Namera, Akira PhD; Makita, Ryosuke PhD; Saruwatari, Tatsuro BSc; Hatano, Aiko MD; Shiraishi, Hiroaki MD, PhD; Nagao, Masataka MD, PhD
Benzodiazepines exhibit various bioactivities such as sedative, hypnotic, anxiolytic, anticonvulsant, muscle relaxant, and amnesic effects. These properties of benzodiazepines have rendered them useful in the treatment of anxiety, insomnia, agitation, seizures, and muscle spasms. Although they have been widely used over a long period as relatively safe drugs, acute poisoning and forensic intoxication cases have been reported due to benzodiazepine overdose.
A short-intermediate acting drug, flunitrazepam is prescribed for the treatment of severe insomnia. Some forensic reports state flunitrazepam overdose is one of the causes of death.1–4 Many cases of alcohol abuse and overdose of other drugs such as barbiturates and tricyclic antidepressants have also been reported to simultaneously find flunitrazepam from the victims. Alcohol increases not only the concentration of benzodiazepines in blood by restraint of its metabolism, but also the binding affinity of a benzodiazepine to its binding site, resulting in significant potentiation of its depressant effect on the central nervous system and respiratory system. Furthermore, the elderly and patients with chronic illness are considerably more susceptible to a lethal overdose of benzodiazepines; because of their susceptibility, even relatively lower doses tend to be fatal in these individuals.
In this study, we report a case of fatal flunitrazepam intoxication; high concentrations of flunitrazepam metabolites as well as those of triazolam were identified in the postmortem blood and urine samples. To identify whether the origin of 7-aminoflunitrazepam was metabolic or bacterial, 8 metabolites of flunitrazepam were identified and quantified by high-performance liquid chromatography coupled with photodiode array and mass spectrometry (LC/PDA/MS).
CASE HISTORY AND AUTOPSY FINDINGS
A 52-year-old woman, who was living away from her family, was found dead on the floor of the living room on the first floor of a house, which belonged to the man with whom she shared the residence. When her family visited her, she did not answer the door. Therefore, they contacted the police station and went to the house along with a police officer. She was found dead, lying face down, on the floor. Her clothes were found to be undisturbed, although press-through packages of flunitrazepam tablets (160 Silece, 2 mg/tablet) and strip packages of triazolam tablets (72 Halcion, 0.25 mg/tablet) were found strewn around the victim. The total amount of flunitrazepam and triazolam ingested was unknown, although the maximum amounts that she took were suggested to be 320 mg of flunitrazepam and 18 mg of triazolam. According to the information provided by her family, the victim was undergoing treatment for depression. An autopsy was performed 18 hours after the body was discovered, to determine the cause of death.
The woman’s height and weight were 149 cm and 44 kg, respectively. Postmortem rigidity was observed in the knee and foot joints. Postmortem hypostasis, dark red in color, was noted on the surface of the precordial region, side of the abdomen, and anterior crura. Discolorations and excoriations were observed on the unclothed parts—face, elbow, and knee. Several impressions of the edge of the carpet were observed on her face, left elbow, precordial region, and abdomen. Internally, congestion of blood vessels on the brain surface and moderate congestion of the blood vessels in the other organs were observed. A small amount of milky white mucus was observed in the trachea and bronchial tubes. Approximately 20 mL of greenish brown mucus was found in the stomach. Pathological investigations revealed signs of congestion in the brain, lungs, liver, and other organs. Both the lungs were congested and edematous, and the left lung showed infiltration of inflammatory cells.
During the autopsy, a drug screening test was performed with the urine sample, using Triage (Biosite, San Diego, Calif), to check for the presence of controlled drugs such as amphetamines and opiates. A positive result was obtained for benzodiazepines. However, no obvious cause of death was found.
