Journal of Chromatography A
jo u rn a l h o m e pa g e :w w w . e l s e v i e r . c o m / l o c a t e / c h r o m a
Trace determination of primary nerve agent degradation products in aqueous soil extracts by on-line solid phase extraction–liquid
chromatography–mass spectrometry using ZrO 2 for enrichment
Bent Tore Røen
a,b,∗, Stig Rune Sellevåg
a, Kjersti E. Dybendal
a, Elsa Lundanes
baNorwegianDefenceResearchEstablishment(FFI),P.O.Box25,NO-2027Kjeller,Norway
bDepartmentofChemistry,UniversityofOslo,P.O.Box1033,Blindern,NO-0315Oslo,Norway
a r t i c l e i n f o
Articlehistory:
Received20October2013 Receivedinrevisedform 19December2013 Accepted4January2014 Available online 10 January 2014
Keywords:
On-lineSPE–LC–MS Zirconiumdioxide Alkylmethylphosphonicacid Lewisbase
a b s t r a c t
Amethodfordeterminationoftheprimarynerveagentdegradationproductsethyl-,isopropyl-,isobutyl- ,cyclohexyl-andpinacolylmethylphosphonicacidinaqueoussoilextractshasbeendevelopedutilizing on-linesolidphaseextraction-liquidchromatographyandmassspectrometry(SPE–LC–MS).Fourdiffer- entstationaryphases(ZrO2,TiO2,polymericmixedmodeanionexchangeandporousgraphiticcarbon) wereinvestigatedfortheirsuitabilityasSPEmaterialsintheon-lineSPE–LC–MSsetup.Zirconiumdioxide waschosenduetoitshighaffinityforthealkylmethylphosphonicacids(AMPAs),anditscompatibil- itywithLC–MS.Aqueoussoilextractswereacidifiedwith0.1%aceticacidandaliquotsof300Lwere injectedona2mm×10mmZrO2column.Separationoftheanalyteswasperformedonareversedphase columnwithacetonitrile/watergradientand15mMammoniumacetate.Methodvalidationwasper- formedwiththeanalytesaddedtoanaqueousextractofaloamsoil,andtheAMPAscouldbedetermined atconcentrationsaslowas0.05–0.5gL−1.Themethodwaslinear(R2>0.995)fromthelimitofquan- tification(LOQ)to100×LOQ,andthewithinassayrepeatabilitywasbelow10%and5%relativestandard deviationatLOQand50×LOQ,respectively.Thedevelopedmethodwasemployedfordetermination oftheAMPAswhichhadbeenaddedtotheaqueousextractsoffivedifferentsoiltypesfromcultivated anduncultivatedareas.Theobtainedrecoveriesshowedthattheanalytescouldbedeterminedatthe sensitivitiesachievedinthemethodvalidationinfouroftheextracts.Forthefirsttime,wehavedemon- stratedamethodcapableofdetectingprimarynerveagentdegradationproductsatsubppblevelsinthe aqueousextractsofvarioussoils.Themethodrequiresnosamplepreparationaftersoilextractionother thanpHadjustmentoftheaqueousextract.
© 2014 The Authors. Published by Elsevier B.V. All rights reserved.
1. Introduction
Theorganophosphorouscompoundsknownasnerveagentsare themostlethaltypeofchemicalwarfareagentscurrentlyknown.
Alldevelopment,stockpilinganduseofthecompoundsarepro- hibitedbytheChemicalWeaponsConvention(CWC)[1],except withinresearchactivitiesthataredeclaredandinaccordancewith theconvention.Incasesofdeliberateorunintentionalspreadof nerveagents,efficientandsensitivetechniquesformeasurementof thecompoundsortheirdegradationproductsareimportant.After
∗Correspondingauthorat:NorwegianDefenceResearchEstablishment(FFI),P.O.
Box25,NO-2027Kjeller,Norway.Tel.:+4763807881;fax:+4763807509.
E-mailaddress:Bent-Tore.Roen@ffi.no(B.T.Røen).
beingreleasedintotheenvironment,thenerveagentsdegradeby hydrolysis totheircorrespondingalkylmethylphosphonicacids (AMPAs)asshowninFig.1.Thesedegradationproductsarespe- cificforeachnerveagentanddonothaveanynaturalsources,and hencetheyarevaluablemarkersforthereleaseofnerveagents.
TheAMPAsmayundergofurtherhydrolysisbylossoftheO-alkyl group,resultinginthenon-specificmethylphosphonicacid(MPA).
Thisprocessisveryslowinwater,butmorepronouncedwhenthe AMPAsareadsorbedtosoil[2].Highsensitivityindetermination oftheprimaryhydrolysisproductsmaythereforebeessentialin ordertogiveforensicproveofthespreadofnerveagents.
Soilhasbeenutilizedassamplematrixforverificationofthe releaseofchemicalwarfareagentsonseveraloccasions[3–7].The highlywatersolubleAMPAscanbeextractedfromsoilinneutral [8,9]or alkaline[10,11]aqueoussolutions. Reversedphase (RP) liquidchromatographyconnectedtomassspectrometry(LC–MS) withelectrosprayionisation(ESI)[12–15]andgaschromatography (GC)–MS[10,16,17]aremostfrequentlyemployedfordetermina- tionoftheAMPAsinaqueoussoilextracts.Thelattertechnique 0021-9673© 2014 The Authors. Published by Elsevier B.V.
http://dx.doi.org/10.1016/j.chroma.2014.01.004
Open access under CC BY-NC-ND license.
Open access under CC BY-NC-ND license.
Fig.1. Structureofselectednerveagentsandtheirprimaryhydrolysisproducts.ThepKaandlogKowvalueswerecalculatedusingAdvancedChemistryDevelopment (ACD/Labs)SoftwareV11.02(©1994–2012ACD/Labs).
requiresderivatisationoftheAMPAstotheirrespectivephospho- nateesterspriortodetermination.Duetotheirioniccharacter(pKa
2.2–2.3),capillaryelectrophoresis(CE)[18,19]andionexchange chromatography[20] havebeenemployed fordetermination of the AMPAsin aqueous soil extracts as well. If the AMPAs are presentatlowppblevels,analyteenrichmentpriortoinstrumen- taldeterminationisrequired.Aqueoussoilextractsoftencontain, however,highamountsoforganicandinorganiccomponentspos- siblyinterferingwithbothanalyteenrichmentandinstrumental determination.MethodsfordeterminationofAMPAsinaqueous soilextractshavethereforeinmostcasesincludedproceduresfor removalofinterferingcompoundswithlowdegreeofenrichment [9,11,12,21,22].
