Jun Nishigami

Screening and determination of methamphetamine and amphetamine in the blood, urine and stomach contents in emergency medical care and autopsy cases

Methamphetamine (MA) and amphetamine (AMP) were screened and their levels were determined using the Toxi-Lab thin-layer chromatography system and gas chromatography-mass spectrometry, respectively, in the blood, urine and stomach contents from 211 emergency medical care and 417 autopsy cases. MA and AMP were detected in 5 emergency medical cases, and the blood MA and AMP concentrations ranged from 0.697-0.041 micromol/100 g and from 0.0944-0.0003 micromol/100 g, respectively. MA and AMP were detected in 19 autopsy cases, in which blood MA and AMP concentration ranged from 14.3-0.123 micromol/100 g and from 0.256-0.0017 micromol/100 g, respectively. The autopsy cases included 5 cases of sudden death with blood MA concentration of less than 3 micromol/100 g. MA and AMP screening and determination in emergency medical care and autopsy cases provide useful information and are indispenable in clarifying the dimensions of MA abuse in Japan.

Postmortem degradation of administered ethanol-d6 and production of endogenous ethanol: experimental studies using rats and rabbits

Deuterium-labeled ethanol-d6 was employed to study the metabolism and postmortem change of ethanol in putrefied organ tissues. First, 4 ml/kg body weight of 25% (w/v) solution of ethanol-d6 was administered orally to each of 15 rats. The heart blood and organs were collected 15-90 min after the administration and the ethanol-d6 was analyzed by head space gas chromatography/mass spectrometry. The ethanol-d6 concentration in the organ tissues reached its maximum at 15 min after the administration and then gradually declined, showing the same pattern as human ethanol metabolism. Ethanol-d6 (3 ml of the same solution/kg body weight) was injected into the vein of a rabbits ear (total of 12 rabbits). The rabbit was killed with carbon monoxide 30 min after the administration and the carcass was allowed to stand for 1-4 days at 30 degrees C in a moist chamber. The concentration of ethanol-d6 decreased moderately. Postmortem ethanol and 1-propanol concentrations, in contrast, showed marked increases 2.5 days and more after sacrifice in line with the degree of putrefaction of each organ tissue including skeletal muscle. This suggests the postmortem activation of micro-organism activity. These results indicate that ethanol concentrations in cadaver tissues must be carefully assessed with due consideration of postmortem degradation and production.

Toxicological analysis of the psychotropic drugs chlorpromazine and diazepam using chemically fixed organ tissues

Toxicological analysis for chlorpromazine and diazepam was performed using chemically fixed organ tissue specimens. After chlorpromazine and diazepam had been injected into rabbits, organ tissues (brain, lung, liver, kidney and skeletal muscle) were collected and fixed in 3 fixative solutions: buffered 10% formalin solution (pH 7.4, 10% BF), non-buffered 10% formalin solution (pH 5.1, 10% non-BF), buffered 4% paraformaldehyde solution (pH 7.4, 4% BPA). Chlorpromazine and diazepam were determined by GC-MS (gas chromatography-mass spectrometry) after 5 different fixation periods, and were detected even after 28 days of fixation. Recoveries of chlorpromazine and diazepam in 10% BF were within the range 48–86% and 68–171%, respectively after 28-day fixation, those in 10% non-BF were 22–54% and 48–78%, respectively, and those in 4% BPA solution were 13–59% and 14–50%, respectively. Thus, 10% BF was found to be the most suitable fixation medium for analysis of chlorpromazine and diazepam.ZusammenfassungChlorpromazin- und Diazepamgehalte aus fixiertem Organmaterial wurden bestimmt. Nach intravenöser Gabe von Chlorpromazin und Diazepam an neun Kaninchen, wurde Organmaterial (Gehirn, Lunge, Leber, Niere und Skelettmuskulatur) entnommen und nach drei unterschiedlichen Verfahren fixiert: 1. gepufferte (pH=7.4), 10%ige Formalin-Lsg., (10% BF); 2. ungepufferte (pH=5.1), 10%ige Formalin-Lsg., (10% nonBF) und 3. gepufferte (pH=7.4), 4%ige Paraformaldehyd-Lsg. (4% BPA). Nach 28 Tagen Fixierung wurden die Chlorpromazin- und Diazepamgehalte gaschromatographisch mit massenspezifischer Detektion (GC-MS) bestimmt. Die Wiederfindungsraten für Chlorpromazin und Diazepam lagen für die erste Variante (10% BF) zwischen 48–86% bzw. 68–171%, für die zweite Variante (10% non-BF) zwischen 22–54% bzw. 48–78% und für die dritte Fixierungsart (4% BPA) zwischen 13–59% bzw. 14–50%. Die Fixierung mit gepuffertem, 10%igem Formalin war die geeigneteste Fixierungsvariante zur anschlißenden Bestimmung von Chlorpromazin und Diazepam aus fixiertem Organmaterial.

