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Fingerprinting polychlorinated biphenyls in environmental samples using comprehensive two-dimensional gas chromatography with time-of-flight mass spectrometry

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A GC × GC-TOFMS installed with a Rtx-PCB (60 m × 0.18 mm × 0.18 μm) in the first dimension and Rxi- 17 (1.5 m × 0.1 mm × 0.1 μm) column in the second dimension was used to separate 188 out of 209 congeners. A further 12 congeners were identified
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   JournalofChromatographyA, 1318 (2013) 276–283 ContentslistsavailableatScienceDirect  Journal   of    Chromatography   A  journalhomepage:www.elsevier.com/locate/chroma Short   communication Fingerprinting   polychlorinated   biphenyls   in   environmental   samplesusing   comprehensive   two-dimensional   gas   chromatography   withtime-of-flight   mass   spectrometry David   Megson a , ∗ ,   Robert   Kalin b ,   Paul    J.   Worsfold a ,   Caroline   Gauchotte-Lindsay c ,Donald   G.   Patterson    Jr. d ,   Maeve   C.   Lohan a ,   Sean   Comber a ,   Thomas   A.   Brown a ,   GwenO’Sullivan e a BiogeochemistryResearchCentre,SoGEES,PlymouthUniversity,Plymouth,DevonPL48AA,UK  b DepartmentofCivilandEnvironmentalEngineering,UniversityofStrathclyde,GlasgowG11XQ,UK  c DivisionofInfrastructureandEnvironment,SchoolofEngineering,UniversityofGlasgow,GlasgowG128LT,UK  d ExponentInc.,OneCapitalCityPlaza,Suite1620,3350PeachtreeRoad,Atlanta,GA30326,USA e DepartmentofEnvironmentalScience,MountRoyalUniversity,4825MountRoyalGateSW,   Calgary,AlbertaT3E6K6,Canada a   r   t   i   c   l   e   i   n   f   o  Articlehistory: Received31July2013Receivedinrevisedform3October2013Accepted4October2013 Available online 10 October 2013 Keywords: PolychlorinatedbiphenylsComprehensivetwo-dimensionalgaschromatographyTimeofflightmassspectrometryAroclor a   b   s   t   r   a   c   t A   GC ×   GC-TOFMS   installed   with   aRtx-PCB   (60   m ×   0.18   mm   ×   0.18    m)   in   the   first   dimension   and   Rxi-17   (1.5   m   ×   0.1   mm   ×   0.1    m)   column   in   the   second   dimension   was   used   to   separate   188   out   of    209congeners.   Afurther   12congeners   were   identified   through   additional   data   processing   resulting   in   theidentification   of    a   total   of    200   congeners.   However,   caution   is   advised   if    these   12   congeners   wereto   be   used   in   quantitative   assessments.   The   remaining   9co-eluting   congeners   were   three   doublets(CB65   +   CB62,   CB160   +   CB163   and   CB201   +   CB204)   and   one   triplet   (CB20   +   CB21   +   CB33).   This   method   wastested   onfive   Aroclors   and   resulted   in   the   separation   of    all   congeners   present   in   the   heavier   Aroclormixtures   A1254   and   A1260.   The   suitability   of    this   method   for   applications   inbiological   matrices   wasdemonstrated   onextracted   whiting   and   guillemot   liver   samples   which   resulted   in   the   identification   of 137   individual   PCBs   in   the   whiting   liver   sample   and   120   in   the   guillemot   sample.   Fingerprinting   was   abletoshow   clear   differences   in   the   PCB   signature   of    the   two   animals.   This   highlights   the   potential   of    thismethod   for   PCB   fingerprinting   in   environmental   forensics   studies   and   other   assessments   that   requirecongener   specific   analysis. © 2013 Elsevier B.V. All rights reserved. 