Sensitive determination of alpha-methyltryptamine (AMT) and 5-methoxy-N,N-diisopropyltryptamine (5MeO-DIPT) in whole blood and urine using gas chromatography-mass spectrometry.

We devised a sensitive and simple method to determine alpha-methyltryptamine (AMT) and 5-methoxy-N,N-diisopropyltryptamine (5MeO-DIPT) in whole blood and urine, using gas chromatography-mass spectrometry (GC-MS). AMT and 5MeO-DIPT were extracted using an Extrelut column with an internal standard, bupivacaine, followed by derivatization with acetic anhydride. The derivatized extract was used for GC-MS analysis of EI-SIM mode. The calibration curves of AMT and 5MeO-DIPT were linear in the concentration range from 10 to 750 ng/ml in both blood and urine samples. The method detection limit (MDL) of AMT and 5MeO-DIPT were 1 ng/ml each in whole blood and 5 ng/ml each in urine. This method should be most useful to accurately determine the presence of these drugs in blood and urine in clinical and forensic cases.

Cerebral concentrations of myo-inositol in rats with induced brain death.

In anaesthetized, mechanically ventilated rats with brain death induced by epidural balloon inflation, we assessed the levels of cerebral myo-inositol in three brain areas 4 h after brain death had been confirmed. The levels were measured using HPLC, along with the water content. Myo-inositol levels were significantly increased in the cerebellum (P<0.05) and decreased in the brainstem (P<0.001), compared to findings in controls. Such changes can serve as hallmarks of brain death.

A fatal case of poisoning by lidocaine overdosage--analysis of lidocaine in formalin-fixed tissues: a case report.

The death of a 76-year-old man with heart disease as a result of the injection of an excessive dose of lidocaine is presented. The patient was given 5 ml of 10% lidocaine hydrochloride (500 mg) intravenously instead of 2.5 ml of 2% lidocaine hydrochloride (50mg) in order to treat repeated paroxysmal ventricular arrhythmia. Immediately following the injection the patient had tonic clonic seizures and complete cardiopulmonary arrest followed. Although resuscitation attempts once successfully restarted his pulse and spontaneous respiration, the patient died on the eighth day after the injection. Toxicological examinations were carried out on the tissues obtained at the time of autopsy and which had been fixed in formalin solution for 40 days, and lidocaine was detected in each tissue examined. The concentrations were (ng/g or ml): parietal lobe, 308.0; occipital lobe, 208.7; temporal lobe, 318.0; frontal lobe, 223.2; cerebellum 200.9; pons 285.7; liver, 109.5; kidney 52.2; skeletal muscle 127.0; and formalin solution 8.4. In an experiment on rats we determined the concentration changes of lidocaine in formalin fixed tissues. The concentrations of lidocaine in these tissues significantly decreased to 1/3-1/4 from the original. This data shows that the cause of death was poisoning by lidocaine overdose.

Changes of the cerebral mannitol concentrations in the course of brain death of a rodent model.

Determining the time of brain death is one of the critical issues in forensic examinations. Few authors have attempted to determine the time of brain death using pharmacokinetic approaches. We investigated cerebral concentrations of mannitol of which a single dose (1 g/kg) was administered in the course of brain death. The inflation of an epidural balloon was adopted as a rodent model of brain death. Brain death was determined using ordinary tests. Specimens were collected 4 h after brain death. Brain water content was higher in brain dead (BD) groups than those in control groups. Cerebral concentrations of mannitol in the BD group were significantly higher than those in the control group (P<0.01). In all areas of brain the concentration was the highest at the time when mannitol was administered during balloon inflation. Interhemispheric difference in the cerebrum was observed, followed by balloon inflation (P<0.05). Significant differences were observed in the average concentration of administered mannitol before and after brain death in the contralateral hemisphere (P<0.01) and in the brainstem (P<0.01). As the concentrations of mannitol in the brain are affected by cerebral trauma and brain death follows, mannitol can be used to determine the time of brain death at forensic examinations.

Simultaneous determination of bromvalerylurea and allylisopropylacetylurea in human blood and urine by gas chromatography-mass spectrometry.