Theaimofthepresentstudywastodevelopamethodfortrace determinationofAMPAsinaqueoussoilextracts.Henceanenrich- mentstepwasconsiderednecessary,andtheperformanceoffour commerciallyavailablesolidphaseextraction(SPE)columnshas beenexploredforthispurpose.Thecriteriaforchoiceofcolumn materialwerehighrecoveryoftheanalytesandcompatibilityinan on-lineSPE–LC–MSsystem.Theefficiencyoftheaqueousextrac- tionoftheAMPAsfromdifferentsoiltypeshasbeenexaminedby others[8,13,22,23]andwasnotinvestigatedinthiswork.Thesta- tionaryphasesinvestigatedwereporousgraphiticcarbon(PGC),a polymericmixedmodeanionexchange(MAX)sorbent,ZrO2 and TiO2.ThePGCsorbenthasbeensuccessfullyemployedinon-line SPE–LC–MSfordeterminationofAMPAsinwatersamplesatsub ppblevels[24].TheMAXcolumnwasincludedforinvestigation duetoreportedhighrecoveriesbyoff-lineSPEincombinationwith GC–MSfordeterminationofAMPAsinaqueoussamples[25].Zir- coniumdioxideexhibitsLewisacidpropertiesandhasaffinityto strongLewisbasesliketheAMPAswhendissociated[26].Kanau- jiaetal.exploredtheenrichmentofseveralAMPAswithzirconia coatedsilicaparticlesandfoundthattheanalyteswereselectively extractedinthepresenceofcarboxylicacids[27].Also,zirconia coatedstirbar[28]andzirconiahollowfibermembrane[29]have beenusedforextractionoftheAMPAsfromwatersamples.Tita- niumdioxidedisplaysLewisacidpropertiessimilartoZrO2 [30].
Nostudy hasbeenreportedfor enrichmentofAMPAsonTiO2, butthematerialhasbeenextensivelyusedfor selectiveenrich- mentofotherorganophosphates[31–33].Otherstationaryphases havebeenusedforenrichmentoftheAMPAsfromaqueousmatri- ces,likestronganionexchange(SAX)columns[17,22,34].Kanaujia
et al. foundthat the efficiencyof SAX waslower compared to usingMAX,however[25].RetentionbasedonRPinteractionsis notsuitedduetothepolarityoftheAMPAs.Hydrophilic–lipophilic balancedpolymershavebeenusedforisolationoftheAMPAsfrom aqueousmatricesafteracidifyingthesamplestoprotonatetheana- lytes[12,16].Therecoveriesobtainedforethylmethylphosphonic acid(EMPA)werebelow35%withthistechniquethough.Reten- tionbasedonhydrophilicinteractions hasalsobeenutilizedfor enrichmentoftheAMPAs[35],aswellastheuseofmolecularly imprintedpolymers[12,21].Thesetwotechniquesrequireachange fromaqueoustoorganicsolventpriortoanalyteenrichment,and arethereforenotsuitedfordirectdeterminationoftheAMPAsin aqueousextracts.
AsaconsequenceoftheSPEcolumnscreening,ZrO2wascho- senforpreconcentrationoftheAMPAs(seeSection3.1).Zirconium dioxideischaracterizedbyseveralsurfaceproperties,andcanact bothasananion-andcationexchangerdependingonpH[36].More importantlyinthiscontext,ZrO2canundergoligandexchangepro- cessesasshownbelow[26].
Zr(OH)(H2O) +L1−Zr(OH)L1−+H2O (1) Zr(OH)(H2O) +L1−Zr(H2O)L1+OH− (2) Zr(OH)L1−+L2−Zr(OH)L2−+L1− (3)
Zr(OH)L1−+OH−Zr(OH)2−+L1− (4) The ligand exchange behavior originate from the presence of strongLewisacidsites onthesurface of unsaturated Zr(IV), and occurs when a Lewis base (L−) is present in the solution.
OrganophosphatesliketheAMPAsarestrongLewisbasesdueto theirelectronegativephosphonategroups,andthisisthereason forZrO2havinghighaffinityfortheAMPAs.Process1isexpected tobethedominantforligandadsorptionbecausehydroxideions aremoretightlyboundbyzirconiacomparedtowatermolecules, butprocess2willcontributeatlowpH[26].TheadsorbedLewis basecanbedisplacedbyintroducingasecondsoluteLewisbase (L2−)fromanaddedsaltorbuffer[26]asshowninprocess3.Also, bypHincreaseL1−canbedisplacedbythehydroxideionwhichis astrongLewisbaseitself[37](process4andreversionofprocess 2).
A Loam 2.3 7.2 31 10.5
B Loamysand 1.8 5.5 10 11.3
C Sandyloam 1.0 6.2 7 8.6
D Sand 0.7 5.1 4 4.2
E Clay 1.6 7.1 27 4.4
Inthepresentwork,wereportforthefirsttimeanautomated SPE–LC–MSmethodfortracedeterminationofprimarynerveagent degradationproductsinaqueoussoilextracts.Theanalyteswere preconcentratedontheZrO2SPEcolumn,followedbyRP–LCsep- arationandESI–MSinnegativemode.Thedevelopedmethodwas employedfordetermination offiveAMPAs(Fig.1)in theaque- ousextractsoffivedifferentsoilsfromcultivatedanduncultivated areas.
