Automated Analysis of Non-Steroidal Anti-Inflammatory Drugs In

Original 環境化学 Vol.１８,No.４［２００８］ ［ JournalofEnvironmentalChemistryVol.１８,No.４,pp.５１１-５２０,２００８］

AutomatedAnalysisofNon-steroidalAnti-inflammatory DrugsinEnvironmentalWaterbyOn-lineIn-tube Solid-phaseMicroextractionCoupledwithLiquid Chromatography-Tandem MassSpectrometry

KurieOHCHO,KeitaSAITOandHiroyukiKATAOKA

SchoolofPharmacy,ShujitsuUniversity (１-６-１ Nishigawara,Okayama,Okayama７０３-８５１６ Japan)

［ReceivedMay７,２００８;AcceptedAugust２２,２００８］

Summary A simpleandsensitivemethodforthesimultaneousdeterminationof１５ non-steroidalanti- inflammatorydrugs(NSAIDs)—acetaminophen,ibuprofen,naproxen,fenoprofen,flurbiprofen, loxoprofen,ketoprofen,mefenamicacid,flufenamicacid,diclofenac,tolfenamicacid,oxaprozin, phenylbutazone,indomethacin,andacemetacin—inenvironmentalwaterwasdeveloped.This method consistsofin-tube solid-phase microextraction (SPME) coupled withliquid chromatography-tandem massspectrometry(LC-MS-MS).TheseNSAIDswereanalyzedwithin １０ min usinganODS-３ column and５ mM aqueousammoniumformate/acetonitrile(６０/４０, v/v)asthe mobilephase.Electrosprayionizationconditionsinthenegativeion modewere optimizedforMS-MS detectionofthesedrugs.Theoptimum in-tubeSPME conditionswere ２０ draw/ejectcyclesof４０μ ofsampleataflowrateof１５０μ/minusingaCarboxen-１００６ PLOTcapillarycolumn asanextractiondevice.TheextractedNSAIDswereeasilydesorbed fromthecapillarybypassageofmobilephase.Usingthein-tubeSPME/LC-MS-MS method, goodlinearityofthecalibrationcurve(r ０．９９９７)wasobtainedintheconcentrationrange from０．１～１０ ng/m forallcompoundsexamined.Thelimitsofdetection(S/N＝３)ofNSAIDs rangedfrom ５～６５ pg/m.Thismethodcouldbesuccessfullyappliedtoanalysisofsurface waterandwastewaterwithoutanyotherpretreatmentorinterferencepeaks.Therecoveriesof NSAIDsspikedintoriverwaterwereabove８０%,andtherelativestandarddeviationswere below ８．３%.AmongtheNSAIDstestedinthisstudy,loxoprofenwasdetectedinhospital wastewaterat４５８ pg/m.

Keywords:in-tube solid-phase microextraction,automated samplepreparation,liquid chromatography-tandem massspectrometry,non-steroidalanti-inflammatorydrugs, environmentalwatersamples

aquaticenvironment１-５）.A widerangeofthesecom- INTRODUCTION pounds,some ofwhichhavethepotentialtoharm Inrecentyears,therehasbeenanincreaseincon- aquaticlifeeveninsmallquantities,hasbeendetected cernregardingtheoccurrence,fate,andtoxicityof atppborpptlevelsinenvironmentalwatersthrough- pharmaceuticalsandpersonalcareproductsinthe outtheworld.Manyoftheseenvironmentalpollutants

