Retrospective screening of synthetic cannabinoids, synthetic opioids and designer benzodiazepines in data files from forensic post mortem samples analysed by UHPLC-QTOF-MS from 2014 to 2018

(1)

Retrospective

screening

of

synthetic

cannabinoids,

synthetic

opioids

and

designer

benzodiazepines

in

data

ﬁles

from

forensic

post

mortem

samples

analysed

by

UHPLC-QTOF-MS

from

2014

to

2018

Per

Ole

M. Gundersen

a,b,

*

,

Sebastian

Broecker

c

,

Lars

Slørdal

b,a

,

Olav

Spigset

a,b

,

Martin

Josefsson

d,e

a

DepartmentofClinicalPharmacology,St.OlavUniversityHospital,Trondheim,Norway

b

DepartmentofClinicalandMolecularMedicine,NorwegianUniversityofScienceandTechnology,Trondheim,Norway

c

BroeckersSolutions,Berlin,Germany

d

DepartmentofPhysics,ChemistryandBiology,LinköpingUniversity,Linköping,Sweden

e

NationalForensicCentre,DrugUnit,Linköping,Sweden

ARTICLE INFO Articlehistory: Received7January2020

Receivedinrevisedform19March2020 Accepted25March2020

Availableonline3April2020 Keywords:

Retrospectivescreening UHPLC-QTOF-MS Postmortembloodsamples Newpsychoactivesubstances Syntheticcannabinoids Syntheticopioids Designerbenzodiazepines

ABSTRACT

Theintroductionofnewpsychoactivesubstances(NPS)ontheillicitdrugmarkethasledto major challengesfortheanalyticallaboratories.Keepingscreeningmethodsuptodatewithallrelevantdrugsis hardtoachieveandtheriskofmissingimportantfindingsinbiologicalsamplesisamatterofconcern. Aiming for an extended retrospective data analysis, diagnostic fragment ions from synthetic cannabinoids(n=251),syntheticopioids(n=88)anddesignerbenzodiazepines(n=26)notincluded inouroriginalanalyticalmethodwereobtainedfromthecrowdsourceddatabaseHighResNPS.comand convertedtoapersonalizedlibraryinaformatcompatiblewiththeanalyticalinstrumentation.Datafiles fromtheanalysisof1314forensicpostmortemsampleswithanAgilent6540ultrahighpressureliquid chromatography quadrupoletime-of-flight massspectrometry (UHPLC-QTOF-MS)performedinour laboratoryfromJanuary2014toDecember2018wereretrievedandretrospectivelyprocessedwiththe newpersonalizedlibrary.Potentiallypositivefindingsweregroupedintwo:Themostconfidentfindings containedMS/MSdataforlibrarymatch(category1)whereasthelessconfidentfindingslackedsuchdata (category2).Fivenewcategory1findingswereidentified:Flubromazepamintwodatafilesfrom2015 and 2016, respectively, phenibut (4-amino-3-phenylbutyric acid) in one data file from 2015, fluorofentanylinonedatafilefrom2016andcyclopropylfentanylinonedatafilefrom2018.Retention timematcheswithreferencestandardsfurtherstrengthenedthesefindings.Alistof35presumably positivecategory2 findingswas generated.Ofthese,onlyonefindingofphenibutwasconsidered plausible after checking retention times and signal-to-noise ratios. This study shows that new compounds can be detected retrospectively in data files from QTOF-MSusing anupdated library containingdiagnosticfragmentions.Automaticscreeningprocedurescanbeuseful,butamanual re-evaluationofpositivefindingswillalwaysbenecessary.

(http://creativecommons.org/licenses/by/4.0/).

1.Introduction

In recent years, there has been a continuously increasing numberofnewpsychoactivesubstances(NPS)appearingonthe Europeanillicitdrugmarket[1].Thediversityandhighnumberof new compounds pose challenges for clinical and toxicological laboratories who strive to keep their drug screening methods

updated. Synthetic cannabinoids, i.e. compounds acting as

cannabinoid receptor agonists and produced as alternatives to

D

-9-tetrahydrocannabinol(THC)representthelargestandmost structurally diverse group [2]. New synthetic opioids, and in particularthefentanylanalogues,havebeenofmountingconcern because of their formidable toxic potential [3–6]. Designer

benzodiazepines is another group in focus due to the high

prevalenceofuse,atleastinourcountry[7],comparedtoother groupsofNPS.

Todevelop,establishandmaintainascreeningmethodcapable of detecting all drugs relevant at any given time is a major challenge.Theuseofhighresolutionmassspectrometry(HR-MS)

* Correspondingauthorat:Dep.ofClinicalPharmacology,St.OlavUniversity Hospital,Postbox3250Torgarden,7006Trondheim,Norway.

E-mailaddress:per.ole.m.gundersen@stolav.no(P.O.M. Gundersen).

http://dx.doi.org/10.1016/j.forsciint.2020.110274

ContentslistsavailableatScienceDirect

Forensic

Science

International

(2)

e.g. quadrupole time of ﬂight mass spectrometry (QTOF-MS)

instrumentation has proven to be an applicable tool when

searching for drugs of abuse in biological samples [8–13]. The

detectionofunknowncompoundsistimeconsumingandhardly

feasible on a routine basis with a large number of samples.

Consequently,themethodmustencompassascreeningortargeted

approach, based on an extensive and comprehensive database

containingmultipletypesofdataforidentiﬁcation.Suchdatacan beretentiontimes(RTs)and fragmentationdatafrom collision-induceddissociation(CID), in silicoorothertheoretical evalua-tions,inadditiontothemolecularformulaofthesubstance.The

database can be created and maintained _{“in-house”} by the

laboratory.Thisrequiresaccesstoahighnumberofwell-deﬁned

reference compounds. Procurement of reference standards is

costly,particularlyifadatabaseshouldbeuptodatewithasmany

new and relevant compounds as possible. Databases are also

commerciallyavailablefromsuppliersofMSinstruments(e.g.the

Forensic Toxicology Personal Compound Database and Library

fromAgilent),butusersaredependentonthefrequencyofnew releasesand/oradditionsbeinguptodate.Therearealsoexamples ofcommercialoperatorsofferingfreedatabases(e.g.themzCloud fromThermoFisher).Anotheropportunityiscrowdsourced data-baseswithinformationsubmittedbyglobalHR-MSusers.Onesuch

example is HighResNPS.com [14]. When performing CID on a

certain compound, different instrument conﬁgurations tend to

generate the same diagnostic fragment ions even though the

relativeabundancemayvary.Thus,fragmentdataacquiredonone instrument canthen be usedas identiﬁcation across platforms [14–16].Inprinciple,thesameistrueforacrowdsourceddatabase withdiagnosticfragmentsacquiredbyinstrumentsfromdifferent

manufacturers, providing that the added fragment masses are

convertedtotheoreticalvalues.

Incontrasttoanalyticalmethodsbasedonsingleionmonitoring ormultiplereactionmonitoring,HR-MSfull-spectrumdataremain availableandpermittheidentiﬁcationofnon-targetcompoundsand retrospectiveanalysis,alsocalledpost-targetanalysis.Fordatafrom

HR-MS instrumentation with fragmentation capabilities, e.g.

