ContentslistsavailableatScienceDirect
Journal
of
Chromatography
B
j o ur na l h o me 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 b
Determination
of
antipsychotic
drugs
in
hospital
and
wastewater
treatment
plant
samples
by
gas
chromatography/tandem
mass
spectrometry
夽
F.
Logarinho
a,1,
T.
Rosado
a,b,1,
C.
Lourenc¸
o
c,
M.
Barroso
d,
A.R.T.S.
Araujo
c,e,
E.
Gallardo
a,b,∗aCentrodeInvestigac¸ãoemCiênciasdaSaúde,FaculdadedeCiênciasdaSaúdedaUniversidadedaBeiraInterior(CICS-UBI),Covilhã,Portugal bLaboratóriodeFármaco-Toxicologia—UBIMedical,UniversidadedaBeiraInterior,Covilhã,Portugal
cInstitutoPolitécnicodaGuarda(IPG),EscolaSuperiordeSaúde,Guarda,Portugal
dServic¸odeQuímicaeToxicologiaForenses,InstitutodeMedicinaLegaleCiênciasForenses-Delegac¸ãodoSul,RuaManuelBentodeSousa,Lisboa,Portugal eUnidadedeInvestigac¸ãoparaoDesenvolvimentodoInteriordoIPG(UDI/IPG),Guarda,Portugal
a
r
t
i
c
l
e
i
n
f
o
Articlehistory: Received29May2016
Receivedinrevisedform12October2016 Accepted23October2016
Availableonline24October2016 Keywords:
Antipsychotics Solidphaseextraction
Gaschromatography–tandemmass spectrometry
Wastewaterssamples
a
b
s
t
r
a
c
t
Thedevelopmentandperformanceevaluationofamethodforthesimultaneousdeterminationofsix antipsychoticdrugsinhospitaleffluentsandwastewatertreatmentplants(WWTP)samplesareherein presented.Themethodinvolvesanoff-linemixedmode(reversed-phaseandstrongcationexchange) solidphaseextraction(SPE)withgaschromatography(GC)coupled totandemmassspectrometry (MS/MS).Thepresentmethodologywasvalidatedfollowinginternationallyacceptedcriteria,andthe studiedparametersincludedselectivity,linearity,limitsofdetection(LOD)andquantitation(LLOQ), instrumentallimits,precisionandaccuracy,stabilityandrecovery.Theprocedurewaslinearfor con-centrationsrangingfrom0.1to10g/L(0.02to2g/Lforhaloperidol),withdeterminationcoefficients higherthan0.99forallanalytes.Intra-andinter-dayprecisionwaslowerthan15%forallanalytesatthe studiedconcentrations,whileaccuracyremainedbetweena±15%interval.Recoveriesrangedfrom31% to83%.LowLODswereachieved,between2and10ng/L,allowingareliableidentificationofallanalytes attracelevels,usingonly50mLassamplevolume.Allstudiedparameterscompliedwiththedefined criteriaandthemethodwassuccessfullyappliedtogatherpreliminaryresultsofthedeterminationof antipsychoticsonhospitaleffluentsandoninfluentandeffluentofWWTPs,openingperspectivesforthe studyoftheirfateintheaquaticenvironment.
©2016ElsevierB.V.Allrightsreserved.
1. Introduction
Pharmaceuticalsareconsideredaclassofnew,socalled “emerg-ing”contaminantsthathaveraisedgreatconcerninthelastyears [1]. They are continuously being released in the environment mainlyduetoinsufficientremovalinwastewatertreatmentplants (WWTPs)(70–80%),whereastheremaining20–30%isduetoother sourcesofpollution,suchaslivestockandindustrialwastes, hos-pitaleffluentsanddisposalofunusedorexpiredpharmaceuticals [2].
夽 Selected paperfromtheXVI Latin-AmericanCongressonChromatography (XVICOLACRO)andthe9thNationalMeetingonChromatography(9ENC),5th–9th January2016,Lisbon,Portugal.
∗ Correspondingauthorat:CentrodeInvestigac¸ãoemCiênciasdaSaúde, Univer-sidadedaBeiraInterior,Av.InfanteD.Henrique,6201-556,Covilhã,Portugal.
E-mailaddress:egallardo@fcsaude.ubi.pt(E.Gallardo).
1 Theseauthorscontributedequallytothispaper.
Asaresult,theamountofpharmaceuticalsandtheirbioactive metabolitesbeingintroducedintotheenvironmentisincreasingly high,whichleadstoharmfulconsequencesduetothetheir recog-nized(eco)toxicity,aswellasunpredictableenvironmentalimpact. Nowadays, a largediversityof pharmaceuticals hasbeenfound intheenvironment,includingclassessuchasanti-inflammatory drugs,analgesics,antibiotics,antiepileptics,-blockers,lipid reg-ulators,antidepressants,anxiolytics,sedatives,contraceptives,etc. [3].
Regarding the psychiatric drugs, anxiolytics, sedatives, hyp-notics and antidepressant groups have been determined in wastewaters and aqueous environmental matrices [4]. Surpris-ingly,theantipsychotics(APs)grouphasnotreceivedpractically anyresearchattention.Furthermore,inarecentstudyofpsychiatric drugsuseinPortugal,itwasverifiedanincreaseoftheconsumption ofthesedrugsintheperiod2000–2012,andparticularly
expres-http://dx.doi.org/10.1016/j.jchromb.2016.10.031
sivefor thegroupofAPs(+171%)[5].Thisrising numbercould beexplainedbytheincreaseoftheprevalenceofpsychosis dis-turbs,thelengtheningofthedurationoftreatment,thebroadening oflicensedtherapeuticindicationsobtainedforsecond-generation APsand therisingproportionofoff-label prescriptionsofthese drugs[6].