2. Experimental
2.1. Chemicalsandsolutions
Pinacolyl methylphosphonic acid (PMPA, 97%), EMPA (98%) and MPA (98%) were purchased from Sigma–Aldrich Chemie GmbH, Steinheim, Germany. Isopropyl methylphosphonic acid (iPMPA),isobutylmethylphosphonicacid(iBMPA,1000gmL−1 in methanol) and cyclohexyl methylphosphonic acid (CMPA, 1000gmL−1 in methanol)weredeliveredby CerilliantCorpo- ration, Round Rock, TX, USA. Ammonium formate (98%) was purchasedfromBDHLaboratorySupplies,Dorset,UK.Acetonitrile (ACN,99.9%),ammoniumacetate(AA,98%),ammoniumcarbon- ate(AC)andammoniumhydroxide(25%)weredeliveredbyMerck KGaA,Darmstadt,Germany.Methanol(LC–MSgrade),formicacid (98%) and acetic acid (99%) were obtained from Fluka Chemie GmbH, Buchs, Switzerland. Laboratory type I water (classified accordingtotheAmericanSocietyofTestingandMaterials,D1193- 91)wasdeliveredin-housebyMaximaultrapurewater system fromELGA,Marlow,UK.
StocksolutionsofEMPA,iPMPAandPMPAwerepreparedat 0.5mgmL−1 bydiluting25mgoftheneatagents in50mLACN.
FurtherdilutionsweremadeinACNortypeIwater,whilethefinal workingsolutionswerepreparedintypeIwaterorinaqueoussoil extracts.Theworkingsolutionswerepreparedtocontainnomore than1%ACN.Allsolutionswerestoredat4◦Cuntiluse.Asolutionof 3.1%(v/v)aceticacidwaspreparedintypeIwater.Fromthis,50L wasaddedto1.5mLofthesamplesdirectlyintheautosamplervials (finalconcentration0.1%,v/v).
2.2. Soilsamplesandextractionprocedure
Thesoiltypesthatweresubjectedtoaqueousextractionare listedinTable1,andwereobtainedfromLUFASpeyer,Germany.
Thesoilsweresampledatadepthof0–20cmfromvariouscul- tivated(soilA–C)anduncultivated(soilDandE)areas.Allsoils weredriedat roomtemperatureuntilsieveable,then sievedto agrainsizeof2mmandcharacterisedbythesupplier.Thetotal organiccarbon(TOC),pHandcationexchangecapacity(CEC)of eachsoilarelistedinTable1.Classificationofthesoilsisgivenon thebasisoftheparticlesizedistribution,accordingtotheUnited StatesDepartmentofAgriculture(USDA).
Soilextractionwasperformedaccordingtoarecommendedpro- cedurefordeterminationofCWCrelatedchemicals[38].Aliquots of5gsoilwereweighedinto30mLfluorinatedethylenepropylene tubes(NalgeNuncInternational,Rochester,NY,USA)andextracted twicewith5mLtypeIwater.Thetubeswereshakenfor10min
at2000rpmona MultiReaxtesttubeshaker(HeidolphInstru- ments,Schwabach,Germany)andcentrifugedat3200×gfor5min onaCentraCL3RfromIEC(Needhamheights,MA,USA).Thesuper- natantswerecombinedin15mLpolyethylenesampletubesfrom SarstedtAG&Co.(Nümbrecht,Germany),andasecondcentrifuga- tionwasperformedat6200×gfor30minonaHeraeusMegafuge 1.0R(DJBLabcare,NewportPagnell,UK).Ifnototherwisedescribed, thesupernatantwasfilteredthroughaMillexPVDF0.22mfilter (Millipore,Carrigtwohill,Co.Cork, Ireland)andadded0.1%(v/v) CH3COOH.
2.3. Instrumentalconfiguration
AnUltimate3000RSLC(DionexCorporation,Idstein,Germany) wascoupledtoaMicroTof-QIImassspectrometer(BrukerDalton- ics,Bremen,Germany).Aschematicdiagramofthefinalsetupfor sampleloadingandchromatographicseparationisshowninFig.2.
TheSPE–LCsystemwaslocatedinsideanFLM-3100flowmanager supportedwithtwo10-ports,two-positionmicroswitchingvalves (onlyoneusedinthefinalmethod),andwithatemperatureof 35◦C.Theloadingflow(P1)wasdeliveredfromaDGP-3600Mdual gradientpumpviaaWPS-3000autosamplerwithvariablevolume split-loopinjectionanda500Lsampleloop.Solventsdelivered byP1were(A)typeIwater;(B)40mMACand0.75%(v/v)NH4OH inwater/ACN(60/40);(C)2%(v/v)CH3COOHinwater/ACN(96/4).
TheLCflowwasdeliveredfromchannel2oftheDGP-3600Mpump (P2).SolventsdeliveredbyP2were(A)typeIwater;(B)acetoni- trile;(C)200mMAA.PreconcentrationwasperformedonaZrO2
column (2mm×10mm,3m)fromZirChromSeparations,Inc., Anoka,MN,USA.SeparationwasachievedwithaNucleodurPyra- mideC18column(2mm×100mm,1.8m)fromMacherey-Nagel GmbH&Co.KG,Düren,Germany.The0.2mpre-filterwasfrom ThermoFisherScientificInc.,Bellefonte,PA,USA.
Aqueous soil extractsof 300L were loadedonto theZrO2
column with 100% (A) at 300Lmin−1, delivered by P1. After 3min,theswitchingvalvewasshiftedto“Inject”positionandthe ZrO2 column wasbackflushedwith15mMAA at200Lmin−1, elutingtheAMPAsontotheseparationcolumn.Atthesametime, thepre-filter wasbackflushedtowastefrom P1 for removalof
Fig.2.Diagramoftheon-lineSPE–LC–MSsetup(seetextfordetails).
particlesatthefilterinlet.Thispre-filterbackflushprocedurewas firstdescribedbySvendsenetal.[39]andisslightlymodifiedin thepresentsetupforusewithoutathirdpump.At4min,thevalve wasswitchedbackto“Load”positionforgradientseparationofthe AMPAsandreconditioningoftheZrO2 column.Gradientelution fromP2 was0%(B)at3–4min,0–50% (B)in 4–12min,50–90%
(B)in12–14minand90%(B)at14–16.5min.Eluent(C)was7.5%
throughout the analysis, ensuring a constant concentration of 15mMAA.Afterreturningtostartgradientconditions,thecolumn wasequilibratedfor11min,givinganinjection-to-injectioncycle timeof28min.Duringgradientseparation,theZrO2columnwas re-conditionedbyP1with100%(B)at4–9.5minand50%(A)/50%
(C) at 10–15.5min. Finally, the preconcentration column was flushedwith100%(A)priortonextinjection.
