Analytical separations of mammalian decomposition products for forensic science: A review

Analytica Chimica Acta 682 (2010) 922

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Analytica Chimica Acta

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Review

Analytical separations of mammalian decomposition products for forensicscience: A review

L.M. Swanna, S.L. Forbesb, S.W. Lewisa,

a Department of Chemistry, Curtin University of Technology, GPO Box U1987, Perth, WA 6845, Australiab Faculty of Sci

a r t i c l

Article history:Received 29 JuReceived in re29 SeptemberAccepted 29 SAvailable onlin

Keywords:DecompositionAnalytical sepForensic scien

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102. Mammalian decomposition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103. Non-chromatographic approaches to chemical studies of decomposition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

3.1. Chemical techniques for locating clandestine human remains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113.2.

4. Chrom4.1.

4.2.

5. ConclAcknoRefer

CorresponE-mail add

0003-2670/$ doi:10.1016/j.Chemical techniques for the identication of adipocere . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12atographic studies of decomposition products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12Vitreous humor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124.1.1. Potassium ions (K+) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164.1.2. Hypoxanthine (Hx) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164.1.3. Amino acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17Decomposition products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174.2.1. Decomposition uid and soil solution analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174.2.2. Volatile organic compounds (VOCs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194.2.3. Adipocere . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

usions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20wledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21ences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

ding author. Tel.: +61 08 9266 2484; fax: +61 08 9266 2300.ress: [email protected] (S.W. Lewis).

see front matter 2010 Elsevier B.V. All rights reserved.aca.2010.09.052ence, University of Ontario Institute of Technology, Oshawa, ON, Canada, L1H 7K4

e i n f o

ly 2010vised form2010eptember 2010e 7 October 2010

chemistryarationsce

a b s t r a c t

The study of mammalian soft tissue decomposition is an emerging area in forensic science, with a majorfocus of the research being the use of various chemical and biological methods to study the fate of humanremains in the environment. Decomposition of mammalian soft tissue is a postmortem process that,depending on environmental conditions and physiological factors, will proceed until complete disin-tegration of the tissue. The major stages of decomposition involve complex reactions which result inthe chemical breakdown of the bodys main constituents; lipids, proteins, and carbohydrates. The rststep to understanding this chemistry is identifying the compounds present in decomposition uids anddetermining when they are produced. This paper provides an overview of decomposition chemistry andreviews recent advances in this area utilising analytical separation science.

2010 Elsevier B.V. All rights reserved.

10 L.M. Swann et al. / Analytica Chimica Acta 682 (2010) 922

L.M. Swann received a BSc(Hons) in forensic sciencefromCurtin University of Technology. She is currently

S.L. Forbes is an Associate Professor and Director ofthe Forensic Science program at University of Ontario

1. Introdu

The studing area in fthe use of vaof human reroles to plaestimation

Decompprocess thaological factissue [3]. Afor investignation of cainterval vercomplex rethe bodys[4]. As decotheir structand glucoseinvolved, aenvironmenined in detacertain chemand potenti

The rstcompoundsthey are prsample to stinsect life,using coloutry can provtechniquesedge neededecompositmatographyoffer this le

positg an

mma

r derpse,tablentalysicathat tith

l goilitynd thambcellute reagato, poortemafteompensicompometntalain sn bed, dehysie 1.rsttagerstterisxia [9r pH4]. The onset of autolysis can be observed by the presence oflled blisters on the skin, and skin slippage [5,11] as cellularranes dissolve, releasing nutrient rich cellular uids into the1]. Tissues containing cells with the highest rate of ATP syn-and membrane transport should typically decompose rst

owing autolysis, the second process in decomposition is thed stage, where the beginnings of putrefaction are seen. Thisdestruction of the soft tissues of the body by the action ofrganisms (bacteria, fungi and protozoa) in a mostly anaer-nvironment. Putrefaction is identiable by a green/purpleuration of the skin and occurs usually between 36 and 72hin the nal stages of a PhD in forensic chemistryunder the supervision of Dr Simon W. Lewis withinthe Department of Chemistry at Curtin. Her thesisresearch involves the chemical characterisation ofcompounds produced in decomposition uid and thedevelopment of new analytical methodologies to dealwith this complex sample matrix.

S.W. Lewis is an Associate Professor of Forensic andAnalytical Chemistry in the Department of Chemistryat Curtin University of Technology, Perth, WesternAustralia. He obtained a PhD in Analytical Chemistryfrom the University of Plymouth, UK before taking upa position as a lecturer at Deakin University, Geelong,Australia (19942005). He currently leads the Foren-sic and Analytical Chemistry Group, with researchinterests focused on chemical techniques applied toforensic analysis, in particular ngerprint detection,decomposition chemistry and trace evidence.

ction

y ofmammalian soft tissue decomposition is an emerg-orensic science,with amajor focus of the research beingrious chemical andbiologicalmethods to study the fatemains in the environment. Such techniques havemajory in locating clandestine gravesites [1], assisting in theof postburial interval [2].osition of mammalian soft tissue is a postmortemt, depending on environmental conditions and physi-tors, will proceed until complete disintegration of then understanding of this process is extremely importantations of suspicious deaths as it complicates determi-use of death and makes the estimation of postmortemy difcult. The major stages of decomposition involveactions which result in the chemical breakdown ofmain constituents; lipids, proteins, and carbohydratesmposition proceeds, these macromolecules degrade toural componentswhich include amino acids, fatty acids,[5]. The precise details of the biochemical pathways

long with a detailed knowledge of the temporal andtal variation thatmaybeobservedhave yet to be exam-il. It has also been suggested that temporal variation ofical species couldbeused to agedecomposing remains

ally aid in the determination of postmortem interval.step to understanding this chemistry is identifying thepresent indecompositionuids anddeterminingwhenoduced. However, decomposition uid is a challengingudy, as it is a complex chemicalmixturewith associatedmicrobial organisms and other debris. While methodsrimetry or fourier-transform infrared spectrophotome-ide useful trend information, more selective analyticalare required to provide the level of chemical knowl-d to fully understand the fundamental chemistry ofion. Separation science techniques such as gas chro-, liquid chromatography and capillary electrophoresisvel of specicity. This paper provides an overview of

decomutilisin

2. Ma

Aftethe copredicronmeThe phstatestimeswies wilvariabstage abodysaffectregulainvestilividitypostmshortlyble decthe for

Dec[10], sronmetwo mtion cabloateof the pin Tabl

Thefresh soccurscharacto anocelluladeath [uid membbody [1thesis[9].

