Narumi Jitsufuchi

Sensitive determination of sulpiride in human plasma by high-performance liquid chromatography

We developed a simple, sensitive and reliable method for the determination of sulpiride, a specific antipsychotic drug, in human plasma using high-performance liquid chromatography. A structurally related benzamide, tiapride, was used as the internal standard. A Sep-Pak C18 cartridge was used to extract a sample from 1 ml of plasma. The extract was dissolved in methylene chloride, and then back-extracted with 0.01 M hydrochloric acid. The aqueous layer was put on a octadecylsilica column with a mobile phase of 50% acetonitrile in 0.01 M phosphate buffer (pH 3.0). A fluorescence detector with excitation at 300 nm and emission at 365 nm was used for detection. The calibration curve was linear in the concentration range of 10-1500 ng/ml, and the lower limit of detection was 1 ng/ml. We used this method to examine plasma levels of sulpiride in 14 inpatients being treated with sulpiride for 6 months. The determined plasma levels were 70.1-1121.2 ng/ml, and the correlation between daily dose and plasma concentration was positive. This simple, reliable method is expected to be put to good use in forensic and hospital laboratories.

Death attributed to the toxic interaction of triazolam, amitriptyline and other psychotropic drugs

A 71-year-old man was found dead in a car into which exhaust fumes had been introduced. His wife who was in the same car recovered consciousness following hospitalization. She claimed that they had both attempted suicide by taking a large number of sleeping pills. Autopsy revealed no significant external injuries or medical disorders that would have led to the husbands death. The concentrations of alcohol and carbon-monoxide hemoglobin in his whole blood were 0.26 mg/ml and < 10%, respectively. Therefore, poisoning by carbon monoxide from the exhaust fumes was ruled out, and further toxicological examinations were undertaken. Triazolam, pentobarbital, amitriptyline and bromazepam were all detected in the tissues of the victim; whole blood concentrations were 45.60, 386.4, 521.2 and 166.7 ng/g, respectively. Triazolam (7.350 ng/g) and pentobarbital (288.2 ng/g) were also detected in the whole blood of the wife, collected 17 h after admission to hospital. When evaluating these results in the light of existing literature, we concluded that the victim and his wife had indeed attempted suicide by taking triazolam and pentobarbital. However, only the man had died of triazolam poisoning due to its apparently lethal combination with amitriptyline and other psychotropic drugs which had been prescribed to treat his depression.

Detection of triazolam in skeletal remains buried for 4 years

Abstract Analyses of the hypnotic triazolam from the remains of two human skeletons buried underground for 4 years were made for purposes of confirmation. The bone marrow and mummified muscle were digested with 2 M sodium hydroxide, efficiently extracted using a 3-step solvent extraction procedure, and selectively analyzed by gas chromatography/mass spectrometry with the negative ion chemical ionization mode. Estazolam was the internal standard used. Triazolam was detected in all the samples; the concentrations were 0.36 ng/g in the bone marrow of one victim, and 0.37 and 5.5 ng/g in the bone marrow and mummified muscle of the other victim. This method should prove useful for determination of triazolam in extensively decomposed bodies.

Distribution of drugs in various tissues in a brain dead man.

We examined the distribution of drugs in a 49-year-old brain-dead man. Our objective was to determine the possibility of diagnosing how and at what point the patient became brain dead. The presence of mepivacaine, pentazocine, lidocaine and thiamylal in various tissues, including seven regions of the brain were confirmed, using gas chromatography/mass spectrometry. Tissue-to-blood concentration ratios of mepivacaine, pentazocine and lidocaine in the brain were higher than these ratios in other tissues, while ratios of thiamylal were lower. Therefore, cerebral blood flow was likely to have ceased between the administration of the former drugs and that of the latter drug, in agreement with clinical records. Among seven regions of the brain, the ratios of the former three drugs were high in occipital and parietal lobes, and were low in the cerebellum and medulla oblongata. On the other hand, the ratios of the latter drug were high in the cerebellum and the medulla oblongata. Therefore, cerebral blood flow presumably ceased first in occipital and parietal lobes, and last in the cerebellum and the medulla oblongata. Based on these results, assessment of concentrations of drugs in human tissues, including various regions of brain is useful to determine the time and progression of brain death.

Selective determination of sultopride in human plasma using high-performance liquid chromatography with ultraviolet detection and particle beam mass spectrometry

We developed a sensitive and selective method for determining levels of sultopride, a neuroleptic drug of the substituted benzamide, in human plasma using high-performance liquid chromatography (HPLC) combined with UV detection and particle beam mass spectrometry (PBMS). Sultopride was extracted with tert.-butylmethyl ether using a salting-out technique. Tiapride served as an internal standard (I.S.). Sultopride and I.S. were separated by HPLC on a silica column with a mobile phase of acetonitrile-0.1 M ammonium acetate (94:6, v/v). The calibration curves were linear over the concentration range from 5 to 1000 ng/ml by HPLC with UV detection and from 10 to 1000 ng/ml with PBMS detection. The limit of quantitation was 5 ng/ml with UV detection and 10 ng/ml with PBMS detection. The absolute recovery was 92% and the within-day coefficients of variation were 2.9-7.1% at plasma concentrations from 50 to 500 ng/ml, determined by HPLC with UV detection. Using this method, we measured the plasma concentrations of sultopride with replicate analyses in four hospitalized patients and steady-state plasma levels were determined to be 161.6 +/- 30.8, 321.1 +/- 93.7, 726.5 +/- 143.1 and 1273.6 +/- 211.2 ng/ml, respectively.

