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volatiles evolved from decaying human
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www.elsevier.com/locate/forsciint
Forensic Science International 153 (2005) 147–155
A study of volatile organic compounds evolved from the
decaying human body
M. Statheropoulosa, C. Spiliopouloub, A. Agapioua,*
aSchool of Chemical Engineering, National Technical University of Athens (NTUA),
Sector I, 9 Iroon Polytechniou Str., 157 73 Athens, GreecebDepartment of Forensic Medicine and Toxicology, Medical School, University of Athens, 115 27 Goudi, Greece
Received 1 October 2003; accepted 13 August 2004
Available online 25 November 2004
Abstract
Two men were found dead near the island of Samos, Greece, in the Mediterranean sea. The estimated time of death for both
victims was 3–4 weeks. Autopsy revealed no remarkable external injuries or acute poisoning. The exact cause of death remained
unclear because the bodies had advanced decomposition. Volatile organic compounds (VOCs) evolved from these two corpses
were determined by thermal desorption/gas chromatography/mass spectrometry analysis (TD/GC/MS). Over 80 substances
have been identified and quantified. The most prominent among them were dimethyl disulfide (13.39 nmol/L), toluene
(10.11 nmol/L), hexane (5.58 nmol/L), benzene 1,2,4-trimethyl (4.04 nmol/L), 2-propanone (3.84 nmol/L), 3-pentanone
(3.59 nmol/L). Qualitative and quantitative differences among the evolved VOCs and CO2 mean concentration values might
indicate different rates of decomposition between the two bodies. The study of the evolved VOCs appears to be a promising
adjunct to the forensic pathologist as they may offer important information which can be used in his final evaluation.
# 2004 Elsevier Ireland Ltd. All rights reserved.
Keywords: Evolved VOCs; TD/GC/MS analysis; Corpse; Volatiles; Odors; Cadaver
1. Introduction
Over the last decade interest has increased regarding the
identification of VOCs for medical [1,2], toxicological [3,4]
and environmental applications [5]. Forensic science uses
headspace analysis for volatile substances in blood and
organ specimen (i.e. brain, lung, liver, kidney, skeletal
muscle); this approach has been a well-established method
[6,7]. The odor of putrefaction is characteristic and familiar
to the front line experts such as police investigators, forensic
pathologists, anthropologists, entomologists, crime scene
technicians and other medical and non-medical profes-
* Corresponding author. Tel.: +30 210 7723109;
fax: +30 210 7723188.
E-mail address: agapiou@cental.ntua.gr (A. Agapiou).
0379-0738/$ – see front matter # 2004 Elsevier Ireland Ltd. All rights r
doi:10.1016/j.forsciint.2004.08.015
sionals. The chemical analysis of the malodorous com-
pounds has not attracted much of research interest. It has
been referred that investigators do not attempt to identify and
quantitate these compounds because routine methods cannot
detect them at their low concentrations [8]. However, these
odors are readily sensed by various insects with their sensi-
tive olfaction. Forensic entomology has been used for
determining time interval since death [9,10]. Furthermore,
it is speculated that the odor of putrefaction comes primarily
from the sulfur compounds and various inorganic gases that
are produced in the bowel.
The breakdown of soft tissues of the body during putre-
faction is aided by microorganisms such as bacteria, fungi
and protozoa. This results in the production of gases, liquids
and simple molecules. Various gases (CO2, H2S, CH4, NH3,
SO2, H2) produced in the process distend the tissues [11].
eserved.
M. Statheropoulos et al. / Forensic Science International 153 (2005) 147–155148
Along with these, a variety of volatile organic compounds
(VOCs) are liberated. These volatile substances are inter-
mediate products of decomposition while the large biologi-
cal macromolecules such as proteins, nucleic acids,
carbohydrates and lipids are broken down into their building
block components (C, H, O, N, P and S). Carbohydrates are
broken down mainly to oxygenated compounds (alcohols,
aldehydes, ketones, acids, esters and ethers) and proteins to
nitrogen, phosphorus and sulfur compounds. Nucleic acids
produce nitrogen and phosphorus compounds. Lipids give
rise to hydrocarbons, nitrogen, phosphorus and oxygenated
compounds.
