Pre-Print Eawag_08766

Nontarget screening with high resolution mass spectrometry: Critical

review using a collaborative trial on water analysis
EmmaL.Schymanski1*,HeinzP.Singer1,JaroslavSlobodnik2,IldikoM.Ipolyi2,PeterOswald2,Martin
Krauss3,TobiasSchulze3,PeterHaglund4,ThomasLetzel5,SylviaGrosse5,NikolaosS.Thomaidis6,
AnnaBletsou6,ChristianZwiener7,MaraIbez8,TaniaPortols8,RonalddeBoer9,MalcolmJ.Reid10,
MatthiasOnghena11,UweKunkel12,WolfgangSchulz13,AmlieGuillon14,NakeNoyon14,Gala
Leroy15,PhilippeBados16,SaraBogialli17,DraenkaStipaniev18,PawelRostkowski19,Juliane
Hollender1,20*.
1
Eawag:SwissFederalInstituteforAquaticScienceandTechnology,berlandstrasse133,8600
Dbendorf,Switzerland.
2
EnvironmentalInstitute,s.r.o.,Okrun784/42,97241Ko,SlovakRepublic.
3
HelmholtzCentreforEnvironmentalResearchUFZ,Permoserstrae15,04318Leipzig,Germany.
4
UmeUniversity,Linnaeusvg6,90187Ume,Sweden.
5
TechnischeUniversittMnchen,ChairofUrbanWaterSystemsEngineering,AmCoulombwall8,
85748Garching,Germany.
6
UniversityofAthens,DepartmentofChemistry,PanepistimiopolisZografou,15771Athens,Greece.
7
EberhardKarlsUniversityofTbingen,EnvironmentalAnalyticalChemistry,Hoelderlinstr.12,72074
Tbingen,Germany.
8
ResearchInstituteforPesticidesandWater,UniversityJaumeI,Avda.SosBaynats/n,12071
CastellndelaPlana,Spain.
9
MinistryofInfrastructureandtheEnvironment(Rijkswaterstaat),Zuiderwagenplein2,8224AD
Lelystad,Netherlands.
10
NorwegianInstituteforWaterResearch(NIVA),Gaustadalleen21,NO0349Oslo,Norway.
11
UniversityofAntwerp,ToxicologicalCenter,Universiteitsplein1,2610Wilrijk(Antwerpen),
Belgium.
12
GermanyFederalInstituteofHydrology(BfG),AmMainzerTor1,56068Koblenz,Germany.
13
BetriebsundForschungslaboratorium,ZweckverbandLandeswasserversorgung,AmSpitzigenBerg
1,89129Langenau,Germany.
14
SuezEnvironnementCIRSEE,38rueduprsidentWilson,78230LePecq,France.
15
VeoliaResearchandInnovation(VERI),1PlacedeTurenne,94417SaintMauriceCedex,France.
16
Irstea,URMALYFreshwaterSystems,EcologyandPollutions,CentredeLyonVilleurbanne,5ruede
laDouaCS70077,F69626VilleurbanneCedex,France.
17
UniversityofPadua,DepartmentofChemicalSciences,ViaMarzolo,1,35131Padova,Italy.
18
CroatianWaters,UlicagradaVukovara220,10000Zagreb,Croatia.
19
NILUNorwegianInstituteforAirResearch,Instituttveien18,2007Kjeller,Norway.
20
InstituteofBiogeochemistryandPollutantDynamics,ETHZurich,8092Zurich,Switzerland.

*CorrespondingAuthor:emma.schymanski@eawag.ch,phone:+41587655537;fax:+41587655893,
juliane.hollender@eawag.ch,phone:+41587655493;fax:+41587655893

Keywords:Nontargetscreening,highresolutionmassspectrometry,LCMS,GCMS,suspect
screening,surfacewater.

1
Abstract

Inthisarticle,adatasetfromacollaborativenontargetscreeningtrialorganizedbytheNORMAN
Associationisusedtoreviewthestateoftheartanddiscussfutureperspectivesofnontarget
screeningusinghighresolutionmassspectrometryinwateranalysis.Atotalof18institutesfrom12
EuropeancountriesanalysedanextractofthesamewatersamplecollectedfromtheRiverDanube
witheitheroneorbothofliquidandgaschromatographycoupledwithmassspectrometric
detection.Thisarticlefocusesmainlyontheuseofhighresolutionscreeningtechniqueswithtarget,
suspectandnontargetworkflowstoidentifysubstancesinenvironmentalsamples.Specific
examplesaregiventohighlightmajorchallengessuchasisobaricandcoelutingsubstances,
dependenceontargetandsuspectlists,formulaassignment,theuseofretentioninformationand
theconfidenceofidentification.Approachesandmethodsapplicabletounitresolutiondataarealso
discussed.Whilemostsubstanceswereidentifiedusinghighresolutiondatawithtargetandsuspect
screeningapproaches,someparticipantsproposedtentativenontargetidentifications.This
comprehensivedatasetrevealedthatnontargetanalyticaltechniquesarealreadyconsiderably
harmonizedbetweentheparticipants,butthedataprocessingremainstimeconsuming.Although
thedreamofafullyautomatedidentificationworkflowremainselusiveintheshortterm,
importantstepsinthisdirectionhavebeentaken,exemplifiedinthegrowingpopularityofsuspect
screeningapproaches.Majorrecommendationstoimprovenontargetscreeningincludebetter
integrationandconnectionofdesiredfeaturesintosoftwarepackages,theexchangeoftargetand
suspectlistsandthecontributionofmorespectrafromstandardsubstancesinto(openlyaccessible)
databases.

Introduction

Theevolutionofaccuratemass(AM)highresolutionmassspectrometry(HRMS)coupledtogasor
liquidchromatography(GCorLC,respectively)hasspurredanewtrendinanalyticaldataprocessing
inrecentyears.Targetedanalyticalmethodsarenowoftencomplementedwithnontargetor
untargeteddataacquisitionandscreeningmethods,wheretandemmassspectrometry(MS/MS)is
usedtoobtainfragmentationinformationtosupportidentification.EarlierarticlesontheuseofAM
HRMS(hereaftershortenedtoHRMS,wheretheaccuratemassisimplied)intheenvironmental
context(e.g.[14]),mentionthethreemainapproachestowardssubstanceidentificationinnon
targetHRMSanalysiswhicharesummarizedherewithsomeadditionalnuances:
(a)targetscreeninginvolvesareferencestandardmeasuredinhouseunderthesameanalytical
conditionssuchthatretentiontime(RT),HRMSand,wherepossible,(HR)MS/MSinformationis
availableforidentificationandconfirmation.Quantitativetargetresultsshouldbedistinguishedfrom
semiquantitativeresultsbyusingthetermquantitativetargetanalysis/screening.
(b)suspectscreeningisperformedwhenpriorinformation(fromvarioussources,discussedin
greaterdetailbelow)indicatesthatagivenstructuremaybepresentinthesample.Thuswhileno
referencestandardisavailable,theexactmassandisotopepatterncalculatedfromthemolecular
formulaandplus/minustheexpectedadduct(s)ofthesuspectsubstancecanbeusedtoscreenfor
thissubstanceinthesample.
(c)nontargetscreeninginvolvesallremainingcomponentsdetectedinasamplewherenoprior
informationisavailable.Asnostructuralinformationisavailableinadvance,afullnontarget
identificationstartingfromtheexactmass,isotope,adductandfragmentationinformationneedsto
beperformed.

2
Thedataanalysisfortargetandsuspectsubstancesonnontargetacquisitiondatacanbeperformed
intwomainways(oracombinationofboth).Traditionallyintargetanalysisthepresenceorabsence
ofeachsubstanceisdeterminedindividuallyusingtheextractedionchromatogram(XIC).However,
theevolutionofnontargetmethodsmeansthatoftenascreeningfortarget/suspectcompoundsis
performedfollowingpeakdetectionwithasuitablealgorithm,suchthattheexactmassesofthe
appropriateadductofthetarget/suspectissearchedwithinagivenmassand(fortargets)RTerror.
Whilethefirstapproachtreatstargetsandsuspectspreferentially(i.e.,theycanbedetectedincases
wherethepeakisofinsufficientqualityforautomatedpeakdetection),inthelattercasethetarget
andsuspectcompoundsareeffectivelyasubsetofallthenontargetcomponents.Here,theterm
componentisusedtorefertothegroupofexactmasses(i.e.adductandisotopologues)associated
withonecompound.Irrespectiveoftheextractiontechnique,evidencefromthemeasurementdata
isneededtoconfirmtheidentification,includingtheisotopepattern,presenceofadditionaladducts,
RT,fragmentationinformationandotherexperimentalevidence(e.g.presenceofrelatedsubstances,
timetrends).Severalpapersdiscussthisingreaterdetail(e.g.[1,35]).Whiletheconceptof
identificationpoints(IPs)givenintheEUGuideline2002/657/EC[6]canbeusedtorepresentthe
evidenceavailableforanidentificationwhereareferencestandard(andthusaRT)isavailable,these
areinurgentneedofrevisionastheywerereleasedbeforeHRMS(/MS)becameasprominentasitis
today.TheconceptofidentificationlevelsinHRMSanalysishasbeendiscussedrecentlyinboth
theenvironmentalcommunityandbeyond(e.g.[79])buildingonearlierattemptsbythe
MetabolomicsSociety[10]todealwiththevaryinglevelsofconfidenceinidentificationsresulting
fromthesethreestrategiesforidentifyingsubstancesusingnontargetacquisitiondata.

