Chromatography 2014, 1, 120-140; doi:10.

3390/chromatography1030120
OPEN ACCESS

chromatography
ISSN 2227-9075
www.mdpi.com/journal/chromatography
Article

A Longitudinal Study of Decomposition Odour in Soil Using

Sorbent Tubes and Solid Phase Microextraction
Katelynn A. Perrault *, Barbara H. Stuart and Shari L. Forbes

Centre for Forensic Science, University of Technology, Sydney, PO Box 123, Broadway, NSW 2007,
Australia; E-Mails: [email protected] (B.H.S.); [email protected] (S.L.F.)

* Author to whom correspondence should be addressed; E-Mail: [email protected];

Tel.: +61-2-9514-2000.

Received: 27 May 2014; in revised form: 14 July 2014 / Accepted: 25 July 2014 /
Published: 30 July 2014

Abstract: Odour profiling of decomposed remains is important for understanding the

mechanisms that cadaver dogs and forensically-relevant insects use to locate decomposed
remains. The decomposition odour profile is complex and has been documented in outdoor
terrestrial environments. The purpose of this study was to perform longitudinal analysis of
the volatile organic compound (VOC) profile in soils associated with decomposed remains
across all stages of decomposition. Two VOC collection techniques (sorbent tubes and
solid phase microextraction) were used to collect a wider analyte range and to investigate
differences in collection techniques. Pig carcasses were placed in an outdoor research
facility in Australia to model the decomposition process and VOCs were collected
intermittently over two months. VOCs of interest were identified over the duration of the
trial, showing distinct trends in compound evolution and disappearance. The collection
techniques were complementary, representing different subsets of VOCs from the overall
profile. Sorbent tubes collected more decomposition-specific VOCs and these compounds
were more effective at characterising the matrix over an extended period. Using both
collection techniques improves the likelihood of identifying the complete VOC profile of
decomposition odour. Such information is important for the search and recovery of victim
remains in various stages of decomposition.

Keywords: volatile organic compounds; decomposition chemistry; gas chromatography-mass

spectrometry; GC-MS; human remains search; VOC collection
Chromatography 2014, 1 121

1. Introduction

During cadaver decomposition a variety of chemical and biological processes contribute to the
evolution of odorous chemicals known as volatile organic compounds (VOCs). The resulting
decomposition VOC profile has important implications in the search and recovery of victims and in
subsequent post-mortem interval (PMI) determination. Forensic search procedures for missing persons
or victims of homicide employ the use of detection dogs [1–4] or handheld detection devices [5–8] that
rely on decomposition VOC recognition. These can also be used in disaster victim recovery where
speed and accuracy are paramount concerns [6,8]. VOCs released during decomposition attract a
predictable pattern of arthropods (referred to as insect succession) to deceased victims, which can
further be used to estimate PMI in forensic casework [9,10]. These areas of forensic practice rely on
the production of volatile organic compounds from a victim. Thus, establishing an accurate
decomposition VOC profile can provide information for improving best practices.
Decomposition odour research over the past ten years has elucidated key consistencies in the
decomposition VOC profile. These trends have been well-documented in [1,3–6,11–20]. Inconsistencies
exist in the VOC profiles reported in published literature, which reflects variations in decomposition
variables (environment, weather, soil type, cadaver/carcass size, geographical location etc.) and/or
analytical methods (collection technique, instrument used, instrument parameters, etc.) used in these
studies. Variation-inducing factors are often associated with outdoor decomposition environments
involving soil. Leached decomposition by-products entering soil exhibit different retention based on
soil characteristics such as temperature, texture, moisture and humidity [4]. Soil can be thought of as a
sink for VOCs where a chemical can coexist in a liquid state between soil particles, as a vapour in the
soil gas, or as a solid sorbed onto soil particles [21]. In addition, leached nutrients can impact
components of the soil community such as microorganisms. Soil organisms produce volatile metabolic
by-products that can be released into the soil thereby further impacting the soil’s VOC profile [5].
Decomposition VOCs released directly into the atmosphere from decomposed remains have been
considered previously in the literature [1,6,14–20,22]; however there are few studies that have
investigated the VOC profile from the soil beneath decomposing remains. A range of collection
techniques, conditions, and instrumentation have been used in decomposition soil VOC studies. Both
Vass [4] and Brasseur et al. [11] identified a reduced number of VOCs compared to traditional
decomposition VOC studies that investigated the headspace above remains. However, Forbes and
Perrault [22] showed that a direct comparison between headspace and soil from the same remains
demonstrated additional compounds in the soil.
The decomposition process changes over time resulting in a dynamic VOC profile. Trends in VOC
evolution during early decomposition (days or weeks) differ from trends in later decomposition
(months or years). While longitudinal VOC studies have been reported for the headspace above
decomposed remains, this has not been a focus for decomposition soil analysis. Longitudinal analysis
involves the repeated measurement of a response over a period of time. Without longitudinal
measurements the dynamicity of VOC production and soil interactions are not portrayed because
components of the profile may not be present at the chosen point of analysis. This may reveal the cause
for some studies citing a reduced decomposition VOC presence in soils [4,11].
Chromatography 2014, 1 122