Many factors affect the decomposition rate of a corpse.
Above ground decomposition is typically faster than burials
because the corpse is exposed to varying environmental
factors (i.e. temperature, humidity, air currents), insects
and carnivores [12]. Burials in many forms slow decom-
position by decreasing gas diffusion, limiting both macro
and microorganisms and increasing the amount of carbon
dioxide leading to anaerobic conditions [13].
Sampling volatile organic compounds onto sorbent tubes
and their subsequent analysis by TD/GC/MS is an efficient
method for qualitative and semi-quantitative determination of
VOCs. The aim of the work presented here is the determina-
tion of the type and amount of VOCs present at a single time
point during the decay process of the human body. Consider-
ing the given set of circumstances of each death case, VOCs
may offer valuable information which could prove useful in
estimating the postmortem interval. Moreover, this data could
also help uncover the etiology of death.
2. Case profiles and autopsy findings
Two human males were found dead in December 2002
near the island of Samos in Greece. The port authorities
enclosed each cadaver in a plastic body bag which was then
placed in a metal case and air sealed with silicone in a
wooden box. Bad weather travel conditions prevailed at the
time. So the bodies were shipped 4 days later to the medical
school mortuary in Athens for medico-legal examination.
2.1. Case study 1
The cadaver belongs to an unknown man, 183 cm tall,
slim and aged about 40 years (between 34 and 44 years old).
Although the cause of death remains uncertain, the victim
couldhavedrownedbecause a largeamountoffluid (seawater)
was found in his chest cavity. Autopsy revealed that there
were neither remarkable external injuries nor acute poisoning.
Most internal organs showed softening by putrefaction.
2.2. Case study 2
The cadaver belongs to an unknown man, 183 cm tall,
robust, and aged about 30 years (between 26 and 36 years
old). The cause of death could not be determined because
decomposition had progressed. Although the external
appearance of the corpse was better preserved in relation
to the first case, putrefaction was more advanced. Decay was
prominent in the internal organs.
3. Experimental
3.1. Chemicals
Five standard mixtures (C = 50 mmol/L) were purchased
from ITChem Hellas. In order to determine the relative
response factors (RRF) for the applied semi-quantitative
method, four different methanolic standard mixtures of 12
substances each (H/C, O-atoms, heteroatoms and aromatic
substances) were used. Furthermore, a standard solution of
deuterated chlorobenzene-d5 99% in methanol was used as
internal standard. Chlorobenzene-d5 is a better standard for
high molecular weight and low volatility compounds. How-
ever, it was chosen for the applied full screening semi-
quantitative method as it complies to the general rules of
EPA TO-17 for gas standards [14]. Furthermore, its elution
occurs in the middle of the chromatographic analysis (mean
retention time R̄t = 28.6).
3.2. Sampling tube preparation
Three layer sampling sorbent glass tubes of 4 mm i.d. and
115 mm length were used for VOC measurement (TD-300,
Alltech Associates). These consisted of 300 mg carbograph
2, 200 mg carbograph 1 and 125 mg carbosieve S-III. The
tubes were conditioned for 2 h at 300 8C at a flow of 150 mL/
min of He to minimize background effects. The above
conditioning procedure was verified for success by blank
measurements. The sampling tubes were sealed with brass
nuts, caps and PTFE ferrules and kept at 4 8C until the day of
sampling. Before sampling, 1 mL of the above methanolic
standard mixture of chlorobenzene-d5 was added by means
of microsyringe to the glass wool supporting the sorbent
material.
3.3. VOCs, CO2 and NH3 sampling
Each wooden box (2.20 m � 0.52 m � 0.60 m) was
opened and the metal case was layed open. A small hole
was made at the body bag at the upper chest level of each
cadaver and a Teflon tube (0.6 cm i.d.) was inserted. The
sorbent tube was placed at the end of the Teflon tube. Five
liters of air (‘‘headspace’’) were drawn from the body bags
through the sampling tube by a sampling pump (VSS-1, A.P.