Schizophrenia is a debilitating and emotionally devastating illness with long-term impacton patients’ lives. Many experts considerschizophreniatobethemostsevereexpressionof psy-chopathology,encompassingsignificantdisruptions of thinking, perception, emotion, and behavior. Schizophrenia is usually a lifelongpsychiatricdisability[7].APsdrugsaretheelective treat-ment and they have been broadly classified into two groups. Theolderagents are referred toastypical orconventional APs or dopamine receptor antagonists, with pharmacologicactivity attributedto the blockade of central dopamine receptors, par-ticularly the D2 receptor subtype. Examples of typical agents include haloperidol (HAL), fluphenazine, thiothixene, chlorpro-mazine(CPZ),cyamemazine(CYA),levomepromazine(LVMP)and thioridazine[8].Neweragents,commonlyreferredtoasatypical, serotonin-dopamineantagonists,orsecond-generationAPs, con-sistofclozapine(CLZ),risperidone,olanzapine,quetiapine(QTP), ziprasidone,aripiprazole,paliperidone,iloperidone,asenapine,and lurasidone,which have demonstratedpostsynapticeffects at 5-HT2AandD2receptors[9,10].ThisnewgenerationofAPslargely overcametheextrapyramidalside-effectsviadecreasedactivityat dopaminereceptorscomparedwiththeirtraditionalcounterparts [11].Theyaretheagentsoffirstchoiceintreatmentof schizophre-niaand evidencesupports that theyhave superior efficacy for treatmentofnegativesymptoms,cognition,andmood[12].
Nowadays,inourcountrysince2000itwasverifiedthattheuse offirst-generationAPsremainedpracticallyunchanged,andthere hasbeenaneffectiveincreaseintheuseofsecond-generationAPs, withQTPbeingthedrugwiththehighestrateofuse[5].
Ingeneral,APsareadministeredatrelativelylowdailydosages andtheyarewidelymetabolizedinthebody.Inconsequence,the concentrationofthesedrugsinhumanspecimensisverylow,and itexhibitsinter-individualvariations,whichsuggeststheneedfor clinicalmonitoringofpatientsundergoingtherapy,inadditionto minimizethesideeffects[13].Inthisregard,APdrugshavebeen determinedinbiologicalmatricesbynumerousmethods,suchas gaschromatography(GC)coupledtoeithernitrogen-phosphorus detector[14,15],massspectrometric(MS)detection[16]or tan-dem massspectrometry(MS/MS) [17],high-performance liquid chromatography(LC)coupledtoUVordiodearray[18–20], coulo-metric[21],chemiluminescence[22],MS[23]orMS/MSdetectors [24–27],capillaryelectrophoresiswithelectrochemiluminescence [28]orUVdetection[29],andelectrochemicalmethods[30,31].
Concerningtheenvironmentfate,tothebestofourknowledge, therearefewreportsthatreferthedeterminationofAPsin aque-ousmatricesinmulti-residueanalysis,allusingLC–MS/MS,namely risperidoneintheUnitedStates[32],olanzapineinSerbia[33],CPZ, CLZ,olanzapineandrisperidoneinGreece [34],risperidoneand haloperidolinEurope[35],andatotalofnineAPs(CPZ, olanzap-ine,CLZ,risperidone,sulpiride,QTP,ziprasidone,aripiprazoleand perphenazine)inChina[36].
Therefore,themain goalofthis workis concernedwiththe development,optimizationandvalidationofananalytical method-ology for the specific and sensitive determination of different antipsychoticdrugs(HAL,CPZ,CYA,LVP,CLZ,andQTP).These com-poundswereselectedbasedonprescriptionandconsumptionrates inourcountry[37].Themethodologyusedherewasbasedon solid-phaseextraction(SPE)usingpolymericStrataX-Ccartridges,afast microwave-assistedderivatizationandGC–MS/MS.Thematrices usedinthisworkarehospitalwastewatersandsamplesandit con-stitutesafirstapproachtoassessthepotentialcontributionofthis
groupofpsychotropicdrugstothepharmaceuticalloadofWWTPs andthereforeitisintendedtodeterminetheoccurrenceandfate ofthesedrugsforabetterunderstandingofthepotential environ-mentalimplications.
