1

Massspectrometricstrategiesfortheinvestigationofbiomarkersof1

illicitdruguseinwastewater2

3

4 FélixHernández1*,SaraCastiglioni2,AdrianCovaci3,PimdeVoogt4,5,ErikEmke4,Barbara5 Kasprzyk-Hordern6, Christoph Ort7, Malcolm Reid8, Juan Vicente Sancho1, Kevin V.6 Thomas8,AlexanderL.N.vanNuijs3,EttoreZuccato2,LubertusBijlsma17 8

1ResearchInstituteforPesticidesandWater,UniversityJaumeI,Castellón9 2Department of Environmental Health Sciences, IRCCS - Istituto di Ricerche10

FarmacologicheMarioNegri,Milan,Italy.11 3ToxicologicalCenter,UniversityofAntwerp,Antwerp,Belgium12 4KWRWatercycleResearchInstitute,Nieuwegein,theNetherlands13 5IBED-UniversityofAmsterdam,Amsterdam,theNetherlands14 6DepartmentofChemistry,UniversityofBath,Bath,UnitedKingdom15 7Swiss Federal Institute of Aquatic Science and Technology (Eawag), Dübendorf,16

Switzerland17 8NorwegianInstituteforWaterResearch(NIVA),Oslo,Norway18 19 20 Runninghead:MSstrategiesforinvestigatingillicitdrugsinwastewater21 22 23 *Authorforcorrespondence:24 Prof.Dr.FélixHernández25 ResearchInstituteforPesticidesandWater,UniversityJaumeI26 Avda.SosBaynats/n27 E-12071Castellón,Spain28 e-mail:felix.hernandez@uji.es29 tel:+3496438736630 31

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2

Tableofcontents32

33

1 Introduction

2 Targetandnon-targetapproaches

3 Samplingapproachesandsamplepreparation

3.1 Sampling

3.2 Monitoring

3.3 Collectionof(meta)information

3.4 StabilityofIDsbiomarkersinthesamplesunderstorageconditions

3.5 Sampletreatment

4 ApplicationsofLow-ResolutionMassSpectrometry

5 ApplicationsofHigh-ResolutionMassSpectrometry

5.1 Wide-scopescreening

5.2 Non-targetanalysis

5.3 Investigationofmetabolitesandtransformationproducts

6 Chiralanalysis

7 Relevantanalyticalparametersandqualitycontrol

8 Generalsummaryandperspectives

34

35

3

Listofacronymsandabbreviations36 37

CBH Cellobiohydrolase

EDDP 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine

EF EnantiomericFraction

EI ElectronIonization

EMCDDA EuropeanMonitoringCentreforDrugandDrugAddiction

ESI ElectroSprayIonization

GC-MS GasChromatographycoupledtoMassSpectrometry

HE HighcollisionEnergy

HRMS High-ResolutionMassSpectrometry

IDB IllicitDrugBiomarker

ILIS Isotope-LabelledInternalStandards

IP IdentificationPoint

LC-MS LiquidChromatographycoupledtoMassSpectrometry

LC-MS/MS Liquidchromatographycoupledtotandemmassspectrometry

LE LowcollisionEnergy

LOD LimitsofDetection

LOQ LimitsofQuantification

LRMS Low-ResolutionMassSpectrometry

MDA 3,4-methylenedioxyamphetamine

MDMA 3,4-methylenedioxymethamphetamine

NPS NewPsychoactiveSubstances

QC QualityControl

QqQ Triplequadrupoleanalyzer

QSRRs QuantitativeStructure-RetentionRelationships

SCORE SewageanalysisCORegroupEurope

SDL ScreeningDetectionLimit

SIM SelectedIonMonitoring

s/n signal-to-noise

SRM SelectedReactionMonitoring

TIC TotalIonChromatogram

TPs TransformationProducts

tR Retentiontime

4

38 39

UHPLC Ultra-HighPerformanceLiquidChromatography

WBE Wastewater-BasedEpidemiology

WWTP WasteWaterTreatmentPlant

5

Abstract40

Theanalysisofillicitdrugsinurbanwastewateristhebasisofwastewater-based41

epidemiology (WBE), and has received much scientific attention because the42

concentrationsmeasuredcanbeusedasanewnon-intrusivetooltoprovideevidence-43

basedandreal-timeestimatesofcommunity-widedrugconsumption.Moreover,WBE44

allows monitoring patterns and spatial and temporal trends of drug use. Although45

informationandexpertise fromotherdisciplines is required to refineandeffectively46

apply WBE, analytical chemistry is the fundamental driver in this field. The use of47

advancedanalyticaltechniques,commonlybasedoncombinedchromatography-mass48

spectrometry, is mandatory because the very low analyte concentration and the49

complexity of samples (raw wastewater) make quantification and identification/50

confirmationofillicitdrugbiomarkers(IDBs)troublesome.51

Wereviewthemost-recentliteratureavailable(mostlyfromthelastfiveyears)52

onthedeterminationofIDBsinwastewaterwithparticularemphasisonthedifferent53

analytical strategiesapplied.Thepredominanceof liquidchromatographycoupledto54

tandemmassspectrometrytoquantifytargetIDBsandtheessencetoproducereliable55

and comparable results is illustrated. Accordingly, the importance to perform inter-56

laboratoryexercisesandtheneedtoanalyzeappropriatequalitycontrolsineachsample57

sequence is highlighted. Other crucial steps in WBE, such as sample collection and58

samplepre-treatment,arebrieflyandcarefullydiscussed.Thearticlefurtherfocuseson59

thepotentialofhigh-resolutionmassspectrometry.Differentapproachesfortargetand60

non-target analysis are discussed, and the interest to perform experiments under61

laboratory-controlled conditions, as a complementary tool to investigate related62

compounds(e.g.,minormetabolitesand/ortransformationproductsinwastewater)is63

treated.Thearticleendsupwiththetrendsandfutureperspectivesinthisfieldfrom64

theauthors’pointofview.65

Keywords:Massspectrometry,Drugsofabuse,UrinaryMetabolites,Wastewater-based66

epidemiology67

68

6

1. Introduction69

Illicit drug use is a global problem with severe consequences, not only for70

people´shealthandwelfare,butalsoasaclearthreattothestabilityandsecurityof71

entire regions and economic and social development. Accordingly, there is a72

considerable financial cost related to illicit drug use, associated with drug use73

prevention,treatmentofaddicts,andonthefightagainstorganizedcrime(EMCDDA,74

2012; Nutt et al., 2007; UNODC, 2014). Policy makers need accurate and reliable75

informationontheuseofthesesubstancesinordertomakeevidence-baseddecisions76

andtoeffectivelyallocateresources.Theprevalenceofillicitdrugusehastraditionally77

been estimatedwith direct subjectivemethods, like general population surveys and78

interviews,andindirectmethodssuchasmonitordrug-relatedcriminality,seizures,and79

hospital records (EMCDDA,2015a).Despiteconsiderable improvementswithmodern80

communication facilities, and the use of complementary methods such as targeted81

studiesandstatisticalmodeling,thesesurveymethodsmainlyrelyonthewillingnessof82

userstoself-reportandtomonitoractions.However,thesocialtaboorelatedtoillicit83

drug use might provoke non-participation and false responses in such surveys that84

makes thesemethods vulnerable and potentially inaccurate. In addition, a common85

challengewithsuchmethodsisthattheyaretime-consuming,expensive,andcomplex86

(Banta-GreenandField,2011).Therefore,thedevelopmentofnewandcomplementary87

approaches is encouraged in order to obtain objective, low-cost, fast, reliable, and88

comparabledata.89

The chemical analysis of illicit drug residues in untreatedwastewater is as a90

valuabletooltocomplementexistingapproachestomonitorspatiotemporalpatterns91

andtrendsofillicitdruguseinlargecommunities(EMCDDA,2015b;Ortetal.,2014c;92

Thomasetal.,2012;Zuccatoetal.,2008).Thisapproachistermedwastewater-based93

epidemiology(WBE),andreliesontheprinciplethatillicitdrugsconsumedbyindividuals94

are excreted, either unchanged or as a mixture of metabolites, into urban sewer95

networks.Thequantitativemeasurementofthesespecificillicitdrugbiomarkers(IDBs)96

inwastewatersamplesreflectsthedrugscollectivelyexcretedbyusersandenablesdata97

tobegatheredondrugusebythecommunitywithinthegeographicalboundarydefined98

bythecatchmentareaofawastewatertreatmentplant(WWTP).Severalcrucialsteps99

7

are involved in this approach (Fig. 1.), which requires significant expertise from100

numerousresearchfields,andtherefore,strongmulti-disciplinarycollaboration(Ortet101

al.,2014a).102

103

104

105

Figure1.MainconsecutivestepsoftheWBEapproachanddatarequiredforeachstep106

(modifiedfrom(Castiglionietal.,2014)).107

108

TheWBEconceptwasinitiallyproposedbyDaughtonin2001(Daughton,2001)109

andfirstappliedin2005throughtheestimationofcocaineuseinItaly(Zuccatoetal.,110

2005). Since then,WBE has expanded to include other illicit drugs, such as heroin,111

cannabis,andamphetamine-likestimulants(Castiglionietal.,2014;EMCDDA,2015b;112

vanNuijsetal.,2011)andnewpsychoactivesubstances(NPS)(Kinyuaetal.,2015;Reid113

et al., 2014b; van Nuijs et al., 2014). Data have also been reported forWBE-based114

analysisofalcohol(Boogaertsetal.,2016;Mastroiannietal.,2014;Reidetal.,2011;115

Rodríguez-Álvarezetal.,2015,2014),tobacco(Castiglionietal.,2015;Rodriguez-Alvarez116

etal.,2014;B.J.Tscharkeetal.,2016),andcounterfeitmedicines(Venhuisetal.,2014).117

An important step in the progression of WBE was accomplished with the118

establishment of a European-wide network (Sewage analysis CORe group Europe –119

Wastewater-based epidemiology

Sample Collection

Sample AnalysisQuantitative determination of biomarkers

Loads of target biomarkers entering the WWTP (g/day)

Amount of substance consumed by the population served by the sewer network

Normalization to the defined population (mg/day/1000 inhabitants)

Amount of substance as doses/day/1000 inhabitants

Flow rate (m3/day)

Human metabolism Correction factors

Mean Dose

Population Estimates

1.

2.

3.

4.

6.

5.

8

SCORE), supportedby the EuropeanMonitoringCentre forDrug andDrugAddiction120

(EMCDDA) (EMCDDA, 2015b). This network has since 2010 standardized the WBE121

approach,coordinatedinternationalstudies(Ortetal.,2014c;Thomasetal.,2012),and122

conductedinter-laboratoryexercisesforqualitycontrolpurposes.Thelatterprovideda123

meanstoestimateandcriticallyassesstheuncertaintyrelatedtothewastewater-based124

estimates (Castiglioni et al., 2013).However, importantWBE researchhas alsobeen125

conductedinothercontinentssuchasAsia(Khanetal.,2014;Kimetal.,2015;Laietal.,126

2013b;Lietal.,2014),Australia(Irvineetal.,2011;Laietal.,2013a;Prichardetal.,2012;127

Tscharkeetal.,2016,2015),NorthAmerica(Banta-Greenetal.,2009;Burgardetal.,128

2013;SubediandKannan,2014),andCentralandSouthAmerica(Bijlsmaetal.,2016;129

Devault et al., 2014; Maldaner et al., 2012; Voloshenko-Rossin et al., 2015).130

Furthermore, a recently published review on neuropsychiatric pharmaceuticals and131

illicit drugs in wastewater treatment plants (Asimakopoulos and Kannan, 2016) is132

recommendedforreadersinterested.133

TheincreasinginterestofWBEisclearlyillustratedbythenumberofpapersand134

citations on the topic (>200 papers in years 2005-2015, >5000 citations; ISIWeb of135

Science),internationalconferencesandworkshopsorganizedonthistopic,andfunding136

received from the European Commission (“http://score-cost.eu/”; “http://sewprof-137

itn.eu/”).138

ChemicalanalysisofIDBsinwastewaterplaysanimportantkeyrolewithinthe139

WBE approach. Advanced analytical techniques and expertise is required to obtain140

accurateconcentrationdataonIDBsinwastewater,becausequantitativedataarethe141

basisofsubsequentback-calculationsofIDBmassloadsanddruguse.Concentrations142

ofIDBsinwastewatersamplesaregenerallyaroundafactor1000lowerthaninhuman143

biological fluids (ng/L versusng/mL),whichpointsout the challenge forquantitative144

analysis.Lowanalyteconcentrationsincombinationwiththecomplexityandunknown145

compositionofthewastewatermatrixmighthampernotonlythesensitiveandaccurate146

quantificationbutalsoasoundidentification.Chromatography-massspectrometryis147

the best-suited approach to obtain the sensitivity, selectivity, and identification148

requirementsinchemicalanalysisdirectedtowardsWBE.149

9

Gaschromatographycoupledtomassspectrometry(GC-MS)(González-Mariño150

etal.,2010;Marietal.,2009)ingeneralprovideshighlevelsofselectivityandsensitivity.151