Collection techniques used for decomposition VOCs also impact the number and types of VOCs
captured and detected. Sorbent-based methods such as sorbent tubes [3,11,17–20] and solid phase
microextraction (SPME) [1,2,12,17] are commonly employed in the field of decomposition VOC
research due to their widespread and standardised use for environmental monitoring. General ranges
for sorbent specificity are often indicated by manufacturers and in the literature, yet the sorbents
commercially available for each technique are not equivalent to each other. The prevalence of
sorbent-based methods in decomposition odour analysis prompts the issue of potential bias of results
associated with the use of a single collection technique or sorbent. Choice of sample collection
technique is often based on availability of technique, suitability with laboratory instrumentation, and/or
amenability to field usage. These two techniques are also used in two different manners. SPME is a
passive technique relying on the establishment of equilibrium between the sample headspace and the
sorbent. Sorbent tubes can be used in either a passive or dynamic manner, however most publications
in this area have used sorbent tubes dynamically via pumped sampling. The known differences in the
sample collection techniques and their associated sorbents will lead to further discrepancies in the
results obtained.
The aim of this study was to characterise the soil VOC profile longitudinally (i.e., at various
intervals throughout the decomposition process) using two VOC collection techniques on the same
odour source. A characterisation of the VOC profile collected using sorbent tubes and SPME fibres
was desired to determine the superiority or complementarity of the techniques, and to provide a more
comprehensive overall VOC profile for decomposition soil. These collection techniques have only
been used independently in previously documented studies without comment on how collection technique
has impacted analytical results. This study aimed to bridge these gaps in analytical knowledge.

2. Experimental Section

2.1. Field Setup

In order to characterise the VOC profile throughout the entire decomposition process, a field trial
was conducted using four pig (Sus scrofa domesticus) carcasses killed by captive headbolt. Pigs are
considered to be suitable models for decomposition studies due to their internal anatomy, fat distribution,
and gut flora [23]. While this is true, their use as human odour analogues is still not conclusive and
literature does not exist providing a direct comparison of odour produced by whole body decomposition
to whole pig carcass decomposition in the field. Although we are aware that minor differences in
decomposition VOCs may exist between the two species [4,12], a literature search shows that the core
VOCs found from decomposing human and pig remains are representative [4,12,22,24,25].
The pig carcasses each weighed approximately 70 kg (to mimic average human body weight) and
were placed directly on the soil surface at a field research facility in Sydney, Australia. Surface
deposition was used in this study due to the common occurrence of aerobic decomposition
environments in homicide and missing persons’ cases. Especially in the case of missing persons, death
may occur during bushwalking or while lost, resulting in surface deposition of the remains. The site is
classified as open eucalypt woodland and possessed sandy clay topsoil. The vegetation at the site was
dominated by various eucalypt trees (Eucalyptus punctata, Eucalyptus eugenoides, Eucalyptus fibrosa
Chromatography 2014, 1 123

ssp. fibrosa), lantana shrubs (Lantana camara), large-leaved rush (Lomanda longifolia) and kangaroo
grass (Themeda australis).
Four additional sites with no carcasses were designated as control sites to monitor fluctuations in
the natural odour profile of the area. Samples were collected periodically during a two month period
(15 January–15 March 2013). Weather variables were recorded using a HOBO® U30 No Remote
Communication (NRC) data logger (OneTemp, Marleston) in close proximity to the carcasses. Hourly
measurements were taken using Hobo® sensors for ambient temperature (°C), relative humidity (%),
solar radiation (W/m2), wind speed (m/s), wind direction (ø) and rainfall (mm). Soil pH and volumetric
water content were measured using a direct soil pH measurement kit (Hanna Instruments) and a soil
moisture sensor (Vernier, LabQuest 2 Interface).

2.2. Sorbent Tubes

Active sampling onto sorbent tubes was performed in situ using 30 cm VOC-Mole™ soil probes
(Markes International Ltd.). In situ sampling with this equipment allowed for the collection of highly
volatile compounds that may otherwise be lost during sample transport and storage. The VOC-Mole™
soil probe was placed into the ground near the torso of each pig carcass at the beginning of the trial and
a sorbent tube was affixed to the cap of the probe during sample collections. The probe contains
subsurface holes that allow the soil vapour phase to diffuse into the probe. Pumped sampling was
performed using a Field and Laboratory Emission Cell (FLEC™). A Tenax TA/Carbograph 5TD
sorbent tube (Markes International Ltd.) was attached to the end cap of the VOC-Mole™ soil probe
and air was drawn from the soil probe through the sampling tube using the air pump. A sample was
collected at a rate of 100 mL/min for 30 min from the VOC-Mole™ soil probe at each experimental
site (with carcass) and control site (no carcass). Sorbent choice was determined based on manufacturer’s
specifications. The range of compounds targeted using this sorbent combination is comparable to
previous research [3,19,20]. Sample contamination and/or loss during transportation and storage was
minimised using brass long-term storage caps according to product usage specifications by the
manufacturer and according to the use of sorbent tubes in previous studies [3,24,25]. Tubes were
capped, wrapped in aluminium foil and stored in an air-tight, sealed glass jar for transport to and from
the field site. Select compounds collected on sorbent tubes have been previously validated in literature
for stability up to 4 days [25] and were analysed within 48 h from collection. Internal standard
injection of 5 µL of 20 ng/µL bromobenzene (GC grade, Sigma Aldrich) in methanol (HPLC grade,
Sigma Aldrich) was performed using an automated syringe pipette. A Unity 2™ Thermal Desorber
(Markes International Ltd.) was used to desorb VOCs for analysis. Each tube was purged under dry
nitrogen for 1 min, followed by thermal desorption at 300 °C for 4 min under a reverse flow of helium
carrier gas. Desorbed VOCs were collected on a general purpose cold trap at −10 °C containing a
dual-sorbent bed of Tenax TA/Carbograph 1TD. Cold trap desorption was performed for 3 min at 300 °C.
A flow path temperature of 120 °C was used. A split flow of 20 mL/min was used at the cold trap.

