Classification of condom lubricants in cyanoacrylate treated fingerprints by desorption electrospray ionization mass spectrometry

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Amsterdam University of Applied Sciences

Classification of condom lubricants in cyanoacrylate treated fingerprints by

desorption electrospray ionization mass spectrometry

van Helmond, Ward; Begieneman, Mark P.V.; Kniest, Roos; de Puit, Marcel

DOI

10.1016/j.forsciint.2019.110005

Publication date

2019

Document Version

Final published version

Published in

Forensic Science International

License

CC BY-NC-ND

Link to publication

Citation for published version (APA):

van Helmond, W., Begieneman, M. P. V., Kniest, R., & de Puit, M. (2019). Classification of

condom lubricants in cyanoacrylate treated fingerprints by desorption electrospray ionization

mass spectrometry. Forensic Science International, 305, [110005].

https://doi.org/10.1016/j.forsciint.2019.110005

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Classi

ﬁcation

of

condom

lubricants

in

cyanoacrylate

treated

ﬁngerprints

by

desorption

electrospray

ionization

mass

spectrometry

Ward

van

Helmond

a,b,c,

*

,1

,

Mark

P.V.

Begieneman

a,1

,

Roos

Kniest

a

,

Marcel

de

Puit

a,c,

*

a

NetherlandsForensicInstitute,DigitalTechnologyandBiometrics,LaanvanYpenburg6,2497GB,DenHaag,theNetherlands

b

AmsterdamUniversityofAppliedSciences,ForensicScience,Weesperzijde190,1097DZ,Amsterdam,theNetherlands

c

DelftUniversityofTechnology,FacultyofAppliedSciences,DepartmentofChemicalEngineering,VanderMaasweg9,2629HZ,Delft,theNetherlands

ARTICLE INFO Articlehistory: Received6August2019

Receivedinrevisedform16October2019 Accepted21October2019

Availableonline23October2019 Keywords:

MassSpectrometryImaging DESI-MSI

Polydimethylsiloxane Polyethyleneglycol Principalcomponentanalysis Lineardiscriminantanalysis

ABSTRACT

Tracesofcondomlubricantsinfingerprintscanbevaluableinformationincasesofsexualassault.Ideally, notonlyconfirmationofthepresenceofthecondombutalsodeterminationofthetypeofcondombrand usedcanberetrieved.Previousstudieshaveshowntobeabletoretrieveinformationaboutthecondom brandandtypefromfingerprintscontaininglubricantsusingvariousanalyticaltechniques.However,in practice fingerprintsoftenappearlatentand needtobe detected first,whichis oftenachievedby cyanoacrylate fuming. In this study, we developed a desorption electrospray ionization mass spectrometry (DESI-MS) method which, combined with principal component analysis and linear discriminantanalysis(PCA-LDA),allowsforhighaccuracyclassificationofcondombrandsandtypesfrom fingerprints containing condom lubricant traces. The developed method is compatible with cyanoacrylate(CA)fuming.Wecollectedandanalyzedarepresentativedataset fortheNetherlands comprising32differentcondoms.Distinctivelubricantcomponentssuchaspolyethyleneglycol(PEG), polydimethylsiloxane(PDMS),octoxynol-9andnonoxynol-9werereadilydetectedusingtheDESI-MS method.Basedontheanalysisoflubricantspots,a99.0%classificationaccuracywasachieved.When analyzinglubricantcontainingfingerprints,anoverallaccuracyof90.9%wasobtained.Fullchemical imagescouldbegeneratedfromfingerprints,showingthedistributionoflubricantcomponentssuchas PEGandPDMSthroughoutthefingerprint,whilestillallowingforclassification.Thedevelopedmethod showspotentialforthedevelopmentofDESI-MSbasedanalysesofCAtreatedexogenouscompounds fromfingerprintsforuseinforensicscience.

1.Introduction

Sexual assault is a major health problem and a violationof human rights [1]. When the identity of the perpetrator is unknown,themostimportanttracesin sexualassaultcasesare oftenofbiologicalorigin,suchasblood,semen,salivaandhair.This isduetothefactthatDNAcanberetrievedand analyzedfrom thesetraces,allowingfortheidenti_ﬁcationofpossiblecriminals. Nowadays,biologicalmaterialbecomesincreasinglyhardertoﬁnd incases ofsexual assault,potentiallybecauseawarenessof the importance ofDNA in forensictechniques and evidence in the justicesystem is growing among criminals [2]. Because of this

awareness, as wellas alertnessto sexual transmitted diseases, criminalsarebecomingmorevigilantinleavingbiologicaltraces andtheuseofcondomsinsexualassaultcaseshasincreasedover thepastdecades[3–6].Othertypesoftraceevidencemaythusbe requiredtoestablisha linkbetweenvictimandcriminal.Inthe past,studieshaveshownthatacondomcanbeacriticalpieceof evidenceinsexual assaultcases[4,7,8]. Theanalysisofcondom derived traces can thus be of signi_ﬁcant associative evidential importance.Ideally,notonlyconﬁrmationofthepresenceofthe condombutalsodeterminationofthetypeofcondombrandused canberetrieved.