TheESIwasoperatedinnegativeionisationmodewithacap- illaryvoltageof 3500V and anend plate offsetof −500V. The collisioncellenergywas5.0eVandcollisionRFpeak-to-peakvolt- agewas150V.Nitrogenfornebulisinggas(1.2bar)anddryinggas (8.0Lmin−1,200◦C)wasprovidedbyahighpuritygenerator(Dom- nickHunter,Durham,UK).CompressedN2 (purity6.0)fromAGA AS,Oslo,Norway,wasusedas collisiongas.Massspectrawere acquiredin them/zrange 50–500,andquantitativecalculations wereperformedwithpeakareasoftheextractedquasimolecular ions[M−H]−±5mDa.
2.4. Solidphaseextraction
Fourcolumnswith differentstationaryphases wereinvesti- gatedforpreconcentrationoftheAMPAsfromaqueoussamples:
HypercarbPGC(2.1mm×10mm,5m)fromThermoFisherSci- entific Inc.; Oasis MAX (2.1mm×20mm, 30m) from Waters Corporation, Milford, MA, USA; ZrO2 (Section 2.3) and TiO2
(2mm×10mm,5m)fromZirchrom.TheperformanceoftheSPE columnswasfirstinvestigatedwiththesetupasdescribedinFig.2, butwithoutseparationcolumn.Instead,the“Waste”and“LC–MS”
outlets were connected to the MS during “Load” and “Inject”, respectively,viathesecondswitchvalveintheflowmanager.In thisway,bothpotentialbreakthroughoftheanalytesduringsam- pleloadingandthedesorptionratecouldbemeasured.Threeofthe AMPAs(EMPA,iPMPAandPMPA)ataconcentrationof20gL−1 intypeIwaterandaqueousextractsofSoilAwereusedforinves- tigationoftheperformance ofthecolumns.Optimisationofthe washingprocedurefortheZrO2andTiO2columnswasperformed withtheMSinthem/zrange300–4000,tomeasurethesignalof elutedhumicandfulvicacids.
TheperformanceofthedifferentSPEcolumnsinthecomplete on-lineSPE–LC–MSsetupwasinvestigatedwithEMPA,iPMPAand PMPAadded toan aqueous extract of Soil A at 20gL−1. The soilextractwasdividedintwoparts,and onepartwasfiltered (0.22m)andadded0.1%(v/v)CH3COOH.Theotherpartofthe extractwaselutedthrougha2.5mLBa/Ag/Hanionprecipitation cartridge(DionexCorporation,Sunnyvale,CA,USA).Thesetupand procedureforanionprecipitationwereasdescribedina former study[24],except thatnoCaCl2 wasadded priortotreatment.
ThepHwasmeasuredbeforeandaftertreatmentwithanOrion2- StarpHmeterfromThermoFisherScientificInc.TheSPEcolumns wereinvestigatedaccordingtotheanalyticalproceduredescribed inSection2.3,butwithuseofaNucleodurGravityC18separation column(2mm×100mm,1.8m)fromMacherey-NagelGmbH&
Coandwith2%ACNintheloadingsolventandasstartgradient.
WhenusingthePGCcolumnforSPE,theswitchingvalveremained in“Inject”positionduringgradientseparation.Aliquotsof300L wereinjectedontheSPEcolumns,andrecoverieswerecalculated bycomparingtheobtainedpeakareaswiththosewherethesame amountsofAMPAsintypeIwaterwereinjected(n=4).
2.5. Methodvalidation
Methodvalidationwasperformedwiththeanalytesaddedto aqueousextractsofSoilA.Thelinearitywasinvestigatedatsixcon- centrationlevels,namelyat1,10,25,50,75and100timesthelimits ofquantification(LOQs).TheSPE–LC–MSmethodrepeatabilitywas investigatedatLOQand50×LOQbyperformingsixanalysesofone extractsubsequently(withinassay),andbyinjectingafreshlymade extractforsixconsecutivedays(betweenassay).
RecoveriesoftheAMPAswereinvestigatedwhenaddedtothe aqueousextractsofthedifferentsoiltypesshowninTable1,atcon- centrationsof50×LOQ.Threeextractionswereperformedforeach soiltypeandeachsubsequentlyspikedextractwasanalysedtwo times.Therecoverieswerecalculatedbycomparingtheobtained peakareaswiththosewheretheAMPAswereaddedtotypeIwater (n=6).Possibleionsuppressionwasinvestigatedbycontinuously introducingtheAMPAsaftertheseparationcolumnwhenanalysing thesoilextracts.At-piecewasmountedbetweentheseparation columnandESI–MS,andcoupledtoa500LsyringefromHamilton BonaduzAG(Bonaduz,Switzerland).Thesyringewasmountedona KDS100syringepumpfromKDScientific(Holliston,MA,USA),and theAMPAswereintroducedasa25gmL−1solution(50gmL−1 forEMPA)at5Lmin−1.
3. Resultsanddiscussion
Aqueoussoilextractsvaryalotincompositiondependingonthe characteristicsoftheextractedsoil.Forexample,agriculturalsoils givehighamountsoforganiccompoundsintheaqueousextract, possiblyinterferingwithfurthersamplepreparationsteps.Inan earlierstudy,weemployedthePGCcolumninon-lineSPE–LC–MS fortracedeterminationofAMPAsinnaturalwatersamples[24].
Themethodworkedwellalsofortheaqueousextractofasandy soilofloworganiccontent,butlowerrecoverieswereobserved whenhandlingagriculturalorclaysoils(resultsnotshown).There- fore,wewantedtoexplorealternativestationaryphasesforon-line SPE–LC–MSin ordertoachieve thehighest possiblerobustness andsensitivityindeterminationoftheAMPAsinawiderangeof aqueoussoilextracts.