2.3. SPME

SPME is less amenable to in situ sampling due to the difficulties associated with sealing the fibre
from external contamination during transport and storage. The SPME fibres are fragile and can be
Chromatography 2014, 1 124

damaged in the field (i.e., for analyzing soil in situ), particularly when sampling in adverse conditions.
Field portable SPME devices are available but were not cost efficient based on the number of daily
replicates required for the study. Field or mobile laboratory access is not always immediately available
meaning that VOC extraction must be performed back in the laboratory. Ex situ sampling was
conducted on approximately 1 g of experimental and control soils collected from each site. Soils were
collected in an airtight 20 mL headspace vial and sealed with a magnetic screw cap containing a
polytetrafluoroethylene/silicone septum of 1.3 mm thickness.
Ideally, SPME sorbent phase would be identical to sorbent tubes. However, commercially available
sorbents between the two techniques are not the same and therefore SPME fibre selection needed to be
performed. SPME fibre choice was assessed using reference decomposition soil in the lab prior to
analysis of samples. Fibres tested were Polydimethylsiloxane (PDMS), PDMS/Divinylbenzene
(PDMS/DVB), and PDMS/DVB/Carboxen (PDMS/DVB/CAR). A PDMS/DVB fibre (65 µm, 24 ga,
Supelco) was chosen based on literature examples [1,2,12] and lab confirmation which allowed for the
widest possible range of compounds to be collected in test samples. While the sorbents used with each
collection technique differed, this was thought to represent how these techniques have been used in
previous literature.
The fibre was exposed in each headspace vial for 20 min at 40 °C. Increasing the length of exposure
beyond 20 min provided no additional benefit. Heating the vials to 40 °C enhanced the release of
volatiles that naturally adhere to soil yet maintained a temperature within range of what cadaver dog,
handheld devices, or portable instrumentation could reasonably be exposed to in practice. Prior to
exposure, each headspace vial was injected with 5 µL of 20 ng/µL bromobenzene (GC grade, Sigma
Aldrich) in methanol (HPLC grade, Sigma Aldrich). Vials were placed in a dry bath heating block
during exposure which allowed for controlled and uniform heating of the soil sample. Fibres were
desorbed for 5 min in the inlet of the gas chromatograph at 150 °C using a 0.75 mm I.D. SPME
injection sleeve in splitless mode.

2.4. Gas Chromatography-Mass Spectrometry (GC-MS) Analysis and Data Processing

A DB-VRX capillary column was used (30 m × 0.250 mm ID and 1.4 µm film thickness) inside of
an Agilent 6890N GC coupled to a 5973N Mass Selective Detector. A helium carrier gas flow rate of
1.0 mL/min was used. The oven temperature was held at 35 °C for 4 min and then increased by
3 °C/min to 80 °C. The temperature was further increased to 120 °C at a rate of 10 °C/min and finally
increased to a temperature of 240 °C at 40 °C/min and held for 4 min. The transfer line and source
temperatures were held at 230 °C and 280 °C, respectively, and the MSD was operated in full electron
ionization (EI) scan mode from 40 to 450 m/z.
Verification of mass spectral identifications and retention time was completed using standards for
32 compounds of interest; heptane, undecane, 2-methylpentane, 1-pentanol, 2-ethyl-1-hexanol, ethanol,
1-hexanol, hexanal, nonanal, heptanal, octanal, 2-heptanone, 2-pentanone, 2-butanone, 2-nonanone,
p-xylene, toluene, ethyl benzene, naphthalene, benzene, o-xylene, styrene, tetrachloroethylene,
dimethyl disulfide, dimethyl sulfide, indole, pyridine, putrescine, cadaverine, hexanoic acid, propanoic
acid, and butanoic acid. Although standards could not be analysed for every compound, this list of
compounds was thought to represent the range of compounds expected based on a literature search.
Chromatography 2014, 1 125

A minimum match threshold of 70% to the NIST library was used for compound identification, in
addition to retention time comparison to standards where possible. A list of compounds of interest was
generated from the decomposition soil samples for each experimental day by removing compounds at
comparable levels in controls.
Each compound was classified as being a(n) sulphur-containing compound, nitrogen-containing
compound, aromatic, carboxylic acid, ester, aldehyde, ketone, alcohol, hydrocarbon, and/or other.
Multiple classifications were assigned where required. Internal standard normalisation was performed
to provide the relative amount of each compound present on the sampling tube. Internal standard
precision was tested (n = 5 on 3 days) and was below 8% for sorbent tubes and below 11.5% for SPME;
therefore it was deemed suitable to be used for providing a measure of relative quantification. The four
experimental sites were treated as replicates and the average of each VOC’s abundance was taken
between pig carcasses. The averages were then summed within each class to provide data to be used
for multivariate analysis. Principal component analysis (PCA) was performed based on the sum of the
normalised peak areas in each chemical class using The Unscrambler X version 10.3 (CAMO Software).

