E.Paglianoa,∗,M.Onorb,E.Pitzalisb,Z.Mesterc,R.E.Sturgeonc,A.D’Ulivob
aScuolaNormaleSuperiore,PiazzadeiCavalieri,7,56126,Pisa,Italy
bCNR,ConsiglioNazionaledelleRicerche,IstitutodiChimicadeiCompostiOrganometallici,ViaG.Moruzzi,1,56124Pisa,Italy cNationalResearchCouncilofCanada,1200MontrealRoad,Ottawa,ONK1A0R6,Canada
a r t i c l e i n f o
Articlehistory: Received4March2011
Receivedinrevisedform27July2011 Accepted29July2011
Available online 5 August 2011 Keywords: Vaporgeneration Trialkyloxoniumtetrafluoroborate Nitrite Nitrate Enrichedisotope
Gaschromatographymassspectrometry Wateranalysis
a b s t r a c t
Triethyloxoniumtetrafluoroboratederivatizationcombinedwithdirectheadspace(HS)orSPME-gas chromatography–massspectrometry(GC–MS)isproposedhereforthesimultaneousdeterminationof nitriteandnitrateinseawateratmicromolarlevelafterconversiontotheircorrespondingvolatileethyl- esters(EtO-NOandEtO-NO2).Isotopicallyenrichednitrite[15N]andnitrate[15N]areemployedasinternal
standardsandforquantificationpurposes.HS-GC–MSprovidedinstrumentaldetectionlimitsof0.07mM NO2−and2mMNO3−.Validationofthemethodologywasachievedbydeterminationofnitriteandnitrate
inMOOS-1(SeawaterCertifiedReferenceMaterialforNutrients,NRCCanada),yieldingresultsinexcel- lentagreementwithcertifiedvalues.Allcriticalaspectsconnectedwiththepotentialinter-conversion betweennitriteandnitrate(lessthan10%)wereevaluatedandcorrectedforbytheuseoftheisotopically enrichedinternalstandard.
© 2011 Elsevier B.V. All rights reserved.
1. Introduction
Nitriteandnitrateareubiquitousandtheavailabilityofana- lyticalmethodsfortheirsimultaneousdeterminationattraceand ultra-tracelevelsincomplexmatricesisimportantforunderstand- ingaspectsofenvironmentalandbiologicalprocesses.Theyreflect thefinalstatesofa varietyofN-compoundsinmanymetabolic pathwaysoccurringinlivingsystems[1,2].Issuesrelatedtotheir speciationinbodyfluidsareexploredinordertounderstandthe roleofNOasaregulatorymoleculeinthevascularsystem,inthe brain,andintheimmunesystem[3].DirectdeterminationofNO gasisachallengeforAnalyticalChemistryandtheuseofnitriteand nitrateasNOmetabolicproductsareexploitableforthispurpose.
Environmentalcontaminationbynitriteandnitratecanoccur throughindustrialanddomesticcombustionandfromagricultural sources;theirmonitoringhasgainedincreasingimportance[1].
Thestateoftheartoftheiranalyticaldeterminationhasbeen reviewedbyMoorcroft[1]andJobgen[4]whofocusedtheiratten- tion onliquid-phase molecular spectroscopy (determinationby UV–vis and chemiluminescence) and electrochemicaldetection. Bothtechniquesareoften usedincombination withchromato- graphicseparationorcapillaryelectrophoresisinordertoperform
∗ Correspondingauthor.Tel.:+393408428735.
E-mailaddresses:[email protected],[email protected](E.Pagliano).
on-linedetectionwhichsuffers,ingeneral,fromlimitedselectivity andsensitivity,especiallyduetohighmatrixeffects.Oneadditional drawbackisrelatedtothederivatizationstep:manyreactionshave beenproposedtoconvertnitritetoasuitablemoleculefordetection whereaslittleattentionhasbeengiventothelessreactivenitrate, whichcanbeonlydeterminedfollowingacriticalreduction.This separationstepsuffersmatrixeffectswhenrealsamplesarepro- cessed,especiallyproteinaceousmaterials.Tsikas[5]presentedan overviewofcurrentlyavailableassaysfornitrateandnitritebased ontheGriessreaction.