2. Experimental
2.1. Reagentsandstandards
Standardmethanolicsolutionsofhaloperidol(HAL),clozapine (CLZ),chlorpromazine(CPZ)wereacquiredfromLGCPromochem (Barcelona, Spain) at the concentration of 1mg/mL. Promazine (PRZ)(IS),levomepromazine(LVP)andcyamemazine(CYA)were acquiredfromSigma-Aldrich(Lisbon,Portugal).Quetiapine(QTP; 98%purity)waskindlydonatedbyAstraZenecaPLC(London,UK).It shouldbepointedthatPRZisnotcommerciallyavailableas thera-peuticdruginPortugal,andthereforeitsappearanceinanauthentic sample,impairingquantitativeanalysis,ishighlyunlikelytooccur. Furthermore,thiscompound’schemicalstructureissimilartothat of studied compounds’, allowing improving linearity, precision andaccuracy,whileminimizinganalytelossesduringthesample preparationprocess.Methanol(MerckCo,Darmstadt,Germany), hydrochloricacid(HCl)(Panreac,Barcelona,Spain)andammonium hydroxide(J.T.Baker,Lisbon,Portugal)wereofHPLCgrade. Potas-siumdihydrogenphosphate(KH2PO4)wasacquiredfromPanreac (Barcelona,Spain),N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA)andtrimethylchlorosilane(TMCS)werepurchasedfrom Macherey-Nagel(Düren,Germany).Ultrapurewaterwasobtained byaMilli-QSystem(Millipore,Billerica,MA,USA).Phenomenex StrataTM-X-Cextractioncartridges (200mg)wereobtainedfrom Tecnocroma(CaldasdaRainha,Portugal).
Workingstandardsolutionswerepreparedbyproperlydiluting thestocksolutionswithmethanoltofinalconcentrationsof0.1,1 and10g/Lofallanalytes,exceptHAL(0.02,0.2and2g/L).The internalstandardworkingsolutionwaspreparedinmethanoltoa concentrationof10g/mL.Allworkingandstocksolutionswere storedintheabsenceoflightat4◦C.
2.2. Samplecollectionandpre-treatment
Thesamplesusedinthisworkwerewastewatersfromahospital andWWTPintheurbanregionofPorto,Portugal.Allsampleswere collectedinamberglassbottles, previouslywashedwith deter-gentandthenrinsedthoroughlywithMilli-Qwater.Everysample wascollectedinduplicate(about0.5L).Thehospitalwastewaters werecollectedattwodistinctsamplingpoints:attheeffluentof theinternmentservicesandatdifferentsitesofthefinaleffluentof thehospitalWWTP.Influentandeffluentwastewatersampleswere collectedfromoneWWTPnetwork.Thesampleswerethenbrought tothelaboratoryinice-packedcontainers.Uponarrival,allsamples wereimmediatelyvacuum-filteredthrougha1-mglass microfi-brefilter(TypeA/EGlassFiberFilters,PallCorporation)andthen througha 0.45-mmixed celluloseesterfilter(GN-6Metricel® MCEMembraneDiscFilters,PallCorporation).Finally,theywere storedat4◦Cuntilanalysis.
2.3. Gaschromatographyandmassspectrometryconditions Chromatographicanalysis wasdoneusing an HP 7890Agas chromatography system equipped with a model 7000B triple quadrupolemass spectrometer,both fromAgilentTechnologies (Waldbronn,Germany),aMPS2autosamplerandaPTV-injector fromGerstel(MülheimanderRuhr,Germany).Acapillarycolumn (30m×0.25-mmI.D.,0.25-mfilm thickness)with5% phenyl-methylsiloxane(HP-5MS),suppliedbyJ&WScientific(Folsom,
Table1
RetentiontimeandGC–MS/MSparameters(quantitationtransitionsunderlined).
Analyte Molecularweight(g/mol) Retentiontime(minutes) Transitions(m/z) Collisionenergy(eV) Dwelltime(s)
PRZ 284.13 10.95 179.2−>179.2 5 50 CPZ 318.10 11.73 271.8−≥257.1 20 19.1 318.5−>233.2 20 10.3 LVP 328.16 11.83 242.0−≥210.2 20 20.4 242.0−>227.1 20 15.0 CYA 323.14 12.21 323.7−≥277.2 20 30.8 323.7−>100.3 20 20.4 CLZ 326.13 14.02 255.6−≥192.1 20 30.6 255.6−>239.0 20 19.9 HAL 375.14 14.69 296.4−≥296.3 10 10.0 296.4−>103.2 10 50.1 QTP 455.21 18.56 320.7−≥210.2 30 79.1 208.8−>139.0 30 79.1
CA,USA),wasusedforthechromatographicresolutionofthe
phar-maceuticalsubstances.
Theinitialoventemperaturewasheldat120◦Cfor2min,then
raisedto300◦Cat20◦C/min(heldfor14min),givingatotalrun
timeof25min.Thetemperaturesoftheinjectionportandtheion
sourceweresetat250◦Cand280◦C,respectively.Thesamplewas
introducedintothegaschromatographbysplitlessinjectionmode
andtheflowofhelium(carriergas)was0.8mL/minataconstant
flowrate.
Themassspectrometerwasoperatedwithafilamentcurrent
of35Aandelectronenergy70eVinthepositiveelectron
ion-izationmode.Nitrogenwasusedascollisiongasataflowrateof
2.5mL/min.Datawasacquiredinthemultiplereaction
monitor-ing(MRM)modeusingtheMassHunterWorkStationAcquisition
Softwarerev.B.02.01(AgilentTechnologies).
Initially,theretentiontimesandmassspectrawereobtained
byindividuallyinjectingderivatizedstandardsolutionsatahigh
concentration(5g/mL).Bothretentiontimesandmass-to-charge
ratio(m/z)wereusedforcompoundidentification.Twotransitions
werechosenforeachcompound(onequantitativeandone
qualita-tive),andtheselectionwasperformedinsuchmannerinorderto
obtainbetterselectivityandsensitivityfortheanalytesandfewer
matrixinterferences.Thechoiceoftheionswasbasedonthehigher
massesandmoreabundantmasspeaks(especiallythemostspecific
massesforeachcompound)tomaximizesignal-to-noiseratioin
matrixextracts.Tandemmassspectrometryconditionswere
opti-mizedbyinjectingastandardsolutionatdifferentcollisionenergies
anddwelltimes.Thetransitionswerechosenbasedonthebest
selectivityand abundance,in ordertomaximizesignal-to-noise
ratioinmatrixextracts.Themostabundanttransitionwasused
forquantitation,whereasthesecondtransitionwasfor
confirma-tion.Table1resumestheprecursor,productions,collisionenergies,
retentiontimesanddwelltimeselectedforeachcompound.
