Forensic Science

Forensic Science International 78 (1996) 119-124

Detection of toluene in an adipoceratous body

Hiroyuki Inoue *, Mineo Iwasa, Yoshitaka Maeno, Hiroyoshi Koyama, Yuki Sato, Ryoji Matoba

Department of Legal Medicine, Nagoya City University Medical School, Kawasumi Mizuho-ku, Nago.va 467, Japan

Received 19 September 1995; accepted 15 November 1995

Abstract

A 24-year-old male was found dead in a car left in a river for about 3 months. The cadaver was almost adipoceratous and autopsy findings revealed that there were neither remarkable injuries nor lethal diseases. Toluene, ethanol, 1-propanol, 2-propanol, 1-butanol, dimethyl sulfide, dimethyl disulfide, isovaleraldehyde and n-butyl n-butyrate were detected in the specimens collected at the autopsy by head space gas chromatography/mass spectrometry (GC/MS). The toluene concentrations (pg/g) were 31.0 in brain, 10.6 in liver, 5.4 in kidney, 15.0 in skeletal muscle and 187.1 in adipose tissue. The presence of diatom in lung, liver and kidney suggested that death was caused by drowning. So far as we know, this is the first report of detection of toluene in an adipoceratous body.

Keywords: Toxicology; Toluene; Adipocere; Postmortem changes; Gas chromatography/mass spectrometry; Drowning

1. Introduction

Toluene is widely used as organic solvent in chemical and pharmaceutical industries and as a major component of paint and lacquer. It is also the common substance in glue and thinner used by abusers for sniffing. For diagnosis of thinner sniffing and/or poisoning, toxicological analyses for volatile substances are per- formed on blood and organ specimens collected at the autopsy mainly using gas chromatography and gas chromatography/mass spectrometry (GC/MS) with head

* Corresponding author.

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120 H. Inoue et al. /Forensic Science International 78 (1996) 119-124

space method [l-6]. When cadavers are putrefied, several kinds of putrefactive gas complicate the profile of the chromatogram [l] and may disturb the detection of inhaled volatile substances. Using GC/MS with a polar fused-silica capillary column, we determined toluene in body materials of an adipoceratous body.

2. Case profile and autopsy findings

In June 1994, a 24-year-old male was found dead in a car left in a river for about 3 months. A bottle of refreshing drink which was plugged tightly and contained colorless liquid was also found left near the cadaver. He was a painter and was missing for about 3 months.

The cadaver was 173 cm tall and weighing 67.0 kg. The gross examination of the body revealed no evidence of any external injuries. The subcutaneous adipose tissue and some part of skeletal muscles were adipoceratous. Autopsy findings revealed that there were neither remarkable injuries nor lethal diseases, though most internal organs showed softening by putrefaction.

Diatoms were detected in lung, liver and kidney by a conventional acid digestion technique.

3. Toxicological analysis

3.1. Chemicals

Toluened8 was purchased from Aldrich Chem. Co. (Milwaukee, WI, USA). Standard substances and other reagents were obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan).

3.2. Specimens for analyses

Toxicological analyses for volatile substances were performed on the brain, liver, kidney, skeletal muscle and adipose tissue collected at the autopsy. Liquid in the bottle of the refreshing drink left near the cadaver was also analyzed qualitatively.

3.3. Extraction procedure

The method used was a minor modification of the procedure described by Kato et al. [7]. Each 0.5 g of tissue sample was minced and placed into a 15-ml vial containing 1 ml of distilled water, 2 g of sodium chloride, 20 ~1 of dimethyl sulfoxide (DMSO) and 20 ,ul of DMSO solution containing toluene-d8, as an internal standard at a concentration of 1 mg/ml. The vial was tightly sealed using a screw cap with a Teflon-coated rubber plug and warmed at 55°C for 20 min with mechanical shaking. One hundred microliters of head space gas was taken by using a gas syringe through the plug, and injected into a gas chromatograph/mass spectrometer.

H. Inoue et al. : Forensic S&nw lnternarional 78 (1996) II%124 121

Calibration curve was made using 0.5 ml of distilled water and 20 ~1 of DMSO solution containing various concentrations of toluene instead of tissue sample and DMSO, respectively.

3.4. Conditions of gas chromatography/mass .spectrometrJl

GC/MS analyses were performed using a Shimadzu QP-5000 (Kyoto, Japan) equipped with a polar fused-silica CBP20 capillary column (25 m x 0.32 mm I.D., film thickness 0.5 /Lrn; Shimadzu). The oven temperature was held at 40°C for 1 min following injection and programmed to 90°C at a rate of S”C/min. The injection port and interface temperatures were 200°C and 23O”C, respectively. Helium was used as the carrier gas (a head pressure of 20 kPa and a total flow rate of 40 ml/min). The mass spectrometer was operated under positive electron impact mode at an ionization energy of 70 eV.

3.5. Qualitative and quantitative GC!A4S analyses

For qualitative analysis, the mass spectometer was operated with scan mass range of 30 to 200 atomic mass units. Peak identification was made by comparing the retention times and mass spectra of the authentic substances with those of unknown samples. For quantitative analysis, the following masses were monitored by selected ion monitoring (SIM): m/z = 91 for toluene, m/z = 98 for toluene-d8. The standard curve for toluene was obtained by plotting the peak area ratio of toluene (m/z 91) to toluene-d8 (m/z 98) versus the amount of toluene.