In thelast decades several studies have beenperformed to develop methods to detect traces of condoms. Most of these studiesfocusedonthelubricantsthatareaddedtothecondomsby manufacturers.Indeed,primarycomponentsoflubricantssuchas polydimethylsiloxane(PDMS)andpolyethyleneglycol(PEG)were foundto bedetectable bydesorption chemical ionizationmass spectrometry[9],pyrolysisgaschromatographymass spectrome-try(pyGC-MS),GC–MS[10],Ramanspectroscopy[11]andFourier

* Correspondingauthorsat:NetherlandsForensicInstitute,DigitalTechnology andBiometrics,LaanvanYpenburg6,2497GB,DenHaag,theNetherlands.

E-mailaddresses:w.van.helmond@hva.nl(W.vanHelmond),m.de.puit@nﬁ.nl

(M.dePuit).

1

Theseauthorscontributedequally.

http://dx.doi.org/10.1016/j.forsciint.2019.110005

ForensicScienceInternational305(2019)110005

ContentslistsavailableatScienceDirect

Forensic

Science

International

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transforminfrared spectroscopy (FT-IR) [9,12]. Another specific componentincaseofspermicidecontainingcondoms, nonoxynol-9(N9),couldalsobeidentifiedbyFT-IR[9],GC–MS[13]andliquid chromatography mass spectrometry (LC–MS) [12]. Multiple studiesexamined thepossibilitytodiscriminatedifferenttypes ofcondoms.Maynardetal.describedatwo-stepmethodusing FT-IRasafirstscreeningtool,followedbyeitherGC–MS,pyGC-MSor LC–MS as a confirmation method, enabling them to uniquely identify 11 types of condoms [12]. Burger et al. showed that capillaryelectrophoresesmayalsobeapromisingtechniquefor classifying both condom and personal lubricants, although it remainedunclearwhichdiscriminatingconstituentswereusedin theanalysis[14].

However,mostoftheseanalytical techniquesrequiresample preparationand/orextractionwhichmaybetimeconsumingand resultinlossoftheinitialtraceevidence.Additionally,themethod itselfmayalsolimittheamountofinformationretrievedfromthe sample. For instance, analysisof silicone lubricants by GC–MS requirespyrolysisofthelubricant[10],thatcancausedegradation ofminorcomponents.Theseminorcomponentswerefoundtobe an important differentiating factor in distinguishing sexual lubricantsandpersonalhygieneproducts,whichcontainsimilar majorcomponentssuchasPDMSandPEG[15].Also,preservation oftheoriginalevidentialtracecanbeofgreatinterestinforensic science. In this respect, the use of ambient ionization mass spectrometrytechniquesismorefavorable.Apopulartechnique thathasbeenusedinrecentyearsisdirectanalysisinrealtime (DART)MS,thathasbeenshowntobeaveryeffectivetoolforthe detectionofboth themajorand minorcomponentsof condom lubricants,withouttheneedtoextensivelypreparethesampleor potentiallossofevidence[16–20].Furthermore,DARTanalysisis highlyeffectiveindiscriminatinglubricants.Baumgartenetal.and Maric et al. successfully discriminated condom and personal lubricantsusing DART-Time-of-Flight (TOF) MSanalysis[19,18]. Using a DART-High Resolution MS (HR-MS) analysistechnique, Coonetal.couldrapidlygeneratediagnosticchemicalﬁngerprint signatures of 110 condoms, enabling them to discriminate condomsof16differentbrands[20].However,adisadvantageof DARTanalysisisthatthistechniqueisunabletoretrievespatial chemical information from the samples, such as ﬁngerprints containinglubricants.

Lubricatedfingerprintsarelikelytobefoundatacrimesceneof sexualassault, ashandlingof a condomwill transfertheouter coating of the condom onto the perpetrator’s fingerprints, potentiallyleaving condomlubricant contaminated fingerprints behind[21,22].Detectionofalubricantfromafingerprintfoundat asexualassaultscene,wouldgreatlyincreasethestrengthofthe evidence,asitnotonlyestablishescontactwithacondombutalso indicatesthepresence of thecriminal atthe crimescene [21]. Bradshaw et al. developed a method for the visualization of condomlubricantwithina_fingerprintbymappingthe_fingerprint ridgepattern using Matrix AssistedLaser Desorption/Ionization Mass Spectrometry Imaging (MALDI-MSI) [21]. In a follow-up studytheyshowedthatdifferentiationof6differentcondomsin lubricated fingerprints was possible using MALDI-MSI, Raman spectroscopyandATR-FTIRimaginginasynergisticmanner[22]. BesidesMALDI-MSI,desorptionelectrosprayionization(DESI)MSI hasalso beenshowntobea powerfulanalytical tool. Whereas MALDIoffersexcellentspatialresolution20

m

m,oneofthemain disadvantagesisthe necessitytoapplymatrix solutiontoyour sample[23].Applyingthematrixsolutionintoolargedropletswill bedetrimentalforthespatial resolutionas diffusionwill occur withinthesedroplets.OneofthemainadvantagesofDESI-MSIis thatsamplesrequire nosamplepreparation andcan bereadily analyzed.UsingDESI,typicallyspatialresolutionsof100

m

mcan beachieved[23].However,thechoiceoftheelectrospraysolvent

compositionisessential,astheinteractionbetweenthe electro-sprayandthesurfacegreatlyinfluencesthesensitivityandspatial resolution [24]. Mirabelliet al. wereable togeneratechemical images of latent lubricated fingerprints deposited on different surfaces and of different ages using DESI-MS [25]. The data acquired from DESI-MS analysis of lubricated fingerprints, combinedwithsupervisedpatternrecognitionstatisticalanalysis (lineardiscriminantanalysis(LDA)andsoftindependentmodeling ofclassanalogy(SIMCA)),enabledMirabellietal.todistinguish10 differentcondomtypeswitha94%predictionabilityforbothLDA andSIMCA[26].

