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14/15 - Rosier et al. - COV restes humains


Development and validation of a new TD-GC/MS method and its applicability in the search for human and animal decomposition products

E. Rosier, E. CuypersM. DekensR. VerplaetseW. DevelterW. Van de VoordeD. MaesJ.
Université de Leuven - Analytical and Bioanalytical Chemistry
, Volume 406, Issue 15, pp 3611–3619


Différencier entre restes humains et animaux au moyen des composés volatiles libérés au cours de la décomposition est impossible tant que reste à établir quels sont les marqueurs volatiles spécifiques à la décomposition des tissus humains. L'identification de tels marqueurs permettrait de dresser les chiens "cadavre" plus efficacement (en fait de manière assez sûre pour que l'alerte d'un chien à un endroit où un corps a été présent, mais n'est plus là soit prise en compte dans les cours de justice), en attendant que soit conçu un dispositif capable d'oeuvrer aussi bien que le chien.
Cette étude décrit le développement et la valisation d'une nouvelle méthode analytique applicable à la recherche de tels marqueurs spécifiques.

 

Introduction

In forensic science, locating the body is one of the biggest challenges for police forces, but obviously of utmost importance for the progress of the investigation. Mostly, a multidisciplinary approach is used. This approach consists of the use of a variety of forensic techniques such as forensic ecology, forensic archeology, geophysical prospection, and several other means of forensic research. One of the investigative methods used is the use of the “dog human remains”. These dogs are trained mostly with nonspecific compounds such as butan-1,4-diamine (putrescine) and pentan-1,5-diamine (cadavérine) by the Belgian Federal Police; there are only two trainers in Belgium in charge of these dogs. Frequently, trained cadaver dogs are used for the location of the buried bodies because of their olfactory capacity [1]. It may take days to find the body, while insects are attracted to the odor of decomposed bodies within minutes. Currently, it is a mystery which feature is responsible for their exceptional odor detection capacity. Depending on the decomposition stage, different insects are attracted [2]. In forensic entomology, the presence of the insects is being used to deduct the postmortem interval [3].
Remarquer que Eddie, contrairement à beaucoup de chiens "cadavre" n'a pas été dressé avec de la cadavérine ni avec de la putrescine, contrairement aux chiens belges dont il s'agit d'améliorer la fiabilité !
Several stages can be discriminated during decomposition of humans and animals [4, 5, 6]. The decomposition begins approximately 4 min after death. The first stage is the autolysis. The visible signs in this stage are fluid-filled blisters on the skin and skin slippage. Lipases, proteases, amylases, and other intracellular enzymes begin to dissolve the cells, which leads to the second stage, the putrefaction. This stage starts after approximately 48–72 h; soft tissue will decompose by the action of microorganisms (bacteria, fungi, and protozoa). A greenish discoloration of the skin appears. Gasses, liquids, and simple molecules are formed during this so-called bloating stage. When the outer layer of the skin breaks and the gasses escape, the active decay begins. Proteins, muscles, and fat break down. During the active decay, the liquefaction starts. This leads to skeletonization. Eventually, in the last stage or diagenesis, the bones degrade.
The decomposition process can be affected by many factors such as the characteristics of the body (e.g., age, body size, cause of death, clothing) or the location (e.g., temperature, moisture, type of soil, and, rodent and carnivore activity) [6].
A wide variety of volatile organic compounds (VOCs) are formed during the decomposition process. A few research groups have already studied these postmortem VOCs. They identified alkanes, alcohols, acids, esters, ketones, aldehydes, cyclic hydrocarbons, aromatic compounds, and sulphur- and nitrogen-containing compounds [7, 8, 9, 10, 11, 12, 13, 14, 15, 16]. Different stages of the decomposition were studied using human cadavers or domestic pigs as human analog. As a result, different VOCs were linked to human decomposition, but only one group (Degreeff et al.) compared VOCs from human and animal remains. Only phenylethene and methyl benzoate were found to be more specific for human remains than for animal remains [15]. Cablk et al. analyzed the VOCs of animal tissue samples and compared their results to the published results for human samples. In comparison with Hoffman et al., they found 11 human specific compounds [16]. Literature evidence on human specific decomposition marker(s) is clearly limited.
In the previously described studies, several different analytical techniques were used. Because of the high volatility of VOCs and the wide spectrum of VOCs that are released during the decay process, a suitable sampling technique is essential in order to capture as many analytes as possible. A first sampling method that was used is thermal desorption (TD) where air is drawn through a sorbent tube and the VOCs can adsorb on the sorbent in the tube [8, 9, 10, 11, 12]. A second sampling technique is solid-phase micro-extraction (SPME). In this method, a fiber is immersed in the headspace and the molecules can adsorb in a passive way on the coating [14, 16]. There are also some rarely used techniques described such as scent transfer unit [15] or sorbent cartridges [13]. For detection and identification, gas chromatography coupled to mass spectrometry (GC/MS) was mostly used [8, 9, 10, 11, 12, 14, 15, 16]; one study used two-dimensional GC coupled to time-of-flight-MS (GCxGC-TOFMS) [13]. Unfortunately, the sensitivity of the published methods is not known and no validation was published to ensure the analytical quality.
In the first step of this study, a setup for human and animal remains was made for both, in a laboratory environment as well as outdoors. Next, the sampling of VOCs was optimized. The analysis and identification of the VOCs was done using TD-GC/MS. After optimizing the setup, sampling, and analysis parameters, the method of choice was validated. In the future, VOCs that are released during the decomposition of human and animal remains will be identified with this method and (a) human specific marker(s) (qualitatively or semiquantitatively) will be searched. The first outdoor experiments were performed in collaboration with the Disaster Victim Identification (DVI) of the Federal Police (Belgium), and further experiments will be performed to search (a) human specific marker(s).
Unlike the literature described above, where no validation data were published, the goal of our work was to develop a validated, highly specific and sensitive method for the detection of VOCs released during human and animal decomposition. Setup, sampling, and analysis steps were optimized accordingly.