Follbloateis themicrooobic ediscoloInstitute of Technology (UOIT) and holds a Tier 2CanadaResearchChair (CRC) inDecompositionChem-istry. She gained her PhD in Forensic Chemistry fromthe University of Technology in Sydney, Australia andcompleted post-doctoral training at the University ofWestern Australia before accepting a position in 2005in the Faculty of Science at UOIT. Her research focuseson the chemical processes that occur in soft tissuedegradation, with applications to both food scienceand forensic science.

ion chemistry and reviews recent advances in this areaalytical separation science.

lian decomposition

ath, many physicochemical processes take place withinwith the resulting changes from these occurring in aorder. The rate of change is strongly inuenced by envi-factors, the most signicant being temperature [69].l principle known as Vant Hoffs rule, or the rule of ten,he velocity of chemical reactions increases two ormoreeach 10 C rise in temperature [9]. All decomposing bod-through the same decomposition process, but it is thein temperature that will determine the length of eache overall velocity of the process [9]. A deviation in theient temperature towarmer or cooler surroundingswilllar metabolism by affecting the enzyme catalysts thatctionswithin the body [9].Within a few hours of death,rs may use the rate of cooling of the body, postmortemtassium levels in vitreous uid, and rigor to estimate

interval. However these methods can only be usedr death, and have limited accuracy [6,7]. Thus, once visi-osition changes commence, the techniques available toscientist are greatly reduced.osition commences almost immediately after deathimes within the rst 4min [5,11], depending on envi-conditions and surroundings. It can be characterised bytages, pre and postskeletonisation [12]. Preskeletonisa-further broken down into four subsequent stages; fresh,cay and dry, as rst described by Reed [13]. A summarycal changes observed in each of these stages can be seen

identiable process is autolysis, occurring during theand literally meaning self-digestion of cells. Autolysisin the most metabolically active cells, i.e. cells wheretically high rates of ATP production are more sensitive]. It is thought to be triggered by the decrease in intra-occurring as a result of the decreased oxygen levels after

L.M. Swann et al. / Analytica Chimica Acta 682 (2010) 922 11

after death [5,9,10]. The discolouration seen on the body is theresult of variously coloured pigments released into the tissues ofthe abdomen by lysing pancreatic cells [9]. The result of putrefac-tion is the catabolism of carbohydrates, proteins and lipids presentin soft tissutrapped volthe carcassbody, often[5,11].

FollowinAdditionalbreakdownproducing ption producin this stageis still occustill being d

The dryIn this staga leathery-is characterof the body[14]. This prtant bone, tthese remais consideragenesis, orsurroundinchanging ra

Althoughdistinctiontively, stage

3. Non-chrdecomposi

3.1. Chemic

Amajorphysical anremains. Inhas a confouNumerousrecovery sitevidence asimaging [16ground penassociated lfor locating

Recentlysiteshasbeedestine graresults in aa cadaver. Aof nitrogen-proteins, pecompoundsis commonluse as a chetion [25]. Inof a bodyw(NRN) and(i.e. soil ass

Their stuin three co

Table 1Overview of mammalian decomposition.

Stage of decomposition Description Reference

Fresh Commences immediately after death [14]

g

decay

assigh thal pundsinat]. Thleuc-foldhe afollosedites [addiet adecomposition in both burial and surface decomposition

ios [24]. The study involved the use of larger swine carcasseslogues for human decomposition. Six carcasses were placedlow gravesites and a sequential destructive sampling regimeed to collect gravesoil samples at specic postburial inter-his study expanded on the study conducted by Carter et al.investigating the lateral diffusion of NRN from the decom-n site. The authors hypothesised that NRN concentrationsincrease asdecompositionproceeded, butwoulddecrease astance from the cadaver increased [24]. To study this hypoth-avesoil samples were collected from the centre and edge ofravesite once a month for a period of 6 months, as well ashe base and walls of the graves at the time of exhumation.nally, ve swine carcasses were placed on a soil surface andd to decompose for 97 days during which time soil sam-ere collected from beneath the decomposing carcasses. Therin analysis was adapted from Carter et al. [22] and theance was scanned from 500 to 640nm using a UVvis spec-er. The results indicated that NRN could detect gravesoil inly postmortem period (2 months postburial) and that thediffusionof nitrogen inuxwasnarrow in a gravesite. In con-he NRN concentration was signicantly higher during bothrly and later postmortem stages for the carcasses decom-on the surface. These results demonstrated the potential ofes into gases, liquids and simplemolecules [5,9,11]. Theatile gases and uid are responsible for the bloating ofand a build up in pressure can result in purging from thesevere enough to cause additional postmortem injuries

g the purging of gases, the active decay stage begins.volatile fatty acids are produced from the continuedof muscle, and the decomposition of protein and fathenolic compounds and glycerols [5,11]. Decomposi-ts such as putrescine and cadaverine are also detectableand insect activity is at a premium [12]. Putrefaction

rring during active decay, as chemical constituents areegraded and released.stage is the nal stage of the decomposition process.e, any remaining moist skin and tissue is converted tolike sheet that adheres to bone [5,11]. Skeletonisationized by the appearance of exposed bone in over 50%, but erosion of the skeletal elements has not yet begunocesswill proceed until only the harder andmore resis-eeth and cartilage remain [10]. Chemical weathering ofins continues but the time taken for this deteriorationbly longer than previous stages of decomposition. Dia-the exchange of ionic species between bones and theirg environment, occurs, but at a rate dependent on thetios of calcium and phosphate in a variety of soils [9].decomposition is divided into these four stages, the

between the stages can be difcult to identify. Alterna-s may be absent altogether.

