Incorporation of five common hypnotics into hair after a single dose and application to a forensic case of drug facilitated crimes.

In order to obtain fundamental information on the disposition of hypnotics into hair after a single oral dose the quantitative hair analysis of triazolam (TZ), etizolam (EZ), flunitrazepam (FNZ), nitrazepam (NZ) and zolpidem (ZP) have been performed using a validated LC-MS/MS procedure. Hair specimens (straight, black) were collected from three subjects about one month and three months after a single 0.25 mg dose of TZ, 1 mg of EZ, 2 mg of FNZ, 5 mg of NZ and 10 mg of ZP tartrate. The subjects ingested just one out of five different hypnotics on each day, each of five days in turn. All ingested hypnotics have been detected in hair from each subject both one month and three months after intake, and their concentrations were in the range of 0.023-0.043 pg/hair strand (0.077-0.36 pg/mg) for TZ, 0.11-0.63 pg/hair strand (0.44-5.2 pg/mg) for EZ, 0.14-2.6 pg/hair strand (0.56-22 pg/mg) for FNZ, 0.33-1.7 pg/hair strand (1.3-17 pg/mg) for NZ and 20-40 pg/hair strand (120-270 pg/mg) for ZP. For FNZ and NZ, not only the parent drugs but also their metabolites, 7-amino-FNZ and 7-amino-NZ, were detected in the range of 2.3-9.2 pg/hair strand (9.2-82 pg/mg) and 2.4-9.1 pg/hair strand (8.0-55 pg/mg), respectively. The calculated incorporation ratios into hair against the dose were found to exhibit similarity between the four benzodiazepines. This finding suggests the ability to apply these quantitative data to approximately estimating the amounts of other benzodiazepines, which have similar chemical structures, in hair although it should be noted that the amounts of drugs in hair varies considerably depending on the hair color. On the other hand, the incorporation ratio of ZP showed 15-29 times higher than that of TZ, indicating that lipophilic ZP was more likely to incorporate into hair than benzodiazepines. In addition, the application of the present data to a drug-facilitated sexual assault was shown.

Detailed quantum mechanical studies on bioactive benzodiazepine derivatives and their adsorption over graphene sheets.

Estazolam (Z1) and related derivatives, adinazolam (Z2), alprazolam (Z3), 4-hydroxyalprazolam (Z4) and triazolam (Z5) have been studied by using various computational tools to analyze their geometry and spectral characteristics. The compounds were found to interact with graphene monolayer results shows that there is enhancement in various physico-chemical descriptors and surface enhanced Raman spectra (SERS). The various reactive descriptors obtained from the FMO analysis predict the reactive nature of the compound. The various lone pair/sigma to pi conjugation was analyzed using NBO formalism, which provides valuable information about intra molecular electron transfer which is vital in predicting the inherent stability of the molecule. Simulated electronic spectra using TD-DFT and CAM-B3LYP functional are discussed in detail with respect to electronic transitions and light harvesting efficiency. Suitability of candidates as a photo sensitizer in dye sensitized solar cells was studied and 4-Hydroxyalprazolam is identified as a suitable candidate. Nucleophilic and electrophilic regions of the molecules are identified using MESP, which adds to the reactivity information. It can be seen that the highest interaction energy has been obtained in the case of the Z5-graphene system, while the lowest interaction energy has been obtained in the case of the Z1-graphene system. Docking indicates that the ligands adsorbed over graphene also form stable complexes with the receptors as indicated by the high binding affinity energy values.

Segmental Hair Testing of Triazolam to Unmask a Suspected Case of Idiopathic Recurrent Stupor.

ABSTRACT: Stupor is a diagnostic challenge at emergency department. Differential diagnosis includes idiopathic recurrent stupor, formerly attributed to "endozepine-4" accumulation. This condition has been recently questioned because many suspected cases resulted in exogenous benzodiazepine intake that eludes the conventional toxicological assay. In case of unexplained recurrent stupor, to extend the benzodiazepine search in nonconventional matrices can allow unmasking of hidden toxic behavior.