3.1. ScreeningofstationaryphasesforSPE
Fourdifferentstationaryphases,includingthePGC,wereinves- tigated as SPE materials in an on-line SPE–LC–MS setup for determination ofexpected low concentrationsof theAMPAsin aqueoussoilextracts.TheSPEmaterialofchoicemustbeableto isolatethehighlypolarAMPAsfromaqueousextractspossiblycon- taininghighamountsofinterferingcompoundssuchashumicand fulvicacids.Rapiddesorptionoftheanalytesshouldsubsequently beachievedusinganeluentthatisMSfriendlyandcompatiblewith theseparationstep.Moreover,ifthereareco-extractedcontami- nantsfromthesoilextractsthatarenotelutedtogetherwiththe analytes,theseshouldbewashedoutfromtheSPEcolumnpriorto subsequentinjections.
Intheformerstudy,wecombinedPGCSPEwithhydrophilic interactionLC(HILIC).The higheramountof organicsolventin HILICcomparedtoRPseparationgiveshighersensitivityinESI–MS duetotheenhancedionisationefficiency[40].However,sincethe SPEcolumnmustbeequilibratedwiththesameamountoforganic solventpriortoHILIC,themethodismorepronetoanalytebreak- throughduringpreconcentration.Hence,inthepresentstudywe haveinvestigatedtheperformanceofthedifferentSPEcolumns withmobilephaseconditionssuitedforRPseparation.First,each ofthecolumnswasinvestigatedforretentionanddesorptionof theAMPAs withoutseparation column. Then, theSPEcolumns
3.1.1. Porousgraphiticcarbon
Theanalyteswereintroducedinaloadingmobilephasecon- taining2%ACN,suitableasstartgradientconditionsforseparation byRPinteractions.FullretentionoftheAMPAswasachievedwhen solvedintypeIwaterandwith3minsampleloadingtime(corre- spondingto33columnvolumes).Backflushdesorptionwaseasily achievedbyintroductionof10mMAAinthemobilephasecontain- ing2%ACN.Hence,thePGCcolumnshowedgoodcompatibility withseparationbasedonRP.Thecompoundsfromthesoilthat showedretentiononthePGCcolumnwheninjectinganaqueous extractofsoilA(300L)wereelutedbyintroductionof10mM AAinbackflushmode.Thus,noreconditioningofthecolumnwas neededpriortothenextinjection,exceptforequilibratingwiththe loadingmobilephase.
3.1.2. Mixedmodeanionexchangecolumn
ThepolymericMAXmaterialexhibitbothstronganionexchange andhydrophobicinteractionproperties.Thus,adsorptionanddes- orptionoftheanalytesaregovernedbypH,ionstrengthandthe amountof organicmodifier. The column showedfull retention oftheAMPAswhen injected intype Iwater, but desorptionof theanalyteswasslowwhen usingmobilephaseadditives suit- ableforLC–MSdetermination.VariousH2O/ACNcompositionsand amountsofammoniumformateandformicacidwereinvestigated forelutingtheanalytes.Longdesorptiontimewasobservedespe- ciallyforPMPA(15–20columnvolumes)and5–10%carryoverwas seenforthecompoundsbetweensuccessiveinjections(resultsnot shown).BecauseofslowdesorptionoftheanalyteswithLC–MS friendlysolutionsandhighcarry-over,furtherinvestigationwas notperformedwiththeMAXcolumn.
3.1.3. ZrO2andTiO2
TheadsorptionofLewisbasesliketheAMPAsonZrO2 ispH dependent[37]andretentionshouldbeachievedatacidictoneu- tralconditions.DesorptionisobtainedbyintroducingaLewisbase intheformofhydroxylionsorotheranionsofanaddedsaltor buffer,competingfortheadsorptionsitesonzirconia.Whenthe AMPAsweresolvedintypeIwater,theywerecompletelyretained ontheZrO2columnafterelutingwith40columnvolumesof2%
ACN.TwoadditiveswereinvestigatedfordesorptionoftheAMPAs, namelyAA(pH7)andAC(pH9).With15mMofbothadditivesin 2%ACN,completedesorptionoftheanalyteswasobtainedafter elutingwith3–4columnvolumesinbackflushmode. Nosignif- icantdifferencewasseeninthedesorptionratewhetherAAor ACwasused.Duetobetterchromatographyfortheearlyeluting AMPAsontheRPseparationcolumn,AAwaspreferredasaddi- tive.However,tofullyre-establishtheretentionoftheAMPAsin subsequentinjections,theZrO2columnneededtobeconditioned inacidicsolution.Forthispurpose,aceticacidwasused.With0.1%
CH3COOHintheloadingmobilephase,morethantwicethedesorp- tionvolumewasneededcomparedtoloadingwithH2O/ACNonly.
Therefore,thecolumnwasconditionedwith1%CH3COOHprior toinjection,whilesampleloadingwasperformedinH2O/ACN.In addition,thesampleswereadjustedtopH3.5–4byadding0.1%
CH3COOH.
The TiO2 column behaved similar to theZrO2 column with respecttoadsorptionanddesorptionoftheAMPAsatdifferentACN concentrationsandtypeofadditiveused.Thus,nofurthermethod developmentwasperformedforthiscolumn. Inadditiontothe AMPAs,theperformanceofthesecondarynerveagentdegrada- tionproduct,MPA,wasinvestigatedontheZrO2andTiO2columns.
Completeretentionwasachievedonbothcolumns,butdesorption ofthecompoundwasveryslowwhenusingAAorACasmobile
0 20 40 60 80
EMPA iPMPA PMPA
% Recove TiO2
Fig.3.RecoveriesoftheAMPAsfromanaqueousextractofsoilAbyon-line SPE–LC–MSwithPGC,ZrO2andTiO2asSPEcolumns,givenasmeanvalues±SD (n=4).
phaseadditives.SinceMPAwasnotconsideredessentialfordeter- minationoftheuseofnerveagents,nofurtherinvestigationwas performedwiththiscompound.