QTOF-MSorlineariontrapOrbitrap,fragmentationdataarealso available.Inprinciple,allcompoundsareavailableforinvestigation at a certain level, but thedata available arelimited bysample extractionrecovery,chromatographicselectivityandthedegreeof ionization and fragmentation. Depending on which acquisition

mode is used, the QTOF-MS data also contain fragment ions

originatingfromthemolecularionsgenerated intheionsource. Based on newknowledge, post-targeted analysis of data cangenerate new findings in a specific toxicological or clinical sample and ultimatelychangetheconclusioninaparticularcase.Aretrospective studyisalsoimportantasaninternalqualitycheckforthelaboratory toassesswhetherthescreeningrepertoireusediscomprehensive andrelevant.Inaddition,newtrendsindrugabusecanbeidentified, as exempli_{fiedinthestudybyKriikkuetal.wherethetoxiclifespanof} U-47700wasexplored[17].

Thenumberofstudiesapplyingsucharetrospectiveapproach inaforensicorclinicaltoxicologysettingarelimited.Nobleetal. processed2339forensicsamplesretrospectivelywithatargeted screeningmethodtodetect504-anilidopiperidine-related fenta-nylanalogues[18].InanothercasestudyU-47700,diclazepamand ﬂubromazepamweredetectedinretrospect[19].Mollerupetal. applieda post-targetedapproach when developing a screening

method for valproate using positive ionisation mode [20].

Retrospectiveanalysisofurine sampleshasbeenusedtodetect metabolitesofpesticides[21].Post-targetedanalysisofdatahas alsobeenusedfordetectionofdrugsandpesticidesinnon-human matricesincludingsewagewater,surfacewaterandfood[22–26]. SinceDecember2013,ourlaboratoryhasutilizedaworkﬂow basedonultra-highperformanceliquidchromatography(UHPLC)

coupledtoa6540QTOF-MSfromAgilent(SantaClara,CA,USA)for

therapeutic drugs and drugs of abuse in post mortem blood

samples.ThesameUHPLCandMSmethodhasbeenappliedfrom

2014tothepresent.Acommercialdatabasesuppliedwithentries addedmanuallyafteranalysingreferencematerialshasbeenused foridentiﬁcation.However,inordertodetectaneworpreviously

unknown drug in a biological sample, additional information

connectedtothecaseorsample(e.g.aseizure)hastobeavailable. In ourexperience, suchinformationis rarelyavailable, andthis mayincreasetheriskofmissingdetectionofNPS.Theconsistency of the screening methodenables retrospective analysissothat

new compounds can be found. The use of HighResNPS for

identifying compounds in samples analysed on Agilent

QTOF-MS has previously been shown, but only ﬁles from data

independent acquisition (DIA) could be investigated with this

approach [14]. Our method was based on data dependent

acquisition(DDA) which, asopposed toDIA,involves acquiring ofMS/MSspectraafterselectionofprecursorionsisolatedbythe quadrupole.Athoroughexplanationof thedifferencesbetween DIAandDDAcanbefounde.g.inthepapersofSundströmetal.[27] andBroeckeretal.[8].TobeabletouseHighResNPS,diagnostic fragmentinformation fromthedatabasehadtobeconvertedto

spectra in the format accepted by the Agilent MassHunter

Qualitative searching tool. In Agilent terminology, a library is

thesumofcompoundsinadatabasecontainingMS/MSspectra

andthesedatabasesandlibrariesarecalledPersonalCompound DatabaseandLibrary(PCDL).

Theaimofthisstudywastore-processdataﬁlesofforensicpost

mortemsamplesacquiredfromJanuary2014toDecember2018

in a PCDL-facilitated search for NPS belonging to the

sub-groups synthetic cannabinoids, synthetic opioids and designer benzodiazepines.

2.Materialsandmethod

2.1.Chemicalsandreagents

Reference substances used in the experiments to calculate recoveriesandmatrixeffectsandexploreinstrumentsensitivities werepurchasedassolidmaterialorstocksolutionsfromeitherof thefollowingsources:CaymanChemicals (AnnArbor,MI, USA), ChironAS(Trondheim,Norway),SigmaAldrich(St.Louis,MO,USA) andLipomed(Arlesheim,Switzerland).Individualstocksolutions intherangefrom0.2to1.0mg/mLwerepreparedandcombined

into working solutions which were spiked into blood. For

conﬁrmationoftentativeﬁndings,referencesubstancesoftilidine,

phenibut (4-amino-3-phenylbutyric acid) and JWH-167 were

purchasedfromSigmaAldrich,ChironASandCaymanChemicals respectively.LC–MSqualityacetonitrile,methanol,LiChrosolve1

waterandARISTAR1formic acidwereallpurchasedfromVWR

Chemicals(Oslo,Norway).AmmoniumacetateofLC_–MSgradewas fromSigmaAldrich(St.Louis,MO,USA).Asolutionoftheinternal referencestandardscodeine-d3,morphine-d3, benzoylecgonine-d3andgriseofulvinwaspreparedbydilutingstocksolutionsin20% methanol(v/v)inwatertoaﬁnalconcentrationof200ng/mL.

D3-codeine, d3-morphine and d3-benzoylecgonine werefrom

Lip-omed whereas griseofulvin was from Janssen Chimica (Geel,

Belgium).

2.2.Validationoforiginalscreeningmethod

2.2.1.Instrumentsensitivityandlimitofidentiﬁcation

The same UHPLC-QTOF-MS instrumental method, sample

preparationand internalreferencestandard concentrationwere used for allthe samplesthroughout theperiod. The peak area resultsandRTsoftheinternalreferencestandardsinonedataﬁle

(3)

per batchwereextracted in orderto illustrate thevariation in responseovertime.Limitofidentiﬁcation(LOI)wasevaluatedfora

selectionof syntheticcannabinoids (MDMB-CHMICA,

AB-CHMI-NACA, BB-22, JWH-018, PB-22 and THJ-018), synthetic opioids

(fentanyl, remifentanil, cyclopropylfentanyl, para-ﬂuorofentanyl, furanylfentanyl, acetylfentanyl) and designer benzodiazepines

(deschloroetizolam, diclazepam, etizolam, ﬂubromazepam,

ﬂu-bromazolam,pyrazolamandmeclonazepam).Bloodsampleswere

spikedat0.1,0.2,0.5,1.0,2.0,5.0,10and20ng/mL,andpreparedin triplicateswiththesamemethodasdescribedforthepostmortem

samples.LOIwasdeﬁned astheminimumconcentrationwhere

thecompound wasidenti_ﬁedandatleastoneMS/MSspectrum

wasacquiredforlibrarysearchinallthreeparallels(seeSection2.5

fordetailsonidentiﬁcation). 2.2.2.Recoveryandmatrixeffects

Recoveries(REs)andmatrixeffects(MEs)werecalculatedfor thesamecompoundsasusedintheLOIexperiment.Subsamplesof pooledwholebloodwerespikedafter(B)orbefore(C)extraction to a ﬁnal concentration of 0.1

m

g/mL. The peak areas in neat standardsolutionofthesameconcentration(A),sampleBandC wereusedtocalculateREandME(Eqs.(1)and(2)).AnMEbelow

100% indicates ion suppression whereas a value above 100%

indicatesionenhancement.