Buck, USA) at a flow rate of 200 mL/min. A reference
sample was also taken for comparison outside the cadaver
bag at a close distance.
A second, smaller Teflon tube (0.1 cm i.d.) was inserted
through the body bag of the cadaver in order to monitor
M. Statheropoulos et al. / Forensic Science International 153 (2005) 147–155 149
carbon dioxide concentrations during VOCs sampling. A
portable IR gas analyzer (Anagas CD 98, Environmental
Instruments, England) was used with a PTFE filter provided
by the instrument manufacturer. The flow rate of the instru-
ment with the front filter was 120 mL/min.
A third Teflon tube was finally inserted through the body
bag of each cadaver to monitor ammonia concentration as
well. A portable ammonia gas sensor without internal pump
(Toximeter TX 2000, Oldham, France) was used. The
ambient temperature and relative humidity during sampling
were measured by a portable thermometer-hygrometer
(model H 270, Dostmann Electronics, Germany).
Actually three variations of sampling were employed for
the qualitative analysis of VOCs. The first method was
described above. In the second method the VOCs were first
placed in a 2 L flexible Tedlar bag and then transferred to the
sorbent tube. This method was chosen because it is widely
used for expired air analysis where preconcentration of the
sample is necessary. In order to avoid sample pass through
the pump, an in house vacuum chamber was used. The
Tedlar bag was filled directly using the negative pressure
provided by the pump [15]. The number of VOCs identified
with this method was slightly lower than the previous one
and quantitative differences among VOCs appeared, as
lower volume of air was sampled. These findings were
expected since this method involved multiple analytical
steps where leaks may occur and VOCs may also be retained
in the bag. In the third method, a sorbent tube was inserted
directly in each cadaver bag and air was allowed to diffuse
into it without a pump. This experiment showed an even
lower number of VOCs and only the higher peaks could be
identified. For the qualitative analysis, the results of the three
sampling methods were taken into consideration. For semi-
quantitation purposes, the first method was used since it
involves fewer steps and experimental conditions were
controlled.
In order to examine if VOCs originate from the plastic
packing material of the body bag, a solid phase micro
extraction (SPME) experiment was carried out. An empty
body bag (1.87 m � 0.87 m, Adamedical, Hellas) was filled
with synthetic air and left for 1 day at ambient temperature.
The SPME (85 mm carboxen/polydimethylsiloxane on a
Stableflex fiber, Supelco) was inserted through the body
bag and the fiber was exposed for 30 min, equal to the actual
time of sampling into the sorbent tubes.
3.4. Chromatographic analysis
Sorbent tubes were thermally desorbed to an HP 5890/
5972 GC/MS system using an in house made thermal
desorption unit (TDU). This unit is designed to stand on
top of the gas chromatograph, featuring its own injection
system, so as to function without heated transfer lines and
without the need to occupy an injection port of the chro-
matograph. Other than the thermal desorption module, it
includes a refocusing trap based on freezing with liquid
nitrogen in order to enhance the chromatographic separation.
Its autonomous injection system provides the ability to inject
the analytes desorbed from the refocusing step in split
(injector purge) or splittless mode (no injector purge)
depending on the load of analytes to the system or the
detection limits required for the current analysis.
Desorption flow of He was set at 30 mL/min, while the
temperature was kept constant at 200 8C. Desorption and
refocusing duration was 20 min in order to maximize recov-
ery. The cryo trap capillary was a 22 cm part of a 0.53 mm
id, AT-Q, Q-PLOT column (Alltech Associates); it was
chosen in order to enhance trapping of ultra-VOCs and
consequently the chromatographic resolution. A 20 s heating
pulse has proved to be adequate for flush desorption of
trapped analytes in the GC column; short enough to prevent
extensive deterioration of the cryo trap column thereby
limiting artifacts in the analysis. Cryogen used was liquid
nitrogen.
