Rom J Leg Med [28] 398-402 [2020]

DOI: 10.4323/rjlm.2020.398
© 2020 Romanian Society of Legal Medicine FORENSIC TOXICOLOGY

TOXICOLOGICAL STUDY OF AN EXHUMED BODY USING ALTERNATIVE MATRICES FOR

THE DETECTION OF PSYCHOACTIVE DRUGS

Cristina Pérez-Martínez1,*, Lucía Fernández-López1, Gemma Prieto-Bonete1, Aurelio Luna1

University of Murcia, School of Medicine, Regional Campus of International Excellence “Campus Mare Nostrum”,
Department of Legal and Forensic Medicine, Murcia, Spain

Abstract: In forensic toxicology, systematic toxicological analyses (STA) and interpretation of results are difficult
when routine matrices such as blood (typically from a central and peripheral source), urine, bile, vitreous humor, stomach or
gastric contents are not available. Furthermore, in instances of limited remains, only muscle, hair, or a skeleton may be available
for toxicological examination to unambiguously identify as many toxicologically relevant compounds as possible to assist in
determining the cause of death. This article describes a case of the exhumed corpse of a 53-year-old male known to have
been addicted to several types of drugs. The STA was carried out on alternative biological matrices, including brain, kidney
and bone marrow, using gas chromatography–mass spectrometry (GC-MS) and high-performance liquid chromatography–
mass spectrometry (HPLC-MS) and subsequently the identified substances were quantified by GC-MS and ultra-performance
liquid chromatography–tandem mass spectrometry (UPLC-MS/MS). The mixture found included multiple agents such as
barbiturates, benzodiazepines (BZD) and anaesthetic agent. We demonstrate the possibility of detection of several drugs in an
exhumed body 2 years after interment. Despite the problematic of interpreting the quantitative toxicological results, the data
could provide complementary information about aetiology of death.

Keywords: forensic toxicology, systematic toxicological analyses, exhumed corpse, alternative biological matrices,
gas chromatography–mass spectrometry, high performance liquid chromatography–mass spectrometry, ultra-performance
liquid chromatography–tandem mass spectrometry, drugs.

INTRODUCTION or corpses buried and later, and after a while, exhumed

[8], due to the uncertainty of finding evidences after a
Forensic toxicology is aiming to detect given post-mortem interval (PMI) [9].
possible xenobiotics, drugs and other potentially toxic Peripheral and cardiac blood, urine, vitreous
compounds in biological autopsy samples, quantify humour, bile, stomach or gastric contents are the
the concentrations of relevant and main compounds typical biological samples collected at autopsy [10-13].
found in samples, and contribute to the investigation However, in cases where routine specimens cannot
of the manner of death [1, 2]. In order to contribute to be analysed due to putrefaction or skeletonization
the understanding and assessment of these test results [12] or in cases when forensic autopsy cannot collect
in a medical-legal context [3], the use of appropriate adequate blood specimens [14], alternative matrices
analytical methods, tools, the technical knowledge of such as brain, kidney, liver, bone marrow and bone are
forensic toxicologists, etc. are necessary [4, 5]. In today’s useful for diagnostic purposes [3, 13]. But care must
society, medical and recreational drugs represent most be taken, because high drug concentrations in blood
of the compounds that cause fatal poisonings [1]. may increase further during the post-mortem period,
The interpretation of the results of a providing erroneous results: in such cases, the use of
toxicological analysis in post-mortem cases alternative specimens (such as brain, kidney, marrow
is complicated [6, 7], especially in the case of bone, bone, etc.) will help improve forensic research
decomposing, poorly preserved, skeletonized bodies and hopefully lead to a more complete understanding