Liquid chromatography-fast atom bombardment mass spectrometry for detection and determination of pentazocine in human tissues.

A reliable and sensitive method was developed for the detection and determination of pentazocine in human solid tissues using liquid chromatography-dynamic fast atom bombardment (FAB) mass spectrometry, combined with a three-step liquid-liquid extraction procedure. Levallorphan tartrate served as an internal standard. The extract was evaporated to dryness and dissolved in the mobile phase, acetonitrile-10 mM ammonium acetate solution (20:80, pH 4.0) containing 0.5% glycerol as FAB matrix. The eluent was pumped at a flow rate of 25 microl/min and split before introduction to FAB mass spectrometer. Quantitative analysis was carried out by means of monitoring quasi-molecular ions with m/z 286 for pentazocine and m/z 284 for levallorphan. The lower limit of detection of pentazocine in each tissue tested was 1 ng/g with scan mode and 0.1 ng/g with SIM mode. Using this method, the concentrations of pentazocine were determined in the tissues of an autopsied individual to perform toxicological evaluation.

Simple and rapid determination of enflurane in human tissues using gas chromatography and gas chromatography-mass spectrometry

A simple, rapid and reliable method was devised to determine the levels of enflurane in human tissues, using gas chromatography and gas chromatography-mass spectrometry. 1,4-Dioxane was used as an internal standard (I.S.). Enflurane and the I.S. were extracted from 0.25 g of body tissues using an automatic headspace sampler and 1 ml of headspace gas was injected into the gas chromatograph. Enflurane was analyzed qualitatively by gas chromatography-mass spectrometry and quantitatively by gas chromatography with a flame-ionization detector. The calibration curves in all tissues examined were linear in the concentration range 1-100 microg/0.25 g. The lower limit of detection was 200-300 ng/0.25 g. The accuracy and precision of this method were evaluated at two different concentrations, 1 and 20 microg/0.25 g. The coefficient of variation ranged from 3.4-13.4%. We used this method to determine the presence of enflurane in tissues from an autopsied individual who died suddenly during extirpation of a malignant tumor.

Sensitive and selective determination of bromisovalum by high-performance liquid chromatography/particle beam mass spectrometry

A specific procedure using high-performance liquid chromatography/particle beam mass spectrometry (HPLUPBMS) has facilitated determination of bromisovalum in human plasma and whole blood. Bromisovalum, a sedative and hypnotic, was effectively extracted with SepPak C,, cartridges and selectively determined by HPLUPBMS of EI mode. An originally synthesized 2-bromohexanoylurea served as the internal standard (IS). The calibration curve was linear over the concentration range 0.5-5.0 &g and the lower limit of detection was 0.1 r&g (10 ng at the time of direct injection). This method could be practically applied to detect bromisovalum in the whole blood of an autopsied individual.

Sensitive determination of bromazepam in human tissues using capillary gas chromatography-mass spectrometry

A reliable and sensitive gas chromatographic-mass spectrometric method was devised to determine the levels of bromazepam in human tissues. Bromazepam was extracted from body tissues using a three-step solvent extraction procedure. N-Desmethyldiazepam served as the internal standard. Selected ion monitoring with m/z 317 for bromazepam and m/z 270 for internal standard was used for quantitation. Calibration curves in all body tissues were linear over the concentration range from 50-500 ng/g. The lower detection limit in body tissues was 2-5 ng/g and the absolute recovery in body tissues was 27.8-68.0%. This method was used to determine the levels of bromazepam in tissues of an autopsied individual who had been prescribed psychotropic drugs and who was found dead in a car.

Determination of isonicotinic acid in the presence of isoniazid and acetylisoniazid Studies on isonicotinic acid formation from isoniazid in isolated rat hepatocytes

In comparison with the hepatocytes obtained from intact rats and rats pretreated with phenobarbital or 3-methylchoranthrene, the amount of isonicotinic acid (INA) formed from isoniazid (INH) increased substantially after incubation at 37 degrees C using the pretreated hepatocytes. This suggests an oxidative pathway for INA formation from INH, apart from hydrolysis. In order to explore the exact mechanism of INA formation in the hepatocytes, an HPLC assay for INA in the presence of INH and acetylisoniazid was developed. In this assay, INA was extracted after the preparation of an ion pair with tetra-n-butylammonium hydroxide, and analysed using an ODS column and a mobile phase consisting of 0.067 M potassium dihydrogenphosphate solution-methanol (96:4, v/v). The method is simple, accurate and especially suitable for INA determination after incubation of INH in isolated rat hepatocytes.