TheconceptsofidentificationstrategyandconfidencearemergedinFigure1,showingthattarget,
suspectandnontargetcompoundsstartbydefinitionatLevels1(referencestandardavailable),3
(tentativecandidate(s))and5(noinformation),respectively.IfsufficientMS(exactmass,isotope,
adduct),MS/MS(i.e.,fragmentation)andexperimentalinformation(e.g.retentionbehaviour,
presenceofrelatedsubstances)isavailable,suspectandnontargetcomponentscangainin
confidencethroughtoLevel2(librarymatchand/ordiagnosticfragments)andevenLevel1following
purchaseofthecorrespondingstandardforidentifications(greenarrowsinFigure1).Compounds
withaconfirmedidentitythenbecometargetcompoundsinfutureinvestigations.However,should
theevidencebetweensampleandreferencestandard(target)ortentativecandidate(suspect)not
match,thenthecomponentassociatedwiththetargetorsuspectshouldbecomeanontargetof
interest(Level5)seeredarrows.IftheHRMSandRTinformationmatchesatargetcompoundbut
HRMS/MSisnotavailableduetolackofintensity,thisisstillconsideredatargetidentification,but
shouldbereportedwithfeweridentificationpoints(e.g.2IPasopposedto4.5IPwhereHRMS/MS
fromanisolatedprecursorisavailable[6]).

Itisimportanttonotethatthiselaboratematrixofstrategy,confidenceandevidenceisnecessary
withsoftionization(SI)HRMS/MSanalysisatthisstageforonemainreason:thelackof
comprehensivespectrallibrariesforsoftionizationtechniquestodatecomparedwiththe
comparativelycomprehensiveGCelectronionization(EI)MSlibrariesavailable[11,12]withover
200,000substances.Thereasonsforthisarevaried,includingthelackofreproducibilitybetweenSI
instrumentsandvarioussettingsandtherelativenewnessofthetechnique,andarediscussedin
greaterdetailelsewhere(e.g.[1315]).Asaconsequence,theidentificationofunknown
environmentalsubstancesofinterestmeasuredwithGCEIMScanoftenbeperformedwitha
spectrallibrary,whilesubstancesmeasuredwithalternativeionizationtechniquesgenerallyrequire

3
theparallelapproachesoftarget,suspectandnontargetscreening.ThisisnotjustlimitedtoLC
basedtechniques;thedependenceofstrategyonionizationtechniqueisshownforthe
environmentalcontextinrecentworkcomparingquadrupoletimeofflight(QToF)GCMSusingEI
andatmosphericpressurechemicalionization(APCI)[16]andGCandultrahighperformanceLC
(UPLC)coupledtoQToFMSforauniversalscreeningapproach[17].

Figure1:Matrixofidentificationapproachversusidentificationconfidence.

Anumberofstrategiesfortheselectionofcandidatesforsuspectandnontargetscreeningof
environmentalsubstancesusingHRMS/MSinformationhavebeendevelopedandputintopractice
recently.Suspectscreeninghasbeenperformedusingpredictedtransformationproducts(TPs)[18
20],registeredpesticidesandtheirTPsinSwitzerland[21],surfactantsinwastewater[3]andfracking
fluids[22],sitespecificchemicals[5]andhighconsumptionpesticidesandpharmaceuticalsin
sediments[23].Otherlists,suchasthepotentiallypersistentandbioaccumulativeorganicsin
commerce[24],pharmaceuticals[25],aswellasimpurities,byandtransformationproducts[26]
fromHowardandMuircanalsobeusedinsuspectscreening.Fornontargetidentification,candidate
discriminationcriteriausedincluderetentiontimeinformation,alsorepresentedaspartitioning
coefficients[19,23]orthechromatographichydrophobicityindex(CHI)[5,27],insilicofragmentation
predictionwithbonddissociationmethodssuchasMetFrag[28]orrulebasedapproachessuchas
MassFrontier[29]andalsothenumberofreferencesavailableforasubstanceindatabasessuchas
ChemSpiderorSciFinderasameasureofrelativeimportanceintheenvironment[30,31].Itisclear
fromthedetaileddataprocessingschemesinrecentnontargetpapers(e.g.Figures1and3in[5],
Figure1in[21])thatnontargetscreeningofenvironmentalsamplesisbecomingincreasingly
complex.

Inresponsetothistrendofincreasingcomplexityandtheneedarticulatedbymemberstocompare
andharmonizenontargetscreeningmethodsinEurope,theNORMANAssociation(www.norman
network.net)instigatedacollaborativenontargetscreeningtrialin2013onasampleextractfrom

4
theRiverDanube.Eachparticipatinginstitutewasrequestedtoanalysethetestsampleusing
establishedMStechniquesintheirlaboratoryanddeclare(1)howmanysubstanceswerepresentin
thesampleand(2)howmanyofthesecouldbeprovisionallyidentifiedusingtarget,suspectand
nontargetscreeningapproaches.Analyticalstandardsforthecalculationofretentionindex
informationwerealsoprovided.Followingthetrial,aworkshopwasheldwithallparticipantsto
developagreementsonharmonizedterminology,workflowsandreportingformats.Thisdataset
formsauniqueopportunitytoreviewthestateoftheartofnontargetacquisitionandidentification
techniquesusinghighresolutionmassspectrometrywithacomparabledatasetfromseveral
environmentalinstitutesinEuropeandcommentonthecurrentandfuturetrends.

MaterialsandMethods

SamplingandTrialParticipation
ThesampleusedinthecollaborativetrialwascollectedfromlocationJDS57,downstreamof
Ruse/Giurgiu(RO/BG;rkm488;coordinatesN43.890150,E26.017067)onSeptember18,2013asa
partoftheThirdJointDanubeSurvey,organizedbytheInternationalCommissionfortheProtection
oftheDanubeRiver(ICPDR)[32,33].Thesamplepreparationincludedalargevolumesolidphase
extraction(LVSPE)of1000litresofwater[34].Briefly,thesamplercartridgewasfilledwith160gof
MachereyNagelChromabondHRX(neutralresin)and100geachofChromabondHRXAW
(anionic)andHRXCW(cationicexchangeresin).Theresinswereextractedwith500mLeachofethyl
acetateandmethanol(HRX),500mLmethanolwith2%7Mammoniainmethanol(HRXAW)or500
mLmethanolwith1%formicacid(HRXCW).Theextractswerethencombined,neutralized,filtered
(WhatmanGF/F)andreducedtoafinalvolumeof1Lusingrotaryevaporation.Aliquotsof1.5mL,
equivalentto1.5Lofriverwater,weretransferredintovialsandevaporatedtodrynessunder
nitrogen.Theseweresenttoeachparticipantalongwithalaboratoryblank,whichwascreatedvia
circularpumpingof5LofLCMSgradewaterthroughtheLVSPEtomimicleachingfrom1000Lof
waterpassingthroughtheLVSPE.Samplestability(atleast3timepointsover1.5months)and
homogeneitytesting(threereplicates)wasperformedusing20and50substancesforGCandLC
MS,respectively,toconfirmthesuitabilityofthesampleforthetrial.Thesubstancesarelistedalong
withtheresultsintheElectronicSupplementaryMaterial(ESM),TablesS1andS2.

ThesamplesweredispatchedonDecember9,2013alongwithstandardmixturesforusetocalculate
retentionindexinformation:C10toC25alkanestandardsforGCMStechniquesand10substancesfor
LCMStechniques.TheLCMSstandardmix(ChemSpider[35]identitynumberandthelogarithmof
theoctanolwaterpartitioningcoefficient(logP)calculatedwithChemAxon[36]giveninbrackets)
consistedofmetformin(3949;1.36),chloridazon(14790;1.11),carbetamide(133997;1.65),
monuron(8470;1.93),metobromuron(17276;2.24),chlorbromuron(24141;2.85),metconazole
(77764;3.59),diazinon(2909;4.19),quinoxyfen(2635909;4.98)andfenofibrate(3222;5.28).All
participantswererequestedtomeasurethesestandardsalongwiththesampleandreportthe
resultsbyMarch15,2014.Assuch,thisdatasetformedatestsetforretentioninformation
comparison,datastorageandreprocessingofrawmassspectraldataforretrospectiveanalysis.Of
the26institutesfrom15countrieswhoreceivedsamples,19submittedresults;7institutesforGC
MSand17institutesforLCMS(5institutessubmittedforboth).Theparticipantsrangedfrom
institutesperformingnontargetmethodsforthefirsttimethroughtoexperiencedresearchgroups.
Theoveralltimecommittedbytheparticipantsvariedfrom2daysto6weeks.Eachinstitutethat
submittedresultsisrepresentedbyanumber.

5
LCHRMS
AnoverviewoftheLCHRMS(/MS)methodsusedisgiveninTable1andTable2.

Table1:SummaryofliquidchromatographicmethodsusedbyLCparticipants.