*Correspondence to: Cristina Pérez-Martínez, PhD, University of Murcia, School of Medicine, Regional Campus of International Excellence
“Campus Mare Nostrum”, Department of Legal and Forensic Medicine, E-30100, Murcia, Spain, E-mail: cristina.perez.mtnz@gmail.com
398
 Toxicological study of an exhumed body using alternative matrices for the detection of psychoactive drugs

of any drug-related event [3]. 009) were obtained from Cerilliant (Round Rock, TX).
Besides variations in the type of sample, it The analytical and chromatographic solvents used were
has been found that the concentrations of drugs in acquired from Sigma Aldrich.
tissues, organs or body fluids can vary significantly
between death and the collection of the specimen Standard and post-mortem samples
[4]. Consequently, several factors must be considered preparation
such as the drug distribution and transformation Regression lines were assessed based on peak
in the perimortem and post-mortem states [15-23], areas ratios of each of the 5 compounds to that of the
the degradation of drugs, accidental or endogenous standards. The method showed linearity, expressed by
production and contamination, which can depend regression coefficient (R2) of above 0.975 in all cases.
on the intake routes and the chemical properties of The LOQ was found to be between 0.005 and 0.01
individual drugs and poisons [24-32]. All this makes it µg/mL depending on the analyte, and the LOD was
difficult to interpret post-mortem levels of drugs. between 0.0008 and 0.05 µg/mL.
In any case, in addition to taking into account Duplicate biological post-mortem samples
the type of sample, the conditions of the sample and were homogenized using an IKA® ULTRA-TURRAX®
the variations that may occur in the concentrations of disperser (Z404519, Sigma Aldrich). The homogenates
the drugs, other factors must be considered such as were subjected to a drugs extraction procedure. The
the scope of the analyses, the suitability of the sample procedure described by Yawney et al. [35] was carried
for the experiments, the stability or otherwise of the out in brain, kidney and liver post-mortem samples;
substances detected and other factors that influence while the protocol described by McIntyre et al. [36]
the variation of the concentrations in the samples [6, was followed in case of the bone marrow samples. The
11, 33-34]. Therefore, despite obtaining positive results extracts obtained from the processing of the samples
in the detection and quantifying drugs in biological were evaporated in nitrogen stream and reconstituted in
samples obtained from an autopsy, it is complicated 60 µl of mobile phase. Control and calibration standards
and sometimes not possible to interpret the results and were prepared by spiking methanol with standards at
extrapolate them to the antemortem state of the subject. concentrations ranging from 0.005 µg/g to 5 µg/g.
This article reports the use of alternative matrices
in post-mortem toxicological analysis in a case of Toxicological analysis
suspected multi-drug abuse death of a patient with The screening of illicit and therapeutic drugs
possible addiction habits. was performed by GC-MS and HPLC-MS after liquid-
liquid extraction (LLE) and the addition of 25 µl of IS.
CASE REPORT The determination of concentrations of compounds
such as propofol and pentobarbital was carried out by
A 53-year-old male was exhumed 2 years after GC–MS and UPLC-MS/MS were used for midazolam.
death following circumstantial proof and evidence The toxins were confirmed by comparing the retention
pointing to possible intentional intoxication. Despite times and mass spectra characteristics of the peaks with
the long burial period, the body was well preserved, those of standards and were quantified by means of
although it was not possible to collect routine biological external calibration curves.
samples. Toxicological analyses were performed on
bone marrow (20.1 g), brain (125 g) and kidney (48 RESULTS
g) at the Legal and Forensic Medicine Department of
the University of Murcia (External Service for Forensic General toxicological screening for common
Science and Techniques (SECYTEF). Analysis of both drugs performed in the alternative matrices available
illicit and therapeutic drugs were called for. - bone marrow, brain and kidney - revealed the
presence of the anaesthetic propofol, the barbiturate
Materials, standards and chemicals pentobarbital and BZD midazolam. These drugs were
Based on the results obtained using GC-MS confirmed and quantified as shown in Table 1 and
and HPLC-MS, the drugs with the highest toxicological since the natural formation of this kind of compound
relevance and deuterium-labeled standards were can be ruled out, the observations clearly pointed to
applied for Midazolam (M-908), Diazepam-D5 (D- the antemortem intake of propofol, pentobarbital and
902), Propofol-D17 (P-077) and Pentobarbital-D5 (P- midazolam.