We devised a sensitive and simple method to simultaneously determine bromvalerylurea and allylisopropylacetylurea in human blood and urine by gas chromatography-mass spectrometry. Bromvalerylurea and allylisopropylacetylurea were extracted using an Extrelut column with an internal standard, 2-bromohexanoylurea, followed by derivatization with heptafluorobutyric anhydride. The derivatized extract was submitted to GC-MS analysis of EI-SIM mode. The calibration curves of both compounds were linear in the concentration range from 0.01 to 10 microg/ml in both blood and urine samples. The lower limits of detection of bromvalerylurea and allylisopropylacetylurea were 0.005 and 0.005 microg/ml, respectively. This method proved most useful in accurately identifying these drugs in blood and urine from an autopsied individual.

HPLC simultaneous determination of glycerol and mannitol in human tissues for forensic analysis.

A method for simultaneous determination of glycerol and mannitol in various human tissues was devised and for this we used high-performance liquid chromatography (HPLC). Specimens were homogenized in a mixture of chloroform and methanol, phosphate buffer (pH 7.0) and pentaerythritol (IS) solution. After centrifugation, an aliquot of the aqueous layer was evaporated to dryness and derivatized with p-nitrobenzoyl chloride at 50 degrees C for 1h, then applied to HPLC with analytical conditions of: column, CAPCELL PAK C18 MG (250 mm x 3.0 mm i.d., 5 microm, Shiseido Co. Ltd., Tokyo, Japan); column temperature, 1-2 degrees C; mobile phase, 75% acetonitrile-distilled water containing 0.05% trifluoroacetic acid, 0.05% heptafluoro-n-butyric acid and 0.1% triethylamine; flow rate, 0.5 ml/min; wavelength, 260 nm. Calibration curves for both substances were linear in concentration ranges from 1 to 500 microg/0.1g and correlation coefficients exceeded 0.99. The relative standard deviation (R.S.D.) of the method was evaluated at concentrations of 10 and 100 microg/0.1g, and ranged from 0.84 to 10.6%. Using this method, we determined the regional distribution levels of glycerol and mannitol in various tissues from an autopsied brain dead man.

Toxicological analysis of chlorhexidine in human serum using HPLC on a polymer-coated ODS column.

A simple and reliable high-performance liquid chromatographic (HPLC) method for analyzing chlorhexidine in human serum was developed. After the addition of an internal standard, levomepromazine, 0.2 mL serum was deproteinized with 10% perchloric acid. The acidic supernatant was neutralized with 1M potassium carbonate solution, and the insoluble salt was removed by centrifugation. An aliquot of the supernatant was applied to HPLC with UV detection (260 nm). HPLC separation was achieved on a polymer-coated ODS column equilibrated with acetonitrile/water containing 0.05% trifluoroacetic acid, 0.05% heptafluorobutyric acid, and 0.1% triethylamine (40:60, v/v). The calibration curve was linear in the concentration range from 0.05 to 50.0 microg/mL, and the lower limit of detection was 0.05 microg/mL. The accuracy and precision of the method were evaluated at concentrations of 0.5 microg/mL and 5.0 microg/mL. The coefficients of variation ranged from 4.0 to 4.5%. The concentration of chlorhexidine in the serum of a patient who died after a suspected intravenous injection of chlorhexidine gluconate was determined.

A sudden death following tetracaine-induced spinal anesthesia.

A 56-year-old Japanese woman died after being given 10 mg of tetracaine to induce spinal anesthesia in preparation for orthopedic surgery. Autopsy findings and microscopic analysis revealed no external injuries or disease, and further toxicological examinations were carried out to determine the cause of death. For this we used gas chromatography-mass spectrometry. Tetracaine was not detected in most tissue samples, but the metabolite, p-butylaminobenzoic acid, was clearly detected in all samples. The concentration of the metabolite in the brain stem (234.7 ng/g) was higher than in the cerebrum (30.5 ng/g), cerebellum (36.7 ng/g), whole blood of the left heart (164.8 ng/ml) and whole blood of the left femoral vein (84.0 ng/ml). These distribution patterns were exactly same as rabbits sacrificed by high spinal anesthesia. Our findings suggest that tetracaine spread to the high regions of cerebrospinal nerves and acted directly on the central nervous system. Therefore, the cause of her death was high spinal anesthesia induced by tetracaine. This is apparently the first toxicologically proven case in which spinal anesthesia affected high brain centers.