MATERIALS AND METHODS
Flunitrazepam and 8 flunitrazepam metabolites (7-aminoflunitrazepam, desmethylflunitrazepam, 3-hydroxyflunitrazepam, 7-acetamidoflunitrazepam, 7-acetamido-3-hydroxyflunitrazepam, 7-acetamidodesmethylflunitrazepam, 7-aminodesmethylflunitrazepam, and 7-amino-3-hydroxydesmethylflunitrazepam) were donated by F. Hoffmann-La Roche (Basel, Switzerland). Triazolam and etizolam were donated by Pharmacia & Upjohn (Tokyo, Japan) and Yoshitomiyakuhinn Co (Osaka, Japan), respectively. α-Hydroxytriazolam and 4-hydroxytriazolam were purchased from BIOMOL Research Laboratories (Plymouth Meeting, Pennsylvania). Each standard was dissolved in acetonitrile (1 mg/mL) and stored under refrigeration. β-Glucuronidase (obtained from Helix pomatia, type H-2, Sigma, St Louis, Mo) was purchased from Sigma-Aldrich Japan (Tokyo, Japan). All the other reagents and solvents were of analytical or high-performance liquid chromatography grade and were purchased from Wako Pure Chemicals (Osaka, Japan).
Liquid chromatography coupled with photodiode array and mass spectrometry was performed using an Agilent LC-1100 system (Agilent Technologies Inc, Palo Alto, California). Separation was achieved using a reversed-phase column (ZORBAX Eclipse Plus C8; 150 × 2.1-mm internal diameter; particle size, 3.5 μm; Agilent Technologies Inc). The mobile phase consisted of a binary mixture of solvents: A (10 mM ammonium acetate) and B (methyl alcohol). The flow rate of the mobile phase and the column oven temperature was set at 0.2 mL/min and 40°C, respectively. The gradient program was as follows: 10% B (0–5 min), 10% to 90% B (5–40 min, linear), and 90% (40–45 min). Absorbance of the analytes was monitored at 200 to 350 nm and at 240 nm for quantitative analyses.
The electrospray ion mass spectrometer was operated in the positive ion mode and scanned from m/z 50 to 500. The drying gas (nitrogen) temperature was set at 350°C; flow rate, 13 L/min; nebulizer gas pressure, 50 psi; and capillary voltage, 2500 V. The fragmentor voltage was set at 150 V. Liquid chromatography coupled with photodiode array and mass spectrometry and LC/MS data acquisition and integration were achieved using the LC/MSD ChemStation chromatographic analysis software (Agilent Technologies Inc).
In a tube, 0.5 g of blood (or 0.5 mL of urine), 1.5 mL of borate buffer (10 mM, pH 10), and 5 μL of etizolam (0.1 mg/mL, internal standard) were mixed. The mixture was poured into an Extrelut extraction column (10 × 150-mm internal diameter), which was washed using diethyl ether before extraction. After standing for 20 minutes at room temperature, the analytes were eluted with 5 mL of ethyl acetate. The eluted ethyl acetate was evaporated to dryness under nitrogen stream. The residue was dissolved in 100 μL of 30% methyl alcohol, and an aliquot (5 μL) was injected into the LC/PDA/MS column. Before extraction, the urine sample was hydrolyzed using β-glucuronidase (5000 U/mL in urine) at 37°C for 18 hours.
Analytical data of this method were validated in accordance with the US Food and Drug Administration guidelines.5 Blood or urine samples spiked with flunitrazepam and triazolam at concentrations of 20 to 5000 ng/g (or mL) were prepared and analyzed in 7 different points and analyzed according to the procedure described above. The calibration curves were obtained by plotting the peak-area ratio of the targets to internal standard against their respective concentrations. The curves exhibited linearity in the concentration range of 20 to 5000 ng/g (or mL). The correlation coefficients of the calibration curves were more than 0.998. The accuracy was from 80% to 112.5%, and the values of relative standard deviation for intraday and interday variations in the concentrations of 100, 1000, and 5000 ng/g (or mL) were from 2.35% and 12.4%, respectively. The lower detection limit was 10 ng/g (or mL) (signal-to-noise ratio = 3). Blank and spiked blood or urine were alternately examined; the results did not affect the next analysis, and carryover was not observed under the concentration of 5000 ng/g (or mL). The detection minimum limit of our method was sufficient for judging whether the sample was poisoned.