The useof GC–MSin this field has alsobeen proposedand recentlyreviewedbyHelmke[3].GC–MSstrategiesfortheanaly- sisofthesetwoanionsincludenitrationusingaromaticcompounds andalkylationwithpentafluorobenzylbromide(PFB-Br).Thefirst approachisapplicableonlyforthedirectdeterminationofnitrate ionasnitritedoesnotreactunderthespecifiedconditions;forits detectionacriticaloxidationstepisrequired.Thelatterapproach isabletoachievesimultaneousdeterminationofbothanions[6].In anycase,derivatizationofnitrateionbyPFB-Brrequiresdemanding reactionconditionssuchaselevatedtemperatureandhighPFB- Brconcentration[3].Tsikas[7]publishedthefirstmethodforthe determinationofnitritebyGC–MSwhichwasfreeofderivatization, butithasnotbeenusedforthedeterminationofnitrate.
Recently, analytical applicationof triethyloxonium tetrafluo- roborate salt (TEOT)has beenproposedby theauthors forthe chemicalvaporgeneration(CVG)ofCl−,Br−,I−,CN−,SCN−,S2−,
2512 E.Paglianoetal./Talanta85 (2011) 2511–2516
NO3−,NO2−[8].Trialkyloxonium“Meerwein”reagents[9,10]have provencapableofaqueousphasealkylationofseveralinorganic substrates,allowingtheirseparationfromthereactionmatrixas volatileorsemi-volatilereactionproductsofdefinedchemicalcom- position[8].TheTEOTmethodhasbeenvalidatedbydetermination ofbromideinBCR-612referencematerialandhasbeenappliedto thedeterminationofiodineandiodate[8].
TEOTconvertsnitriteandnitratetotheircorrespondingethyl estersthroughanO-alkylationreaction.Thesemoleculespresent suitablevolatilityandstabilitytobedeterminedbyGC–MS.
Inthisworkwepropose,forthefirsttime,applicationofaCVG- GC–MSmethodologyforthesimultaneousdeterminationofnitrite andnitrateatmicromolarlevel.Theprocedureissimpleandthe useofisotopicallyenrichedinternalstandardsconfersruggedness tothemethod.Validationhasbeenachievedbyanalysisofa“Sea- waterCertifiedReferenceMaterialforNutrients”,MOOS-1.Results reportedheredemonstratetheanalyticalpotentialitiesanduseful- nessofaqueousphasealkylationwithTEOTforthedetermination ofanionicspeciesbyCVG.
2. Experimental
2.1. Reagentsandmaterials
Aqueoussolutionsofisotopicallyenrichednitriteandnitrate werepreparedbydissolution ofsodiumnitrite(15N,98%+)and potassium nitrate (15N, 99%), respectively (Cambridge Isotope Laboratories,Inc.)inultrapurewaterinordertoobtainfinalcon- centrationsof200mg/L15NO
3−and20mg/L15NO2−.Analkylating solutionofTEOT(triethyloxoniumtetrafluoroborate)wasprepared bydissolvingthereagent(Fluka:TEOT>97%)inultrapurewater; thequantity of solid salt required to ensure40mg of reactant in 100mLsolution.Aqueousoxonium saltsare unstable,sothe solutionis prepared just prior tosample derivatization. Work- ingsolutionsofnitriteandnitratewerepreparedbydilutionof Flukaconcentrates(Fluka:nitrate,certifiedanionstandardsolution 1001mg/L±2mg/L; nitrite standard for IC 1000mg/L±4mg/L) withultrapurewater.Allstandardsolutionswerestoredat4◦C. 2.2. GC–MSmethods
GC–MSexperimentswereperformedusinganAgilentGC6850 equippedwithanAgilent5975CMSdetector.FortheHS-GC–MS procedure,manualsampleinjectionwasemployedusinga250mL sample volume. The oven program consisted of the following: isothermal(10min) at 30◦C, 5◦C/minto 100◦C maintained for 4min,25◦C/minto240◦Cmaintainedfor10min.Inletsetup:inlet modeispulsedsplitwithsplitratio8:1,pulsedpressure200kPa andtemperature150◦C.AmodelJ&W122-1364EDB-624column (6%Cyanopropyl-phenyl94%dimethylpolysiloxane)wasusedin constantflowmodeat0.7mLHe/min.Massspectrometrydetec- tion(70eVelectronimpact,transferline260◦C,ionsource250◦C) wasperformedinSIMmodemonitoringm/z30–31–60–61(dwell time 100ms for each m/z) for nitrousacid ethylester and m/z 46–47–76–77fornitricacidethylester(samedwelltime).
