Detection of Butane Metabolites As an Indicator of Butane Abuse

Detection of butane metabolites as an indicator of butane abuse

Hiroshi Kinoshita1,*, Naoko Tanaka1, Ayaka Takakura1, Mitsuru Kumihashi1, Mostofa Jamal1, Asuka Ito1, Shoji Kimura1, Kunihiko Tsutsui2, Yasushi Nagasaki3, Shuji Matsubara4, Kiyoshi Ameno1

______Abstract: We have identified 2-butanol, 2-butanone, and t-butanol with a faint peak of n-butane in a postmortem blood sample from an inhalant abuser by using head-space solid-phase microextraction coupled with gas chromatography- mass spectrometry (HS-SPME/GC-MS). The compounds 2-butanol, 2-butanone, and t-butanol are metabolites of n-butane and isobutane. We concluded that the victim had abused volatile gases prior to death. The identification of metabolites of gases in the blood may be a good biomarker of volatile gas abuse. Key Words: biomarker, abuse, volatile substance, head-space solid-phase microextraction with gas chromatography- mass spectrometry (HS-SPME/GC-MS).

olatile substance abuse is one concerning Vproblem in youth and adults today [1, Case report 2]. Forensic toxicologists need to identify gaseous components in blood or tissue samples as evidence for A man in his 30s (height, 173 cm; weight, 96 kg) suspected volatile gas abuse both in clinical situations was found dead in his room. He had a history of volatile and at autopsy [3]. However, this requires pre- gas abuse. Subsequent investigation by the authorities analytical and analytical precautions with regard to revealed that there were six empty butane gas canisters proper sample collection, storage, and handling [3-5] meant for a portable cooking stove found around him. as some kinds of gaseous components disappear easily, The containers were labeled as containing n-butane, even at room temperature. The contents of n-butane isobutane and propane. in blood samples have been shown to decrease by 50% A medicolegal autopsy revealed no evidence within 3 days when stored at room temperature [6]. of external injury or findings of natural disease. Both Most inhaled n-butane and isobutane are believed to be lungs showed edema and congestion (left and right eliminated unchanged by the pulmonary route, and a lungs weighed 558 g and 635 g, respectively). Marked small portion of each component is metabolized by the congestion was observed in each organ. The postmortem liver [7-10]. We report here that the detection of n-butane interval was estimated to be approximately 1 day at the or isobutane metabolites may be a good biomarker for time of autopsy. gas abuse. A drug screening test using an Instant View®

1) Kagawa University, Faculty of Medicine, Department of Forensic Medicine, Ikenobe, Miki, Kita, Kagawa, Japan * Corresponding author: Kagawa University, Faculty of Medicine, Department of Forensic Medicine, 1750-1, Ikenobe, Miki, Kita, Kagawa 761-0793, Japan, Tel: +81-87-891-2140, Fax: +81-87-891-2141, Email: [email protected] 2) Kagawa University, Faculty of Medicine, School of Nursing, Health Sciences, Ikenobe, Miki, Kita, Kagawa, Japan 3) Hyogo Medical Examiners Office, Arata-cho, Hyogo-Ku, Kobe, Japan 4) Community Health Care Education Support Center and Postgraduate Clinical Education Center, Kagawa University Hospital, Ikenobe, Miki, Kita, Kagawa, Japan