However,_{fingerprints}inpracticeoftenneedtobedetected_first, as they appear latent. One of the most used visualization techniques for latent fingerprints on non-porous substrates is cyanoacrylate (CA) fuming [27,28]. Fingerprints from a sexual assault scene that have been analyzed at a forensic lab, are potentiallytreatedwithCA.Inthisregard,theaimofourstudywas todevelopamethodtoanalyzelubricatedfingerprints,whichwas compatible with CA fuming and able to differentiate between differenttypesofcondoms.Thedevelopedmethod,basedon DESI-MSanalysis,is capableofgeneratingchemical images,mapping common lubricant components in a lubricated fingerprint. Additionally, using this method, combined with a statistical approach, PCA followed by LDA, we were able to differentiate between32typesofcondomsfrom21differentbrands.

2.Materials&methods 2.1.Materials

UPLC-gradeacetonitrileandformicacidwerepurchasedfrom Biosolve(Valkenswaard,Netherlands).UPLC-grademethanolwas purchased at Merck (Darmstadt, Germany). Cyanoacrylate was purchasedfromBVDA(Haarlem, Netherlands).Microscopeglass slides were purchased from Thermo Fischer Scientiﬁc (Breda, Netherlands). 24-wells slides were purchased from Prosolia (Indianapolis, USA). Reference masses purine (5mM) and hex-akis(1H,1H, 3H-tetraﬂuoropropoxy)phosphazine)(HP-0921, 2.5 mM)werepurchasedfromAgilentTechnologies(SantaClara,USA). Mitramicrosamplertips(10

m

L)werepurchased fromNeoteryx (Torrance,USA).Arangeof32differentcondomswerepurchased atonlinepharmacyandcondomwebsites(Table1).

2.2.Lubricantsamples

Lubricantofeachcondomwascollectedbyswipingtheinterior ofthecondompackageandbothsidesofthecondomwitha10

m

L microsampleruntilsaturation.Themicrosamplerisavolumetric absorptionmicrosamplingdevice(VAMS),thatonlyabsorbs10

m

l of sample. Because of this, it provides more control over the amountoflubricanttobesampled,incomparisontocottonswabs, that absorbed too much lubricant. The condom lubricant was carefullytransferredontothe24-wellsslidebyslightlytouching each well once with the lubricated microsampler tip. Each lubricantwas spotted 12times (n=12) onseparate wells. Asa control,eachsamplewellwasfollowedbyablankwell.Slideswere left to dry for at least 1h at room temperature (RT). Next, cyanoacrylate(CA,0.5gheatedto120C)fumingwasperformed onallslides,inaMVC1000fumingsystem(FosterandFreeman LTD,Worcestershire,UK)for10minat80%humidity.Slideswere thenlefttodryovernightatRTbeforeanalysis.

2.3.Fingerprintsamples

Fingerprintsweredonatedvoluntarilybya femaleandmale donor, after giving informed consent. No ethical approval was

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obtainedasthematerialwasgatheredinanoninvasivemanner anddidnotinfringeonanyprivacyofthedonors.Allexperiments werecarriedoutfollowinginstitutionalguidelinesandaccording torelevantlaws.Allcondoms(n=32)werehandledbyeachdonor (n=2).Aftertouchingthecondomandtheinsideofthepackaging, thelubricantwasdistributedoverthefinger.After5minofdrying, fingerprintsofeachcondomlubricantweredepositedon micro-scopeslides.Fromeachdonor,ablankfingerprintwasusedasa control(n=2).Slideswerelefttodryforatleast1hatRTfollowed byCAfuming,asdescribedabove.Fingerprintswerethenleftto dryovernightatRT.

2.4.DESI-Q-TOFMS

Desorptionelectrosprayionizationmass spectrometry (DESI-MS)datawereacquiredusinganAgilenttechnologies(SantaClara, USA)6530quadrupoletime-of-ﬂight(TOF) MSequippedwitha Prosolia(Indianapolis,USA)2D-DESI.24-wellssampleslideswere analyzedindwellmodeusingpositivepolaritywiththefollowing parameters:sprayvoltage,5kV;nitrogensheathgaspressure,6.0 bar;dryinggasﬂow,8L/minute,sourcegastemperature,300C; acquisition time, 200ms; mass range, m/z 100–1200; inlet-to-surface distance, 1mm and tip-to-surface distance, 3mm. Combinationsofseveralsprayincidentangles(52,45 and35) and tip-to-inletdistances(4, 5,6, and8mm)weretested.Best results were achieved with an angle of 45 and a tip-to-inlet distanceof6mm.Alargertip-inletdistanceledtodecreased carry-over,alsodescribedbyMirabellietal.[26].Adwelltimeof20swas used,withapost-acquire-delaytimeof30sinbetweenthewells. Tofurtheravoidcarry-overafteranalysisofeachsamplewell,the nextblankwellwasdwelledfor5minbeforemeasuringthenext sample.Additionally,theMS-inletwascleanedafteranalysisof3– 4slidestoavoidcarry-over,asalsoindicatedbyMirabellietal.[26]. Different spraying solvents were tested, namely a mixture of