Correction for isotopic interferences between analyte and internal standard in quantitative mass spectrometry by a nonlinear calibration function.

Stable isotope-labeled internal standards are of great utility in providing accurate quantitation in mass spectrometry (MS). An implicit assumption has been that there is no "cross talk" between signals of the internal standard and the target analyte. In some cases, however, naturally occurring isotopes of the analyte do contribute to the signal of the internal standard. This phenomenon becomes more pronounced for isotopically rich compounds, such as those containing sulfur, chlorine, or bromine, higher molecular weight compounds, and those at high analyte/internal standard concentration ratio. This can create nonlinear calibration behavior that may bias quantitative results. Here, we propose the use of a nonlinear but more accurate fitting of data for these situations that incorporates one or two constants determined experimentally for each analyte/internal standard combination and an adjustable calibration parameter. This fitting provides more accurate quantitation in MS-based assays where contributions from analyte to stable labeled internal standard signal exist. It can also correct for the reverse situation where an analyte is present in the internal standard as an impurity. The practical utility of this approach is described, and by using experimental data, the approach is compared to alternative fits.

Sensitive UPLC-MS-MS assay for 21 benzodiazepine drugs and metabolites, zolpidem and zopiclone in serum or plasma.

This paper reports an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS-MS) method to quantitate 21 benzodiazepines, zolpidem and zopiclone in serum and plasma. After liquid-liquid extraction, an Acquity UPLC with a TQ Detector and BEH C18 column was used (Waters, Milford, MA). The injection-to-injection run time was 7.5 min. Forty-eight authentic serum and plasma patient specimens were analyzed and results compared to those obtained using a previously published method. Average r(2) values for linearity (1 to 1,000 ng/mL over five days) were all above 0.995, except α-hydroxytriazolam (0.993). Intra-day and inter-day relative standard deviation values were within ± 15% and the percent deviation from the expected concentrations were within ± 11%. Recovery ranged from 62 to 89%. Matrix effects ranged from -28% to +6%. The limits of detection were 1 ng/mL, except for lorazepam, nordiazepam, oxazepam and temazepam (5 ng/mL). Ion ratios were ± 15% for all analytes. For authentic patient specimens (n = 48, 76 positive results), there was excellent correlation between the UPLC-MS-MS results and the previous method. The best least-squares fit had an equation of y = 1.0708x + 1.6521, r(2) = 0.9822. This UPLC-MS-MS method is suitable for the quantification of benzodiazepines and hypnotics in serum and plasma, and offers fast, reliable and sensitive results.

Acute intoxication caused by overdose of flunitrazepam and triazolam: high concentration of metabolites detected at autopsy examination.

A 52-year-old woman 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 house. On visiting the site, her clothes were found to be undisturbed. Packages of flunitrazepam (Silece, 2 mg/tablet) and triazolam (Halcion, 0.25 mg/tablet) were found strewn around the victim. Toxicological analysis was performed, and the concentrations of flunitrazepam, triazolam, and their metabolites in the victim's blood and urine were measured by high-performance liquid chromatography coupled with photodiode array and mass spectrometry. A high blood concentration of 7-aminoflunitrazepam was detected (1,270 ng/g), and further metabolites such as 7-acetamidoflunitrazepam, 7-acetamidodesmethylflunitrazepam, and 7-aminodesmethylflunitrazepam were detected in the blood and urine samples. In addition, 4-hydroxytriazolam and α-hydroxytriazolam were detected in her urine at a concentration of 950 and 12,100 ng/mL, respectively.On the basis of the autopsy findings and toxicology results of high concentrations of both flunitrazepam and triazolam derivatives, the cause of death was determined to be acute intoxication from flunitrazepam and triazolam.

A drug rape case involving triazolam detected in hair and urine.

