Experimental estimation of postmortem interval using multivariate analysis of proton NMR metabolomic data.

Nuclear magnetic resonance (NMR) spectroscopy has recently been applied to metabolic studies. In particular, metabolic profiles of tissues or of the whole body can easily be acquired through multivariate analysis of NMR spectra. The present study investigates metabolic changes after death in rat femoral muscles using pattern recognition of proton NMR spectra. Rats were killed by suffocation, cocaine overdose and induced respiratory failure, and then low molecular weight metabolites extracted using perchlorate from excised tissues were measured using proton NMR. All spectral data were processed and assessed by multivariate analysis to obtain metabolic profiles of the tissues. The results of principal component analysis (PCA) score plots soon after death showed that the metabolic profiles of the tissues differed according to the mode of death. The principal component (PC) scores of the data varied hourly and correlated with postmortem interval. The present results showed that NMR-based metabolic profiling could provide useful information with which to estimate postmortem intervals and causes of death.

GC/MS analysis of an herbal dietary supplement containing ephedrine.

An unconscious 20-year-old female was admitted to hospital with a heart rate of 164, a blood pressure of 132/90 mmHg, and hypokalemia. "Triage" urine screening tests were negative on arrival and 12 h later. The next day, her SGOT and SGPT levels rose remarkably; however, on the third day, the patient regained consciousness. Two Japanese OTC drugs and an American herbal dietary supplement ("7th heaven") were found in her room. The supplement and the patient's samples were analyzed using GC/MS. Ephedrine (2.32 mg/g) and caffeine (17.96 mg/g) were detected in the supplement and in the patient's serum (0.627 mg/L, 383 mg/L, respectively), as well as acetaminophen, bromvalerylurea, and etenzamide, which are constituents of the OTC drugs. The serum ephedrine concentration was above the therapeutic level but did not reach the fatal level. The acetaminophen concentration was sufficient to cause liver damage. Although a prescription is necessary to obtain products containing ephedrine in Japan, this patient had no prescription. Thus, how the patient obtained the drug and the amount ingested were unclear. Information about acquisition of drugs via the Internet or magazine advertisements is constantly changing and unreliable. Thus, it is indispensable to analyze unfamiliar supplements found with patients.

An accidental case of aconite poisoning due to Kampo herbal medicine ingestion.

An accidental case of aconite intoxication occurred after a patient took a therapeutic dose of Kampo herbal medicine containing Aconiti tuber, Uzu but had used the wrong decoction procedure. The poisoning was likely caused by an increased level of Aconitum alkaloids in the decoction; the patient developed aconite intoxication due to incomplete decoction. Aconitum alkaloid levels in the leftover solution which the patient had drunk and in the decoction extracted from 3g Uzu were determined. It was found that decoction makes the medicine safer to drink. Older individuals, especially those with dementia, have a higher risk of aconite poisoning because they sometimes do not boil the medicine appropriately.

Effects of chronic administrations of aconitine on body weight and rectal temperature in mice.

Aconitine, a major Aconitum alkaloid, is well known for its high toxicity that induces severe arrhythmias leading to death. However, aconitine has been used as one of the most popular compounds in Shino-Japanese traditional herbal medicine. Little has been reported concerned with the long-term effects of aconitine. Therefore, the authors investigated the physiological effects of chronic administrations of aconitine by determining the changes in body weight and rectal temperature of mice, compared with the concentrations of aconitine and its metabolites (benzoylaconine and aconine) in the liver and kidneys. The concentration ratio of aconitine to the total Aconitum alkaloids (from day 0 to 22; 90 min after the last administration) gradually decreased, whereas its metabolites increased until day 22. The body weight gain in aconitine-administered group was less than that of the control group until day 22. Transient rectal hypothermia occurred within 30 min after the last administration of aconitine. Then the rectal temperature gradually increased to normal level in respect to time. This study might reveal the possibilities that the drug metabolism of aconitine increased and the toxicity of aconitine decreased due to long-term administrations of aconitine.

Diagnostic Performance of Triagetrade mark for Benzodiazepines: Urine Analysis of the Dose of Therapeutic Cases.

We evaluated the diagnostic performance of Triage for benzodiazepines in 74 urine specimens from outpatients given therapeutic doses of benzodiazepines and compared the results of EMIT assays. Results obtained in all urine samples were confirmed using liquid chromatography-mass spectrometry (LC-MS). Overall agreement between results of Triage and EMIT assays was 73%. All of the Triage-positive samples were also positive by EMIT assays. Results of Triage and EMIT assays were different for 20 samples obtained from patients given thienodiazepines (etizolam, brotizolam, and clotiazepam) and nitrobenzodiazepines (nitrazepam, flunitrazepam, and clonazepam). LC-MS confirmed parent drugs in urine specimens, consistent with the prescriptions of drugs. The low agreement between Triage and EMIT results in this study might be due to low sensitivity of Triage for thienodiazepines. Thienodiazines are frequently prescribed benzodiazepines, and Triage panel is the most frequently used screening kit in Japan. It should be noted that negative results obtained by a Triage test might not mean the absence of thienodiazepines.