omatographic approaches to chemical studies oftion

al techniques for locating clandestine human remains

areaof investigationwithin theeld is the applicationofd chemical techniques for locating clandestine humanmany instances, bodies are concealed by burial, whichnding effect on the search and location of the remains.techniques have been applied to forensic search anduations including theuseof cadaverdogs [15], botanicalsociated with grave sites [16,17], probing [18], thermal], and the use of geophysical equipment such as theetrating radar [1921]. Each of these techniques hasimitations and is best applied in a sequential mannerburied human remains [16]., chemical analysis of soils related to decompositionnproposedas analternativemethod fordetecting clan-ves [2224]. The process of soft tissue decompositionsignicant pulse of nutrients into the soil surroundingproportion of these nutrients result from the releasecontaining compounds within the body, which includeptides, aminoacids, andamines [10]. Thesenitrogenoushave the ability to reactwithninhydrin, a chemical thaty available in police and forensic laboratories due to itsmical enhancement technique for ngerprint visualisa-2008, Carter et al. hypothesised that the decompositionould result in an increase in ninhydrin reactive nitrogenmay provide a new technique for identifying gravesoilociated with a decomposing cadaver) [22,26].dy involved the burial of juvenile rats (Rattus rattus)ntrasting soil sites followed by sequential harvesting

Bloatin

Active

Dry

at pre-beneatchemiccompodetermtry [27with a1.42.2ples. Tin soilto be ugraves

AnCartercarcassscenaras anain shalwas usvals. T[22] bypositiowouldthe disesis, greach gfrom tAdditioalloweples wninhydabsorbtrometthe earlateraltrast, tthe eaposingAutolysis [6,7]Fluid lled blisters on the skin, skinslippage

[6,7,12]

Marbling of skin due to livor mortisPutrefaction destruction of softtissues by microorganisms

[7,11]

Greenish discolouration of the skin [6,7,12]Gas and uid accumulation followedby purging

[12]

Anaerobic fermentationBloating has ceased [14]Skin usually broken in one or moreplaces

[7]

Rapid leaching from bodyLarge numbers of aerobic andanaerobic bacteria

[7,11,15]

Extensive insect activityPossible carnivore activity [15]Collapse of abdominal cavityLoss of internal organs through insectactivity or autolysis

[12,15]

Possible adipocere formation [15]Diagenesis [12,14]No carrion fauna remaining [14]Small amount of decaying tissueMummication of remaining skin [15]Bone exposure and skeletonisation [11,15]

ned postburial intervals (PBI). Gravesoil collected frome decomposing cadavers was analysed using an NRNrotocol which involved the extraction of nitrogenous-from the soil, reaction with ninhydrin reagent, and

ion of the absorbance at 570nmusing spectrophotome-e concentration of NRNwas determined by comparisonine standard. The gravesoil samples demonstrated aincrease in NRN when compared to the control sam-

uthors suggested that the signicant increase of NRNwing decomposition of a cadaver had the potentialas an investigative technique for locating clandestine22].tional study applied the NRN technique proposed byl. [22] to an investigation of swine (Sus domesticus)


the technique for detecting the original site of decomposition forcadavers that have been scavenged or subjected to postmortemrelocation.

A case summary presented in 2009 details the application of theNRN technithe remainThe colourithe locationwere collecprimary deand hand, ssumptive dconcentratithat decommethod as aposedhuma[23]. Howevdecomposit

A novelbeen propoa motorizelayer opencarcasses psoil, then slected by dwere piercelids. The adsolution thrhydrin reagabsorbancearray UVvdetecting Naverage recand buriedNRN was nbutwas det6, 10, and 20higher maspared to thdiffered conural environand this resThe study wtechnique f

3.2. Chemic

Adipocefrom thenedecomposecially in buto forensictrations indspectroscople of softStuart et altransform (believed toinvolved mstrating thebands in theacteristic odegree of fshown to beterising adialso succes

environments [31]. The chemical composition of pig and humanadipose tissue degradation was investigated over a period of 21days. The lipidprole forbothpig andhuman tissuedemonstratedadecrease in carbonyl bands relating to triglycerides and a concomi-

creased. Ted toalterd hass andd speno snt anystalhe foyl reluabrate

oma

ile theenpositvironmicamet

tativlatin

ial stis ofle oesti

rker wing ae estir ow60s, ftion il sturom, whmad

atograttemrsofd639s dec

treou

he erepl ise (Htreouterioortemed [4timeputs huparque to an outdoor death scene following disturbance ofs, most likely through the activity of scavengers [23].metric technique was used to assist with determiningof death and the origin of decomposition. Soil samplested from several locations including the presumptivecomposition site (head and torso), the lower body, armcalp, and control sites. The soil collected from the pre-ecomposition site demonstrated a signicantly higheron of NRN than the other sites and it was concludedposition had occurred in this location. The value of thisquantitative chemical alternative for locating decom-n remains anddecomposition siteswas thus conrmeder, exactlywhat constitutesNRN in the context of theseion studies has yet to be established.NRN technique using headspace collection has recentlysed [28] and is based on a purge and trap method usingd pipetter and an activated, alumina-coated, poroustubular (PLOT) column. The study utilized feeder ratlaced on either the soil surface or buried within theealed in pressed wooden boxes. Samples were col-rawing the headspace through PLOT columns, whichd through septum ports placed on the compartmentsorbed compounds were eluted by forcing a 2M KClough the PLOT column and into a vial containing nin-ent. Following dilution with an ethanol solution, theof the sample was measured at 570nm using a diode-is spectrophotometer. The method was successful atRN with only a 5min headspace collection period. Theovered masses of NRN were higher for both exposedcarcasses when compared to the blank (soil) samples.ot detected during the rst 4 weeks of decompositionected in the headspace of the compartments inweeks 5,. In contrast to the study byVan Belle et al. [25], slightly

ses of NRNwere detected for the buried carcasses com-e exposed carcasses. However, the experimental setupsiderably between both studies (i.e. one was in a nat-ment and the other was in a controlled environment)ult may not be representative of all forensic scenarios.as useful in proposing an alternative use of the NRN

or locating clandestine graves.

al techniques for the identication of adipocere

re, a postmortem decomposition product that formsutral fats present in soft tissue,may be founddirectly ond remains or in the surrounding soil environment, espe-rials. Spectroscopic techniques have also been applieddecomposition studies to identify fatty acid concen-icative of adipocere formation. The use of infraredy as a qualitative tool for determining the lipid pro-tissue has been well documented [2931]. In 2000,. [30] proposed a diffuse reectance infrared FourierDRIFT) spectroscopy technique to study soil samplescontain adipocere from human remains. The techniqueinimal sample preparation and was useful in demon-degradation of triglycerides into fatty acids. Carbonyl17401700 cm1 regionwere identied as being char-

f adipocere formation and assisted in determining theormation that had occurred. DRIFT spectroscopy wasa rapid and effectivemethod for detecting and charac-

pocere formation in soils [30]. The same technique wassfully applied to adipocere samples formed in aqueous

tant inincreasappear

Anmethoin soilinfrarelittle toa solveATR crising tcarbonalso vaon the

4. Chr

Whhave bdecoming enbiochearationquantibase resics.