―５１１― areproducednotonlybyveterinaryandhumanexcre- plicationshavealsobeenreviewed３０）. tionbutalsobythedisposalofmedicinesinhospital Inthepresentstudy,wedevelopedanautomated andhouseholdwaste. on-linein-tubeSPME/LC-MS-MS methodforthesi- Non-steroidalanti-inflammatorydrugs(NSAIDs) multaneousdeterminationof１５ NSAIDsinenviron- arewidelyusedinhuman andanimalhealthcare, mentalwaterstoachievehighthroughputanalysis.We mainlyasantipyreticandpainkillingdrugs.Thereare alsousedthismethodfordeterminationofNSAIDsin morethan５０ differenttypesofNSAIDonthemarket severalenvironmentalwatersamples. inJapan,some ofwhichhaveconsumptionratesof over１０ tperyear.NSAIDshavebeendetectedinen- MATERIALSAND METHODS vironmentalsamples,includingwastewaterandsurface water６-２０）,anda numberofstudieshaveshownthat Materials NSAIDsarenoteliminatedinsewagetreatmentplants Fig.１ showsthestructuresofthe１５ NSAIDs becauseoftheirpolarstructuresandhighstability.In usedinthisstudy.Acetaminophen,ibuprofen,naproxen, addition,thereisagreatdealofconcernregardingthe fenoprofen,flurbiprofen,loxoprofen,ketoprofen,mefe- behaviorandfateofNSAIDsinenvironmentwaters namicacid,flufenamicacid,diclofenac,tolfenamicacid, duetotheirpharmacologicalactivity.Therefore,itis oxaprozin,phenylbutazone,indometacin,andacemetacin necessarytodevelopasimple,rapid,andsensitiveana- werepurchasedfromSigma-Aldrich(SaintQuentinFal- lyticalmethodformonitoringofNSAIDsatnaturallyoc- laviers,France).Eachcompoundwasdissolvedin curringlevels. methanolto makeastocksolutionataconcentration AnalysisofNSAIDsinenvironmentalsampleshas of１ mg/m.Thesestocksolutionswerestoredat４℃ beencarriedoutbygaschromatography-massspec- anddilutedtotherequiredconcentrationswithpure trometry(GC-MS)１３，１４）,highperformanceliquidchroma- waterpriortouse.LC-MS gradeacetonitrileandwa- tography(HPLC)withUV１５，１６）,LC-massspectrometry terasthe mobilephasewerepurchasedfromKanto (LC-MS)１６）,andLC-tandem massspectrometry(LC- Kagaku(Tokyo,Japan).Allotherchemicalswereofana- MS-MS)１７-２０）.However,GC-MS methodsrequiretime- lyticalgrade. consumingderivatizationofthesecompounds.Although LC-MS-MS methodsarehighlysensitiveandselec- Samplecollectionandpreparation tive,thesensitivitiesofHPLC-UVandLC-MS meth- Surfacewaterandwastewatersampleswerecol- odsareinsufficientforapplicationtoanalysisof lectedin １- pre-cleanedamberglassbottleswith environmentalwatersamples.Furthermore,mostof polytetrafluoroethylene(PTFE)linedcaps,andfiltrated thesemethodsrequirerelativelylargesamplevolumes through０．２-μm nylonsyringefilters１３ mm indiame- andlaboriouspretreatmentofsamplespriortoanaly- ter(Tosoh,Tokyo,Japan)ifnecessary.Samplingwas sis.Thedetailsoftheseanalyticalmethodsfordeter- performedatsixsitesintheareaaroundAsahiRiver miningNSAIDsinenvironmentalsampleshavebeen andSasagaseRiverinOkayamaCity,includingwastewa- summarizedinrecentreviews２１-２３）. terfrom hospitalsandwastewatertreatmentplants Thein-tubesolid-phase microextracation(SPME) (WWTP),andtapwaterfromourlaboratory.Thesam- technique２４）,usinganopentubularfused-silicacapillary pleswerestoredinthedarkat４℃ andthenanalyzed withaninnersurfacecoatingastheSPME device,is within４８ h.Samples(０．５～０．９ m)wereaddedto simpleandcanbeeasilycoupledon-linewith HPLC ０．１ m of２００ mM Tris-HClbuffer(pH ８．０),and andLC-MS.In-tubeSPME allowsconvenientautoma- madeuptoafinalvolume of１ m withwaterin２-m tionoftheextractionprocess,whichnotonlyreduces screw-cap autosampler vialsequipped with theanalysistime,butalsoprovidesbetteraccuracy, silicon/PTFEsepta,andthevialswerethensetonto precision,andsensitivitythanoff-line manualtech- thesampletrayintheautosampler. niques.We havereportedthedevelopmentofthein- tubeSPME methodfordeterminationofvariouscom- Instrumentandanalyticalconditions pounds,suchasdrugsandendocrinedisruptors,by The HPLC system wasa Model１１００ series couplingwithHPLC２５）,LC-MS２６，２７）,andLC-MS-MS２８，２９）. (AgilentTechnologies,BoeblingenGermany),whichcon- Thedetailsofthein-tubeSPME techniqueanditsap- sistedofabinarypump,anon-line-degasser,anauto-

