Application of a chemiluminescence immunoassay system and GC/MS for toxicological investigations on skeletonized human remains

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Application

of

a

chemiluminescence

immunoassay

system

and

GC/MS

for

toxicological

investigations

on

skeletonized

human

remains

P. Basilicata,

M. Pieri

*

,

A. Simonelli,

D. Faillace,

M. Niola,

V. Graziano

DepartmentofAdvancedBiomedicalSciences,LegalMedicineSection,UniversityofNaples“FedericoII”,Naples,Italy

ARTICLE INFO

Articlehistory:

Received4February2019

Receivedinrevisedform27April2019 Accepted1May2019

Availableonline8May2019 Keywords:

Chemiluminescenceimmunochemical assay

Non-Conventionalbiologicalmatrices Cocaine

Morphineandbenzodiazepinedetection Gaschromatography/massspectrometry

ABSTRACT

Hair,larvaeandcardiacmuscle,theonlybiologicalsamplespresentonaskeletonizedhumanbodyfound

inaruralarea,wereusedforforensictoxicologicalanalysesinordertodeterminepossiblecausesof

death.Sincenoinformationaboutthevictimorthecircumstancesofdeathwasavailable(exceptforthe

placewherethecorpsewasfound,knowntobeagatheringplacefordrugaddicts),theﬁrstapproachfor

theanalysisofnon-conventionalmatricesinvolvedthescreeningofdifferentclassesofactiveprinciples,

usingachemiluminescence-basedscreeningassaydesignedforwholeblood.Theimmunoassaytest

resultsshowedpositivitytoamphetamines,cocaineandopiatesonwater/methanolextractfromcardiac

tissue,larvaeandhairsamples.Gaschromatography–massspectrometry(GC/MS)analysesconﬁrmed

theimmunoassayresults,exceptforamphetamines.Theminimalsamplepreparation(hydrationand

extractioninanultrasonicbath),thereducedsamplevolumerequiredfortheanalyses,togetherwiththe

correctnessofresultsasconﬁrmedbyGC/MS,showedthesuitabilityofthescreeningtestforforensic

applicationsonnon-conventionalmatrices.QuantitativeanalysesinGC/MSallowedthecauseofdeathto

beascertainedonthebasisoftheratiobetweenparentdrugsandmetabolites.

1.Introduction

Itcanbeextremelychallengingtoascertainthecauseofdeath whenthebodyistotallyorpartiallyskeletonized,especiallyifthe causesofdeatharetobefoundinsoft tissue,whichcommonly applies in the case of sudden death. In such cases, traditional biologicalspecimensarecommonlynotavailableandalternative matrices,suchaslarvae,fragmentsoftissuesororgansandhair, assumeextremeimportance.Determinationof thecolonization intervalofacorpse(“postmorteminterval”)hasbeenamajorissue forforensicentomologists since the19thcentury [1,2]. Forthe above“classical”purpose,larvaemaynowbeseenasthesubstrate for toxicological analyses: in recent decays, sensitivity and speciﬁcity of mass spectrometry-based methods have allowed the analysis of larvae and/or insects to evidence the possible presenceofdrugsthatcouldbecontributorycausesofdeath[3–5]. Larvaeareofparticularinterest,sinceadrugcanstillbepresent

anddetectedininsectsthathavefedonacorpse,whileitmayno longerdetectableinorgansortissueremains.Duringtheperiod between 1977–2016 over 60 papers were published on the detection of toxicants, as reviewed by Da Silva et al. [6]. Nevertheless,thepossibilityofreferringﬁndingsfromalternative matricestocausesofdeathare,inacertainway,limitedtothe availabilityofpublisheddatathatcanprovidesupportwiththe interpretationofresults, withparticularattentionto pharmaco-logicalandphysiologicalmechanismsregulatingdrugdisposition inthespeciesofinterest.