2.4. Samplepreparation
ForAPsextraction,thefollowingprocedurewasused:a50mL ofwatersamplewasfortifiedwith25LoftheISsolution.After stirred-mixedfor15min,themixturewasloadedontotheSPE car-tridgespreviouslyconditionedwith2mLof methanoland2mL ofacidifiedultrapurewater (pH5).Interferenceswereremoved fromthe sorbentwith2mLof 0.1M HCl in water and2mLof 0.1MHCl inmethanol.Afterwashing,thecartridgesweredried underfullvacuumfor15mintoremovealltracesofwater.Finally, theretainedanalyteswereeluted with2mLof5%ammonia in methanol.Theelutionsolventwasevaporatedtodrynessundera gentlestreamofN2 andthedryextractswerederivatizedusing 65L ofMSTFA/TMCSin a microwavereactor at800Wduring 2min.Aftercoolingdowntoroomtemperature,theseextractswere
transferredtoautosamplervialsanda2Laliquotoftheresulting solutionwastheninjectedintheGC/MS–MSsystem.
3. Resultsanddiscussion
3.1. Derivatizationprocedure
Forthederivatizationprocedure,adomesticdigitalmicrowave ovenwithanominalpowerof800W,wasusedinthisstudy.A com-parisonwasmadewithaconventionalheatingblockat85◦Cduring 45min.Forthispurpose,twolotsofsampleswithalltheanalytes atconcentrationof1and10g/L(0.2and2g/LfromHAL)were prepared.Allthesampleswereextractedbytheaforementioned methodandderivatizationwasmadeusingbothheating proce-durestocomparetheirability.Similarresultswereobtainedfor bothsetsofexperiments(Fig.1).AfterapplicationofF-Testand t-Test witha significancelevel of0.05 itwasverifiedthatboth derivatizationproceduresarecomparable.Thisscorerepresentsa majorimprovementwhencomparedtotheclassicderivatization process,namelyinthereductionoftheanalysistime.
3.2. Validationprocedure
Thedescribedmethodwasfullyvalidatedaccordingtothe guid-ingprinciplesoftheFoodandDrugAdministration(FDA)[38]and oftheInternationalConferenceonHarmonization(ICH)[39].The validation wasperformedfollowing a5-day validationprotocol andincludedselectivity,linearityandlimits,intra-andinter-day precisionandaccuracy,recoveryandstability.
3.2.1. Selectivity
The method’s selectivity was evaluated by analyzing blank wastewater samples of ten different origins, to investigate the potentialinterferencesattheretentiontimesandselected tran-sitions of the studied compounds. Samples were pooled and separatedin20aliquots(tenanalyzedasblanksandtenspikedwith alltheanalytes),allspikedwiththeIS.Qualitycontrols(QC) sam-ples wereprepared and analyzedsimultaneously. Identification criteriaforpositivityincludedanabsoluteretentiontimewithin 2%or±0.1minoftheretentiontime ofthesameanalyteinthe controlsampleandthepresenceoftwotransitionspercompound. Toguaranteeasuitableconfidenceinidentification,themaximum allowedtolerancesfortherelativeionintensitiesbetweenthetwo transitions(asapercentageofthebasepeak)wereasfollows:ifthe relativeionintensityinthecontrolsamplewashigherthan50%, thenanabsolutetoleranceof±10%wasaccepted;ifthisvaluewas between25and50%,arelativetoleranceof±20%wasallowed; ifit wasbetween5 and25%,an absolutetoleranceof±5%was accepted;and,finally,forrelativeionintensitiesof5%orless,a relativetoleranceof±50%wasused[40].
Fig.1. Comparisonbetweenclassicalderivatizationusingathermalblockandtheuseofmicrowave-assistedderivatization.
Fig.2. IonchromatogramofaspikedsampleattheLOD(A)andablank(B)sample.
Usingtheabovementionedcriteriaforpositivity,alltheanalytes weresuccessfullyand unequivocally identifiedinallthespiked samples,whereasintheblanksamplesnointerferingpeakscould bedetectedand/ormisidentifiedasbeingtheanalyte.Therefore, the methodwas considered selective for the selected antipsy-choticsinwastewatersamples.Representativeionchromatograms ofaspiked(attheLOD)andablanksampleareshowninFig.2.