For the SPME-GC–MSprocedure, a 57310-Udf 65mm poly- dimethylsiloxane/divinylbenzene(PDMS/DVB)SupelcoSPMEfiber assemblywasused.Thefiberwasexposedtotheheadspaceofthe vialfor10minat ambienttemperature whereasthedesorption processrequired1.5minintheGCinlet.Theovenprogramcon- sistedof:isothermal(10min)at30◦C,10◦C/minto80◦C,25◦C/min to200◦Cmaintainedfor10min.Inletsetup:inletmodeispulsed
modeat1mLHe/min.Massspectrometrydetection(70eVelec- tronimpact,transferline260◦C,ionsource250◦C)wasperformed inSIMmodemonitoringm/z30–31–60–61(dwelltime100msfor eachm/z)for nitrousacidethylester andm/z46–47–76–77for nitricacidethylester(samedwelltime).
2.3. Samplepreparation
A2mLsamplewasintroducedwithoutanypretreatmentinto a4mLvialandisotopeenrichedspike(100mL)added.Afterseal- ing,100mLaqueousTEOTwasinjectedthroughtheseptum.GC–MS analysiswasperformedafter3hreactiontimeat23◦C;theHSpro- cedurerequiredtheinjectionof250mLofheadspace;SPMEentails theexposureofthefiberintheheadspacefor10min.
3. Resultsanddiscussion 3.1. Identification
Nitriteandnitrateareconvertedtotheircorrespondingethyl estersbyTEOT.TEOTisastrongalkylatingagentwhichisableto performalkylationinaqueoussolutionandtoreactwithanionsin neutralsubstrates.Inthepresentcase,O-alkylationofthesubstrate occurswiththeformationofnitrousandnitricethylester:
O N O O N O O
Novolatileproductsarising fromN-alkylation ofnitrite and nitratehave beenobserved. Nitriteand nitrateethylesters are suitableforGC–MSanalysisandtheircorrespondingEImassspec- traarereportedinFigs.1–2.Inthecaseofthenitriteethylester (Fig.1),wenotetheabsenceofamolecularionandthefragmenta- tionproduced[CH2NO2]+ion(m/z60)whichcorrespondstothe lossof 15amu (methyl loss) andtheion [NO]+ (m/z 30) which correspondstolossof45amu(ethoxyloss).Forthenitrateethyl ester (Fig.2), theinterpretation of themass spectrum is simi- lartothenitriteethylester,andtheEIspectrumisthesameas reportedintheNIST05library.Unfortunately,thereisnopossibil- ityofmakingasimilarcomparisonforthenitriteethylester.Mass spectrareportedinourpreviouswork[8]wereobtainedusinga Varian3400CXGCequippedwitha1077split–splitlessinjector directlyinterfacedtoaSaturn3iontrapmassspectrometer(Var- ian).Mass spectrapresented hereareobtainedwithadifferent apparatusandtunesetting.Asaconsequence,somesmalldiffer- encesin relative intensitiesof thefragmentionscan benoted. WiththeactualsettingsoftheMS,themassspectraareofbet- terqualitywithaviewtoanalyticaldeterminationofnitriteand nitrate.