4. Results

The total ion chromatogram and the mass chromatogram obtained from vapor of standard mixture are shown in Fig. 1. The peaks of toluene, alcohols and other organic solvents were well separated from each other on the chromatograms using the present method.

The calibration curve for toluene was linear in the concentration range from 1 pug/g to at least 200 ,ug/g, with a correlation coefficient of 0.999. Table 1 shows the relative recovery rates of toluene from body materials to the standard value obtained from a water sample. The calculated recovery at concentrations of 4 and 100 @g/g ranged from 83 to 97% and from 93 to 98%, respectively.

Fig. 2 illustrates the chromatograms from the brain specimen collected at the autopsy. Similar chromatograms were obtained from the other organs. Qualitative observation revealed that the specimens included at least nine volatile substances; toluene, ethanol, I-propanol, 2-propanol, 1 -butanol, dimethyl sulfide, dimethyl disulfide, isovaleraldehyde and n-butyl n-butyrate (Fig. 2b. and Table 2). Only toluene was detected as a volatile substance in liquid in the bottle left in the car.The toluene concentrations (pg/g) were 31.0 in brain, 10.6 in liver, 5.4 in kidney, 15.0 in skeletal muscle and 187.1 in adipose tissue.

122 H. Inoue et al. 1 Forensic Science International 78 (1996) 119-124

16937117

a.

16

- ,....I.. , . . . . ..(....,....,...,,,_._ ,, 1 2 3 4 5 6

--“--- 7 6 9

9539943 -1 b.

Fig. 1. Total ion chromatogram (a) and mass chromatogram (b) from vapor of standard mixture. Peaks: 1, acetone; 2, ethyl acetate; 3, methanol; 4, 2-propanol; 5, ethanol; 6, 2-butanol; 7, toluene-d8; 8, toluene; 9, 1-propanol; 10, 3-pentanol; 11,2-pentanol; 12, p-xylene; 13, m-xylene; 14, I-butanol; 15, o-xylene; 16, l-pentanol.

5. Discussion

The polar fused-silica capillary column used in the present method was able to separate various organic solvents and alcohols quite easily even if the specimens included products in the process of putrefaction. As pointed out by Seto [8], combination of GC/SIM and a deuterium-labeled internal standard, toluene-d8,

Table 1 Relative recoveries of toluene from tissue samples

Sample Percent recovery of toluene

100 m/g 4 KaP

Brain 97.9 + 7.6 82.7 + 4.2 Liver 93.0 * 1.2 83.7 + 4.1 Kidney 98.3 5 4.2 96.5 f 9.2 Muscle 97.9 k 2.8 96.9 + 5.1 Fat 96.1 f 2.8 91.9 f 7.5

Data represent the mean k S.D. of three values.

H. Inoue et al. / Forensic Science hter~~ational 78 (1996) 119-124 123

31 45 47 58 71 91 98

Fig. 2. Total ion chromatogram (a) and mass chromatogram (b) from the brain specimen of the cadaver. Peaks: 1, dimethyl sulfide; 2, isovaleraldehyde: 3. 2-propanol; 4, ethanol; 5, toluene-d8 (IS): 6. toluene: 7, I-propanol; 8, dimethyl disulfide; 9, I-butanol; 10, n-butyl n-butyrate.

was good for their reliability of quantification of toluene regardless of sample matrix.

Qualitative analysis showed that the specimens included at least nine volatile substances but the bottle left in the car only included toluene. These results suggest that the individual had been sniffing thinner, probably in the bottle, and I-butanol was a product in the process of putrefaction, though it was well known to be one of components of thinner. Tomita [9] reported that experimental dead bodies under

Table 2 Retention times and principal ions of the authentic substances

Substance Retention time (min) Principal ions (m/z)

Dimethyl sulfide 1.06 47, 62, 45 Isovaleraldehyde 1.93 44,41, 58 2-propanol 2.10 45. 43, 31 Ethanol 2.18 31, 45, 46 Toluene 3.41 91, 92 I -propanol 3.56 31, 42. 59 Dimethyl disulfide 4.02 94, 45, 79 1 -butanol 5.64 31, 56, 41 n-Butyl n-butyrate 7.17 71, 43, 56

124 H. Inoue et al. /Forensic Science International 78 (1996) 119-124

water produced various alcohols such as methanol, ethanol, 1-propanol, 1-butanol, occasionally 2-propanol and so on. In the present case, we also detected most of these alcohols clearly except methanol, which appeared barely on the mass chromatogram at m/z 31.

Toluene is a lipophilic low-molecular-weight volatile substance and the concentra- tions in body fluids and organs are altered by diffusion after death [lO,ll]. However, these reports also described that postmortem concentrations in brain were relatively constant during the 24-h [lo] or 48-h [l l] postmortem interval. In fatal cases of toluene poisoning by inhalation, toluene concentrations of brain ranged from 297 to 740 pug/g [5,12,13], while those in toxic cases of toluene (their direct causes of death were asphyxia, drowning, general contusions and sudden cardiac death), from 5 to 80 ,ug/g [2-4,12,13]. Although attention should be directed to postmortem diffusion and re- distribution of toluene occurring in the body in the interval between death and auto- psy sampling when interpreting results, the toluene concentration of brain in our case (31.0 pg/g) was thought to be toxic level, but not so high as to be lethal. The presence of diatoms in lung, liver and kidney suggested that death was caused by drowning.

So far as we know, this is the first report of detection of toluene in an adipoceratous body. Postmortem changes of toluene concentrations in body for long time periods and possible influences caused during adipocere formation must be clarified.

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