Massspectrometry-basedmethodsallowuniqueand irrefut-ableidentification ofcertain chemicals,whileimmunochemical methodsofferthepossibilitytoperformrapidandlessexpensive screening of several classes of substances [4,7]. Moreover, immunoassay screening tests are less sample-consuming and require reduced sample pretreatment. Consequently, immuno-assays focusing onthe accuracy of determinations of different drugsintypicalforensicmatriceshavebeenwidelytested[4,6–11]. Thepossibilityofperformingforensictoxicologicalscreeningon alternativematricesislimitedduetothedifficultyintestingtheir efficacyonrealsamples.

Recently,thelegalauthoritiesorderedtheforensicautopsyand toxicological analysisofskeletonized humanremains. Thiscase gaveustheopportunitytoverifytheapplicabilityofananalytical strategyfortheanalysisofnon-conventionalmatrices,basedon

*Correspondingauthorat:DepartmentofAdvancedBiomedicalSciences,Legal MedicineSection,UniversityofNaples“FedericoII”,ViaS.Pansini,5,80131Naples, Italy.

E-mailaddresses:pbasilic@unina.it(P. Basilicata),maria.pieri@unina.it

(M. Pieri),angela.simonelli@unina.it(A. Simonelli),danila.faillace@libero.it

(D. Faillace),Massimo.niola@unina.it(M. Niola),vincenzo.graziano2@unina.it

(V.Graziano).

https://doi.org/10.1016/j.forsciint.2019.05.003

ContentslistsavailableatScienceDirect

Forensic

Science

International

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chemiluminescence immunoassay for the rapid screening of differentclasses ofdrugs, followedby conﬁrmationanalyses in gaschromatography/massspectrometry.GC/MSdataalsoallowed thecauseofdeathtobedetermined.

2.Casereport

Skeletonizedremainsofanunknownsubjectwerefoundina ﬁeld,knowntobeagatheringplacefordrugaddicts.Thediscovery of the body followed an anonymous call to police. During inspectionofthearea, thepolice recoveredan emptywoman’s bagwornoverthebody.Inoneoftheoutsidepocketsofthebag therewasanemptyjarofhandcreamcontaininganeedle.Thebag alsocontaineda pinkplasticwatch.Twobracelets(oneof dark rubberandoneofsteel),togetherwithaclaspknife,twolighters andasyringe(ﬁttedwithaneedle),werefoundonthesceneafter bodyremoval.

External examination revealed the absence of appreciable injury.Thecorpsewasinanadvancedstateofdecompositiondue to the action of Diptera larvae still present on the skeletal remains. Results of anthropological analyses (data not shown) evidenced that the corpse was that of a young adult male (between18and25yearsold),ratherthick-set,ofmedium-low height (172cm3.5cm). The corpse presented few dental elements in both arches, all characterized by peculiar erosion of the mesial andlateral margins of the upper anteriorteeth (centralincisors),presumablyduetodentaldecayortoanerosive processof anothernature.Thebodypresenteda deformedleft collarbone,probablyduetoapreviousfracturingprocess,lacking theacromialportion.

Thecorpsepresentedonlyafewcutaneousshredsontheback, wheresmallchannels,compatiblewiththeactionoflarvae,were clearlyvisible.Furthermore,therewerefragmentsof paraverte-bral, parasternal, and proximal femoral quadriceps, strongly alteredbydecompositionprocesses.Theheartwithina residual pericardialsacwastheonlyrecognizableorganandpresentedan alteredﬂattenedanddriedshape.Thealmostcomplete skeleto-nizationandtheactionofmicroandmacrofaunadidnotallow accurate estimation of the post-mortem interval, presumably datingbackatleast30days,alsointhelightofclimateconditions (the corpsewas foundin late summer). Completetoxicological analyseswereperformedonhair,larvaeandonthecompletely dehydratedcardiacmuscle.

3.Materialandmethods

Certified standard solutions of drugs of abuse used for confirmationanalysisingaschromatography/massspectrometry (GC/MS)werefromCerilliant(Sigma-Aldrich,Milan,Italy),N,O-bis (trimethylsilyl)trifluoroacetamide(BSTFA)andheptafluorobutyric acid(HFBA)derivatizingagentfromAcros(MorrisPlains,NJ,USA), andHPLCgrade-solventsfromCarloErba(Milan,Italy).