―５１２― 環境化学 Vol.１８,No.４［２００８］

Fig.１ Structuresofthe１５ non-steroidalanti-inflammatorydrugsexaminedinthisstudy sampler,acolumn compartment,adiodearraydetector, In-tubesolid-phasemicroextraction andanHP ChemStation.AnODS-３ column (１００ mm AsshowninFig２,aCarboxen-１００６ porouslayer ×２．１ mm,particlesize５ μm;GLScience,Tokyo,Ja- opentubular(PLOT)capillarycolumn (６０ cm×０．３２ pan)wasusedforHPLCseparation.Chromatography mm i.d.,１７μm filmthickness;Supelco,Bellefonte, wasperformed byisocraticreversephaseseparation PA,USA)wasusedasthein-tubeSPME deviceand with ５ mM aqueousammonium formate/acetonitrile wasplacedbetweentheinjectionneedleandtheinjec- (６０/４０ v/v)ataflowrateof０．３ m/min andcolumn tionloopretainedinthesystemtoavoidfoulingofthe temperatureof４０℃ . meteringpump.Capillaryconnectionswerefacilitated ElectrosprayMS-MS forthe１５ NSAIDswasper- byuseofa２．５ cmsleeveof１/１６-inpolyetheretherke- formed onanAPI４０００ triplequadruple massspec- tone(PEEK)tubingateachendofthecapillary.PEEK trometer(AppliedBiosystems,FosterCity,CA,USA), tubingwithaninternaldiameterof３３０ μm was equippedwithaturboionsprayinterfaceoperatedin shown tobesuitabletoaccommodatethecapillary negativeionmodeat４５００ V and４５０°C.Nitrogenasa used.Standard１/１６-instainlesssteelnuts,ferrules, nebulizinganddryinggaswasgeneratedfromcom- andconnectorswereusedtocompletetheconnec- pressedairusingaKakenN２ generator(SystemInstru- tions.Theautosamplersoftwarewasprogrammed to mentsCo.,Ltd.,Tokyo,Japan).Theionsourcesgas１ controlthein-tubeSPME extraction,desorption,andin- (GS１)and２ (GS２)weresetat４０ and５０/min,re- jection.(A)Samplingandextraction:vials(２ m)were spectively.Thecurtaingas(CUR)flowwassetat４０ filledwith１ m ofsampleforextraction,andsetinto /minandthecollisiongas(CAD)at３．０/min.Quan- theautosamplerprogrammedtocontroltheSPME ex- tificationwasperformed by multiplereaction monitor- tractionanddesorptiontechnique.Inaddition,２-m ing(MRM) ofthedeprotonatedprecursor molecular autosamplervialswithaseptum,onecontaining１．５ ions[M-H]－ andtherelatedproductionforNSAIDs. m ofmethanolandanothercontaining１．５ m ofwa- QuadrupolesQ１ andQ３ weresetonunitresolution. ter,weresetintotheautosampler.Thecapillarycol- MRM inthenegativeionization modewasperformed umn waswashedandconditionedbytworepeated usingadwelltime of１５０ ms pertransitiontodetect draw/ejectcycles(４０μ each)ofthesesolvents,and ionpairs.Table１ showstheoptimizedMS-MS condi- thena５０-μ airplugwasdrawnpriortotheextrac- tionsforeachcompound.LC-MS-MS datawereproc- tionstep.Theextractionof１５ NSAIDsontothecapil- essedusingAnalystSoftware１.３.１ (AppliedBiosystems). larycoatingwasperformedby２０ repeateddraw/eject