acetonitrile and water(90:10 v/v),acetonitrile, methanol anda mixtureofmethanolwithwater(90:10v/v).Allsolventscontained 0.4% formic acid, 0.02mM Purine and 0.025mMHP-0921. Best result were achieved witha mixture of acetonitrileand water (90:10v/v)whensprayedataconstantvolumetricﬂowrateof3

m

L/minute, delivered by a syringe pump (Fusion 100, Chemyx, Stafford, USA).The64lubricatedand 2blankﬁngerprintswere analyzedusingthesamesettings,butinsteadofdwelling,5scans of3mmweremeasuredwithineachﬁngerprint,usinga150

m

m/ secondscanrate(totalingto20s)andastepsizeof1mm.MSfull scandatawereacquiredwithAgilentMassHunterData Acquisi-tionsoftware(versionB.08.00).Beforedataanalysis,theﬁrstline of each analyzed ﬁngerprint was removedfrom the results, as theseoftencontainedspectrawithlowintensities.Thiswaslikelya resultofsamplewetting,asdescribedbyBodzon-Kulakowskaetal. [29].Lubricantcomponentswereputativelyannotatedusingthe online METLIN mass spectral metabolite database [30] and comparisonwithpreviouslyobtainedresultsfromliterature. 2.5.Fingerprintimaging

Chemicalimages(1220mm)ofacyanoacrylatedblankand EXSlubricatedﬁngerprintwereacquiredusingthesame param-eters as described above. The MS-inlet was cleaned after acquisitionofeachchemicalimage.Imageswereacquiredusing a150

m

m/secondscanrate,resultingina30

m

mpixelwidthand stepsize(totalingto400rows).DatawereconvertedtoimzML, using FireFly (v.3.0.1.1, Prosolia, Indianapolis, USA),and subse-quentlyanalyzedusingMSIReader(v1.01)[31].

2.6.Statistics

DatawereconvertedtomzXMLandthe5000mostabundant peakswereﬁlteredusingmsConvert[32].Dataweresubsequently

Table1

The32condomsusedinthisstudy,theabbreviationused,themanufacturerandtheircountryoforigin.

Condoms Abbreviation Manufacturer Country

BillyBoyExtraLubricated BBEL MapaHealthCare Germany

BalanceCondoom BC Condoom-anoniem Netherlands

BeppySoftComfort BSC Beppy Netherlands

DurexClassicalNatural DCN Durex UK

DurexExtraSafe DES Durex UK

DurexFeelingSensitive DFS Durex UK

DurexOrgasmic DO Durex UK

DurexPerforma DP Durex UK

DurexRealFeeling DRF Durex UK

DurexXLPower DXLP Durex UK

Euroglider EU AshaInternational/Euroglider Netherlands

EXSRegular EXS LTCHealthCare UK

FairSquaredOriginal FSO FairSquaredGMBH Germany

GlydeUltraNaturelle GUN GlydeHealth Australia

JustSafeStandaard JSS Safe Netherlands

KruidvatClassic KC Kruidvat Netherlands

KruidvatExtra KE Kruidvat Netherlands

KruidvatSensationBanana KSb Kruidvat Netherlands

KruidvatSensationChocolate KSc Kruidvat Netherlands

KruidvatSensationStrawberry KSs Kruidvat Netherlands

KruidvatUltra KU Kruidvat Netherlands

LELOHEXCondooms LH Lelo Sweden

LevelPopular LP YourLevelsBV Netherlands

MoreAmoreSoftSkin MASS BizzyDiamondBV Netherlands

MySize MS R&SGermany Germany

MatesSKYNOriginal MSO LifestyleHealthcare Australia

ONNaturalFeeling ON R&SGermany Germany

PlayboyLubricated PL Playboy USA

PasanteNaturelle PN PasanteHealthcareLtd/Karex UK

Startex ST ForeSeeline Belgium

UniqPull UP UniqInternational Colombia

Wingman WI Wingman Netherlands

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processedwithR(version3.4.2)usingRstudio(Version1.1.456), usingtheMALDIquantpackage[33].Massspectrawere square-roottransformedandnormalizedusingthetotalioncurrent(TIC). Afteraligning and averaging the spectra, peaks were detected usingthecorrespondingMALDIquantfunctions.Principal compo-nent analysis (PCA) was then executed to reduce the data dimensionality. After splitting the data in a 75% training and 25%testset,theﬁrst12PCs (explaining90% ofthecumulative proportionofvariance)wereusedtogeneratealineardiscriminant analysis(LDA)model,usingtheMASSpackage[34].Classiﬁcation accuracywasevaluatedbygeneratingconfusionmatricesusingthe caretpackage[35].