3.2.2. Calibrationmodelandlimits
Linearityofthemethodwasestablishedonspikedsamples pre-paredandanalyzedusingthedescribedextractionprocedure(five replicates)intherangeof0.1–10g/Lforallcompoundsexcept
forHAL(0.02–2g/L).Calibrationcurveswereobtainedby plot-tingthepeakarearatiobetweeneachanalyteandtheISagainst analyteconcentration.Theacceptancecriteriaincludeda determi-nationcoefficient(R2)valueofatleast0.99and thecalibrators’ accuracywithin±15%(exceptatthelowerlimitofquantification (LLOQ),where±20%wasconsideredacceptable).Themethodwas linearwithintheadoptedcalibrationrangesforallanalytes; how-ever,sincetheadoptedcalibrationrangeswerewideandinorder tocompensateforheterocedasticity,weightedleastsquares regres-sionshadtobeadopted.Sixweightingfactorswereevaluatedfor eachanalyte(1/√x,1/x,1/x2,1/√y,1/y,1/y2),andtheoneforwhich thesumofrelativeerrors(dataobtainedduringtheassessmentof
Table2
Linearitydata(n=5).
Analyte Weight Linearrange(g/L) Linearity R2* LLOQ(g/L) LOD(ng/L) IQL(g/L) IDL(ng/L) Slopea Intercepta CPZ 1/x 0.1–10 0.055±0.016 −0.013±0.0013 0.995±0.001 0.1 10 0.02 2 LVP 1/x 0.1–10 0.066±0.016 0.002±0.0004 0.994±0.003 0.1 10 0.04 4 CYA 1/x 0.1–10 0.029±0.006 −0.001±0.0001 0.993±0.003 0.1 10 0.03 3 CLZ 1/x 0.1–10 0.009±0.003 0.006±0.001 0.993±0.004 0.1 10 0.03 3 HAL 1/x 0.02–2 0.048±0.009 −0.001±0.003 0.994±0.004 0.02 2 0.008 0.8 QTP 1/x 0.1–10 0.003±0.001 0.002±0.001 0.993±0.001 0.1 10 0.03 3
aMeanvalues±standarddeviation.
inter-dayprecisionandaccuracy)wasthelowest,presenting simul-taneouslyameanR2valueofatleast0.99waschosen(Table2).This factorwas1/xforallanalytes.
Bymeansoftheseweightedleastsquaresregressions,linear relationshipswereobtained,andthecalibrators’accuracy(mean relativeerror(bias)betweenthemeasuredandspiked concentra-tions)waswithina±15%intervalforallconcentrations,exceptat theLLOQ(±20%).CalibrationdataareshowninTable2.
Togetherwitheachcalibrationcurve,azerosample(blank sam-plewithIS)andthreequalitycontrol(QC)samplesatlow[LQC: 0.8g/L;0.16g/L(HAL)],medium[MQC:3g/L;0.6g/L(HAL)] andhigh[HQC:6g/L;1.2g/L(HAL)]concentrations(n=3)were alsoanalyzed.
TheLLOQwasdefinedasthelowestconcentrationthatcouldbe measuredwithadequateprecisionandaccuracy,i.e.witha coef-ficientofvariation(CV,%)lessthan20%andarelativeerror(RE, %)within±20%ofthenominalconcentration.Themethod’slimits ofdetection(LOD)weredeterminedasthelowestconcentrations thatshowedadiscretepeakclearlydistinguishablefromtheblank withasignal-to-noiseratioofatleast3,inwhichtheanalytescould beunequivocallyidentified,andweredeterminedbyanalyzingsix replicatesofspikedsamples.
The LLOQs were 0.1g/L for all antipsychotics except HAL (0.02g/L). The LODswere10ng/Land 2ng/L for (HAL).These limitswereconsideredsatisfactory,especiallywhencomparedto thoseobtainedbyotherauthors.Asitcanbeobserved,theLODs obtainedbyourmethodwerecomparabletothosereportedby Borova et al. [34]. Indeed, these authors report LODs between 2.5–4.9ng/LforclozapineandchlorpromazinebyLC/MS/MS. Fur-thermore,andtothebestofourknowledge,GC–MS/MShasnot beenusedyetfortheremainingantipsychoticsdeterminationin thiskindofsample.Additionally,inthis work,even thoughthe derivatizationproceduremaybealimitingstepwhencomparing liquidchromatography(LC)andGCmethods,weconsiderthatthe GCtechnique(coupledtotandemMS)exhibitsasprincipal advan-tagethefactthatitislesssusceptibletomatrixeffects,thatrequires specialattentionwhenit isusedaLCtechniqueandevenmore whendealing withhighcomplexity samplesasthewastewater samples.
Instrumentaldetectionlimits(IDL)andinstrumental quantifica-tionlimits(IQL)werecalculatedusingsignal-to-noiseapproachin standardspreparedinthereagentofderivatizationasthe concen-trationsofeachcompoundcorrespondingtosignal-to-noiseratio of3:1and10:1,respectively.ThesevaluesareresumedinTable2.
3.2.3. Intra-andinter-dayprecisionandaccuracy
Precisionwasexpressedintermsofcoefficientofvariation(CV, %)andcalculatedbytheone-wayanalysisofvariation(ANOVA).
Intra-dayprecisionwasevaluatedbyanalyzinginthesameday 5replicatesofblanksamplesspikedwiththestudiedanalytesat 4concentrationlevels(0.5,1,5and10g/L)and(0.1,0.2,1,and 2g/LforHAL).Inter-dayprecisionwasevaluatedataminimumof sixconcentrationlevelswithina5-dayperiod.Theaccuracyofthe
methodwascharacterizedintermsofthemeanREbetweenthe measuredandthespikedconcentrations;theacceptedlimitwas ±15%forallconcentrations,exceptattheLLOQ,where±20%was accepted.