216 Romanian Journal of Legal Medicine Vol. XXIV, No 3(2016)

M-1 panel (Alfa Scientific Designs, Inc, Poway, CA, putrefaction in forensic practice, [14] were not detected USA) and ethanol examination by routine head-space in the present sample. Therefore, the chemicals detected gas chromatography were performed. A blood sample in the present case are consistent with either gas or its was collected for toxicological examination. metabolites inhaled prior to death. The components of the gas in the canister abused Equipment for Toxicological Examination in the present case were n-butane, isobutane, and propane. Analysis of volatile hydrocarbons and the above- However, the absence of butane in the postmortem mentioned metabolites was performed in accordance sample does not always mean a death is not butane related with previous reports, with slight modification [10]. In [15]. Diagnosis of volatile substance abuse prior to death brief, a gas chromatography-mass spectrometry (GC- would be based on a combination of circumstances and MS) model QP-2010 Plus (Shimadzu, Kyoto, Japan) was clinical and toxicological findings, rather than on any used. The gas chromatograph-mass spectrometer was one factor alone [3]. From the circumstances, autopsy fitted with a DB-5MS column (30 m × 0.25 mm I.D., findings and results of the toxicological examination, film thickness, 0.25 µm; Agilent Technologies, Santa we concluded that the cause of this subject’s death was Clara, CA). Helium was used as the carrier gas (1.78 mL/ butane related. min). Identification of each compound was determined Detection of these metabolites may be dependent by retention times and confirmation ions (2-butanol, on the pattern of inhalation before death [10]. It could 2-butanone, and t-butanol for m/z=45, 43, and 59, be more detectable in a person with a longer history respectively [10]). of duration of abuse or chronic misuse but may not Subsequent toxicological analysis using a 6890N be detected in a person after a single inhalation [10]. gas chromatograph combined with a 5973 MS mass Although the details of this man’s history of abuse were spectrometer (both Agilent Technologies) was performed as described previously [11].

Sample preparation The procedure for head-space solid-phase microextraction method (HS-SPME) was carried out according to a previous report [12]. In brief, 0.5 mL of postmortem blood with 0.5 mL of distilled water was placed into a 20-mL glass vial and sealed with a silicone rubber septum and aluminum cap. The SPME fiber assembly (polydimethylsiloxane, 100 µm thickness; Sigma-Aldrich Japan, Tokyo, Japan) was inserted into the head space of the glass vial through a silicone rubber septum. The vial was heated to 60¯C on a heating block, and the fiber was exposed to the head space for 15 min.

Results and Discussion

HS-SPME/GC-MS analysis identified 2-butanol, 2-butanone, and t-butanol with a faint peak of n-butane in the blood sample (Fig. 1). No other chemicals or pharmaceuticals were identified by subsequent toxicological analysis using GC-MS. Ethanol was not detected in the blood. The compounds 2-butanol and 2-butanone are metabolic products from n-butane, and t-butanol is yielded from isobutane by ω-1 oxidation in liver microsomes through a major metabolic pathway for hydrocarbons [8]. It has been reported that 2-butanone is produced postmortem, during the early phase of decomposition [10, 13]. In the present case, postmortem putrefactive formation was not suspected to have occurred yet because neither ethanol, a common core Figure 1. Total ion and mass chromatogram for each ion in substance found in the decomposition process [13] nor blood sample (1:2-butanone (m/z=43), 2:2-butanol (m/z=45), n-propanol, which is one of the reliable markers for 3: t-butanol (m/z=59), respectively).

217 Kinoshita H. et al. Detection of butane metabolites as an indicator of butane abuse unknown, simultaneous identification of 2-butanol, HS-SPME is a simple and reliable procedure for 2-butanone, and t-butanol in the blood strongly suggest simultaneous analysis of multiple volatile substances, that the victim chronically inhaled gas containing and its combination with GC-MS (HS-SPME/GC-MS) n-butane and isobutane before death. has been applied in forensic practice [5, 19-22]. HS- Degradation or disappearance of the parent drug SPME analysis allows easy and simultaneous detection or chemical could occur during the postmortem interval of multiple volatile components, with simple sample for various reasons, such as bacterial activity, postmortem preparation. It is important to keep in mind that as the biotransformation, or natural evaporation [15-17]. In volatile chemicals in blood samples may be markedly such cases, the presence of metabolites in postmortem decreased, samples should be stored in gas-tight samples would be an indicator of the use of each of the containers with minimum headspace and kept under parent chemicals [16, 17]. Moreover, as the boiling point cold conditions in cases of suspected volatile substance of 2-butanol, 2-butanone, and t-butanol, detected in abuse [3-5, 15, 23]. the present case, are higher than that of n-butane [18], postmortem diffusion of those metabolites may have Conflict of interest. The authors declare that been less than the parent chemicals. they have no conflict of interest concerning this article.

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