WhenaqueoussoilextractswereintroducedontheZrO2and TiO2 columns, many of the compounds with retention on the stationaryphaseswerenotcompletelyelutedbyintroductionof 15mM AA.Whenthecolumnswerewashedwith50mMACin 50%ACNbetweeninjections,acontinuoussignalform/z500–4000 wasmeasured(maximumatm/z1000–1200),probablycausedby elutedhumicandfulvicacids.Thecompoundsweremosteffec- tivelyelutedfromthecolumnswithACNconcentrationsbetween 30% and 60%. This is consistentwith what hasbeen foundfor retention ofaromaticcarboxylic acidsatdifferent ACNconcen- trationsonZrO2andTiO2[41,42].Theadditionof50mMAC(pH 9) wasmore effective for eluting the compoundscompared to adjustingthepHto11withNH4OH.Inorganicphosphate,how- ever,adsorbsstronglytoZrO2andisreportedtoberemovedonly underalkalineconditions[43].Thewashingsolutionwasthere- foreadded40mMACandthenadjustedtopH10with0.75%v/v NH4OH.
3.1.4. RecoveriesfromdifferentSPEcolumns
TofindthemostsuitableSPEmaterialforthecurrentapplica- tion,therecoveriesofEMPA,iPMPAandPMPAonthePGC,ZrO2 and TiO2 columnswerecompared. Theanalytes wereaddedat 20gL−1eachtoanaqueousextractofSoilA.Extractionwasper- formedaccordingtotheproceduredescribedinSection2.2.The columnsweremountedintheon-lineSPE–LC–MSsetupasshown inFig.2,andaliquotsof300Lwereinjected.Theanalysiscon- ditionswereasdescribedinSection2.3,exceptthataseparation columnwithaslightlydifferentC18stationaryphasewasused,and 2%ACNwasaddedintheloadingandstartgradientmobilephase.
ThesamplesthatwereinjectedontheZrO2andTiO2columnswere acidifiedwith0.1%CH3COOHpriortoanalysis.ForthePGCcolumn, itwasfoundinanearlierstudythatremovalofinorganicanions fromtheaqueoussamplessignificantlyimprovedtherecoveriesof AMPAs[24].Thesoilextractwasthereforetreatedwithaprecipi- tationcolumnonBa-,Ag-andH-formtoremovemajorinorganic anionspriortoinjectiononthePGCcolumn.ThepHintheextract was8.2,andwasloweredtopH3.7and3.8afteradditionofacetic acidandtreatmentbytheanionprecipitationcolumn,respectively.
TherecoveriesobtainedfromthedifferentSPEcolumnsarepre- sentedinFig.3.
TherecoveriesofEMPAandiPMPAweresignificantlyhigher withuseof theZrO2 columncompared to thetwo others.The recoveryabove100%(114±3%)foriPMPAisnotfullyunderstood, butmaybeduetoionreinforcementfrominterferingcompounds.
AlsoforPMPA,thehighestrecoverywasobtainedwiththeZrO2
column, though less evident. When using the ZrO2 and TiO2 columnsforpreconcentration,theadditionofaceticacidwassuffi- cientforpreparingthesamples.Hence,thesetwocolumnsoffered
Table2
Methodvalidationdata.
EMPA iPMPA iBMPA CMPA PMPA
LOD(gL−1) 0.5 0.3 0.05 0.3 0.05
LOQ(gL−1) 1.5 0.9 0.15 0.6 0.15
Linearity(R2),LOQ-100×LOQ 0.996 0.998 0.997 0.996 0.998
Repeatability(%RSD),n=6
Withinassay LOQ 8 1 8 8 5
50×LOQ 2 3 3 1 4
Betweenassay LOQ 13 12 22 12 13
50×LOQ 9 5 12 8 9
anadvantageoverthePGCcolumnintermsoflesslabordemand- ingandlessexpensivesamplepreparationpriortoSPE–LC–MS.In conclusion,theZrO2columnwaschosenforfurtherinvestigations dueitshighrecoveriesandminimalneedforsamplepreparation.
3.2. Methodoptimisation
In thescreening testsof theSPE columns,a C18 separation columnwithnon-polarendcappingwasused.Atthelowestrec- ommendableamountoforganicmodifier(2%ACN),theretention ofEMPAandiPMPAontheC18columnwasstilllow,givingpoor refocusingofthesecompounds. Therefore,a separation column withpolarendcappingwaschosen,whichwasstableandfunction- ingin100% aqueousmobilephasesystems.Whenstartingwith pureaqueousand15mMAA mobilephase, betterrefocusingof themorepolarAMPAswasachieved,givingmoresymmetricand higherpeaks.
The loading capacity of the ZrO2 column is an important issue as a higher injection volume will increase the sensitiv- ityofthemethod.The autosamplerwasconfiguredforvariable volume split-loop injection with 500L as the highest injec- tionvolumepossible.With500L injected,theloadingvolume needed for complete elution of the sample from the injection loopwas750L,which correspondsto33 voidvolumesofthe ZrO2column.WhentheAMPAsweresolvedintypeIwaterwith 0.1%CH3COOH(500Linjected at300Lmin−1), breakthrough occurredafterelutingwith70–110columnvolumesintheorder PMPA<iPMPA<EMPA.Withtheanalytessolvedinanextractof SoilA with0.1% CH3COOH(500L injected),the breakthrough volumewasreduced to30 columnvolumesfortheleastreten- tivecompound. Hence,breakthrough of PMPA occurredbefore theanalytes were completely introduced on the ZrO2 column.
Reducingtheloadingflowto200Lmin−1 didnotincreasethe breakthrough volume. To ensure high method robustness and repeatability,aninjectionvolumeof300Lwaschosen.Theload- ingtimeandloadingflowratewassetto3minand300Lmin−1, respectively, which correspond toa loading and wash volume ofapproximately 40 column volumes.Atthese conditions, and withcleaningandregenerationoftheZrO2columnbetweeneach injection, no reduction in retention of the AMPAs was found afterinjectingmorethan fiftyaqueoussoil extracts.The stabil- ityoftheZrO2 columnwasconfirmedbycomparingpeakareas of the analytes measured in spiked aqueous extract of soil A at different times during method validation. It was, however, observedthattheelectrosprayionsourceshouldbecleanedregu- larlyduetothedepositionofwhatwereprobablysaltsofinorganic ions.