A 60 m SPB-624 capillary column with 1.4 mm station-
ary phase and an internal diameter of 0.25 mm (Supelco)
was utilized for high-resolution chromatographic separation.
Column head pressure of helium purge gas was set to 25 psi.
GC program was selected as follows: 35 8C initial for 5 min,
ramp of 4 8C/min up to 180 8C, hold for 20 min. MSD mass
range was limited from 35 to 350 amu due to the expected
detection of VOCs, but with the benefit of 1.8 scans s�1.
3.5. Data processing
Chromatographic peaks were initially identified with the
help of Wiley 138 library, using similarity indexes higher
than 80%. In order to enhance peak identification, a sub-
stance database was constructed using ‘‘Easy-Id’’ tool of HP
Productivity Chemstation. A compound database for VOC
analysis has been used. The compound database consists of
the compounds recorded with reference samples and/or the
original ones. The record for each substance in the database
has several fields. Among them the retention time, most
abundant mass and three qualifier ions are the most impor-
tant. Quantitative results were generated with the application
of internal standard method [16], using the following equa-
tion:
Ci ¼Ai
AISTD� 1
RRFi�CISTD
where Ci = concentration of substance i in the vapor phase
expressed in nmol/L, Ai = peak area of substance i, in counts,
AISTD = peak area of internal standard, in counts calculated
for each run, RRFi = (Ai/AISTD) � (Ci/CISTD), relative
response factor of substance i, from standard mixture,
relative to chlorobenzene-d5; expresses the different ioniza-
tion of substance i compared to the internal standard,
CISTD = calculated concentration of internal standard in
the total air volume sampled in nmol/L.
The concentration calculation for the internal standard
has been done by reducing the known molar quantity
M. Statheropoulos et al. / Forensic Science International 153 (2005) 147–155150
injected on the tube to the 5 L of air sampled. This con-
centration was calculated to be 10 nmol/L. This semi-quan-
titative full screening method was selected in order to offer
fast and reliable quantitative results in a variety of complex
substances without the need of constructing many calibra-
tion curves.
4. Results
Over 15 volatile substances were identified common in
all sampling experiments employed; over 60 substances
from the first two experiments. A variety of substances have
been identified, including almost all types of organic sub-
stances: hydrocarbons, aldehydes, ketones, alcohols, esters,
aromatics and sulfides. Among them dimethyl disulfide,
toluene, hexane, benzene 1,2,4-trimethyl, 2-propanone, 3-
pentanone, 2-pentanone and 2-methyl pentane showed the
highest concentrations. Table 1 presents the concentrations
(semi-quantitative analysis) of identified volatile substances
along with the mean retention time. A typical chromatogram
of VOCs identified during putrefaction is presented in Fig.
1a. Fig. 1b shows the reference sample taken at a close
distance. The mean ambient temperature measured during
sampling for cadaver one was 13.75 8C and the relative
humidity 69.32%. For cadaver 2 the mean temperature was
17.45 8C and humidity 51.37%, respectively. The ambient
temperature during the blank ‘‘body bag’’ SPME experiment
was 31.1 8C and humidity was 29.9%. The most prominent
VOCs identified migrating from the ‘‘body bag’’ were
phenol, 1-hexanol-2-ethyl and cyclohexanone.
5. Discussion
So far in the literature, there is not enough work to relate
VOCs with human decomposition. However, this has been
carried out in pathological conditions in living humans,
where similar experimental methodology has been widely
applied. Thus, in order to study VOCs during putrefaction
this type of available information was used in an attempt to
interpret the results of the present study.
It seems that there are three possible sources of VOCs in
this study. Atmospheric air trapped during packaging the
cadaver appears to be the first source. To examine this
possible contribution, a reference field blank sample was
taken outside the body bag and it did not show any measur-
able VOCs (Fig 1b). The next possible source is that VOCs
could migrate from the plastic ‘‘body bag’’ used to encase
the cadavers. This was tested by the SPME experiment
which was performed at a higher temperature than during
sampling. It appeared that phenol, 1-hexanol-2-ethyl and
cyclohexanone were found at very low concentrations.