RE ð%Þ ¼C_B100 ð1Þ

ME ð%Þ¼ B_A100 ð2Þ

2.3.Originalanalysisofthebloodsamples

Dataﬁlesincludedinthisstudywerefromtheanalysesofpost

mortem blood samples from forensic autopsies sent to our

laboratory in theperiod fromJanuary 2014toDecember 2018. Inalimitednumberofcaseswherebloodwasnotavailable,spleen tissuewasused.Samplesfromatotalof1314caseswereanalysed in this period. Permission to re-process the data _ﬁles (in this contextmeaningopeningthedataﬁleandrunthealgorithmwith thenewPCDL)wasgivenbytheRegionalCommitteeofMedical andHealth ResearchEthics inMidNorway (approvalNo. 2018/

2157).The dataﬁleswere anonymized and theanalyst had no

information about the original ﬁndings when doing the

re-processing.Asecondpersoncomparedthenewﬁndingswiththe analytical report originally attached to the relevant cases. Accordingtothepermissiongrantedfromtheethicscommittee,

re-analysis of the sample specimens as such could not be

performed. The samples were originally processed with the

commercially available Forensic Toxicology Personal Compound DatabaseandLibraryfromAgilent(SantaClara,CA,USA)withmore

than3000compoundscontainingMS/MSspectracomplemented

withbetween250and300compoundswithRTs.

2.3.1.Samplepreparation

Each blood sample was thawed at room temperature and

200mg was weighed into a micro tube and 50

m

L solution of

internalreferencestandardand800

m

Lice-coldacetonitrilewere added.Thetubewasthenmixedonavortexmixerfor30sand centrifugedat7000gfor10min.before500

m

Lofthesupernatant was transferred to a 96-well plate, evaporated todryness and reconstituted in 50

m

L of 30% acetonitrile (v/v) in 0.03mg/mL ammoniumformate.Inthecaseswhereonlyspleenwasavailable, samplepreparationwasadjustedaccordingtotheconditionofthe tissue.Ifablood-likematerialcouldbeobtainedfromthespleen,it

washandledasabloodsample.Intheothercasesasubsampleof tissuematerialwashomogenizedwithanequalvolumeofH2O, and200mgofthismaterialwereprocessedlikeabloodsample.

The samples were prepared in weekly batches by the same

procedurethroughouttheperiod. 2.3.2.Instrumentation

Instrumentalanalysiswas performedusinga 6540QTOF-MS

(Agilent,SantaClara,CA,USA)withelectrosprayionization(ESI) coupledwitha1290InﬁnityUHPLCsystemfromAgilentequipped withanAcquityHSST3column(100mm2.1mm,1.8

m

m)from Waters(Milford,MA,USA).Aninjectionvolumeof2

m

Lwasused. Separationwasachievedusingamobilephaseconsistingof0.05%

formic acidin 10mMammoniumformate(A) and0.05%formic

acid in acetonitrile (B). A gradient witha ﬂow of 0.50ml/min startingat5%Bincreasingto50%in10min.andcontinuingto100% overthenext6min.wasused.Aftera4-minuteholdat100%Bthe columnwasre-equilibratedfor2min.at5%B,givingatotalcycle timeof22min.Autosamplerandcolumntemperaturesweresetto 10Cand50C,respectively.

PositiveESI was usedand withfragmentorvoltageat 120V, capillaryvoltageat3500V,gastempat320_C,_gas_ﬂow_at₈_L/min, nebulizerpressureat40psigandsheathgastemperatureat380C.

Data was acquired in data dependent Auto MS/MS mode. MS

spectraandMS/MSspectrawerebothacquiredinthemassrange of50–1000m/zatarateof6Hz.Thedetectoroperatedin2GHz extendeddynamicrangegivingaresolution(m/

D

matFWHM)of approx.20,000atm/z322.0481.Precursorselectionwasbasedon abundanceandanintensitythresholdof1000countswasapplied. Afteronespectrumfroma precursorwasacquired,thisspeciﬁc precursorwasexcludedfor0.03min.Precursorswerefragmented inthecollisioncellusinganelectronvoltageaccordingtoEq.(3):

CollisionenergyðeVÞ¼4þð0:06m=zof precursorÞ ð3Þ

Thecomputercontrollingtheinstrument wasequippedwith theMassHunterAcquisitionsoftware(Acq)B.05.01(Agilent,Santa Clara, CA, USA). The acquireddata ﬁlesconsisted of MS1 (full spectrumMS-only)ofallionizedcompoundsandMS/MSspectra of theprecursorsselectedforfragmentation.Them/z massesof 121.0509and922.0098wereappliedforautomatedmass correc-tioninallMSspectra.Adailyperformancesampleofamphetamine

(0.74ng/mL), diazepam (0.35ng/mL), 7-amino-ﬂunitrazepam

(0.35ng/mL),morphine(0.35ng/mL)and

D

9-tetrahydrocannabi-nol(0.5ng/mL)inMeOHwasinjectedatthebeginningofevery

analytical run to monitor important instrument parameters.

SampleswerenotanalysediflargedeviationsinRTs(morethan 0.2min.),massaccuracies(morethan5ppm)orpeakareasfrom thehistoricalaverageswereobservedforthecompoundsinthe dailyperformancesample.

2.4.CreatinganewPCDL

HighResNPS (highresnps.com) is a free, online, spreadsheet-format,crowdsourcedHR-MSdatabaseforNPS-screeninginitiated and managedby a group of researchers at Section of Forensic ChemistryattheUniversityofCopenhagen[14].Several

contrib-utors worldwide submit fragmentation data when new drugs

(referencestandardsorseizuresetc.)aredetectedandanalysedby aHR-MSinstrument.Also,diagnosticionsderivedfromtheoretical dissociationsofthemoleculesaresupplied.FromthisHighResNPS database(totalnumberofentriesinMay2019was1782including duplicates,and1304containedatleastonediagnosticfragment

ion), 374 unique compounds with minimum one diagnostic

fragment primarily belonging to the drug classes synthetic

cannabinoids,syntheticopioidsordesignerbenzodiazepineswere

(4)

implementedin2014wereﬁlteredout.Basedonthisselectiona PCDLwasdeveloped.Forthispurposeeachcompoundwasadded asanindividualdatabaseentry.Thenthesoftwaretool“Spectrum Generator”createdbyBroeckersSolutions(Berlin,Germany)was usedtoconvertthetext-basedinformationofdiagnosticionsfrom theHighResNPSdatabaseintotheAgilent“cef”ﬁleformatwhich allowsanimportoflibraryspectraforeachPCDLentry.Table1

shows the resulting HighResNPS subset PCDL content. By this

approachthediagnosticfragment ions werestoredasa library

spectrum. Relative abundance of the ions was not taken into

account even though this would be possible by the software

“SpectrumGenerator”.Thecollisionenergyofthelibraryspectra waschosenbythesoftwareas20eVjusttohaveanyvalueinthe PCDL.Anexampleofthelibraryentryofﬂubromazepamisshown inFig.1.Acompletelistofthe374uniquecompoundsisgivenin thesupplementarymaterial(TableS1).

2.5.Dataprocessing

Ofthe1314dataﬁlesavailable,batchesofapprox.250were

re-processed using MassHunter DA Reprocessor software B.09.00

(Agilent,SantaBarbara,CA,USA).There-processingwasrelatively fast,approximately 1min. per sample,when usinga computer equippedwitha2.67GHzprocessorand8GBofRAM.Thisprocess wasrunninginthebackgroundallowingre-processeddataﬁlesto beopenedandevaluatedinbatchesof50–80simultaneouslyin MassHunterQualitativeAnalysisSoftware(version10.0)(Agilent, SantaClara,CA,USA).

