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简介在环境研究领域,鉴定新兴污染物在地表水样品中的存在越来越被关注.
许多该类污染物是由市政污水排放进入地表水而产生的.
在这些新兴的污染物中,地表水中的药物和个人护理药品(PPCPs)的测定浓度已经分别达到百万分之一和几兆分之一.
PPCPs的存在,说明当前污水处理系统对于去除该类物质是低效的.
应用文章高效液相色谱仪/质谱联用仪UHPLC-TOF分析河水样品中的药物及个人护理产品作者SharanyaReddyPerkinElmer,Inc.
Shelton,CTUSA我们目前研究了一种利用UHPLC-TOF分析美国东北部水样品中目标和非目标PPCPs化合物的方法.
三重四级杆是多反应监测模式,仅仅根据预定的目标进行分析筛选,与三重四级杆不同,时间飞行质谱仪提供全谱的精确质量数,这可被用来分析和鉴定无限量的化合物,无需提前知道被分析化合物的信息,或者当没有参考标准物质的时候.
在本研究中,我们展示了PerkinElmerAxION2TOF能够提供准确质量信息,且当其与AxIONECID软件联用时,可用于鉴定地表水中的未知化合物.
2试验条件样品采集和制备:采集水样品(400mL)于1L的琥珀瓶中,样品通过Whatman的玻璃纤维过滤器过滤(GF/C,1.
2μm),然后储存在4°C温度下,待分析.
分析之前,样品经过C-18的固相萃取小柱(SPE)萃取.
SPE萃取:本应用文献采用的是PhenomenexStrata-XSPE小柱(500mg/6ml)进行样品萃取.
萃取之前,小柱依次通过甲醇(5mL)、水(5ml)清洗.
将经过滤的水样品加于SPE小柱上,每个水样中加入PPCPs标准品(100ng)或未加标,以5-6mL/min的流速流过SPE小柱,真空抽干约20min,然后使用甲醇(4mL)和酸化甲醇(含有2%的甲酸,4mL)进行淋洗,淋洗液氮气吹干至约0.
3mL,水定容至0.
5mL.
液相色谱条件:泵:PerkinElmerFlexarFX-10泵流动相A:含有0.
1%甲酸的水溶液流动相B:含有0.
1%甲酸的乙腈溶液梯度洗脱:在5min之内B由10%增加至90%进样体积:3μL,部分填充进样模式使用的色谱柱:PerkinElmerBrownleeSPPC-18,2X50mm,2.
7μm质谱仪条件:质谱仪:PerkinElmerAxION2飞行时间质谱仪离子源:PerkinElmerUltraspray2(双ESI源)离子化模式:正离子模式脉冲模式:100-800m/z光谱采集速率:1.
5谱图/s毛细管出口电压:100V捕集模式:100-800m/z(D7:42,D8:63)内标校准:利用m/z分别为118.
08625和622.
02896两个离子作为锁定离子进行内标校准.
结果在当前的研究中,AxION2TOFMS被用于分析水样中存在的九种目标PPCPs化合物及一些未知的化合物.
九种目标化合物在色谱柱上3min之内获得分离(图1).
SPE萃取程序回收率约为75%或者更高(数据以加入100ppb标准的MilliQ水样测定结果为依据).
使用AxION2TOFMS高灵敏度的TrapPulse(捕集)模式,对于大多数分析化合物,具有低于ppb级优越的仪器检出限(测定咖啡因和可可碱的检测限分别是4.
0和8.
0ppb),很宽的线性范围(0.
5-500ng/mL).
2ResultsInthepresentstudytheAxION2TOFMSwasusedtoanalyzeninetargetedPPCPsandseveralunknowncompoundspresentintheriverwatersamples.
Theseparationoftheninetargetanalyteswasachievedunder3minsoncolumn(Figure1).
TheSPEextractionprocedureresultedinanestimated75%orgreaterrecoveryoftheanalytes(basedon100ppbstandardspikedinMilliQwater).
UsingtheAxION2TOFMSinthehighersensitivityTrapPulsemode,showedexcellentsubppbinstrumentdetectionlimitsformostanalytes(detectionlimitsforcaffeineandtheobrominewere4.