Hexanol-2-ethyl has been detected in serum of hemodialysis
patients and in people suffering from liver disease [17]. It is
believed to be a metabolite of di-2-ethylhexyl phthalate
(DEHP), a widespread plasticizer often used in plastics
for medical use. Cyclohexanone found in the blank body
bag experiment was 50 times less than the amount found in
the air pumped in the experiments. It is then assumed that the
VOCs determined in the cadaver bags mainly evolved from
the dead bodies in putrefaction.
Dimethyl disulfide (DMDS) was found to be the sub-
stance with the highest concentration on both cadavers. It is
probably a byproduct of protein breakdown by bacteria.
Some studies in living humans suggest that the production
of many volatile sulfur compounds result from impaired
hepatic metabolism of sulfur-containing substances includ-
ing methionine [18].
DMDS, along with other mercaptanes, have been found
during headspace analysis in the urine of diabetic persons.
DMDS was supposed to result from enterobacterial degra-
dation of methionine in hepatic encephalopathy [19]. DMDS
was also identified in a study investigating sulfur compounds
from gingival crevicular sites in humans [20]. Generally
speaking, volatile sulfur compounds are commonly asso-
ciated with anaerobic decomposition of organic matter and
are well known for their characteristic strong disagreeable
and permeable odors in foods and drinks. Furthermore,
Ochiai et al. reported the identification of DMDS at low
ppbv in the expired air of humans using a three-stage
cryogenic trapping preconcentration system [21]. Tanger-
man et al. identified also DMDS in the breath of cirrhotic
patients [22]. Moreover, volatile sulfur compounds have
been suggested as one of the endogenous factors responsible
for hepatic coma [23]. Indicatively, the odor in liver failure
has been described as musty, rotten egg-like (corpse-like).
Although toluene was not predicted to be among the
most abundant substances, it was identified in both cases. It
has to be pointed out that the amount found was excep-
tionally high. The circumstances of death for both victims
are still unknown. One wonders if the large amount of
toluene is a result of poisoning. Inoue et al. identified
toluene in a headspace analysis of an adipoceratous body
mainly in adipose tissue, brain, skeletal muscle, liver and
kidney. Toluene is highly lipophilic and is thus found
abundantly in cadavers who previously were exposed to
toluene. Other volatiles also reported in their study and
also identified in the present work included dimethyl
disulfide, ethanol and 1-butanol [7]. Both alkyl benzenes
and trimethyl benzenes were found in low concentration
and this indicates that the deceased may have had some
contact with gasoline [24].
Acetone is produced by lipolysis during fat catabolism.
The breakdown of triglycerides in adipose tissue liberates
increased amounts of ketone bodies [25]. Acetone is formed
by decarboxylation of acetoacetate, which derives from
lipolysis or lipid peroxidation. High concentrations of acet-
one are found in expired air of living humans with uncon-
trolled diabetes mellitus [26]. In diabetic or starvation
ketoacidosis the breath has a characteristic odor that has
being described as the fruity aroma of old apples.
