Brian Waters

Autopsy report for a caffeine intoxication case and review of the current literature

Caffeine (1,3,7-trimethylxanthine) is a popular mild central nervous system stimulant found in the leaves, seeds and fruits of various plants and in foodstuffs such as coffee, tea, and chocolate, among others. Caffeine is widely used and is not associated with severe side effects when consumed at relatively low doses. Although rarely observed, overdoses can occur. However, only a few fatal caffeine intoxication cases have been reported in the literature. Herein, we report the pathological examination results and information on caffeine concentrations in the blood, urine and main organs in a fatal caffeine intoxication case. Even though high caffeine concentrations were found in the systemic organs, no caffeine-related pathological changes were detected.

Combination of a short middle-bore capillary column with a thicker stationary phase and a short narrow-bore separation column with a thinner stationary phase for the rapid screening of non-volatile drugs by gas chromatography–mass spectrometry

Dear Editor, An increase in requests for drug analysis has led to techniques that can analyze many samples in a short amount of time. For the purpose of enhancing the efficiency of analysis and increasing sample throughput, we looked at increasing the analysis speed of gas chromatography–mass spectroscopy (GC–MS), currently the most widely used technique for forensic drug analysis. The fast GC–MS techniques and the difficulty of their practical implementation have been reviewed in the literature [1]. Our group reported high-throughput chiral analysis of urinary amphetamines by fast GC–MS [2]. It seems very easy to establish a fast GC–MS method with a short narrow-bore capillary GC column and fast temperature programming, if using a modern GC–MS instrument that enables the rapid scanning of mass spectra and powerful pumping to maintain vacuum against the relatively large gas inflow into the ionization chamber. There are short narrow-bore capillary GC columns for fast GC–MS in the presence [3–5] and absence [2, 6, 7] of retention gaps, which are sometimes useful for obtaining narrow peak widths and symmetrical peak shapes. The retention gap is usually a short and deactivated capillary column without stationary phase. In this communication, we have tried to create a drug screening method by fast GC–MS using a short middle-bore capillary column with a thicker stationary phase in place of the inactive retention gap and a short narrow-bore capillary column. Our results showed that more than 20 drugs in whole blood could be screened within 4.1 min of GC–MS analysis. In this study, we mainly selected 30 psychotropic drugs [8, 9] as target compounds for analysis. Pure powder standards of fenfluramine-HCl, imipramine, diphenidolHCl, clomipramine-HCl, diazepam, chlorpromazine-HCl, nordiazepam, and trazodone were purchased from Sigma Aldrich (St. Louis, MO, USA); diphenhydramine-salicylate and nortriptyline-HBr from Wako (Osaka, Japan); and ethenzamide from Nacalai Tesque (Kyoto, Japan). Other drugs in the powder form were obtained from the corresponding pharmaceutical companies. Lidocaine-d10 was purchased from C/D/N Isotopes (Québec, Canada); diazepam-d5 and nordiazepam-d5 (both 1 mg/ml methanol solution) from Cerilliant (Round Rock, TX, USA). Caffeine-d3 (7-methyl-d3-theophylline) and 7-ethyltheophylline were synthesized in our laboratory. Other common reagents were of the highest purity commercially available. Blank whole blood was obtained from a healthy volunteer under permission; it was stored at -20 C until use. Methanolic stock solutions were made of the powder standards at a concentration of 1.0 mg/ml. A working stock solution of the 30 drug standards listed in Table 1 was made up in n-propyl acetate at a concentration of 20 lg/ml. Calibrators were prepared by adding enough of the standard working stock solution to 1 g of blank blood to yield the final concentrations of 0.10, 0.25, 0.50, 1.0, and 5.0 lg/ml in 16 9 125-mm screw cap tubes. A working internal standard (IS) solution containing caffeine-d3 (100 lg/ml), This article is for the special issue TIAFT2012 edited by Osamu Suzuki.

Methamphetamine and amphetamine concentrations in survivors of body-packer syndrome in Japan

There are few reports from Japan on the analysis of fluids in survivors of body-packer syndrome. We analyzed the concentrations of stimulants in the serum, plasma and urine collected from three patients suspected of being body packers at immigration that were referred to hospitals between 2010 and 2011. The drugs were extracted with solid-phase columns and analyzed by gas chromatography-mass spectrometry (GC-MS). In all cases, wrapped, cylindrical packets of foreign bodies were detected in the intestinal tract on plain X-ray (X-P) and computed tomography (CT), and they were eventually removed surgically. In case 1, the patient presented with convulsions and tachycardia at admission to the hospital and one of the packets was found to have ruptured. In case 2, although the subject appeared to have an intestinal obstruction caused by the packets on the third day, he exhibited no symptoms on arrival and the packets did not appear to have ruptured. In case 3, the patient exhibited restlessness on the first day and one of the removed packets had ruptured. In all cases, methamphetamine (MA) and amphetamine (AP) were detected in serum, plasma and urine. In this study, we report the variation in MA and AP concentrations in survivors of body-packer syndrome. The serum and plasma concentrations of MA were high in subjects that exhibited symptoms of MA intoxication. MA and AP were also detected in the case in which the patient exhibited no symptoms of intoxication and the packets had not ruptured. These results suggest either that the stimulants may have seeped through the wrap of the packets, or that the subject had been abusing the drugs.