Initanalysprincipgive anbiomaprovidreliablon thethe 19centraseveraaway fsciencestudieschromin anmarke[5,12,3varioubelow.

4.1. Vi

In twidelyintervaanthinThe vi(or pospostmconnlongerity andvitreouers come in bands relating to free fatty acids as immersion timehe formation of fatty acids in the human tissue samplesoccur more rapidly than in the pig tissue samples.

native Fourier transform infrared (FTIR) spectroscopybeen applied to the investigation of adipocere formedmock cofns [29]. Attenuated total reectance (ATR)ctroscopy is a surface analytical technique that requiresample preparation [32]. Samples can be extracted usingd cast as a lm on a slide, then placed directly onto the. This method also proved to be effective in character-rmation of adipocere in soils based on changes in thegion of the spectrum. Analysis by ATR spectroscopywasle in demonstrating the affect of the burial environmentof adipocere formation.

tographic studies of decomposition products

e non-chromatographic approaches described abovevaluable in providing a qualitative prole of theion uid as it is formed and released into the surround-ment, further information is required regarding thel pathways involved in the process. For this reason, sep-hods are being investigated with the goal of providinge data that will assist in strengthening the knowledgeg to decomposition chemistry as is applies to geoforen-

udies into the use of chromatographic methods for thedecomposition products were very much based on thef identifying chemical markers that could be used tomation of postmortem interval. The use of a chemicalould aid in the elimination of examiner bias as well as

n orthogonal method that would aid in offering a moreimate of postmortem interval than existing techniquesn. The earliest reports of this approach were made inocusing on themeasurement of potassium ion (K+) con-n vitreous humor by ame photometry [33,34], Whilstdies have continued this work, there has been a shiftthe use of these early techniques towards separationich are less prone to matrix interferences [35]. Initiale use of gas chromatography, but more recently, liquidaphy and capillary electrophoresis have been appliedpt to utilise the temporal variation of the chemicalecomposition inorder toestimatepostmorteminterval]. A summaryof chromatographic techniquesapplied toompositionmatrices is shown in Table 2, and discussed

s humor

arly stages of postmortem interval, one of the mostorted biochemical methods to estimate postmortemthe determination of potassium ion (K+) and hypox-x) concentrations in vitreous humor [33,34,41,57,58].s humor refers to the uid that lls the vitreous bodyr chamber) of the eye [44]. Its potential usefulness forchemical analysis results from it being anatomically

5] and thus somewhat protected and preserved forperiods frombacterial contamination, enzymatic activ-refactive changes [34,43]. It has also been stated thatmor has advantages for chemical analysis for biomark-ed to the use of blood and cerebrospinal uid (CSF) due


Table 2Summary of chromatographic methods used in the analysis of mammalian soft tissue decomposition products and to aid in the estimation of postmortem interval.

Authors Analysismethod

Matrix Postmorteminterval ofsamples

Analytes Analytical conditions/samplepreparation

Application

Patrick and Logan [40] TechniconTSM aminoacidanalyser

Vitreoushumor

Up to 96h Free aminoacids

1mL vitreous humor+50mgsalicylsulphonic acid. Centrifugefor 10mins.

Estimation ofpostmortem interval ininfants

Rognum et al.[41]

HPLC-UV

Vitreous humor


Table 2 (Continued )




Application

Vass et al. [5] GCMS Soil solution Up to 3 weeks Amino acids,putrescine,cadaverine

Column: DB-5MS, 30m0.25mmo.d. 0.25m thickness

Estimation ofpostmortemintervalIonisation energy: 70eV

Source temp: 174 CInjection port temp: 175 CTransfer line temp: 280 CFinal column temp: 280 CHead pressure: 15psi

Forbes et al. [48] GCMS Adipocere Not given Long chain fattyacids, methylester derivatives

Column: db5-ms,30m0.25mm0.25m

Detection ofadipocere ingrave soilsCarrier gas: helium

Column pressure: 100kPaInitial column temp: 100 CFinal column temp: 275 CSplitless mode

Vass et al. [49] TD/GCMS Headspaceanalysis

Up to 1.5 years VOCs Column: DB-1 phase, 1.0m lmthickness, 0.32mm i.d.60m

Cadaver dogtraining, odourdatabaseInitial temp: 35 C

Final temp: 280 CCarrier gas: heliumFlow rate: 1.5mLmin1

Run time: 50minsStatheropouloset al. [50]

TD/GCMS Headspaceanalysis

34weeks

VOCs Column: SPB-624, 60m0.25mmi.d., 1.4m stationary phase

Characterisationof VOCs fromhumandecomposition

Head pressure: 25psiFinal column temp: 180 CCarrier gas: heliumRun time: 40mins

Munoz et al.[51]

HPLC-UV

Vitreous humor Not given Hx Column: RP 3.9mm300mm Estimation ofpostmorteminterval

Mobile phase: 0.05M KH2PO4 pH3, containing 1% MeOHFlow rate: 1.5mLmin1

Detection: 200400nmDetection limit: 0.02MLimit of quantitation: 0.08M

Zhou et al. [52] LPIC-conductivitydetection

Vitreous humor 127h K+ Eluent: 0.96mM HNO3 Estimation ofpostmorteminterval

Flow rate: 0.8mLmin1

Column: LPIC (self-made),7 cm0.6 cm, 2025m i.d.Column temp: ambientRun time: 6.7mins (K+ elutiontime)Detection limit: 1mMLimit of quantitation: 2mM

Statheropouloset al. [53]

TD/GCMS Headspaceanalysis

Approx 4 days VOCs Column: SPB-624, 60m0.25mmi.d., 1.4m stationary phase

Characterisationof VOCs fromhumandecomposition

Head pressure: 25psiFinal column temp: 180 CCarrier gas: heliumRun Time: 40mins

Girela et al. [54] HPLC-uorometricdetection

Vitreous humor,CSF

560h Amino acids(OPAderivatives)

Column: C-18 Cause of deathand estimationof postmorteminterval

Mobile phase: sodiumphosphate buffer(solvent A),acetonitrile:water(50:50) (solvent B)Gradient: 90:10 (A/B) to 100 (B)Run Time: 25mins

Notter et al. [55] SPE-GCMS Pig adiposetissue

Not given Long chain fattyacids

Column: DB-5MS,30m0.25mm0.25m.