1.Introduction Polychlorinatedbiphenyls(PCBs)werefirstdiscoveredinenvi-ronmentalsamplesin1966[1,2].However,duetotheirstructural similarity,theseparationofall209PCBsstillpresentsasignifi-cantanalyticalchallenge.Inordertoachievepositiveidentificationandaccuratequantification,alltargetcompoundsmustbesuffi-cientlyresolved.IntoxicologicalstudiesitisimportanttoseparatetheWorldHealthOrganisation12dioxinlikePCBs(WHO12)[3].However,inenvironmentalforensicsstudiesagreaternumberof congenersoftenneedtobeseparatedtoidentifyprocessessuchasmicrobialdegradation[4],volatilisation[5]andbiotransformation inhumans[6].Havingtheabilitytoseparateandidentifyspecific PCBcongenersisessentialinenvironmentalstudiesthatrequirePCBfingerprinting. ∗ Correspondingauthor.Tel.:+441752584567. E-mailaddress: dpmegson@hotmail.co.uk(D.Megson). EarlyanalyticaltechniquessuchasEPAmethods8082and608focusedoncalculatingtotalPCBconcentrationsasspillstotheenvi-ronmentoccurredfromtechnicalmixturessuchasAroclorsratherthanfromindividualcongeners.Asmorewas   understoodaboutPCBsitbecameevermoreimportanttoidentifyandquantifyindi-vidualPCBcongeners.In1997Frame[7]documentedtheretention timesandelutionordersofall209congenerson20GCcolumnswhichsignificantlyimprovedtheabilityofanalyticalchemiststoconfidentlyidentifyandquantifyPCBsinanunknownsample.However,nosingleGCcolumniscurrentlyabletoseparateall209congenersandsoitwasnecessarytomanagedatasetswithco-elutionsoranalysesamplesonmultipleGCcolumns.Thedevel-opmentofcomprehensivetwo-dimensionalgaschromatographycansignificantlyincreasepeakcapacityandthereforepotentiallyremovetheneedtoundertakerepeatsampleinjectionsonmulti-pleGCcolumns[8].Thedemandforcongenerspecificanalysislead tothedevelopmentofEPAmethod1668cwhichstatesthatover180individualcongenerscanbeseparatedusingadualcolumnsystem. 0021-9673/$–seefrontmatter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.chroma.2013.10.016  D.Megsonetal./J.Chromatogr.A 1318 (2013) 276–283 277 RecentlyPCBanalysisusingGC × GC-TOFMShasbeenusedtoseparatemorethan190individualPCBcongenersalongwithsimul-taneousidentificationofotherorganohalogenatedcontaminants[9,10].Separationof198PCBswasundertakenbyHarjuetal.[11] in2003usingGC × GC-  ECDequippedwitha60mDB-XLBinthefirstdimension( 1 D)and2.25mBPX-70intheseconddimension( 2 D).Thisresultedinco-elutionsofonetripletandfourdoublets.Noco-elutionswererecordedforeitherthe12‘dioxinlike’congeners(WHO12)orEuropeanindicatorPCBs(EC7),inadditiontheruntimewasupto4hlong.Separationof194congenerswas   reportedbyFocantetal.[12]in2004usingGC × GC-TOFMSequippedwitha60mDB-XLBin  1 Dand2.5mBPX-50in  2 D;Howeverthisresultedinco-elutionsofonetripletandsixdoublets.Oneco-elutionwasrecordedforaWHO12congener(CB123)andtwoco-elutionswererecordedfortheEC7congeners(CB101andCB153).Separationof 196congenerswasreportedbyZapadloetal.[13]in2011usingGC xGC-TOFMSequippedwitha30mSPB-Octylin  1 Dand1.8mSLB-IL59in  2 D.Howeverthisresultedinco-elutionsofonetripletandfivedoublets.Noco-elutionswererecordedfortheWHO12con-genersandtwoco-elutionswererecordedfortheEC7congeners(CB101andCB153).GC × GC-TOFMShasthereforebeenshowntobeapowerfultechniqueforresolvingPCBcongeners.SeveralpapershavebeenpublishedonPCBseparationusingavarietyofcolumncombina-tions[9–13];howevernoneofthesestudiesinvestigatedtheuse oftherecentlydevelopedRtx-PCBcolumn(commerciallyavail-ablesince2005).ThispaperreportstheseparationachievedusingaPCBspecificlowpolaritycolumnin  1 Dwithastationaryphasewhichispredominantlydimethylpolysiloxane(Rtx-PCB),coupledwithamid-polaritycolumnin  2 Dwithastationaryphasewhichis50%diphenyl,50%dimethylpolysiloxane(Rxi-17).ThismethodwastestedonthefivemostcommonAroclorsolutions[14]andits applicationtobiologicalmatriceswasdemonstratedthroughanal-ysisofextractedliversamplestakenfromawhitingobtainedfromtheEnglishChannelandaguillemotrecoveredfromthecoastof GreatBritain. 