Parti Instrumentand Column Dimensions Solvent Inj. Flow;Run

cipan Model [mmxmm, vol time
t m] [L] [mL/min];
[min]
ABSciexTripleTOF Phenomenex
1 5600 LunaC18(2) 2.0x150;3.0 H2O/ACN(FA) 10 0.2;33
ThermoQExactive WatersXbridge
2 Orbitrap C18 2.1x50;3.5 H2O/MeOH(FA) 20 0.2;30
ZICHILIC, 2.1x150;5.0 0.050.4;
3 AgilentToF6230 PoroshellC18 3.0x50;2.7 H2O/ACN(NH4ac) 10 58
WatersAcquity
Agilent6550 UPLCHSST3and
iFunnelQTOF ZORBAXECLIPSE 2.1x150;1.8
4 LC/MS PLUSC18RRHD 2.1x100;1.8 H2O/MeOH(NH4ac) 100 0.4;20
WatersMicromass AcquityUPLCBEH
5 XevoG2QTOF C18 2.1x100;1.7 H2O/MeOH(FA) 50 0.3;18
PhenomenexAQ
C18(Guard),
ABSciexTripleTOF AgilentZorbax 2.0x4.0;n/a
6 5600 EclipsePlusC18 2.1x150;3.5 H2O/ACN(FA) 5 0.3;37
WatersXevoG2S WatersAcquity 2.1x150;1.8 H2O/ACN(NH4fa,FA); 0.40.45;
7 QTOF HSSC18,BEHC18 2.1x100;1.7 H2O/MeOH(NH4ac) 5 15
ThermoLTQ WatersAtlantis
8 OrbitrapDiscovery HSST3 2.1x150;3.5 H2O/MeOH(FA) 10 0.2;19
ThermoLTQ Phenomenex
9 OrbitrapXL KinetexC18 3.0x100;2.6 H2O/MeOH(FA) 10 0.2;41
ThermoLTQ ThermoHypersil
10 OrbitrapDiscovery Gold 2.1x100;3.0 H2O/MeOH(FA) 20 0.2;35
Agilent6530AM
11 QTOFLC/MS EclipsePlusC8 2.1x150;3.5 H2O/ACN(NH4ac,FA) 10 0.25;29
AgilentAMQTOF Phenomenex 2.1x100;1.7
12 LC/MS6520 KinetexC18 2.1x100;2.6 H2O/ACN(FA,NH4OH) 40 0.3;38
Agilent6550
iFunnelQTOF
13 LC/MS ZorbaxExtendC18 2.1x50;1.8 H2O/ACN(FA) 2 0.5;46
Brukermaxis H2O/MeOH(NH4fa,FA, 0.20.48;
14a impact DionexAcclaim 2.1x100;2.2 NH4ac) 10 19
ThermoQExactive H2O/MeOH(none,FA,
15 Orbitrap HypersilGoldaQ 2.1x100;1.9 NH4fa) 5 0.3;35
WatersXevoG2S H2O/MeOH(NH4fa,pH
16 QTOF WatersC18BEH 2.1x100;1.7 5) 3 0.45;13
Agilent6550QTOF AgilentPoreshell 2.1x5;2.7 H2O/MeOH(FApos
17 LC/MS HPHC18(+Guard) 2.1x150;2.7 only) 5 0.3;35
ACN=acetonitrile,H2O=water,FA=formicacid,MeOH=methanol,NH4ac=ammoniumacetate,NH4OH=
ammoniumhydroxide,NH4fa=ammoniumformate.Oventemperaturesrangedfrom22and50C.n/a=not
available.aQuantitativetargetanalysiswasperformedusingaThermoTSQAccessTripleQuadinstrument.

6
Table2:SummaryofmassspectrometricanddataprocessingproceduresusedbyLCparticipants.

Partici Scan Resol(mz) Ionizn Fragment Target Suspect,NTProcedure

pant Range Method Software
PeakView,Markerview.MS,
30,000 ESI+ Peakview, isotopes,RT,MS/MS,manual
1 1001200 (m/z400) only CID,40 Multiquant peakcheck
HCD various, ExactFinder2.0,nontarget,
50(DD); Xcalibur,Trace MetFusion,internallists.MS,
140,000 mergedHCD Finder, RT,MS/MS,library,
2 1001000 (m/z200) ESI 3070(DIA) nontarget prediction,interpretation
MassHunterQual,Profinder
Insource (B.06.00).MS,RT,internal
12,000 fragmentation MassHunter list,MS/MS,library,STOFF
3 1001700 (m/z1000) ESI at100V Qual(B.06.00) IDENTDB,pred.isotopes
MassHunterQuantandQual
(vB.06.00,Build6.0.633.0).
56,014 CID,10,20, MassHunter/ FindByFormula,MassProfiler,
4 1001000 (m/z922) ESI 40.AllIons Quant MS/MS,isotope,PCDL
ChromaLynxXS,homemade
20,000 CID,1540 ChromaLynx database,MassBank.MS,
5 501000 (m/z556) ESI Ramp,MSE XS MSE,prediction
CID,40; STOFFIDENT,DAIOS,internal
31,000 merged25, listofsubstances.MS,(RT,
6 1001200 (m/z327) ESI 40,55.DDA MasterView MS/MS)
50700, 22,500 CID,1055 Unifi.RT,MS,MS/MS,
7 501200 (m/z956) ESI Ramp Unifi Prediction
30,000 ESI+
8 501000 (m/z400) only Notused mzMine,ChemSpider
Xcalibur.MS,RT,MS/MS,
100,000 RT(CHI),fragment(MetFrag)
9 1001000 (m/z400) ESI HCD,variable Xcalibur prediction,manualinterp.
30,000
10 801500 (m/z400) ESI CID,35 ExactFinder ExactFinder.MS,RT,MS/MS.
8000
11 501300 (m/z600) ESI CID,10,20 MassHunter Pragstlibrary.MS,MS/MS
18,000 MassHunter Metlindatabase(pesticide,
12 502000 (m/z311) ESI CID,15,40 QualB04.00 forensic).MS,MS/MS
ForensicsTox,Pesticides,
20,000 ESI+ MassHunter MassBank,DAIOS.MS,RT,
13 501200 (m/z622) only CID,20,40 B6.0 MS/MS,prediction
LCQuan, TargetandDataAnalysis.MS,
Target isotopes,MS/MS,RT(KNN
40,000 Analysis,Data GASVM),fragment(MetFrag,
14a 501000 (m/z431) ESI CID,25 Analysis SmartFormla3D)prediction
TraceFinder,Sieve,inhouse
library,Thermolibrary(with
70,000 andwithoutRT).MS,RT,
15 701000 (m/z200) ESI HCD,50 TraceFinder MS/MS,library&prediction
20,000 ESI+ CID,1045 MassLynx, MassLynx/ChromaLynx,in
16 501000 (m/z556) only Ramp.MSE ChromaLynx houselibrary.MS,RT,MS/MS
MassHunter.MS,RT,MS/MS,
23,000 MassHunter prediction,Agilent
17 501500 (m/z119) ESI CID,variable B06SP1 ForensicToxlibrary
ESI=Electrosprayionization,CID=collisioninduceddissociation,HCD=higherenergyCID,DD=data
dependentacquisition.DIA,Allions,MSe=fragmentationwithoutprecursorisolation,RT=retentiontime,DB=
database(s).aQuantitativetargetanalysiswasperformedusingaThermoTSQAccessTripleQuadinstrument.
7
Oneofthe17LCparticipantsusedaserialcouplingofzwitterionichydrophilicinteraction(HILIC)and
reversedphase(RP)chromatography(LCLC;HILICC18)[37],whiletheremainderusedC18reversed
phasecolumns(oneC8)witheitherhighperformanceliquidchromatography(HPLC)orlongultra
HPLC(UHPLC)runs.Thesolventwasgenerallywater/methanolorwater/acetonitrile,withnoor
variedmodifiers(formicacidorammoniumacetate,formateorhydroxide).Between2and100Lof
thesamplewasinjected.Electrosprayionization(ESI)onlywasused,withdifferentcollisioninduced
dissociation(CID)orhigherenergyCID(HCD)energies,whileparticipantswhomeasuredinboth
positiveandnegativemodedidthisinseparateruns.Allionapproaches(fragmentationwithout
precursorselection)wereusedbysomeparticipants.Thescanrangesstartedbetweenm/z50and
100,endingbetweenm/z1000and2000,whiletheresolutionrangedbetween~18,000(atm/z300)
and140,000(atm/z200).FiveinstrumentsfromtheOrbitrapfamilywereused,while12werefrom
theToFfamily(differentvendors).Thedataprocessingforthetargetcompoundswasgenerally
performedusingvendorsoftware,whilethepeaksthatwerenotassignedastargetswereidentified
throughsuspectandnontargetscreening,performedwithamuchwidervarietyofsoftwareand
methods.Whilemanyparticipantsalsoquantifiedtargetsubstances,thisisnotthefocusofthe
currentarticle.

Manydifferentinformationsourceswereusedbyparticipants,showninTable3,includingopenand
vendorlibrarieswithmassspectraavailable,extensivecompounddatabases,selectivecompound
databasescontainingwaterbodyrelevantsubstances(STOFFIDENTandDAIOS)aswellassuspect
listsfromliterature.Theseweregenerallysupportedorrejectedthroughacombinationofaccurate
mass(includingisotopeand/oradductinformation),RT,MS/MS,librarymatchandpredicted
properties.ApproachestoconsiderretentiontimeinformationincludedthelogPmethodusing
standardsprovidedtoeachparticipant(S.Grosseetal.inprep.),oftenincombinationwithSTOFF
IDENT[38],theCHIapproach[27]andaQSPRapproach(R.Aalizadehetal.,inprep)usingsupport
vectormachines(SVM)andknearestneighbours(kNN).Softwareusedforfragmentationprediction
includedMetFrag[28],MetFusion[39],MassFrontier[29],MassFragmentinUNIFIandMassLynx
(Waters[40]),SmartFormula3D(BrukerDaltronics[41])andMolecularStructurecorrelator)[42,43].

GCMS
AsummaryoftheGCMSmethodsisgivenintheESM,TablesS3andS4.Oneparticipantsubmitted
highresolutiondata,whilethreeofsevenGCparticipantsusedGCxGC.Twousedchemicalionization
(CI)methodsinadditiontoEI,APCIaswellaspositiveandnegativeCI.Fourparticipantschosealarge
volumeinjectiontoobtainsufficientsignalintensity(seeresults)andthescanrangestartedbetween
m/z3550,endingbetweenm/z350and800.Arangeofdifferentsolventswereused.Target
compoundidentificationwasgenerallyperformedusingvendorsoftware.Nontargetidentification
wasperformedusingtheNISTdatabase(seeTable3)eitherwithorwithoutAMDIS[44],combinedin
mostcaseswiththeKovatsretentionindex.