Quantitation offatty acids inadipocereCarrier gas: helium

Initial oven temp: 100 CFinal over temp: 290 CSource temp: 230 CSplit ratio: 13:2:1Split ow: 20.0mLmin1

Pressure: 100kPaRun time: 26minsDetection limit: 5mgmL1

Limit of quantiation: 10mgmL1


Table 2 (Continued )




Application

Hoffman et a[15]

Notter et al. tty

Dekeirsschieet al. [56]

Swann et al.

Swann et al.

to accessibiblood to rafrom enviro

The follopotassiumvitreous huTo date, nobased on mdeveloped.and potassthere is awi[34,41,43,5measurememortem intin an attempostmortemand the poin linear reusing potasl. SPME-GCMS Headspaceanalysis

Not given VOCs

[31] GCMS Pig adiposetissue, humanskin andsubcutaneousfat

Not given Long chain faacids

ter TD/GCMS Headspaceanalysis

Approx 2months

VOCs[36] GCMS Decompositionuid

Up to 60 days Volatilefatty acids

Long chainfatty acids

[37] CE-UVvis Decompositionuid

Up to 60 days Biogenicamines

Aminoacids

lity [34], ease of sampling [43], less susceptibility thanpid chemical changes [58] and relative independencenmental inuences [59].wing section discusses the most prominent analytes,ions and hypoxanthine, which have been analysed inmor in an attempt to estimate postmortem interval.reliable method for postmortem interval estimationeasurement of either Hx or potassium ions has beenThere has been signicant disagreement on how Hxium ion concentrations vary in vitreous humor, andde variability inmeasurements taken between samples7,58,60,61]. Thisunreliability limits theuseof individualnts of both potassium ions andHx as indicators of post-erval. Various statistical approaches have been appliedpt to improve the accuracy in estimation. Typically, theinterval has been used as the independent variable

tassium ion concentration as the dependent variablegression analysis. Recently, it has been suggested thatsium ion concentration as the independent variablewill

improve ththe suggestval. Howev492 cases, owith the reSeveral autpotassium ipostmortemHowever, pspective, mfor the vitreas serumoremployed tand betweemination offactors incl[61,64,65],of terminalconsumptioColumn: DB5-MS, 15m0.25mmi.d., 0.25m lm thickness

Characterisationof VOCs fromhuman remainsfor cadaver dogtraining

Injection port temp: 250 CCarrier gas: heliumFlow rate: 1.5mLmin1

Initial oven temp: 35 CFinal oven temp: 260 CSplitless modeRun time: 23.5minsColumn: DB-5MS,30m0.25mm0.25m

Characterisationof adipocere

Carrier gas: heliumInitial oven temp: 100 CFinal oven temp: 290 CSource temp: 230 CSplit ratio: 13:2:1Split ow: 20.0mLmin1

Pressure: 100kPaColumn: RTX/MXT -1301/624,60m0.32mm1.8m

Comparison ofVOCs fromcarcasses in 3biotopes

Initial oven temp: 35 CFinal oven temp: 245 CCarrier gas: helium

Flow rate: 1.0mLmin1

Column pressure: 8.7psiTransfer line temp: 250 CRun time: 36minsColumn: DB-wax Characterisation

ofdecompositionuid

Carrier gas: heliumIonisation energy: 70eV

Spilt ratio: 10:1Injector temp: 240 CSource temp: 160 CFinal column temp: 265 CRun time: 25minsCapillary: unfused silica, 75mi.d., L: 65 cm, E: 56 cm

Characterisationofdecompositionuid

Buffer: 70mm boric acid, 32%MeOH, pH 9.5Temperature: 30 C

Voltage: 30kVCurrent: 26ADetection: 200nmRun time: 12minsDetection limit: 5.88.6mgL1

e postmortem estimation [47,62]. This has also beenion for the use of Hx in determining postmortem inter-er, a review in 2006 found that in a random sample ofnly 153werewithin the predicted postmortem intervalmaining 339 having a systematic overestimation [62].hors have suggested that a combination of both Hx andondeterminationwouldbemoreeffective in estimatinginterval than either individual measurement [41,43].

roblems arise for two reasons. From an analytical per-ethod development needs to be established specicallyous humormatrix andnot based onothermatrices suchurine [63] andconsistent analytical protocolsneed tobeo ensure reliable, reproducible results between samplesn laboratories. From a forensic viewpoint, each deter-postmortem interval needs to take into account severaluding cause of death [47], environmental conditionsbody temperature [64], age of deceased [64], durationepisode [61,64], chronic illness [34,57,61] and alcoholn [65]. The combination of all of these factors makes


biochemical indicators in vitreous humor a potential complemen-tary technique rather than a stand-alone method for postmorteminterval estimation.

4.1.1. PotasA correl

postmortemshowed antions up tousefulness ohave since b

Potassiupresent intion at theto detect vels of totalas alkalineconcentrati(sodium, chstable for loas indicatoronly analytwhich allow[58].