3.Results

3.1.Detectionofcondomlubricants

As a _ﬁrstscreening of thechemical components of condom lubricants,adetectionmethodwasdevelopedbasedontheanalyses of the 24-wells cyanoacrylated sample slides. Typical scans of lubricantsfrom4differentcondomsareshowninFigs.1_–4.Ascanbe deducedfromthesespectra,distinctivepatterns,originatingfrom polymersthatmakeupalargepartofthelubricants,werefound. Closeranalysisofthedetectedm/zvaluesthatformtheseionseries leadtotheputativeannotationofthemajorcomponentsofthe condomlubricants(Table2).Alargecomponentofmanylubricants

appearedtobepoly(ethyleneglycol)(PEG),whereas m/zvalues correspondingtopolydimethylsiloxane(PDMS)weredetectedas well. In some lubricants, the polyethoxylated phenol nonionic surfactants octoxynol-9ornonoxynol-9, servingasspermicides, were observed (Table 2). Next to these chemical components, multipleionseriescorrespondingtothefattyalcoholethoxylates PEGdecyletherandPEGdodecyletherwerefound,oftenusedas non-ionicsurfactants(Table2)[36].Poly(propyleneglycol)(PPG) was putatively annotatedin someof the lubricants as well.In addition to the ion series resulting from the polymers largely presentincondomlubricants,afewmolecularionspecieswerealso detectedandputativelyannotated.Anexampleisthedetectionof benzocaine,alocalanestheticusedintwooftheDurexcondoms (Performa and Orgasmic) (Table 2). Furthermore, masses corre-spondingtoundecylamineanddodecylaminewereobserved.Asall samplesweresubjectedtocyanoacrylatefuming,acommonlyused detection technique for latent ﬁngerprints within the forensic setting,amasscorrespondingtoacyanoacrylate(CA)fragmentwas alsofound(Table2).Finally,incontrols,exceptCA,noneoftheabove mentionedchemicalcomponentswerefound(datanotshown). 3.2.Differentiationofcondomlubricants

Todifferentiatebetweenthe32condoms,principalcomponent analysis (PCA) and linear discriminant analysis (LDA) were performed,as both areshown tobeeffectivein discriminating

Fig.1.SpectrumobtainedfromDESI-MSanalysisofspotsoflubricantfromBSCcondom,showingionseriescorrespondingtoPDMS()andnonoxynol-9[M+Na]+_(*)_and

[M+K]+

(_).

Fig.2.SpectrumobtainedfromDESI-MSanalysisofspotsoflubricantfromMSOcondom,showingionseriescorrespondingtopoly(ethyleneglycol)decylether(*), octoxynol-9[M+K]+₍₎

and[M+Na]+

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condom lubricants based on mass spectra [26,18,19]. PCA was performedtoreducedatadimensionality,usingthemassspectra acquiredfromthe32differentcondomlubricants(Fig.5).Using only the first two principal components (PCs), a distinction betweenmajorlubricantclassescouldalreadybemade.Togain moreinsight inwhichcomponentscan beusedtodifferentiate condomlubricants,theloadingsofthefirst5PCswereanalyzed (Fig.6).ThefirstPCcontainsm/zvaluescorrespondingtothe[M +Na]+_and_[M₊_K]+_ion_series_of_PEG,_whereas_in_the_second_PC_m/z

valuesofnonoxynol-9(both[M+Na]+_and_[M₊_K]+₎_are

incorpo-rated.InthethirdPC,putatively,octoxynol-9(both[M+Na]+_and

[M+K]+₎_was_found_to_be_the_major_component._Undecylamine_and

anunidentifiedm/zof848.6672arethemajorcontributorstothe fourth PC. In the fifth PC, dodecylamine, PEG, PPG and an unidentified m/zof 125.9863werethestrongest differentiating factors.

Next,lineardiscriminantanalysis(LDA)wasusedtogeneratea classificationmodelusingthefirst12PCs(explaining90%ofthe cumulativeproportionofvariance(Fig.S1)),basedonthetraining data(75%oflubricantdataobtainedfromanalysisofthe24-wells slides). The generated model was subsequently evaluated by classificationofthetestdata(25%oflubricantdataobtainedfrom analysisofthe24-wellsslides).Analysisoftheresultingconfusion matrixshowsthatthemodelisabletoclassifycondomlubricants withhighaccuracy(99.0%)(TableS1).Only1samplewaspredicted

incorrectly;asamplecontainingKSbwas predictedbythe PCA-LDAmodelasKSs(TableS1),whichbothoriginatefromthesame brandofflavoredcondoms(KruidvatSensations).Theselubricants likely contain the same basis, while different colorants and flavorings are added. The fact that many components of the lubricantarelikelytobeidentical,couldexplainthe missclassi-ficationofthegeneratedmodel.Subsequently,wegeneratedthe PCA-LDA classification model based on the data from the lubricatedcyanoacrylatedfingerprintsinthesamemanner.Using thisdata,anoverallaccuracyof90.9%wasachieved(TableS2).A fewmissclassificationwerepresentbutseemtobe comprehensi-ble,suchasthepredictionofDESasDCN(bothDurexcondoms)and the prediction of DP as DO (both Durex condoms that contain benzocaine).However,themodelperformedpoorforonespecific condomlubricant,namelyPL(sensitivityof40%).Furtheranalysis ofthePLdatarevealedthelowintensityofmanyofthechemical components, possibly explaining the poor performance of the modelinthiscase.

3.3.Imagingofcondomlubricantsinﬁngerprints

Visualisationofthepresenceofcondomtraceswithin ﬁnger-printswouldgreatlyenhancethestrengthof theevidence,asit bothestablishesthepresenceofthesuspectatthecrimesceneand contact with a condom. Therefore, full chemical images were

Fig.3.SpectrumobtainedfromDESI-MSanalysisofspotsoflubricantfromKCcondom,showingionseriescorrespondingtopoly(ethyleneglycol)dodecylether[M+Na]+

(*) andpoly(ethyleneglycol)decylether[M+Na]+_().