2.Materialsandmethods  2.1.Chemicals,standardsandsamples IndividualPCBcongenerswereidentifiedusingninePCBcon-generstandardcalibrationmixtures(CS1toCS9;AccuStandard)containing10  gmL  − 1 ofeachPCBin1mL    ofisooctane.Approx-imately50  LofCS1–CS9werecombinedtoproduceasolutioncontainingall209congeners(209PCBsolution).FivecertifiedPCBtechnicalmixtures(99%purity)wereobtainedataconcentrationof1000  gmL  − 1 including;Aroclor1016,Aroclor1254andAroclor1260(FisherChemicals),Aroclor1242andAroclor1248(Grey-houndChromatography).Priortoanalysis,allAroclorsweremadeuptoconcentrationsof10mg   L  − 1 throughserialdilutionwithhex-ane(fordioxins,furansandPCBs;Sigma–Aldrich).OnewhitingandoneguillemotliversamplewerepreparedusingestablishedtechniquesforextractionoforganicbiomarkersinmarinetissuereportedbyBrownetal.[15].Brieflythisinvolved;freezedrying andgrindingsamplesintoapowderbeforesonicationandextrac-tionwithdichloromethane/methanol.Extractswerefiltered,driedandre-suspendedinhexaneandthenon-polarfractionseparatedbycolumnchromatography(SiO 2 ).Sampleswereevaporatedtodrynessandreconstitutedwith10  Lofhexanepriortoanalysis.  2.2.GC  × GC-TOFMSanalysisanddataprocessing  Sampleswereanalysedonatime-of-flightmassspectrometer(Leco,St.Joseph,MI   Pegasus4D)coupledtoatwodimensionalgaschromatograph(AgilentTechnologies7890A)equippedwithathermalmodulator(Leco,St.Joseph,MI).Thegaschromatographwas   installedwithaRtx-PCB(60m × 0.18mm   × 0.18  m)  1 Dcol-umnandaRxi-17(1.5m × 0.1mm × 0.1  m) 2 Dcolumn.One  Lof samplewas   injectedinsplitlessmode,analyticalblankswererunwitheachbatchofupto10samplesandthesame209PCBmixsolutionwas   analysedintriplicate.TheGCinlettemperaturewassetat280 ◦ Cwithaheliumcar-riergasflowrateof1mL    min − 1 andapurgetimeof120sataflowrateof20mL    min − 1 .Theprimaryoventemperaturewasinitiallysetat90 ◦ Cfor1min,thenincreasedto150 ◦ Cat30 ◦ Cmin − 1 ,thento300 ◦ Cat1 ◦ Cmin − 1 .Thesecondaryoventemperaturewassetat20 ◦ Chigherthantheprimaryoventemperature.Themodula-tortemperaturewasalsosetat20 ◦ Chigherthantheprimaryoventemperature,themodulationperiodwas   3swithahot-pulsedura-tionof700ms   andthecoolingtimewas800ms.   Thetransferlineanddetectortemperaturewassetat300 ◦ C.Themassspectrome-terwas   operatedwithasourceionisationenergyof70eV,detectorvoltageof1800Vandthedataacquisitionrateof100spectras − 1 for100–550Daltons.Theruntimeforeachsamplewas   153min.AlldatafileswereprocessedusingChromaTOFsoftware.Theprocessingmethodwassettoidentify10,000peakswithasignal-to-noiseratioofgreaterthan10:1.ThroughoutthispaperPCBsarereferredtousingtheGuitartetal.[16]numberingsystem.  2.3.Qualitycontrolprocedures Analyticalblankswererunwitheachbatchofapproximately10samples.Allsampleswerespikedwitha  13 C 12  internalstandard.Standardmixtureswereanalysedintriplicatetocheckthatsepa-rationwasconsistentlyachieved.Standardsolutionsindecreasingconcentrationswereanalysedtoestablishthelimitofdetectionforindividualcongeners;thelowestconcentrationofanindividualcongenerthatcouldbeconsistentlydetectedwas   1pg  L  − 1 . 3.Resultsanddiscussion  3.1.Identificationofall209congeners CalibrationsolutionsCS1–CS9wererunindividuallyandreten-tiontimeswerecomparedwithliteraturevaluesonelutionorderproducedbyLecoCorporation[17]toidentifyeachcon- gener.Fig.1showsthetwo-dimensionalretentiontimesforall209PCBcongeners,groupedbytheirdegreeofchlorina-tion.Analysisofthe209PCBsolutionresultedintheseparationof188individualPCBswithninedoubletsandonetripletthatcouldnotberesolvedbycomprehensivetwodimensionalchro-matographywithmassspectrometricdetection.Co-elutingPCBswereinvestigatedtoseeiftheycouldbeidentifiedbyfurtherdatainterpretation.Thisincludedcomparingslicesoftheonedimen-sionalchromatogramaswellasspectraldeconvolutionwhichwasundertakenusingtheChromaTOFsoftware.Theresultsofwhicharepresentedinthesupplementarymaterial.Usingfurtherdatainterpretation,sixoftheco-elutingdoubletswereidentifiedinallthreetriplicatesamples(CB4+CB10,CB67+CB58,CB95+CB88,CB84+CB89,CB101+CB90andCB175+CB182).Thisresultedintheidentificationofatotalof200ofthe209PCBsinthemix-ture(Table1).Althoughtheseparationwassufficienttoallowfor