While eetry [33,57is well dostudies in tpotassiumlinear equa[34,41,43,5thereby, alstudies alsofrom4.7 [3cies includealcohol conanalytical iof vitreouspostmortem

The rstanalysis ofmortem incompared c214nm, witminationofhandling sa[6769] astion sampleimproved sdetection lionly requirsis time wammol L1, wISE methodtion (r2 =0.9by both CE

The disacomplex buchemical coether and dthe 18-crowpotassiumwas essentipotassiumas a chelatcapillary in

The addition of HIBA was reported as affecting the complete res-olution of all the cations studied [45]. UV-absorbing additives,such as 4-ethylbenzylamine and imidazole, were added as theirelectrophoretic mobilities match that of the analytes. They were

re sion ms comies [etry

, resun co

ion lithan904)tionsubjer nimatas reiuml foterm, theed foinat

attempotastectiosing

ed ss repthe tedictage orequd hadalua007ion wtratiint

withing amol Llventve th

Hypooxanelevas patogrticals Hxnumstratl (r=less tdy ttimarevitectistratan pdedsium ions (K+)ation between concentration of potassium ions andinterval was rst reported by Jaffe [33]. This study

increase in potassium concentration in 36 determina-125h postmortem [33]. Several studies regarding thef this as a biochemical indicator of postmortem intervaleen reported [34,5760].m was chosen as an analyte over other componentsvitreous humor for a variety of reasons. Instrumenta-time of this work (1960s) was not sensitive enoughery low levels of some constituents, such as the lev-proteins and albumins [58]. Other components suchphosphotase were uniformly negative with respect toon and associated error. Of the electrolytes analysedloride and potassium), sodium and chloride remainedngperiods of time after death [34,58], discounting thems of postmortem interval. Vitreous potassium was thee that showed an increase in concentration after death,ed it to be expressed by a linearmathematical function

arly work focused on methods using ame photom-,58,60] and ion selective electrodes (ISE) [65,66], itcumented that several discrepancies exist betweenhe estimation of postmortem interval using vitreousion concentrations [61,64,65]. Several papers reporttions for the determination of postmortem interval7,58,60,61], however, the intercept varies markedly,tering the estimation of postmortem interval. Thesehave a wide range of associated uncertainty, ranging4] to34h [61]. Suggested reasons for thesediscrepan-ambient temperature [61,64], elevatedurea levels [65],sumption [65], sample acquisition and storage [45] andnstrumentation [45,64]. Such variation limits the usepotassium ion concentration as a reliable indicator ofinterval.reported shift away from ame photometry for the

potassium ions in vitreous humor to determine post-terval was published by Ferslew et al. [44]. Ferslewapillary electrophoresis (CE) [44], with UV detection ath an ion selective electrode (ISE) method for the deter-vitreouspotassium in25human subjects. CE is useful atmples that are particularly complex or dirty in naturecan often be the case with work involving decomposi-s. The use of CE allowed for rapid analysis time (


by HPLC showed a smaller range of scatter when compared withthe determination of potassium by ame photometry for the samesamples, particularly in the rst 24h. The gradient for the determi-nationofHxbecomes steeperwith increasingambient temperature[41]. This isthat tempeof decompomortem int

In 1994,vious literapostmortema method bsmaller mapotassiumanalysis ofsium ion coions and Hxandnot aftesimilar analauthors as aof hypoxiacompare thies of Madewith UV detometrywa[43]. It wasboth Hx andval. Howeveand themakthe observeestimate pobined resulto arrive atpostmortemthe mean oby the twothe overall

In 2002,using the hHx in the vHPLC and othe same (dwith a knowdetection linot given). Tbothpotassusing an invtionmethodhumor fromdecrease inno informathe 2002 pation is signito 0.02mo

More recapproach fotial injectiodeterminatSIA is an aalyte deterlimits of 1.0ions.wereaear correlations, whichreports [41,tianalyte de

widely available and requires customising for specic applications[72].

4.1.3. Amino acidseousnd Vut ts wialysamiith thn to pganowinacidexchalyseyear7 Amortemrkwpostt 24008,s hualdehples). Noe or

s clatratitiont thi

com

Decoethopositf su

ic entionognitial atheir[77].certons ifactraturnt, thes dien cthatrs, pdecptab, fat10,1all oC2Ctry oby Vase be fatt-valin accordancewith the reported literature,which statesrature is the factor with the greatest impact on ratesition and subsequently, the determination of post-erval [8,12,14].Madea et al. published results that contradicted pre-ture reports of the linear relationship between Hx andinterval [42]. VitreousHxwas analysed byHPLC, using

ased on the work of Rognum, but concluded that thergin of error was associated with the determination ofions by ion selective electrodes rather than the HPLCHx. It should be noted that Rognum determined potas-ncentrations using ame photometry. Also, potassiumwere shown to rise linearly immediately postmortemr24hashadbeen reported [41]. Sinceboth studies usedyticalmethods, this discrepancy is accounted for by thedifference in sample collection itself, and the duration

prior to death [42]. In 1997 [43], an effort was made toe linear equations provided by the three separate stud-a et al. [42], Sturner [34] and Rognum et al. [41]. HPLCtection was used to determine Hx whilst ame pho-s used tomeasure vitreouspotassium ion concentrationconcluded that there was a linear correlation betweenpotassium ion concentration with postmortem inter-r, the experimental method used, sampling techniquese-upof the samplegroup itselfwere the reasonsbehindd differences in the resulting linear equations used tostmortem interval [43]. The result of this work com-ts from both Hx and vitreous potassiummeasurementsa new equation that claimed improved accuracy forinterval determination. This was achieved by taking

f the individual estimates of postmortem interval givenseparate equations, reducing the standard deviation ofestimate [43].the estimation of postmortem interval was improvedypothesis that cause of death is a factor in the level ofitreous humor [47]. The analysis determined Hx usingnly investigated cases where the cause of death waseath by hanging). Using 206 samples from 176 subjectsn postmortem interval ranging from 1.08 to 28.91h,

mits in mol L1 range were obtained (exact numbershe linear correlationbetweenpostmortem interval andium ions (r2 =0.818) andHx (r2 =0.757)was reportedbyerse prediction statistical method. The inverse predic-was again used for the determination of Hx in vitreous134 samples [51]. A revised HPLC method showed aanalysis time and improved reliability [51]. Althoughtion is given in regards to the separation conditions inper, in 2006,Munoz et al. state that the sample prepara-cantly faster and detection limits are improved downl L1 for Hx.ently Passos et al.moved away froma chromatographicr the determination of Hx and instead used a sequen-n analysis (SIA) system that enabled the simultaneousion of both potassium and Hx in vitreous humor [71].utomated ow based technique that allows multian-minations within a single instrument [72]. Detection2mol L1 for Hx and 5105 mmol L1 for potassiumchieved respectively. TheHx levels showedagreater lin-ion to postmortem interval (r2 =0.998) than potassiumwas in agreement with some of the earlier literature43]. While this approach shows great promise for mul-terminations of this kind, SIA instrumentation is not