Fig.4.SpectrumobtainedfromDESI-MSanalysisofspotsoflubricantfromDOcondom,showingionseriescorrespondingtopoly(ethyleneglycol)[M+H]+

(*),[M+K]+₍₎

and [M+Na]+

().

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obtainedfromfingerprintsthat handledan EXS condomand a blank(natural)fingerprint,bothtreatedwithCAfuming(Fig.7).As expected,CA(m/z556.1794)waspresentinbothfingerprints,and revealsthefrictionridgepatternofthefingerprintinbothcases

(Fig.7CandD).WhenrenderingthechemicaldistributionofPDMS (m/z 445.1200) for both ﬁngerprints, only in the lubricated ﬁngerprintadistinctiveimagewasacquired,thatwas absentin theblank(Fig.7AandB).A similarresultwasobtainedforthe

Table2

Detectedm/zvalues,theirputativeannotationandcorrespondingformula.

Putativeannotation Experimentalm/zvalues Formula n Ref

Benzocaine 166.0862 C9H11NO2 [M+H]+ – [30,37,18] Undecylamine 172.2058 C11H25N [M+H]+ – [30] Dodecylamine 186.2217 C12H27N [M+H]+ – [30] Ethylcyanoacrylate 556.1794 (C6H7NO2)n [M+H-C4H8N]+ n=5 – Poly(ethyleneglycol) 195.1226,239.1489,283.1753,327.1017,371.2279,415.2540, 459.2808,503.3059 H(C2H4O)nOH [M+H]+ n=4...11 [38,12] 217.1046,261.1308,305.1572,349.1836,393.2098,437.2359, 481.2622,525.2883,569.3160,613.3404,701.4077 H(C2H4O)nOH [M+Na]+ n=4...14 233.0785,277.1041,321.1298,365.1567,409.1832,453.2097, 497.2360,541.2620,585.2889,629.3150 H(C2H4O)nOH [M+K]+ n=4...13 Poly(ethyleneglycol) decylether 313.2348,357.2611,401.2873,445.3135,489.3393,533.3649, 577.3712 C10H21(C2H4O)nOH [M+Na]+ n=3...9 – Poly(ethyleneglycol) dodecylether 341.2662,385.2923,429.3183,473.3441,517.3681,561.3913, 605.4167 C12H25(C2H4O)nOH [M+Na]+ n=3...9 [39] Poly(propyleneglycol) 273.1674,331.2093,389.2514 H(C3H6O)nOH [M+Na]+ n=4...6 [38] 347.1857 H(C3H6O)nOH [M+K]+ n=5 Poly(dimethylsiloxane) 371.1013,445.1200,519.1382 (C2H6SiO)n [M+H]+ n=5...7 [38] 429.0882 (C2H6SiO)n [M+H-CH4]+ n=6 Octoxynol-9 449.2877,493.3136,537.3400,581.3661,625.3919,669.4184, 713.4439,757.4698 C14H21(C2H4O)nOH [M+Na]+ n=5....12 [38,40] 509.2876,553.3138,597.3399,641.3662,685.3925,729.4182, 773.4447,817.4707 C14H21(C2H4O)nOH [M+K]+ n=6...13 Nonoxynol-9 419.2772,463.3031,507.3291,551.3552,595.3811,639.4076 C15H23(C2H4O)nOH [M+Na]+ n=4...9 [38,40,12,21] 347.1982,391.2244,435.2509,479.2771,523.3033,567.3295, 611.3555,655.3815,699.4078,743.4352,787.4599 C15H23(C2H4O)nOH [M+K]+ n=2...12

Fig.5. Principalcomponentanalysisscoreplotbasedontheanalysisof32condomsandblank(n=12)usingtheﬁrsttwoprincipalcomponents.Separateclusteringofseveral lubricantgroupsisobserved.

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majorlubricantcomponentPEG(m/z585.2889,Fig.7EandF).PEG was foundto behighly abundantin the lubricatedfingerprint, whileonlyminorabundancewasfoundintheblank.Importantly, incaseofthelubricatedfingerprint,classificationusingasubsetof thedata(to getthesamenumber ofaveraged scans),correctly predictsthesourceofthelubricantasEXS(TableS3).

4.Discussion

Tothebestofourknowledge,thisisthe_firststudydescribinga method for the differentiation of condom lubricants from CA treated fingerprints using DESI-MS combined with a PCA-LDA classi_ficationmodel.Thegeneratedmodelshowedhighaccuracy forbothdirectanalysisofcondomlubricantspots(99.0%),aswell aslubricatedfingerprints(90.9%).Moreover,sincealargerangeof differentbrands and types of condoms, commonly soldin the Netherlands, were analyzed, a representative database was collected.

The detection, discrimination and visualization of condom derived traces from fingerprints is of significant evidential importance in sexual assault cases, as it provides crucial information onthe presence of a criminal at a crimescene as wellascontactwithacondomandtypeofcondomused,thereby greatlyincreasingthestrengthoftheevidence.Inpreviousstudies, ithasalreadybeenshownthatcondomlubricantscanbedetected anddiscriminated,solelyorwithin_{fingerprints,}usingseveralMS techniques, includingDART-MS [18–20], MALDI-MS [21,22] and DESI-MS[25,26].However,inthesestudiestheeffectof cyanoac-rylate (CA)was not examined, while in forensics CAfuming is frequentlyperformedtovisualizefingerprintsastheyoftenappear latent. We now show that the current described method is compatiblewithcyanoacrylatefuming,renderingitmoresuitable for applicationto forensic casework.Additionally, full chemical imagescouldbeacquiredfromCAtreatedlubricatedfingerprints, showingthespatialdistributionoflubricantcomponentssuchas PEGandPDMSthroughoutthefingerprint,whichcanbecombined withclassificationofcondomlubricants.Thespatialinformation provided bychemical imaging, confirms thatthe lubricantwas

transferred by ﬁngerprint contact as it links the presence of condomlubricanttotheﬁngerprintridgedetail,makingitofmore evidentialvaluethanthesoleanalysisandcomparisonofcondom components.

