On-column derivatization for determination of amphetamine and methamphetamine in human blood by gas chromatography-mass spectrometry

A simple determination method of amphetamine (AP) and methamphetamine (MA) in human blood was developed using on-column derivatization and gas chromatography-mass spectrometry (GC-MS). AP and MA were adsorbed on the surface of Extrelut and then derivatized the N-propoxycarbonyl derivatives using propylchloroformate. Pentadeuterated MA was used as an internal standards. The recoveries of AP and MA from the spiked blood were 89.7 and 90.3%, respectively. The calibration curves showed linearity in the range of 12.5-2000 ng/g for AP and MA in blood. The coefficients of variation of intraday and interday were 0.42-4.58%. Furthermore, this proposed method was applied to some medico-legal cases of MA intoxication. MA and its metabolite AP were detected in the blood samples, and the correlation of the blood level of amphetamines and the behaviors of the victims was in good agreement with the criteria proposed by Nagata [Jpn. J. Legal Med. 37 (1983) 513].     

Suicide in Japan: present condition and prevention measures

This article introduces the reader to present conditions and suicide prevention measures in Japan. The suicide rate has increased gradually since the early 1990s, reaching a postwar peak in 1998. The number of suicides has remained at about 30,000 every year since 1998. Middle-aged (55-59 years) and elderly men have especially high suicide rates. In 2002, The Council of Learned People on Measures Against Suicides (organized by the Japanese Ministry of Health, Labor, and Welfare) released its report on national suicide prevention strategies. Although national suicide prevention strategies have just begun to be established, some prefectures or regions have undertaken unique suicide prevention measures.     

Miniaturized sample preparation method for determination of amphetamines in urine

A simple and miniaturized sample preparation method for determination of amphetamines in urine was developed using on-column derivatization and gas chromatography-mass spectrometry (GC-MS). Urine was directly applied to the extraction column that was pre-packed with Extrelut and sodium carbonate. Amphetamine (AP) and methamphetamine (MA) in urine were adsorbed on the surface of Extrelut. AP and MA were then converted to a free base and derivatized to N-propoxycarbonyl derivatives using propylchloroformate on the column. Pentadeuterated MA was used as an internal standard. The recoveries of AP and MA from urine were 100 and 102%, respectively. The calibration curves showed linearity in the range of 0.50-50 microg/mL for AP and MA in urine. When urine samples containing two different concentrations (0.50 and 5.0 microg/mL) of AP and MA were determined, the intra-day and inter-day coefficients of variation were 1.4-7.7%. This method was applied to 14 medico-legal cases of MA intoxication. The results were compared and a good agreement was obtained with a HPLC method.     

A new molecular approach to help conclude drowning as a cause of death: Simultaneous detection of eight bacterioplankton species using real-time PCR assays with TaqMan probes

We developed a novel tool for concluding drowning as a cause of death. We designed nine primer pairs to detect representative freshwater or marine bacterioplankton (aquatic bacteria) and then used real-time PCR with TaqMan probes to rapidly and specifically detect them. We previously cultured the genus Aeromonas, which is a representative freshwater bacterial species, in blood samples from 94% of victims who drowned in freshwater and the genera Vibrio and/or Photobacterium that are representative marine bacteria in 88% of victims who drowned in seawater. Based on these results, we simultaneously detected eight species of bacterioplankton (Aeromonas hydrophila, A. salmonicida; Vibrio fischeri, V. harveyi, V. parahaemolyticus; Photobacterium damselae, P. leiognathi, P. phosphoreum) using three sets of triplex real-time PCR assays and TaqMan probes labelled with fluorophores (FAM, NED, Cy5). We assayed 266 specimens (109 blood, 157 tissues) from 43 victims, including 32 who had drowned in rivers, ditches, wells, sea or around estuaries. All lung samples of these 32 victims were TaqMan PCR-positive including the lung periphery into which water does not readily enter postmortem. On the other hand, findings in blood and/or closed organs (kidney or liver) were PCR-positive in 84% of the drowned victims (except for those who drowned in baths) although the conventional test detected diatoms in closed organs in only 44% of the victims. Thus, the results of the PCR assay reinforced those of diatom tests when only a few diatoms were detectable in organs due to the low density of diatoms in the water where they were found. Multiplex TaqMan PCR assays for bacterioplankton were rapid, less laborious and high-throughput as well as sensitive and specific. Therefore, these assays would be useful for routine forensic screening tests to estimate the amount and type of aspirated water.     

Technical and interpretive pitfalls of postmortem CT: Five examples of errors revealed by autopsy

Image acquisition of dead bodies, particularly using postmortem computed tomography (PMCT), has become common in forensic investigations worldwide. Meanwhile, in countries such as Japan which have an extremely low rate of autopsy, PMCT is being increasingly used in the clinical field to certify the cause of death (COD) without performing an autopsy or toxicological tests, even in cases of unnatural death. Additionally, these PMCT images are predominantly interpreted by clinical personnel such as emergency physicians or clinicians who are not trained in PMCT interpretation and who work for the police, that is, the so-called police doctors. Many potential pitfalls associated with the use of PMCT have been previously described in textbooks and published papers, including the pitfalls of not performing a complete forensic pathology investigation, and the use of physicians without appropriate PMCT training to interpret PMCT and direct death investigation and certification. We describe five examples in which apparent misdiagnosis of COD based on PMCT misinterpretation was revealed by autopsy. Here are the five examples of errors: (1) Postmortem changes were misinterpreted as COD, (2) resuscitation effects were misinterpreted as COD, (3) COD was determined after an incomplete examination, (4) fatal findings caused by external origin were wrongly interpreted as ‘of internal origin’ based on PMCT, and (5) non-fatal findings on PMCT were wrongly interpreted as fatal. Interpretation of PMCT by appropriately trained physicians and an accompanying complete forensic investigation, including autopsy when indicated, is necessary to prevent significant errors in COD determination and related potential adverse medicolegal consequences.     