However,derivatizationof thetargetcompounds isoftennecessary formost IDBs in152

order to make them compatible with GC. Consequently, sample treatment and153

measurement is generally laborious and time-consuming. Liquid chromatography154

coupled tomass spectrometry (LC-MS) is amore-versatile technique that allows the155

determinationofpolar,low-volatility,and/orthermolabilecompounds,whichmostIDBs156

are,withlesssampletreatmentandshorterchromatographicanalysistimes.Besides,157

thesamplematrix(i.e.,water)iscompletelycompatiblewiththistechnique.LC-tandem158

massspectrometry(LC-MS/MS),e.g.atriplequadrupole(QqQ)analyzer,isevenmore159

powerful,andhasbecomethetechniqueofchoiceforthequantitativedetermination160

of(known)IDBsinwastewatersamples(vanNuijsetal.,2011).161

Despite the predominance of LC-MS/MS in WBE studies, the use of high-162

resolution mass spectrometry (HRMS) has been recently explored and opens new163

perspectivesintheanalyticalfield.Itsstrongpotentialtoscreenandforidentification164

purposesoriginatesfromtheacquisitionofaccurate-massfull-spectrumdata.LC-HRMS165

is a powerful technique that allows the wide-scope screening of many illicit drugs,166

metabolitesandtransformationproducts,aswellastheinvestigationofNPS(Alechaga167

etal.,2015;Badeetal.,2015c;Baz-Lombaetal.,2016;Bijlsmaetal.,2013b;Hernández168

et al., 2014; Ibáñez et al., 2014; Reid et al., 2014a). There are several illustrative169

examples(aspresentedlaterinthismanuscript)inthemost-recentliteraturethatshow170

thatmodern analytical chemistry is essential in order to increase our knowledge on171

trendsinsubstanceuseinthegeneralpopulation.172

In this article, an exhaustive review of the existing literature is not the goal.173

Rather,theobjectiveistopresentanoverviewoftheapproachbyusingthemost-recent174

literatureavailable,withspecificemphasisonthedifferentanalyticalstrategiesapplied175

forWBE.Mostcitedpapershavebeenpublishedwithinthelastfiveyears,exceptfor176

somethathavealsobeenincludedbecauseoftheirrelevanceinthedevelopmentofthe177

WBEapproach.ThepredominanceofLC-MS/MStoquantifypriority,well-known,target178

IDBs and the essence to produce reliable and comparable results are illustrated.179

Accordingly, the importance to perform inter-laboratory exercises and the need to180

10

analyze appropriate quality controls in each sample sequence is highlighted. Other181

crucialstepsrelatedtoWBE,suchassamplecollectionandsamplepre-treatment,are182

discussed. The article further focuses on novel analytical approaches, such as183

enantiomericprofiling,thedifferentstrategiesfortargetandnon-targetanalysiswith184

LC-HRMS,andontheinteresttoperforminvivoorinvitrometabolismexperimentsand185

degradation laboratory experiments, as a useful tool to investigatemetabolites and186

transformationproductsinwastewater.Thearticleconcludeswiththetrendsandfuture187

perspectivesinthisdisciplinefromtheauthors’pointofview.188

189

2. Targetandnon-targetapproaches190

The terms target and non-target analyses are widely employed in analytical191

chemistry.Otherexpressions,suchasinvestigationofunknownsorsuspectscreening,192

arealsofrequentlyused,andillustratetheanalyticalchallengesincomplexfields,such193

as environmental analytical chemistry (Bletsou et al., 2015; Krauss et al., 2010;194

Schymanskietal.,2014b).Withthe implementationofLC-HRMS inseveralanalytical195

fields,thetermpost-targetanalysishasbeenalsoemployed,andillustratetheworking196

modeinwhichfull-spectrumaccurate-masstechniquesareapplied(Hernándezetal.,197

2005). Clarification of these terms is, however, necessary to fully understand the198

different strategies that can be applied to investigate the presence of IDBs in199

wastewatersamples.200

Target methodologies are commonly analyte-dependent; i.e., compound-201

specific information is required beforemeasurement. Based on this analyte-specific202

information,highly sensitiveand selectiveanalyticalmethods canbedeveloped,but203

othercompoundsthatmightbepresentinthesampleswillremainundetected.Target204

analysisistypicallyappliedinmethodsbasedonLCand/orGCcoupledtoMS(Selected205

IonMonitoring(SIM)mode)ortandemMS(SelectedReactionMonitoring(SRM)mode).206

Alimitedlistoftargetcompoundsisincludedinthescopeofthemethod,andonlythose207

previously selected ions/transitions are monitored. Reliable identification and208

quantification is commonly themain objective pursued in this type of analysis. This209

identificationisachievedthroughacquisitionofatleastthreeionsintheSIMmodeor210

twoMS/MStransitionsintheSRMmode,andevaluationofretentiontime(tR)andion-211

11

intensityratios(SANTE/11945,2015).Theuseofreferencestandardsiscompulsorywith212

this methodology in order to optimize the mass spectrometric measurement213

parametersandforquantitativemethodvalidation.214

ThismethodologycanalsobeappliedwithHRMSinstruments.However,thanks215

to the accurate-mass full-spectrum acquisition at good sensitivity, HRMS offers the216

possibilitytoinvestigatethepresenceofmanyothercompounds,notonlythoseinitially217

targeted.Databasesdevelopedin-housearecommonlyusedtofacilitatescreeningofa218

large number of compounds with HRMS. The information included in the database219

dependsontheavailabilityofareferencestandard.Whenavailable,theinformationis220

verycomplete(i.e.,tR,exactmassofthe(de)protonatedmoleculeand/oradducts,and221

themainfragmentions)tohighlyfacilitatetheanalyticalresearch.Whenthereference222

standardisnotavailable,onlylimitedinformationonthetargetanalytescanbeincluded223

inthedatabase(e.g.,molecularformulaandexactmass,theoreticalisotopedistribution,224

predictedtR).Informationreportedintheliteratureonproductionscanalsobetaken225

intoaccounttofacilitatecompoundidentification.Eveninthisworst-casesituation(i.e.,226

whennostandardisavailable),accurate-massfull-spectrumdatacanbeusedforthe227

tentative identification of the compounds in samples (Hernández et al., 2015a), a228

processwhere the interpretation and justification of the fragment ions observed is229

crucial.Inanycase,thefactthatasearchisdirectedtowardsalistofcompoundsimplies230

atargetapproach, independentof theavailabilityofreferencestandards.This target231

approachiscommonlyknownassuspectscreening(Hugetal.,2014;Kraussetal.,2010)232

whereasotherauthorsusethetermpost-target(Hernándezetal.,2005;Ibáñezetal.,233

2008).234

TargetanalysisbasedonHRMScommonlydetectsandidentifiesthecompounds235

(i.e., qualitative analysis) inwastewater, becauseHRMS really takes advantageof its236

excellent performance for this type of application (Hernández et al., 2014, 2011a).237

However,recentstudieshavealsobeendirectedtowardsthequantitativeanalysisof238

IDBsinwastewater(Bijlsmaetal.,2013b;Fedorovaetal.,2013;González-Mariñoetal.,239

2012;Heuettetal.,2015;vanderAaetal.,2013),andanotableincreaseinthenumber240

of quantitative applications of HRMS that pursue a complete analysis (i.e., sensitive241

detection,reliableidentification,accuratequantification)isexpectedinthenearfuture.242

12

Asignificantadvantageoffull-spectrumacquisitionsisthattheyalsoallowthe243

investigationofanyothercompound,notonlythe listofselectedcontaminants, ina244

non-targetmethodology. In contrast to the (post-) target approach, a genuinenon-245

targetanalysisdoesnotuseanypreviousinformationonthecompoundstobesearched246

inthesamples.Someauthorsusethetermnon-targetscreeningwhentheysearchfor247

unknowns,whichinastrictsensestartswithoutanyinformationonthecompoundsto248

be investigated. The term unknown does not necessarily mean that the compound249

discovered in the analysis is a new or an unreported compound. Following the250

elucidationprocesses, theunknownmightturnouttobeaknowncompound,which251

alreadyhasbeenreportedintheliterature,butunexpectedornotspecificallysearched252

forinthesamples.Inagenuinenon-targetanalysis,thereisnoanalyteselectionapriori253

(neither before nor after MS acquisition). Under these circumstances, compound254

identificationattracelevelsinwastewaterisachallenge,andcommonlymorethanone255

elementalformulaandseveralplausiblestructuresareobtainedforagivenunknown256

detectedinasample(Ibáñezetal.,2008;Kraussetal.,2010;Schymanskietal.,2014a).257

Moreinformationonnon-targetanalysisappliedintheenvironmentalfieldcanbefound258

elsewhere (Chiaia-Hernandezetal., 2014;Hogenboometal., 2009;Hugetal., 2014;259

Schymanskietal.,2015,2014b;vanLeerdametal.,2014).260

Most IDBsareof (medium)highpolarity; therefore,LC-HRMScommonlyuses261

TOFandOrbitrapanalyzers.TheabsenceofstandardizedmassspectrallibrariesinLC-262

MS isanadditionaldifficulty innon-targetanalysis,opposite toGC-MSwithelectron263

ionization (EI), where the availability of commercial libraries (e.g., NIST) offers the264

possibilitytoidentifycompoundsbymatchingexperimentalandlibraryspectra.265

In order to have amore-realistic and complete overview on the presence of266

organiccontaminantsingeneral,andonIDBsinparticular,acombinationoftarget(both267

withorwithoutstandards(i.e.,suspectscreening)andnon-targetmethodologiesseems268

tobethemost-attractiveapproach(Hugetal.,2014).Furthermore,thecombinationof269

GC-HRMSandLC-HRMS(e.g.,usethesameQTOFinstrumentforbothconfigurations)270

movestowardsamore-comprehensivescreeningoforganiccontaminantsintheaquatic271

environmentindependentlyoftheirpolarityandvolatility(Hernándezetal.,2015b).272

273

13

3. Samplecollectionapproachesandsamplepreparation274

Measurementswithhigh-endinstrumentsandsophisticatedstatisticalanalysis275

cannot compensate or reveal deficiencies in sample collection. Depending on the276

desiredlevelofrepresentativenessandaccuracy,samplecollectionisasimportantas277

any subsequent steps. A sound understanding of the investigated environment is278

imperativeandmustbedocumentedindetail.FormostapplicationsinWBE,theend279

user relies on 24-hour composite influent (i.e., raw) wastewater samples collected280

routinely at the inlet of a WWTP. We briefly outline how such a sample is ideally281

collected (sampling) and how many samples are needed (monitoring) for specific282

applications.283

284

3.1. Sampling285

The number of consumers of a specific substance, pharmacokinetics, the286

populationconnected toaWWTP,and thehydraulicpropertiesof thesewersystem287

determinei)thenumberoftoiletflushesthatcontainthesubstanceofinterest,andii)288

over which period an individual toilet flush expands when it passes the sampling289

location.Thesetwofactorsdeterminethetemporalvariabilityonthescaleofminutes,290

whichinturndeterminestherequiredsamplingfrequencytocollectarepresentative291

sample(i.e.,howmanysamplesmustbecollectedandpooledoveracertainperiodfor292

analysis).Duetothepotentiallysmallnumberofrelevanttoiletflushes–atleastforone293

of multiple substances analyzed in the collected sample – it is recommended that294

sampling intervalsdonotexceed5-10minutesat the influentof largeWWTPs.Even295

shorter sampling intervalsof 1-5minutesarenecessary for composite samples from296

effluentsofindividualpremises.Attheeffluentofe.g.aprison,thetoiletflushesextend297

overshorterperiodsandtheabsolutenumberofsubstance-relatedflushesistypically298

muchsmallerthanattheinfluentofa(large)WWTP,bothleadingtoasubstancepattern299

that fluctuatesmuchmore at the scale of a fewminutes. This is also applicable to300

influentsofverysmallWWTPs,whereitmaybebeneficialtosamplefromtheeffluent301

ofaprimarytreatmenttank,whichattenuatestemporalfluctuationstosomeextent.302

However,pleasenote that the latteralso removessomeparticulatematterand that303

WWTP-internalrecirculationmayinfluencemassloadsthere.Shortersamplingintervals304

14

arealsorequiredforthecollectionofrepresentativesamplesthatextendoverperiods305

<24h,e.g.hourlycompositesamplestoreliablyassessdiurnalvariations.Pleasenote,306

grabsamplestodeterminediurnalvariationsarenotsuitablebecauseofpotentialhigh307

short-term variations. In addition, intra-day variations in wastewater flows require308

samples to be collected in a flow- or volume-weighted manner. More details and309

peculiaritiesforwastewatersamplinginsewers,anoverviewofrelevantliterature,and310

alistofrequirementsforaseriesoftypicalapplicationsisdescribedelsewhere(Coutu311

etal.,2016;DeKeyseretal.,2010;Ort,2014;Ortetal.,2010a,2010b).312

313

3.2. Monitoring314

Ideally,onewouldanalyze365dailycompositesamplesperyear.Duetolimited315

resources, this amount of samples is usually not feasible. Different316

(research/epidemiological) questions require different monitoring designs, and317

obviously more samples provide higher accuracy. Random day-to-day variability,318

systematic weekly cycles, and seasonal fluctuations influence baseline variation.319