3.2. Quantificationmethodandinter-conversionofnitriteand nitrate
Withtheproposedmethod,theproblemarisingfromthepoten- tialchemical inter-conversion of nitrite and nitrateduring the derivatizationstephasbeenevaluated.Ithasbeenproventhatno detectableconversionofnitratetonitriteariseswhereasthecon- versionofnitritetonitrateisobservedandoccurstotheextentof about10%atthe0.2mMNO2−concentrationlevel.Thisproblem likelyoriginateswiththepresenceofdissolvedatmosphericoxy- gen[11,12]andthestrongacidicconditionsthatarisefromtheacid hydrolysisofTEOT.Thisinterferingeffectcanbecorrectedforby
E.Paglianoetal./Talanta85 (2011) 2511–2516 2513
Fig.1.Nitrousacidethylester,CH3CH2ONO.(a)Fullscanmassspectra(m/z30–100)ofthestandardofnaturalorigin.(b)Fullscanmassspectra(m/z15–100)ofthe15N
2514 E.Paglianoetal./Talanta85 (2011) 2511–2516
useforclassicalisotopedilutionmethodology,butheresomecon- siderationshavebeengiventocorrectfortheproblemofconversion ofNO2−toNO3−andtheinstrumentalmassbiaswhichresultsin isotoperatiosdifferentfromtheirvaluesexpectedfromthenatural andenrichedabundancesof14Nand15N.
DefiningNxand Ny asthesignals(m/z30and 31)measured from10mg/lNO2−standardofnaturaloriginandExandEyasthe signals(m/z30and31)measuredfrom10mg/lNO2−isotopically enrichedspecies,itispossibletodefinethefollowingparameters (xA N,xAE,yAN,yAE): xA N= Nx Nx+Ny xA E= Ex Ex+Ey yA N= Ny Nx+Ny yA E= Ey Ex+Ey (1)
IftheMSsignalsarenotaffectedbymassbias,theabovedefi- nitionsofxA
N,xAE,yAN,yAEwillbesimplytherelativeabundances of14Nand15Ninthenaturalandenrichedsamples.
DefiningX1andY1asthesignals(m/z30and31)measuredfrom theblendofthesamplespikedwithisotopicallyenrichedstandard andN1andE1asthefractionofanalytefromnaturalandenriched origininthisblend,wecanwrite:
X1=xAN·N1+xAE·E1
Y1=yAN·N1+yAE·E1 (2)
TheconcentrationoftheanalyteCAinthesampleisrelatedto theconcentrationofenrichedstandardCEandisgivenby: CA=CE·
N1 E1
(3) AnapproximatevalueforCAshouldbeobtainedbasedonthe value of CE calculated from the gravimetric preparation of the enrichedstandard. Inanycase,thebestresultsareobtainedby measuringthevalueofCEusingthesamemethodinordertotake intoconsiderationtheeffectswhicharisefromtheconversionof nitriteandfrommassbias.Inotherwords,CEbecomesanempirical quantificationparameter.
Itsvaluecanbecalculatedbyreverseisotopedilution,forwhich asecondblendpreparedfromastandardofnitritewithaknown concentrationC0
AclosetothatofthesampleCA,andspikedwith thesameamountofenrichedstandardisprocessed.Ifthereare nosignificantmatrixeffects,theresponseofthissecondblendand ofthesamplemustbesimilar.Additionally,thechemicalbehavior andinterferenceswillalsobesimilar.
