceoftheselectedionsaccordingwiththatofthecalibrationstandardatcomparablecon-centrations,withinthetolerances(seetable2).Iftheconfirmationcannotbemade,repeattheinjec-tion,orchangethequalifierions,orchooseotherconfirmationmethodswithhighersensitivity.Un-dertheaboveHPLC-MS/MSoperatingconditions,theselectedionchromatogramoftheethoxyquinstandardsolutionwereseeinannexCfigureC.1Relativeabundance/%>20～50>10～20Permittedtolerances/%Theoperationoftheblanktestisthesameasthatdescribedinthemethodofdetermination,butwithomissionofsampleaddition.6.4CalculationandexprThecalculationofethoxyquinresiduesinthesampleiscarriedoutbyHPLCandHPLC/MSdatapro-cessororaccordingtothefollowingformula:Where:X,—Ethoxyquincontentinthesample,mg/kg;A—Peakareaofethoxyquininthesamplesolution;c?—Peakareaofethoxyquininthestandardworkingsolution,μg/mL;V—Finalvolumeofsamplesolution,mL;As—Concentrationofethoxyquininthestandardworkingsolution;m—Massoftestsample.g.Note:Theblankvalueshouldbesubtractedfromtheaboveresultofcalculation.Thelimitofdeterminationofethoxyquinresiduesinpigmuscle,pigliver,pigkidney,chickenmuscle,SN/T2539—2010fishmusclechickenegg,beehoneyandmilkbythismethodis0.01mg/kg.Accordingtotheexperimentaldata,thefortifyingconcen