0and8.
0ppb,respectively)andlinearityoveraverywideconcentrationrange(0.
5to500ng/mL).
ExperimentalConditionsSamplecollectionandpreparation:Riversamples(400mL)werecollectedin1Lamberbottles.
SampleswerefilteredthroughWhatmanglassfiberfilters(GF/C,1.
2μm)andstoredat4°Cuntilanalysis.
Priortoanalysis,thesampleswereextractedthroughC-18solidphaseextraction(SPE)cartridges.
SPEextraction:PhenomenexStrata-XSPEcartridges(500mg/6mL)wereusedforextraction.
Thecartridgeswereinitiallyconditionedwithmethanol(5mL)followedbywater(5mL).
ThefilteredriversamplewasloadedonthecartridgeswithorwithoutaspikeofstandardPPCPs(100ngeach)andextractedthroughthecartridgeat5-6mL/min.
Thecartridgewasdriedundervacuumfor~20minspriortoelutingwithmethanol(4mL)andacidifiedmethanol(containing2%formicacid,4mL).
Theeluatewasdriedundernitrogento~0.
3mLanddilutedto0.
5mLwithwater.
Liquidchromatographyconditions:Pump:PerkinElmerFlexarFX-10pumpMobilephaseA:Watercontaining0.
1%formicacidMobilephaseB:Acetonitrilecontaining0.
1%formicacidGradientconditions:10%Bto90%Bin5minInjectionvolume:3μLinpartialfillmodeColumnused:PerkinElmerBrownleeSPPC-18,2X50mm,2.
7μmMassspectrometryconditions:Massspectrometer:PerkinElmerAxION2TOFIonizationsource:PerkinElmerUltraspray2(DualESIsource)Ionizationmode:positivePulsemode:100-800m/zSpectralacquisitionrate:1.
5spectra/secCapillaryexitvoltage:100VTrapmode:100-800m/z(D7:42,D8:63)Internalcalibration:Internalcalibrationwasdoneusingtwoionsm/z118.
08625and622.
02896aslockmassions.
Figure1.
ShowstheseparationofPPCPsinunder2.
5min.
图12.
5min之内PPCPs的分离河水样品中目标和非目标化合物的鉴定从污水处理厂的下游采集的地表水水样品,按照上述方法进行处理.
检测结果显示,该水样品中含有卡巴西平、二苯基羟胺、磺胺甲噁唑、氟西汀(图2).
检测得到的化合物通过与标准物质的精确质量和保留时间是否匹配来进行确认.
3Identificationoftargetandnon-targetanalytesinriverwaterThesurfaceriverwatersampleswerecollecteddownstreamfromasewagetreatmentplantandwereprocessedandanalyzedasdescribedabove.
Thisanalysisrevealedthepresenceofcarbamazepine,diphenylhydramine,sulfa-methoxazoleandfluoxetineintheriverwatersamples(Figure2).
Thepresenceofthesecompoundswasconfirmedbyaccuratemassandretentiontimematchingwithstandards.
3Figure2.
Identificationofcarbamazepine,diphenylhydramine,sulfamethoxazoleandfluoxetineinriversampleusingaccuratemassandretentiontimematchingwithstandards.
图2利用精确质量和标准物质的保留时间鉴定水样品中的卡巴西平、二苯基羟胺、磺胺甲噁唑、氟西汀.
44Figure3.
Showsamajorunknowntargetanalyteelutingat~2.
7mins.
Figure4aandb.
AccuratemassandisotoperatioofunknownanalytewasenteredintoAxIONECIDsoftwaretodetermineelementalcomposition.
TheAxIONECIDsoftwaresearchesagainstthePubChemdatabaseandbysortingfordecreasingAIDs(chemicallistedformostnumberofassays),thefirstpossiblecandidateforthegivenelementalcompositionwasgivenasN,N-Diethyl-m-toluamide(DEET),acommoningredientininsectrepellents.
ab图3约2.