Datareportingandcomparison
Participantswererequestedtosubmittheirresultsinadatacollectiontemplate(DCT),amultitab
spreadsheet,toensuresufficientinformationwasavailableforevaluation.Pretreatmentstepsto
increasecomparabilityincludedreclassificationofmethodsandreportedidentificationstobe
consistentwiththedefinitionoftarget,suspectandnontargetprovidedintheintroduction.Several
substanceidentifierswereusedtoperformthecomparison,includingtheSMILEScode[45],
ChemicalAbstractServices(CAS)number,names,molecularformulasandtheInChIKey[46].
OpenBabel[47]wasusedtoperformconversions,whileChemSpider[35]andPubChem[48]were
8
usedtofilldatagaps.Entriesforthesametargetrepeatedmultipletimesperparticipantwere
mergedtocreateoneentryonly,whilemultipleidentificationsforthesamesubstanceandpeakasa
target,suspectand/ornontargetselectedaccordingtothehierarchytarget>suspect>nontarget.
However,multipleidentitiesforthesamepeak(e.g.coelutingtargets)werenotmerged.

Table3:Summaryofinformationsourcesusedbycollaborativetrialparticipants.

Stateasusedduringthetrial CurrentState
Database/LibraryName TotalCompounds Compounds Compoundsat
withSpectra March2015
ChemSpider[35] 32million 32million
DAIOS[49,50] 1,404 >1,000a 1,404
PubChem[48] 63,105,228 68,479,719
STOFFIDENT[38] 7,864b 7,864
MassBankMS/MS[5153] 3,350 3,350
mzCloud[54] 1,956 2,510
NISTEIMS[11,55] 212,961c 242,477
NISTMS/MS[11,55] 4,628 8,171
WileyRegistryofMassSpectralData(EIMS)[56] 289,000[12] 638,000
AgilentBroecker,Herre&Pragst 8,998c 3,497 8,998
Toxicology/Forensicsf[57,58]
AgilentPesticideLibraryLC/QTOFMS/MSf[59] 1,664 ~700c 1,664
AgilentPesticideLibraryGC/QTOFEIMSf 750 750 750
AgilentMETLINSyntheticSubstanceLibraryg 64,092c ~10,000c 64,092
AgilentMETLINScrippsOnlineDatabasef,g[60,61] 83,135 12,171c 240,566
AgilentVeterinaryDrugLibraryf 1,684 770 1,684
BrukerToxScreener(incl.PesticideScreener)g [62] 704ad 1753
Sciex/ABSciexLC/MS/MSMetaLibraryg[63] 2,381c 2,381
ThermoEnvironmentalFoodSafety(EFS)g 447p;278n; 732
withretentiontime(RT)g 454dp;90dn
Thermotoxicologyg 618p;36n 654
WatersdatabasewithRTg 730de 730
InhouseLibrarieswithoutspectra(twoparticipants) 2,000;1,600[17] 2,000;1,600
InhouseLibrarieswithspectra(twoparticipants) 526d;63d 526;63
d
InhouseLibrarieswithspectraforsomesubstances 2,200 835ad 2,200
7,815 1500ap; 7,815
500an
3,000 350d 3,000
SurfactantList[3] 394 394
a
indicatesthatfragmentinformationbutnotspectrawithintensitieswereavailable(e.g.Q1,Q2,Q3,);
b
retentiontimeinformationandmeasuredstandardsusedtoselectbestmatchesbylogDvalues;
c
maximumnumberofsubstances,participantsuseddifferentversionswithslightlyvaryingnumbers;
d
indicatesretentiontimeinformationincluded,measuredonthesamesystemoranidenticalsetup;
e
retentioninformationtransformedusing40standards;fconfirmedbyAgilentTechnologies(T.Faye,pers.
comm.);gnumbersprovidedbyparticipants;ppositiveionizationmode;nnegativeionizationmode.

ResultsandDiscussion

StabilityTesting

Differentbatchesofthesampleusedinthetrialweresubjectedtostabilityandhomogeneitytesting
todeterminewhetherthesamplewassuitableforsuchatrialandprovideabaselineforcomparison,
tablesofresultsareprovidedintheESM,TableS1(GC)andS2(LC).ForGCMS,20substanceswere
selectedtocoverthefullruntimeandpolarityrange.HomogeneitytestingwasperformedonLots
9
15,33and50(3replicatesofeach)withacoefficientofvariationgenerallybelow1%andmaximum
of7%.Forthestability(testedonDay1,10and30),thehighestrelativeconcentrationwas1.033on
Day30.ForLCMS,50substanceswereselectedandwhilethehomogeneity(3aliquots)showed
coefficientofvariationbetween1and16%,verysimilarvalueswereshownforreproducibility
testing(3injections).StabilitywastestedonDays1,7,15and48andshowednodegradationofany
compoundselected.Generallyanincreasingtrendtohigherfactors(DayX/Day1)wasfound.The
ratherhighvariationofthestabilityfactors(between0.5and2wasduepredominantlyto
measurementsensitivityandisconsideredsufficientforthetrialasthemainfocushereisnota
comparisonofthequantitativeresults.

LCHRMSOverview
Intotal,17institutessubmittedLCHRMSresultsand15ofthesereportedtargetsubstances(for
whichtheidentitywasconfirmedwithareferencestandard).Thebreakdownoftargets,suspects
andnontargetsisgiveninTable4,includingadditionalcategories(whetherthetargetwas
quantifiedornot,isomermix,identifiednontargetandformulaonly)toreflecttheidentification
confidenceandhowthesubstanceswereidentified.Participants6and13,withlargenumbersof
assignedformulas,wereabletodothisautomaticallythoughtheirsoftware.Thedistributionreflects
thetargetandsuspectlistsaswellasdatabasesavailableatthedifferentinstitutesandshowsthe
hugeamountofinformationthatcanbegainedbyincreasingtheexchangeofknowhowbetween
institutes.Thetimeinvestedbytheparticipants(2daysto6weeksofthetotal3monthsgivenfor
thetrial)isalsoreflectedintheseresults.

Table4:ResultssummaryforLCHRMSparticipants,brokendownintocategoriesandidentificationconfidence[7].

Partic Quantified Semi/non Suspect Isomermix, Identified Formula Nontarget

ipant Target quantified (Level23) unspecified nontarget only peaks
(Level1) Target structure (Level23) (Level4) remaining
(Level1) (Level3) (Level5)
1 25 68 6,776
2 92 14 8 40 1 8,535
3 4 70
4 125 43 61 9 1
5 18 5 8 17
6 53 7 57 1,316 712
7 33 3,174
8 30 3
9 68 4 14 2 10 26
10 1 11 4
11 4 7 8
12 4 4 3 26 5 57
13 9 66 12 2 1,218 133
14 23 53 78 2 9 1,649
15 33 13 1 273
16 9 18 3
17 7 37

Intotal625targetresultswerereported,correspondingwith347uniquecompounds,andthe
majority(451results)werequantified.Atotalof631suspectandtentativelyidentifiednontarget
substanceswerereported,553ofwhichhadadefinedstructureassociatedwiththepeak(the

10
remainingsubstanceswerereportedasasubstanceclassorunspecifiedisomer).Altogether649
uniquecompoundswereidentifiedastarget,suspectornontarget.Themostfrequentlyreported
included(targets+suspects)carbamazepine(13+2),atrazine(9+4),sulfamethoxazole(11+1),DEET
(6+6),metformin(10+2),terbutylazine(6+6),caffeine(7+5),atrazinedesethyl(8+3)andtramadol
(7+3).Metoprolol,terbutylazinedesethyl,phenazone/antipyrine,4&5methylbenzotriazole,
atrazine2hydroxy,venlafaxine,isoproturon,telmisartan,perfluorooctanesulfonate(PFOS)and
metolachlorwereallreported9times.Oneinstituteadditionallyreportedanisobaricsubstance
(propisochlor)asasuspectinsteadofmetolachlor.Afurther7and4substanceswerereported8and
7times,respectively.Theseresultsindicatethatthewellknowncompoundsareobviouslymore
commontargetcompoundsandfoundmoreoften,butthefactthatmanyparentandTPsoccurin
thislistindicatesthatmanygroupsconsidertheformationofTPsalreadyintheirtargetscreening.
Terbutylazinewasthemostfrequentlyreportedofsomeisobariccompounds;seeFigure2andtext
belowformorediscussiononcoelutingisobars.Ofthe24substancesprovidedassuspectswiththe
trial(detectedatthislocationinprevioussurveys[64]andgivenintheESM,TableS5),allbutthree
weredetectedatleastoncebytheLCparticipants;oneofthesemissingthreewasreportedbyaGC
participant(bisphenolA),whiletheremainingtwowerenotreportedbyanyparticipant(naproxen
andN,Ndimethyl1decanamide).

TheparticipantswererequestedtoreportintensitiesinthesampleandtheblankwiththeDCTand
differentapproacheswereusedtoconsidertheresultsoftheblanksample.Allbutoneparticipant
reportedintensitiesinthesample,whileallbutfourreportedintensitiesintheblank.
Dibutylphthalateand/orisomerswasreportedeleventimesbysevenparticipants;threeparticipants
reportedthisinboththesampleandblank,twointhesampleonly,oneintheblankonlyandonedid
notreporteitherintensity.Oftheotherfrequentlyreportedsubstances,metforminand
terbutylazinewerereportedintheblankonce(severalordersofmagnitudelower),carbamazepine,
desethylatrazineandatrazinetwice(twoormoreordersofmagnitudelower),DEETthreetimes(one
totwoordersofmagnitudelower)andcaffeinefourtimes(afactorof2600lowerthanthesample).
Mostofthereportedalkylphosphatespecies(e.g.tributylphosphateandtriphenylphosphate,see
TableS5)werefoundinbothsampleandblank,aswerethepolyethyleneglycolsuspects.The
participantswhoappliedablanksubtractionusedintensitycutoffsatdifferentratiostodetermine
whetherthesubstancewasconsideredpresentintheblankornotandthisaffectedthenontarget
peaklistsprovided.Asanexample,oneparticipantsubtractedallnontargetpeakswherethesame
peakwasdetectedintheblankandtheintensitywaswithintwoordersofmagnitudeoftheintensity
measuredintheblank.Asonlyoneeachofblankandsamplewasprovided,itwaschallengingto
determinewhethertheintensitiesweresamplespecific,especiallyassampleenrichmentwasused.
Assuch,bothfalsepositiveandfalsenegativeresultsarepossible.