Thequalitativemethodusedinthere-processingwasbasedon thealgorithm “Find byformula” together witha librarysearch, bothusingtheHighResNPSsubsetPCDL.The_“Findbyformula_” searchleadtopositiveﬁndingsthatwerebasedonMS1spectral information.Thecriterionwasamasserrorlessthan5ppmanda

scoreabove 80 where thescoringwas taking themass match,

isotopespacingandisotopeabundanceintoaccount.Inthecase

when MS/MSspectrawere acquiredfor the precursor ion of a

detectedcompound, theseMS/MSspectrawerecompared with

those in thePCDL. The comparison was done both by reverse

search (the peaks in the PCDL are compared with theMS/MS

spectra)andbyforwardsearch(thepeaksintheMS/MSspectraare comparedwiththePCDL).Thethresholdlibrarymatchwassetto1 (ofmax.100)forbothforwardandreversescore.Asthemaximum

number of fragment ions per library spectrum was three, the

lowestresultingreversescoreofamatchwas33.

A ﬁlter in the software was applied in order to distinguish

compoundswithMS/MSspectra(category1)andwithoutMS/MS

spectra (category 2). Category 1 compounds found by the

algorithm “Find by Formula” could be evaluated further by

comparingtheacquiredMS/MSwiththelibraryspectrum.Ifthere

wasnoagreementbasedontheMS/MScomparisonthecompound

was considered a false positive. If there was a match, a visual

evaluation comparing the acquired spectrum with the library

spectrumwasundertakentoruleoutfalsepositivematchesdueto

fragments of low abundance e.g. from contaminants. The LOIs

estimatedforthecompoundsselectedinthevalidationappliesfor category1compounds.

Forcategory2compounds,noMS/MSdatahadbeenacquired andfragmentconﬁrmationcouldnotbedone.Thus,onlytheMS signalcouldbeusedtoevaluatethequalityoftheﬁndings.Without

the MS/MS spectra identiﬁcation parameter the number of

potential positives would have been large and included noisy signalsandbadpeakshapes.Apeakareathresholdof5104_was applied to limit the number of findings to investigate. Conse-quently, higher detection limits were expectedfor these com-poundscomparedtocategory1compounds.Inordernottomiss anyimportantfindings,amassaccuracylimitof10ppmandmass matchscoreabove80was_firstapplied(criteriona).Thiswastested with42randomdatafilesandgave74findings.Afterinvestigating the results and filtering out findings due to interferences and

backgroundsignal, onlycompounds withmass accuracy better

than5ppmandmassmatchscoreabove95wereleft.Thesetwo thresholdswereconsequentlyusedascriterionb.Finally,athird factorwasaddedtocriterionb,anRTrestrictionof1.5min,asthe compoundsinthegroupsunderinvestigationarehighlylikelyto eluteafterthistimeperiod(criterionc).Thenumberoffindingsin the42randomdatafilesasafunctionof criteriona,b orcare illustratedinFig.2.Criterionc(massaccuracybetterthan5ppm, massmatchscorehigherthan95 andRT1.5min.or more)was appliedforallcategory2compounds.Acompoundappearingin several data filesin the samebatch was consideredan isomer originatingfromthechemicalsusedorasendogenousmolecules withequaltheoreticalmasses.Theriskofacceptingfalsepositives ishigherforcategory2thanforcategory1_findings,especiallyif thresholdsandlimitsaresettoowide.

Anynewﬁndingwasfurtherevaluatedbycomparingacquired MS/MSspectrawithothersources(e.g.mzCloud1)oralternatively byanalysingareferencestandard, ifavailableatthelaboratory. DuetovariationsintheRTsoverthetimeperiodthesampleswere

Table1

NumberofnewcompoundsincludedintheHighResNPSsubsetPersonalCompoundDatabaseandLibrary(PCDL)groupedaccordingtodrugclassandsourceofdiagnostic fragmentions. Synthetic cannabinoids Synthetic opioids Designer benzodiazepines Total Libraryspectrabasedondiagnosticionsfromstandards 126 47 22 195 Libraryspectrabasedondiagnosticionsfromtheoreticalevaluation 116 40 0 156 Libraryspectrabasedondiagnosticionsfromseizures 4 2 4 10 LibraryspectrabasedondiagnosticionsfromRESPONSEprojecta

(seizuresortestpurchase on-line)

13 – – 13

Totalnumberofuniquecompounds(databaseentries) 259 89 26 374

a_A_European_project_named_Response_to_challenges_in_forensic_drug_analysis._{https://www.policija.si/apps/nﬂ_response_web/seznam.php}_.

Fig.1.Libraryspectrumofﬂubromazepam. 1

(5)

originallyanalyzed, RTdeviationsupto0.5min.weretolerated

when comparing these samples to reference standards. If a

consistencyin fragmentsor RTswas observed, theﬁndingwas reportedtoapersonwithaccesstotheoriginalcasereport.Ifa presumablynovelmoietywasidentiﬁedandareferencestandard

was available, this standard was analysed and RTsand MS/MS

spectrawerecompared. 3.Resultsanddiscussion

3.1.Validationoforiginalanalyticalmethod

3.1.1.Instrumentsensitivityandlimitofidentiﬁcation

The instrument response and RT variation over time was

expressedbyplottingthepeakareaandRToftheinternalreference standardsextractedfromonecalibratorfromeachanalyticalrun (Fig.S1insupplementarymaterial).Morphine-d3showedanRT

difference(maximumminimum)of0.28min.andameanpeak

areaof 2.7105_(standard _deviation_(SD)_1.3₁₀5_)._Codeine-d3 showed anRTdifferenceof 0.35min.and a mean peakareaof

4.4105 _(SD _1.6₁₀5_). _{Benzoylecgonine-d3} _showed _an _RT

difference of 0.32min. and a mean peak areaof 7.8105 _(SD 3.9105_)._Finally,_griseofulvin_showed_a_RT_difference_of_0.44_min. andameanpeakareaof2.8105_(SD_1.4₁₀5_)._The_peak_areas_of internalreferencestandardsinthedataﬁlesarenotonlyreﬂecting

the variation in instrument response but also variation in

extraction efﬁciencyand matrix effects over time. This gives a morerelevantexpressioncomparedtoadirectinjectionofaneat performancetestsample.

LOIswereestimated for a representativegroup ofsynthetic cannabinoids, synthetic opioids and designer benzodiazepines (Table 2). LOIs are unknown for new compounds but the

experiment indicated that synthetic cannabinoids could be

detectedifpresentaboveapproximately10–20ng/mL,synthetic opioidsabove1ng/mLanddesignerbenzodiazepinesabove10ng/

mL. Electrospray ionization is best suited for analysis of

compoundswithmedium-to-highpolaritybutisnotoptimalfor allcompounds[28].TheLOIsinTable2areonlyestimatesofthe instrumentsensitivitythroughtheacquisitionperiod.Asseenby theresultsfromtheinternalreferencestandards,thepeakareas varied during theperiod due to e.g. instrument condition and periodicmaintenance.HowthisinturnaffectedtheLOIsisdifﬁcult todetermine,asthevalueisnotonlyaresultofsignalintensity,but alsotheautomaticselectionofprecursorionsbasedontheDDA settings.Ifthecompoundstillisamongtheprecursorsselectedfor fragmentationitwillprobablybeidentiﬁed.Giventhepeakarea

threshold applied to detect category 2 substances, a higher

concentration must be present in order to detect them as

compared to category 1 substances. A review of the data ﬁles

from the LOI experiments shows that a peak area of 5104

generallycorrespondstotwo-orthreefoldtheconcentrationofthe

LOI of category 1 substances (see Table S2 in supplementary

material).Thesedataalsoindicatethatmassmatchscoreof95is achievedformostcompoundswhenapeakareaaround5104_is measured.