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 Pérez-Martínez C. et al.

Table 1. Summary of findings of toxicological analyses.

Biological material
Analyte Bone marrow ppm (µg/g) Brain ppm (µg/g) Kidney ppm (µg/g)
Pentobarbital 0.2398 n.d. 0.0445
Propofol n.d. n.d. 0.8123
Midazolam 0.0055 0.2190 0.1050
n.d.: Not detected.

Our results evidenced of the existence of drug deposits after death [46], PMI, sample type,
propofol at a concentration of 0.8123 µg/g in the kidney dissemination, redistribution in body cavities and
sample, which may be due to rapid redistribution and drug metabolism after death [47, 48]. In addition, it is
metabolism by conjugation in the liver to glucuronide necessary to add the characteristics of the medications
and sulfate inactive metabolites, which are excreted by themselves - including lipid solubility, plasma protein
the kidney with elimination half-life of 23 hours [37]. binding and molecular size, all of which influence the
The amount of pentobarbital found in the volume of distribution [40].
bone marrow sample was of 0.2398 μg/g and in kidney Bibliographic references found on toxicological
sample a concentration of 0.0445 μg/g. It was found in studies in non-conventional samples of exhumed
the kidney sample because is cleared by the kidneys [38] corpses are scarce. The matrices used in these works
and its chemical stability, which allows it to be stored in have been putrefied material (abdominal area, muscles,
the bone marrow. This also may reflect chronicity since etc.), skin, tissue, hair, nail, ulna and radius; the range
it was not found in other samples, but further studies are of the PMI is between 10 days and 4.5 years and the
needed to determine aspects such as the dose required compounds that had been detected are opiates, BZD,
and the frequency of use of the drug necessary for it to morphine and 6-acetylmorphine, and other types of
be deposited in the bone [39]. drugs [8, 42, 49-51].
Midazolam was detected in three matrices, The most recent toxicological investigations
finding the highest concentration in brain (0.2190 μg/g) carried out into accidental deaths, suicides, homicides
possibly due to its high lipophilicity at physiological and cases of drug-facilitated sexual assault involving
pH, which explains for midazolam’s rapid absorption the compounds identified in our case (propofol,
and crossing of the blood-brain barrier [40, 41] BZDs pentobarbital and midazolam) showed higher
and their metabolites are distributed throughout the concentrations in normal matrices such as femoral,
body and accumulate mainly in lipid-rich areas such as cardiac and peripheral blood, head and pubic hair,
adipose tissue and the central nervous system, where it urine and tissue samples of medico-legal autopsies [43,
exerts its action [42, 43]. On the other hand, we found 52-71] than our results.
this compound in lower concentrations in the kidney Interpretation of the toxicological results
(0.1050 μg/g) coinciding with other study which reports obtained in different biological matrices of an exhumed
that it finally goes to the excretory organs [44]. corpse 2 years after death is difficult because the
processes of decomposition and putrefaction introduce
DISCUSSION substantial changes in the biological material.8 In
addition to the changes in the matrices, we found
The tasks carried out by forensic sciences substantial changes in the structure and composition
such as the identification and analysis of remains, of the drugs and toxicological problems arise as a
especially when it comes to decomposed or incomplete consequence of the instability of the chemical in this
bodies, is a complicated job [45]. Among the tests type of situation [72-74]. Together with the absence of
to be performed, we find the toxicological studies, routine material (blood, urine or bile), this may lead
which must be interpreted with great caution because to misinterpretation [72]. Despite these caveats, data
the pharmacodynamic and kinetic properties of the from this study point to the relevance of toxicological
compounds according to the different matrices are not analyses of alternative matrices to complement forensic
clearly known. On the other hand, the concentrations investigation.
found in biological samples will also depend on several In conclusion, the detected substances,
factors, which will cause them to be modified, such as Midazolam, a type IV sedative agent of moderate
post-mortem redistribution [4], which is affected by action, would has a synergistic effect when
cell death, decomposition, passive drug release from administered with propofol (used as an intravenous
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