The Triage immunoassay was positive for benzodiazepines. Because packages of flunitrazepam (Silece, 2 mg/tablet) and triazolam (Halcion, 0.25 mg/tablet) were found on the floor around the victim, toxicological screening was performed for these suspected drugs by LC/PDA/MS. 7-Aminoflunitrazepam was detected in the blood and urine, and triazolam metabolites (4-hydroxytriazolam and α-hydroxytriazolam) were detected in the urine. No other drug or alcohol was found in the blood, urine, and gastric contents of the victim. Flunitrazepam, triazolam, and their metabolites were quantified by LC/PDA. The concentration of each compound is summarized in Table 1. The concentration of 7-aminoflunitrazepam (1270 ng/g) in the blood was higher than that reported in previous cases1–3 and was within the range of fatality.6,7
Postmortem examinations conducted as a part of the previous studies have revealed that 7-aminoflunitrazepam is produced from flunitrazepam by the action of bacteria.8 Moreover, the redistribution of these basic drugs from the stomach, lung, and other organs has also been reported.9,10 Thus, a high concentration of flunitrazepam or 7-aminoflunitrazepam in the stomach is an indication of drug redistribution in the body. However, in this case, flunitrazepam was not detected in the stomach, and the concentration of 7-aminoflunitrazepam in the victim’s stomach was the same as that in her blood. In addition, the volume of gastric contents was only 20 mL, and the calculated amount of 7-aminoflunitrazepam was 57.4 μg. These basic drugs diffused probably from the blood into the stomach in a concentration-based gradient.11 These results indicate that the redistribution process could be a reason for the high concentration of 7-aminoflunitrazepam and other metabolites. To determine the origin of 7-aminoflunitrazepam and other metabolites, which may have been produced by bacterial or metabolic action, these compounds were analyzed by LC/PDA/MS. The concentrations of the further metabolites of 7-aminoflunitrazepam (7-acetamidoflunitrazepam and 7-acetamidodesmethylflunitrazepam) were relatively high. Previous pharmacological studies12,13 have reported that the concentrations of 7-aminoflunitrazepam and 7-aminodesmethylflunitrazepam gradually increased for up to 12 to 24 hours after the ingestion of flunitrazepam. It is surmised that these compounds are further metabolized to acetamido derivatives in the body. Therefore, the 7-aminoflunitrazepam, detected in this case, was converted from flunitrazepam mainly by the metabolic activities of the liver. Thus, we believe that quantification of these metabolites can be a useful method for the identification of the metabolic pathway—bacterial or hepatic.
Although the 7-aminoflunitrazepam concentration is the chief parameter used during forensic investigation of the cause of death, desmethylflunitrazepam and 7-acetamidodesmethylflunitrazepam are also active metabolites.14 As shown by the present study, it is difficult to evaluate toxicity only on the basis of 7-aminoflunitrazepam concentration because it is plausible that 7-aminoflunitrazepam is produced by bacteria after the victim’s death. Quantitative measurement of the active metabolites, such as desmethylflunitrazepam and 7-acetamidodesmethylflunitrazepam, is essential to determine the cause of death.
No traces of triazolam were found in the victim’s blood and urine. However, triazolam metabolites—4-hydroxytriazolam and α-hydroxytriazolam—were found in the urine, at a concentration of 950 and 12,100 ng/mL, respectively. The half-life of triazolam is only 2 hours, it is metabolized in the liver via oxidative pathways, and the metabolites are immediately excreted into the urine. It is assumed that after the ingestion of these drugs, the victim fell onto the floor and moved around in the same area, because several impressions of the edge of the carpet were observed on her face, left elbow, precordial region, and abdomen (Fig. 1). A previous report stated that the ratio of 7-aminodesmethylflunitrazepam to 7-aminoflunitrazepam in urine increases with time, independent of the dose, and that it may be used to estimate the time of ingestion after an oral dose.15 If this hypothesis is applied to the present case, the victim must have died 5 to 6 hours after flunitrazepam intake. She survived for a few hours after the ingestion of the drugs, and the respiratory depression gradually produced hypoxia, which ultimately proved fatal.
On the basis of the autopsy findings and the results of toxicological examination, we concluded that the death was mainly due to the combined toxicity of flunitrazepam and triazolam.
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