In recent years, there has been heightened awareness regarding the use of drugs to modify a person's behavior to facilitate crime. A drug rape case involving the potent, short-acting sedative triazolam will be presented. On three occasions, the victim consumed green tea and chocolate before being massaged and ultimately sexually abused. Screening for alcohol, commonly used drugs and illicit substances in blood and urine sampled during the forensic examination 20 h after the last incident, was negative. Consequently, hair samples for chemical analysis were taken from the assaulted individual 34 days after the last incidents. The hair was cut into three 2-cm segments (0-6 cm) that were washed, dissolved in extraction solvent and screened and verified by ultra performance liquid chromatography coupled with time of flight mass spectrometry (UPLC-TOF-MS) and with tandem mass spectrometry (UPLC-MS/MS), respectively. In the 2-cm hair segment corresponding to the period of the alleged assaults, the presence of the sedative triazolam was revealed at a concentration of 1.0 pg/mg hair. The preserved urine sample, taken 20 h after the last incident, was reanalyzed by UPLC-MS/MS for metabolites of triazolam, and 39 µg/l α-hydroxytriazolam was detected in the hydrolyzed urine. This case illustrates that hair is a valuable forensic specimen in situations where natural processes have eliminated the drug from typical biological specimens due to delays in the crime being reported. Furthermore, it was possible to verify the hair finding with a urine sample by detection of a metabolite of triazolam.

Imprinted functionalized silica sol-gel for solid-phase extraction of triazolamin.

A triazolam-imprinted silica microsphere was prepared by combining a surface molecular-imprinting technique with the sol-gel process. The results illustrate that the triazolam-imprinted silica microspheres provided using γ-aminopropyltriethoxysilane and phenyltrimethoxysilane as monomers exhibited higher selectivity than those provided from γ-aminopropyltriethoxysilane and methyltriethoxysilane. In addition, the optimum affinity occurred when the molar ratio of γ-aminopropyltriethoxysilane, phenyltrimethoxysilane, and the template molecule was 4.2:4.7:0.6. Retention factor (k) and imprinting factor (IF) of triazolam on the imprinted and non-imprinted silica microsphere columns were characterized using high performance liquid chromatography (HPLC) with different mobile phases including methanol, acetonitrile, and water solutions. The molecular selectivity of the imprinted silica microspheres was also evaluated for triazolam and its analogue compounds in various mobile phases. The better results indicated that k and IF of triazolam on the imprinted silica microsphere column were 2.1 and 35, respectively, when using methanol/water (1/1, v/v) as the mobile phase. Finally, the imprinted silica was applied as a sorbent in solid-phase extraction (SPE), to selectively extract triazolam and its metabolite, α-hydroxytriazolam, from human urine samples. The limits of detection (LOD) for triazolam and α-hydroxytriazolam in urine samples were 30 ± 0.21 ng mL(-1) and 33 ± 0.26 ng mL(-1), respectively.

Determination of triazolam and alpha-hydroxytriazolam in guinea pig hair after a single dose.

A sensitive liquid chromatography-tandem mass spectrometry method is presented for determination of triazolam and alpha-hydroxytriazolam in guinea pig hair after a single dose of triazolam. Eighteen guinea pigs were divided into three dosage groups (10, 100, and 500 microg/kg) and administrated a single dose of triazolam intragastrically. Before administration, drug-free hair was shaved from their back. Newly grown hair in shaved area was collected every seven days after administration. About 20 mg of decontaminated hair was cut into small segments and incubated in 2 mL of phosphate buffer (pH 8.4) at 45 degrees C overnight. Triazolam-d(4) and alpha-hydroxytriazolam-d(4) were used as internal standards, and liquid-liquid extraction was performed with 3 mL of ethyl ether. The sample was separated on an Allure propyl PFP column with a mobile phase of acetonitrile/20 mM ammonium acetate (7:3, v/v). Detection was implemented with multiple reaction monitoring mode by an API4000 triple-quadrupole tandem mass spectrometer. Limits of detection for triazolam and alpha-hydroxytriazolam were 1 and 5 pg/mg, respectively. Triazolam and alpha-hydroxytriazolam could only be detected in the first week, and 100 microg/kg was the minimal dosage detectable in guinea pig hair. The concentration of triazolam in hair was related with administration dosage and hair color. alpha-Hydroxytriazolam has a higher concentration than triazolam in guinea pig hair.