Togetherwith thedesired level of sensitivity and significance, thebaseline variation320

determinestherequirednumberanddistributionofsamples.Todate,twomonitoring321

design studies have investigated different approaches that aim to answer different322

questions.Theyarebasedonafewavailablelong-termtimeseries;i.e.,datasetswith323

observationsovermorethan28consecutivedays.Inbrief,itwasfoundthatthebaseline324

variation of almost all investigated drug residues in five different catchments325

(populationsizesthatrangefromapprox.7000to1.3millionpeople)didnotexceed326

80%(ascoefficientofvariation),notaccountingforanytemporalcorrelation(EMCDDA,327

2016).Basedon thesedata,Ortetal. (Ortetal., 2014b)proposed that56 stratified328

random samples (10working days and 4weekend days per quarter) are suitable to329

estimateanannualmeanwithanaccuracyof10%.Humphriesetal.(Humphriesetal.,330

2016) evaluatedmore informedapproaches,which relyon knowledgeaboutweekly331

cycles.Inthepresenceofstrongweeklycycles(e.g.,cocaineorMDMA),itisefficientto332

distributemonitoringdayssystematicallybyconsideringpeak,mid,andthroughusage333

days. The identificationofweekly cycles requires an intensive,monitoringperiod. In334

return,theaccuracy,particularlyofintra-annualtrends,improvestheinformedroutine335

15

monitoring compared to an uninformed schemewith the same reduced number of336

samples. In thecontext toassesseffectsof interventions, thebaselinevariation, the337

magnitudeoftheeffecttobeshown,andtheconfidenceleveldeterminethenumber338

ofrequiredsamples.339

340

3.3. Collectionof(meta)information341

Anexampleofaquestionnairetocollect(meta)informationrelevanttoevaluate342

theappropriatenessofsamplingandtofacilitatetheinterpretationofresultsiscanbe343

found(Ortetal.,2014c).Thispaperencompassescatchmentproperties(e.g.,hydraulic344

residencetime,populationsize,andhowitwasestimated),aswellasdetailsonsample345

collectionandhandling.346

347

3.4. StabilityofIDsbiomarkersinthesamplesunderstorageconditions348

ItisoftennotpossibletoimmediatelyanalysesamplesforthepresenceofIDBs.349

Itis,therefore,necessarytostorewastewatersamplesuntilanalysisunderconditions350

that avoid transformationof theanalytes,becausebiotransformationduring storage351

wouldleadtofalseinterpretationsofWBEdata,evenifanalyticalmethodsareaccurate.352

For most IDBs, experiments have been performed to evaluate stability for353

different storage conditions, including various temperatures, pH values, and354

preservation agent additions, and for different time frames (reviewed in detail by355

(McCalletal.,2016).Storageofwastewatersamplesat-20°Censuresstabilityofmost356

IDBsforatleast3weeks.ForsomeIDBs(i.e.,theamphetamine-typestimulantsand11-357

nor-9-carboxy-delta-9-tetrahydrocannabinol (THC-COOH)),experimentshaverevealed358

thattheyareevenstableat-20°Cformorethan15weeks(McCalletal.,2016).Stability359

isensuredforonlyseveraldaysat4°C,whereasotherIDBs(e.g.,cocaine)arewithin360

severalhourstransformedatthistemperature(McCalletal.,2016).361

Acidification of the samples increases stability of the majority of IDBs, but362

significantly enhances biotransformation of THC-COOH. Because multi-analyte363

determinationsareoftenappliedinWBEstudies,itis,therefore,notadvisabletoacidify364

thesamplesbeforestorage.Theeffectoftheadditionofpreservationagentssuchas365

16

sodiummetabisulfite(Na2S2O5)hasalsobeenstudied,andrevealedhigherstabilityfor366

cocaineanditsmetabolitebenzoylecgonine(McCalletal.,2016).However,fortheother367

IDBs,moreexperimentsneedtobeperformedinordertoevaluatetheefficacytoadd368

preservationagentstowastewatersamplesforstoragepurposes.369

370

3.5. Sampletreatment371

Inordertoremovesolidparticlesfromthewastewatersamples,twostrategies372

canbefollowed:(i)afiltrationstepwithmembraneofglassfiberfilterswithporesizes373

as lowas0.1µm,or (ii)acentrifugationstep. Inaddition,becauseconcentrationsof374

mostIDBsinwastewaterareintheng/L-µg/Lrange,apreconcentrationstepisgenerally375

required prior to analysis in order to achieve the necessary quantification limits.376

However, some modern analytical instruments allow direct injection of filtered or377

centrifuged wastewater due to the high sensitivity provided (Berset et al., 2010;378

Biscegliaetal.,2010;Boixetal.,2015;Chiaiaetal.,2008;Laietal.,2011).379

A sample-preparation step is commonly needed not only to preconcentrate380

analytes,butalsotoremovematrixcomponentsthatmightinterferewiththeanalytical381

measurement(e.g.,ionizationsuppression/enhancementprocessesinLC–MS)ofIDBs.382

Byfar,themost-usedprocedurereportedintheliteratureisoff-linesolid-phase383

extraction (SPE)withsamplevolumesbetween50mLand1000mL (vanNuijsetal.,384

2011).PopularSPEsorbentsusedforareproducibleextractionofIDBsfromwastewater385

arebasedonapolymericbackbonewithreversed-phaseorcation-exchangeproperties.386

Some methods demonstrated that off-line SPE sample preparation could also be387

incorporatedinafullyautomatedon-lineSPEapplicationthatisdirectlylinkedtothe388

LC-MSanalysis(Fedorovaetal.,2013;Heuettetal.,2015;Postigoetal.,2008).389

Some alternativeways of sample preparation can be found in the literature.390

González-Mariñoetal. (González-Mariñoetal.,2009)appliedcommerciallyavailable391

molecular imprinted polymers (MIPs) to extract and concentrate amphetamine-type392

stimulantsfromwastewater,andreportedbetterperformanceoftheMIPsintermsof393

selectivity, sensitivity, accuracy, and precision compared to off-line SPE. The main394

drawbacks of this approach were no possibility for multi-class analysis, and higher395

17

analysistimeandcost.Theusefulnessofsolid-phasemicroextraction(SPME)forsample396

preparationtoanalyzeamphetamine-typestimulantsorTHC-COOHinwastewaterhas397

beendemonstrated(Racamondeetal.,2013,2012).SPMEishighlycompatiblewithGC-398

MS, and shows good performance; however, the main drawback was the limited399

applicabilitytodeterminatemulti-classcompounds,whichisdesiredforWBEpurposes.400

401

4. ApplicationsofLow-ResolutionMassSpectrometry402

Low-resolutionMSsystems,witheitheriontraporQqQanalyzers,arethemost403

widely applied for the quantitative determination of IDBs in wastewater. TheseMS404

systems typicallyoperate in theMS/MSmode,whereoneormoreproduct ionsare405

monitored by selecting appropriate precursor-to-product ion transitions (i.e., SRM).406

Evenwiththisapproach,therearesomeprobabilitiesthatothercompounds,notrelated407

to the analyte, can share the same transition. Therefore, at least two transitions is408

required to be monitored, and the presence of a compound is considered to be409

confirmed ifbothtransitionsproduceachromatographicpeakat thesameretention410

time, that corresponds to that of the injection of a reference standard. Identity411

confirmationisoftheutmostimportancebecauseitgivestherequiredconfidenceto412

the reported results and reduces the likelihood of reporting false positives. The413

confirmationofidentityisespeciallyrelevantwhenanalyzinghighlycontaminatedand414

complexmatricessuchasinfluentwastewatersamples.Forconfirmation,notonlythe415

acquisitionof several transitions is needed, but also the complianceof the ion ratio416

betweenreferencestandardsandsamples.Thisimportantaspectisdiscussedinmore417

detailinsection7“Relevantanalyticalparametersandqualitycontrol”.418

Although ion-trap analyzers have been used in this field (Bones et al., 2007;419

Gheorgheetal.,2008;MartínezBuenoetal.,2011;Postigoetal.,2008),LC-MS/MSwith420

QqQhasbecomethemost-populartechniqueduetoitsexcellentperformanceinterms421

ofrobustness,dynamicrange,sensitivity,andselectivity.Thesecharacteristics,together422

withthecompatibilityofLC-MS/MSwithaqueoussamplesandmostlypolaranalytes423

targeted inWBEstudies,allowone tonotably simplify sample treatment. LC-MS/MS424

withQqQcannowadaysbeconsideredastheworkhorseinanalyticallaboratoriesthat425

dealwithWBE.Thisfacthasalsobeenillustratedinmonitoringstudiesofillicitdrugsin426

18

wastewater,wherethemajorityoflaboratoriesappliedthistechnique(Castiglionietal.,427

2008;Ortetal.,2014c;Thomasetal.,2012).428

Someoftheearlystudies,usedonlyonetransitionforquantification(Bartelt-429

Hunt et al., 2009; Chiaia et al., 2008; Gheorghe et al., 2008;Metcalfe et al., 2010).430

However, it isnowadayswidelyaccepted thatconfirmationofanalyte identity inLC-431

MS/MS-based methodologies requires a minimum of two transitions (European432

Commission, 2002). Yet, the acquisition of more transitions per compound would433

obviouslygivemoreconfidence to theconfirmationprocess,and is feasiblewith the434

latestanalyticalinstrumentswithfasteracquisitiontimes(Bijlsmaetal.,2014a,2009;435

Boledaetal.,2007).ThelimitedfragmentationinESI,frequentlywithlow-abundance436

secondary and tertiary transitions, can limit the absolute number of useful MRM437

transitionsforidentificationinsomeparticularcases.Nevertheless,withtheexcellent438

sensitivity of the new instruments, the acquisition of, at least, two transitions is439

commonlynotaproblem.Typicallythemost-abundanttransition isselectedtofavor440

quantificationatlowconcentrations(quantifier,Q),andtheotheronesareacquiredfor441

confirmation (qualifier, q). At this point, it is also important to understand the442

fragmentationpatternoftheanalytesunderexperimentalconditionstoallowselection443

ofanalyte-specificfragmentsinordertominimizepotentialinterferencesofthematrix444

and/or background noise. Hence, it might occur that the most-sensitive transition445

presentsmorematrixinterferenceand/orbackgroundnoise,andmightthereforenot446

bethemostappropriateforquantification.Asanexample,THC-COOH,ametaboliteof447

the active ingredient found in cannabis, was measured in the positive electrospray448

ionization(ESI)mode.Inthismode,thetransitionm/z345>327isoccasionallyselected449

forquantification(Table1).However,thistransitioncorrespondstoanon-specificloss450

ofwater,whichmightbemorepronetointerferenceswhenanalyzingcomplexmatrices,451

suchaswastewater (Pozo, Sancho, Ibáñez,Hernández,&Niessen,2006). This fact is452

demonstratedinFigure2,wherethreedifferenttransitionswereacquiredtomeasure453

THC-COOH in influent wastewater. The transitions 345 > 327 and 345 > 299,454

correspondingtolossesofH2OandHCOOH,respectively,presentedhighernoisethan455

theless-abundant,butmore-selective,transition345>193.Thesematrixinterferences456

19

hasalsobeenobservedinthenegativemode;whenanon-specificlossofCO2(343>457

299)wasmeasured.458

459

460

461

Figure2.SelectivityofTHC-COOHtransitions.462

Table1showsthequantification(Q)andconfirmation(q)transitionsusedduring463

the monitoring campaign 2015 coordinated by SCORE to measure the most widely464

studiedIDBsininfluentwastewaterinWBEresearch.Ingeneral,theQtransitionis,in465

moststudies,thesamewithfewexceptions.However,moredifferencesareobserved466

inthequalifiertransitionusedforconfirmation.467

468

469

470

471

472

473

Time3.50 4.00 4.50 5.00 5.50

%

0

100

3.50 4.00 4.50 5.00 5.50%

0

100

3.50 4.00 4.50 5.00 5.50

%

0

100

DOA0057 5: MRM of 4 Channels ES+ 345 > 193.2

3.66e5Area

4.0216423

DOA0057 5: MRM of 4 Channels ES+ 345 > 299.3

1.86e6Area

DOA0057 5: MRM of 4 Channels ES+ 345 > 327.3

1.10e7Area

Time3.50 4.00 4.50 5.00 5.50

%

0

100

3.50 4.00 4.50 5.00 5.50

%

0

100

3.50 4.00 4.50 5.00 5.50

%

0

100

DOA0064 5: MRM of 4 Channels ES+ 345 > 193.2

3.74e5Area

4.0218042

DOA0064 5: MRM of 4 Channels ES+ 345 > 299.3

6.00e5Area

4.0226048

DOA0064 5: MRM of 4 Channels ES+ 345 > 327.3

1.10e6Area

4.0243030

O CH3

O

OH

H3C

H3C

O CH3

OH

H3C

H3C

-H2O

-H2O,-CO

H2C

HO CH3

OH -C9H12O2

Solvent Influentwastewater

4.0223494

4.0230205

4.021.80x103

4.022.60x103

4.024.30x103

4.021.64x103

4.022.35x103

4.023.02x103

20

Table1.SRMtransitionsmostoftenusedwithLC-QqQ-MSinstrumentstodetermine474 IDBsinwastewaterduringthemonitoringcampaign2015ofSCORE(“http://score-475 cost.eu/”).476