GC/MSanalyseswereperformedusingaDSQIIsingle quadru-polemassspectrometerdirectlylinkedtoaTraceGC2000series gaschromatographequippedwithasplit-splitlessautosamplerAS 3000, all from ThermoFisher (San José, CA, USA). Gas chro-matographic separations were performed with a Rxi1-5MS (30m0.25mm0.25

m

m)capillarycolumn(Restek,Bellefonte, PA,USA).DatawereprocessedusingtheXcalibursoftware(version 2.0.7)fromThermoFisher.

ImmunochemicalscreeningtestswereperformedonRandox Evidence Investigator(Randox Toxicology.Country Antrim,UK), usingDoAI+WBSQandDoAIIWBwholebloodtestsforAMP/ MAMP/MDMA, barbiturates, benzodiazepines, buprenorphine, cannabinoids, cocaine, methadone, opiates, phencyclidine,

tricyclicantidepressants,fentanyl,ketamine,LSD,methaqualone, oxycodoneandpropoxyphene.

3.1.Immunochemicalscreeningtest

3.1.1.Samplepreparationforchemiluminescenceimmunoassay

3.1.1.1.Larvae. Larvaeweregentlywashedwithbidistilledwater toremovedirtandsoildebris.Aliquotsof150mgwereaddedwith 2mL of methanol and put in ultrasonic bath overnight. After ﬁltration, organic extract, dried under nitrogen stream, was reconstituted using 200

m

L of the specimen dilution buffer of theimmunoassaykit.

3.1.1.2.Cardiactissue. Inanattempttoobtainabloodsurrogate, afteradditionof250mLofbidistilledwatertheentireorganwas placedinultrasonicbathfor15mininordertodissolvethedried bloodpresentontheorgan.Theliquidwasﬁlteredundervacuum, storedat 20Candusedforsubsequenttoxicologicalanalyses. Immunoassaytestswereperformedon100

m

Laliquot,processed accordingtoimmunoassaysystemspeciﬁcations.

3.1.1.3.Hair. Hairwas washed byadding water until complete coverage;samplesweregentlyvortexed,andtheliquiddischarged. Theprocedurewasrepeateduntilclearwastewaterwasobtained. Thewashingstepwasrepeatedwithacetone.Hairwasleftatroom temperatureuntildriedandﬁnelycut.Aliquotsof50mgwereused for immunoassaytests;thesample wasprepared followingthe same procedure used for larvae (2mL methanol, overnight in ultrasonic bath, organic extract dried and reconstituted in specimendilutionbuffer).

For all thetested matrices, screening tests were performed according tothe manufacturer’s speciﬁcations for wholeblood samples, after specimen preparation as speciﬁed above. The immunochemical system used an enhanced chemiluminescent substratewithahorseradishperoxidaselabelforthedetectionof antibodiesoranalytesboundtothebiochipsurfaceofthedevice. Although the immunoassay results were in terms of semi-quantitative data,given the nature of specimens analyzed, the resultsarereportedintermsofpositive/negative.

3.2.GC/MSanalysis

3.2.1.SamplepreparationforGC/MSanalyses

3.2.1.1.Larvae. Larvaeweregentlywashedwithbidistilledwater toremovedirtandsoildebris.Aliquotsof360mgwereaddedwith 2mLbidistilled water, and placed in ultrasonic bath overnight. After filtration, the liquid fraction was recovered, spiked with internalstandardandpH-adjusted.Finally,sampleswerepurified bysolidphaseextraction(SPE)speci_fictoamphetamines,cocaine andopiates(seebelow).

3.2.1.2.Cardiactissue. GC/MSanalyseswereperformedon_“blood surrogate”,obtainedasspecifiedabove(seecardiactissuesample preparation for chemiluminescence immunoassay). Five-mL aliquots of filtered liquid spiked with deuterated internal standards were purified by SPE for detecting amphetamines, cocaineandopiates(seebelow).