TargetandsuspectscreeningwasperformedusingbothpeaklistsandXICs.Generally,theuseofXICs
gavemorehitsasthisalsocapturedtarget/suspectsubstanceswherethepeakwasnotsuitablefor
thepeakpickingalgorithmused.However,thepeakpickerandtheparametersusedhavealarge
influenceonthequalityofpeakpickingresultsandthiswouldbeinterestingtoinvestigatecloserin
thefuture.Inthistrial,thestrategyandresultsdependedhighlyonthesoftwareusedbythe
participant,asmostusedvendorsoftwaretoperformtargetandalsosuspectscreening.Many
participantsmentionedusingmanualorvisualconfirmationofthetargetand/orsuspectresults.

AnalyticalMethodsinLCHRMSNontargetScreening
TheresultssummarizedinTable4showthatnontargetacquisitionandscreeninghasdevelopedat
11
anincrediblyrapidpaceinrecentyears,withmanyinstitutesnowregularlyscreeningseveral
hundredcompoundsusingmultiresiduemethods.Theparticipantsagreedthatgenericmethods,
basedonC18columns,genericgradientsandeitherHPLCorlongrunUHPLCwerebestfornontarget
approaches,toallowsufficienttimeforfragmentacquisition.Manyinstitutesalsoanalysedthe
sampleseveraltimestogainadditionalinformation,runninge.g.positiveandnegativeseparately
ratherthanperformingpolarityswitching,acquiringMSandallionMS/MSdatatogainallfragments
atonce,runningwithandwithoutinternalstandardoracquiringdatadependentMS/MSoftarget
compoundsfirst,followedbyremeasurementstoacquireMS/MSofsuspectsandnontargetsof
interest.Thisshowsthatthenumberofcompoundsbeinginvestigatedisalmostontheedgeofthe
instrumentcapabilitiesandthereisagreatinterestinthedevelopmentofappropriatedataanalysis
strategiesforallionMS/MSdata.

OneparticipantusedHILICcombinedwithRPLCtoimprovetheseparationofthehighlypolar
substancesandthepeakdistributionshowninFigureS1(a)showsthepotentialforgreater
separationofthepolarsubstancesthatotherwisemayeluteinthedeadvolumeofRPcolumns,
showninFigureS1(b).TheidentifiedsubstancesreportedbythisparticipantfromtheHILICcolumn
(metforminat16.85min,melamineat7.48min)werealsodetectedbyotherparticipantswiththe
C18column(12and4timesintotal,respectively)between0.3and2.9min.ThisshowsthatHILIC
methodsmayhelpaddressthelimitationsinnontargetscreeningofverypolarcompoundswithRP
columnsalone,e.g.interferencesobservedwithnontargetsinthelowRTrange[3]andinregionsof
highmatrixinterference[23],butthishastobeexploredfurther.

Althoughtherewasagreatvarietyinthescanrangeusedbytheparticipants,allbutoneidentified
substancefellbetweentherangeofm/z100900.Thetargetcompoundpiperazine([M+H]+m/z
87.0921)wasreportedbyoneparticipant.Thelowestm/zsuspectswerefivesinglehitsubstances
betweenm/z100and114,whilethefirstmultiplyreportedsubstance(benzotriazole)wasdetected
atm/z[M+H]+120.0556.Thehighestm/ztargetreportedwasiohexol(m/z821.8879)andone
suspectwasreportedabovethis,tilmicosin(m/z869.5690).Theconclusiontoharmonizethenon
targetmethodswastosuggestaminimumscanrangeofm/z1001000,butmeasurewiththelargest
possiblescanrangewithoutnegativelyaffectingtheoverallanalyticalperformanceandtheresults
showthatthiswouldhavecapturedthemostrelevantsubstancesinthissample.

Severalotherinterestingaspectscouldbeinvestigatedwiththiscomprehensivedataset.Although
manyoptionsarenowavailableforionization,manyparticipantschosetoreportsubstances
exclusivelyinpositiveESImode(excludingnegativemode),whilenoLCparticipantusedalternative
ionizationmethodssuchasAPCIorAPPI.Adesireforbetterfunctionalitytocompareandmerge
peaklistsmeasuredusingcomplementaryionizationtechniqueswasexpressedtoincreasetheease
ofuseofmultipleionizationtechniquesfornontargetscreening,asthisisnotyettimeefficientfor
routineanalysis.Similarfunctionalityisalsoneededtomergeresultsfromcomplementary
chromatographicapproaches(e.g.HILICRP).Thedelicatebalancebetweensamplevolumeinjected
andchromatographicperformancewasalsovisible.Whiletheparticipantwiththehighestsample
injectionvolume(200L)alsoreportedthemosttargetcompounds,neithermelaminenor
metforminwereincluded,indicatingthatthehighamountofsolventmayhavehinderedthe
detectionofthesehighlypolarsubstancesforchromatographicreasons.Withtheavailabledata,itis
clearthatcomprehensivenontargetscreeningapproachescanbeappliedwithgreatsuccessusing
eitherOrbitraporToFbasedinstruments.Instrumentspecificaspects,suchaswhichcompoundscan
onlybemeasuredsuccessfullywiththehigherresolutionoftheOrbitraps,whichionizebetteror
12
worseinspecificinstruments,whichmethodsperformbestforlowintensitysubstances,which
solventsandmodifiersarebetterandtheinfluenceofdetectionlimitsondetectionetc.would
requireatailormadespikedenvironmentalsampletocreateknowncasestudies.Theparallel
submissionofaspikedsamplewasencouragedbyallparticipantsforfuturetrials,toinvestigate
someofthesephenomenacloser.However,thenatureofthedatasetavailableallowsadetailedlook
atanumberofspecifictopicsinnontargetscreening,whichareillustratedwithseveralexamplesin
thefollowingtext.

Example:IsobaricSubstances
Isobaricsubstances(whereisobaricisusedtorefertosubstanceswiththesameexactmass)form
someofthemostchallengingcasesinhighthroughputscreening.Withhighaccuracydata,isobaric
substancesoftenhavethesamemolecularformula(butnotalways,assomecanhavethesame
exactmasswithintheinstrumentalaccuracy[65])andinenvironmentalscreeningthesecanoftenbe
verysimilarsubstancesstructurally.Thecaseofterbutylazine,sebutylazine,simazine,propazineand
theirTPs,showninFigure2,formsagoodexample.Fragmentationinformationcanbeusedto
distinguishpropazine(2xC3H7)fromterbutylazineandsebutylazine(bothC2H5,C4H9)inthetop
rowofFigure2,orsimazine(2xC2H5)fromthedesethylTPs(bothC4H9)inthesecondrow.Dueto
thestructuralsimilarity,thesesubstancesalsooftencoelute,especiallyingenericchromatographic
methodssuchasthoseusedinnontargetmethods.Twoparticipantsreportedbothsimazineand
desethylterbutylazineforthesamem/zandRTbutwithdiagnosticfragmentsindicatingthepresence
ofboth.Inthissample,neitherfragmentationnorretentioninformationcouldseparateterbutylazine
andTPsfromsebutylazineandTPswiththegenericchromatographyused,althoughclearlymore
institutesreportedtheterbutylazinerelatedsubstances.Thisislogicalintermsofsubstanceuse,as
terbutylazineiswidelyusedwhileneithersebutylazinenorpropazinearecurrentlyregisteredinthe
EU.However,propazineispresentasabyproductinothertriazinesandcouldbeobservedwhen
othertriazinecompoundsarepresentifconcentrationsweresufficient.Itisalsointerestingtoreflect
ontheprioritizationthatthenumberofreferenceswouldgivehere:mostinstitutesreported
terbutylazine(10)anditsTPterbutylazinedesethyl(9)insteadoftheisobaricsimazine(4);the
formerwerealsoprovidedassuspects(seeTableS5);terbutylazinedesethylwasalsoreportedby
oneGCparticipant.Withouttheknowledgeofterbutylazinebeingpresent,sortingcandidatespurely
bythenumberofreferenceswouldclearlyfavoursimazine(518referencesversus92inChemSpider,
seeFigure2).Thus,caremustalsobetakenwiththisstrategy,asadditionalknowledge(especially
presenceofrelatedsubstances,fragmentationinformationandretentiontime)shouldalsobe
considered.Thisreinforcesthepointmadebymanyparticipantsthatabetterintegrationof
identificationstrategiesisnecessaryfornontargetscreeningapproachesandtheincorporationof
usedataratherthanthenumberofreferencesaloneisdesirableinthefuture.

13

Figure2:Isobaricparentsandtransformationproductsinthecollaborativetrialsample,thenumberoftimesreported
andthenumberofreferencesinChemSpider[35].