GC-PCI-MS/MS and LC-ESI-MS/MS databases for the detection of 104 psychotropic compounds (synthetic cannabinoids, synthetic cathinones, phenethylamine derivatives).

Designer psychotropic compounds continue to be a major problem in Japan and all around the world. Electron impact mass spectrometry (GC-EI-MS) and liquid chromatography with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) data on these compounds have been widely reported. In this report, we present a detection method that has been rarely utilized to analyze these types of compounds, gas chromatography with positive chemical ionization and tandem mass spectrometry (GC-PCI-MS/MS). We report on the development of GC-PCI-MS/MS and LC-ESI-MS/MS databases of 104 psychotropic compounds, including 32 cannabinoid derivatives, 29 cathinone derivatives, 34 phenethylamine derivatives, and several other designer compounds. Using this database, we were able to detect 5 psychotropic compounds in an actual forensic autopsy case. If GC-PCI-MS/MS is used together with the more established methods of GC-EI-MS and LC-ESI-MS/MS, we believe the forensic toxicology community could be better prepared to deal with the challenges of these ever-changing compounds.

Development of a preparation method to produce a single sample that can be applied to both LC-MS/MS and GC-MS for the screening of postmortem specimens

Simple and efficient extraction methods have been developed for the screening of a wide array of drugs in postmortem autopsy specimens. Acidic and basic compounds were targeted with two extraction methods that can be applied to both GC-MS and LC-MS/MS instrumentation. The extractions were achieved by utilizing lipid-removal and solid-phase extraction cartridges while carefully monitoring the pH of the samples to ensure the adequate removal of interfering substances like lipids and amino acid derivatives. These methods were applied to actual autopsy cases, with 94 and 124 compounds detected by GC-MS and LC-MS/MS, respectively. The developed methods could easily be incorporated into a forensic laboratorys daily routine for screening many different compounds from postmortem samples.

Application of high-throughput chiral analysis of amphetamines by GC–MS to whole blood specimens

Dear Editor: Chiral analysis of amphetamine-type stimulants (ATSs) has become essential to distinguish between their illicit and legitimate uses. This is because Selegiline (L-deprenyl) used for treatment of Parkinson’s disease produces L-methamphetamine (MAMP) and its metabolite L-amphetamine (AMP), while Gewodin (famprofazone) used for pain control also produces both D-MAMP and L-MAMP and their metabolites in the human body. Furthermore, in some countries, Adderall containing both D-AMP and L-AMP is frequently prescribed by doctors for treatment of attention-deficit hyperactivity disorder [1]. Chiral analysis of ATSs has been achieved by gas chromatography–mass spectrometry (GC–MS) without using an expensive GC column coated with b-cyclodextrin [2]; the enantiomers of ATSs can be separated after derivatization with trifluoroacetyl-L-prolyl chloride (TPC) or R-(–)-a-methoxy-a-trifluoromethylphenylacetyl chloride [3] with a conventional capillary column. However, the retention times of the derivatized compounds usually become much longer than those of the free compounds, because of the much larger molecular sizes of the derivatives. Furthermore, chiral analysis usually needs a long analysis time to achieve good separation of enantiomers. In our previous article [1], we reported a very rapid and simultaneous chiral analysis of ATSs in human urine. It seems that many more reports [1, 2, 4–7] on chiral analysis of ATSs have dealt with urine specimens than with whole blood specimens [3], probably because urine gives higher recovery rates and clean extracts with fewer impurity peaks than does whole blood. In this brief communication, we demonstrate that rapid chiral analysis can be also applied to whole blood specimens. D-MAMP was purchased from Dainippon Pharmaceutical (Osaka, Japan). Three standards (D-AMP, L-AMP, and L-MAMP) and deuterated internal standards (L-AMP-d3 and L-MAMP-d6) were synthesized in our laboratory [2]. D-AMP, L-AMP, and L-AMP-d3 were sulfate salts, while all others were hydrochloride salts. TPC was purchased from Sigma-Aldrich (St. Louis, MO, USA); triethylamine (TEA) and diatomaceous earth (granular) from Wako Pure Chemical (Osaka, Japan). Other common reagents used were of analytical grade. Control drug-free whole blood samples were obtained from laboratory personnel volunteers (n = 5) with informed consent. The whole blood (0.2 ml) was spiked with internal standards to give the final concentration of 50 ng/ml each, and 0.8 ml of 1 M hydrochloric acid was added. The mixture was vortexed for 30 s, and centrifuged at 3,600 rpm for 3 min. The supernatant was adjusted to pH 12.6 with 5 M sodium hydroxide solution, and then 0.01 ml of TEA was added. This alkaline sample was immediately poured into a polypropylene filtration tube containing 2 g of granular diatomaceous earth. A small disc of filter paper was used to prevent the granular diatomaceous earth passing through the tube. After 15 min, the analytes were extracted with 5 ml of n-hexane containing 20 ll of 0.1 M TPC dichloromethane solution and 0.1 ml of propyl acetate. The samples were evaporated to dryness under a nitrogen stream and reconstituted in 100 ll of ethyl acetate for GC–MS analysis. H. Fujii K. Hara M. Kashiwagi A. Matsusue B. Waters S. Kubo (&) Department of Forensic Medicine, Faculty of Medicine, Fukuoka University, 7-45-1 Nanakuma, Jonan-ku, Fukuoka 814-0180, Japan e-mail: kuboshin@fukuoka-u.ac.jp