3.2.1.3. Hair. Hair was washed as described for the chemiluminescenceimmunoassayand50mgaliquotswereused for GC/MS analyses, involving the use of deuterated internal standardsandspecifichydrolysisandSPEpurification(seebelow). Biological samples, spiked with the appropriate deuterated internalstandardsandpretreatedasspecifiedabove,underwent

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SPEpuriﬁcationspeciﬁctococaineandbenzoylecgonine,opiates, amphetaminesandbenzodiazepines(seebelow).

3.2.1.4. Cocaine,benzoylecgonine and free morphine. Aliquots of larvae and cardiac tissue, pretreated as described above, were spikedwithinternal standards (d3-cocaine, d3-benzoylecgonine

and d3-morphine), and pH-adjusted to 8–9 with 10M KOH

saturatedwithKHCO3priortoSPEextraction.

Aliquotof washedhairsample,withtheadditionof 1mLof 0.1HCl,underwentacidichydrolysisat45Covernightforcocaine andbenzoylecgoninequantiﬁcation.Thesamplewasaddedwithto 2mLof0.1Mphosphatebuffer(pH6)and2NNaOHuntilpH8_–9.It wasthencentrifuged(2000rpmfor5min)priortoSPEextraction. SPEextraction,performedwithScreen-Ccartridges(200mg/ 3mL),involveddrop-to-dropelutionat5mmHgandthefollowing extractionprocedure.Conditioning:2mLmethanoland2mL0.1M phosphatebuffer(pH6).Sampleloading.Washing:6mLbidistilled water;3mL0.1NHCl;9mLmethanolandcartridgeslefttodryfor 5min.Elution:4mL(dichloromethane/methanol=8/2,2% ammo-nia, v:v). Eluted fraction, dried under nitrogen stream, was derivatizedbyadding50

m

LBSTFAandthesamplesleftat75C for20min.

3.2.1.5.Totalmorphine. Aliquotsofpretreatedlarvaeandcardiac tissue,spikedwithinternalstandard(d3-morphine),underwent

acidichydrolysis.Afteradditionof200

m

Lof37%HClthesamples wereleftat 120C for 20min.Theywerethen cooledto room temperatureandpH-adjustedto8–9byadding800

m

L2MTris buffer(pH 8)and200

m

L10MKOHsaturatedwithKHCO3.The

sampleswerecentrifuged(4000rpmfor40min)andpuriﬁedby SPEasdescribedabove(see“Cocaine,benzoylecgonineandfree morphine”subsection).

Aliquotofhairsamplewashydrolyzedandpuriﬁedasdescribed above (see “Cocaine, benzoylecgonine and free morphine” subsection)fortotalmorphinequantiﬁcation.

3.2.1.6. Amphetamines. Aliquots of larvae and cardiac tissue, pretreated as described above, were spiked with internal standards (d6-amphetaine, d5-methamphetamine and d5

-MDMA),andpH-adjustedto6with0.1Mphosphatebuffer,and thencentrifuged(4000rpmfor20min).PuriﬁcationthroughSPE extractioninvolved theuse of BondEluteCertify (200mg/3mL) cartridges, with the drop-to-drop elution at 5mmHg and the followingextractionprocedure.Conditioning:2mLmethanoland 2mL0.1Mphosphate buffer (pH 6).Sample loading. Washing: 1mL1M acetic acidand cartridges weredried for 5min.6mL methanoland cartridgesweredriedfor5min.Elution:in atest tube with the addition of 50

m

L (methanol/HCl=9/1): 4mL (isopropanol/dichloromethane/ammonia=78/22/2, v:v). Eluted fraction,driedundernitrogenstream,wasderivatizedbyadding 100

m

LHFBAandthesampleswereleftat70_C_for₂₀_min.

3.2.1.7. Benzodiazepines. Aliquots of larvae and cardiac tissue, pretreatedasdescribedabove,werespikedwithinternalstandard (d5-prazepam),andpH-adjustedto6with0.1Mphosphatebuffer.