7min淋洗出峰的主要未知目标分析物图4a和b未知分析化合物的精确质量和同位素比值被输入AxIONECID软件,进行元素组成的测定.
采用PubChem数据库检索,以降序AIDs(大量化合物编号)编号排序,给定元素组成的第一可能的候选化合物为N,N-二乙基-m-甲苯酰胺(DEET),是杀虫剂的常规成分.
4Figure3.
Showsamajorunknowntargetanalyteelutingat~2.
7mins.
Figure4aandb.
AccuratemassandisotoperatioofunknownanalytewasenteredintoAxIONECIDsoftwaretodetermineelementalcomposition.
TheAxIONECIDsoftwaresearchesagainstthePubChemdatabaseandbysortingfordecreasingAIDs(chemicallistedformostnumberofassays),thefirstpossiblecandidateforthegivenelementalcompositionwasgivenasN,N-Diethyl-m-toluamide(DEET),acommoningredientininsectrepellents.
ab该水样品被用于进一步检测,进行非目标化合物的分析.
利用AxIONECID软件来分析在约2.
7min被淋洗出的一个主要色谱峰.
精确的单一同位素质量和同位素的比值信息被AxIONECID用于进行PubChem数据库检索(或其它的数据库检索),进行可能的分子式匹配,并提供可能的匹配汇总,以及对于一个给定元素组成的化合物提供可能化合物的结构建议.
在这个例子中的元素组成,C12H17NO得到了最高的匹配分值(图4a),最可能的候选物是N,N-二乙基-m-甲苯酰胺(DEET),该化合物是基于已知的活性AIDS编号(检测IDs)(图4b).
DEET是杀虫剂的常规成分,未知化合物的精确分子质量与DEET预期的分子质量之间的误差小于2ppm(图5).
通过与标准物保留时间的匹配进一步确证了河水样品中含有DEET化合物.
利用类似的方法,河水样品中还测出了对乙酰氨基酚化合物,该化合物是一种常用的止痛药(未给出数据).
Foracompletelistingofourglobaloffices,visitwww.
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Allrightsreserved.
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Allothertrademarksarethepropertyoftheirrespectiveowners.
010131A_01PerkinElmer,Inc.
940WinterStreetWaltham,MA02451USAP:(800)762-4000or(+1)203-925-4602www.
perkinelmer.
comFigure5.
PresenceofDEETwasconfirmedbyaccuratemassdataTheriversampledatawasfurtherexaminedforunknownnon-targetanalytes.
Amajorchromatographicpeakelutingat~2.
7min(Figure3)wasanalyzedusingAxIONECIDsoftware.
AccuratemonoisotopicmassandisotoperatioinformationisusedbyAxIONECIDtosearchthePubChemdatabase(orotherdatabases)forpotentialmolecularformulamatchesandprovidearankedsummaryofthepotentialmatchesaswellassuggestionsforpossiblecompoundstructuresforagivenelementalcomposition.
Inthisexample,theelementalcomposition,C12H17NOwaslistedwithhighestscore(Figure4a)andthetopcandidatewasidentifiedasN,N-Diethyl-m-toluamide,(DEET)basedonthenumberofactiveAIDS(AssayIDs)reported(Figure4b).
DEETisacommoningredientininsectrepellentandtheaccuratemassoftheunknowncompoundwasverifiedtobewithin2ppmmasserroroftheexpectedmassofDEET(Figure5).
ThepresenceofDEETintheriversamplewasfurtherconfirmedbyretentiontimematchingwiththestandard.
Usingasimilarapproach,acetaminophen,acommonlyusedanalgesicdrugwasalsoidentifiedintheriverwater(datanotshown).
ConclusionsThePerkinElmerAxION2TOFoperatedinthehighersensi-tivityTrapPulsemoderesultedininstrumentdetectionlimitsof<1ppbformajorityofthePPCPs.
Thesurfaceriverwatersamplewasscreenedforbothtargetandnon-targetPPCPs.
TheaccuratemassandisotoperatioprovidedbytheTOFconfirmedpresenceofseveralofthetargetPPCPsincludingantidepressantdrugsinthesurfacewaters.