DefiningX2andY2asthesignals(m/z30and31)measuredfrom thisstandardspikedwithisotopicallyenrichedcompoundsandN2 andE2asthefractionofanalytefromnaturalandenrichedorigin present,itispossibletowrite:
X2=xAN·N2+xAE·E2
Y2=yAN·N2+yAE·E2 (4)
TheconcentrationofthestandardC0
Aisaccuratelyknown,sothe concentrationofenrichedstandardCEisgivenby:
CE=CA0· E2 N2
(5)
Table1
SummaryofresultsfordeterminationofnitriteandnitrateinMOOS-1. Nitrite(mM) Nitrate(mM) HS-GC–MS SPME-GC–MS HS-GC–MS SPME-GC–MS 3.15 3.19 21.7 21.3 3.15 3.42 22.3 23.5 3.20 3.48 22.3 23.8 3.15 3.21 22.4 23.4 3.16±0.03 3.3±0.2 22.2±0.3 23±1 Certifiedvalues:nitrite3.06±0.15mM;nitriteandnitrate:23.7±0.9mM. Boldlinereportsthemeanoffourindependentmeasurementsperformedusing HSandSPME.Theresultsarecoherentandprovideevidencethattheprecision achievedwiththeHSmethodisbetterthanthatwithSPME.Theoverallmean betweenthesedataisgivenby:nitrite3.2±0.1mM;nitrate22.6±0.9mM;nitrite andnitrate:26±1mM. CA=CE· N1 E1 =C0 A· E2 N2 ·N1 E1 =C0A· yA N·X2−xAN·Y2 yA N·X1−xAN·Y1 · yA E·X1−xAE·Y1 yA E·X2−yAE·Y2 (6) Defining: N= xA N yA N E= xA E yA E (7)
theformalismof(6)becomes: CA=C0A· X2−N·Y2 X1−N·Y1 ·X1−E·Y1 X2−E·Y2 (8) Theabovediscussionisfocusedonnitrite,butthesameisvalid fornitrate(m/z 46and 47).Equation(8) presentsthefinal for- mulausedforthecalculation.Thisisnotaconventionalwayof employinganisotopicallyenrichedstandardforquantificationpur- poses;indeed,equation(8)doesnotdependontheconcentration of enriched standard or ontherelative abundancesof 14N/15N in thesample and in the internal standard. Fromthis point of view,thisprocedureisquitedifferentfromarigorousisotopedilu- tionmethodology.Here,calibrationisachievedthroughthevalues of N, E and X2, Y2, which are empirical parameters obtained frommeasurementsperformedinaparticularinstrumentalsetting characteristic of the experiments.In all measurements, a solu- tioncontaining2.17mMNO2−and16.1mMNO3−wasusedinthe reverseexperimentforcalibrationpurposes(X2,Y2value). 3.3. QuantificationofnitriteandnitrateinMOOS-1
Quantitative datarelated toanalyticalcontent ofnitrite and nitrateinMOOS-1(SeawaterCertifiedReferenceMaterialforNutri- ents)are summarized in Table 1.The related chromatogram is presentedinFig.3.Inordertoperformquantification,ionextrac- tionatm/z30–31andatm/z46–47wascarriedoutfornitriteand nitraterespectively;peakheightswereemployedforallcalcula- tions.Alldatareportedareobtainedapplyingequation(8).Other experimentsnotreportedhere,basedontheclassicalinternalstan- dard(IS)andonstandardadditions(SA),providedresultsinaccord withthosesummarizedinTable1.Furthermore,theslopesofboth SAandISplotswereconsistent,suggestingthatmatrixeffectswere accountedforbytheuseoftheenrichedstandard.
Regardinganalytical figures ofmerit, instrumental detection limits(concentrationoftheanalytethatproducesapeakwitha
E.Paglianoetal./Talanta85 (2011) 2511–2516 2515
Fig.3.HS-GC–MSchromatogramcollectedinSIMmodeduringanalysisofMOOS-1sample.Nitriteion:convertedintoethylesternitrousacid,retentiontime8.61min,mass extracted30.Nitrateion:convertedintoethylesternitricacid,retentiontime22.02min,massextracted46.Thepeakelutinginfrontofethylnitriteismethoxyethane,an impurityarisingfromthetriethyloxoniumtetrafluoroborate.
Table2
RecoverytestperformedonMOOS-1.