3. Results

3.1. Carcass Decomposition

All pig carcasses decomposed at similar rates throughout the study and were classified based on
the stages of decomposition adapted from those first described by Payne [26]. Accumulated degree
days (ADD) was used to represent time to account for the effect of temperature on the rate of
decomposition [27]. ADD was calculated by summing the average daily temperature (°C) measured by
the weather station at the research facility. The fresh stage immediately followed death until the onset
of bloat on day 2 (48.16 ADD) when distension of the torso and skin discoloration were observed.
Putrefaction, liquefaction of tissues, and tissue digestion by insects were characteristic of active decay
which began on day 4 (106.95 ADD). Differential decomposition occurred in the later stages
(advanced decay and skeletonisation) where different regions of the carcass exhibited different stages
of decomposition at the same time. Localised areas of advanced decay (reduced soft tissue presence
and insect activity) began on day 10 (244.28 ADD) and complete advanced decay was seen by day 14
(340.85 ADD). Regions of bone exposure were observed from day 10 onwards (244.28 ADD).
The skeletonised stage was characterised beyond day 17 (413.73 ADD) when the majority of the
carcasses’ skeletal structure was exposed and remaining tissue had mummified. The study concluded
on day 59 (1315.46 ADD).
The average overall temperature during the trial was 22.3 °C with an average daily temperature
between 16.0 °C to 31.8 °C. The total cumulative rainfall was recorded as 324.2 mm. There was no
rainfall during days 0–11, creating a dry initial environment that promoted the onset of
mummification. Desiccation of soft tissue initiated during the active decay stage and all subsequent
stages exhibited varied extents of mummification. Although a large amount of rainfall was experienced
during the season, it occurred in short periods with more than 50% of the total rainfall occurring on the
combination of days 13, 39, 45. Moisture released during decomposition tended to increase the soil’s
volumetric water content (VWC), however a stronger correlation to heavy rainfall was seen. The site
Chromatography 2014, 1 126

experienced an average humidity of 81% and average solar radiation of 93.7 Watts per square metre
(W/m2). Average wind speed was negligible (0.009 m/s). Soil at the site exhibited a naturally acidic pH
of 5–6 whereas carcass enrichment caused a basic shift in pH (above 7) beyond day 10.

3.2. Decomposition VOC Database

A compiled list of compounds present in at least two of the carcasses during each measured day is
represented in Table 1. Of these, 47 VOCs were only identified using sorbent tubes, 48 VOCs were
only identified using SPME, and 36 compounds were identified by both techniques. The compounds
found in common between the techniques varied across compound classes. Relative quantitative data is
available for each compound class in the supplementary information deposit.
Sulfur-containing compounds and nitrogen-containing compounds were identified predominantly
using sorbent tubes (Figure 1). The major compounds in these two categories were dimethyl disulfide,
dimethyl trisulfide, trimethylamine, 3-methylpyridine, and indole. These compounds were identified
throughout the majority of decomposition. Sorbent tubes more frequently collected oxygenated
compounds in low carbon ranges when compared to SPME. Short chain esters (C3 and most C4),
short chain ketones (C4–C6), short chain alcohols (C2–C3 and most C4), and short chain aldehydes
(3-methylbutanal) were identified predominantly when using sorbent tubes and were identified
throughout the full duration of decomposition.

Figure 1. Number of compounds identified by each collection technique by individual

chemical classes over the entire duration of decomposition.
Chromatography 2014, 1 127

Table 1. Decomposition volatile organic compounds (VOCs) identified in soil using sorbent tubes (■) and solid phase microextraction
(SPME) (□) for collection (n ≥ 2). Literature references to decomposition odour research are given for each compound. Asterisks (*) mark
compounds previously identified by literature in decomposition soils.
Day 2 Day 4 Day 6 Day 8 Day 10 Day 14 Day 17 Day 21 Day 24 Day 31 Day 45 Day 59
48.2 ADD 107.0 ADD 149.6 ADD 196.8 ADD 244.3 ADD 340.9 ADD 413.7 ADD 490.5 ADD 557.8 ADD 712.62 ADD 1078.5 ADD 1315.5 ADD
Sulfur-containing
Dimethyl sulfide * [1,4,14,17–20,28] ■ ■
Dimethyl disulfide * [1,2,11,14,17–20,28] ■/□ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■
Dimethyl trisulfide * [11,13,14,17,18,20,28] ■/□ ■ ■ ■/□ ■ ■ ■ ■ ■ ■ ■
2,4-dithiapentane [28] ■
3-methylthiophene [14] ■
Nitrogen-containing
Trimethylamine [2,3,6,12,14] ■ ■ ■ ■ ■ ■ ■
3-methylpyridine [14] ■ ■ ■ ■ ■ ■
Benzonitrile * [3,11,14,15,19] ■
Benzylnitrile [14] ■
Aromatics
Indole * [1,2,12,15,16] ■ ■ ■/□ ■/□ ■ ■/□ ■/□ ■
4-methylindole □
2-ethylfuran [17] ■ ■
2-butylfuran ■
2-pentylfuran [1,2,12] □ ■/□ ■/□ ■/□ ■/□ ■/□ ■/□ □ ■/□ ■/□ ■/□ ■/□
2-heptylfuran □ □ □ □ □
2-methyltetrahydrofuran ■ ■ ■ ■ ■ ■
2-butyltetrahydrofuran ■
Carboxylic Acids
Acetic acid [3,6,13] ■ □ □
Propanoic acid [1–3,16] □ □ □
2-methylpropanoic acid [14,16,17] □ □ □
Butanoic acid [1–3,14,15] □ □ ■/□ □ □
2-methylbutanoic acid [3,12,14,15] □ □ □ □ □ □ □ □ □
3-methylbutanoic acid [3,6,12,14,15] □ □ □ □ □ □ □ □ □
Pentanoic acid [1–3,14,16] □ □
4-methylpentanoic acid □ □ □ □ ■
Hexanoic acid [1–3,14,16] □ □ □ □ □
2-methylhexanoic acid [12] □ □
Benzoic acid [14] ■
Chromatography 2014, 1 128