―５１３― Fig.２ Schematicdiagramofon-linein-tubeSPME/LC-MS-MS system. (A)Samplingandextraction;(B) desorptionandLC -MS-MS analysis cyclesof４０μ ofsampleataflowrateof１５０μ/min pattern,collisionenergy,andcollisioncellexitpoten- withthesix-portvalveintheLOADposition.Afterex- tial.Table１ showstheoptimum MS-MS conditionsof traction,thetipoftheinjectionneedlewaswashedby the１５ NSAIDs.Deprotonatedions[M-H]－ (Q１ mass) onedraw/ejectcycleof２ μ ofmethanol.(B)Desorp- wereobtainedforallNSAIDs,andwereusedaspre- tionandLC-MS-MS analysis:theextractedcom- cursorionsin MS-MS experiments.The majorfrag- － poundsweredesorbedfromthecapillarycoatingwith mentions [M-H-CO２] wereobserved in most themobilephaseandtransportedtotheLCcolumn by NSAIDsexceptsome compounds.Theproductionsof switchingthesix-portvalvetotheINJECTposition, loxoprofen,oxaprozin,phenylbutazoneandacemetacin anddetectedwiththe MS-MS system.Duringanaly- areconsideredtobe m/z=８３ (oxocyclopentylpart), － － sis,theCarboxen-１００６ PLOT capillarywaswashed m/z=２２０[M-H-CH２CH２CO２],m/z=２７９[M-H-CH２CH２] andconditionedwith mobilephaseforthenext and m/z=７５ (-OCH２COOH),respectively.Thesefrag- extraction. mentionsweresufficientlyseparatedbyQ３ inunit resolution,andthese MRM transitionswereselected forconfirmationandquantification.Thesefindingsare RESULTSANDDISCUSSION ２３） inagreementwithpreviousreport.Todeterminethe LC-MS-MS analysisofNSAIDs optimalcomposition,differentmobilephasesconsisting ForMS-MS operation,ESInegativeionmodewas ofaqueousammo niumformate-acetonitrileweretested. mosteffectiveforionizationofthe１５ NSAIDsexam- Thebestsignalswereachievedusing５ mM ammo - inedinthisstudy.A solutionof１００ ng/m in５０% niumformate/acetonitrile(６０/４０ v/v).LCseparationof methanolcontaining５ mM ammo niumformateinfused the１５ NSAIDswasperformed usinganODS-３ col- ataflowrateof１０μ/minproducedasignalofappro- umn.Thedevelopmentofthechromatographicsystem priateabundanceinESInegativeionmodeforthede- focusedonshortretentiontimes andco-elutionof protonatedprecursorion[M-H]－ usingtheturboion theseNSAIDs,payingattentionto matrixeffectsas spray.Parameters,includingnebulizergasstream,cur- wellaspeakshapes.Thus,anincreasedflowrateof taingas,andionsprayvoltage,wereoptimizedbyflow ０．３ m/min producedagoodpeakshapeandmadea injectionanalysiswithamobilephaseflowrateof０．３ runtime of１０ minpossible(Fig. ３). m/min.Tuningwasperformedusinganautomatictun- ingtoolincludedintheanalystsoftwaretodetermine thedeclusteringandfocusingpotentials,fragmentation

―５１４― 環境化学 Vol.１８,No.４［２００８］

Table１ Optimum conditionsforMS-MS (ESInegativeionmode)analysisofnon-steroidalanti-inflammatory drugs

Fig.３ MRM chromatograms obtainedfrom１０ ng/m ofeachstandardcompoundbyin-tubeSPME/LC-MS- MS innegativeionizationmode. Peaks:A=acetaminophen,B=loxoprofen,C=naproxen,D=ketoprofen,E=fenoprofen,F=flurbiprofen,G=oxaprozin, H=diclofenacsodium, I=indometacin,J=phenylbutazone,K=ibuprofen,L=acemetacin,M=mefenamic acid,N= flufenamicacid,O=tolfenamicacid.SeeExperimentalsectionforLC/MS/MS conditions.