Nitrite(mM) Nitrate(mM)
Added Expected Found Rec.(%) Added Expected Found Rec.(%) 0 3.06 3.21 105.0 0 20.6 22.2 107.6 0 3.06 3.17 103.5 0 20.6 22.4 108.5 2.72 5.78 5.77 99.8 20.2 40.8 44.8 109.8 2.72 5.78 5.75 99.6 20.2 40.8 43.2 105.7 5.43 8.49 8.09 95.2 40.4 61.0 62.9 103.1 5.43 8.49 8.10 95.3 40.4 61.0 62.1 101.7 10.87 13.93 12.91 92.7 80.8 101.4 106.5 105.1 10.87 13.93 13.27 95.3 80.8 101.4 106.3 104.8 Therecovery(Rec.,%)hasbeencalculatedastheratioofmeasuredresponse(found) andconcentrationexpected(expected).Abiasoflessthan10%isevident.
thetemperatureoftheHS,theamountofHSinjectedandtimeof exposureoftheSPMEfibertotheHS.
FromTable1,itisevidentthattherearenosignificantdiffer- encesbetweentheconcentrationsofnitriteandnitrateobtained usingHS-GC–MSandSPME-GC–MSprocedures.Theoverallmean oftheresultsfortheconcentrationofnitriteis3.2±0.1mM(RSD 4.0%,n=8)andfortheconcentrationofnitrateis22.6±0.9mM(RSD 4.0%,n=8).Thesumofnitriteandnitratecanbecalculatedtobe 26±1mM(RSD4.0%,n=8).Theseresultsareinexcellentagree- mentwiththecertifiedvaluesforMOOS-1,i.e.3.06±0.15mMfor nitriteand23.7±0.9mMfornitriteandnitrate.
Table2reportsresultsforarecoverytestperformedonMOOS-1; thevaluesobtainedconfirmnotonlytheaccuracyofthisanalytical methodbutthatitisabletocorrectfortheconversionofnitrite tonitrate.Finally,Table3presentsrecoverydatafornitritefrom amineralwatercontainingnodetectableconcentrationofnitrite. Fouradditionsatdifferentconcentrationsofnitritewereperformed spanningmorethanthreeordersofmagnitudeofconcentration. DespitecalculationofX2andY2fromequation(8)beingbasedinall casesonthereverseexperimentperformedonasolutionof2.17mM NO2−and16.1mMNO3−,noerrorsareevidentfortherecoveryof
Table3
Recoverytestperformedonmineralwater.
Nitrite(mM) Nitrate(mM) Added Found Rec.(%) Found
0 n.d. – 121.3
0.22 0.20 92.3 117.5
2.15 2.15 99.8 120.7
21.52 21.06 97.9 116.8 215.21 203.95 94.8 107.7
Calibrationinbasedinallcasesonreversecalibrationperformedwithasolutionof 2.17mMNO2−and16.1mMNO3−.
instrumentalandchemicalproblemspossiblyarisingwiththispro- cedurearewellcorrectedforbytheuseoftheenrichedstandard andbytheselectedmethodofquantification,whichdemonstrates theruggednessthattheuseofanenrichedstandardimpartstoan analyticalprocedure.
4. Conclusion
AqueousphasealkylationwithTEOThasbeenappliedtothe determinationofnitriteandnitrateinasampleofseawatercer- tified reference material (MOOS-1) by CVG-GCMS. Nitrite and nitratewereconvertedtotheircorrespondingvolatileethylesters andcouldbesimultaneouslydetectedanddeterminedusingdif- ferent approaches, including HS-GC–MS and SPME-GC–MS.The two procedures provide similaranalytical figures of merit and directHSispreferredforanalysiswhenmanualinjectionisper- formed, while SPME should be selected when the automation of the procedureis required. The resultsobtained for MOOS-1 using an isotopically enriched internal standard for quantifica- tionprovidedresultsinexcellentagreementinternallyandwith thecertifiedvalues.Theproblemofchemicalinter-conversionof nitritetonitrate(lessthan10%)iswellaccountedfor usingthe present method.Tothe bestof ourknowledge,this is thefirst
2516 E.Paglianoetal./Talanta85 (2011) 2511–2516
figuresofmeritwhileseparatingthederivatizedanalytesfromthe samplematrix.Moreover,useofenrichedisotopesmakesavail- abletheultimateprimarycalibrationtechniqueofisotopedilution. A reverse spike against a primary natural abundance standard affordstraceability,makingthemethodsuitableforcertification purposes and promising for application tosamplesof different origin.
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