M. Statheropoulos et al. / Forensic Science International 153 (2005) 147–155 151
Table 1
Concentrations of VOCs identified on two decaying human males
Substance R̄ta (min) Concentration (nmol/L) Mean concentrationb (nmol/L)
Cadaver 1 Cadaver 2
1 Dimethyl disulfide 22.22 7.27 19.51 13.39
2 Toluene 23.10 6.85 13.37 10.11
3 Hexane 11.31 1.28 9.88 5.58
4 Benzene, 1,2,4-trimethyl 35.40 6.52 1.55 4.04
5 2-Propanone 7.97 2.04 5.64 3.84
6 3-Pentanone 19.84 0.95 6.22 3.59
7 2-Pentanone 19.41 1.70 4.90 3.30
8 Pentane, 2-methyl 9.31 0.17 5.04 2.61
9 Benzene, 1,3,5-trimethyl 34.90 2.54 – 2.54
10 Benzene, 1-ethyl, 3-methyl 34.02 2.71 2.06 2.39
11 Cyclopentane, methyl 12.99 – 2.11 2.11
12 Pentane, 3-methyl 10.12 0.07 3.64 1.86
13 Pentanal 19.58 – 1.78 1.78
14 Heptane, 2,4-dimethyl 23.19 – 1.68 1.68
15 Heptane 17.45 0.73 2.55 1.64
16 Benzene 1-ethyl, 2-methyl 35.07 1.83 1.40 1.62
17 Butanoic acid, ethyl ester 25.09 0.18 3.03 1.61
18 Hexanal 25.86 0.24 2.94 1.59
19 Octane 23.41 0.39 2.48 1.44
20 Benzene, 1,2,3-trimethyl 37.42 1.56 1.27 1.42
21 Ethanol 7.09 0.13 2.42 1.28
22 Propanoic acid, 2-methyl, ethyl ester 22.32 – 1.23 1.23
23 Furan, 2-ethyl 18.50 – 1.14 1.14
24 Disulfide, methyl ethyl 27.60 1.02 0.90 0.96
25 Carbon disulfide 8.30 0.47 1.22 0.85
26 1-Butanol 18.89 – 0.82 0.82
27 1-Heptene 17.13 0.36 1.20 0.78
28 Trisulfide, dimethyl 35.64 0.77 0.56 0.67
29 Benzene 16.55 0.98 0.32 0.65
30 2-Butanone 13.72 0.34 0.97 0.66
31 Acetic acid, propyl ester 20.30 0.29 0.96 0.63
32 m-Xylene 29.69 0.98 0.12 0.55
33 Cyclohexane 14.99 – 0.55 0.55
34 1-Hexene 11.16 0.36 0.67 0.52
35 Butane, 2,3-dimethyl 9.05 0.09 0.94 0.52
36 2-Hexanone 25.58 0.17 0.75 0.46
37 1-Hexanol 30.12 – 0.45 0.45
38 Acetic acid, ethyl ester 14.11 0.22 0.66 0.44
39 Benzene, propyl 33.71 0.42 0.33 0.38
40 p-Xylene 29.46 0.36 0.30 0.33
41 1-Pentanol 24.67 – 0.31 0.31
42 1-H-Indene, 2,3-dihydro 38.38 0.31 0.28 0.30
43 Pentane 6.13 0.02 0.52 0.27
44 2-Heptanone 30.78 – 0.26 0.26
45 Pentane, 2,4-dimethyl 12.75 0.16 0.33 0.25
46 Hexane, 2-methyl 15.61 0.24 – 0.24
47 Benzene, ethyl 28.56 0.28 0.09 0.19
48 Hexane, 3-methyl 15.94 0.20 0.17 0.19
49 Cyclohexanone 32.44 0.15 0.13 0.14
50 Benzene, 1,2-diethyl 38.25 – 0.12 0.12
51 Acetaldehyde 4.14 0.08 0.16 0.12
52 2-Pentene, 4-methyl 12.14 0.11 0.10 0.11
53 Butane, 2,2,3-trimethyl 12.53 0.05 0.14 0.10
54 Pentane, 2,3,4-trimethyl 20.75 0.06 0.13 0.10
55 Carbon oxide sulfide 2.80 0.04 0.15 0.10
56 2-Octene 24.02 0.06 0.12 0.09
M. Statheropoulos et al. / Forensic Science International 153 (2005) 147–155152
Table 1 (Continued )
Substance R̄ta (min) Concentration (nmol/L) Mean concentrationb (nmol/L)
Cadaver 1 Cadaver 2
57 Alpha-pinene 31.77 0.08 0.09 0.09
58 Pentane, 2,3-dimethyl 15.28 – 0.08 0.08
59 Heptane, 3-methyl 21.34 – 0.08 0.08
60 Heptane, 2-methyl 21.13 – 0.07 0.07
61 Butanoic acid, 2-methyl ester 20.30 – 0.07 0.07
62 1-Pentanol, 2-methyl 20.81 – 0.07 0.07
63 Pentane, 2,3,3-trimethyl 21.04 0.07 0.06 0.07
64 Methane, chlorodifluoro 2.91 0.02 0.01 0.02
65 di-Limonene 36.75 – 0.06 0.06
66 Tetrachloromethane 15.73 0.08 0.04 0.06