―５１５― Optimizationofin-tubesolid-phasemicroextraction (１３．２%) ofnaproxen,３．０ ng(２９．９%) ofketoprofen, anddesorption １．２ ng(１２．１%)offenoprofen,１．７ ng(１６．９%)offlurbi- Tooptimizeextractionofthe１５ NSAIDsbyin- profen,１．２ ng(１１．６%)ofoxaprozin,２．０ ng(１９．６%) tubeSPME,severalparameters,suchasthestationary ofdiclofenac,０．９ ng(９．５%) ofindometacin,０．１ ng phaseofthein-tubeSPME capillarycolumn andnum- (１．５%) ofphenylbutazone,２．６ ng(２６．１%) ofibupro- berandvolume ofdraw/ejectcycles,wereinvestigated. fen,０．２ ng(１．６%) ofacemetacin,０．５ ng(４．９%) of In-tubeSPME conditionswereoptimizedwithstandard mefenamicacid,０．３ ng(３．５%)offlufenamicacid,and solution(１０ ng/m ofeach)usinganMS-MS detector ０．５ ng(５．４%)oftolfenamicacidwereextractedonto (ESInegative).Fourdifferentcapillarycolumns(DB-１, theCarboxen-１００６ PLOTcolumn byin-tubeSPME.Al- DB-１７,Supelcowax,Carboxen-１００６ PLOT)wereevalu- thoughtheextractionyieldsofthesecompoundswere atedforextractionefficiency.AsshowninFig.４,the low,theirreproducibilitywasgood(relativestandardde- porouspolymer-typecapillarycolumn (Carboxen-１００６ viationsRSD＜１０%)duetotheuseofanautosampler. PLOT)showedbetterextractionefficiencythanthe Themobilephasewasfoundtobesuitableforde- otherliquid-phasetypecapillarycolumns(DB-１,DB- sorptionofNSAIDsextractedintothestationaryphase １７,andSupelcowax).AstheCarboxen-１００６ PLOTcol- ofthecapillarycolumn.Dynamicdesorptionofthese umn hasalargeadsorptionsurfacearea,theamount compoundsfromthecapillarywasreadilyachievedby extractedwasgreaterthanthoseobtainedwiththe switchingthesix-portvalve.ThedesorbedNSAIDs otherliquid-phasetypecolumns.Tomonitortheextrac- weretransportedtotheLCcolumn by mobilephase tiontime profilesofNSAIDsbyin-tubeSPME,the flow.Nocarryoverwasobservedbecausethecapillary numberofdraw/ejectcycleswasvariedfrom ０ to２５ column waswashedandconditionedbydraw/ejectcy- usingaCarboxen-１００６ PLOTcapillary.Extractionequi- clesofmethanolandmobile-phasepriortoextraction. libriumofNSAIDsbyin-tubeSPME wasobtainedwith TheextractionanddesorptionofNSAIDsbythein- ２０ draw/ejectcyclesatarateof１５０μ/min.Theef- tubeSPME methodwereaccomplishedautomatically fectsofpHofthesamplematrixontheextractionof within３０ min,andautomatedanalysisofabout４８ sam- NSAIDsbyin-tubeSPME wereexaminedusingsev- plesperdaywasalsopossiblebyovernightoperation. eralbuffersolutions.Tris-HClbuffer(pH ８)wasthe mosteffectiveforextractionofthesecompounds,and Linearity,reproducibilityanddetectionlimit theoptimalbufferconcentrationwas２０ mM. Totestthelinearityofthecalibrationcurve,vari- TheabsoluteamountsofNSAIDsextractedinto ousconcentrationsofthe１５ NSAIDsrangingfrom０．１ theSPME capillarycolumn werecalculatedbycompar- to １０ ng/m wereanalyzedbyabsolutecalibration ingpeakareacountswiththecorrespondingdirectin- method.AsshowninTable２,alinearrelationshipwas jectionofthesamplesolutionontotheLCcolumn.At obtainedforeachcompoundinthisrange(six-pointcali- sampleconcentrationsof１０ ng/m,０．６ ng(５．８%)of bration),andthecorrelationcoefficientsrangedfrom acetaminophen,０．６ ng(６．５%) ofloxoprofen,１．３ ng ０．９９９７ to１．００００． TheRSDsineachpointranged