Visualizing latent fingerprints on color-printed papers using ultraviolet fluorescence

Laser detection of latent fingerprints on a white paper has been performed, previously. Ultraviolet fluorescence from various kinds of printer toner and ink used for home printers were measured to study fluorescence imaging of fingerprints on a color-printed white paper. The experimental system consisted of a nanosecond pulsed tunable laser and a cooled CCD camera. Excitation wavelengths are 230 and 280 nm. Fourteen printers consisting of three color laser printers, three color inkjet printers, five monochrome laser printers, two monochrome copy machines, and a color copy machine were tested. Toner and ink of most printers exhibited fluorescence in the region from 360 to 550 nm. In most cases, clear fluorescence images were obtained by time-resolved imaging with a band-pass filter and 280-nm excitation. However for toners from laser color printers that showed strong fluorescence, better results were obtained with 230-nm excitation. Latent fingerprints on a photograph page and a black-character page of a newspaper were also imaged.     

Detection of marine and freshwater bacterioplankton in immersed victims: Post-mortem bacterial invasion does not readily occur

We previously applied our method of detecting marine or freshwater bacterioplankton (bacteria) in the blood of immersed victims as a marker of drowning. However, we did not confirm the absence of post-mortem bacterial invasion during immersion. Here we examined the nature of bacterioplankton in blood samples from 21 immersed and 4 non-immersed cadavers. We found only freshwater bacterioplankton in the blood of two victims that were retrieved from the sea or an estuary inhabited by marine bacterioplankton even though one victim was highly putrefied. The results of diatom testing suggested that these two victims had drowned in fresh or brackish water with low salinity and then flowed out to the estuary or the sea. Two others were submerged in water, but representative bacterioplankton were undetectable in their blood although one victim was highly putrefied. Autopsy findings and the results of diatom tests did not indicate that the cause of death was drowning. As in previous studies, we identified freshwater bacterioplankton in the blood of seven other victims that had drowned in freshwater, marine bacterioplankton in the blood of four victims that had drowned in seawater and none in four victims found on land that had died by means other than drowning. Bacterioplankton in the blood of drowned victims appears to reflect the type of water aspirated and blood does not become easily contaminated with bacteria post-mortem even in decomposed bodies.     

Should providers be allowed to extra bill for uncovered services? Debate, resolution, and sequel in Japan

Japan has managed to provide universal coverage at relatively low cost by containing prices and restricting the conditions for which services can be billed in the compulsory social health insurance (SHI) program. However, decline in Japan's economic growth ushered in new actors backed by the prime minister who proposed that the providers should be allowed to extra bill for services not covered by the SHI. In 2004 they took the strategy of drawing the attention of the public to areas where the rigidity of the current prohibition appeared to be unfair and ridiculous. They were opposed by the Ministry of Health, Labor, and Welfare and the Japan Medical Association, who strongly objected on the grounds of safety and equity. The compromise reached by the two ministers in charge led to a clarification of the services that are to be covered and of the process for extending coverage to new procedures and drugs. The prohibition on extra billing has remained essentially unchanged, but the momentum for deregulation has been lost. In 2005 an alternate proposal was made to contain SHI expenditures by introducing a global cap on health expenditures and increasing out-of-pocket payment. Although this proposal was not fully adopted, the gradual decline in SHI benefit levels could lead to a renewed move to allow extra billing.     

Risk factors of sudden death in the Japanese hot bath in the senior population

A series of experiments were carried out to clarify the cause of death and the risk factors related to sudden death in the Japanese senior population while bathing in a Japanese style "hot bath." The biodynamic changes while bathing were carefully monitored under actual bathing situations occurring in both the winter and summer seasons. We observed double product (DP), total peripheral blood vessel resistance (TPR), cardiac output (CO), and blood vessel compliance (COMP) by measuring blood pressure, heart rate, pulse wave, and electrocardiogram (ECG). The finding of a high level of DP in the elderly suggests that more myocardial oxygen consumption is needed than for young adults, particularly in subjects with arrhythmia. Although the values for TPR and CO changed somewhat during bathing, the changes were considered normal and to be expected. However, more significant and substantial changes were observed during the winter experiment than during the summer experiment, no doubt owing to lower temperature of the bathing room. The value of COMP did not vary significantly between winter and summer subjects. Twelve subjects in the elderly developed ECG changes while bathing such as supraventricular extrasystole or ventricular tachycardia. No clinical significance was found in the biochemical analyses of the blood obtained before and after bathing. In conclusion, some subjects in the elderly showed risky changes in the above parameters and ECG, factors which may partially explain some of the causes of the many reported cases of lapse of consciousness and unexpected sudden death in the elderly while bathing especially in the winter season. Cold climate, hot water immersion, and hydrostatic pressure may affect their physiological compensation along with existing of coronary stenosis or weakness of respiratory function as a normal consequence of advanced age.