VitrErdei aacids, btrationacid ansuchasdeal wrelatioand Lotion shaminocationacid anthan 196h. 2postmthis wotion ofthe rs

In 2vitreouphthal58 sam560hysis timauthorconcencorrelasuppor

4.2. De

4.2.1.A m

decomcases oforensestimathe recsequenbles ofdeathwith asituatition, ortempean evebecom

Whnotedcadavehumanan acceweightology [

Smacids (chemisWorkuid phvolatiland isohumor has also been analysed for free amino acids.ass used paper chromatography to identify 13 aminohis early research did not attempt to correlate concen-th postmortem interval [73]. Prior to this, most aminois had been performed using non-separative methodsnoacidnitrogenanalysis [7476]. Very fewpublicationse amino acid content in vitreous humor, particularly inostmortem interval. The work done in 1988 by Patrick

[40] was the rst report of a chromatographic separa-g a relationship between postmortem interval and freeconcentration in vitreous humor [40]. Analysis was byange chromatography using a Technicon TSM aminor. This workwas completed on 120 cases of infants lessold, with samples having a postmortem interval up toino acids showed a logarithmic linear relationship withinterval although at different rates. The conclusion of

as that samples showed a reliable result for the estima-mortem interval but only if they were collected withinh.HPLC was used to determine free amino acids in bothmor and cerebrospinal uid [54]. This work used an o-yde (OPA) derivatisationprocedureprior to analysis forwith an average postmortem interval of 23.9h (rangeinformation is given in regards to detection limit, anal-error in estimation of postmortem interval. While theim a linear relationship between selected amino acidons in vitreous humor and postmortem interval, theco-efcients presented (0.3191 r0.4508),wouldnots contention to any great extent.

position products

mposition uid and soil solution analysisd with application to both early and late stages ofion would be invaluable to forensic investigators inspicious deaths. In the later stages of decomposition,tomology has been found to be a useful tool in theof postmortem interval. Forensic entomology involvestion of arthropods (mostly insects), coupled with theirrrival times on a corpse and the developmental timeta-offspring, to estimate the time and sometimes cause ofThis technique has been extensively studied and usedain amount of success [13,7780]. There are, however,n which the corpse has reached complete skeletonisa-orshaveaffected thedecayprocess (suchasburial or lowes, insect access and presence of drugs [8184]). In suche estimation of postmortem interval via entomologyfcult, if not impossible [85].onsidering the following body of work, it should bedue to the ethical issues involved in the use of humanig (Sus dometica) carcasses are often used to model theomposition process [85,86]. They are considered to bele substitute due to their similarity to human torsos into muscle ratio, hair coverage, biochemistry and physi-6].rganic molecules, such as short chain volatile fatty5), were the primary focus of early studies into thef decomposition in the mid-late postmortem interval.ass et al. [5,12] used soil solution analysis (the liq-etween particles) to analyse ve microbially producedy acids (VFAs); propionic, butyric, iso-butyric, valericeric acid, and their variation over time, with regards to


Fi

accumulatethat they ca[39], thus einterval. Andeterminedgravesites i

The anachromatogr[8790] forvolatile andand reproduing and ghoseparation

Vass ideusing a relato prepareied prior tionisation dreproducibiimproved tbecame modegree-daypropionic, bmate givencurrently

In morea soil matrhuman cadmass spectadvantagesfaster and mlution as weto the analylished [12],was shownducibility opreparationto analysis.

An increalso allowecompoundsin the analylong chain a2-piperidonsignicancepatterns infatty acids (trends that

hromd peacid (4ic acid), oleic

s etsmittcine)ealino 3 wtisatiave

rograer tol deurese pohoweicator woren oxethiod linheseen toinedo-eluy the47 (gile thpostsh the relpropthe fn for the compounds in decomposition uid remain largelyen, with most suggested pathways being based on previousg. 1. Structures of short chain volatile fatty acids (VFAs).

d temperature (Fig. 1). An advantage of using VFAs isn remain detectable for a considerable amount of timextending their use potentially into the late postmortemearly application of this approach was by Tuller, whoVFAs in samples collected from known execution and

n the former Yugoslavia [39].lysis of VFAs has typically been carried out using gasaphy (GC). GC provides a rapid and reliable methodthe analytical separation and subsequent analysis ofthermally stable mixtures, showing good sensitivitycibility. Early studies attempted to deal with peak tail-sting as well as nding the most effective column for[89,91].ntied and quantied 5 VFAs from human cadaverstively simple aqueous dilution and ltration methodsamples for analysis [12]. Each sample was then acid-o being analysed using packed column GC with ameetection (FID). The addition of formic acid increasedlity by minimising peak tailing and ghosting andhe analyte volatility [87,91]. Vass also stated that VFAsre volatile at pH


Fig. 3. Degradation pathway of phenylalanine to produce phenylacetic and phenyl-propionic acids.

ucts, thus allowing a clearer understanding of the chemistry ofdecomposition.

The limitations associated with the use of GC resulted in thedevelopment of new analytical methods to deal with the varietyof compounds present in decomposition uid. The dirty nature ofthe sample, lends itself to frequent instrument maintenance, thusslowing down sample throughput [98]. Also, GC is unable to detectboth long chain and short chain acids (both shown to be present ina sample of decomposition uid) in a quantitative manner due tothe polaritycompoundsof more coproblem [9times. In adare both vo

In an effpounds in dUVvis detto the detematrices, ppotential foextremely uwith analytproviding mwhilst still

The simtyramine, b(tryptophanuid [38].