MALDI-MS was previously shown to have the potential to discriminatebetweendifferentcondombrandsortypes,combined withchemicalimaginginamultidisciplinaryanalyticalapproach, byBradshawetal.[22].However,theadvantageofusingDESI-MS ascomparedtoMALDI-MStechniques,isthatnomatrixorsample preparationisneededandanalysiscanbeperformedatambient pressure.DART-MSanalysisoffersstraightforwardanalysis with-outtheneedforsamplepreparation,andwasshowntobeableto achievehighclassi_ﬁcationaccuraciesbasedoncondomlubricant spectra [18–20], but lacks the capability to generate chemical images.WenowfoundthatDESI-MScombinestheeasyanddirect analysisofcondomlubricantsampleswiththeabilitytoperform chemical imaging resulting in high accuracy detection and discriminationofcondomtraces.AlthoughMALDI-MSiscapable of achieving higher spatial resolutions, the chemical images generatedusingDESI-MSshowclearridgedetail,whichwefound tobesufﬁcientforthepurposeofthismethod.

Using the developed DESI-MS method, we found multiple condom lubricant components. Among the most commonly encountered compoundswereion seriescorrespondingtoPEG, PDMS,nonoxynol-9,octoxynol-9andPEGdodecylether.Basedon theloadingsofthe_firstPCs,PEG,nonoxynol-9andoctoxynol-9 seemtobethemostdiscriminatorylubricantcomponents.Being an essential part of many lubricant bases, PDMS, PEG and nonoxynol-9 havebeenanalyzed fromcondomlubricanttraces using various analytical techniques, and have, not surprisingly, been included in many recent condom lubricant classification studies [18–20,25,26]. The detection of octoxynol-9in condom lubricantsislesscommonlyencountered,buthasbeendescribed by Thomas et al. [40] and Bradshaw et al. [21]. The putative annotationoftwofattyalcoholethoxylates(PEGdecyletherand PEGdodecylether),thatpossibleserveasethoxylate lubricants, are inagreementwithfindingsbyMirabelliet al.,whoalready mentionedthepossiblepresenceofethoxylatelubricantincertain

Fig.6.PlotoftheabsolutevaluesofcomponentloadingsoftheDESI-MSspectrafromcondomlubricantfortheﬁrst5principalcomponents.

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types of condoms [26]. The m/z values used for the putative annotationof poly(ethyleneglycol) dodecylether in ourstudy, correspondtopreviously describedpolymer fragmentsfroman unknownethoxylated polymer species by Mirabelli et al. [26]. Additionally,Musahetal.reportedthedetectionofoctylalcohol ethoxylatefromSkyncondoms,afterDART-MSanalysis[16].We also detected m/z values corresponding to undecylamine and dodecylamine, which, to our knowledge, are not commonly detectedincondomlubricants,althoughoctylaminewasidentiﬁed in many of the previous studies, mainly used as emulsiﬁer, dispersantorlubricant[16,25,18–20].

There are numerous alternative approaches available to generateclassification models based onanalytical data. In our approach,weusedPCAasafirststep,toreducedata dimensional-ity,making the data easier to perceive. LDA was subsequently chosenasclassi_ficationmethodasitshowedtobeaneasyandfast classificationmethod,whichhadalreadyproventobeeffectivein discriminating condom lubricants based on mass spectra in previousstudiesbyMaricetal.,Baumgartenetal.,andMirabelli

et al. [18,19,26]. In terms of classification accuracy based on lubricantspectra using DART-MS,Maricet al.achieved a98.7% accuracy based on classi_fication of 90 lubricants to one of 12 distinctive groups [18], Baumgarten et al. acquired a 88.9% accuracywhenclassifying18differentlubricants[19],whileCoon etal. discriminated110condomtypesfrom16differentbrands witha 97.4% accuracy [20]. Classification of lubricants from10 differentcondomsusingDESI-MSbyMirabellietal.resultedina 94% accuracy [26]. Our results are largely in line with these previousstudies,aswegaineda99.0%accuracywhenanalyzing condomlubricantspots,anda90.9%accuracybasedonanalysisof lubricantcontainingfingerprints.Additionally,theseresultsshow thatthepresenceofCAdoesnotinterferewiththedetectionand discriminationofcondomlubricants,andhighaccuracyclassi fica-tion of CAfumed lubricanttraces using DESI-MSand PCA-LDA analysisisattainable.

Someofthemisclassiﬁcationsinourstudyseemtobecaused duetolubricantsoriginatingfromthesamecondombrand.When analyzinglubricantspots,asamplecontainingKSbwaspredicted

Fig.7.Chemicalimages(1220mm)showingthedistrubutionofPDMS(m/z445.1200,AandB),cyanoacrylate(m/z556.1794,CandD)andPEG(m/z585.2889,EandF) throughoutaﬁngerprintcontainingEXSlubricant(A,CandE)andanaturalblank(B,DandF)ﬁngerprint.