Compound Q-transition(#Labs/#totalLabs)a

q-transition(#Labs/#totalLabs)b

Amphetamine 136>91(12/17) 136>119(10/12)136>65(2/12)

136>119(5/17) 136>91(5/5) Methamphetamine 150>91(13/17) 150>119(11/13)

150>65(2/13) 150>119(4/17) 150>91(4/4) Methylenedioxymethamphetamine(MDMA) 194>163(15/17) 194>105(11/15)

194>135(3/15)194>77(1/15)

194>135(1/17) 194>105(1/1) 194>133(1/17) 194>105(1/1) Cocaine 304>182(15/16) 304>82(9/15)

304>150(2/15)304>105(2/15)304>77(2/15)

304>82(1/16) 304>182(1/1) Benzoylecgonine 290>168(17/17) 290>105(13/17)

290>77(3/17)290>82(1/17)

THC-COOH(+)mode

345>299(6/10) 345>245(4/6)345>327(1/6)345>193(1/6)

345>327(2/10) 345>299(1/2)345>193(1/2)

345>193(1/10) 345>299(1/1) 345>41(1/10) 345>327(1/1)THC-COOH(-)mode

343>299(3/5) 343>245(2/3)343>191(1/3)

343>245(2/5) 343>299(2/2)aNumberoflaboratoriesthatselectedtheSRMtransition(Q)forquantification/the477

totaloflaboratoriesthatusedLRMSanddeterminetheIBD.478 b Numberof laboratories that selected theSRM transition (q) for confirmation/ the479

totaloflaboratoriesthatselectedthesameQ-transition(seealsoa)480 481 Note: More-detailed information regarding the analytical procedures can be found482 elsewhere:(Andrés-Costaetal.,2014;Bersetetal.,2010;Bijlsmaetal.,2014a;Borova483 et al., 2014; Castiglioni et al., 2006; Castrignanò et al., 2016; Devault et al., 2014;484 Fedorovaet al., 2013;Kankaanpääet al., 2014;Karolaket al., 2010; Lai et al., 2011;485 Postigoetal.,2008;Sentaetal.,2013;Tscharkeetal.,2015;vanNuijsetal.,2009).Itis486 noteworthythatnotallanalyticalmethodologiesusedduringthemonitoringcampaign487 werepublished.488 489

21

TheQ-transitionsselectedforcocaineanditsmetabolitebenzoylecgonine(BE)490

areinpracticallyallcases304>182and290>168,respectively.Bothcorrespondtothe491

neutrallossofbenzoicacid,specificforcocaineanditsmetabolites(Bijlsmaetal.,2011;492

Castiglionietal.,2008).Inrelationtotheq-transition,somedifferenceswereobserved,493

but304>82and290>105weremostfrequentlyselected.TheQ-andq-transitionsfor494

amphetamineareinmostcases136>91and136>119,andformethamphetamine,495

150>91and150>119,respectively.Morevariationoccursintheselectionoftransitions496

forMDMA,althoughmostlaboratoriesuse194>163forquantification.497

THC-COOH has been measured in the negative- and positive-ESI modes.498

Obviously, different transitions/ions are selected in each case. The determination of499

THC-COOH is more problematic than other drugs investigated. The use of different500

ionizationmodes,thepoorersensitivityforthiscompound,andthematrixinterferences501

thataffectLC–MS/MSanalysismakeitsdeterminationmoretroublesome(Bijlsmaetal.,502

2014b;Ortetal.,2014c;Vazquez-Roigetal.,2013).Moreover,othernon-instrumental503

factors,suchaspossiblesorptiontosolids(Harmanetal.,2011),in-sewerandin-sample504

stability(McCalletal.,2016),mightalsoplayanimportantroleinthedifficultytoget505

satisfactoryresultsforthiscompound.Althoughsomedifficultiesmightberelatedtoits506

differentphysico-chemicalproperties(lowerpolarity)comparedwithotherillicitdrugs507

andmetabolites,anunambiguousexplanationhasnotyetbeenfound.Theproblems508

associatedtothedeterminationofTHC-COOHhavebeencorroboratedwiththeresults509

ofinter-laboratoryexercises,wheredataforthiscompound indicatethatrecoveriesof510

spikedamountsofTHC-COOHtowastewaterinvariablyarelow;thosedatasuggesta511

systematic underestimation of the true concentrations of THC-COOH in this type of512

matrix.513

The examples, shown above, illustrate that the selection of appropriate514

transitionsisnotabanalaspect,anditrequiresadetailedstudybefore,consideringnot515

only the abundance of the ions (used as common criterion), but also specificity and516

associatedissuessuchasbackgroundnoise.517

518

5. ApplicationsofHigh-ResolutionMassSpectrometry519

22

HRMS is a powerful technique with many different applications in the520

investigationofIDBsinwastewater,fromthescreeningoflargenumberofcompounds,521

the elucidation of unknowns, the identification of new metabolites and522

degradation/transformationproducts(TPs),toquantificationoftargetanalytesatlow523

concentrations.SeveralreviewshavebeenpublishedontheuseofHRMStodetermine524

of licit and illicit drugs in environmental analysis, and are recommended for those525

researchers interested in this field (Farré et al., 2012;Hernández et al., 2014, 2012;526

Kaufmann,2014;Petrovicetal.,2010;Vazquez-Roigetal.,2013;WongandMacLeod,527

2009).528

529

5.1. Wide-scopescreening530

HRMSallowstheefficientscreeningofalargevarietyofcompounds,including531

IDBs, in wastewater. Its potential comes from the acquisition of accurate-mass full-532

spectrumdata(Kaufmannetal.,2010).Thesedataallowonetoscreenofcompoundsin533

apost-targetwaywithouttheneedtopre-selecttheanalytesformethoddevelopment,534

as stated inprevious sections. Furthermore, thepresenceof compounds initiallynot535

considered,suchasnewsubstancesandmetabolites/TPs,canbealsoinvestigatedfrom536

dataacquiredinaretrospectivewaywithouttheneedforadditionalanalysis(Bijlsmaet537

al., 2013b; Hernández et al., 2011a). This ability is advantageous, because in some538

occasions,samplesmightalreadyhavebeendiscardedortheanalytesaredegraded,so539

additional sample injectionsmightnotbepossible. In thisway, thescreeningcanbe540

furtherwidenedbyreprocessingrawdatawithouttheneedtoperformnewanalysis.541

The most-used HRMS analyzers are undoubtedly TOF and Orbitrap. Both542

instrumentscanbeefficientlycoupledwithLC,althoughTOFMShastheadvantageof543

easy coupling with ultra-high performance liquid chromatography (UHPLC). On the544

contrary,restrictionsduetothelowerscanspeedcanlimittheapplicabilityofcoupling545

anOrbitrapwith UHPLC (Hernández et al., 2014). As a consequence of the intrinsic546

characteristics of these analyzers, the use of LC-HRMS enables screening for a large547

numberofIDBsatsatisfactorysensitivitywithinoneanalysis.Obviously,therestrictions548

derived from the chromatographic and ionization processes and from sample pre-549

treatmenthavetobetakenintoaccountinasearchforcontaminants.TOFanalyzers550

23

havebeenwidelyusedtoscreenlicitandillicitdrugs,andtheirpotentialhasbeenwell-551

documented (Hernández et al., 2014, 2011a).Mass resolution typically ranges from552

20,000uptorecently80,000FWHM,whereasmassaccuracy<2ppmandquantitative553

linearrangesstarttobecomeusual.FollowingtheinventionbyMakarov(Hardmanand554

Makarov,2003;Huetal.,2005;Makarovetal.,2006),theOrbitraphasgainedpopularity555

intheinvestigationofemergingcontaminants(deVoogtetal.,2011;Fedorovaetal.,556

2013). An Orbitrap possesses high mass resolution (>100,000 FWHM), high mass557

accuracy(<5ppm)andacceptabledynamicrange(5·103).Thenewestinstrumentscan558

reachupto450,000FWHMatm/z200andsub-ppmmassaccuracy.However,themain559

drawbackisitsscanningspeed,whichisinversetomassresolution.Thus,acompromise560

betweenachievableresolutionandadequatechromatographymustbefound(Kellmann561

etal.,2009;MakarovandScigelova,2010).562

Hybrid configurations increase the potential of these analyzers for screening563

purposes.Themost-commonareQ-TOFandLIT-Orbitrap,althoughotherpossibilities564

exist, such as IT-TOF and Q-Orbitrap. These hybrid instruments provide relevant565

structural information by obtaining accurate-mass product-ion spectra afterMS/MS566

experiments. InformationobtainedwithMS/MS is highly useful to confirmpotential567

positivesrevealedby,forexample,HRMSorQqQanalysis,andtoelucidatestructures568

ofunknownsorsuspectcompounds.However,thepre-selectionoftheprecursorionis569

required forMS/MS product-ion generation, and, therefore, a second injection is in570

principleneeded. Inorder toovercomethese limitations,product-ionspectracanbe571

collectedwithdata-dependentacquisition (DDA). In thismode, the first scanusually572

worksasthesurveyscan,wheredataareprocessed‘on-the-fly’tosearchforpotential573

compoundsofinterestbasedonpredefinedselectioncriteria;e.g.,intensitythreshold574

orasuspectinclusionlist.Iftheselectioncriteriaaremetortheincludedionisobserved,575

thenasecondMS/MSscan(data-dependent)isperformed.Themajoradvantageofthis576

approach is thecollectionof“clean”structural information in justone injection.The577

mainlimitationsaretheintensitythresholditself,aswellasthesizeoftheinclusionlist578

(numberof suspects searched);bothcannegativelyaffect theachievabledutycycle.579

Hence,adecreaseinthenumberofdatapoints(i.e.,thenumberofscans),affectsthe580

detectabilityofchromatographicpeaks.581

24

Advantageously,mostcurrentinstrumentsalsoallowtheacquisitionoffull-scan582

spectra at different collision energies in just one injection. As a function of the583

instrumentand/ormanufacturer,thisoperationmodefortheQTOFanalyzerisknown584

asMSE(Castro-Perezetal.,2002;Díazetal.,2011;Hernándezetal.,2011a;Plumbetal.,585

2006)inthecaseofWaters,broadbandcollision-induceddissociation(bbCID)(Dasenaki586

etal.,2015)inthecaseofBruker,orall-ionsMS/MS(Kinyuaetal.,2015)inthecaseof587

Agilent.LikewiseforQ-OrbitrapinstrumentsfromThermo,thistypeofacquisitionisalso588

possible,andknownasAllIonFragmentation(AIF)(Berendsenetal.,2015;Coscollàet589

al.,2014),orvariableData-IndependentAnalysis(vDIA)(ZomerandMol,2015).These590

approachesarepossiblethankstotheavailabilityofcollisiongasinsidethecollisioncell591

of the hybrid instruments. With the application of low energy in the collision cell,592

fragmentationisminimized,andtheinformationobtainedcorrespondsnormallytothe593

parent molecule ((de)protonated and/or adducts in some cases). At high collision594

energy,fragmentationofthemoleculeisfavored.Inaddition,thehigh-energyfunction595

providesnotonly fragmentationspectra similar toMS/MSexperiments,butalso the596

isotopicpatternofthefragments,anditconservesadductand/ordimerinformationas597

the quadrupole works as an ion guide. In this way, (de)protonated molecule and598

fragmentiondatacollectionarebothenabledinasingleacquisitionwithouttheneed599

toselecttheprecursorion;therefore,notaffectingnegativelythedutycycle.600

Oneofthemaindifficultiesinwide-scopescreeningistoensurethatthemethod601

candetectandidentifyallcompoundsincludedinthetargetlist.Referencestandards602

are obviously required for a final confirmation of the identity, but also needed to603

performmethodvalidation.Qualitativevalidationofthescreeningisakeyaspect,but604

alaboriousandtime-consumingtask.Theobjectiveistoensurethatthemethoddetects605

agivencompoundatanestablishedminimumconcentration;therefore,thescreening606

detectionlimit(SDL)isthemainparameterevaluated.Tothisaim,watersamplesspiked607

at different levels need to be tested to establish the SDL as the lowest analyte608

concentrationtestedthatcanbedetected(wihtthemostabundantion;i.e.,normally609

the(de)protonatedmolecule).Inabsenceofguidelinesintheenvironmentalfield,the610

approachusedinotherfields,suchaspesticideresidueanalysis(SANCO,2013)ordoping611

control analysis (Pozo, Van Eenoo, Deventer, & Delbeke, 2007) can be adopted. To612