Theobtainedvaluesforintra-dayprecision(CV<12.97%)and bias(±14.34%),aswellastotheininter-dayprecision(CV<11.46%) andbias(±12.75%)wereacceptableatallconcentrations.These resultsaresummarisedinTable3.
Also,intermediateprecision(combinedintra-andinterday)was evaluated using theQC samplesprepared and analyzed simul-taneously with the calibration curves on 5 different days (15 measurementsforeachconcentration)[38].TheCVsweretypically below13%forallcompoundsatallconcentrations,whileaccuracy waswithin±12%ofthenominalconcentration(Table3).
3.2.4. Extractionrecovery
Forrecoverystudies,blanksampleswerespikedwithboth ana-lytesatthreeconcentrations(0.5,1and10g/L)and(0.1,0.2,and 2g/LforHAL)andanalyzedbythedescribedmethod(n=3),after whichtheIS wasadded.Atthesametime,blankextractswere spikedwiththesamequantityoftheanalytesandISafterelution. Theobtainedpeakarearatioswerecomparedbetweenextracted andnon-extractedsamples(thelatterwereusedasneatstandards). Usingtheaforementionedapproach,theabsoluterecoveryvalues rangedfrom31to83%forthestudiedanalytes(Table4).
Theserecoveryvalueswerefoundadequate,despiteofthefact thattheywerenotsohighinabsolutevalue.Yet,lowlimitsof detec-tionandquantificationwereobtained,meaningthatthemethodis sensitiveenoughtodetectlowlevelsofthecompounds.Ourvalues arelowerthanthoseobtainedbyotherauthors,namely concern-ingCPZandCLZ[34,36].However,Borovaet.al[34]havecalculated recoveriesusingauthenticsamples,whileinourcasespiked sam-pleswere used,sinceauthentic samplespositivefor allstudied compoundswerenotavailable.
3.2.5. Stability
Inordertostudyshort-termstability,blanksampleswerespiked attheabove-mentionedLQC,MQCandHQClevelsandleftatroom temperaturefor24h[38].Duringtheentirestabilityprocedurethe analyzedsampleswerecomparedwithsamplesfreshlyprepared andanalyzedinthesameday(bothsetsofsampleswere quan-tifiedinthesamecalibrationcurve,theobtainedconcentrations werecomparedandtherespectiveREwascalculatedrelativelyto thetheoreticalconcentrations;theCVsbetweenthetwosetsof sampleswerecalculated aswell).Theanalyteswereconsidered stableifboththeCVbetweenthetwosetsofsamplesandthe cal-culatedREswereinaccordancewiththeabove-mentionedcriteria forprecisionandaccuracy(CVsbelow15%andREswithina±15% interval)[38](Table5).
F. Logarinho et al. / J. Chromatogr. B 1038 (2016) 127–135 Table3
Intra-interdayandintermediateprecisionandaccuracy.
Analyte Intradayprecisionandaccuracy(n=5) Interdayprecisionandaccuracy(n=5) Intermediateprecisionandaccuracy(n=15)
Concentration (g/L) Measured Concentration (g/L)a CV(%) RE(%) Concentration (g/L) Measured Concentration (g/L)a CV(%) RE(%) Concentration (g/L) Measured Concentration (g/L)a CV(%) RE(%) CPZ 0.1 0.10±0.00 1.01 −4.49 0.5 0.57±0.00 0.33 14.34 0.5 0.55±0.02 4.44 9.48 0.8 0.83±0.03 3.80 4.15 1 0.96±0.12 12.97 −3.02 1 1.01±0.07 7.07 1.45 3 2.84±0.22 7.92 −5.39 2 1.75±0.04 2.09 −12.75 5 4.46±0.19 4.31 −10.36 5 5.26±0.09 1.70 5.16 6 5.68±0.44 7.71 −5.37 8 7.75±0.06 0.80 −3.10 10 9.58±0.15 1.57 4.52 10 10.12±0.09 0.90 1.23 0.83±0.03 3.80 4.15 LVP 0.1 0.10±0.01 9.59 −2.52 0.5 0.55±0.01 2.23 9.74 0.5 0.54±0.01 1.19 7.47 0.8 0.89±0.02 2.53 11.43 1 1.11±0.11 9.90 11.95 1 1.03±0.08 7.57 3.07 3 3.01±0.18 5.85 1.59 2 1.82±0.01 0.28 −8.99 5 4.42±0.18 4.17 −10.83 5 5.14±0.13 2.55 2.72 6 6.16±0.67 10.89 1.49 8 7.89±0.56 7.04 −1.32 10 9.55±1.18 12.37 2.21 10 10.19±0.66 6.47 1.92 CYA 0.1 0.10±0.01 11.04 1.13 0.5 0.52±0.03 5.66 4.68 0.5 0.50±0.01 2.70 −0.32 0.8 0.79±0.04 5.36 −0.72 1 0.98±0.08 8.50 1.16 1 1.00±0.02 1.76 −0.46 3 2.79±0.16 5.89 −6.87 2 1.79±0.11 6.17 −10.40 5 4.82±0.37 7.64 −3.60 5 5.23±0.19 3.65 4.64 6 5.90±0.16 2.63 −1.68 8 7.85±0.35 4.43 −1.81 10 9.73±1.02 10.45 −2.70 10 10.03±0.47 4.66 0.31 CLZ 0.1 0.11±0.00 1.92 6.93 0.5 0.50±0.05 9.51 −2.82 0.5 0.50±0.01 1.89 0.87 0.8 0.76±0.04 4.94 −4.85 1 1.05±0.03 4.75 5.02 1 1.00±0.08 8.10 −0.36 3 3.21±0.28 8.68 6.93 2 1.86±0.08 4.51 −6.90 5 5.63±0.02 0.35 11.78 5 4.97±0.19 3.91 −0.54 6 6.00±0.35 5.81 0.03 8 7.89±0.50 6.31 −1.33 10 10.71±0.02 3.70 7.10 10 10.46±0.45 4.28 4.65 0.76±0.04 4.94 −4.85 HAL 0.02 0.02±0.00 0.83 7.54 0.1 0.11±0.06 13.2 10.15 0.1 0.10±0.01 6.27 −0.40 0.16 0.16±0.05 6.38 2.80 0.2 0.22±0.03 11.9 12.40 0.2 0.19±0.01 4.71 −7.22 0.6 0.59±0.38 12.96 −1.45 0.4 0.37±0.02 4.34 −6.33 1 1.69±0.08 1.42 13.46 1 1.03±0.01 1.39 2.67 1.2 1.23±0.46 7.52 2.61 1.6 1.59±0.03 1.67 −0.59 2 2.75±0.90 8.39 7.49 2 2.02±0.00 0.11 0.97 QTP 0.1 0.10±0.01 5.59 0.07 0.5 0.47±0.05 10.29 −5.03 0.5 0.49±0.02 5.02 −2.92 0.8 0.77±0.06 7.51 −3.65 1 1.01±0.03 3.15 5.11 1 1.03±0.12 11.46 2.81 3 2.67±0.19 7.16 −7.71 2 1.79±0.06 3.57 −10.66 5 5.61±0.16 2.81 12.18 5 4.84±0.33 6.77 −3.26 6 5.36±0.42 7.86 −8.53 8 8.08±0.12 1.43 0.96 10 11.40±0.01 0.08 13.97 10 10.25±0.20 1.94 2.51
CV—Coefficientofvariation;RE—Relativeerror(measuredconcentration-spikedconcentration/spikedconcentration)×100. aMeanvalues±standarddeviation.
Table4
Absoluterecovery(n=3).
Analyte Concentration(g/L) Recoverya(%)
CPZ 0.5 56.7±3.8 1 45.3±6.7 10 43.2±5.4 LVP 0.5 45.0±2.7 1 34.1±1.4 10 30.5±4.6 CYA 0.5 62.6±5.1 1 67.5±3.8 10 64.6±1.2 CLZ 0.5 66.5±11.7 1 46.2±1.1 10 56.3±7.0 HAL 0.1 83.1±1.7 0.2 75.8±7.5 2 78.9±6.1 QTP 0.5 83.3±6.7 1 72.6±6.6 10 81.3±4.2
aMeanvalues±standarddeviation.
Table5 Short-termstability(n=3). Analyte Concentration (g/L) Measured Concentration (g/L)a Measured Concentrationfresh samples(g/L)a CPZ 0.8 0.80±0.06 0.85±0.02 3 2.65±0.06 2.59±0.10 6 6.18±0.72 5.36±0.07 LVP 0.8 0.82±0.11 0.91±0.01 3 2.70±0.15 2.58±0.14 6 5.37±0.08 5.18±0.11 CYA 0.8 0.78±0.04 0.75±0.03 3 2.56±0.09 2.58±0.47 6 6.11±0.04 5.48±0.31 CLZ 0.8 0.77±0.12 0.85±0.06 3 2.68±0.07 2.92±0.12 6 6.15±0.62 5.73±0.60 HAL 0.16 0.15±0.09 0.16±0.01 0.6 0.50±0.12 0.68±0.00 1.2 1.13±0.30 1.22±0.09 QTP 0.8 0.67±0.07 0.70±0.06 3 2.63±0.18 2.66±0.54 6 5.51±0.12 5.25±0.01
aMeanvalues±standarddeviation.
3.3. Identificationandquantificationofantipsychoticsinhospital wastewaterandWWTPsamples
Thepresentedanalyticalmethodwasdevelopedwiththe ulti-matepurposeofaccuratelymeasurea representativelistofthe availableantipsychoticsoneffluentsfromanurbanhospitaland oninfluentandeffluentwastewatersfromWWTP.
TheresultsobtainedaresummarisedinTable6,andan elucida-tivechromatogramofasampleispresentedinFig.3.
Asexpected,thehighernumberofantipsychoticswasfound atthe hospitaleffluentof theinternment services,namelyCPZ (<LLOQ)CYA,CLZ,LVP,probablyduefromtheiruseinthe man-agementofemergencyepisodes.Regardingthefinaleffluentofthe hospitalWWTP,indifferentsamplingsites,thesamecompounds werefound,exceptforCPZ,generallyatlowerlevelsthanthose foundattheeffluentofinternmentservices.Inaddition,QTPhas alsobeendetected.
Infact,theconcentrationsofAPsinthesesampleswerelower than1g/L(excludingQTPinonehospitaleffluentsample).Yuan etal. [36]reportedconcentrations of5.5–12.8g/Lfor CLZand 2–5g/LforQTP,butinpsychiatrichospitalWWTPsamples.
The results obtained for the hospital wastewaters couldbe explainedbytheeliminationoftheemergencyservices-admitted
Table6
Concentrationvaluesofthetargetcompounds.