Analiquotofwashedhairsample,spikedwithinternalstandard (d5-prazepam) was added with 1mL methanol and underwent

hydrolysisat45Covernight.Allsamplesweredilutedwith3mL 0.1M phosphate buffer, then mixed/vortexed for 5min and centrifuged(4000rpmfor40min).

PuriﬁcationthroughSPEextractioninvolvedtheuseofScreenC (200mg/3mL) cartridges, with the drop-to-drop elution at 5mmHg and the following extraction procedure. Conditioning: 3mLmethanoland3mL0.1Mphosphatebuffer(pH6).Sample loading.Washing.3mL(5%acetonitrilein0.1Mphosphatebuffer (pH6),v:v)andcartridgesweredriedfor5min.3mLhexaneand

cartridges were dried for 5min. Elution. 6mL (ethyl acetate/ ammonia=98:2,v:v).Elutedfraction,driedundernitrogenstream, wasderivatizedbyadding50

m

LBSTFAandthesampleswereleft at75Cfor20min.

All samples were analyzed in GC/MS full scan mode and quantiﬁedby MS-Single Ion Monitoring(MS-SIM) mode (three ionscharacteristicofeachanalytewereacquired).

4.Results

4.1.Forensictoxicologicalanalyses

Resultsofscreeningtestsonlarvae,cardiactissueandhairare reportedinTable1.Thelarvaeresultedpositive to benzodiaze-pines,cocaineandopiates;cardiactissueshoweda positivityto cocaineandopiates;hairwaspositivetobenzodiazepine,cocaine and opiates. Moreover, immunochemical assay evidenced a positivitytoamphetaminesforalltestedmatrices.

GC/MS-SIM analyses confirmed positivity to cocaine and morphineonallmatrices;positivitytotemazepamanddiazepam was also recorded, while immunoassay positivity to amphet-amineswasnotsupportedbyGC/MS-SIMconfirmation. Quantita-tiveanalysis,performedinSIMmodebyusingaspecificcalibration curve,evidencedresultsreportedschematicallyinTable2.Inthe case of morphine, GC/MSanalyses onlarvaeand cardiactissue wererepeatedwithandwithoutacidichydrolysistoquantifytotal and free morphine, respectively. Results obtained with and without hydrolysis are almost superimposable both for larvae and cardiac tissue (see Table 2), thus evidencing that in both specimenspositivitytomorphinecanbetracedbacktoalmostfree morphine.

GC/MSfullscananalysesonallspecimensshowednofurther toxicologicallyrelevantanalyte.

5.Discussion

Theneedtoinvestigatethepresenceofdrugsofabuseorother chemicalsinhighlydecomposingbodyorskeletalremains,aswell asascertaintheirroleasacontributoryfactortothecauseofdeath of the subject, represents the drive to perform toxicological analysesonlarvaeandothernon-conventionalmatrices,suchas tissuesororganremains.Afterencouragingresultsobtainedfrom theearly 1980sonwards, in 1991Pounder [5] coined theterm “forensic entomotoxicology” to deﬁne the branch of forensic toxicology focusing on the useof insect specimens as indirect

Table1

Resultsofchemiluminesceimmunoassaytestsonlarvae,cardiactissueandhair. Larvae Cardiactissue Hair

AMP + + – MAMP + + + MDMA + + + Barbiturates Benzodiazepines1 + + Benzodiazepines2 + Buprenorphine Cannabinoids Cocaine + + + Methadone Opiates + + + Phencyclidine TricyclicAntidepressants Fentanyl Ketamine LSD Methaqualone Oxycodone Propoxyphene

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matricestoelucidatedrug-relateddeathsinallthosecaseswhere ﬂuidswerenolongeravailable.AsrecentlyreviewedbydaSilva etal.[6],oneofthemainaimsofforensicentomotoxicologyisto verifythepossiblerole ofa certain drugincontributing tothe cause of deathby performingtoxicological analyses of insects/ larvae foundon the body. Moreover,verifying the presence of drugs is extremely useful also for the “traditional” forensic application of entomotoxicology, i.e. estimation of time-since-death.Drugscan,indeed,inﬂuencethedevelopmentrateofinsect species [12,13]. While several authors have reported results of toxicologicalanalysesperformedonlarvaebychromatographic/ mass spectrometric methods and a considerable database is available[6,14],thepossibilityofapplyingrapidimmunochemical screening to alternative matrices has been less extensively investigated.

