Chizuko Sasaki

Case report: Etizolam and its major metabolites in two unnatural death cases

A simultaneous analytical method for etizolam and its main metabolites (alpha-hydroxyetizolam and 8-hydroxyetizolam) in whole blood was developed using solid-phase extraction, TMS derivatization and ion trap gas chromatography tandem mass spectrometry (GC-MS/MS). Separation of etizolam, TMS derivatives of alpha-hydroxyetizolam and 8-hydroxyetizolam and fludiazepam as internal standard was performed within about 17 min. The inter-day precision evaluated at the concentration of 50 ng/mL etizolam, alpha-hydroxyetizolam and 8-hydroxyetizolam was evaluated 8.6, 6.4 and 8.0% respectively. Linearity occurred over the range in 5-50 ng/mL. This method is satisfactory for clinical and forensic purposes. This method was applied to two unnatural death cases suspected to involve etizolam. Etizolam and its two metabolites were detected in these cases.

Analysis of the sarcomere protein gene mutation on cardiomyopathy - Mutations in the cardiac troponin I gene

Developments in the molecular genetic studies of cardiomyopathy (CM) have led to discovery of a large number of mutations in the genes encoding the sarcomeric proteins. In this study, comprehensive screening of TNNI3 was performed in 36 consented autopsy cases diagnosed as CM, in order to evaluate the prevalence of gene mutations in sudden death caused by CM. In DCM cases, a new missense mutation Pro16Thr was detected. A single nucleotide polymorphism at -8 position of intron 3 (IVS 3 -8 T>A) was identified, which had a significant difference in allele frequency between DCM and control cases. From these results, it was indicated that this study contribute to genetic based diagnosis, risk stratification and prevention of sudden death caused by CM.

Experimental study on external contamination of hair by synthetic cannabinoids and effect of hair treatment

Abstract We recently reported a fatal case of poisoning by N-1-naphthalenyl-1-pentyl-1H-indole-3-carboxamide (NNEI). In this case, NNEI was detected from 13 sections of a 40 cm length of hair after washings. The concentrations of NNEI were generally similar among the hair segments. These results strongly suggested that externally contaminated NNEI could not be removed from the hair by washings. The aim of the present study is to demonstrate the adsorption and removal by washings for NNEI and [1-(5-fluoropentyl)-1H-indol-3-yl]-(4-methyl-1-naphthalenyl)-methanone (MAM-2201) using liquid chromatography–tandem mass spectrometry. NNEI and MAM-2201 were detected in black hair even after a single immersion in NNEI and MAM-2201 aqueous solutions, followed by washings. However, their adsorption seemed less pronounced for dyed or bleached hair. Therefore, hair analysis cannot be considered effective in testing of synthetic cannabinoid abuse, because the external synthetic cannabinoid(s) can be easily adsorbed to hair of a non-user by passive exposure.

ABO genotyping by TaqMan assay and allele frequencies in a Japanese population

ABO genotyping have become common tools for forensic casework. We developed a new rapid ABO genotyping method using a fast real-time PCR system with the TaqMan® Sample-to-SNP™ Kit. Eight single nucleotide polymorphism (SNP) sites in the ABO gene (nt 261, 297, 467, 657, 703, 829, 930 and 1061) were selected to determine the ABO genotypes. ABO genotypes were easily determined by examining allelic discrimination patterns. This method enabled analyses to be completed in about 1h per plate with no postmortem change influences. The detection limit in each SNP site was examined as 100pg per reaction. ABO genotyping from 1000 Japanese individuals was also examined to determine the distribution of ABO genotypes and allele frequencies. Thus, 31 genotypes were clearly identified, and these were controlled by four common and seven rare alleles. The power of discrimination, heterozygosity and polymorphism information contents were 0.913, 0.775 and 0.812, respectively. Therefore, selecting these eight SNP sites could be useful for high specific ABO genotyping. This rapid, sensitive and accurate genotyping method is useful for forensic casework.

Simultaneous determination of 5 psychotropic drugs of various types in an autopsy case of acute multiple drug poisoning.

We attempted the simultaneous determination of 5 drugs, mirtazapine, sertraline, chlorpromazine, amoxapine and zolpidem, detected in a gas chromatography-mass spectrometry screening test in an autopsy case. The solid-phase extraction of the analytes from biological samples was achieved using Oasis(®)HLB cartridges (Waters, Milford, MA, USA). Gas chromatography was performed on a HP-5MS fused silica capillary column (30 m × 0.25 mm i.d., 0.25 μm film thickness, Agilent Technologies). The mass spectrometer was operated with an electron energy of 70 eV in electron impact mode. The qualitative and quantitative analyses were performed in full-scan mode and the selected ion monitoring mode, respectively. The total ion chromatogram showed good separation of these drugs. Linear graphs were obtained with good correlation coefficients for these drugs from 0.001 to 2.0 μg/mL (r(2)=0.9909-0.9986) using imipramine-d6 as an internal standard. The recoveries of these drugs were found to be 62.8-88.0% in spiked whole blood. Mirtazapine, sertraline, chlorpromazine, amoxapine and zolpidem were found in post-mortem samples of the deceased at concentrations of 2.67, 0.07, 0.25, 0.32 and 0.68 μg/mL, respectively. The concentration of mirtazapine was within the lethal level and those of amoxapine and zolpidem were within the toxic level. We diagnosed that the cause of death was acute multiple drug poisoning. The simple and practical procedure used in this study is useful for the simultaneous determination of psychotropic drugs of various types in post-mortem biological samples.

