Determinación de quince compuestos hormonales en aguas depuradas usando extracción en fase sólida combinada con cromatografía líquida de ultra resolución con detección de espectrometría de masas (SPE-UHPLC-MS/MS)

Programa de Doctorado en Gestión Costera Junio de 2013 Memoria para la Acreditación de la Etapa de Investigación (AEI) Doctorando: Rayco Guedes Alonso Directores: José Juan Santana Rodríguez Zoraida Sosa Ferrera La presente memoria recoge la actividad investigadora realizada por el doctorando Rayco Guedes Alonso durante los últimos años, para obtener la Acreditación de la Etapa de Investigación, dentro del Programa de Doctorado en Gestión Costera de la Universidad de Las Palmas de Gran Canaria. Dicha actividad investigadora se resume en forma de artículo científico, en el que se recogen las investigaciones realizadas desde septiembre de 2012, fecha de matriculación en el presente Programa de Doctorado. Además, se muestran también actividades científicas realizadas anteriormente pero ligadas a la investigación principal, conducente a la obtención del título de Doctor. Dicha investigación principal se centra en la “Optimización y desarrollo de metodologías analíticas para la determinación de residuos hormonales en muestras de aguas, lodos, sedimentos y organismos de la isla de Gran Canaria”. Esta investigación comenzó con la realización del Máster en Ciencia y Tecnología Química, mención en Química Analítica, impartido por la UNED durante el curso 2011-2012. Para la obtención de dicho título, se presentó la Tesina de Máster denominada: “Determinación mediante SPE – LC - MS/MS de compuestos hormonales en aguas depuradas procedentes de estaciones depuradoras de aguas residuales (EDARs)”. Además, en los últimos dos años se han presentado tanto artículos científicos como comunicaciones a congresos sobre los avances en dicha investigación. A continuación se muestra el artículo científico que recoge las actividades investigadoras realizadas durante el curso académico 2012-2013: 1  Artículocientífico Determinacióndequincecompuestoshormonales enaguasdepuradasusandoextracciónenfase sólidacombinadaconcromatografíalíquidade “ultra”resolucióncondeteccióndeespectrometría demasas(SPE‐UHPLC‐MS/MS) Rayco Guedes-Alonso, Zoraida Sosa-Ferrera, José Juan Santana-Rodríguez DepartamentodeQuímica,UniversidaddeLasPalmasdeGranCanaria,35017LasPalmasdeGranCanaria,España e‐mail:[email protected]  Resumen Loscompuestoshormonalessonconsideradoscontaminantesemergentesdebidoaque diversosestudioshandeterminadosupresenciaenmuestrasdeaguasprocedentesde estacionesdepuradorasdeaguasresiduales(EDARs).Enesteestudiosehadesarrolladoun métodoparaladeterminaciónsimultáneadequincehormonas(cincoestrógenos,tres andrógenos,cuatroprogestágenosytreshormonasadrenocorticales)enmuestrasde influentesyefluentesdeestacionesdepuradoras.Debidoalasbajasconcentracionesde estoscompuestosenelmedioambiente,sehaelegidolaextracciónenfasesólidacomo procedimientodeextracciónypreconcentracióndelosanalitos.Ladeterminacióny cuantificaciónseharealizadomediantecromatografíalíquidade“ultra”resolución, acopladaaundetectordeespectrometríademasas(UHPLC‐MS/MS).Elmétodo desarrolladopresentaunoslímitesdedetecciónsatisfactorios(entre0.04y1.63ng∙L‐1), buenasrecuperaciones(porencimadel80%paratodosloscompuestos)ybajas desviacionesestándarrelativas(inferioresal13%).Utilizandoelmétodopropuesto,se analizaronmuestrasprocedentesdeinfluentesyefluentesdedosestacionesdepuradoras deaguasresidualesdeGranCanaria(España),lascualespresentaronconcentracionesde algunashormonasenelrangode5a190ng∙L‐1.    