Fig.４ Effectsofcapillarycoatingsonin-tubeSPME of１５ NSAIDs.Eachcompoundwasextractedby２０ draw/ejectcyclesof４０μ oftherespectivestandardsolution(each１０ ng/m)ataflowrateof１５０ μ/min.

―５１６― 環境化学 Vol.１８,No.４［２００８］ from ０．３ to８．４%. Instrumentalprecisionwasas- riverwater.Loxoprofenwasdetectedselectivelyat４５８ sessedbyreduplicateinjection(n=５)ofstandardsolu- ± ５９ pg/m inhospitalwastewater.However,noneof tionsonthesame dayandondifferentdays.As theotherNSAIDsweredetectedinanyofthesam- showninTable２,within-dayandbetween-dayRSDs plestestedinthepresentstudy.Ibuprofen,naproxen, forquantitativeanalysisof１０ ng/m standardsolutions ketoprofen,anddiclofenachavebeendetectedinvari- wereintherangeof１．５～８．４% and３．６～１０．５%,re- ousenvironmentalwatersamplesfrom aroundthe spectively.Ontheotherhand,detectionlimitsofthe worldatconcentrationfrompg/m tong/m level７，１３，１５-１７）. １５ NSAIDsrangedfrom５ to６７ pg/m withasignal-to- Althoughco-elutingmatrixcomponentsmaycausesup- noiseratioof３.Thein-tubeSPME methodshowed３～ pressionoftheanalytesignalduringelectrosprayioniza- ５８ times highersensitivitythanthedirectinjection tion,noseveresuppressionwasnoticedforanyofthe method(５ μ injection),becausethesecompoundsin compoundsinthechromatogram ofwatersamples thesamplesolutionwereconcentratedinthecapillary usedinthisstudy.Theseresultsindicatethatthepro- column during draw/ejectcycles.The developed posed methodremoveinterferencecompoundsfrom methodhasalmostsame sensitivitywithpreviousGC- thesamplematrixbyin-tubeSPME andcanmeasure MS andLC-MS-MS methods[２１-２３],andcanbeap- tracelevelsofNSAIDsinrealenvironmentalsamples. pliedtothequantitativeanalysisofenvironmentallev- Superiorpointofthedevelopedmethodincomparison elsofthesecompounds. withtheprevious methods２１-２３） isautomatedoperation fromsampleextractiontodataanalysis,anditcanana- Applicationtotheanalysisofenvironmentalwa- lyze１５ NSAIDsinenvironmentalwatersampleswith- ters outanyotherpre-treatment.Therefore,the method Themethodwasappliedtoseveralenvironmental representsausefultoolformonitoringanddetermina- watersamples.Toconfirmthevalidityofthismethod, tionofNSAIDsinenvironmentalwaters. knownamountsofthe１５ NSAIDswerespikedinto riverwater,andtheirrecoverieswerecalculated.As ACKNOW LEDGEMENTS showninTable３,theoverallrecoveriesofthesecom- poundswere８１．９～１００．４%,andtherelativestandard ThisworkwassupportedbygrantsfromtheShi- deviationswere０．３～８．３%．Fig.５ showsMRM chro- madzuScienceFoundation,theYakumo Foundationfor matograms obtainedfrom wastewater,tapwater,and EnvironmentalScience,JapaneseSocietyforFoodSci-

Table２ Linearity,reproducibilityanddetectionlimitsofnon-steroidalanti-inflammatorydrugsbyin-tube SPME/LC-MS-MS

―５１７― Table３ Recoveriesofnon-steroidalanti-inflammatorydrugsspikedintoriverwater

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