Inthepresentpaper,animmunochemiluminescentassaywas applied tothe rapid screening of extracts from larvae, cardiac tissue and hair, the only biological matrices available for the forensictoxicologicalanalysesofaskeletonizedhumanbody.For allthespecimensconsidered,samplepretreatmentwasreducedto theminimum,sincethewater/methanolicextractsweredirectly appliedtothechipofthescreeningdevice.Althoughthedevice offeredsemi-quantitativedata,resultswereconsideredintermsof positive/negative.

Asexpected,apositivitytowardsamphetamineswasrecorded for all tested matrices and not conﬁrmed by GC/MS analyses. Putrefactive amines (phenylethylamine, putrescine, tryptamine, tyramine)maycross-reactwithimmunoassaytestsfor amphet-amines [15]. Moreover,different medication classes present in clinic formulary, such as antihistamines (brompheniramine, phenulpropanolamine or nonprescription nasal inhalers), anti-depressants(bupropion,trazodone),antipsychotics (chlorproma-zine,promethazine),orotheragents(ranitidine,ephedrine),have been identiﬁed as potential sources of cross-reactivity with amphetamine/methamphetaminescreeningtests[16].

Immunoassay positivity recorded for hair extract to the benzodiazepine test was confirmed by GC/MS, evidencing the presenceofdiazepamandtemazepam.Confirmationanalysesfor benzodiazepineswerealsorepeatedoncardiactissueandlarvae samples, and resulted negative. Positivity to the BENZO1 test recordedforhairsample,withanegativeresulttoBENZO2,isin linewith assayspecificities declared by the manufacturer. The maintargetofBENZO1isoxazepam(100%cross-reactivity,%CR); whileBENZO2isdesignedforlorazepam (100%CR).Amongthe testedcongeners,diazepamandtemazepamshowhighaffinityto BENZO1 alone (256% CR and 382% CR, respectively), while no significantcross-reaction(<0.1%CR)isreportedforBENZO2[17]. Thusourresultsshowedthesuitabilityoftheimmunoassayforthe rapidscreeningofbenzodiazepines,whosepresencewascorrectly

evidencedinhairextracts.Moreover,performingbothbenzotests givesusefulindicationsaboutthespeciﬁcdrug(s)presentinthe sample.

Inthecaseofcardiactissueextract,GC/MSresultsconﬁrmed thatoftheimmunoassay,thussupportingthehypothesisthatthe maninquestionwasnotundertheeffectofbenzodiazepineswhen he died.GC/MSanalysesonlarvaeextractsdidnotconﬁrmthe immunoassaypositivities.Suchadiscrepancycouldbeattributed totheinadequacyoftheimmunotestforthespecimenortothe presenceofmetabolites(mainlyconjugates)thatcanbedetected by the screening test and not evidenced by GC/MS, since no enzymatichydrolysiswasperformed.