25

accept the empirical value of SDL, it is necessary to have at least 95% of positive613

detections in the spiked samples tested. Another key parameter is the limit of614

identification; i.e., the lowest concentration tested for which the compound can be615

detected(onlyoneion)andidentified(atleasttwoaccurate-massions,withacceptable616

masserrors).Foridentification,otherparametershavetobealsoconsidered,suchas617

retentiontimeandion-ratios(seebelow).618

Qualitativevalidationisnormallyperformedwithselectedcompoundsfromthe619

target list that are taken as amodel, due to the extreme difficulties to validate the620

methodforthehugenumberofcompoundsthatmightbeincludedinthetargetlist.621

Once themethodology is validated, the screening is applied to sample analysis. The622

occurrence of false positives is drasticallyminimized if strict criteria are applied for623

identificationonthebasisoftheaccurate-massdata;however,onecannotignorethe624

possibilityoffalsenegativesforthosecompoundsthatwerenotpreviouslyvalidated.625

AlthoughthequalitativepotentialofHRMSisevident,quantitativeapplications626

havebeenmorelimiteduntilnow,mainlybecauseHRMSanalyzerstypicallyshowlower627

sensitivity and narrower dynamic range thanQqQ instruments that operate in SRM628

mode.Thus,mostresearchuntilnowhasbeenfocusedonidentificationandelucidation629

purposes. However, Orbitrap and the latest TOF instruments show improved630

performancetoprompttheirusealsoforquantificationofIDBsinwastewater(Bijlsma631

etal.,2013b;González-Mariñoetal.,2012).632

ThestrategyusedforHRMSscreeningstronglydependsontheavailabilityof633

reference standards, as described previously. Nevertheless, when dealing with634

thousandsofcompounds,itisalmostimpossibletohaveallreferencestandardsinthe635

laboratory.Oneof themainbenefitswithHRMS is that reference standardsarenot636

strictly required in a first step of the process, because a tentative identification of637

suspectcompoundscanbemadeonthebasisoftheobtainedinformation.Obviously,638

reference standards highly facilitate the analytical task in the screening, and are639

required forultimateandunambiguous confirmation (Fig. 3); however, theymaybe640

acquiredonlyinafinalstagewhensolidwell-foundedevidenceexistsonthepresence641

of the compound in the sample. In thisway, laboratoriesdonotneed toacquireall642

26

referencestandardsbeforeanalysis,withthesubsequentproblemsofavailability(e.g.,643

TPs),costs,andexpirydates(Ibáñezetal.,2014).644

645

646

Figure3.(Post)-targetscreeningstrategy.647

648

In the absence of reference standards, HRMS data might be sufficient for a649

tentativeidentification(Fig.3).Anexample,identificationof2-ethylidene-1,5-dimethyl-650

3,3-diphenylpyrrolidine(EDDP),ametaboliteofmethadone,inwastewaterbyLC-QTOF651

MS,isshowninFigure4.Bycombiningtheinformationobtainedinthelow-energy(LE)652

function on the protonated molecule and in the high-energy (HE) function on the653

fragment ions, tentative identification of this metabolite was feasible without any654

reference standard. The use of UHPLC also facilitated the assignment of the655

chromatographic peaks that corresponded to this compound (note that some ions656

presentintheHEspectradidnotcorrespondtoEDDP;markedasxinthefigure).657

(post)-Target screeningAnalysis after MS acquisition

Database of target compounds• LC-MS amenable• Reported data

Tentative identification(highly reliable, but laborious

and time-consuming

Standard available Standard unavailable

Information obtained from HRMS• Accurate mass (mass error)• Isotope pattern• Retention time• Fragment ions

Identification/Confirmation

Information in database(exact mass, fragment ions, retention time, isotope pattern)

Information obtained from HRMS• Accurate mass (mass error)• Isotope pattern• Retention time• Fragment ions

Information in database(exact mass, theoretical isotope pattern, predicted retention time)

Confirmation with reference standard (final stage)

27

658

Figure 4. Tentative identification of EDDP, a methadone metabolite. A) LE mass659

spectrum (bottom)HEmass spectrum (top).B)XICsof theprotonatedmoleculeand660

severalfragmentions(XindicatesthatthefragmentionisnotrelatedtoEDDP).661

662

Successful identification depends on the quality of the information provided663

(e.g.,morethanoneaccurate-massisrequired,lowmasserrorsgivemoreconfidence664

to the process) and on the knowledge ofmass spectrometry fragmentation rules to665

properlyjustifythefragmentionsobserved.Previousdatareportedintheliteratureon666

fragmentions(innominalandaccuratemass),whichcanalsobeavailableindatabases667

suchasMassBank (Horaietal.,2010; “http://www.massbank.jp/”), areuseful to the668

analyst. Another interesting tool is retention-timeprediction,whichhelps to discard669

potentialfalsepositivesandtofocustheelucidationprocessononlythosepeaksthat670

fit thepredictedtR.ThesetRpredictorsarebasedonquantitativestructure-retention671

relationships(QSRRs)thatvaryfromverysimple,whichincorporateasingledescriptor672

(Badeetal.,2015b;Kernetal.,2009),tomorecomplex,whichusealargenumberof673

descriptors(Badeetal.,2015a;BarronandMcEneff,2016;Gago-Ferreroetal.,2015;674

Milleretal.,2013;Munroetal.,2015);however,allofthesepredictorsarebasedona675

EDDPC20H24N+

-0.3 mDa

C18H19N+·0.0 mDa

C17H16N+

- 0.2 mDa

C13H16N+

- 0.1 mDa

- 0.3 NH2

NH2

NH

mDaC6H12N+

NH

278.1903 LE

249.1512

234.1277

186.1277

150.0918

108.0811

98.0964

XICHE

A B

X

X

XX

NH

28

singlecolumn,commonlyareversedphase(C18)column.Adirect-mappingtechnique676

(PredRet)abletopredictretentiontimesacrossdifferentchromatographicsystemshas677

also recently been developed (Stanstrup et al., 2015), but is limited to compounds678

havingtobewithinthePredRetdatabase.Althoughthisareaisrapidlyadvancing,the679

principal limitation is the use of a single column for most predictions, with further680

optimizationneededforatrulytransferablepredictiontechnique.681

One of the issues that still remains without broad consensus is the criteria682

appliedforconfidentidentityidentification/confirmation.Itisclearthatacombination683

ofparametersisrequiredforthisaim,includingretentiontimeandMSdata.Accurate-684

massmeasurements givemore confidence for a reliable identification than nominal685

massdata,andthisfactorisrecognizedinseveralguidelines,which,forexample,give686

moreidentificationpointstoHRMSionsthanLRMSions(EuropeanCommission,2002).687

In addition, the ion intensity ratio is commonly used as a key parameter in the688

identificationprocess. It iswidely accepted that at least twoaccurate-mass ions are689

requiredforaconfidentidentificationwithHRMS.However,twomainissuesneedtobe690

considered:1)what istheacceptablemasserror?2)what isthemaximumdeviation691

acceptableintheionratio?Moreover,anotherparameterhelpfulintheprocessisthe692

isotopicdistribution,especiallywhenabundantisotopeionssuchaschlorine,sulphur,693

orbrominearepresent.Severalsituationsmightoccurthatleadtodifferentdegreesof694

confidenceinidentification.Forexample,Schymanskietal.(Schymanskietal.,2014a)695

proposeupto5levelsofconfidenceinanon-targetanalysis.Theselevelsrangefrom696

only exact mass to unequivocal molecular formula, and then tentative candidate(s)697

followedbyprobablestructuretoafullyconfirmedstructurewithareferencestandard.698

Figure5summarizesthekeyparametersinthedetectionandidentificationofa699

compoundwithHRMS.As shown in this figure, different scenariosmight occur as a700

function on the information provided, and on the availability of reference standard701

(Badeetal.,2015c;Hernándezetal.,2015a;Nácher-Mestreetal.,2016).702

703

704

705

29

706

Figure 5. Detection and identification criteria in screening of illicit drugswithHRMS707

(modifiedfrom(Nácher-Mestreetal.,2016)).708

709

When a reference standard is available, compounds can be detected or710

identified. Detection is considered satisfactory when the most-abundant ion (Q),711

commonlythe (de)protonatedmolecule, is foundat theexpectedtR (±0.1min,)and712

masserror<5ppm(SANTE/11945,2015).Anotherlikelysituationfordetectionistofind713

tworepresentativeions(i.e.,themost-abundantion(Q)andafragment/adductions(q)714

attheexpectedtR),butwithmasserrorsbetween5-20ppm.Thelattersituationseems715

tooccurwhenthesignalintensityislow(favoredatlowanalyteconcentrations).Inthat716

case, an additional effort is recommended to investigate more accurate-mass ions717

and/orrepeatsampleinjection. Identificationisbasedonthepresenceofatleasttwo718

representativeions(Q,q)attheexpectedtRwithmasserrors<5ppm.Additionally,q/Q719

ratios should fit with those for reference standards within tolerance limits720

(SANTE/11945,2015).Identificationundertheseconditionsishighlyreliableandcanbe721

consideredastheidealsituation.722

Whenthereferencestandardisnotavailable, atentativeidentificationcanbe723

madewhenanexpected ionwithmasserror<5ppm isobserved, togetherwith its724

Screening with HRMS, key parametersRetention time (Rt)Accurate masses

Mass errorsIsotope pattern (Cl, Br…)

• Expected ion (Q), mass error < 5 ppm)

• Compatible isotope pattern (Cl, Br...)

• One or more fragment ions (q), - in agreement with data reported - compatible with the chemical structure

of the candidate (mass error < 5 ppm)

Tentative identification

Identification required withreference standard

Detection

Option 1

•One ion (Q)•Rt agreement •Mass error < 5 ppm

Option 2

• Two ions (Q and q)•Rt agreement •Mass error > 5 ppm

Identification

Standard available

• Two ions (Q and q)

•Rt agreement

•Q mass error < 5 ppm

• q mass error < 5 ppm

• Isotope pattern

Standard unavailable

30

characteristicisotopicpattern.Subsequently,thefragmentionsshouldbeevaluatedby725

comparingthedatawith,e.g.,datareportedintheliteratureorjustifiedbytheaccurate-726

mass fragments taking into account the structure of the molecule. However, for727

structureconfirmation,injectionofthereferencestandardiseventuallyrequired.728

729

5.2. Non-targetanalysis730

Full-spectrum accurate-mass acquisition provided with HRMS also opens the731

possibilitytoinvestigatenon-targetcompoundsinwater(Díazetal.,2012;Hernández732

etal.,2011b;Hugetal.,2014;Ibáñezetal.,2005;Kraussetal.,2010;Schymanskietal.,733

2015).Atrue“unbiased”non-targetscreening,withoutanyaprioriinformationonthe734

compoundstobedetected,isananalyticalchallenge.Thisprocessneedsexpertise,and735

iscomplexandtime-consuming.Themaindifficultiesassociatedwiththisprocesswhen736

applied to environmental orwastewater samples come from: (i) the complexity and737

unknowncompositionofthesamplethatisinvestigated,(ii)thepresenceofmanypeaks738

in the total ion chromatogramwith themost-abundant corresponding commonly to739

compoundsotherthantheanalytes,and,(iii)thelowanalyteconcentrations.Thus,the740

mainproblemistoprioritizethemost“relevant”chromatographicpeaksinthesample,741

becausethemajoritywillnotbeassociatedtodrugs,inordertofocusthesubsequent742

elucidation process on those compounds. From the HRMS information, a complex743

process has to be applied that establishes the empirical formula of the unknown744

compound,searcheschemicaldatabasesforpotentialcandidates,andfinallyassignsthe745

chemicalstructureofthediscoveredcompound.746

Ina truenon-targetanalysis, themaximumnumberof compounds fromvery747

different physico-chemical characteristics should be investigated. Therefore, a748

combination of GC-HRMS and LC-HRMS seems to be the most-appropriate way to749

achievethisaim.Duetothecomplementarityofthesetwotechniquesthisapproach750

canbe seenas themost-comprehensive toadvance towards thedesired “universal”751

screening(Hernándezetal.,2015b).Obviously,some“difficult”compoundswouldnot752

likely be included in a wide-scope screening. For example, very polar/ionic analytes753

wouldrequirespecificchromatographicseparation(e.g.,HILIC).Thus,acombinationof754

C18andHILICchromatographiccolumnswouldrenderawiderscopeforLC-amenable755

31

compounds.Itmustbetakenalsointoaccountthatuniversalityofthescreeningshould756

notreferonlytothetechniquesofmeasurementbutalsotothesampletreatment.From757

thispointofview,agenericextraction,orevenbetter,directanalysisofsamplesinto758

theMS system,would be the best option to avoid compound losses during sample759

manipulation.760

Because no pre-selection of analytes is made in non-target analysis, the761

compoundsdiscoveredinwatersamplesmightbelongtotheillicitdrugsgroup,butalso762

toanyotherfamilyoforganiccontaminantsortheirmetabolites.ThefactthatmostIDBs763

areofmediumtohighpolaritymakesLC-HRMSthemost-attractiveapproachfortheir764

potentialidentification.However,theabsenceofstandardizedmassspectrallibrariesin765

LC-MSisanadditionaldifficultyinnon-targetanalysis.Advancesinthecreationofmass766

spectra libraries for LC-MS/MSanalysiswill beofhelp in thenear future,but at the767

momenttheanalystdoesnotcountontheaidofstandardizedlibrariestofacilitatea768

non-targetanalysis.769

An intermediate situation between target and true non-target analysis is the770

applicationof“biased”non-targetapproaches,where,forexample,theformationof771

“unknown”metabolites/TPs from a given parent compound is investigated with “in772

vitro”or“invivo”experiments,degradationlaboratoryexperiments,orin-silicomodels773

(Reid et al., 2014a). Hence, the investigation is focused on chemically related774

compounds(e.g.,shareacommonfragment,moiety,ormassdefect)oroncompounds775

thathavespecificatomsintheirstructurethatgiveadistinctiveisotopicsignature(e.g.,776