A case of V-A shunt catheters migration into the pulmonary artery

A man in his sixties, who developed CPA at home, was transferred to the emergency center. Since CT images revealed a tube-shaped foreign body in the pulmonary artery, pulmonary embolism was initially suspected; however, this did not lead to a definite diagnosis. Autopsy revealed that the foreign body in the cadaver was a fragment of a V-A shunt catheter implanted about 30 years previously for the treatment of hydrocephalus. Although fibrous adhesion of a part of the catheter to the pulmonary artery wall was seen, suggesting that a fracture of the catheter had occurred a long time before, it was not known when the fracture had occurred. Since no pulmonary arterial obstruction secondary to the catheter or new thrombi, which had been initially suspected, were observed, the cause of death was determined to be ischemic cardiac failure. A fracture of a shunt catheter may be typically associated with some clinical manifestations, which are often found and treated. In this case, however, no symptoms appeared and the fracture of the shunt catheter remained untreated for a long time. This case was therefore considered to be extremely rare, and is an example of how a serious iatrogenic disease could occur.

Simultaneous determination for oxicam non-steroidal anti-inflammatory drugs in human serum by liquid chromatography–tandem mass spectrometry

A high-performance liquid chromatography-tandem mass spectrometry (LC/MS/MS) technique was developed for the simultaneous determination of five non-steroidal anti-inflammatory oxicam drugs (ampiroxicam, tenoxicam, piroxicam, meloxicam and lornoxicam) in human plasma. These five oxicam drugs and isoxicam (internal standard) were extracted from human plasma with an Oasis(®) MAX cartridge column and analysed on a Unison UK-C18 column (2.0 mm × 100 mm, 3 μm) with an acetonitrile:10mM formic ammonium buffer (pH 3.0) (50:50) mobile phase at 0.20 ml/min at 37°C. The analytes were detected using a tandem mass spectrometer, equipped with an electrospray ion source (ESI). The instrument was used in multiple-reaction-monitoring (MRM) mode. The extraction yields from a 200 μl human plasma sample (containing 10 ng of each drugs) with the Oasis(®) MAX cartridge column were 93.3-102.5%. The detection limits were 0.01-6.5 ng/ml (S/N=3). Our developed method is very useful for the simultaneous determination of five oxicam (non-steroidal anti-inflammatory) drugs in human plasma by LC/MS/MS.

A fatality due to inhalation of 1,1-difluoroethane (HFC-152a) with a peculiar device

A man aged in his thirties was found dead in his room, wearing a gas mask connected with two plastic bags. An empty canister of Air Duster was in one of the plastic bags. Toxicological analysis by gas chromatography (GC)-mass spectrometry and GC-flame ionization detection revealed the presence of 1,1-difluoroethane (HFC-152a), which is a component of the Air Duster aerosol. Quantitative analysis showed that the concentration of 1,1-difluoroethane in his heart blood was as high as 546 μg/ml. The cause of death was diagnosed as acute poisoning by 1,1-difluoroethane. It was supposed that he abused the Air Duster product to obtain euphoria and inhaled 1,1-difluoroethane at excessive concentrations with the peculiar tight-fitting gas inhalation device, resulting in his death.

Population genetics of Y chromosomal STR haplotypes in Japanese population

Abstract Five Y chromosomal short tandem repeat (STR) loci DYS389I, DYS389II, DYS439, DYS438 and DYS392 have been analysed to obtain the allele and haplotype frequencies data in 129 unrelated Japanese male individuals living in Kanagawa by means of multiplex PCR with Y-PLEX™ Primer Mix, followed by capillary electrophoresis using the ABI PRISM 310 Genetic Analyzer. The gene diversity values ranged from 0.555 (DYS439) to 0.737 (DYS389II). A total of 72 haplotypes was detected, of which 51 haplotypes were unique and the others occurred at least two times. The most common haplotype (DYS389I, DYS389II, DYS439, DYS438, DYS392) was 14–30–12–13–13 with a frequency of 10%, and the haplotype diversity was calculated as 0.969. The combined resolutions of the five Y STR multiplex system using the Y-PLEX™ 5 was applicable to routine case work.