Example:CoelutingSubstances
Thecaseof4and5methylbenzotriazolesummarizesmanyissueswiththedataevaluationinone.In
thiscase,allparticipantsreportedthetargetsubstancestogether,agreeingthattheyrequiredspecial
methodstobeseparated(e.g.[66])andcouldnotbeseparatedchromatographicallywithageneric
(multiresidue)screeningmethod.Whilethedatacouldnotbecomparedbyname(entriesincluded
4/5MethylBenzotriazole,4&5MethylBenzotriazole,4and5methylbenzotriazolethroughto
Tolyltriazole),orCASnumbers,SMILESandInChIKeys(givenforeitheroftheisomers,e.g.CAS136
856and29385431),intheend,thecomparisonwasperformedbysortingbymolecularformula
C7H7N3asnoothersubstanceswerereportedwiththisformula.Thelimitationinrepresenting
structuraluncertaintiesinInChIKeysiscurrentlythesubjectofanewworkinggroupwithinIUPACto
improvedatacomparisonofsuchcasesinthefuture.Othercasesincludedtheexampleofsimazine,
terbutylazineandrelatedcompoundsshowninFigure2andthe(often)coelutingsubstances
tramadolandOdesmethylvenlafaxine.Tramadolwasreportedmuchmorefrequently;two
participantsreportedbothsubstancesforthesamepeak,whileatleastoneotherparticipantruled
outthepresenceofOdesmethylvenlafaxinemanuallybasedontheabsenceofthediagnostic
fragmentsm/z133.0649and107.0492inthemeasuredspectrum.Anotherinterestingcaseis
dibutylphthalate,wheresevenparticipantsreportedatleastoneofthreeisomers(dinbutyl
phthalate,diisobutylphthalate,pdibutylterephthalate)forbetweenoneandthreepeaksper
participant,usingdifferentstrategies(reportoneisomermultipletimesversustryingtoguess
whichisomerwaswhich,throughtoreportingwithagenericname).Again,thisshowsagreatneed
forreportingstructuraluncertaintiesinawaycompatiblewithhighthroughputscreening.Thisalso
showstheneedforaretentiontimeindexinLCMS/MS;thequestionofwhichphthalateisomerwas

14
whichwassolvedelegantlybytheGCMSparticipantswithacombinationofchemicalionizationand
retentionindexinformation(seebelow).

DependenceonTargetandSuspectLists
Theresultsofthetargetandsuspectscreeninginthecollaborativetrialshowedacleardependence
onthetargetandsuspectlistsused,whichwereinturnalsohighlydependentonthemainfocusof
eachlaboratory.Thisisshownbythelargerangeoftargetcompoundsreported(354of622targets
werereportedonceonly,whilethemaximumreportedbyoneinstitutewas167),whichisavery
largenumberofcompoundsconsideringthelowconcentrationsobservedinthesample.Several
participantsreportedmultiplesuspectidentitiesforonepeak,suchthatthenumberofsuspect
substancesandnontargetpeaksinTable4maybeanoverestimationinsomecases,especially
wheresuspectmatchingwasperformedontheexactmassandisotopepatternonly.Many
participantstookthesuspectscreeningthenecessarystepsfurther,performingaselection/rejection
ofthematchingsuspectsusingretentiontimeandfragmentationinformation(measuredversus
libraryorversuspredictedfragmentswherenolibraryspectrumwasavailable)beforereportingthe
(tentative)identification.Thedangerofscreeningbasedonmassonlywasdemonstratedbyafew
caseswherethesuspectlistcontainedtheexactmassofthecompoundrepresentedasasalt
althoughthiscancertainlynotbedetectedassuchintheinstrument.Forexample,tris[4
(diethylamino)phenyl]methyliumacetate,C31H42N3.C2H3O2,
CCN(CC)c1ccc(cc1)[C+](c2ccc(cc2)N(CC)CC)c3ccc(cc3)N(CC)CC.CC(=O)[O]wasreportedatm/z
516.3565byoneparticipant,whilethemonoisotopicmassofthesubstancewithouttheacetatesalt
is456.3379Da.AnotherexampleisasubstancewiththeformulaC7H8N4O2,whichwasreportedonce
bythreeparticipants,butwithadifferentidentityeachtime(theophylline,1,7dimethylaxanthineor
theobromine).Whiletheforemostwasreportedasatargetconfirmedbyreferencestandard,the
otherstwocompoundsweredetectedadditionallybyoneGCparticipantwithdistinctEIMSspectra
andgoodNISTlibrarymatchvaluessimilarity700,probability87%for1,7dimethylxanthineand
similarity823,probability80%fortheobrominealthoughonlythelatterwasreportedofficiallyasa
cutoffof800wasused).Thisagainshowstheneedforspectraandotheradditionalinformationto
supportsuspectscreening.Theconceptofperformingsmartersuspectscreening(screensmart,
carefullyselectingthecompoundstobescreenedtosuittheinvestigation)wasdiscussedasoften
beingmoreefficientthanscreenbigwithlargeunselectivesuspectlists.Althoughthelatterhasthe
advantageoflargecoverage,thisisattheexpenseofahighrateoffalsepositivesandthusoftena
largerburdenofproof.Participantsalsoagreedthatitwouldbeusefulinthefuturetoexchange
targetandsuspectcompoundlistsbetweeninstitutesusinganopenplatformsuchasSTOFFIDENT
[38];theNORMANAssociationisinvestigatingthisasaplannedactivityfor2015.

MolecularFormulaAssignment,AdductDetectionandHomologousSeries
Movingalongfromtargetandsuspectscreening,animportantaspectofnontargetidentificationis
thedetectionoftheadductstateandassignmentofmolecularformulastocomponentsofinterest
andthisisillustratedusinghomologousseriesidentifiedbyparticipantsinthetrialsample.Whereas
theGCparticipantscoulduselibrarysearchingtoidentifythesecommonsubstances,theLC
participantsperformedsuspectscreeningusing(i)listsofsurfactantspublishedpreviously[3],(ii)
tentativeidentificationthroughrecognitionofdiagnosticfragments(e.g.89.0593and133.0857,
correspondingtoC4H8O2andC6H12O3,respectively)throughto(iii)retrospectivescreeningfor
surfactantsfollowingthediscoveryofdiagnosticfragmentsofsurfactantsduringnontarget
identificationefforts.Thepolyethyleneglycol(PEG)homologuesprovideaninterestingexampleto

15
showadductdetectionandmolecularformulaassignment.Theseweredetectedasthe[M+H]+,
[M+NH4]+and/or[M+Na]+speciesbyseveralparticipants,seeFigureS2forthedistributionofthe
[M+H]+and[M+NH4]+speciesmeasuredbyoneparticipantandFigureS3andS4fortheadductsand
isotopesdetectedforsomeofthesespecies.Whereatleasttwoadductspeciesarepresent,the
neutralmassofthecompoundcanbedeterminedunequivocally;ifonlyonespeciesispresentthe
adductstatemustbeassumedwhencalculatingthemolecularformula.Table5showsthecalculation
ofthemolecularformulasforthreePEGspeciesdetectedasdifferentadductsbyoneparticipant.

Table5:Calculationofmolecularformulasfornontargetmassesassociatedwithvariousadductsandexhibiting
diagnosticfragmentsforpolyethyleneglycolspecies,m/z89.0593and133.0857;columns16providedbyone
participant,columns79(Yes,No)showswhethertheformulawouldhavealsomatchedgivenfragmentinformation.

MatchesFragmentInformation
Detected Adduct Neutral Molecular Score Mass 89,133 177,221a 265,309a
m/z Mass Formula diff. (C4H9O2, (C8H17O4, (C12H25O6,
(ppm) C6H13O3) C10H21O5) C14H29O7)
388.2550 NH4+ 370.2212 C17H30N4O5 98 1.2 Yes Yes No
C16H34O9 98 2.2 Yes Yes Yes
C15H28N7O4 97 1.0 Yes No No
C23H32NOS 91 0.7 No No No
432.2813 NH4+ 414.2472 C19H34N4O6 98 1.5 Yes Yes No
C17H32N7O5 98 2.0 Yes Yes No
C18H38O10 98 1.5 Yes Yes Yes
C18H28N11O 97 1.4 No No No
481.2629 Na+ 458.2734 C20H42O11 98 1.4 Yes Yes Yes
C19H36N7O6 97 1.7 Yes Yes No
C21H38N4O7 97 1.4 Yes Yes Yes
C20H32N11O2 95 1.0 No No No
a
Fragmentinformationform/z177.1122,221.1385,265.1647and309.1909(shownasunitmassabovefor
spacereasons)takenfromtheNIST2014MS/MSlibraryspectrumofoctaethyleneglycol(NIST#1229846).
MolecularformulaswerecalculatedwiththeelementsC,H,N,OandS,thescore(calculatedusingMassHunter
WorkstationSoftwareB.06,AgilentTechnologies)considersmass,isotopematchandisotopespacing.

Thisexamplehighlightstheimportanceofconsideringadductsandapplyingacomprehensivepeak
groupingpriortonontargetidentification,whichisafeaturenowofferedbymanyopensourceand
vendorsoftware,tovaryingdegreesofcomprehensiveness.Oncethecorrectneutralmasswas
identified,molecularformulaswerecalculatedwiththeelementsC,H,N,OandS.Between5and7
formulaswithahighscore(>90)wereproposed;thetop4areshowninTable5.Thescoreconsiders
themass,isotopematchandisotopespacing,howeverthelastthreecolumnsofTable5showthat
thesefourformulascouldbereducedtoonlyoneortwopossibleformulasiffragmentation
informationwasalsoconsideredintheselectionofthemolecularformula.Althoughseveral
approachesnowincludefragmentinformationinformulaselection,includingSIRIUS[67],MOLGEN
MS/MS[68]andmzMine[69],thesewerenotusedinthetrial,whereasofthevendorsoftwareused
byvariousparticipants,onlySmartFormula3DfromBrukerassociatedfragmentationinformation
withtheprecursorionsforformulacalculation.