Cl,Br,S).Here,thenumberofchemicallymeaningfulstructures,whichcanbeassigned777

toanunknownpeak, is limited to structures that showaclose relationshipwith the778

parentcompound(Kraussetal.,2010).Thisissuewillbrieflybetreatedinthefollowing779

section.780

781

5.3. Investigationofmetabolitesandtransformationproducts782

The investigationofmetabolitesandTPsof illicitdrugs inwater samples isa783

current topic of research (Bletsouet al., 2015).WBE is basedon the analysis of key784

biomarkersofdrugs.TheseIDBscanbetheparentcompoundand/orthemajorurinary785

32

metabolite(s).Forthemostknownandwidelyconsumeddrugs,informationonhuman786

metabolismisalreadyavailable,andhasallowedestablishmentofbenzoylecgonineas787

themainmetabolite and IDB of cocaine, THC-COOH asmainmetabolite and IDB of788

cannabis,orthatmethamphetamine,amphetamine,andMDMAaremainlyexcretedas789

unchanged compounds. However, this information is usually scarce for NPS and,790

therefore,themainbiomarkerofuseisgenerallynotwell-established.791

AlthoughWBEissolelybasedonthemeasurementofappropriatemetabolites792

thatresultfromhumanexcretion(commonlythemajorandmost-stableones),thereis793

alsoaconcernaboutthepresenceofmanyothermetabolitesandTPsofillicitandlicit794

drugs in the aquatic environment. Especially, possible long-term (chronic) effects on795

organismsandeffectsofcombinedexposuretomultiplecompoundsisofconcern(van796

derAaetal.,2013).Thedetectionandidentificationofthesecompoundsisachallenge797

forenvironmentalanalyticalchemists,anddifferentapproachescanbefollowedtothis798

aim.InthecaseofknownmetabolitesandTPs,alreadyreportedintheliterature,an799

inclusionlistoftargetanalytescanbemade.Fromananalyticalpointofview,theycan800

be treated similarly to their parent compound with the above-mentioned target801

methodologies. Furthermore, retrospective analysis is also feasible by reviewing the802

acquired MS data. For example, metabolites of drugs have been retrospectively803

investigatedinwastewatersamplespreviouslyanalyzedforparentcompoundsonly.In804

thisway,severalmetabolitesweretentativelyidentifiedwithouttheneedofadditional805

analysis, and illustrate the potential of HRMS in this field (Bijlsma et al., 2013b;806

Hernándezetal.,2011a).807

Regardingunknownmetabolites andTPs, theuseof common fragmentation808

pathways between the parent compound and metabolites/TPs might discover809

new/unexpected compounds. In this strategy, a common behavior in their810

fragmentation is assumed. Thepresenceof additional chromatographicpeaks at the811

accurate masses of the fragments might reveal the presence of analyte-related812

compounds. The accurate-mass spectra and appropriate study of the fragmentation813

mightfinallyallowthe(tentative)identificationofmetabolites/TPs(García-Reyesetal.,814

2007;Hernándezetal.,2011b;Thurmanetal.,2005).Thisapproachcanbeextended815

33

basednotonlyonthefragmentationpathwayoftheparentcompound,butalsoonthat816

ofmetabolites/TPsdetectedinsamples(Hernándezetal.,2009).817

A simpler approach is prediction of possible metabolites or TPs with818

computational(insilico)predictiontools(Kirchmairetal.,2015;Reidetal.,2014a).Many819

different methodologies to predict metabolites or sites of metabolism have been820

reportedrecently.Themetabolicfateofamoleculedependsonitschemicalreactivity821

towardsmetabolicprocessesthatcanoccur,aswellasonitsinteractions(affinityand822

binding orientation) with the biotransformation enzymes involved. The prediction823

systemshould,therefore,properlybeselectedafterconsiderationoftheorganismor824

the system where metabolites/TPs are formed. Commercially available and freely825

accessibleprogramshavebeenapplied in thisprediction step.Kirchmairetal,2015,826

reviewedtendifferentsoftwareforpredictingmetabolites,butonlytwoarewithout827

costsandanadditionaloneissolelyavailableforacademia(Kirchmairetal.,2015).This828

free-availabilityisprobablythemainreasonthattheUniversityofMinnesotaPathway829

PredictionSystem(UM-PPS:(“http://eawag-bbd.ethz.ch/”)isoneofthemost-common830

predictiontoolsinsuspectmetabolite/TPsscreening(Kernetal.,2009).831

Predictionofmetabolites/TPsisfollowedbyHRMSanalysis;theexactmassfor832

eachofthepredictedcompoundisextractedfromthechromatogramandcheckedbya833

comparisonwithcontrolsamples.TheplausibilityofthechromatographictR, isotopic834

pattern,andionizationefficiencyareusedasfurtherfilterstonarrowdownthenumber835

ofcandidatepeaks.Thestructuresofsuspectedcompoundsaretentativelyidentified836

basedontheobservedfragmentationpattern.837

AsuitablestrategytoinvestigatemetabolitesandTPsmakesuseof“invitro”or838

“invivo”metabolismexperiments,andoflaboratoryorfield-degradationexperiments839

undercontrolledconditions,whichcanidentifyknownandunknownmetabolitesorTPs840

ofselecteddrugs,respectively.Intherecentliterature,severalexamplescanbefound841

thatdealwiththeinvestigationofdrugmetaboliteswithinvitroorinvivoexperiments842

(Holmetal.,2015;Ibañezetal.,2016;Meyeretal.,2015;Pozoetal.,2014;Takayama843

etal.,2014).Theseexperimentsareveryusefulandallowthediscoveryofmetabolites,844

whichmight be expected and detected inwastewater, and are, therefore, potential845

targetIDBsinfutureWBE-basedstudies.Thediscoveryofmetabolitesisofparticular846

34

relevancefornewdrugs;i.e.,NPSwhosemetabolismisnotwellknown.Otherpapers847

study the degradation of illicit drugs, after spiked samples have been subjected to848

processes,suchashydrolysis,photodegradation,chlorination,biodegradation,orany849

otherprocessof interest(Bijlsmaetal.,2013a;Boixetal.,2014,2013;Postigoetal.,850

2011). In all cases, HRMS plays a key role in the tentative identification of the851

compoundsformed.Furthermore,theuseofspecializedsoftwareiscriticalforarapid852

andefficient comparisonbetween the full scandata set fromuntreatedand treated853

samples,becausemanualinspectionofTICchromatogramtolookforvisiblepeakscan854

easilyfailincomplexmatriceswithlowanalyteconcentrations.855

856

6. Chiralanalysis857

The determination of specificmetabolic excretion products of illicit drugs in858

wastewater is not always possible, andmakes difficult the differentiation between859

consumptionofdrugsanddirectdisposalofunuseddrugs. In such situations, chiral860

analysis can be applied because most illicit drugs are chiral, and are subject to861

stereoselectivehumanmetabolism (Emkeetal., 2014;EvansandKasprzyk-Hordern,862

2014).863

MDMA (3,4-methylenedioxymethamphetamine) and MDA (3,4-864

methylenedioxyamphetamine)profiling inwastewater isanexcellentexampleof the865

importanceofchiralenantioselectiveanalysistodistinguishbetweenconsumptionand866

directdisposalofunusedMDMAandMDA.Bothdrugshaveoneasymmetriccarbon867

centerandthereforetheycanexistintheformoftwoenantiomericpairs,whichdiffer868

bothquantitativelyandqualitativelyinpharmacologicalactivity:S(+)-enantiomersare869

more amphetamine-like stimulants and R(−)-enantiomers are more hallucinogenic870

(Kasprzyk-HordernandBaker,2012a).BothMDMAandMDAhavenomedicalusageand871

are synthesized and abused in racemic forms. Their human metabolism is872

stereoselective and leads to the enrichment of excreted drugs with their R(−)-873

enantiomers.However,ifthepresenceofMDAinurineisduetoMDMAabuseandnot874

directMDAuse,anenrichmentofMDAwithS(+)-enantiomertakesplace(Mooreetal.,875

1996).Itis,therefore,expectedthatafterconsumption,bothdrugswillbepresentin876

urineandwastewaterenrichedwithR(-)-enantiomers. Indeed,Kasprzyk-Hordernand877

35

Baker(Kasprzyk-HordernandBaker,2012a)reported,inafirststudyofthiskind,that878

MDMAwasenrichedwiththeR(-)-enantiomerduetopreferentialmetabolismofS(+)-879

MDMA in humans. Furthermore, the identified MDA was enriched with S(+)-880

enantiomer,tosuggestthatitspresencemightbeassociatedwithMDMAconsumption881

anditssubsequentmetabolismintoS(+)-MDAandnotintentionalMDAuse(ifthelatter882

weretrue,MDAinwastewaterwouldbeenrichedwithR(-)-enantiomer).883

Surprisingly,inseveralinstances,duringEurope-widemonitoringundertakenby884

theSCOREgroupin2011-13(Emkeetal.,2014;Ortetal.,2014c;Thomasetal.,2012),885

unexpectedlyhighloadsofMDMAwereobserved.Forexample,in2011,aberrantlyhigh886

massloadsofMDMAwereobservedinthewastewaterofUtrechtintheNetherlands.887

These loads highly deviated from the results observed in the previous monitoring888

campaign in 2010 (Bijlsma et al., 2012). Enantiomeric profiling as shown inFigure 6889

revealedthatMDMAwasracemic(enantiomericfraction(EF)=0.54),whichindicated890

its direct disposal in the sewage system and further explains high loads of MDMA891

quantifiedinUtrechtwastewaterduringthesamplingweekin2011(averageloadwas892

20-timeshigherthanin2010).Incontrast,thesamplesfrom2010(greenlineinFig.6)893

showedanaverageEFof 0.65 that corresponded toexcretionprofiles inurine after894

consumptionofMDMA(Emkeetal.,2014).Thisdirectdisposalcouldbetheresultofa895

police raid into an illegal production facility that took place two days before the896

monitoringstarted(Emkeetal.,2014).Thepoliceestimatedthat30kgofrawMDMAor897

tabletshadbeendisposedunderthepressureofthepoliceraid.898

Enantiomericprofilingofwastewaterrepresentsapowerfultoolthatallowto899

determineifmassloadsofstudieddrugsactuallyoriginatedfromconsumption,disposal900

ofunuseddrugs,orproductionwaste.901

902

903

36

904

Figure6.MDMA loadsduring two separateweeks sampled in2010and2011 in the905

sewage treatment plant of Utrecht, the Netherlands, and their corresponding906

enantiomericfractions(EF)(Emkeetal.,2014).907

908

Cellobiohydrolase (CBH) is the most widely used stationary phase for909

enantiomericprofilingofamphetamineswithchiralliquidchromatographycoupledwith910

tandemmassspectrometry.Thiscellulaseenzymeisimmobilizedonto5µmsilicabeads911

withanisoelectricpoint(pI)of3.9.Itcontainsmultiplechiralcentresandmechanisms912

forionic,hydrophobic,andhydrogenbonding.Ithasbeensuccessfullyappliedwithan913

isocraticmobilephaseofH2Owith10%2-propanoland1mMNH4OH,toamphetamine-914

likecompounds(Fig.6and7)(Bagnalletal.,2012;Emkeetal.,2014;Kasprzyk-Hordern915

andBaker,2012a,2012b).Thereareseveralfactorsthathavetobetakenintoaccount916

toachievesatisfactorychiralrecognitiononCBH.Theseareprimarilytemperature,pH917

andmobile-phasecomposition.Mobile-phasepHplaysakeyroleinchiralrecognition918

becauseitinfluencesionizationofbothanalytesandCBH.Duetotheisoelectricpointof919

3.9,CBHisnegativelychargedatpH>pI,soincreasingthemobilephasepHwillfacilitate920

ionicinteractionswithpositivelychargedanalyte(e.g.,amphetamineorMDMA).This921

interaction will facilitate longer retention times and higher enantioselectivity.922

Hydrophobicinteractionsandhydrogenbondingcanbeinfluencedwithmobilephases923

thatcontaindifferentnatureandpercentageoforganicmodifiersand ionicstrength.924

Lessthan20%oforganicmodifier isallowedtoavoiddenaturationofthisenzymatic925

chiralselector.Lowerpercentagesoforganicmodifiersleadtohigherretentioninthe926

37

case of amphetamine-like compounds. Isopropanol, methanol, or acetonitrile are927

usuallyusedasorganicmodifiers.Theyarecharacterizedbydifferentelutionstrengths;928

e.g.,methanol has a lower elution strength than isopropranol. Other factors, which929

should be considered are (Camacho-Muñoz et al., 2016): (i) correct sampling and930

sample-preparation protocols that do not introduce stereoselectivity (e.g.,microbial931

metabolicdegradationofanalytesmightleadtoincorrectestimationofenantiomeric932

fractions, or useof chargedeluting agents during SPE (e.g.,methanolmodifiedwith933

ammoniumhydroxide)mightleadtolossofchiralrecognitionintheCBHcolumn),and934

(ii)eliminationofmatrixeffects(viarobustsamplepreparationapproachesanduseof935

deuteratedor13C-labelledinternalstandards).936

EnantiomericprofilinghasbeenusedinWBEasacomplementarytoolalongside937

non-chiralmulti-residuemethods that use reversed phase (C18) stationarymaterials.938