3.1.2.Recoveryandmatrixeffects

MajordifferenceswereobservedintheestimatedRE(%)ofthe syntheticcannabinoids,withvaluesrangingfrom32%(THJ-018)to 91%(AB-CHMINACA)(Table2).TheremainingcompoundshadREs

above82%.AllcompoundsshowedanMEbetween69%and127%

demonstrating that both ion-suppressionand ion-enhancement

occur.MEvalueswithrelativelylittledeviationfrom100%forthe studiedcompoundsindicatethatsevereionsuppressionisunlikely forothercompoundsinthesegroups.

3.2.Retrospectivedataﬁleanalysis

Atotalnumberof1314dataﬁles(242,252,273,242and305, respectively,fromtheyears2014to2018)wereprocessedwiththe newPCDL.Theretrospectiveanalysisrevealedsixnew_ﬁndingsof

category 1 in addition to two compounds (ﬂuorofentanyl and

cyclopropylfentanyl)thathadbeenreportedwhenthedatafiles wereprocessedwiththeoriginalmethod,but onlyafterseized materialhadbecomeavailable(Tables3and4).Inadditionthere were35possiblefindingsofcategory2(Table5)notreportedwhen thedatafileswereprocessedwiththeoriginalmethod.

3.2.1.Category1ﬁndings

Flubromazepamwasdetectedintwodataﬁlesfrom2015and 2016respectively.Therewasamassmatchscoreinbothdataﬁles higherthan95,amassaccuracybetterthan3.46ppmandanRT deviationoflessthan0.07min.Themassmatchcanbevisualized

bytheresemblance ofthespectrumof_{ﬂubromazepam}andthe

theoretical pattern indicated by the boxes in Fig. 3. The three diagnosticfragmentsinthelibraryspectrumwerealsofoundinthe MS/MSdataacquiredfromtheprecursorinthetwodataﬁles(see

Fig.4A).AnadditionalcomparisonoftheMS/MSspectrafromthe data ﬁleand theanalysisofa referencestandard showed good

Fig.2.Numberofcategory2ﬁndingsin42randomdataﬁlesasafunctionof criteriona(peakareathresholdof5104

,massaccuracylimitof10ppmandmass matchscoreabove80),criterionb(massaccuracylimitreducedto5ppmand massmatchscoreabove95)orcriterionc(massaccuracybetterthan5ppm,mass matchscorehigherthan95andRT1.5min.ormore).

Table2

Retentiontime(RT),limitofidentiﬁcation(LOI),recovery(RE)andmatrixeffect (ME) fora selectionof compoundsinthe threegroups of newpsychoactive substancesincludedinthepresentstudy.

Substance RT[min] LOI[ng/mL] RE[%] ME[%] Syntheticcannabinoids MDMB-CHMICA 14.0 10 68 97 AB-CHMINACA 11.9 20 91 107 BB-22 14.3 10 57 86 JWH-018 14.4 2 51 85 PB-22 13.8 10 68 89 THJ-018 14.8 10 32 69 Syntheticopioids Fentanyl 7.1 1 87 132 Remifentanil 5.4 1 94 123 Cyclopropylfentanyl 7.5 1 82 128 Para-ﬂuorofentanyl 7.2 0.5 88 124 Furanylfentanyl 7.3 0.5 100 124 Acetylfentanyl 6.0 1 100 127 Designerbenzodiazepines Deschloroetizolam 8.8 2 107 119 Diclazepam 10.7 5 87 110 Etizolam 9.3 2 110 121 Flubromazepam 9.1 10 110 72 Flubromazolam 8.5 5 113 121 Pyrazolam 6.4 10 114 122 Meclonazepam 9.3 10 110 105

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agreement also for additional fragment masses (see Fig. 4B). Flubromazepamwasﬁrstdescribedin1962andisahighlypotent andincompletelyevaluatedbenzodiazepinestructurallyrelatedto phenazepam[29,30].Flubromazepamstartedtoemergeinonline shops in Europe in 2012. In Norway it was detected in seized

material by the Norwegian National Criminal Investigation

(KRIPOS)fortheﬁrsttimein2013.

Phenibutwasdetectedinadataﬁlefrom2015andshoweda massmatchscorehigherthan85,amassaccuracyof1.63ppm andanRTdeviationof0.12min.comparedtoareferencestandard analysedin2018.EvaluationoftheRTovertimeshowedthat a

deviation up to 0.5min. could be expected due to change of

analyticalcolumnlotandtubing.Phenibutisaneuropsychotropic drugwithpossiblecognitionenhancingeffectsthatwasdiscovered andintroducedintoclinicalpracticeinthe1960sSovietUnion[31]. ThedrugiswidelyusedinRussiaandisclaimedtohavevarious clinicaleffects,e.g.torelievetensionandanxietyandtoimprove sleep. Phenibut can cause dependency. It is not scheduled or classiﬁedasamedicinaldruginNorwayandisnotforlegalsale. Privateimportisprohibitedbylaw.KRIPOSdidnotdetectphenibut in any cases before2019.Our laboratory reporteddetection of phenibutinseized materialand biologicalsamplesfor theﬁrst time in 2016, and it has since then been part of the routine analyticalrepertoireatourlaboratory.

Fluorofentanylwasdetectedinonedataﬁlefrom2016witha massmatchscorehigherthan97,amassaccuracyof0.21ppm andgoodagreementinthediagnosticions.Analysisofreference

materialshowedanRTdeviationoflessthan0.05min.Moreover,a compoundwithmolecularformulaC23H28N2Owasdetectedina dataﬁlefrom2018withmassmatchscorehigherthan96andmass accuracyof2.87ppm.Thediagnosticfragmentsofm/z105.0699

and 188.1434showedthatthecompound mostprobablywas a

fentanyl analogue and the software suggested either

cyclo-propylfentanyl,methacrylfentanylorcrotonylfentanyl.Thesethree

compounds share the same formula and diagnostic fragments.

Consequently,theyarenotpossibletodistinguishfromeachother basedoncategory1criteriaonly,butanalysisofreferencematerial

showed good RT agreement (deviation 0.01min.) with

cyclo-propylfentanyl.Infact,fluorofentanylandcyclopropylfentanylhad alreadybeenconfirmedbytargetedanalysisofthedatafilesbased

upon information from analysis of seizures from the scene

requestedby thepolice [32,33]. However, asthese compounds

would not have beendetected originally ifwe had not known

which substances to suspect, theyare included in thepresent material.

Identificationofflubromazepam,phenibut,fluorofentanyland

cyclopropylfentanyl (of category 1) was based on the mass

accuracyof themonoisotopicMS signal,presenceof diagnostic fragment ions and, finally, RT agreement. Fulfilment of these criteriagavethehighestlevelofcon_fidencethatcanbeachievedin aretrospectivereviewwhenre-analysisoftheactualspecimenis notpossible.Detectionandconfirmationofcompoundswith HR-MS can be divided in different levels of confidence based on informationavailablefromthedataacquisition,assuggestedby

Fig.3.MS1-spectrumofflubromazepamextractedfromadatafile(redlines)withtheoreticalisotopicpatternillustratedbytheblackboxes.(Forinterpretationofthe referencestocolourinthisfigurelegend,thereaderisreferredtothewebversionofthisarticle).