 

The Search for a Volatile Human Specific Marker in the Decomposition Process

E. Rosier, S. Loix, W. Develter, W. Van de Voorde, J. Tytgat, E. Cuypers
PLOS - September 16, 2015

In this study, a validated method using a thermal desorber combined with a gas chromatograph coupled to mass spectrometry was used to identify the volatile organic compounds released during decomposition of 6 human and 26 animal remains in a laboratory environment during a period of 6 months. 452 compounds were identified. Among them a human specific marker was sought using principle component analysis. We found a combination of 8 compounds (ethyl propionate, propyl propionate, propyl butyrate, ethyl pentanoate, pyridine, diethyl disulfide, methyl(methylthio)ethyl disulfide and 3-methylthio-1-propanol) that led to the distinction of human and pig remains from other animal remains. Furthermore, it was possible to separate the pig remains from human remains based on 5 esters (3-methylbutyl pentanoate, 3-methylbutyl 3-methylbutyrate, 3-methylbutyl 2-methylbutyrate, butyl pentanoate and propyl hexanoate). Further research in the field with full bodies has to corroborate these results and search for one or more human specific markers. These markers would allow a more efficiently training of cadaver dogs or portable detection devices could be developed. 

Introduction

During the decomposition of human and animal remains, a wide spectrum of volatile organic compounds (VOCs) is emitted in the environment. The past few years, the research to characterize this ‘smell of death’ has increased and a wide variety of compounds has already been identified: alkanes, alcohols, acids, esters, ketones, aldehydes, cyclic hydrocarbons, aromatic, sulphur- and nitrogen-containing compounds [1–19]. Pig remains are often used as human analogues (Table 1) because of their similarity in hair coverage, weight, fat to muscle ratio, gut fauna and biochemistry [5, 16, 20]. However, the VOC-profiles of human and animal remains were hardly compared, notwithstanding the fact that they could be interesting to find a human specific marker. The following research groups compared human and animal remains. Degreeff et al. reported that phenylethene and methyl benzoate were more specific for human than animal remains [3]. Cablk et al. compared their experimental results of animal remains with literature results of human remains. They found 11 compounds published on human research which they could not detect in their animal study [2]. Vass suggested that carbon tetrachloride, pentane, decane and undecane appeared to be human specific. Additionally, he saw that 2-methylbutanal was always greater than 3-methylbutanal in the animal remains he studied (pig, deer, dog, cat, squirrel and sheep). However, in human remains he noted that this phenomenon was reversed or that both compounds were equal to each other [19]. Clearly, there are still inconsistencies in literature of the human specific compounds and more research has to be done.

Ultimately, a variety of forensic disciplines could benefit from these human specific markers. Mainly in the search of human bodies or remains. Thanks to their good olfactory capacity, cadaver dogs are able to locate bodies [17, 21]. At this moment, mostly nonspecific compounds such as cadaverine and putrescine are used to train these dogs. They can find human cadavers with this training, but the use of artificial scents is highly debated. Cadaver dogs trained with these scents did not always react on real cadaver samples (data derived from dog handlers of the Federal Police in Belgium). Training aids appear to be an oversimplification of the decomposition odor [22]. A human specific marker can be used to train cadaver dogs more efficiently and therefore win time to locate a body. Moreover, when a human specific marker is found, it might be possible to develop a portable device that is sensitive enough to locate human remains.

The decomposition can be influenced by many environmental factors such as temperature, humidity, soil type, submersion of the body [23]. Therefore, it is difficult to compare results of research groups that study the decomposing remains outdoors. In this study, we sampled the headspace of 6 human and 26 animal remains that decomposed for 6 months. This study was conducted in glass jars in laboratory environment to pre-concentrate and therefore easily sample the released VOCs. It is also a manner to standardize the methodology with control of the parameters such as temperature and moisture, as much as possible. These samples were collected and analyzed with a validated method using thermal desorber combined with gas chromatography coupled to mass spectrometry (TD-GC/MS) [12]. When the VOC-profiles of human and animal remains were identified, principal component analysis (PCA) was applied on the results to search for (a) human specific marker(s). The aim of our study was to identify VOCs specific for human decomposition.