Thisadditionalanalysisrequiredanadhocsamplepreparation,whichmeantahigher939

quantity of sample, and a more time-consuming and less-cost effective analysis. A940

recentlydevelopedmulti-residuemethodcombinedchiralrecognitioncapabilityofthe941

CBH-basedstationarymaterialswithmulti-residueseparationpotentialoftheC18-based942

materials.Themethodologyenableddetectionandquantificationofalltargeted(chiral943

andnon-chiral)humanbiomarkersinwastewateralongwithsatisfactoryenantiomeric944

separationsof18analytesandauniquesinglesample-preparationstep(Castrignanòet945

al.,2016).946

947

38

948

Figure7.Masschromatogramsshowchiraldrugs:amphetamine(AMPH),MDMA,MDA949

andmethamphetamine (METH) inwastewaterobtainedwithCBHcolumnandHPLC-950

QqQMS(modifiedfrom(Kasprzyk-HordernandBaker,2012b)). 951

952

7. Relevantanalyticalparametersandqualitycontrol953

Theuseofadvancedanalyticaltechniquesandtheexpertiseoftheanalystare954

essential to obtain accurate quantitative data for IDBs in wastewater samples. In955

addition,appropriatemeasuresforqualitycontrolarerequiredtoobtainreliabledata.956

1

2

S(+)-MDA

R(-)-MDA

R(-)-MDMA

S(+)-MDMA

S(+)-METH

R(-)-METH

R(-)-AMPH

S(+)-AMPH

39

Obviously,priortoitsapplication,theanalyticalmethodologyneedstobefully957

validatedforallanalytesintermsoflinearity,trueness/accuracy(evaluatedbymeans958

ofrecoveryexperiments)andprecision(asrepeatabilityRSD),selectivity/specificity,and959

limitsofdetection(LOD)andquantification(LOQ).Oneofthemaindrawbacksinthis960

fieldisthelackofguidelinesspecificallydirectedtowardsanalysisofIDBsinwastewater.961

In absence of such guidelines, recommendations in other fields, such as pesticide962

residue analysis (SANCO, 2013; SANTE/11945, 2015), residues in products of animal963

origin(EuropeanCommission,2002)bioanalyticalmethods(EMA,2012),orclearwater964

actprograms(EPA,2007)canbeusedasguidelines.965

Commonly,aminimumof5replicatesarerequiredtochecktheaccuracyand966

precisionatthetargetedLOQ,andatleastoneotherhigherlevel,forexample,10times967

the targeted LOQ. A quantitative analytical method should be demonstrated at the968

initialvalidation,butalsolaterwithsamplebatchestoperformqualitycontrols(QCs)969

thatprovideacceptablerecoveryateachspikinglevelandforeachanalyte.Acceptable970

mean recoveries for IDBs inwastewaterare typically in the range70–120%,withan971

associated repeatability RSD ≤ 20%, (e.g., as established for pesticides in972

SANCO/12571/2013(SANCO,2013)).973

Aspreviouslydescribedinthisreview,LC-MSsystemsaremostwidelyappliedin974

WBE studies. Yet,matrix effects are one of themain problems associatedwith the975

correct determination of IDBswith these techniques.Matrix effects result from the976

competition between matrix co-eluting components and analytes in the ionization977

process(Trufellietal.,2011).Thiscompetitionaffectsquantificationoftheanalytesand978

must,therefore,beremoved,minimized,orcorrectedfor.Quantificationwithmatrix-979

matchedcalibrationissuitableandisfrequentlyusedinsomeresearchfields,wherea980

representative blank matrix can be easily obtained. However, the variability of the981

chemicalcompositionofwastewaterandthecommonpresenceofsomecompoundsin982

thesamplesusedas“blank”posesdifficultiestousethisapproachinWBE.Instead,the983

standardadditionsmethodmightbeused,butitwouldimplymanymoreinjectionsin984

theLC-MSsystem,apartfromtheneedtoadjusttheadditionsaccordinglytotheanalyte985

concentration in the sample for appropriate application of this methodology.986

Furthermore, the high level of some compounds makes it problematic to maintain987

40

linearityinthecalibrationcurve.Hence,theuseofisotope-labelledinternalstandards988

(ILIS)isthemostcommonwaytocorrectformatrixeffects,butalsoforpotentialerrors989

associatedwithsamplemanipulationandstorageiftheILISisaddedtothesamplejust990

aftersamplecollection(i.e.,assurrogate).Whenavailable,theuseofalabelledanalyte991

isrecommendedtoensureasatisfactorycorrectionofmatrixeffects.However,itshould992

be noted that, during method validation, there is an absolute need to thoroughly993

evaluateifthelabelledISaccuratelycorrectsformatrixeffects.994

Theestimationoflimitsofdetection(LODs)andlimitsofquantification(LOQs)995

isusuallyperformedbasedonasignal-to-noise(s/n)ratioof1:3and1:10,respectively.996

More-realisticLOQscanbeestimatedats/n10forthequantitationtransition,butalso997

at s/n 3 for the qualification transition to ensure not only the quantification of the998

compoundbut also its reliable identification (Bijlsma et al., 2014a). InOrbitrap data999

processing,noiseisfilteredout.Therefore,thecommons/napproachtoderiveLOQand1000

LODdoesnotapply.Instead,LOQsinOrbitrapMSaredeterminedbasedonthelowest1001

concentrationofanILISinpurewaterthatproducesanappreciablesignalthatmeets1002

theidentificationcriteria(Bijlsmaetal.,2013b;deVoogtetal.,2011).Thematrixeffect1003

iscalculatedfromtheratiooftheresponsesoftheILISinthematrixandinpurewater1004

and used to calculate the actual LOQ in that matrix. Some guidelines, like SANCO,1005

definedthemethodLOQas the lowestspiking levelof thevalidationthatmeets the1006

methodperformanceacceptability–thatdefinitionisastrictercriterionthatcanalso1007

be applied in WBE. Anyway, an estimation of LODs and LOQs in wastewater is1008

complicated, because notable variations in chemical composition between samples1009

occur.Representativematrix-matchedstandardscannotbeeasilyprepared,duetothe1010

presenceofanalytesinmostinfluentsamples,andbecauseofthevariationsinsample1011

composition from one sample to the other. This fact makes problematic a rational1012

comparison of these parameters among different published methodologies. An1013

estimation of LODs/LOQs from standards in solvent at least could make a fair1014

comparisonbetweeninstrumentalperformances,althoughnotanalyticalmethods.An1015

efficientandrealisticapproachtoestimatetheseparametersinahomogeneouswayis1016

frominter-laboratoryexerciseswhereallparticipantsanalyzethesamesampleswith1017

theirownanalyticalmethodology.1018

41

Confirmation of positives is an essential aspect in the analysis. To this aim,1019

guidelinessuchasthatfromtheEuropeanCommission(EuropeanCommission,2002)1020

canbeadopted,wheretheconfirmationofpositivefindingsisbasedonthecollection1021

ofidentificationpoints(IPs).ThenumberofIPsearneddependsonthemassanalyzer1022

used.Thus,forQqQlow-resolutioninstrumentsusedintandemMS,aminimumoftwo1023

transitionsshouldbemonitoredforasafepositivefinding,whereasinhigh-resolution1024

instrumentsatleasttwoionsmustbemonitored.Inaddition,theaccomplishmentof1025

theionratiobetweenrecordedtransitionsandaretentiontimewithinthemaximum1026

tolerancesallowedarerequired.1027

Notonlyquantification,butalsoconfirmationoftheidentitycouldbeaffected1028

bymatrixinterferences.Asstatedbefore,twotransitions(Q,q)arenormallyacquired1029

inLC-MS/MSanalysis.Oneofthemost-controversial issues istheaccomplishmentof1030

theionratiobetweenthesetransitions(q/Qratio)requiredforconfirmation.Ionratios1031

insamplesmightbeaffectedbymatrixcomponents;e.g.,whenatleastoneofthetwo1032

selectedSRMtransitionsissharedwiththematrix,whichmightleadtounexpectedion1033

ratioswithdeviationsthatexceedthemaximumallowed.Thissituationismoreprone1034

tohappenwhenthespecificityoftheneutrallossesinvolvedintheselectedtransitions1035

isnotevaluatedandcommonlosses,suchasthelossofH2OorCO2,areimplicated.Non-1036

specific transitions are a weakness not yet tackled with the current guidelines and1037

shouldbeconsideredinthisresearchfield(Delatouretal.,2007).Inthissituation,the1038

acquisitionofallavailabletransitionsisrecommendedtofacilitateconfirmationofthe1039

positivesbytestingtheadditionaltransitionsacquired.Incasesofnon-compliance,the1040

samplemightbereportedaspositive,butincludeacommentonthenon-complianceof1041

theratio,andreporttheactualq/Qratiodeviationobtained.Additionalworkwouldbe1042

necessarytotestifanyinterferingcompoundactuallyaffectedtheq/Qratio.1043

Duringarecentinter-laboratorystudyundertakenbytheSCOREgroup,oneof1044

thequestionsraisedrelatedtothesignificantvariationsinq/Qratiosreportedbythe1045

differentparticipants(Castiglionietal.,2013),evenwiththesameSRMtransitions.As1046

anexample,forcocaine,with304>182(Q)and304>82(q),ionratiosfromdifferent1047

laboratoriesrangedfrom0.12to0.38,andseemedtonotablydependontheinstrument1048

used and on the parameters optimized in each laboratory (e.g., cone and collision1049

42

voltages). In addition, some variations from batch to batch were also observed.1050

Nevertheless, these variations were not relevant to confirm identity, because it is1051

recommended that q/Q ratios are measured and evaluated for accomplishment by1052

everylaboratorywithineachsamplebatchwiththestandardsincludedinthesequence1053

ofsampleanalysis.1054

Apart fromthevalidationofthemethod ineach laboratorybefore itsroutine1055

use,theuseofinternalqualitycontrols(QCs)isofprimaryimportancetotrackpossible1056

dailymethodvariations.Intheory,aninternalQCisafinalcheckofthecorrectexecution1057

ofalltheprocessesthatareincludedintheanalyticalprotocol.Duetothedifficultiesto1058

obtainblankwastewatersamples,thosewiththelowestconcentrationsexpectedare1059

preferredtopreparespikedsamplesusedasinternalQCs.Thismeansthatsamplesfrom1060

locationswithlowdruguseand/orcollectedinthemid-week,wheretheconsumption1061

ofillicitdrugsisexpectedtobelowerthanintheweekends,wouldfitbettertoprepare1062

theQCs.DespitethisfactthatlowconcentrationsofIDBsareexpected,samplesselected1063

should be analyzed to accurately know the potential analyte concentration in these1064

“blank”samples,inordertobesubtractedfromtheQCprepared.Althoughthereisno1065

guidelineinthisfield,QCsindividualrecoveriesfrom60to140%mightbeacceptablein1066

wastewater analysis, as suggested in the SANCO guidelines (SANCO, 2013). Other1067

guidelinesrecommendanacceptablerangeof80-120%attheLOQlevel(EMA,2012).1068

WhenQCrecoveriesareoutofthisinterval,thesamplesequenceofanalysisshouldbe1069

repeated.Iftheproblempersists,thenappropriatemeasuresshouldbetakentoensure1070

theaccuracyofthemethod.1071

The implementation of quality assurance practices to analytical chemistry is1072

recognized as a prerequisite to produce data with known metrological qualities.1073

Regardlessofthetargetanalyteorsampletype,qualityassuranceandqualitycontrol1074

are the cornerstones to analytical data validation. In addition to daily internal1075

performanceverificationsviaQCmaterials,agoodqualityassuranceplanshouldinclude1076

regularexternalperformanceevaluationsforanindependentassessmentofanalytical1077

proficiency.Thegoalofinter-laboratorystudiesistodemonstratethatparticipationin1078

thisexercise leads to improvedqualityofanalytical results.Theresultsareofcrucial1079

interestforlaboratoriesbecausetheseprovideclearinformationoftheirmeasurement1080

43

capabilities.Ithastobepointedoutthatparticipationiseithervoluntaryorforcedby1081

external requirements (e.g., legal, accreditation, control bodies). Inter-laboratory1082

exercisesinvolvecomparisonofparticipants’resultsonasetofspikedwatersamples.1083

Careneeds tobetaken, toensureconcentrationsareequal inallaliquots,especially1084

whenparticulatemattershouldalsobeanalyzed.Thisalsoholdstruewhensubsampling1085

fromcompositesamples(CapelandLarson,1996).Theseparateresultsarecompared1086

withthemedianoftheentiregrouptodeterminetheaccuracyofthemethodused.By1087

usingasetoftwosamplesspikedatdifferentlevelswiththesamecompoundasocalled1088

Youdenpairiscreated.VisualizationoftheseresultsinaYoudenplotshowwithin-and1089

between-laboratory variability. Also individual constant errors become visible, and1090

randomerrorsandsystematicalerrorscanberecognized.1091

Several inter-laboratory studies have been organized by the SCORE group on1092

selectedIDBsregularlymonitoredinWBE.Intheperiod2011-2015fiveconsecutivesets1093

ofinterlaboratoryexerciseshavebeenorganized,initiallywithmethanolicsolutionsof1094

analytes that gradually moved to more-complicated matrices such as extracts and1095

genuinewastewatersamples.Thesewerethefirstinter-laboratoryexercisesorganized1096

todetermineIDBsinwastewater.Theresultsofthisstepwiseinter-laboratorytesting1097

and the improvements made during this five-year period can help to optimize the1098

analyticalproceduresofparticipatinglaboratoriesandaidinareliableinterpretationof1099