RetentionTimeInformationinLCHRMS
AnimportantaimofthetrialwastoassesstheuseofretentiontimeinformationintheLCscreening
approaches.Mostparticipantsmeasuredthesubstancesprovided(giveninMethods)andusedthe
resultstocalculatearetentiontimeindex(RTI)fortheLCdata.SevenparticipantsreportedtheseRTI
forsomeoralloftheirreportedvaluesintheDCTs.Theresultsof26targetcompoundsthatwere
detectedatleast4timeswereusedtoinvestigatetheRTIfurther.ThecalculatedRTIwasfoundto
16
havebelow3%rootmeansquaredeviation(RSD)forelevenofthesetargets,between3and6%
RSDforanothereleventargetsandabove6%RSDfortheremaining4ofthe26targetcompounds.
TheRTIsoftargetsubstancesreportedbytwoparticipantswereusedtocalculatelogDvaluesthat
wereinturnusedtosearchtheSTOFFIDENTdatabase[38],wherewithempiricalmatchingcriteria
areincluded.Above80%(50of60,81of96)ofthetargetcompoundsthatwereinSTOFFIDENT
werealsowithinthecalculatedlogDrangeusingtheRTIstandards.Whilethemajorityofsubstances
matched,thenonmatchof10and15manuallyvalidatedtargetsubstances(perparticipant,
respectively)showsthataconsensusapproachshouldbeconsideredwhenusingretentiontime
predictionapproaches[70].

Adifferentapproachwasusedbyanotherparticipant(14)torejectsuspectsonthebasisofretention
time(andother)information.Theirinitialsuspectscreeningyielded1060and538suspecthitsin
positiveandnegativemode,respectively,whichwasreducedto81and75suspectmatches,
respectively,usingadditionalmassaccuracy,isotopeandpeakscorecriteria.Ofthese,afurther27
and40suspectswererejectedusingretentiontimemodelsdevelopedusingaSVMkNNQSPR
approach(R.Aalizadehetal.,inprep),whileafurtherfourandfivewererejectedastheywerenot
ESIamenable.Finally78suspectswerereported,asgiveninTable4(48positive,30negative).

Whileallparticipantsagreedthattheretentioninformationisusefulandessentialforidentification
efforts,manyparticipantsexpressedaneedtohavethisinformationbetterintegratedintotheir
currentsoftware.Additionally,thenontargetsfoundbysomeparticipantswereclearlycompounds
suchassulfonatedsurfactantsandpolyfluorinatedcompounds,whichoftenperformpoorlyin
retentiontimepredictionandotherQSPRcalculations[27,71].Afurtherexampleofhowuseful
retentioninformationcanbetoconfirmtheidentificationofsuspectcompoundsisgivenbelowfor
GCMS.

NontargetIdentificationExample
Severaltentativelyidentifiednontargetcompoundswereproposedbyparticipants,despitethe
limitedtime(seeTable4)andthesecompoundscouldbefoundretrospectivelyintherawfilesfrom
otherparticipants.Oneparticipantreportedadoublepeakatm/z[MH]199.0431at19.48(small)
and19.98(large)minutesofa40minuterun,tentativelyidentifiedasmesitylenesulfonicacid(2,4,6
trimethylbenzenesulfonicacid,ChemSpiderID69438)orisomer,molecularformulaC9H12SO3,with
thefragmentsm/z184.0196and135.0815.Anotherparticipantfoundasignalatm/z199.0428with
adoublepeakat5.91and6.15minutesofa25minuterun(smallandlargeintensity,respectively)
withpeaksatm/z79.9566(SO3),80.9644(SO4H),119.0500(C9H11),135.0811(C9H12O,orSO2),
183.0115(CH4)and184.0194(CH3)withHCD90.Atotalof135candidateswiththisformulaarein
ChemSpider,butusingMetFragtorankaccordingtotheinsilicofragmentationofthecandidates,
trimethylbenzenesulfonicacidisomersandacoupleofethylisomersareallrankedequallywitha
scoreof1and4explainedpeaks.Althoughthelargerpeakisalsopresentintheblankandappearsto
comefromthesorbentsusedinthelargevolumesampling,thisistwoordersofmagnitudelower
thantheintensitiesobservedinthesamplesforbothparticipants.However,thestandardfor
mesitylenesulfonicacidwasavailableatthesecondinstituteandalthoughtheretentiontime
matchedwell(5.96minutes),thefragmentsdidnotmatch;onlytwofragments(m/z79.9573and
135.0824)wereseeninthestandardatvaryingcollisionenergiesincludingHCD90.Thusthe
tentativeidentificationcannotbeconfirmed.However,thepeakseenbythesecondparticipantat
m/z183.0115isdiagnosticforlinearalkylbenzenesulfonicacid(LAS)species[3]andthestructure
belowmaybeabletoexplainthespectra.Althoughnotyetconfirmed,thisexampleshowshowthe
17
measurementsofthesamesamplefromtwodifferentlaboratoriescanbeusedtohelpinthe
identification.

Figure3:Spectrumofthenontargetfoundat6.15minutesbyoneparticipant,atentativelyidentifiedcandidate(4
isopropylbenzenesulfonicacid,insert)andtheresultingfragments.Theoriginaltentativeidentificationofmesitylene
sulfonicacidcanberejectedonthebasisofmismatchingfragments,seeFigureS5andS6.

GCMS

FortheGCMSresults,fourinstitutesreportedtargetcompounds(confirmedwithreference
standards),with64resultsreportedfor55uniquecompounds.Thetargetsubstancesdetected
multipletimeswereallpolycyclicaromatichydrocarbons(PAHs):phenanthrene(4),anthraceneand
naphthalene(3each),fluorene,fluorantheneandpyrene(twiceeach).Theremainingidentifications
wereperformedusinglibraryinformation,generallytheNISTMSlibrary[11]versions02(175,214
spectraof147,350compounds),08(220,460EIMSspectraof192,108compounds)and11(243,893
EIMSspectraof212,961compounds),althoughoneparticipantalsousedtheWileylibrary,7th
Edition[12].Matchfactorsbetween700and800(ofmaximum999)wereusedasthelowerlimitfor
librarymatches(somealsoincombinationwiththeprobabilityestimates)andmostparticipantsalso
confirmedthesematchesmanually.Anadditional295compoundswerereportedfromlibrary
matches(identifiedbutnotconfirmedwithreferencestandards),suchthatinall,255unique
compoundswerereportedwithasingleidentityasatargetsubstanceorlibrarymatch.Afurther47
peakswerereportedaslibrarymatchesbutwithisomerunidentifiedwhile35reportedsubstances
remainedunknown(manyparticipantsdidnotreportunknownsubstances).Mergingthetargetand
librarymatchcompoundlistsrevealedthatphenanthrenewasdetectedbysixofsevenparticipants,
whiledibutylphthalate,diethylphthalate,tris(1chloro2propanol)phosphate(TCPP)andcaffeine(5
timeseach)and4oxoisophorone,benzothiazole,cyclohexylisocyanateandtriphenylphosphineoxide
and1,1(1,4phenylene)bisethanone(4each)werealsodetectedfrequently.Severalsubstances
overlappedwiththeLCMStargetsandsuspects,notablytheseveralphthalateandalkylphosphate
species,caffeineandcarbamazepineintotal46substanceswerereportedasidentifiedinbothLC
andGC,includingthesuspectsandlibrarymatches,ofthe858uniquecompoundsreportedintotal
(~5.4%).

ThesamplepreparationwasnotidealforGCanalysisandtheparticipantsalsouseddifferent
solvents.Asaresult,therelativelyfewsubstancesdetectedandlimitedcomparabilitybetweenGC
participantsisnotcompletelyunreasonable.AseparatesampleforGCMSanalysisisrecommended

18
forfuturetrials.Whilesomeparticipantsalsoquantifiedtheirtargetsubstances,thisdataisnot
presentedhereasthedatasetwastoosmallforameaningfulevaluation.

Figure4:(a)Useofcomplementaryionizationtechniques(EI,PCI),librarymatchandretentioninformation(experimental
KRIversusmeasuredvaluesinNIST)byoneparticipanttoidentifytwodibutylphthalateisomersandreversetheorderof
proposeddatabasematchesonthebasisofretentioninformation.(b)ThecorrespondingAPCIQToFMS/MSspectrumof
thesuspectdibutylphthalatereportedbyanotherparticipant,whichcouldbeeitherisomer(bothstructures:seeinsert).

HighResolutionGCMS
OneparticipantusedhighresolutionGCAPCIQToFMSduringthetrial,combiningtheresultswith
EIMSinaninterestingapproach.Asmentionedabove,theworkflowtoidentifysubstanceswiththe
GCAPCIQToFMSapproacheffectivelyfollowedthetarget,suspect,nontargetapproachesusedfor
LCHRMSscreening,ratherthantheclassictargetscreeningplusNISTdatabasesearchusedgenerally
inEIMS.FurtherparticipantsusedGCxGC,whileanotherparticipantusedthecombinationof
chemicalionizationandEIMStoidentifythemolecularmasscorrespondingwiththeEIMS
spectrum,showninFigure4.Thebasepeakm/z149(indicativeofphthalates)wasseenthreetimes
butwasonlyassociatedwiththem/z[M+H]+279twice,supportingtheproposedNISTmatchesof
dibutylphthalateisomers.However,considerationoftheexperimentalKRIwiththeliteraturevalues
inNISTpromptedaswitchofthetentativelyproposedidentitiesinFigure4(shownbythedashed
boxesandbluearrows),asdiisobutylphthalate(shownleft)hadalowerretentionindexthan
dibutylphthalate(shownright).Finalconfirmationviaareferencestandard,however,isstill
necessary.TheHRMS/MSspectrumobtainedviaAPCIQToFMS/MSisshowninFigure4(b).

AsfortheLCparticipants,theGCparticipantsmentionedthatthedataprocessingisalimitingfactor
forusingGCxGCand/orsoftionizationincombinationwithEIMSandtheneedforexactmass
spectra,especiallyMS/MSspectrawasexpressed.Thisreinforcestheobservationthattheworkflow
appliedforidentificationisdeterminedbytheionizationtechniqueused(EIversussofterionization
methods)andthesizeofthespectrallibraryavailableratherthanthechromatography.