Sample Origin Compounda Concentration(g/L)a
#1 Hospitaleffluentof internmentservices CPZ <LLOQ CYA 0.10±0.02 CLZ 0.50±0.06 LVP 0.10±0.01 #2 Hospitaleffluent LVP <LLOQ #3 Hospitaleffluent CYA 0.10±0.02 #4 Hospitaleffluent CYA 0.85±0.03
CLZ <LLOQ QTP 2.32±0.09 #5 Hospitaleffluent LVP <LLOQ
CLZ <LLOQ QTP <LLOQ #6 WWTPinfluent CLZ 0.15±0.02 QTP <LLOQ #7 WWTPeffluent CLZ 0.10±0.01 #8 WWTPeffluent CLZ <LLOQ
aTheotherstudiedcompoundsarenotdetectedinthesesamples.
patients’treatmentthroughthehospitalsewagesystem,andalso inthecaseofdaypatients,wholeftthehospitalaftertreatment.
Furthermore, thecontribution of this group of psychotropic drugstothepharmaceuticalloadofWWTPswasalsoaddressed. AttheinfluentoftheurbanWWTPnetwork,CLZandQTP(<LLOQ) weredetermined,whileattheeffluentonlyCLZ(100ng/L) was detected, probably due to the efficient removal of QTP by the WWTP.ThesefindingsconfirmedtheAPseffectiveeliminationin theWWTPandeventoitseffluentatrelevantconcentrations.
Inamulti-residuemethoddevelopedbyBorovaetal.[34]for thedeterminationofahighernumberofpsychoactive pharmaceu-ticals,illicitdrugs,andrelatedhumanmetabolitesinfiveWWTPs, CPZwasnotdetectedinanysampleandCLZwasonlydetermined inoneWWTPataconcentrationlowerthan50ng/L.Yuanetal. [36]determinedCPZ(<LLOQ),CLZ(<50ng/L)andQTP(<10ng/L)in municipalWWTPs.
Undoubtedly,theuseofpharmaceuticalsisgrowing,in agree-ment with the occurrences of pharmaceutical residues at low g/LintheWWTPeffluents [41],andnamelytheconsumption ofAPs,andinthissensetheirinputtotheaquaticenvironment isincreasingmakingthemofincreasingenvironmentalrelevance. Thenumberofstudiesonitisatthemomentlimited.Moreover, in thisstudy,toreally confirmhospital loads,it wouldbe nec-essarytomeasuretheAPsnotonlyasparentcompounds,butin theirconjugatedformaswell;thereforetheactualoccurrenceof thosecompoundsinwastewatersmayhavebeenunderestimated. Infact,theseconjugateswhenpassingthroughthesewageplant couldbeconvertedbacktotheparentcompounds,whichcould leadtohigherconcentrationsintheeffluentthaninits correspond-inginfluent[42].Additionally,besidesthetransformationproducts, theanalysisofthemetabolitesshouldbealsoassessed,togivemore informationontheiroccurrenceinhospitalwastewaterandtheir fateinsubsequentWWTPs.
4. Conclusions
An analytical method using solid-phase extraction and gas chromatography/tandemmassspectrometrywasdevelopedand optimizedforthequantificationofsixantipsychoticsin wastewa-tersanditwassuccessfullyappliedtohospitaleffluentsandWWTP residualsamples.Thevalidatedmethodwaslinearintherange of0.1–10g/L(0.02–2g/LforHAL),withadequateprecisionand accuracyandreallowlimitsofdetection(≤0.01g/L)usingonly 50mLofsample.Furthermore,thefactthatmicrowave-assisted derivatizationresumestheprocessto2minmakesthewhole pro-cedurequickandlesstime-consuming.
Fig.3.Ionchromatogramofanauthenticsample(CLZ<LLOQ,CYA0.85andQTP2.32g/L).
Ourworkfocusedin afirstinstanceonthedeterminationof theseAPsinhospitalwastewaterseffluentsandsomepreliminary resultsfromfieldmeasurementswerereported.Itwasexpectable thathospitaleffluentwillcontributetosomeextenttothe pharma-ceuticalloadintheinfluententeringaWWTP.Theanalysisofthe receivingWWTPconfirmedthepresenceofthedrugsand there-foreprovidedmoreanalyticalinformationaboutthepathwayof theseemergingpollutants,pointingouttheneedoftheassessment oftheirecotoxicology(evaluationofeffectsonnon-target organ-isms)andalsothattheremoval/degradationprocessesefficacyof WWTPsshouldbeurgentlyimproved.Inconclusion,thepresent workpointsoutthenoteworthyimportanceoftheevaluationof thefate,theoccurrence,thepersistenceandbioaccumulationand thepotentialtoxicityoftheAPsintheaquaticenvironment,with possiblenegativeconsequencestotheaquaticecosystemsandin generaltothehealthpublic.
Acknowledgments
This work is supported by FEDER funds through the POCI—COMPETE2020—Operational ProgrammeCompetitiveness andInternationalisationin AxisI—Strengthening research, tech-nological development and innovation (Project POCI-01-0145-FEDER-007491) and National Funds by FCT—Foundation for ScienceandTechnology(ProjectUID/Multi/00709/2013).T.Rosado acknowledgestheProgramaOperacionalRegionaldoCentro 2007-2013QREN(Programa“MaisCentro”)intheformofafellowship (CENTRO-07-ST24-FEDER-002012),referenceTDM-2-006).
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