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asKSs(bothKruidvatcondoms),whileinfingerprintscontaining lubricants,DESwaspredictedasDCN(bothDurexcondoms)and DP was predicted as DO (both Durex condoms that contain benzocaine).Themisclassificationofcondomlubricantoriginating fromtwo differentDurex sources was also experienced in one occasion by Mirabelli et al. [26], likely being the result of similaritiesbetweencondomlubricantsoriginatingfromthesame brand. This was shown by Maric et al. and Coon et al., who classifiedcondomlubricantstoamajorlubricantgroup/brandwith highaccuracy[18,20].Predictingthecondomlubricanttracesby brandonly,insteadofbrandandtype,wouldpresumablyleadtoan increased classi_fication accuracy in our study as well. For one particular condom (PL), we found a low sensitivity (40%) in lubricatedfingerprints,whichseemedtobetheresultoflowion intensities, possibly explaining the poor performance of the statisticalmodelinthiscase.

As the majorcomponents of condom lubricants are known contaminantsinmassspectrometry[38],weencountered carry-overproblemsduringmethoddevelopmentandoptimization,that weresimilartotheeffectsdescribedbyMirabellietal.[26].Intheir study,itwasfoundthatthemostrelevantparametersdetermining the‘memoryeffect’werethedistancebetweenthespraytipand iontransferlineandbetweentheiontransferlineandsample.Too shortdistancesresultedincontaminationoftheiontransferline, assamplematerialcouldbesuckedintotheMSinlet[26].Indeed, we also found that increasing the ion transfer line-to-surface distanceandspraytip-to-iontransferlinedistance,togetherwith cleaningtheMSinletafter3–4samples,resultedinavoidanceof samplecarry-over,indicatingthatthesearecrucialsettingsand actionsforreliableresultswhenanalyzingcondomlubricanttraces withDESI-MS.Also,whenimaging lubricatedfingerprintsusing DESI-MS,wefoundthathighamountsofcondomlubricantinthe fingerprintsdidnotgeneratehighqualitychemicalimages,dueto a decrease in clear ridge detail as a consequence of high abundances of PDMS and PEG ion signals. However, the classification model still predicted the source of the lubricant correctly,indicatingthatadiscriminationcouldstillbemade.

In this paper, we solely focused on theanalysis of condom lubricant traces in CA treated fingerprints. However, the main componentsofthesecondomlubricants,suchasPEGandPDMS, canalsobefoundinmanypersonalcareproducts[15].Asaresult, analysisoffingerprintsthatpossiblycontaintracesofanyofthese personalcareproducts,mayleadtomisclassifications.Although theability todiscriminatebetweenpersonal careproductsand condomlubricantsinfingerprintswasnotanalyzedinthepresent study,arecentstudyperformedbyMoustafaandBridgeshowed thatdiscriminationbetweentheseclassesofproductsispossible using DART-MS and LDA [15]. The addition of discriminating factorsfromotherclassesofpersonalcareproductstothecurrent developedmodelwouldfurtherincreasetheforensicapplicability of the generated method. Furthermore, we only measured fingerprintswithcondomlubricanttracesfromglasssubstrates, while in practice, fingerprints can be found on all available substrates. Furtheroptimization of the analysis of _{fingerprints} containingcondomlubricanttracesonseveraldifferentsubstrates wouldalsobenefitthedevelopedmethod.Indeed,Mirabellietal. showed that chemical analysis and imaging of fingerprints containing condom lubricant is possible on metal and paper surfaces[25,26].However,spectraobtainedfrompapersurfaces hadlowersignalintensitiesduetosorptioneffects,andawash-out effectwasencounteredwhenanalyzingonmetalsurfaces[25,26]. 5.Conclusion

We developed a DESI-MS method for the detection and discriminationofcondomlubricanttracesfromﬁngerprintsthat,

combined with a PCA-LDA classification model, has an overall accuracyof 90.9%andis compatiblewithCAfuming,making it moreapplicableforforensiccasework.Additionally,fullchemical imagesoffingerprintscontainingcondomlubricanttracescouldbe acquired,visualizingthespatialdistributionofcondomlubricant compounds,suchasPDMSandPEG.Thisconfirmsthatthecondom lubricantisoriginatingfromthefingerprintandnotthesubstrate, therebyincreasingevidentialstrength.Theseresultsarepromising leadsforfurtherdevelopmentofDESI-MSmethodstoqualitatively analyze exogenous compounds from fingerprints for use in forensicscience.

CRediTauthorshipcontributionstatement

WardvanHelmond:Conceptualization,Methodology,Formal analysis,Investigation,Writing-originaldraft,Visualization.Mark P.V.Begieneman:Conceptualization,Methodology,Investigation, Writing - original draft. Roos Kniest: Investigation, Writing -review&editing.MarceldePuit:Conceptualization,Methodology, Writing - review & editing, Project administration, Funding acquisition.

DeclarationofCompetingInterest Therearenoconﬂictstodeclare. Acknowledgement

WvHacknowledgesaRAAK-PROresearchgrant(no. 2014-01-124PRO),theNetherlands.

AppendixA.Supplementarydata

Supplementarymaterialrelatedtothisarticlecanbefound,in theonline version,at doi:https://doi.org/10.1016/j.forsciint.2019. 110005.

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