Experimental studies on postmortem diffusion of ethanol-d6 using rats

In an investigation of postmortem ethanol diffusion deuterium-labeled ethanol-d6 was instilled by peroral gavage immediately after death by CO into the stomach of rat carcasses which were subsequently kept for 12-72 h at 5 or 30 degrees C. The heart blood, abdominal fluid and several tissues were collected and analyzed by head space gas chromatography-mass spectrometry. Rat carcasses showed no macroscopic changes until at least 72 h at 5 degrees C, and 12 h at 30 degrees C. At 30 degrees C, slight macroscopic change was observed after 24 h, moderate change after 48 h and marked change after 72 h. In the abdomen ethanol-d6 diffused gradually into neighboring organs (hepatic left lobe, left kidney and spleen) at 5 degrees C, with ethanol-d6 reaching a peak concentration of 0.75-2.38 mg/g at 24 h. At 30 degrees C, ethanol-d6 was also detected in neighboring organs and reached a peak concentration of 1.06-2.61 mg/g at 12 h. Thereafter, the ethanol-d6 concentration in the liver, kidney and spleen decreased, with concentrations ranging from 0.30 to 0.61 mg/g at 30 degrees C and 0.05 to 1.47 mg/g at 5 degrees C at 48 h. In the femoral skeletal muscle, ethanol-d6 was not detected until 24 h or more storage at 30 degrees C and never detected at 5 degrees C. In the brain and the organs in the thoracic cavity ethanol-d6 was detected after 12 h or more at 5 or 30 degrees C. Comparison of these results of direct peroral gastric instillation with those when ethanol-d6 was injected into the stomach through a laparotomy incision suggest that the brain and thoracic cavity changes were a result of diffusion from the mouth and esophagus. After 24 h at 30 degrees C, the postmortem ethanol production (0.33-0.85 mg/g) was comparable to those in previous reports. These results indicate that the assessment of ethanol concentration in the heart blood and organs in autopsy cases must be carefully conducted in comparison with the alcohol content of the stomach.

Toxicological analysis of drugs and poisons in formalin-fixed organ tissues. 2. Volatile substances

Diethylether, chloroform and toluene were administered by inhalation and ethanol intravenously to rabbits. As soon as possible after death, tissue specimens were collected from the brain, lung, liver, kidney and skeletal muscle and fixed in non-buffered 10% formalin at room temperature (10–20° C) for 4 different periods (1, 2, 5 and 14 days). The volatile substances were analyzed and identified by gas chromatography/mass spectrometry (GC-MS). The measured concentrations of ethanol, diethylether, chloroform and toluene in the brain tissue 1 day after fixation decreased to 8, 23, 73 and 84% respectively compared with those in the non-fixed brain tissue (100%). The rank order of the rate of decrease in the fixed state was: ethanol > diethylether ≫ chloroform > toluene. These volatile substances could be detected clearly in all the tissue specimens, even after a 14-day fixation period. These results provide useful toxicological information that will help to differentiate whether volatile substances have been administered antemortem or postmortem.ZusammenfassungKaninchen erhielten intravenös oder per inhalationem Ethanol, Diethylether, Chloroform und Toluol. Gewebsproben von Gehirn, Lunge, Leber, Niere und Skelettmuskulatur wurden entnommen und in ungepuffertem 10-prozentigem Formalin bei Raumtemperatur (10–20°) über 4 verschiedene Zeiträume (1, 2, 5 und 14 Tage) fixiert. Die flüchtigen Substanzen wurden mit Hilfe der Gaschromatographie/Massenspektrometrie (GC/MS) analysiert und identifiziert. Die gemessenen Konzentrationen von Ethanol, Diethylether, Chloroform und Toluol im Hirngewebe nahmen einen Tag nach der Fixation auf 8, 23, 73 bzw. 84%, — der Konzentrationen im nicht-fixierten Hirngewebe (100%) ab. Die Reihenfolge der Konzentrationsabnahme in fixiertem Material gegenüber dem nicht-fixierten Material lautet: Ethanol > Diethylether ≫ Chloroform > Toluol. Diese flüchtigen Substanzen konnten auch nach einer 14-tägigen Fixationsperiode eindeutig in allen Gewebsproben nachgewiesen werden. Diese Resultate ergeben eine nützliche toxikologische Information zur Differenzierung, ob flüchtige Substanzen vor dem Tode zugeführt wurden oder nicht.

Screening of volatile substances and determination of toluene (a thinner component) in the blood and urine in emergency medical care and autopsy cases by the pulse heating method

Screening of volatile substances was performed by pulse heating gas chromatography - mass spectrometry (GC-MS) using a GS-Q column in 211 emergency medical care and 342 autopsy cases. At least 36 standard substances could be separately detected. Six kinds of volatile substances were screened in a total of 553 cases. Toluene and/or hippuric acid were detected in the blood and/or urine in respectively, 4 emergency medical care and 8 autopsy cases. There were 11 abusers (9 males and 2 females) in these 12 positive cases. The ages of the abusers ranged from 13-26 years. There was no particular pattern to the monthly frequency distribution of identification of thinner (toluene) abuse cases, which occurred throughout the year. It is believed that these data at least partly reflect the present status of thinner/glue abuse in Japan. We conclude that pulse heating GC-MS is useful in the screening and quantitative determination of volatile substances including toluene and other thinner/glue components.