WBEdata.Tothisend,concentrationsofIDBsinwastewaterprovidedbylaboratories1100

thatdidn’tperformwellintheinterlaboratoryexerciseswerenottakenintoaccount;1101

however,togetherwithexpertsinthefield,solutionsfortheanalyticaldeviationsare1102

sought.1103

1104

8. Generalsummaryandperspectives1105

The investigation of IDBs in wastewater is a subject of current interest in1106

analyticalchemistry.Thecomplexityofthematrixunderinvestigation,thelowanalyte1107

concentrations commonly found, and the need to detect and quantify not only the1108

parentcompoundsbutalsothemainmetabolitesofdrugs,makethistaskananalytical1109

challenge. In addition, humanmetabolism is not alwayswell known, and the target1110

biomarkersmightbeunknown.Inthisfield,liquidchromatographycombinedwithlow-1111

44

andhigh-resolutionMSisanindispensabletool.Differentstrategiescanbeappliedto1112

investigateIDBsinwastewater(discussedinthisreview)fromquantificationoftarget1113

compoundstothedetectionand(tentative)identificationofunknownmetabolitesand1114

TPs.1115

QuantificationofIDBsinwastewaterisarequisiteinWBEinordertoestimate1116

druguseinpopulations.However,thepresenceofdrugresiduesinwastewateralsohas1117

other implications. For example, there is a possible environmental impact when1118

compounds are not completely removed byWWTPs. Indeed, low removal rates for1119

certain illicit drugs, such asMDMA, ketamine, andmethadone, have been observed1120

(Bijlsmaetal.,2012).Thuslowremovalratesimpliesthatdrugresiduescanbepresent1121

in effluent wastewater, and finally reach the aquatic environment. Therefore, the1122

determinationofillicitdrugsandmetabolitesineffluentwastewaterandsurfacewater1123

isalsoofinterest,aswellastheinvestigationofpotentialTPsthatcanbeformedinthe1124

environment.Here,concentration levelsaremuch lowerthan in influentwastewater1125

and,therefore,excellentsensitivityisrequiredfortheanalyticalmethods.1126

In this field,undercontinuousdevelopment,several trendsandneedscanbe1127

highlighted:1128

1) Thereisaneedtoincreasetheknowledgeofmetabolitesthatmightbepresent1129

in the aquatic environment. Especially forNPS, there is a lack ofmetabolism1130

studies,andtargetcompoundsarestillnotfullyidentified.Here,theuseofHRMS1131

is highly promising due to its potential to detect and identify drug-related1132

compounds to make use of different approaches, such as the “common1133

fragmentationpathway”,whichisveryusefultofindparent-chemicallyrelated1134

compounds.Advantageously,aretrospectiveanalysiscanbemadeatanytime1135

fromHRMS data to facilitate the search of compounds of interest (e.g., new1136

discoveredmetabolites)insamplespreviouslyanalyzed.Furthermore,fromthe1137

perspectiveofenvironmentalchemistry,itisofinteresttoextendthisresearch1138

toTPs,whichinmanyoccasionsarestillunknown.1139

2) Thedevelopmentofwide-scopescreeningwillsurelybeoneofthekeyfeatures1140

ofHRMSinthenextfewyears.Theuseof longlistsofselectedcompoundsis1141

essential to facilitate target screening with or without (suspect screening)1142

45

reference standards. Tomaintain the “universality” of this approach, generic1143

sample treatments would be required in order to avoid potential losses of1144

analytesduringsamplehandling.Someproblematiccompounds,mainlythose1145

presentinionicform,willalwaysrequirespecific/individualmethods,because1146

theycannotbeincludedinmulti-residue/multi-classmethodologies,duetotheir1147

specialphysico-chemicalcharacteristics.1148

3) Inlinewiththepreviouspoint,itisexpectedtowidenfuturescreeningtoinclude1149

NPS. However, it will not be easy to obtain satisfactory results in this case1150

because of twomain limitations: i) very low concentrations expected in the1151

samplesasaconsequenceofthelimiteduseofthesecompoundsincomparison1152

with “conventional” drugs, such as cocaine, cannabis, or amphetamines; ii)1153

continuousappearanceofnewNPSwithchangesinthechemicalstructurethat1154

complicatetheirdetectionandidentification.Relatedtothelastpoint,HRMSwill1155

beapowerfultooltoidentifyNPSandunknownmetabolites.1156

4) Thecombinationofnon-biasedandbiasednon-targetanalysiswithHRMSwill1157

beoneofthekeyresearchfields inthenearfuture.Hopefully, thiscombined1158

approach will allow one to discover new compounds of interest, such as1159

unknown or non-searched metabolites, TPs and/or new drugs. Their low1160

concentrations inmost caseswill be an extra difficulty to obtain satisfactory1161

results.1162

5) LC-MS/MS with triple quadrupolemass analyzers will still be the reference1163

techniquetoquantifyselectedIDBsintargetmethodologies,duetoitsexcellent1164

sensitivity and selectivity. However, new instruments with improved1165

performanceswillwidenthenumberofcompoundsincludedinthemethodup1166

to several hundreds. The incomparable sensitivity reached with this1167

instrumentationwillobtaindataonthepresenceofNPSinwastewaters,with1168

expectedconcentrationsatthepg/Llevel.1169

6) A common protocol of action should be used in WBE studies to produce1170

homogeneous and comparable data at different sites and provide the most1171

reliableestimatesofdruguse.Uncertaintyfactorsassociatedwiththedifferent1172

stepsinvolvedinWBE(sampling,chemicalanalysis,stabilityofdrugbiomarkers1173

insewage,back-calculationofdruguse,estimationofpopulationsize)haveto1174

46

beconsideredinordertoreduceand/orminimizetheuncertaintyoftheentire1175

procedure(Castiglionietal.,2013).Abestpracticeprotocolhasbeenrecently1176

proposedandadoptedbyEurope-widestudies.Threephasesoftheapproach1177

areincluded:samplingandsamplehandling,storagetreatmentduringsampling,1178

chemicalanalysis-quality control (EMCDDA,2016).Currently, themost-urgent1179

needs for future researchare:1) improve thequalityofchemicalanalysesby1180

followingspecificqualityrequirements;2)improvetheknowledgeonstabilityof1181

IDBs in-sewer; 3) plan additional pharmacokinetic studies to produce reliable1182

humanexcretionprofilesforIDBs;4)explorenovelpossibilitiestoestimatethe1183

populationsizeinacatchment.1184

7) TheimprovedcharacteristicsofHRMSinstruments,mainlysensitivityandlinear1185

dynamicrange,willmakethistechniqueattractiveforquantitativeanalysisas1186

well.Thesearch for thedesired“All-in-One”methodand instrumentwill still1187

continueinthecomingyears,becausecombinationofalldesiredfeaturesinjust1188

one method/instrument is an exciting issue: qualitative and quantitative1189

analysis, with possibilities for structural elucidation of unknowns. Future1190

developmentsofHRMSwillsurelyberelatedtoscan-speedimprovementsfor1191

Orbitrap,whichforexamplewillallowmoreefficientcombinationtoUHPLC,or1192

GC,whereasimprovementsinTOFMSanalyzerswillincreasethemass-resolving1193

power.Advanceswillincludedevelopmentoftribridanalyzersthatincorporate1194

newpossibilitiessuchasIonMobilitySpectrometry.Improvementsinaccurate-1195

mass full-acquisition data processing with more-powerful and user-friendly1196

softwarearealsoexpected. Inthenearfuture,wewillseeagrowthofHRMS1197

applications,notonly inWBE,butalso inother fields, suchasenvironmental1198

research,food-safety,toxicologyanddopingcontrolanalysis1199

8) It will be necessary to harmonize the criteria for reliable1200

identification/confirmation of compounds detected in samples. Relevant1201

parameters,asthenumberandspecificityofionsrequired,maximumion-ratio1202

deviationsallowed,masserrorsacceptedareneededtofacilitatecomparisonof1203

dataandtoavoidfalsepositivesornegativesduetotheuseofdissimilarcriteria1204

amongdifferentauthors.1205

47

9) The limitsofdetectionand/orquantificationofthemethods isarequisiteto1206

performrealisticcomparisonofdatainWBEstudies.Thenon-detectionmight1207

betranslatedtofiguresthatdependontheLOD;i.e.,themethodsensitivity.The1208

comparison of drug consumption among populations makes use in some1209

occasions of very low concentration data for less-consumed drugs; this1210

comparisonmightbedistortedwithunrealisticLOD/LOQdata.Thereisaneed1211

toharmonizecriteriafortheirestimationinWBE.1212

10) Further development of chiral analytical methods and wider application of1213

enantiomeric profiling ofwastewater are expected in the coming years. This1214

expectation is because enantiomeric profiling of illicit/abused drugs in1215

wastewater representsapowerful tool toallowone todifferentiatebetween1216

consumption and disposal of unused drugs or production waste in WBE.1217

Advancesareneededinthedesignofnew,more-robuststationarymaterialsto1218

separatemulti-residuemixturesofchiraldrugswithinashortermethodtime.1219

11) Awastewatervalidationguideline,basedontheexistingguidelines, isdesired1220

includingkeyaspectsrelatedtoqualitycontrolthatmustbecarefullyconsidered1221

inWBE. Internal and external quality controls are both needed for a reliable1222

analytical methodology. The organization of interlaboratory exercises are1223

imperative from twoperspectives: i) toproduce reliableWBEdata, and ii) to1224

improveanalyticalperformanceofparticipatinglaboratories.1225

12) The ultimate goal ofWBE is to provide results in real-time. This data can be1226

achieved with biosensors. Biosensors have already been applied in WBE to1227

screenPSA, prostate cancerbiomarker (Yanget al., 2015c),DNA (Yanget al.,1228

2015a),andcocaine(Yangetal.,2016).Thereare,however,severalissuesthat1229

need to be addressed;mainly the relatively low sensitivity of biosensors and1230

most probably low selectivity. Due to fast advancements in the field, wide1231

applicationofbiosensorsinWBEisenvisaged(Yangetal.,2015b).1232

13) ThepotentialofWBEwillbeexpandedtootheraspectsofpublichealth.Sewage1233

contains a hidden wealth of highly complex chemical information about1234

biologicalprocesses thatoccur in thehumanbody (Daughton,2011). Specific1235

biomarkerscouldserveasindicatorsofexposure,stress,vulnerabilitytodisease,1236

acquired disease, or health. Biomarkers include endogenous compounds1237

48

producedinresponsetostressorindicativeofhealth,adductsofendogenous1238

chemicals and xenobiotics, or metabolites of detoxification or intoxication1239

processes from xenobiotic exposure (Daughton, 2012). Theoretically, such1240

biomarkers could serve as collective measures of community-wide health or1241

disease,andcouldprovideameanstoconductepidemiologyinnear-realtime.1242

Ithas,therefore,thecapabilitytoserveasanearly-warningsysteminpandemics1243

(Ortetal.,2014a).Quantitativeanalysisof‘healthbiomarkers’insewagemight1244

allowone tomonitor changesover time; forexample, in response to specific1245

campaigns, identification of trends and, inter-community comparisons in1246

relationtotheirhealth,diet,lifestyle,andenvironment(ThomasandReid,2011).1247

14) Itisessentialtowidentheknowledgeonselectionofthemost-appropriateIDB1248

inWBEandtotakeintoaccountspecificrequirementsthataproperbiomarker1249

shouldfulfil inordertoensurethereliabilityoftheback-calculatedestimates.1250

Themain requirements for an IDB are: 1) excretion in consistent amounts in1251

urine; 2) detectable in urbanwastewater; 3) stable inwastewater; 4) human1252

excretion as unique source. These characteristics are essential to perform a1253

reliablequantitativeanalysisofthesubstanceunderinvestigationandshouldbe,1254

therefore,carefullyconsideredinfuturestudiestoselectnewIDBs.1255

1256

Acknowledgments1257

Financial support by the European Union's Program for research, technological1258

development,anddemonstrationSEWPROF(projectno.317205)andtheCOST-action1259

SCORE (Action no. ES 1307) are gratefully acknowledged. Dr. Felix Hernandez1260

acknowledges the Spanish Ministry of Economy and Competitiveness for financial1261

support inthefieldof illicitdrugresearch(RefCTQ2015-65603-P).Dr.Alexandervan1262

Nuijs acknowledges the Research Foundation Flanders (FWO) for his post-doctoral1263

fellowship. Dr. Lubertus Bijlsma acknowledges NPS-Euronet1264

(HOME/2014/JDRUG/AG/DRUG/7086),co-fundedbytheEuropeanUnion,forhispost-1265

doctoral fellowship. This publication reflects the views only of the authors, and the1266

EuropeanCommissioncannotbeheldresponsibleforanyusethatmightbemadeofthe1267

informationcontainedtherein. 1268

49

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