Theresultsobtainedforthecocaineimmunoassayevidenced positivityinalltestedmatrices.Checkingthetestcross-reactivity, itmaybenotedthatthecocaineimmunoassayisdesignedtoreact withbenzoylecgonine,BE,(100%CR),withpoorafﬁnitytococaine (2.9% CR). Results of GC/MS analyses evidenced higher BE concentrationsinallspecimens.Thus,alsococaineimmunoassay issuitablefortheanalysisofalternativematrices.Dataobtainedon larvae and cardiactissue strongly suggestthat the subjectwas undertheeffectofcocaineshortlybeforehedied.GC/MSdatacan only be discussed as a ratio betweenthe parentdrug and the metabolite,duetothewellknownintra-individualvariabilityto cocainetoxicity,theabsenceofstandardizedprotocolstoprepare theavailablematricesand,inthecaseofcardiactissueextract,the completeabsenceofanyliteraturedata.Ratiosbetweencocaine (COC)andBEwere0.309and0.644forlarvaeandcardiactissue, respectively. Rees et al. [18] reported cocaine concentrations measured in cardiac and femoral muscles in nine cases, and compared results to blood (femoral and cardiac) and vitreous humor. Ratios betweenCOC and BE in the range 0–0.32 were obtainedforskeletalmuscle(median0.14),whilevaluesbetween 0–0.47 (median 0.09) were obtained for cardiac muscle [18]. Consideringthesevaluesasindicativeofacocaine-relateddeath (bloodratiowas:0–0.53,median0.12forcardiacblood;0.02–0.1, median0.07forfemoralblood)[18],itmaybeassumedthatthe casediscussedhereinconcernsadeathresultingfromcocaine.

Resultsoftheimmunoassaytestsevidencedapositivityofall specimenstoopiates,whichwasconfirmedbyGC/MSanalyses.In thecaseoflarvaeandcardiactissueextracts,confirmationanalyses performedwithandwithoutacidichydrolysisevidencedsimilar free- and total-morphine concentrations in all samples, thus showing that morphine (the target molecule of the opiate immunoassay) was the more abundant species. Such results, similarlytothoseobtainedfor cocaine,suggestthatthesubject survivedforashorttimeafterdrugassumption.Asreportedby DarkeandDuflou,lowerbloodconcentrationsofmorphine3-and 6-glucuronides,andlowerratiosoftheseconjugatestofreemorphine, aresuggestiveofamorerapiddeath,astherehasbeenlesstimeforthe metabolismofmorphinetooccur[19].

Finally,theresultsobtainedforhaircon_ﬁrmedthatthesubject waspolydrug-addicted.Consideringalsocircumstantialdata(the bodywasfoundclosetoadistrictknowntobeagatheringplacefor drug addicts),the medical examinerconcluded that deathwas probablycausedbyanoverdoseofcocaineandopiatesina poly-addictedsubject.

6.Conclusions

Tothebestofourknowledge,thepresentpaperistheﬁrstto reportresultsofachemiluminescence-basedimmunoassayforthe forensic screeningof extractsof larvae, cardiac tissue and hair sampled from a skeletonized body. Although conﬁrmation analyses, performed in GC/MS, were mandatory, data from immunotesting wereuseful toset upthe GC/MS-SIM analyses. Nofurtheranalytesweredetected,exceptforthoseresultingfrom

Table2

GC/MS-SIMquantitativeresultsforcocaine,morphineandbenzodiazepineson larvae,cardiactissueandhair.

Larvae(ng/mg) Cardiactissuea

(ng/ml) Hair (ng/mg) Cocaine Cocaine 5.35 328 43.24 Benzoylecgonine 17.32 509 0.74 Benzodiazepines Temazepam N.D. N.D. 0.02 Diazepam N.D. N.D. 0.03 Morphine Free-morphine 2.98 208 – Total-morphine 3.15 211 1.73 a

Analyseswereperformedonaqueousextractoftheentireorgan,inorderto attempt a“blood surrogate”,see“Sample preparationforchemiluminescence immunoassay”section.

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screening, evidencing good specificity of the screening tests. Finally,commentsontheparentdrug/metaboliteratio,quantified bygaschromatography/massspectrometry,allowedidentification ofthepossiblecauseofdeath, attributedtococaineandopiate acutetoxicityinapoly-addictedsubject.

Conﬂictofinterests None.

CRediTauthorshipcontributionstatement

P. Basilicata: Formal analysis, Conceptualization. M. Pieri: Conceptualization, Writing - review & editing. A. Simonelli: Formal analysis. D. Faillace: Investigation. M. Niola: Funding acquisition.V.Graziano:Investigation,Conceptualization.

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