Fig.4showstheextractedionchromatograms(EICs)ofanaque- ous extract of Soil A withthe AMPAs added at concentrations offiftytimestheLOQs(determinedinSection3.3).Nomemory effectswereobservedfortheanalyteswhenintroducingablank sample immediatelyafter the spikedsoil extract. The negative
ionESI–MSspectraweredominatedbythedeprotonatedionsat thelow collisioncellenergy of 5.0eV.Theaccuratemass mea- surementsofthetime-of-flight(TOF)MSprovidehighselectivity indeterminationoftheAMPAswithoutemploymentoftandem MS.
3.3. Methodvalidation
ThemethodvalidationwasperformedwiththeAMPAssolved intheaqueousextractofSoilAtorepresentanauthenticsample matrix.DatafromthemethodvalidationaresummarisedinTable2.
Thelimitsofdetection(LODs)weredeterminedastheconcentra- tionoftheanalytesgivingasignalintensityforthequasimolecular ionsof200–300countsatthreerepeatedinjectionswhenextracted atanaccuracyof±5mDa.Thisisfourtofivetimesthesignalheight ofthearbitrarybaselinenoisepresentwhenextractedatthishigh massaccuracy.ThesignalofCMPAwasdisturbedbyabackground contaminantwithamassdifferenceof20mDa,givingabiasinthe measuredm/zatlowintensities.Duetothisinterference,theLOD ofCMPAneededtobesetataconcentrationgivinganintensity ofapproximately900counts.Extractedionchromatogramsatthe determinedLODsareshowninFig.5.Priortothepresentstudy, LODshavenotbeenreportedfordeterminationoftheAMPAsin soilextractsbyLC–MS.Lagarrigueetal.employedtransientisota- chophoresispreconcentrationandCEseparationcoupledtoESI–MS fordeterminationofthefiveAMPAsinsoilextractswithreported LODsof 4–70gL−1 [18]. Nassaret al. have obtainedLODsfor EMPA,iPMPAandPMPAof25–50gL−1inaqueousleachatesof soilsamplesusingCEwithelectrokineticinjectionandUVdetec- tion[19].ComparedtowhatwasachievedbytheCEtechniques,
0 2 4 6 8
0 2 4 6
8 4
3 5 2
Intensity, counts (x104)
Time, min 1
1 EMPA 2 iPMPA 3 iBMPA 4 CMPA 5 PMPA
Fig. 4.EICs ([M–H]−±5mDa) from on-line SPE–LC–MS determinationof the AMPAs,addedtoanextractofaloamsoilatconcentrationsof50×LOQ.Analiquot of300Lwasloadedonthe(2mm×10mm)ZrO2column,andgradientseparation with0–90%ACN(15mMAA)wasperformedonthe(2mm×100mm)C18column withpolarendcapping.
0 400
800 m/z 177.068 ± 0.005
0
200 m/z 151.052 ± 0.005
0
200 m/z 137.036 ± 0.005
Intensity, counts
10 8
6 4
2 0
0
200 m/z 123.021 ± 0.005
Time, min
Fig.5. EICs([M–H]−±5mDa)oftheAMPAsatthedeterminedLODsinanaqueous extractofaloamsoil.Fromtop:PMPA,CMPA,iBMPA,iPMPAandEMPA.
theobtainedLODswiththecurrentmethodarelowerbyafactor ofatleast40.
TheLOQswerecalculatedasthreetimestheLODsexceptfor CMPA,where LOQwassetattwo timestheLOD.Linearitywas investigatedintherangeofLOQto100×LOQ,andhighlinearcor- relation(R2>0.995)was foundfor allcompounds. Goodwithin assayrepeatabilitywasobtainedbothatLOQ(<10%RSD)andat 50×LOQ(<5%RSD).Thesomewhathigherbetweenassayvariabil- itywasprobablycausedbybetweendayvariationsintheESI–MS response.ItmayalsobeduetominoradsorptionoftheAMPAs tocolloidalmaterialsinthesoilextracts,varyingbetweendays [23,44].Internalstandardcouldbeusedtocorrectforvariationsin instrumentalresponseandmatrixbehaviorifquantitativedetermi- nationisofhighimportance.Themainfocusinthepresentstudy wasonmethodsensitivity,andhencetheobtainedbetweenday repeatabilitywasconsideredacceptable.
Notracesof theAMPAswereobservedwhenblankaqueous extractsofthefivesoiltypeslistedinTable1wereanalysed.Hence, theanalyteswereaddedtotheextractsat50×LOQforinvesti- gationoftherecoveriesfromtheon-lineSPE–LC–MSprocedure.
Table3showsthatrecoverieshigherthan85%wereobtainedfor iPMPA,iBMPA,CMPAandPMPA,exceptwhenanalysingtheextract ofsoilE(clay).TherecoveriesofEMPAweresignificantlylower comparedtotheothercompoundsforallsoiltypes.Thisiscon- trarytowhat wasobserved fortherelative retention ofEMPA, iPMPAandPMPAontheZrO2columninSection3.2.SinceEMPA elutednearthecolumnvoid,observedreduced recoverieswere probablycausedbyionsuppression.HigherrecoveryofEMPAwas foundfromtheextractofsoilAwhenscreeningforSPEstationary
Table3
RecoveriesoftheAMPAs(addedat50×LOQ)fromaqueoussoilextractsbyon-line SPE–LC–MS,givenas%recovery±SD(n=6).