Diagnostic performance of Triage for benzodiazepines: urine analysis of the dose of therapeutic cases.

We evaluated the diagnostic performance of Triage for benzodiazepines in 74 urine specimens from outpatients given therapeutic doses of benzodiazepines and compared the results of EMIT assays. Results obtained in all urine samples were confirmed using liquid chromatography-mass spectrometry (LC-MS). Overall agreement between results of Triage and EMIT assays was 73%. All of the Triage-positive samples were also positive by EMIT assays. Results of Triage and EMIT assays were different for 20 samples obtained from patients given thienodiazepines (etizolam, brotizolam, and clotiazepam) and nitrobenzodiazepines (nitrazepam, flunitrazepam, and clonazepam). LC-MS confirmed parent drugs in urine specimens, consistent with the prescriptions of drugs. The low agreement between Triage and EMIT results in this study might be due to low sensitivity of Triage for thienodiazepines. Thienodiazines are frequently prescribed benzodiazepines, and Triage panel is the most frequently used screening kit in Japan. It should be noted that negative results obtained by a Triage test might not mean the absence of thienodiazepines.

Effects of long-term administrations of aconitine on electrocardiogram and tissue concentrations of aconitine and its metabolites in mice.

Aconitum alkaloids are well known for their acute and high toxicity, for example, in the causation of severe arrhythmias leading to death. Aconitine, one of the major Aconitum alkaloids, is a highly toxic compound from the Aconitum species. However, there has been no studies reported on the influence of the chronic administration of aconitine. Thus, this study was conducted to investigate the influence of chronic administration of aconitine in experimental animal models. A dose of 1mg/kg per day was administered to the experimental animal models. We determined the concentration of aconitine and its metabolites (benzoylaconine and aconine) in organs and blood with gas chromatography/selected ion monitoring (GC/SIM). In addition, we concurrently recorded the electrocardiogram (ECG). Fifteen minutes after administration on day 0, the early aconitine administered group (acute group) revealed peak organ and blood concentration levels of aconitine with a gradual decrease, thereafter. The concentration of aconitine in organs and blood (from days 0 to 22; 90 min after the last administration of aconitine) gradually decreased according to repeated administration, whereas benzoylaconine and aconine increased. ECG revealed various types of arrhythmias. However, the frequency of arrhythmias remarkably decreased with time and repeated administration of aconitine. These results indicate two possibilities. First, the increase in the activity of aconitine metabolism. Secondly, the decrease of effectiveness to the heart due to long-term (chronic) administration of aconitine.

[GC-MS analysis of methamphetamine and amphetamine in hair of Thai drug addicts].

UNLABELLED: Psycho-stimulant dependence in young individuals has become a serious problem in Thailand, where consumption of the so-called YaBa methamphetamine tablets has become a fashionable trend. Due to its easy availability in the form of a tablet, young individuals abuse methamphetamine. Methamphetamine tablets are known to be potently addictive and its difficulty in cessation of drug use and to be abstinent from the drug. We herein report the results obtained from GC-MS analysis of methamphetamine and amphetamine in 33 samples of urine and hair from patients with psycho-stimulant dependence. These samples were collected from patients registered at the outpatient clinic in the Department of Psychiatry, Chiang Mai University Hospital and were sent to Nippon Medical School, Department of Legal Medicine (Tokyo, Japan) for further analysis by Dr. Werawan Ruangyttikarn, Department of Forensic Medicine, Chiang Mai University. Sample preparation: Hairs samples were cropped near the hair root. After washing, they were cut into 1.2-cm sections and extracted with methanol/5N HCl (2:1) for an hour and then, solid-phase extraction was conducted using Bond-Elut Certify. Following extraction, GC-MS analysis was performed. Urine samples were subjected to GC-MS analysis after preparation with Bond Elut Certify. RESULTS AND DISCUSSION: In 6 samples, both urine and hair samples analyzed were negative for detection of the stimulant drugs. In those cases individuals might stop taking drug for about 5 months. In 18 samples, urine samples were negative whereas hair samples were positive. These results suggest that individuals might stop using drugs for a few days before they went to the hospital but they abuse drugs continuously. In 9 samples, both urine and hair samples were positive. These results show that individuals always abuse drugs. In order to treat drug dependence effectively it is necessary to obtain the patient history of drug use and to evaluate and determine short-term and long-term drug use with urinalysis and hair analysis, respectively. Our present data revealed that useful information concerned with the long term drug abuse can be obtained from hair analysis, and that this method of analysis is applicable not only to forensic cases but also for evaluating clinical cases.