Fig.4.(A)AcquiredMS/MS-spectrumofﬂubromazepamwithdiagnosticfragmentsmarkedwithasterisk(atthetop),libraryspectrumfromPCDL(atthebottom)anda comparison(inthemiddle).(B)AcquiredMS/MS-spectrum(atthetop),fullMS/MS-spectrumfromaﬂubromazepamreferencestandard(atthebottom)andacomparison(in themiddle).

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Schymanskietal.[34].Inthatapproach,level5throughlevel1 requiresincreasinginformationfromtheMSsignaltodiagnostic fragmentsandRTs[34].Findingsofcategory1inourretrospective methodcanbecomparedtoa situationclosetolevel1.Level1 requiresconfirmationwithareferencestandard,whichwas the casewithournewfindings,butaslongasthesampleandstandard are notanalysed simultaneously, a definite confirmationis not achieved.

Inaretrospectiveapproachco-identificationofmetabolitescan furtherstrengthen theconfidenceof afinding.Searchesfor the majormetabolitesofthedetectedcompoundsweredoneinthe relevantdatafiles.Metabolitesfrompublishedinvivoandinvitro studieswereselected[29,35–37]. Neitherof themetabolites of

ﬂuorofentanyl were detected in the data ﬁle containing this

compound. In the data ﬁle containing cyclopropylfentanyl the N-dealkylatedmetaboliteandtwohydroxylatedmetaboliteswere detected.Themetabolitesofﬂubromazepam foundin literature

to be the most abundant (hydroxylated ﬂubromazepam and

debrominatedﬂubromazepam)werenotdetected in anyof the

two positive samples. The metabolism of phenibut has to our

knowledgenotbeenstudied,andnoputativetargetmetabolites havebeendescribedintheliterature.

Threeotherpositivecategory1ﬁndingscouldberefutedafter furtherinvestigation(Table4).FormethoxyacetylfentanyltheRT deviationcomparedwiththereferencestandardwassigniﬁcant,

indicating thatthecompound ratherwasanisomer of methox-yacetylfentanyl withsimilarfragmentationpatterns.Therewere nootherdescribed fentanylanalogues withidenticalmolecular formula.Thepresenceoffragmentsofm/z105.0699and188.1434 washoweverastrongindicatorthatthecompoundconsistedof thepiperidineandphenylmoietycharacteristictofentanylitself

as well as many fentanyl analogues. Metabolites of fentanyl

hydroxylated at the alkyl or phenetyl moeity have the same

monoisotopicmassasmethoxyacetylfentanylandthediagnostic fragments 105.0699 and 188.1434 will be the same (Fig. 5).

Fig.5.Fragmentationofhydroxyfentanyl(left)andmethoxyacetylfentanyl(right). Thesuperimposedareaindicatespositionofhydroxyl-group.

Table3

Newcompoundsfoundafterapplyingcategory1criteria,includingidentiﬁcationdataandcaseinformation. Compound(year) Molecular

formula Retentiontime sample/reference standard(Dmin) Mass match score Diagnostic fragment Mass (calculated) Mass accuracy [ppm] First reported inNorway Caseinformation Flubromazepam (2015)

C15H10BrFN2O 9.08/9.15(0.07) 95.55 314.0049 3.46 2013a Male,approx.30yrs.old.Historyofdrug

abuse,founddeadafterdruguse. Ethanol,amphetamine,metamphetamine, methylenedioxymetamphetamine, metylenedioxyamphetamine,diazepam, desmetyldiazepam,7-aminoclonazepam, alprazolam,pregabalin,mephedrone, buprenorphine,norbuprenorphineand gamma-hydroxybutaratefoundinblood. C14H11FN2 226.0901 3.29

C7H7BrN 183.9756 5.25

C14H11N2FBr 305.0084 7.10

Phenibutc

(2015)

C10H13NO2 1.53/1.65(0.12) 85.36 180.1019 1.63 2016b Samesubjectasabove.

C9H9 117.0699 1.81

C10H9O 145.0648 16.67

Flubromazepam (2016)

C15H10BrFN2O 9.21/9.15(0.06) 97.45 333.0033 0.24 2013a Female,approx.50yrs.old.Historyofdrug

abuse,founddeadathome.

Ethanol,paracetamol,gabapentin,pregabalin, tramadol,O-desmethyltramadol,

amitriptyline,nortriptyline,sertralineand chlorprothixenefoundinblood. C14H11FN2 226.0901 3.48

C7H7BrN 183.9756 0.95

C14H11N2FBr 305.0084 16.4

Fluorofentanyld

(2016)

C22H27FN2O 7.18/7.17(0.01) 97.73 355.2180 0.21 2016b Male,approx.20yrs.old.Founddeadathome

withdrugparaphernalia. 7-aminoclonazepam,diazepam, desmethyldiazepam,alprazolam, tetrahydrocannabinoland gamma-hydroxybutyratefoundinblood. C13H18N 188.1434 5.42

C8H9 105.0699 3.56

C14H17FNO 234.1289 9.92

Cyclopropylfentanyld

(2018)

C23H28N2O 7.46/7.47(0.01) 96.53 349.2274 2.87 2017a Male,approx.30yrs.old.Founddeadathome

withpillsonsite.

Morphine,morphine-3-glucuronide, morphine-6-glucuronide,buprenorphine, norbuprenorphine,pregabalin,amphetamine, methylenedioxymetamphetamine,

metyhlenedioxyamphetamine,

benzoylecgonine,7-aminoclonazepamand tetrahydrocannabinolfoundinblood. C13H18N 188.1434 2.52

C8H9 105.0699 1.05

C15H18NO 228.1383 4.46

a

DetectedinseizedmaterialbytheNorwegianNationalCriminalInvestigation.

b _Detected_in_seized_material_by_our_department. c_{4-amino-3-phenylbutyric}_acid.

d

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Fentanylwasreportedintheoriginalanalysisofthesample,which explainsthepresenceofametabolite.Thus,itcouldbeconcluded thattheﬁndingwascausedbyfentanylintake.

Category1findingsofJWH-167andtilidineweredetectedin onedatafileeach,from2014and2015,respectively.Thefragments intheMS/MSspectrawereinrelativelygoodagreementwiththe diagnosticfragmentsfromthelibraryspectrum,andinaddition them/zClouddatabasewasconsultedandshowedagreementwith oneadditional fragment. Reference standards wereacquired to compareRTsandsignificantRTdifferences clearlyshowedthat neitherJWH-167nortilidinewerepresent.Theseexamplesoffalse positiveresultsillustratetheimportanceofhavingaccesstothe referencesubstanceinordertocheckRTconformity.