2  ResearchArticle Simultaneousdeterminationoffifteenhormonal compoundsintreatedwatersusingsolidphase extractionwithultra‐highperformanceliquid chromatography‐tandemmassspectrometry(SPE‐ UHPLC‐MS/MS) Rayco Guedes-Alonso, Zoraida Sosa-Ferrera, and José Juan Santana-Rodríguez DepartamentodeQuímica,UniversidaddeLasPalmasdeGranCanaria,35017LasPalmasdeGranCanaria,Spain e‐mail:[email protected]  Abstract Hormonalcompoundsareconsideratedasemergingpollutantsbecauseofseveralstudies haveconfirmedtheirpresenceinwatersamplesofwastewatertreatmentplants(WWTPs). Inthisstudy,aquantitativemethodforthesimultaneousdeterminationoffifteen hormones(fiveoestrogens,threeandrogens,fourprogestogensandthreeadrenocortical hormones)hasbeendevelopedtodeterminethesecompoundsininfluentandeffluent wastewatersamples.Duetotheverylowconcentrationsoftargetcompoundsinthe environment,asolidphaseextractionprocedurehasbeenusedtoextractand preconcentratetheanalytes.Determinationandquantificationwereperformedbyultra‐ highperformanceliquidchromatography‐tandemmassspectrometry(UHPLC‐MS/MS). Themethoddevelopedpresentssatisfactorylimitsofdetection(between0.04and1.63 ng∙L‐1),goodrecoveries(over80%forallcompounds)andlowrelativestandarddeviations (under13%).Samplesfrominfluentsandeffluentsoftwowastewatertreatmentplantsof GranCanaria(Spain)wereanalyzedusingtheproposedmethod,findingseveralhormones ininfluentsampleswithconcentrationsrangedfrom5to190ng∙L‐1.     3 1. Introduction Duetothehumandevelopment,itissupposedthat morethan100,000differentchemicalcompounds canbeintroducedintheEnvironmenteveryday, manyoftheminverysmallquantities.Manyof thesecompounds,namedemergingpollutants,are notregulatedaspollutants;theyprobablywillbein thefuturebecauseoftheirpotentialnegativeeffect intheecosystem.For20years,manyarticleshave reportedthepresenceofthese“newcompounds” inwastewater.[1‐2]. Theemergingpollutantoriginismainly anthropogenic,consideringthatthemajorityof thesecompoundsarebiologicallyactivesubstances thataresynthesizedtousetheminagriculture, industryandmedicine.Themainsourceofthese emergingpollutantsaretheresidualurbanwaters andthewastewatertreatmentplantseffluents becausemanyoftheseWWTPsarenotdesignedor optimizedtotreatthiskindofcompounds[3].The maincharacteristicofthesepollutantsisthatitis notnecessarytoremainintheenvironmentto causenegativeeffects,inviewofthefactthattheir constantintroductioninitoffsetstheirremovalor degradation[4]. Oneimportantgroupofthesecontaminantsisthe hormonalresidues,whichareconsideredEndocrine DisruptingCompounds(EDCs).Thesecompounds aredefinedaschemicalsubstancescapableof alteringthenaturalhormonalequilibriumproducing harmfuleffectsinanimals,humansandtheir progeny[5].Hormonescanbedifferentiatedin sexualhormones(oestrogens,androgensand progestogens)whicharesynthetizedinthe reproductiveorgans,andadrenocorticalhormones. Someofthemhavelimitsintheiruse,butnota specificlegislation[6]. Thesteroidhormoneshelpcontrollingthe metabolism,inflammations,immunological functions,waterandsaltbalance,sexual developmentandthecapacityofwithstand illnesses[7].Themainmedicalapplicationistobirth control.Thesyntheticoestrogenmostusedtothis is17α‐ethinylestradiol(EE)[8].Progestogensare alsousedashormonalcontraceptivethatcanbe combinedwithoestrogens.Androgensare frequentlyusedbysomesportsmentoincrease theirstrength,massandmuscularsize.Finally, adrenocorticalhormonesareusedintreatmentsof collagendiseases,inflammatoryandallergicupsets andotherillnesses.Thetermsteroidcanbeused fornaturalhormonesproducedbythebodyaswell asforartificiallyproducedmedicinesthatincrease thenaturalsteroideffect. Inthelast50years,thenaturalandsynthetic hormoneworldwideconsumptionhasgrown,as muchasinhumanmedicineasincattlefarming, andtheybecomethemostprescribedmedicines [9]. Asignificantquantityofconsumedoestrogens leavestheorganismthroughexcretions.For example,17β‐estradiol(E2)isoxidizedrapidly, becominganestrone(E1)thatcanturnintoestriol afterwards(E3).Besides,the17α‐ethinylestradiol (EE)isexcretedasconjugated[10]. Asregardstoemissionsources,inthefirstplaceare thewastewatertreatmentplants(WWTPs)[11], andsecondarily,cattlewastesuchasthose leachatesfromdunganduncontrolleddumping [12].SeveralstudiesmadeintheWWTPshave 4 reportedthatthetreatmentplantsarecapableof eliminatearound60%ofhormones[13‐15]. Measurementofhormoneresiduesisavery difficulttasknotonlyduetothedifficultyin measuringverylowconcentration,butalsoduetoa verycomplexityofthesamples.Therefore,useof massspectrometer(MS)asdetectorcoupledwith chromatographytechniqueshasbecomeapowerful methodfortheanalysisofthesetypesof compoundsattracelevels[5,16‐18].Consequently, LC‐MS/MSistheprincipallychosentechnique.One ofthemainadvantagesofLC‐MS/MSisitsabilityto analyzehormoneswithoutderivatization(necessary inGC)ortheneedofhydrolyzetheconjugated form. Duetolowlevelconcentrationofthesecompounds inenvironmentalwater,itisnecessarytoapplyan extractionandpreconcentrationmethodpriortoLC analysis.Themostusedtechniqueofextractionand preconcentrationmethodforliquidsamplesisthe solidphaseextraction(SPE)[19‐21]. Theobjectiveofthisstudyistodeveloparapidand simpleprocedureofextraction,preconcentration anddeterminationoffifteensteroidhormones (Table1),basedonsolidphaseextractionandultra‐ highperformanceliquidchromatographytandem massspectrometry(SPE‐UHPLC‐MS/MS).The developedmethodisappliedtotheidentification andquantificationofthesecompoundsin wastewatersamplesobtainedfromtheinfluents andeffluentsoftwowastewatertreatmentplants (WWTPs)ofGranCanaria(Spain).Theypresent differentmethodsofwastewatertreatments: WWTP1presentsatraditionalmethodbasedon activatedsludge,whileWWTP2usesamembrane bioreactortechnique.  2. Materialsandmethods 2.1.Reagents Allofthehormonalcompoundsusedwere purchasedfromSigma–Aldrich(Madrid,Spain). Stocksolutionscontaining1000mg∙L‐1ofeach analytewerepreparedbydissolvingthecompound inmethanol,andthesolutionswerestoredinglass‐ stopperedbottlesat4ºCpriortouse.Working aqueousstandardsolutionswereprepareddaily. UltrapurewaterwasprovidedbyaMilli‐Qsystem (Millipore,Bedford,MA,USA).HPLC‐grade methanol,LC‐MSmethanol,andLC‐MSwateras wellastheammoniaandtheammoniumacetate usedtoadjustthepHofthemobilephaseswere obtainedfromPanreacQuímica(Barcelona,Spain).  2.2.Samplecollection Watersampleswerecollectedfromtheeffluentsof twowastewatertreatmentplantslocatedinthe northernareaofGranCanariainMayof2013. WWTP1usedaconventionalactivatedsludge treatmentsystem,whileWWTP2employeda membranebioreactortreatmentsystem.The sampleswerecollectedin2Lamberglassbottles thatwererinsedbeforehandwithmethanoland ultrapurewater.Sampleswerepurifiedthrough filtrationwithfibreglassfiltersandthenwith0.65 µmmembranefilters(Millipore,Ireland).The sampleswerestoredinthedarkat4°C. 5 Table1.Listofhormonalcompounds,pKavalues,chemicalstructure,andretentiontimes. CompoundpKa[25] Structure tR(min) E3Estriol10.32.12 PREDPrednisone12.42.29 CORCortisone12.42.32 PREDNLPrednisolone12.52.45 BOLBoldenone15.12.87 NANNandrolone15.12.93 NORETNorethisterone13.12.95 E217β‐estradiol10.32.97 E1Estrone10.32.99 EE17α‐ethinylestradiol10.33.00 DESDiethylstilbestrol10.2  3.02 TESTestosterone15.13.15 NORNorgestrel13.13.29 MGAMegestrolacetate‐‐ 3.54 PROProgesterone‐‐ 3.71  6 2.3.Instrumentation FortheSPEoptimization,theinstrumentusedwas anultra‐highperformanceliquidchromatography withfluorescencedetector(UHPLC‐FD)system consistingofanACQUITYQuaternarySolvent Manager(QSM)usedtoloadsamplesandwashand reconditiontheanalyticalcolumn,anautosampler, acolumnmanagerandafluorescencedetectorwith excitationandemissionwavelengthsof280and310 nmrespectively,allfromWaters(Madrid,Spain). Theanalysisofwastewatersampleswasperformed inaUHPLC‐MS/MSsystemfromWaters(Madrid, Spain),similartothedescribedabove,witha2777 autosamplerequippedwitha25μLsyringeanda traytohold2mLvials,andaACQUITYtandem triplequadrupole(TQD)massspectrometerwithan electrosprayionization(ESI)interface.AllWaters components(Madrid,Spain)werecontrolledusing theMassLynxMassSpectrometrySoftware. Electrosprayionisationparameterswerefixedas follows:thecapillaryvoltagewas3.5kVinpositive mode,and‐2.5kVinnegativemode,thesource temperaturewas150°C,thedesolvation temperaturewas500°C,andthedesolvationgas flowratewas1000L/hr.Nitrogenwasusedasthe desolvationgas,andargonwasemployedasthe collisiongas. ThedetailedMS/MSdetectionparametersforeach hormonalcompoundarepresentedinTable2and wereoptimisedbythedirectinjectionofa1mg∙L‐1 standardsolutionofeachanalyteintothedetector ataflowrateof10µL∙min‐1.  