eachcongenertobevisuallyidentified,quantificationofthesecon-generswouldneedtobeundertakenusingfurtherdataanalysis.TherewerenoisobaricoverlapsbetweenPCBsandfragmentsof higherhomologues-2Cl(e.g.hexa-CBfragment-2Clandtetra-CB).However,therewereseveralco-elutionswithinhigherhomologous  278  D.Megsonetal./J.Chromatogr.A 1318 (2013) 276–283  Table1 Elutionorderandretentiontimesofall209congenersrecordedusingaLecoPegasus4DGC × GC-TOFMSequippedwitha60mRtx-PCBand1.5mRxi-MS.Congenersinboldare   presentat>1%inthefivemainAroclors,congenersinitalicsarepresentat<0.01%inthefivemainAroclors.WHO12congenersaremarkedwith*,EC7congenersaremarked   with  + .  D.Megsonetal./J.Chromatogr.A 1318 (2013) 276–283 279 Table1( continued )  280  D.Megsonetal./J.Chromatogr.A 1318 (2013) 276–283 Table1( continued )Representsco-elutingcongeners,;representscongenersidentifiedbyfurtherprocessing,. series-1Cl(e.g.hexa-CBfragment-1Clandpenta-CB).Theseshouldalsobeconsideredwhenundertakingquantitativeanalysis.Whenincludingthecongenersthatwereresolvedbyfurtherdatainterpretation,noco-elutionswererecordedthatinvolvedeithertheWHO12orEC7congeners.Fig.2presentsthenineco-elutingdoubletsandonetripletthatcouldnotbeseparatedbycomprehensivetwo-dimensionalchromatography,alongwiththeirpercentageabundancesinthefivemajorAroclors[18].Theco-elutionsfromthisstudyarepresentedinTable2along withco-elutionsforvariouscolumncombinationsreportedbyotherauthors.Thisisintendedtohelpfutureresearchersdecideonthecolumncombinationthatwouldbestsuittheirneeds.  3.2.IdentificationofPCBsin5Aroclors PCBswereproducedascommercialmixturessuchasAroclors.Duringthemanufacturingprocessthereareseveralcongenersthatdonothavefavourablechlorinesubstitutionpatterns,suchasPCBswithoneheavilychlorinatedbiphenylandoneun-chlorinatedbiphenyl[18].ThesePCBsarethereforeunlikelytobepresent indetectableconcentrationsinenvironmentalandanimaltis-suesamples.AnalysisofthefivemostcommonAroclors(A1016,A1242,A1248,A1254andA1260)wasundertakentoidentifyco-elutionsinthesecommercialmixtures.ResultswerecomparedwiththedatabaseofPCBsinAroclorscompiledbyFrame[18]whichwas   basedondatafromFrameetal.[19].AllPCBspresent intheheavierAroclormixturesA1254andA1260wereidentified,alongwith113ofthe115congenersinA1248(co-elutionsof CB88+CB95),96ofthe99congenersinA1242(co-elutionsof CB20+CB21+CB33)and63ofthe66congenersinA1016(co-elutionsofCB20+CB21+CB33).Aswiththe209PCBsolution,thismethodwasunabletosep-arateCB33+CB20+CB21inanyAroclor.However,thesampleof A1248didnotcontaindetectableconcentrationsofCB20whichallowedCB33toberesolvedfromCB21.  3.3.IdentificationofPCBsinanimaltissuesample Theapplicationofthismethodtobiologicalmatriceswasdemonstratedthroughanalysisofextractedliversamplestakenfromawhitingandguillemot.Thesesampleswereusedasanexampleofthecomplexmatricesthatareoftenexperiencedinenvironmentalforensicsinvestigations.Thismethodidentified137individualPCBsinthewhitingliver,withafurther18tentativelyidentifiedwithasignal-to-noiseratio<10.Thismethodalsoiden-tified120PCBsintheguillemotliverwithafurther11tentativelyidentifiedwithasignal-to-noiseratio<10.Thesame112PCBswerepositivelyidentifiedinbothsamples.Therelativeproportionsofthepeakareasofthese112congenersclearlyshowedadifferentPCB Fig.1. Plottedpeakapexesrepresentingthetwo-dimensionalretentiontimesforall209PCBcongeners.
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