3.2.2.Category2ﬁndings

Atotalof35possiblecategory2findingswastheresultwhen applyingcriterionc(betterthan5ppmmassaccuracy,massmatch scorehigherthan95andRT1.5min.orlater).Theinitialfindings arepresentedinTable5.AfurtherevaluationofRT,signal-to-noise ratioandchromatographicpeakshapeforeveryfindingwasdone. ThemetaboliteAB-FUBINACAM3(#1315),carfentanil(#17and 18),tilidine(#35)andthreeoffourfindingsofphenibut(#31,32

and 34) could be disproved due to large RT deviations from

reference standards. Based on the RTs of other synthetic

cannabinoidsanalysedwiththesamechromatographicconditions (seeTable2)_ﬁndingsofsyntheticcannabinoidswithRTslessthan 5min.wereregardedashighlyunlikelyandremovedfromthelist. Thiswasthecasefor5-ﬂuoro-PY-PINACA(#3and4),5-

ﬂuoro-3,5-AB-PFUPPYCA (#5), AB-BICA (#9 and 10) and MA-CHMINACA

(#20).5-ﬂuoro-AB-PINACAN-(4-hydroxypentyl)(#2),a

metabo-lite and presumably more polar compound than its parent

substance,is likely to have a shorter RT. Still, it will probably noteluteasearlyas3.6min.Asimilarlimitof4min.wasappliedon the synthetic opioids which lead to the rejection of 3-ﬂuoro methoxyacetylfentanyl(orocfentanyl)(#1)andtwoﬁndingsof N-methylnorcarfentanil(#25and26).Thesignal-to-noiseratiowas3

or less for AB-CHMINACA 3-carboxylindazol (#11 and 12),

N-methylnorcarfentanil(#27)andPB-223-carboxyindole(#30).The

initial _ﬁnding determined as benzyl carfentanil (#16) was

disprovedduetopoorpeakshape.Acategory2compoundfound inoneormoredatafiles,andalsofoundwiththesameRTinother datafileshavingMS/MSspectraacquiredbutnolibrarymatch,was likewiserejected.Onesuchexamplewasohmefentanyl,whichwas foundintwodatafileswithRTsof7.8min.Theioncouldalsobe

found in other data ﬁles withthe same RT but with acquired

MS/MS spectranot in agreement with thePCDL. This strongly

indicatedthatthetwoﬁndingsofohmefentanyl(#28and29)were falsepositives.Thesamewasthecasewithpresumableﬁndingsof AB-FUBINACA(#6–8),JWH-200analog1(orA-796260)(#19)and methoxyacetylfentanyl(#21–24).

Thus,afterreviewingthe35suggestedcategory2findings,only onefindingofphenibut(#33)remained.AsnoMS/MSspectrawere availableforlibrarycomparison,thisfindingcould,however,not

be conﬁrmed with the same degree of conﬁdence as those of

category1.

3.3.Strengthsandweaknesses

ThePCDLconstructedinthisstudyisbasedondataacquiredon instrumentsfromdifferentmanufacturersandbasedondifferent principles.Apreviousstudyhasshownthatlibrariesconstructed

from data acquired on either Orbitrap or QTOF can be used

interchangeably by both instruments providing that suitable

collisionenergiesareapplied[38,39].Anessentialfeatureofthe

PCDL is the mass accuracy of the diagnostic fragments. In

HighResNPS the masses of the fragments are added by either

typing theformula, selecting thecorrect formula from a drop-downlistofcommonfragmentsortypingthetheoreticalmassof the acquiredfragment. This ensures that masserrors from the acquisitionarenottransferredtothedatabase.Asecondimportant settingisthechoiceofcollisionenergyappliedwhenacquiringthe diagnosticfragmentsthatareaddedtothedatabase.Thecollision energyappliedcan eitherbediscrete (e.g.10,20 and40eV) or ramped,providingacombinedresult.Informationonthechoiceof strategy used in the individual entry was not present in the

database. In the Auto MS/MS method used in this study, the

collision energy was a voltage correlated to the mass of the precursor. Potentially this can result in differences in relative

abundancewhencomparingalibraryspectrumandanacquired

MS/MS spectrum. However, the settings in the retrospective

reprocessing algorithm ensure a hit even if only one of the

diagnosticfragmentionscouldbefoundintheacquiredspectrum. Theriskoffalsenegativesampleswillalwaysbepresentwhen searchingforcompoundsthat havenotbeensubjecttospeciﬁc evaluation of LOI, which is the case for the majority of the compoundsinthePCDL.Inaddition,theinstrumentresponsehas beenshowntoﬂuctuatetosomeextentduringtheperiodofdata acquisition. Due totherelatively highLOIs and low recoveries

Table4

Newcompoundsfoundafterapplyingcategory1criteria,butrefutedas“falsepositive”ﬁndings. Compound(year) Molecular

formula Retentiontime(RT) sample/reference standard(Dmin) Mass match score Diagnostic fragment Mass (calculated) Mass accuracy [ppm] Comment Methoxyacetylfentanyl (2016)

C22H28N2O2 5.13–5.75(0.62) 91.36 353.2224 0.20 RTnotinagreementwithreferencestandard.

Monoisotopicmassanddiagnosticfragmentssuggest fentanylhydroxylatedatthealkylorphenetylmoiety C13H18N 188.1434 3.8

C8H9 105.0699 17.23

C9H12N 134.0964 Notfound

JWH-167 (2014)

C21H23NO 11.82–13.86(2.06) 95.32 306.1852 0.90 RTnotinagreementwithreferencestandard

C14H16NO 214.1226 7.94

C7H7 91.0542 9.63

C13H18Na 188.1434 11.05

Tilidine (2015)

C17H23NO2 9.24–5.56(3.28) 90.87 274.1802 1.65 RTnotinagreementwithreferencestandard

C15H17O 229.1223 Notfound

C12H11 155.0855 5.88

C7H7 91.0542 45.1 a

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amongthesynthetic cannabinoidsin thevalidation,therisk of falsenegativesappearstobemorelikelyinthisgroup.Itshould alsobeemphasisedthatthetwolargeNPSgroupscathinonesand phenetylamineswereleftoutofthisstudyinordertolimit the extentofinvestigatedcompounds.

Applying the method on our data ﬁles has shown that

identification of ions that were not selected for fragmentation (category2) clearly requires a manual re-evaluation. Thelist of category2findingswassignificantlylongerthancategory1findings, butstill35potentialpositivesoutof1314datafilesisamanageably lownumber.Thepeakareathresholdof5104

wasimportantto keepthenumberofpotentialcategory2findingslow,butwillatthe sametimeresultinhigherdetectionlimitsforthesecompounds.All exceptoneofthepotentialcategory2findingscouldbedisproved afteracarefulevaluationoftheRTsandsignal-to-noiseratiosinthe chromatogram.Theneed fora manual evaluationof category2 findingsisalimitationoftheDDAapproach.IfDIAhadbeenused

therewouldhavebeenfewpresumable_ﬁndingswheretheMS1

signalwasdetectedbutnofragmentionswereavailable.DIA,onthe otherhand,islimitedbyco-elutingcompoundsbeingfragmentedat thesametimeresultingincomplicatedhighenergyspectra.The patterncanbeevenmorecomplexbyco-elutingcompoundssharing thesamefragments.DDAgeneratesMS/MSspectrafromaknown precursorwhichminimizestheriskof“contaminating”fragments fromco-elutingcompounds.Ontheotherhand,thereisalimittothe

number of co-eluting precursors which can be isolated and

fragmented.Themanycategory2findingsalsoshowtheimportance ofhavingthefragmentationinformationinordertodoanefficient retrospectiveanalysis.Re-analysisofcasesampleswasnotpossible inthisstudyduetoethicalrestrictions.Consequently,the presum-able category 2 finding of phenibut could not be confirmed. In realforensiccaseworkthesamplecouldhavebeenre-analysed

with a targeted MS/MS method where the precursor ion of

phenibutisprioritizedforfragmentationexperiments.Ifamatch withalibraryspectrum wasachievedtheﬁndingwouldhavebeenof category1.

Table5

The35suggestedﬁndingsafterapplyingcategory2criteria,withretentiontime(RT)andanevaluationofwhethertheidentiﬁcationwascorrectornotbasedontheRTand thesignal-to-noiseratio(S/N).