In ordermethod wa

Fig. 5. Electropherogram at optimised running condition showing compounds pro-duced for a decomposition uid sample with identied peaks: tryptamine (1),tyramine (2), neutral (3), tryptophan (4), tyrosine (5), phenylalanine (6) and uniden-tied components (*) [38].

approach. A screening design was carried out followed by a centralcomposite design, using peak resolution and total analysis time asresponse factors. The method was applied to the analysis of sam-ples from a decomposition eld trial to investigate the temporal

on old tri

Volatatilen haaid

ost,50,5tabathetiontifyd fron ord intandcomisces e

MSf thedifference in the molecules [94,98]. Detection of theserequires either derivatisation of samples, or the use

mplex methods, such as tandem GC to deal with this4], thereby signicantly increasing sample preparationdition to this, analysis by GC requires that compoundslatile and thermally stable [32].ort to further characterise non-volatile chemical com-ecomposition uid, an analytical method using CEwithection has been developed [38]. CE has been appliedction of nitrogenous compounds in a wide variety ofarticularly for forensic analysis [99,100]. CE offers ther highly efcient and rapid separations and thus isseful when analysing complex mixtures. CE can copees not suitable for GC due to thermal instability, whileore rapid separations than are achievable with LC,

maintaining high resolution [101].ple CE method determined ve amines (tryptamine,enzylamine, aniline, indole) and three amino acids, phenylalanine, tyrosine) (Fig. 4) in decomposition

to improve resolution and total analysis time, thes subjected to optimisation utilising a chemometric

variatithe e

4.2.2.Vol

positiodogs toThe m[15,49ysis daduringdesorpto idenevolvepositiogroupecyclictainingother/m

VOCTD/GCBoth oFig. 4. Structures of amino acids and biogenf these compounds. A sample electropherogram fromal can be seen in Fig. 5.

ile organic compounds (VOCs)organic compounds (VOCs) associated with decom-ve long been used as a means of training cadaverin the detection of clandestine graves [15,49,102,103].abundant compounds reported from several studies3,56] can be seen in Fig. 6. A decomposition odor anal-se was established in 2004 to identify VOCs produceddecomposition process [49]. This work used thermalgas chromatography-mass spectrometry (TD/GCMS)424 compounds (and several more unidentied) thatm the cadavers with either no external signs of decom-in the very early stages. These compounds were theno classes for ease of data analysis. These classes includenon-cyclic hydrocarbons, nitrogen and oxygen con-pounds, acids/esters, halogens, sulfur compounds, andllaneous compounds [49].volved from a decaying body were analysed byto provide a chemical prole of decomposition [50,53].se studies yielded a chemical prole of decompositionic amines.


mpoun

but with siprevious wplace overinterval of 4[49] as wellRecent worclasses evoTD/GCMSpatternchaliterature [4

4.2.3. AdipoWhilst n

interval, onis adipocerein studyingamore detarapid andmajor complarly myristpresent in c

There ahydroxysteincluding liacids. The dment for saFig. 6. Structures of volatile organic coFig. 7. Structures of long chain fatty a

gnicantly less compounds (30 [53] and 80 [50]) thanork [49]. This is possibly due to sampling only takinga 24h period for cadavers with a known postmortemdays [53] and 34weeks [50] comparedwith 1.5 yearsas preliminary data from a 12-year-old gravesite [49].k in this area showed 35 core compounds from 5 mainlved from the decaying body, with analysis again by[56]. The results of this work showed that the volatilengesover time,which is inaccordancewith the reported9].

cereot directly related to the estimation of postmorteme of the most widely studied decomposition products(see Section 3.2). While FTIR analysis has been usefultrends, analysis of adipocere using GCMS has allowediled understanding of its composition [97,104] throughsimple methods with good reproducibility [55]. Theonents identied were long chain fatty acids particu-ic (C14:0), palmitic (C16:0) and stearic (C18:0), which areharacteristic ratios [55,97,104] (Fig. 7).re reports in the literature of trace levels of 10-aric acid [104] and other unsaturated fatty acids,noleic (C18:2) [55], palmitoleic (C16:1) and oleic (C18:1)isadvantage of the analysis of adipocere is the require-mple pre-treatment, which is necessary to achieve a

reliable andMethods sumatographfrom interfaffected byoxidation othe ambien

5. Conclus

A greatevide the negravesites ahuman remnew avenution. Analytthe decompdirection wpathways tin the analproducts ofmass specttechnique wcurrently cawill be pubds (VOCs).cids.

accuratequantitativedeterminationbyGCMS[31,55].ch as thin layer chromatography and column chro-

y [105,106] have been utilised to separate adipocereering compounds, however, these methods are oftenlow recoveries of lipids, large solvent requirements andf unsaturated fatty acids due to long term exposure tot environment [31,55].

ions

r knowledge of decomposition chemistry will pro-cessary tools to assist in the detection of clandestinend the estimation of postmortem intervals in retrievedains. Advances in analytical separations have openedes in the study of mammalian soft tissue decomposi-ical separations have enabled amore detailed picture ofosition process to be constructed. Further work in thisill allow the construction of the various biosynthetichat occur during decomposition. A surprising omissionytical separation techniques applied to studies of themammalian decomposition is liquid chromatography-rometry (LC-MS), and it would be expected that thisould be particularly useful for this application. We arerrying investigations in this area and our initial resultslished in due course.


There is a real need to develop viable alternatives to ento-mology for the estimation of postmortem intervals. While studiesusing analytical separations show promise for the estimation ofpostmortem interval through measurement of specic chemicalspecies, thea single chepostmortemdetermine pdevelop hartuted collabpotential dability to upossible. Itticularly seprogress.

Acknowled

The authChemistry,on the draftPostgradua

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. Turnese to date have yet to be fully validated. In all likelihoodmical test will not be accurate enough to determineinterval [64]. If the potential for chemical methods toostmortem interval is to be realised, there is a need tomonised analytical protocols through properly consti-orative trials. It will also be necessary to examine theifferences between human and animal models, as these human subjects on a large scale is not going to bewould be for seen that analytical chemistry, and par-paration science will be essential for these studies to

gments

ors wish to thank Dr Kathryn Linge (Department ofCurtin University of Technology) for useful commentsmanuscript. Lisa Swann is supported by an Australian

te Award.

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Analytical separations of mammalian decomposition products for forensic science: A reviewIntroductionMammalian decompositionNon-chromatographic approaches to chemical studies of decompositionChemical techniques for locating clandestine human remainsChemical techniques for the identification of adipocere

Chromatographic studies of decomposition productsVitreous humorPotassium ions (K+)Hypoxanthine (Hx)Amino acids

Decomposition productsDecomposition fluid and soil solution analysisVolatile organic compounds (VOCs)Adipocere

ConclusionsAcknowledgmentsReferences

Analytical separations of mammalian decomposition products for forensic science: A review

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