Thenon-targetPPCPsinthesamplewereidentifiedusingaccuratemassinformationalongwithpowerfuldatabasesearchtoolssuchastheAxIONECIDsoftware.
References1.
Daughton,C.
G.
,Jones-Lepp,T.
L.
,Pharmaceuticalsandpersonalcareproducts(PPCPs)intheenvironment.
ScientificandRegulatoryIssues,ACSSymp.
Ser.
791,OxfordUniversityPress,Washington,U.
S.
(2001).
2.
Daughton,C.
G.
,Environ.
ImpactAssess.
Rev.
24(2004)711.
结论PerkinElmerAxION2TOF采用高灵敏度的TrapPulse操作模式,对于大多数的PPCPs化合物而言,仪器的检出限<1ppb.
利用该仪器进行了地表水样品中目标和非目标PPCPs化合物的监测.
TOF提供的精确质量和同位素比值,确证了地表水中存在一些目标PPCPs化合物,包括抗抑郁药物.
采用准确的质量信息与强大数据库检索软件工具的联用,如AxIONECID软件,来实现样品中非目标PPCPs化合物的鉴定.
参考文献Foracompletelistingofourglobaloffices,visitwww.
perkinelmer.
com/ContactUsCopyright2012,PerkinElmer,Inc.
Allrightsreserved.
PerkinElmerisaregisteredtrademarkofPerkinElmer,Inc.
Allothertrademarksarethepropertyoftheirrespectiveowners.
PerkinElmer,Inc.
940WinterStreetWaltham,MA02451USAP:(800)762-4000or(+1)203-925-4602www.
perkinelmer.
comFigure5.
PresenceofDEETwasconfirmedbyaccuratemassdataTheriversampledatawasfurtherexaminedforunknownnon-targetanalytes.
Amajorchromatographicpeakelutingat~2.
7min(Figure3)wasanalyzedusingAxIONECIDsoftware.
AccuratemonoisotopicmassandisotoperatioinformationisusedbyAxIONECIDtosearchthePubChemdatabase(orotherdatabases)forpotentialmolecularformulamatchesandprovidearankedsummaryofthepotentialmatchesaswellassuggestionsforpossiblecompoundstructuresforagivenelementalcomposition.
Inthisexample,theelementalcomposition,C12H17NOwaslistedwithhighestscore(Figure4a)andthetopcandidatewasidentifiedasN,N-Diethyl-m-toluamide,(DEET)basedonthenumberofactiveAIDS(AssayIDs)reported(Figure4b).
DEETisacommoningredientininsectrepellentandtheaccuratemassoftheunknowncompoundwasverifiedtobewithin2ppmmasserroroftheexpectedmassofDEET(Figure5).
ThepresenceofDEETintheriversamplewasfurtherconfirmedbyretentiontimematchingwiththestandard.
Usingasimilarapproach,acetaminophen,acommonlyusedanalgesicdrugwasalsoidentifiedintheriverwater(datanotshown).
ConclusionsThePerkinElmerAxION2TOFoperatedinthehighersensi-tivityTrapPulsemoderesultedininstrumentdetectionlimitsof<1ppbformajorityofthePPCPs.
Thesurfaceriverwatersamplewasscreenedforbothtargetandnon-targetPPCPs.
TheaccuratemassandisotoperatioprovidedbytheTOFconfirmedpresenceofseveralofthetargetPPCPsincludingantidepressantdrugsinthesurfacewaters.
Thenon-targetPPCPsinthesamplewereidentifiedusingaccuratemassinformationalongwithpowerfuldatabasesearchtoolssuchastheAxIONECIDsoftware.
References1.
Daughton,C.
G.
,Jones-Lepp,T.
L.
,Pharmaceuticalsandpersonalcareproducts(PPCPs)intheenvironment.
ScientificandRegulatoryIssues,ACSSymp.
Ser.
791,OxfordUniversityPress,Washington,U.
S.
(2001).
2.
Daughton,C.
G.
,Environ.
ImpactAssess.
Rev.
24(2004)711.
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cn图5精确质量确认DEET的存在