SNT 2440-2010進(jìn)出口蜂王漿中咖啡因含量的測(cè)定方法液相色譜-串聯(lián)質(zhì)譜法

中華人民共和國(guó)出入境檢驗(yàn)檢疫行業(yè)標(biāo)準(zhǔn)進(jìn)出口蜂王漿中咖啡因含量的測(cè)定方法液相色譜-串聯(lián)質(zhì)譜法Determinationofcaffeineinroyaljellyforimportandexport—LC-MS/MS2010-01-10發(fā)布I本標(biāo)準(zhǔn)由國(guó)家認(rèn)證認(rèn)可監(jiān)督管理委員會(huì)提出并歸口。1進(jìn)出口蜂王漿中咖啡因含量的測(cè)定方法液相色譜-串聯(lián)質(zhì)譜法草酸鉀。3.920%甲醇水溶液：取20ml甲醇用水定容到100pL。3.1060%甲醇水溶液：取60mL甲醇用水定容到100mL。3.110.1%甲酸溶液：準(zhǔn)確取1|mL甲酸用水定容到1000mL。3.12咖啡因(caffeine,C?H?N?O?,CAS編號(hào)5743-12-4,相對(duì)分子質(zhì)量194.19):純度大于等于3.14標(biāo)準(zhǔn)工作溶液：臨用時(shí)用60%甲醇水溶液將標(biāo)準(zhǔn)儲(chǔ)備溶液稀釋成含咖啡因5μg/L、10μg/L、3.15固相萃取柱：C固相萃取小柱(250mg,3mL)或相當(dāng)者。使用前用5mL甲醇和5mL水預(yù)洗。4.1高效液相色譜-四極桿串聯(lián)質(zhì)譜聯(lián)用儀：配電噴霧(ESI)離子源。26.2測(cè)定6.2.1液相色譜-四極桿串聯(lián)質(zhì)譜條件表1流動(dòng)相和洗脫程序時(shí)間/min乙腈/%線性范圍內(nèi)。校正溶液及樣液等體積進(jìn)樣測(cè)定，在上述色譜條件下，咖啡因的保留時(shí)間分別約為表2定性確證時(shí)相對(duì)離子豐度的最大允許誤差士25SN/T2440—20106.3空白試驗(yàn)除不加試樣外，均按上述測(cè)定步驟進(jìn)行。6.4結(jié)果計(jì)算和表述按照式(1)計(jì)算樣品中咖啡因含量，計(jì)算結(jié)果需扣除空白。 (1)式中：X——試樣中咖啡因的含量，單位為微克每千克(μg/kg);A——樣液中咖啡因的峰面積或峰高；c?——標(biāo)準(zhǔn)工作曲線液上查得的標(biāo)液中咖啡因的濃度，單位為納克每毫升(ng/mL);V—樣液最終定容體積，單位為毫升(mL);A?——標(biāo)準(zhǔn)工作液中咖啡因的峰面積或峰高；m——最終樣液所代表的試樣質(zhì)量，單位為克(g)。7.1測(cè)定低限本方法測(cè)定低限為0.01mg/kg。7.2回收率蜂王漿中添加0.01mg/kg、0.02mg/kg、0.05mg/kg濃度水平時(shí)，回收率范圍見(jiàn)表3。表3回收率試驗(yàn)結(jié)果添加濃度/(mg/kg)回收率/%96.6～101.394.2～103.093.1～100.44API4000LC-MS/MS系統(tǒng)電噴霧離子源參考條件”a)氣簾氣(CUR):68.95kPa(10.00psi);b)霧化氣(GS1):379.23kPa(55.00psi);c)輔助加熱器(GS2):344.75kPa(50.00psi);d)離子源噴霧器電壓(IS):800f)ihe:OFF;g)碰撞氣(CAD):6.00V;h)定性離子對(duì)、定量離子對(duì)、去簇電壓、碰撞能量、碰撞室出口電壓見(jiàn)表A.1.待測(cè)物DP/VEP/V咖啡因a為定量離子對(duì)。5圖B.1咖啡因標(biāo)準(zhǔn)品的提取離子色譜圖6istrationofthePeople'sRepublicofChina.ThisstandardwasdraftedbyHunan,HubeiandZhejiangEnofthePeople'sRepublicofChina.ThemaindraftersofthisstandardareWangMeiling,WangPeng,WeiYing,ChenXiaomeiThisstandardisaprofessionalstandardpromulgatedforthefirsttime.7ThisstandardspecifiesthemethodsdeterminatiThisstandardisapplicabletothedeterport.ThecaffeineisextractedfromthetestsamplewitAllreagentswereofanalyticalgradeunlessotherwisespecifiedanddistilledwaterwereused.3.2Methanol:HPLCgrade.3.3Formicacid:Guaranteedgrade.tilledwater.3.8Potassiumoxalate-disodiumhydrogenphosph7gdisodiumhydrogenphosphate,thendiss83.920%methanolaqueoussolution:20mLmewater.3.110.1%formicacidaqueoussolution:1mLofformicacidwasdilutedto1000mLvolumewithdistilledwater.3.12Caffeine(C?H?N?O?,CASNo.:5743-12-4,Molecularweight:194.19):purity≥99.8%,3.13Standardstoresolution:Accuratelyweightappropriateamountofcaffeinestandard,thendis-solvewithdistilledwaterto100μg/mL,storedbelow4℃.Thisstandardsolutionwasstableinsixmonth.3.14StandardworkingsolutiongDilutethestandard-store-solutionwith60%methanolaqueousso-vance.3.160.45μmfiltratemembrane:organictype.4.1Hightperformanceliquidchromatographytriple-huadrupoletandemmassspectrometere-quippedwithelectrosprayionizatiònsource(ESl),4.2Centrifuge:4000r/min.4.4UItrasonicwasher.4.5Balance:Readability0.001gand0Weightabout500gofrepresentativesample.Putthepreparedsampleintoacleancontainer,sealand9samplepreparation,precautionsmustbetakentoavoidcontaminationoranyfactorsthatmaycausethechangeofresiduecontent.solutionand2.5mLpotassiumoxalate-thenelutewith5mLdistilledwaterand5mL20%methanolaqueoussolution.Keeptheelutionspeedas1drop/s.VacuumdryandelutewiforLC-MS/MSdetermination.6.2.ILC-MS/MSoperatingconditiona)Chromatographiccolumn:Qetadecylsilica(bDS)(4.6×150mm,5μm)orequivalentcolumn;b)Columntemperature:c)MobilephaseandgradieTablè1—Mobilephaseand0.1%formicacidaqueoussolution/%Acetonitril/%SN/T2440—2010Transitions(m/z)andotherinstrumentalsettingsmaybeadjusted,ifnecessary,tooptimize6.2.2LC/MS-MSdeterminationMaximumpermittedtolerancesforThecalculationofcaffeineinthetestsampleiscarriedoutbyLC-MS/MSdataprocessororaccordingtotheformula(1),theblankvalueshouldbesubtractedfromtheresWhere:X—thecontentofcaffeineinthetestsample,μg/kg;c?—theconcentrationofcaffeineinthestandardworkingsolution,ng/mL;V—thefinalvolumeofthesamplesolution,mL;m—thecorrespondingmassofthetestsampleinthefinalsamplesolution,g.Accordingtotheexperimentdata,thefortifyingconcentrationsofcaffeineinroyAnnexA(informativeannex)c)Auxiliarygas(GS2):344.75kPa(50.00psi);d)lonsprayvoltage(IS):4800.00V;e)Probetemperature(TEM):550.00C;g)Collisiongas(CAD):6.00V;h)Transitions,DP,CE,CXPseetableA.1TableA.1—Transitions,DP,CE,CXP-and