67 3-Octene 25.90 – 0.05 0.05
68 Methane, chloro 3.38 0.08 0.02 0.05
69 2-Butylfuran 29.45 – 0.05 0.05
70 1-Propanol, 2-methyl 9.30 0.05 – 0.05
71 Disulfide, methyl propyl 33.34 0.04 – 0.04
72 Benzene, 1-ethyl, 4-methyl 32.33 0.04 – 0.04
73 Hexane, 2,3-dimethyl 20.90 – 0.04 0.04
74 Cyclohexane, 1,4-dimethyl 24.01 – 0.04 0.04
75 Octane, 2-methyl 26.90 – 0.03 0.03
76 Octane, 3,6-dimethyl 31.31 0.03 – 0.03
77 Butane, 2,2-dimethyl 7.41 – 0.02 0.02
78 Cyclohexane, 1-ethyl, 4-methyl 30.24 0.02 0.02 0.02
79 Isoprene 7.73 – 0.02 0.02
80 Cyclohexane, 1,2,4-trimethyl(1-alpha) 27.14 0.008 0.02 0.01
81 1-Pentene 5.80 – 0.01 0.01
82 1-Propene, 2-methyl 3.60 – 0.01 0.01
83 1-Propene 2.78 0.006 0.01 0.008
84 Cyclohexane, 1,3,5-trimethyl 26.02 0.008 – 0.008
85 Cyclohexane, 1,1,2,3-tetramethyl 33.08 – 0.007 0.007
86 1,3-Pentadiene 6.84 – 0.006 0.006
a Rt = retention time.b The mean concentration was taken in order to point out the most prominent VOCs.
Although the source of many VOCs evolved in humans is
not yet known, some of them have been correlated with
various pathological conditions and clearly related to meta-
bolic pathways in living humans: acetone from glucose
metabolism, alkanes from oxygen free radical-mediated
lipid peroxidation of fatty acids and isoprene from the
mevalonic acid pathway of cholesterol synthesis [27]. Some
VOCs that are produced in elevated concentrations in patho-
logical conditions in living humans are also present in
putrefaction, derived possibly from similar or different
metabolic pathways.
In the present study many alkanes and benzene deriva-
tives were found. Phillips et al. identified similar alkanes and
benzene derivatives in the breath of patients with lung cancer
[28]. As he states, part of the explanation may involve
increased oxygen free-radical activity in cancer cells. Oxy-
gen free radicals degrade cell membranes by lipid peroxida-
tion and convert these polyunsaturated fatty acids to volatile
alkanes. Alkanes are cleared from the body by excretion
through the lungs or by oxidation to alkyl alcohols by the
cytochrome P450 mixed-oxidase system.
Volatile fatty acids (i.e. propionic and butyric acid) are
primarily breakdown products of muscle and fat. Muscle
protein is degraded to component amino acids. Further
aerobic or anaerobic bacterial action leads to volatile fatty
acids. Further protein and fat decomposition yields phenolic
compounds and glycerols [13]. Although, these fatty acids
that are produced initially in the gut were not identified in the
present study, their esters were detected.
Many esters were also identified on both cadavers. These
included butanoic acid ethyl ester, propanoic acid 2-methyl
ethyl ester, acetic acid propyl ester, acetic acid ethyl ester
and butanoic acid 2-methyl ester. These are likely a result of
the saponification process. Saponification was more
advanced in the second cadaver. This was confirmed by
autopsy and volatile substance profile is in agreement (more
esters and volatiles in higher concentrations in cadaver 2).