Tissue Distribution of Suvorexant in Three Forensic Autopsy Cases

Suvorexant (Belsomra®) is a relatively new insomnia medication that has been available in USA and Japan since 2014. It is a dual orexin receptor antagonist that promotes sleep by inhibiting the binding of orexin neurons to the OX1R and OX2R receptors. In this report, we describe the detection and quantitation of suvorexant from the postmortem specimens of three separate autopsy cases handled by our department. Suvorexant was identified by fast gas chromatography/mass spectrometry during routine screening, and quantitated by a fully validated liquid chromatography-tandem mass spectroscopy method. Quantitation was achieved by positive electrospray ionization in the selected reaction monitoring mode. Monitored transitions were m/z 451 > 186 for quantitation and m/z 451 > 104 for qualification. To our knowledge, this is the first instance of suvorexant being quantitated from actual autopsy cases. It is likely that this compound will be encountered more often by the forensic toxicology community going forward.

The global distribution of the p.R1193Q polymorphism in the SCN5A gene

The SCN5A (sodium channel, voltage-gated, type V, alpha subunit) gene encodes the cardiac sodium channel, a member of the voltage-gated sodium channel family. The p.R1193Q (c.3578G>A) polymorphism in SCN5A is known to accelerate inactivation of the sodium channel current, and has been identified in patients with Brugada and long QT syndromes. In the present study, we investigated the frequency of the p.R1193Q substitution in more than 4000 genomic DNA samples from 34 Asian, European, and African populations using TaqMan and/or APLP (amplified product length polymorphism) assays. Allele A (p.1193Q) was detected in most Asian populations, but was sporadically observed or absent in European and African populations. These results demonstrated that the p.R1193Q substitution is characteristic of Asian populations.

An unexpected death due to massive ascites and a giant mucinous ovarian cystadenoma

A female in her thirties fell face down in her room. She was motionless when her sister found her. She was transported to the hospital by ambulance and was in a state of cardiopulmonary arrest on admission. She did not respond to resuscitation. Her abdomen had started to swell 3years before her death. An autopsy was performed to clarify the decedents cause of death. She was 172cm tall and weighed 146kg. Her maximum abdominal girth was 172.1cm. A subcutaneous hemorrhage measuring 4.5cm in diameter was observed in the epigastric region. The abdominal cavity contained brownish ascites (54.1L). The left ovary was markedly swollen, and the combined weight of the uterus and right ovary was more than 13.0kg. A left ovarian tumor consisting of serous and mucinous cysts was detected. There were no metastatic lesions in the peritoneum or other organs. She might have suffered circulatory disturbance caused by the ascites and ovarian tumor. Moreover, being in a prone position would have resulted in an increase in intra-abdominal pressure, further exacerbating her circulatory problems. Therefore, her cause of death was considered to be circulatory failure caused by significant ascites and a large ovarian tumor.

An autopsy case of sudden unexpected nocturnal death syndrome with R1193Q polymorphism in the SCN5A gene

SCN5A (sodium channel, voltage-gated, type V, alpha subunit) gene encodes the cardiac sodium channel, a member of the voltage-gated sodium channel family. SCN5A mutations have been associated with a variety of inherited arrhythmias, including long QT syndrome and Brugada syndrome. We report an autopsy case of sudden unexpected nocturnal death syndrome. A man in his thirties died at night while sleeping. At autopsy, no traumatic injury, disease or drug intake was observed as a possible cause of death. We examined mutations in the SCN5A gene and identified a heterozygous mutation causing an R1193Q amino acid substitution. It was reported that the R1193Q polymorphism in the SCN5A gene destabilizes channel inactivation and may be a risk factor for Brugada and long QT syndrome. It may be considered that the cause of death in this case was sudden cardiac death.