Table2.Massspectrometerparametersforthedeterminationoftargetanalytes CompoundPrecursorion (m/z) Capillaryvoltage (Ionmode) Quantificationion,m/z (collisionpotential,V) Confirmationion,m/z (collisionpotential,V) E3287.2‐65V(ESI–)171.0(37)145.2(39) PRED359.330V(ESI+)147.0(15)237.0(20) COR361.330V(ESI+)163.0(25)121.0(45) PREDNL361.320V(ESI+)147.1(20)173.1(25) BOL287.230V(ESI+)121.0(28)135.1(15) NAN275.235V(ESI+)109.1(20)83.0(30) NORET299.230V(ESI+)109.1(25)91.0(40) E2271.2‐65V(ESI–)145.1(40)183.1(31) E1269.2‐65V(ESI–)145.0(36)143.0(48) EE295.2‐60V(ESI–)145.0(37)158.9(33) DES267.1‐50V(ESI–)237.1(29)251.1(25) TES289.238V(ESI+)97.0(22)104.0(21) NOR313.238V(ESI+)109.0(26)245.1(18) MGA385.530V(ESI+)267.3(15)224.2(30) PRO315.330V(ESI+)97.0(18)109.1(25)   7 2.4.Chromatographicconditions FortheSPEoptimization,theanalyticalcolumnwas a50mm×2.1mm,ACQUITYUHPLCBEHWatersC18 columnwithaparticlesizeof1.7μm(Waters Chromatography,Barcelona,Spain)operatingata temperatureof30°C.Analytesseparationwas carriedoutemployingthefollowinggradient:starts at55:45(v/v)water:methanolfor1minute.During 3minutes,itchangedto50:50(v/v)andstayedfor 2.5minutesmore.Finally,camebacktoinitial conditionsin1minute,andstayedfor1.5minutes. Therefore,theanalysistook9minutesataflowof 0.5mL∙min‐1. Fortheanalysisofrealsamples,aUHPLC‐MS/MS systemwasused.Theanalyticalcolumnwasthe same,andthemobilephasewaswateradjusted withabufferconsistingin0.1%v/vammonia,and 15mMofammoniumacetateandmethanol.The analysiswasperformedingradientmodeataflow rateof0.3mL∙min‐1.Thegradientstartedat80:20 (v/v)mixtureofwater:methanol,whichchangedto 40:60(v/v)in1.5minutes,andto25:75in1.25 minutes.Following,stayedat25:75for1minute andreturnedto80:20in2.25minutes.Finally, stayedcalibratingforanother0.5minutesmore. Thewholeanalysistook6.5minutes.Thesample volumeinjectedwas10µL.  3. Resultsanddiscussion 3.1.Solid‐PhaseExtraction(SPE)optimization Thereareanumberofparametersthataffectto SPEproceduresuchustypeofsorbent,pH,ionic strength,sampleanddesorptionvolumesandwash step.Tooptimizetheseparameters,itwasused Milli‐Qwaterspikedwithasolutionoffluorescence oestrogens(estriol,17β‐estradioland17α‐ ethinylestradiol)toobtainafinalconcentrationof 250µg∙L‐1. Thefirstparametertooptimizeisthechoiceof sorbent,sinceitcontrolstheselectivity,affinityand capacityoveranalytes.Inthisstudy,theSPE cartridgesusedwereOASISHLB,SepPakC18(both fromWaters,Madrid,Spain)andBondElutENV (fromAgilent,Madrid,Spain).Fromtheresults obtained,bettersignalswerefoundforSepPakC18 cartridge Afterchoosetheoptimumcartridge,weusedan initialexperimentaldesignof23(threeparameters attwolevels),tostudytheinfluenceofpH(3and 8),ionicstrength(0and30%(w/v)ofNaCl)and samplevolume(50and250mL)overextraction process,obtainingtheinfluenceofeachparameter andthevariablecorrelationtoeachother.The experimentaldesignwasobtainedusing StatgraphicsPlussoftware5.1andthestatistics studywasdonewithIBMSPSSStatistics19. Itwasobservedthattheionicstrengthandsample volumehadthemajorinfluenceontherecoveries oftheanalytes.Forthat,a32factorialdesignto optimizethesetwovariablesatthreelevelsper parameter(0,15and30%(w/v)ofNaClforionic strengthand50,100y250mLforsamplevolume) wasused.Theresultsobtainedshownthatan incrementoftheionicstrengthdidnotproducean increaseintheresponseareaofthecompound,and theoptimumvolumewas250mL.Finally,the desorptionvolume(1mLofmethanoland2mLof methanolinoneandtwosteps)andwash‐step(5