Makoto Nihira

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.

Application of a computer-assisted high-performance liquid chromatographic multi-wavelength ultraviolet detection system to simultaneous toxicological drug analyses

An emergency drug screening system for the separation and identification of toxic drugs, MULTI-HPLC, is presented. Chromatographic peaks, which were impossible to identify with a conventional high-performance liquid chromatographic UV detection system, became distinguishable by the spectral search and retention prediction of the data-processing program MCASYST. Sixty-five toxic drugs, frequently identified in drug poisionings in Japan, were selected as references in the drug library. Retention time, optimum detection wavelength, detection limit and recoveries from serum and urine were listed. Possible applications of the system are demonstrated, using gastric contents, sera and urines in cases of multiple drug ingestion. Quantitative analysis was sufficiently sensitive and precise to permit clinical diagnosis with increased accuracy.

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.

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.

Histopathological study on acute poisoning of methamphetamine, morphine or cocaine

Methamphetamine, morphine or cocaine was injected intraperitoneally into Wistar rats (male, 6 weeks old) at a dose of 50 mg/kg, 125 mg/kg, or 50 mg/kg body weight, respectively. These doses of drugs were determined to ensure that the rats would show signs of drug intoxication and survive for a day. Over the period from 5 min to 18 hr after injection, concentrations of the drugs and metabolites in blood were analyzed by the GC/MS method, and the histopathological changes of the heart, lungs, liver and kidneys were examined by light microscopy. The time of maximum drug concentration in the blood after injection was 5 min in the methamphetamine-treated group, 1 hr (total morphine) in the morphine group and 5 min in the cocaine group. These blood concentrations decreased with time. In the liver at 2.5 hr after injection of methamphetamine, centrolobular vacuolation and diffuse eosinophilic changes of hepatocytes appeared. At 6 hr this damage spread to the midzone of the liver and partial necrosis of the centrolobe was found. At 18 hr the liver damage became worse and necrosis was also found in the midzone of the liver. In the heart, from 2.5 hr, eosinophilic changes of the myocardium were observed diffusely. Furthermore, at 18 hr, partial inflammatory cell infiltration and contraction bands were observed. The degree of histopathological damage did not coincide with the time of maximum drug concentration. In the morphine group, centrolobular and midzonal vacuolation, diffuse fatty degeneration and eosinophilic changes were observed in the liver from 2.5 hr to 18 hr after the administration. No necrosis was found, perhaps because the rats did not suffer the hyperthermia observed in the methamphetamine group. The degree of histopathological damage became more serious with time, as it did in the methamphetamine group. In the cocaine group, no histopathological changes were observed, probably because the doses of cocaine were too small to cause histopathological damage, and because cocaine is more rapidly metabolized than methamphetamine or morphine. These results suggest that in histopathological investigations necessitated by drug intoxication, measuring drug concentrations in the blood might be useful in determining the causes of histopathological changes more clearly.