DiscussionandPerspectives

DataProcessingAspects,HRMS/MS
Theparticipationofmanyinstitutesinanalysingtheonesampleshowsthatnontargetscreening

19
methodshavecomealongwayinashorttimeandthatacomprehensivetargetscreening
complimentedwithsuspectscreeningformsanessentialprerequisitetonontargetidentification.
Thevastmajorityofsubstancesreportedwereidentifiedthroughtargetorsuspectscreeningofdata
acquiredwithnontargetedacquisition.Tentativelyidentifiednontargetcompoundswerereported
byafewparticipants,eventhoughnontargetidentificationisstillaverytimeintenseprocess.Many
participantsincludedMS/MSandretentiontimeinformationwherepossibletosupportthe
proposedidentityofsuspectcompounds,addressingthepitfallswithperformingsuspectscreening
onexactmassonly.Suspectscreeningprovedausefulsteptoproposetentativeidentificationsfor
severalcompoundsandeasetheburdenofidentification.Thelargeoverlapbetweentargetand
suspectcompoundsprovesthatthiswassuccessfulinmanycases.Theselectionofthepeaksof
interestfornontargetidentificationremainsverysubjective;itisclearthatnotalloftheupto
severalthousandunknownpeakscanbeidentified.Oneoptionistheprioritizationofmasses
occurringinseveralsamples(e.g.[3,9],andalthoughonlyonesamplewasavailableforthetrial,
severalparticipantsonlyconsideredpeakswhentheyoccurredinatleasttworepeatedanalytical
runs,toreducetheinfluenceofnoiseorotherinterferencesontheresults.Intermsofnontarget
identification,whilesomeparticipantsdeterminethemolecularformulafirst,followedbyelucidation
onthebasisoftheone(orseveralpossible)molecularformulas,otherparticipantswentstraightto
identificationwithanexactmass.Thelatterstrategy,althoughresultinginmorecandidates,has
beenshownrecentlytobemoreeffectivefromadataprocessingpointofview[72](itisforexample
easiertoselectcandidateswithoneorsomeformula(s)fromanexactmasssearchthantomergethe
resultsofseveralseparatecalculations).

Anewtrendrevealedduringthetrialisthemovetowardsfindingrelatedsubstances,withseveral
strategiesused.Theseincludedsearchingforcommonfragmentsorneutrallosses,suspectscreening
forhomologuesandevenhomologueseriesdetection.Otherstrategiesthathavebeenused
elsewhereincludedselectionbymassdefecttoavoidmatrixinterferencesinsediments[23]and
whiletheKendrickmassdefectisoftenusedfornaturalorganicmatterorpetroleumanalysis(e.g.
[73,74]),thiswasnotmentionedbyanyoftheparticipantsanditremainstobeseenwhetherthis
mayhaveanapplicationtoenvironmentalsamples.Asdiscussedabove,challengesstillremainwith
regardtothereportingofsubstancesthatcannotfullycharacterizedstructurallyusingthe
informationavailablealone(e.g.longchainsurfactants,lipidspecieswithundefinedbranching
and/ordoublebondlocations)andalsothelevelofconfirmationrequiredbeforebeingabletoreport
thesesubstancesininvestigations.Asdiscussedintheintroduction,acombinationofarevised
versionoftheEUIPsystem(toe.g.betterreflecttheavailabilityofmuchhigherresolution
instrumentsthanthe10,000intheoriginalguideline[6])withthelevelsystemshowninFigure1
couldbeawaytomoveforwardhere.Afurtherimprovementinthefuturewouldbebetter
processingofdataindependentacquisitiondata,suchthatfragmentsareavailableforallsubstances,
notjustselectedprecursors.Anotherinterestingapproachcouldbetospeedupnontarget
identificationusingalternativedatabasessuchasthoseincludingnaturalproductstoassistinfinding
backgroundnaturalcompoundsthatarenotnecessarilyofinterestfornontargetidentificationof
potentiallyhazardousanthropogenicsubstances.

Thenumberoftargetsandsuspectsdetectedonlyonceindicatesthattheresultsarehighly
dependentontheinstituteknowledgeandfocusarea,aswellastargetandsuspectlistsused.This
reinforcestheneedforadditionalevidencebeforereportingsuspectidentificationandanimproved
representationofidentificationconfidence,suchasupdatedIPs.Itisclearfromthenumberof

20
differentsoftwareandapproachesusedthatthedataprocessingtechniquesareverydifferent,with
vendorversusopensourceapproachesandmanydifferentsourcesofinformationusedbythe
participants.Theneedtoharmonizeinformationsourceswasexpressedandfromtherecognition
thatoneparticipantstargetisanotherparticipantssuspectorunknown,anagreementwasmadeto
exchangetargetandsuspectlistsinacentralizedplatform,wherethedevelopmentofsearchcriteria
andprioritizationmethodsoftheselistswillbeessentialtoallowapplicationofthescreensmart
approach.Additionally,theresultsofthisstudyhavebeenprovidedassupportinginformationfor
useinfutureinvestigations.Enhancingthiswiththeuploadofmassspectraoftargetcompoundsto
anopenaccessdatabasessuchastheNORMANMassBank[52]wouldhelpimprovethesuccessof
target,suspectandevennontargetscreeningimmensely.Anotherneedclearlyexpressedbythe
participantswasforbetterdataprocessingofcomplementaryionizationmodesandmethods(e.g.
positive/negative,APCIandAPPI)aswellasdataindependentacquisitiontechniques,suchthat
fragmentationcanbeobtainedforallsubstancesratherthanselectedprecursormassesandabetter
integrationofretentioninformation.

LessonsLearnedfromtheTrial
ThefirstcollaborativenontargetscreeningtrialrunbytheNORMANAssociationwasavery
ambitiousendeavourcoincidingwiththeThirdJointDanubeSurveyinresponsetotheneed
expressedbymembersinlate2012.Theresponsefromparticipantswasverypositiveandtheaimto
discusswaystoharmonizenontargetscreeninginenvironmentalsampleswithinEuropewere
fulfilledwithaseriesofthreewellattendedworkshops[7577].Theanalyticalmethodsarealready
reasonablyharmonized,whilethemaindifferencesresultingfrominstrumentspecificsettingsthat
participantscannotreasonablybeexpectedtochange.Whilethedataprocessingworkflowswere
notasharmonized,withmanyparticipantsexpressingtheneedforbetterintegrationofinformation,
workflowsandsoftware,agreatdealcanbelearntbyreflectingontheresultsoftheother
participants.Whileparticipantsweregivenalotoffreedomforthiscollaborativetrial,therewasalso
interestinamoretightlydefinedinterlaboratorycomparisoninthefuture,withanadditionalspiked
sampleandamorecomprehensivesuspectlistfromgreatercomparability.

Allparticipantsprovidedtherawdataoftheirmeasurementsforthistrial,suchthataretrospective
analysisoftheresultswillbepossibleandfindingasuitableenvironmentalrepositoryforthisdata
willbethesubjectoffutureefforts.Manyparticipantswerekeentoinvestigatetheirresultsfurther,
usingtheinformationgainedduringthetrial,toperformretrospectiveidentification.Thetrendfor
retrospectiveanalysis,usedalreadyduringtheevaluationoftheseresults,isincreasingandthe
importanceofthisinthefuturecannotbeunderestimated.

Acknowledgements

Theauthorsgratefullyacknowledgethecontributionsofmanyotherswhohelpedduringthe
analysis,reportinganddiscussionoftheresultspresentedhere:PhilippLongree,MartinLoos,
MatthiasRuffandJenniferSchollee(Eawag),MargitPetre,AnettKloandWernerBrack(UFZ),Reza
Aalizadeh,NikiforosAlygizakisandPabloGagoFerrero(UniversityofAthens),SylvainMerel
(UniversityofTbingen),AlexandervanNuijs,AlinIonasandAdrianCovaci(UniversityofAntwerp),
MartijnPijnappels(Rijkswaterstaat),ChristelleMargoum(Irstea),ClaudioBortolini(Universityof
Padua),MarieHlneDevierandHlneBudzinski(UniversityofBordeaux,France),Christophe

21
TondelierandMathildeChachignon(VERI),TobiasBaderandThomasLucke(Zweckverband
Landeswasserversorgung),MichaelSchlsener(BfG),MarEsperanza(SuezEnvironnement),Sinia
Repec(CroatianWaters),PeterTarabek(SlovakNationalWaterReferenceLaboratory),Christoph
Ruttkies(IPB,Halle,Germany),theChemSpidersupportteamandThierryFayefromAgilent.The
watersampleoftheDanubewaskindlyprovidedbytheInternationalCommissionfortheProtection
oftheDanubeRiver(IPCDR)aspartoftheJointDanubeSurvey3.Authorcontributions:ELS,HPS,JS,
IMI,PO,MK,TS,PH,TL,SG,NST,CZandJHwereinvolvedinthecoreteampreparingthetrial,
evaluation,workshopandpublication;MI,TP,RdB,MR,MO,UK,WS,AG,NN,GL,PB,SB,DS,PR
participatedinthetrialandpublicationefforts.

ThistrialwasinitiatedbytheNORMANAssociationwithinkindcontributionsfromparticipants;no
feewaschargedforparticipation.ThisworkwassupportedinpartbytheSOLUTIONSproject,which
receivedfundingfromtheEuropeanUnionsSeventhFrameworkProgrammeforresearch,
technologicaldevelopmentanddemonstrationunderGrantAgreementNo.603437.

CompliancewithEthicalStandards:Theauthorsdeclarethattheyhavenoconflictofinterest.No
violationofhumanandanimalrightsoccurredduringthisinvestigation.

ElectronicSupplementaryMaterial:

AdditionalTablesandFiguresareprovidedasOnlineResource1(ESM_1.pdf,includingFiguresS17
andTablesS16).Azipfile(ESM_2.zip)isalsoprovidedcontainingspreadsheets(asxlsandcsv)with
allidentifiedtarget,suspectandnontargetsubstancesfortheLCMS,GCMSandthesubstances
reportedinboth.

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