Table 1. Cont.
Day 2 Day 4 Day 6 Day 8 Day 10 Day 14 Day 17 Day 21 Day 24 Day 31 Day 45 Day 59
48.2 ADD 107.0 ADD 149.6 ADD 196.8 ADD 244.3 ADD 340.9 ADD 413.7 ADD 490.5 ADD 557.8 ADD 712.62 ADD 1078.5 ADD 1315.5 ADD
Esters
Acetic acid, methyl ester ■ ■
Acetic acid, ethyl ester ■ ■
Acetic acid, propyl ester [14,17] ■
Propanoic acid, 2-methyl-, ethyl ester [17] ■
Propanoic acid, ethyl ester ■
Butanoic acid, 1-methyl-, ethyl ester ■ ■ ■
Butanoic acid, ethyl ester [1,17] ■ ■
Butanoic acid, 2-methyl-, ethyl ester [17] ■
Butanoic acid, 2-methyl-, pentyl ester □ □
Butanoic acid, 3-methyl-, butyl ester [14] ■/□ □
Butanoic acid, 3-methyl-, methyl ester □ □
Butanoic acid, methyl ester [14] □ ■/□ ■/□ ■
Butanoic acid, propyl ester ■ ■ ■
Butanoic acid, butyl ester [1,14] ■ ■
Butanoic acid, pentyl ester □
Butanoic acid, hexyl ester □ □ □
Butanoic acid, octyl ester □ □
Pentanoic acid, ethyl ester ■ ■ ■
Pentanoic acid, methyl ester □ □
Pentanoic acid, 4-methyl-, methyl ester □
Hexanoic acid, methyl ester □ □ □ □ □ □ □ □ □
Hexanoic acid, ethyl ester [1] □ □
Hexanoic acid, pentyl ester [1] □
Heptanoic acid, methyl ester □ □ □ □
Octanoic acid, methyl ester [12] □ □ □
Nonanoic acid, methyl ester □ □
Phenylpropanoic acid, methyl ester □
Chromatography 2014, 1 129

Table 1. Cont.
Day 2 Day 4 Day 6 Day 8 Day 10 Day 14 Day 17 Day 21 Day 24 Day 31 Day 45 Day 59
48.2 ADD 107.0 ADD 149.6 ADD 196.8 ADD 244.3 ADD 340.9 ADD 413.7 ADD 490.5 ADD 557.8 ADD 712.62 ADD 1078.5 ADD 1315.5 ADD
Aldehydes
3-methylbutanal [3,6,18,20] ■ ■ ■ ■ ■ ■ ■ ■ ■
Hexanal * [1,2,4,12,16,17,20] ■/□ ■ □ ■
Heptanal * [1,2,12–14,16,20] □ □ □
2-heptenal [1,12] □
Octanal [1,3,8,16,20] □ □ □ □ □ ■/□ □ □ □
2-octenal [1,12] □ □
2-butyl-2-octenal □
Nonanal * [4,12,13,20] ■/□ ■/□ □ ■/□ ■/□ ■/□ □ ■/□ □
2,4-nonadienal [1,12] □
2-decenal [3] □
Benzaldehyde * [1,3,6,11–14,16,19] □
2-phenylethanal □ □ □ □
2-phenylpropenal ■ □ □ □
2-methyl-prop-2-enal ■
Ketones
2-butanone [3,4,6,17,18] ■ ■ ■ ■ ■ ■ ■
3-methyl-2-butanone [3,6] ■ ■ ■
2-pentanone [3,6,17] ■ ■ ■ ■ ■ ■ ■ ■
2-hexanone [17] ■ ■ ■
2-heptanone [1,3,12,15,17] □ ■/□ □ ■ ■/□ □ ■/□ ■/□
2-octanone [3,15] ■/□ □ □ ■/□ ■/□
3-octanone [3,15] □ □ □ □ ■/□ ■/□
3-octen-2-one □ □ □
3,5-octadien-2-one [12] □ □ □
2-nonanone * [3,11,12,15,18,20] ■ □ ■/□ □ ■/□ ■/□
2-decanone [3,12,20] □
2-undecanone * [3,11,12,15] ■ □ ■/□ □
2-nonadecanone ■
1-phenylethanone * [6,11,14,15] ■ □ □ ■/□ □ ■/□ ■/□
Chromatography 2014, 1 130

Table 1. Cont.
Day 2 Day 4 Day 6 Day 8 Day 10 Day 14 Day 17 Day 21 Day 24 Day 31 Day 45 Day 59
48.2 ADD 107.0 ADD 149.6 ADD 196.8 ADD 244.3 ADD 340.9 ADD 413.7 ADD 490.5 ADD 557.8 ADD 712.62 ADD 1078.5 ADD 1315.5 ADD
Ketones (continued)
3-methyl-3-buten-2-one ■
2-piperidinone [14] □
1,7,7-trimethylbicyclo[2.2.1]heptan-2-one □ □ □ □ ■
6,6-dimethylbicyclo[3.1.1]heptan-2-one □
2,6,6-trimethylbicyclo[3.1.1]heptan-2-one □ □ □
Alcohols
Ethanol * [4,14,17,18,20] ■ ■ ■ ■ ■ ■ ■
1-propanol [3,15] ■ ■ ■ ■ ■ ■
2-propanol [15,18] ■ ■ ■
2-methyl-1-propanol [6,14,17] ■ ■ ■ ■ ■ ■ ■ ■
2-propen-1-ol ■
1-butanol [3,6,15–17] ■ ■ ■ ■ ■
2-butanol [3,6,14] ■
2-methyl-1-butanol ■ ■ ■ ■ ■ ■
3-methyl-1-butanol [3,14] ■ ■/□ ■ ■/□ ■
1-pentanol [1–3,6,12,14,17] ■ ■ ■ ■
2-pentanol [6,17] ■ ■
2-hexanol ■ □ □ ■
1-heptanol [3,20] □
2-heptanol ■ ■
1-octanol [1,3,16] □ ■ ■
3,5-octadien-2-ol □
1-nonanol ■/□ ■ ■ ■ ■
Phenol * [3,6,11,13–15,18] ■ □ □ □ ■/□
4-methylphenol [3,14,15,18] ■ ■ ■ ■/□ ■/□ □
2-phenylethanol [14,16] ■ □ □ □ □
Chromatography 2014, 1 131