Ultrastructural changes during in situ early postmortem autolysis in kidney, pancreas, liver, heart and skeletal muscle of rats.

Many morphological studies of the postmortem interval were carried out under conditions in which the tissue was incubated in vitro after extirpation. However, the extirpation affects cell viability. We examined the ultrastructural changes in the kidney, pancreas, liver, heart and skeletal muscle of male Wistar rats occurring postmortem in situ. In each organ, cell edema (cell swelling), appearance of amorphous dense deposits in the mitochondria, loss of glycogen granules, dilation of the endoplasmic reticulum, clumping and margination of nuclear chromatin, and/or condensation of nuclear chromatin were observed, but the duration of the period of ultrastructural change was organ specific. Most of the ultrastructural changes occurred earlier in kidney. In hepatocytes, the morphological degeneration occurred later than in the renal tubule epithelium and earlier than that in the myocardium. Of the five organs we examined, skeletal muscle showed the greatest delay in postmortem change. In the distal tubule epithelium and pancreatic acinar cells, two forms of nuclear change were seen: one resembled necrotic change and the other resembled apoptotic change. The effect of lysosomes and hydrolytic enzymes was not as great as previous findings.

Sensitive determination of tetrodotoxin using column-switching liquid chromatography-mass spectrometry with electrospray ionization in mouse serum.

A liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) method coupled with a column-switching technique was developed for the determination of tetrodotoxin (TTX) in serum. An on-column, column-switching technique was employed to analyze TTX without pretreatment of the serum. The combination of a multimode column with reversed phases and cation exchange for TTX provided successful separation and MS determination in the ESI-positive mode. A 100-microL serum sample was injected directly into a precolumn. For TTX monitored at m/z 320.1 in the selective ion monitoring mode, the calibration curve was linear within the range 0.1-100 ng/mL, and the limit of detection was 0.5 ng/mL. Recoveries were around 90% for TTX. The present method allows successful analysis of TTX in serum. In conclusion, this new method is simple, accurate, and useful for the determination of TTX and should be of benefit to both forensic and clinical toxicology.

A column-switching LC/MS/ESI method for detecting tetrodotoxin and Aconitum alkaloids in serum.

A liquid chromatography-mass spectrometry-electrospray ionization (LC/MS/ESI) method coupled with a column-switching technique has been developed for the determination of tetrodotoxin (TTX) and Aconitum alkaloids and their metabolites, such as aconitine, mesaconitine, hypaconitine, jesaconitine, benzoylaconine, benzoylmesaconine, benzoylhypaconine and 14-anisoylaconine, in serum. An on-column column-switching technique was employed to analyze TTX and Aconitum alkaloids and their metabolites without pretreatment of the serum. Combination of a multimode column with reversed phases and cation exchange for TTX, and use of a multimode column with reversed phases and a hydrophobic polymer column for Aconitum alkaloids and their metabolites provided successful separation and MS determination in ESI positive mode. A 100 microl serum sample was directly injected into a precolumn. For TTX monitored at m/z 320.1 in the selected ion monitoring mode, the calibration curve was linear within the range 0.1-100 ng/ml and the limit of detection was 0.1 ng/ml. For aconitine, mesaconitine, hypaconitine and jesaconitine, linear calibration curves were obtained up to 500 ng/ml and the limit of detection ranged from 0.2 to 1 ng/ml. For benzoylaconine, benzoylmesaconine, benzoylhypaconine and 14-anisoylaconine, linear calibration curves were obtained up to 500 ng/ml and the limit of detection ranged from 2 to 50 ng/ml. Recoveries from serum samples were within the range 78-119% for all the compounds studied.

[Analysis of MDMA and PCP by GC-MS from patients admitted to the critical care medical center].

In our country, abuse of methamphetamine has increased. Furthermore, dealings of other drugs by using internet have increased. But, the poison cases of 3,4-methylenedioxymethamphetamine (MDMA) and phencyclidine (PCP) have never been reported in our country. We report an MDMA poison case and a PCP poison case. We could detect MDMA, MDA or PCP by GC-MS from urine and serum of patients admitted to the critical care medical center of Nippon Medical School. Case 1: A 23-year-old foreign female was admitted to our hospital because of disturbance of consciousness. Her friend said that she had been found lying on the floor of the bathroom after taking a tablet. The screening test by Triage showed AMP positive. Not methamphetamine but MDMA and MDA were detected from urine and serum of the patient by GC-MS. Case 2: A 27-year-old foreign female was admitted to our hospital because of restlessness and excitement. Her friend said that she had become restless and excited after taking 15-30 tablets of Tylenol. The screening by Triage showed BZO and PCP positive. Not acetaminophen but PCP was detected in the patient's sample by GC-MS. Drug abuse has expanded to Japan over the border. New responses to abuse drugs with respect to medical treatment and drug analyses should be established.