Soilextract EMPA iPMPA iBMPA CMPA PMPA
A 48±1 92±3 104±3 96±1 94±4
B 55±5 89±3 104±7 100±3 101±3
C 68±3 103±5 103±3 98±4 99±3
D 38±2 87±7 91±5 95±5 93±7
E 18±1 46±3 72±4 91±4 66±8
changeofseparationcolumnmayalsohaveresultedinco-eluting compoundswithEMPA,givingmoreionsuppression.Therecov- eriesfromsoilB,CandDwereinthesamerangeasthatofthe extract (of soil A) used in methodvalidation. Thismeans that the methodsensitivity described in Table2 could beexpected also for these samples.The recoveries from the clay soil were 38–95%comparedtowhatwasobtainedfromtheextractofsoil A,andhencethecorrespondingpoorermethodsensitivitycould beexpected.Claysoilscontainlargeamountsofmineralswithcol- loidalproperties(<0.001mm)thatcanbedistributedinthewater phasewhenperformingaqueousextraction.Thelowerrecoveries obtainedfromsoilEmaybecausedbyanalyteadsorptiontothese colloidalminerals[23,44].Nocorrelationwasseenbetweenthe recoveriesfromtheaqueousextractsandtheorganiccontentof thesoils(Table1).ThisindicatesthattheenrichmentofAMPAson ZrO2 isnotvulnerabletohighamountsoforganicmatterinthe extracts.
Toinvestigatepossibleionsuppression,theAMPAswerecon- tinuouslyintroducedaftertheseparationcolumnwhenanalysing blanksamplesofthesoilextracts.Asignificantsuppressionofthe EMPAsignal wasobservedin theregionwhere this compound eluted,andmostseverewhenanalysingtheextractofsoilE.The signalinfrontofthechromatogramwasdominatedbyabroad,tail- ingpeakofthesulphateionoverlappingwithEMPA.Thedegreeof signalsuppressionofEMPAcouldbecorrelatedtotheintensityof thesulphatepeak.SulphateisknowntoberetainedonZrO2[45]
andisamongthemajorinorganicanionsinsoil.Coelutingsulphate wasalsoobservedbyZhouandLucyinon-lineSPE–LCdetermi- nationofphosphonicdiacidsinwatersamples,usingZrO2forSPE [46].Inthatcase,theproblemwaspartiallysolvedbyincreasing theloadingtimetowashoutmostofthesulphateionspriortoLC separation.ThiswasnotpossiblefordeterminationoftheAMPAs sincethesulphateionhadstrongerretentiononZrO2comparedto theanalytes.
Therelationship betweentheobtainedLODsinthe aqueous soilextractsand thesensitivityfordeterminationoftheAMPAs in soil is dependenton theaqueous extraction efficiency. In a comprehensive study by Kataoka et al. including 21 different soil types, recoveries of the AMPAs after aqueous extraction variedfromapproximately20%andupto100%[23].Othershave reportedrecoveriesoftheAMPAsfromsoilintherangeof45–96%
[8,13,20,22]. Provided a conservative estimate of 20% aqueous extractionrecoveryfromsoil,thesensitivityofthepresentmethod wouldbeintherangeof0.5–6ngg−1forsoilA–Dand0.8–13ngg−1 forsoil E.Theinjectionvolumeof theaqueoussoilextract was 300L.Hence,asoilsampleof1gissufficienttoattaintheamount ofextractneededfordeterminationoftheAMPAsattheobtained methodsensitivity.DetectionlimitsfordeterminationofAMPAs insoilhavebeenreportedonlyafewtimes;Kataokaetal.have employedaqueousextractionofEMPA,iPMPAandPMPAfromsoils followedbyderivatisationandGC–MSdeterminationaftertreat- ingtheextractsdifferentways,achievingLODsof0.1–0.2gg−1 [9,47].In1994,Blacketal.reportedthepresenceofiPMPAamongst othercompoundsinauthenticsoilsamplesfromIraqatlevelsfrom 200ngg−1anddownto6ngg−1[4].Thesamplesweresubjectedto aqueousextractionfollowedbysolventchangeandderivatisation priortoGC–MSdetermination,wheresingleionmonitoring(SIM) was necessary for identification of iPMPA at these low levels.
Certainly, the use of SIM, for example with a quadropole MS, wouldhaveenhancedthesensitivityalsoforthepresentmethod.
ThehighresolutionfullscanMSusedinoursetup,however,makes it possibletoscreenfor AMPAsof structuresothers thanthose employed in the methodvalidation without compromising the
sensitivity.Moreover,thecurrentsamplepreparationprocedure aftersoilextraction(additionofaceticacid)ismuchfasterandless labordemandingcomparedtothemethodsusingGC–MSdeter- mination.ThismakesourfullyautomatedSPE–LC–MSprocedure wellsuitedforscreeningaqueoussoilextractsforthepresenceof primarynerveagentdegradationproducts.
4. Conclusions
Wehaveforthefirsttimedemonstratedanon-lineSPE–LC–MS methodcapableofdeterminingprimarynerveagentdegradation productsatsubppblevelsinaqueoussoilextracts.Zirconiumdiox- idewaschosenforpreconcentrationoftheAMPAsfromaqueous soilextractsratherthanTiO2,PGCandMAXduetohighrecoveries, compatibilitywithLC–MSandminimalneedforsampleprepara- tionpriortoanalysis.ThestrongLewisacidsitesonZrO2makeit abletoretaintheAMPAs,evenwhensolvedinsoilextractscon- taininghighamountsoforganicandinorganicinterferences.The analytescouldbedesorbedwiththeadditionofanLC–MSfriendly additivesuch as AA, and this made the ZrO2 stationary phase applicableinanautomatedSPE–RP–LC–MSsetup.Bywashingand reconditioningtheZrO2columnbetweeneachinjection,noreduc- tioninretentionoftheanalyteswasseenafterinjectingmorethan fiftysoilextracts.Detectionlimitsof0.05–0.5gL−1wereachieved fortheAMPAsinanaqueousextractofaloamsoil.Nomorethan 1gofthesoilisneededtoachievethissensitivity(300Lextract injected).Theonlysamplepreparationneededaftersoilextraction wastheadditionof0.1%aceticacid.Hence,theestablishedmethod iswellsuitedforscreeningaqueoussoilextractsforthepresence ofprimarynerveagentdegradationproducts.
Acknowledgement
This workwas funded by the Norwegian Defence Research Establishment(FFI).
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