Table 1. Cont.
Day 2 Day 4 Day 6 Day 8 Day 10 Day 14 Day 17 Day 21 Day 24 Day 31 Day 45 Day 59
48.2 ADD 107.0 ADD 149.6 ADD 196.8 ADD 244.3 ADD 340.9 ADD 413.7 ADD 490.5 ADD 557.8 ADD 712.62 ADD 1078.5 ADD 1315.5 ADD
Hydrocarbons
Pentane [4] ■ ■ ■ ■ ■ ■ ■ ■ ■ ■
Hexane * [4,6,12,17,18,20] □ □ □ ■ ■ ■ ■/□ ■/□ ■/□ ■ ■/□
1-hexene [6,17] ■
Heptane * [4,6,17–19] ■ ■ ■ ■ ■ ■ ■/□ ■ ■
1-heptene [6,17] ■ ■
4-ethyl-3-heptene □
Octane [3,4,17,18] ■ ■ ■ ■/□ ■ ■ □ ■/□ ■ ■
1-octene [3] □
1-decene ■
5-undecene ■ ■ □ ■
Tridecane ■ ■/□ ■ ■/□
1-tridecene □ □
Pentadecane [6,13] □ □ □ ■/□ ■/□ ■/□ □
1-pentadecene ■/□ □
Heptadecane □ □
8-heptadecene □ ■ ■/□
α-pinene [6,17] ■ ■ ■ ■ ■ ■ ■ ■
β-pinene ■
Copaene □ □
1-methyl-2-pentylcyclopropane ■ □ □
1,3-Hexadiene, 3-ethyl-2-methyl- □
1-methyl-4-(1-methylethyl)-1,4-cyclohexadiene □ ■ ■ ■/□
2,2-dimethyl-3-methylene-bicyclo[2.2.1]heptane □
Chromatography 2014, 1 132

Carboxylic acids (C2–C6 volatile fatty acids), longer chain acid esters (C6–C8), and monoterpenoid
ketones were almost exclusively identified using SPME collection. Major compounds in these groups
were 2-methylbutanoic acid, 3-methylbutanoic acid, hexanoic acid methyl ester, and heptanoic acid
methyl ester. SPME and sorbent tubes collected a range of aldehydes; however, saturated aldehydes
were exclusively identified using SPME. During the bloat stage (day 2), SPME collected more
compounds across the VOC profile than sorbent tubes. However, from day 4 onwards both techniques
collected comparable numbers of VOCs demonstrating their complementarity. Indole, 2-pentylfuran,
nonanal, 2-heptanone, 1-phenylethanone and hexane were the most frequently identified compounds in
common between techniques. Occasionally a compound was not detected on a single experimental day
using one technique, but was identified using the other technique. For example, 2-heptanone was
consistently identified from day 8 onwards when the techniques are viewed collectively, however was
identified intermittently by each collection technique. This suggests that the field conditions may have
been unfavourable for one technique over the other on that particular day.
When the profiles from both collection techniques are combined, overall trends become identifiable
in several compound classes. Esters were predominantly identified in early decomposition (i.e., the
first two weeks). As decomposition progressed, esters with fewer carbons decreased and longer chain
esters remained. Ketones and hydrocarbons exhibited the opposite trend. Short chain ketones and
hydrocarbons found early in decomposition remained stable while the number of more complex
ketones and hydrocarbons increased in later decomposition.

3.3. PCA Analysis

PCA is a multivariate technique that is used to reduce dimensionality of the data set thereby
assisting in data visualisation. Once the principal component scores were plotted, this technique allowed
for similarities and differences between samples on each experimental day to be visualised, producing
groupings of days that were most similar. Loadings were used to give a representation of the chemical
classes providing the most discrimination for each experimental day or grouping of days on the biplot.
The results of the PCA analysis (Figures 2 and 3) show the distribution of experimental soil
samples on each day based on the sum of the normalised peak area for each chemical class. Control
sites grouped very closely due to low variation of background compounds between sites. Distribution
of experimental sites between days was more dispersed because of increased variation between these
samples. In order to improve clarity the data points for controls were removed and a dashed circle on
the plot indicates the area they were present. Data points found outside of this circle represent days that
the decomposition VOC profile can be discriminated from the control soils. For both SPME (Figure 2)
and sorbent tubes (Figure 3) the experimental soils at the beginning and end of the field trial were
similar to control soils, likely due to reduced VOC production at these times.
Chromatography 2014, 1 133

Figure 2. Principal component analysis (PCA) biplot of the calculated PCA scores and
loadings using SPME data. Each point for scores represents the average of the four
replicate carcasses on each experimental day (i.e., D6 = day 6). The average abundance of
each compound in the carcasses were summed for each chemical class on each day and
input for PCA analysis. A circle indicates the range of control soils from corresponding
experimental days.