Suggested ﬁnding#

Compound Year RT[min] Correctidentiﬁcation? 1 3-ﬂuoro-methoxyacetylfentanyl(or

ocfentanyl)

2015 3.77 No,fentanylanaloguewithRTunder4minisnotlikely 2 5-fluoro-AB-PINACAN-(4-hydroxypentyl) 2018 3.59 No,syntheticcannabinoidwithRTunder5minisnotlikely 3 5-fluoro-PY-PINACA 2016 2.58 No,syntheticcannabinoidwithRTunder5minisnotlikely 4 5-fluoro-PY-PINACA 2018 2.63 No,syntheticcannabinoidwithRTunder5minisnotlikely 5 5-fluoro-3,5-AB-PFUPPYCA 2014 3.52 No,syntheticcannabinoidwithRTunder5minisnotlikely

6 AB-FUBINACA 2016 10.30 No,referencestandardshowedRTof10.3min.Alargenumberofadditionaldataﬁles containthesameionwithsameRTbutwithfragmentionsnotinagreementwith libraryspectra

9 AB-BICA 2014 3.13 No,syntheticcannabinoidwithRTunder5minisnotlikely 10 AB-BICA 2017 3.79 No,syntheticcannabinoidwithRTunder5minisnotlikely 11 AB-CHMINACA3-carboxylindazol 2014 4.12 No,chromatogramshowsS/N<3

12 AB-CHMINACA3-carboxylindazol 2014 3.92 No,chromatogramshowsS/N<3

13 AB-FUBINACAM3 2014 4.89 No,referencestandardshowedRTof11.0min 14 AB-FUBINACAM3 2015 4.77 No,referencestandardshowedRTof11.0min 15 AB-FUBINACAM3 2017 3.54 No,referencestandardshowedRTof11.0min 16 Benzylcarfentanil 2015 10.91 No,poorchromatography

17 Carfentanil 2015 11.87 No,referencestandardshowedRTof7.7min 18 Carfentanil 2016 11.77 No,referencestandardshowedRTof7.7min

19 JWH-200analog1(orA-796260) 2017 6.15 No,otherdataﬁlescontainthesameionwithsameRTbutwithfragmentionsnotin agreementwithlibrary

20 MA-CHMINACA 2014 2.86 No,syntheticcannabinoidwithRTunder5minisnotlikely

21 Methoxyacetylfentanyl 2014 6.34 No,referencestandardshowedRTof5.7min.Otherdataﬁlescontainthesameion withsameRTbutwithfragmentionsnotinagreementwithlibraryspectra 22 Methoxyacetylfentanyl 2018 6.59 No,referencestandardshowedRTof5.7min.Otherdataﬁlescontainthesameion

withsameRTbutwithfragmentionsnotinagreementwithlibraryspectra 23 Methoxyacetylfentanyl 2018 6.58 No,referencestandardshowedRTof5.7min.Otherdataﬁlescontainthesameion

withsameRTbutwithfragmentionsnotinagreementwithlibraryspectra 24 Methoxyacetylfentanyl 2018 6.59 No,referencestandardshowedRTof5.7min.Otherdataﬁlescontainthesameion

withsameRTbutwithfragmentionsnotinagreementwithlibraryspectra 25 N-Methylnorcarfentanil 2014 2.90 No,fentanylanaloguewithRTunder4minisnotlikely

26 N-Methylnorcarfentanil 2016 2.72 No,fentanylanaloguewithRTunder4minisnotlikely 27 N-Methylnorcarfentanil 2018 6.20 No,chromatogramshowsS/N<3

28 Ohmefentanyl 2018 7.84 No,otherdataﬁlescontainthesameionwithsameRTbutwithfragmentionsnotin agreementwithlibraryspectra

29 Ohmefentanyl 2018 7.85 No,otherdataﬁlescontainthesameionwithsameRTbutwithfragmentionsnotin agreementwithlibraryspectra

30 PB-223-carboxyindole 2016 6.61 No,chromatogramshowsS/N3.6 31 Phenibut 2014 6.39 No,referencestandardshowedRTof1.65min 32 Phenibut 2014 2.94 No,referencestandardshowedRTof1.65min

33 Phenibut 2016 1.58 Yes,probablysincereferencestandardshowedRTof1.65min 34 Phenibut 2016 4.11 No,referencestandardshowedRTof1.65min

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4.Conclusion

DataﬁlesfromUHPLC-QTOF-MSanalysisof1314forensicpost

mortemsamplesfromtheperiod 2014to2018were

retrospec-tivelyre-evaluated.There-evaluationwasperformedusingaPCDL

withcompounds within the groups of synthetic cannabinoids,

syntheticopioidsanddesignerbenzodiazepines.Intotal,fivenew substanceswereidentifiedwiththehighestdegreeofconfidence possiblewitharetrospectiveapproach.Theidentificationreliedon availableMS/MSspectrafromtheacquisitionandmatchingwith thediagnosticfragmentionsinthelibraryspectrum.Inaddition, RTagreementwithareferencesubstancewasdecisiveinorderto filteroutfalsepositives.Thenumberofnewfindingswerelower thanexpectedandmainlyoriginatedfromthefirsthalfofthetime periodinvestigated,indicatingthatourlaboratoryhasbeenableto keeptheanalyticallibraryfairlyuptodate.

Itisimportant toemphasisethatnew andhighlypotentdrugslike fluorofentanylandcyclopropylfentanylcanescapeattentionifnot specificallysearchedfor.Detectioninbiologicalsamplesisinmany casesdependentoninformationaboutthelikelydrugcandidates -either from indirect sources such as labelling on seized drug packages,whichmaybeimprecise,orpreferablyfromdirectanalysis ofthe ingested substance. In Norway, biological samples are analysed bytoxicologylaboratories,whereasimpoundeddrugsaresubmitted foranalysisatacentralpolicelaboratory.Therearenoorganizational connectionsortraditionsforexchangeofinformationbetweenthese twotypesofinstitutions.Ifithadnotbeenfortheavailabilityof seizures in the two cases involving fluorofentanyl and

cyclo-propylfentanyl these would not have been detected with our

originalscreeningmethod.

The presented method proved to be a relatively easy and

convenientapproachtosearchfornewcompoundsretrospectively. Theuseisnotlimitedtoretrospectiveanalysisandcaneasilybe appliedasasupplementto thestandard screeningmethodwithlittle extraeffort,especiallywhentheroutinescreeningworkﬂowgivesa negativeresult but thecircumstances suggesta more thorough investigation.ThePCDLcanbeupdatedatregulartimeintervalsor

whenimportantcompoundsareaddedtoHighResNPS.com.

Note

AfterthecompletionofthisstudyaPublicCompoundDatabase andLibraryversionofthecompletehighresnpsdatabasehasbeen madeavailablefordownloadfromthewebsitehighresnps.com.

CRediTauthorshipcontributionstatement

Per Ole M. Gundersen: Conceptualization, Methodology,

Formalanalysis,Investigation,Writing-originaldraft,Writing -review & editing. Sebastian Broecker: Software, Methodology, Writing-review&editing.LarsSlørdal:Resources,Datacuration, Writing - review & editing. Olav Spigset: Conceptualization, Supervision, Resources, Writing - review & editing, Project administration.MartinJosefsson:Conceptualization,Supervision, Methodology,Writing-review&editing.

AppendixA.Supplementarydata

Supplementarymaterialrelatedtothisarticlecanbefound,inthe onlineversion,athttps://doi.org/10.1016/j.forsciint.2020.110274. References

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