Furthermore, CO2 concentration was greater in the sec-
ond cadaver by approximately 30%. In the first cadaver the
mean concentration of carbon dioxide during VOCs sam-
pling was 152 mmol/L whereas in cadaver 2 it was
196 mmol/L. However, measurements of ammonia concen-
M. Statheropoulos et al. / Forensic Science International 153 (2005) 147–155 153
Fig. 1. (a) A typical chromatogram of VOCs identified during putrefaction. The numbers in peaks are presented as indicated in Table 1; (b)
reference sample loaded with 1 mL internal standard taken at a close distance.
tration were not possible, due to limitations of the portable
gas sensor used (diffusion and sensitivity limit 0–100 ppm).
Ammonia, if present, in both cadavers was probably below
the detection limit of the sensor (<1 ppm). It should be
pointed out that the odor of ammonia is sharp and pungent.
Carbon dioxide and ammonia along with other gases (H2S,
CH4, SO2, H2) are present during putrefaction with resultant
distension of tissues. It should be emphasized that hydrogen
sulfide odor is like rotten eggs and sulfur dioxide odor is
pungent and irritating.
Tomita reported that dead bodies placed under water for
experimental purposes produced various alcohols such as
methanol, ethanol, 1-propanol, 1-butanol, occasionally 2-
propanol and others [29]. Furthermore, O’Neal et al. in a
review article reported a variety of volatiles found in animal
and human corpses under a variety of conditions: ethanol,
acetone, acetaldehyde, n-butanole, sec-butanole, n-propa-
nol, isopropanol, isoamyl alcohol, isobutanol, isopentanol,
ethyl ether, formaldehyde, phenyl ethanol and p-hydroxy-
phenylethanol. As he stated, the alcohols produced are
dependent on the bacteria present and the substrates avail-
able [30]. The first five volatile substances were also identi-
fied in the present study.
Other volatile substances reported in the literature and
considered significant during decomposition are indole and
skatole (fecal, nauseating odors), cadaverine (putrid, decay-
ing flesh odor) and putrescine (putrid, nauseating odor).
These were not detected in the present study, possibly due
to their low volatility [11]. Putrescine can be formed from
the decarboxylation of L-ornithine and cadaverine from
decarboxylation of L-lysine [31].
In this kind of experiments, environmental conditions are
critical and need to be considered. Temperature and humid-
ity not only affect the breakdown of proteins and carbohy-
drates, but may also modify the function of insects and
bacteria [13]. Huckenbeck found different bacterial species
producing different profiles of putrefactive volatiles. For
example C. aminovalericum produced acetone, ethanol and
1-butanol, whereas C. cadaveris produced acetone, ethanol,
1-butanol, isobutanol, n-butanol, isoamyl alcohol and n-
amyl alcohol. Temperature and humidity during putrefaction
will influence the profile of volatiles produced postmortem
[32]. However, volatile substances found in the present study
were identified under the given conditions (i.e. victims in the
sea, winter). Furthermore, volatile esters were likely formed
due to the presence of water.
6. Conclusion
The present study identifies volatile organic compounds
from two cadavers during putrefaction and raises a possible
role for them in forensic science. Over 80 VOCs were
identified and quantified. Among them dimethyl disulfide,
M. Statheropoulos et al. / Forensic Science International 153 (2005) 147–155154
toluene, hexane, benzene 1,2,4-trimethyl, 2-propanone and
3-pentanone were found in higher concentrations. Although
this is one of the first systematic analyses of VOCs produced
during putrefaction from the entire body, it has to be inter-
preted in relation to the given conditions where the two men
were found in sea water. It is of note that these findings
represent measurements at a single time point in the decay
process. Increased esters found in the second cadaver may be
consistent with advanced saponification. Further qualitative
and quantitative study of volatile organic compounds pro-
duced under different known conditions may provide infor-
mation that can be used in the future to more accurately
estimate the postmortem interval for death cases under
unknown situations. SPME and other similar methods could
be used for further investigations of the putrefaction che-
mical profile.
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