The use of sorbent tubes distinguished the experimental soils from control soils for a longer
duration when compared to SPME. The loadings demonstrate that a larger number of chemical classes
contributed to the dispersion of the experimental soil data points using sorbent tubes. Ketones,
aromatics, and esters were the classes detected in common between the two techniques and were
responsible for discrimination. The loadings of the PCA biplot for sorbent tube sampling indicated that
this technique recovered additional nitrogen-containing compounds, alcohols, hydrocarbons, and
sulfides that were specific to experimental soils and important in showing discrimination between
experimental days. Figure 3 demonstrates that the suite of compounds for sorbent tubes more
effectively discriminated experimental soil from control soil over the entirety of the trial and therefore
sorbent tubes were more successful at collecting a valuable range of decomposition VOCs.
Chromatography 2014, 1 134

Figure 3. PCA biplot of the calculated PCA scores and loadings using sorbent tube data.
Each point for scores represents the average of the four replicate carcasses on each
experimental day (i.e.., D6 = day 6). The average abundance of each compound in the
carcasses were summed for each chemical class on each day and input for PCA analysis.
A circle indicates the range of control soils from corresponding experimental days.

4. Discussion

4.1. Decomposition VOC Profile in Soil

Many of the compounds present in the overall profile have been previously identified in
literature [1,3–6,11–20], suggesting that major compounds and trends are comparable regardless of
methodology or location. A distinct signature of decomposition odour was identified in the soil from
day 4 onwards using both sampling techniques which corresponded to the onset of active decay. The
headspace above decomposing remains typically exhibits a variety of decomposition VOCs during the
bloat stage. Few compounds were detected during bloat which suggests that VOCs present in the air
required an equilibration period with the soil before they entered into soil gas or became adsorbed onto
soil particles. Skin ruptures associated with active decay will also cause fluids to be leached resulting
in higher VOC loading in the soil.
Chromatography 2014, 1 135

Although a reduction in decomposition VOCs in soils have been reported [4,11], more recent
research has demonstrated that in an increased number of decomposition VOCs are identified in soil
than above the carcass or cadaver [22]. Due to their composition, soils tend to exhibit a natural
background VOC profile with VOC fluctuations based on living components in the soil community
(i.e., fungi and bacteria). The asterisks in Table 1 demonstrate that only a few of the VOCs identified
in this study have been found in decomposition soils previously, yet many of these VOCs have been
consistently cited in the air above human and animal remains. The techniques used in the current study
were highly effective to collect the range of compounds expected in decomposition odour and
additional compounds (i.e., esters) that could be attributed to soil composition.

4.2. Collection Technique Considerations

The SPME fibre coating has a large influence on the types of compounds that can be collected.
The PDMS/DVB fibre used in the current study is generally used for the collection of volatiles, amines
and nitroaromatic compounds with molecular weight range of 50–300. Low molecular weight VOCs
(<90 MW) tend to be more efficiently collected on fibres containing carboxen. This explains the low
recovery of lighter sulfides and lighter oxygenated compounds using the PDMS/DVB fibre. The fibre
is bipolar which extends its range to collect polar and nonpolar compounds. However, targeted
alcohols analysis is traditionally performed using a carbowax fibre which also explains the reduced
number of alcohols collected using SPME. The wider target range using PDMS/DVB is advantageous
due to the number of compound classes typically reported in decomposition odour. Due to the nature
of non-target and forensic analyses, widening the range of analytes that can be collected using a single
fibre allows for more compounds of interest to be collected while ensuring high throughput. The
majority of studies on decomposition odour have used the PDMS/DVB fibre to obtain successful
results [1,2,12]; however comparable results have been obtained using a Carboxen/PDMS (CAR/PMDS)
fibre [17]. Although sulfur-containing compounds are consistently reported as major constituents of
decomposition odour (especially dimethyl disulfide and dimethyl trisulfide) [1,3,6,15,17–19],
the PDMS/DVB fibre is not suited to the collection of this compound class. This is a significant
drawback of this type of analysis, especially during early decomposition where sulfur-containing
compounds are prominent.
The dual sorbent combination used for the sorbent tubes increased the range of compounds
collected. Tenax TA is suitable for C7–C30 and typically targets aromatics, semi-volatiles, nonpolar and
polar compounds, while the Carbograph 5TD increases the ability to collect VOCs in the C3/4 to C6/7
range. The combination of sorbents is often used in decomposition odour analysis to widen the range
of analytes that can be collected [3,19,20]. The sorbent combination was suitable for collecting
decomposition-specific compounds for most compound classes. Carboxylic acids were not identified
in decomposition soil using sorbent tubes but were identified in the soil when analysed using SPME.
Many of the carboxylic acids collected with SPME in this study (propanoic acid, 2-methylpropanoic
acid, butanoic acid, 2-methylbutanoic acid, 3-methylbutanoic acid, pentanoic acid, and hexanoic acid)
have previously been identified in the headspace of decomposed remains using sorbent tubes and other
pumped sorbent-based collection techniques [3,15]. Carboxylic acids were not detected in a previous
gravesoil study and the researchers suggested that this was likely due to sampling and/or sample
Chromatography 2014, 1 136

preparation [11]. It is notable that many esters of the corresponding acids were identified in the sorbent
tube samples. This may have been attributed to the sorbent selection for the sorbent tubes and SPME.
However, this could also point to microbial processing causing conversion of carboxylic acids to
esters. Esters of C4 to C28 carboxylic acids have been identified as markers of shifts in soil microbial
community and indicated that non-amended surface soils exhibited a lack of esters [29]. In fact, the
overwhelming presence of esters in early decomposition (using both techniques) is not a trend
previously documented for decomposition odour in the headspace of remains or in soil. Decomposition
of soft tissue introduces a pulse of nutrients and moisture into the soil below the remains that can
stimulate microbial metabolism. This can have a direct effect on the proliferation of soil bacteria and
microbial community structure, consequently altering bacterial metabolism VOCs and producing large
number of esters.
Sorbent tube collection was performed using a dynamic approach (pumped sampling) while SPME
is a passive technique performed in a static vial. For this reason, it was anticipated that sorbent tubes
would collect more decomposition VOCs than SPME. However, a similar number of compounds were
identified using both techniques. Although VOC extraction was performed using different mechanisms
(dynamic vs. passive), both allow for a pre-concentration of the sample on the sorbent. This is
beneficial for GC-MS analysis as it increases the potential for identifying low level components of the
overall profile. In comparison to techniques such as dynamic headspace (which inject a small amount
of sample headspace into the instrument without pre-concentration) these techniques are ideal for the
analysis of decomposition soil.

4.3. Practical Considerations

Both sampling techniques provided practical advantages and disadvantages. Sorbent tube sampling
using the VOC-Mole™ soil probe was advantageous in providing an in situ approach to VOC
collection. In situ sampling allows for minimised losses from transport and storage in addition to
providing a better representation of VOCs contributed by the living soil community. Good
characterization of the soil vapour phase would be expected from in situ analysis. On the other hand,
this approach means that the odour source is not available for re-testing if required.
Although SPME collected fewer compounds that distinguished experimental soils from control
soils, there are circumstances in which this approach is desirable. In cases where field access is not
possible, collection of soil into vials may provide a more practical approach. If a thermal desorption
unit is not available in the laboratory, SPME can be employed without specialised instrumentation. It
must be noted that components of the vapour phase will be lost during sample transportation and any
associated long term storage should be minimised. With ex situ analysis there is better characterisation
of the adsorbed soil VOC phase, especially where heating of the sample is performed. Replicate
sample analysis is also more convenient. Sample retesting could, however, produce slight variations
because the septum on the headspace vial is previously pierced.

4.4. Longitudinal VOC Data in Forensic Casework

The VOC profile was highly dynamic over the process of decomposition. If a single experimental
day from this study was chosen, it is apparent that a large portion of the profile would be
Chromatography 2014, 1 137

uncharacterised. Therefore, the longitudinal study of decomposition odour in soil was valuable in
depicting a comprehensive overview of compounds of interest.
Previous studies have suggested using the pattern of VOC production during decomposition for
PMI estimation [17,18]. The dynamic nature of the VOC profile depicts the difficulty in establishing a
reproducible pattern of VOC evolution for these purposes. While certain compounds followed general
timeframes, they were not specific to a short reproducible interval that could be quantified for PMI
estimation. Although the decomposition VOC profile is becoming increasingly characterised, it is still
not possible to predict the exact number and types of VOCs for a particular stage of decomposition.
However, decomposition VOCs have the ability to attract insects in a reproducible pattern. Such insect
succession patterns are more reliable for estimating PMIs. The data obtained from longitudinal studies
can provide information on potential compounds that could be signaling insect attraction during
extended PMIs.
Improved characterisation of the dynamicity of the decomposition VOC profile has valuable
implications for the search and recovery of victim remains. This information can be used as a reference
profile to be input into field-portable GC-MS instrumentation to provide a positive reference for
decomposition odour. Electronic nose equipment generally use one or several target compounds for
detection and therefore these target compounds need to be identified prior to instrument development.
Similar handheld devices have been developed based on gases and VOCs released during
decomposition [7,8]. However, these instruments have yet to be employed functionally. Field
instrumentation will likely never replace cadaver dogs completely, largely due to their high efficacy,
sensitivity and their ability to search large area or rough terrain effectively [12]. However, they may
provide valuable complementary search tools in the future.

5. Conclusions

The complexity of the decomposition odour profile has been identified by many past researchers
and is made more complex in soil environments. The interaction between soil and decomposition
VOCs has been underrepresented in literature, yet its importance in forensic science cannot be
disregarded. This study documented two VOC collection techniques that have previously been used in
decomposition research. While the profiles collected from decomposition soil were complementary
and widened the range of analytes identified, sorbent tubes collected more discriminatory compounds
than SPME. Although most literature in this field relies heavily on established collection techniques,
this is the first published study to document and compare their use to collect decomposition odour
from the same odour source. Due to a difference in analytical results obtained using the two collection
techniques, researchers must be aware of potential bias in results based on the use of a single technique
in research studies. Documenting longitudinal analysis of decomposition VOCs in soil also depicts the
necessity for analysing the entire profile of decomposition rather than a subset of the process.
Improving collection methods and documenting the profile in a longitudinal manner will serve to
improve knowledge for forensic disciplines relying on decomposition VOC recognition.
Chromatography 2014, 1 138

Acknowledgments

The authors wish to thank Markes International Ltd. for providing guidance on the use of
VOC-Mole™ soil probes during the early project stages. They also thank David Bishop for instrument
support in the UTS Centre for Chemical Technologies. This research was funded by the Australian
Research Council (ARC) and by the University of Technology, Sydney (UTS).

Author Contributions

Katelynn A. Perrault: Project design, experimental setup, sample collection, sample analysis, data
analysis, and manuscript preparation, Barbara H. Stuart: Project guidance, experimental setup and
manuscript revision, Shari L. Forbes: Project design, experimental setup, sample collection, data
review, and manuscript revision.

Conflicts of Interest

The authors declare no conflict of interest.

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