The detection of sedatives in hair and nail samples using tandem LC-MS-MS.

Drug screening methods were developed to detect alprazolam, clobazam, clonazepam, diazepam, midazolam, oxazepam, temazepam, triazolam, zopiclone, and selected metabolites in human hair and nail samples employing liquid-liquid extraction and tandem liquid chromatography-mass spectrometry (LC-MS-MS). Hair and nail samples were obtained from patients who had recently discontinued or were currently prescribed one or more of the targeted drugs. Prazepam was used as the internal standard for all compounds. Some components in the hair matrix gave the same transitions as some of the analytes but did not compromise the analyses because their retention times differed from those for the target compounds. The analytical run time was 8-10min. Results of the hair analysis of a DFSA victim are also presented.

Simple method for determination of triazolam in human plasma by high-performance liquid chromatography/tandem mass spectrometry.

Triazolam was analyzed from human plasma samples by high-performance liquid chromatography (HPLC)-tandem mass spectrometry (MS/MS) with an MSpak GF polymer column (50 mm x 4.6 mm i.d., particle size 6 microm), which enabled direct injection of crude biological samples. Separation of triazolam, and lorazepam as the internal standard (IS) was carried out using 10mM ammonium acetate (pH 3.56)-0.1% formic acid and an acetonitrile gradient elution. Both compounds formed base peaks due to [M + H]+ ions by HPLC/ESI-MS, and product ions were produced from each [M + H]+ ion as seen by HPLC-MS/MS. Quantification of triazolam and the IS in plasma samples was made by selective reaction monitoring using each base peak of product ions of HPLC-MS/MS. The recovery range of triazolam spiked into plasma was 86.4-92.7%. The regression equation for triazolam showed excellent linearity in the range of 0.25-20 ng/mL, and the detection limit was 0.1 ng/mL. Intra- and inter-day precisions for triazolam in plasma samples were not greater than 12.4%. Accuracy for the drug was in the range of 88.0-101.4%. Data obtained after oral administration of triazolam in male and female subjects are also presented.

Criminal injury case using vegetable juice intermixed with triazolam.

This article explores an injury case in which a middle-aged male offered two female colleagues vegetable juice containing triazolam. The concentration of triazolam in one subject's vegetable juice was of a higher concentration than the other. This subject suffered from such symptoms as numbness, sleepiness, visual disturbance and anterograde amnesia. Conversely, despite having consumed a considerable quantity of vegetable juice, the other subject, with a relatively lower concentration of triazolam in her drink carton, exhibited no symptoms. The differences in the amount of triazolam ingested by each subject, particularly with respect to the expression of symptoms in just one subject, sparked considerable medico-legal discussion. The advice of a medico-legal expert was thus required to explain the reason for these differences.

A fatal poisoning caused by methomyl and nicotine.

A 35-year-old male was found lying in a prone position in his room. He was in cardiopulmonary arrest on arrival to hospital and was pronounced dead. There was no attempt at resuscitation. No miosis was observed on admission. At post-mortem his stomach contained 170 g greenish liquid with a small amount of shredded tobacco leaves. The serum cholinesterase activities were 47-90 IU (normal range for male: 200-440 IU). GC and GC-MS analyses showed nicotine (21.8 mg), methomyl (304 mg), and triazolam (1.69 mg) in his stomach. He had consumed tobacco leaves, Lannate containing water soluble methomyl (45%), and Halcion tablets containing 0.25 mg triazolam. Methomyl concentrations in blood were 3-8 ng/ml. Substantial amounts of methomyl (2260-2680 ng/ml) were detected in cerebrospinal fluid and vitreous humor. Nicotine concentrations in blood ranged from 222 to 733 ng/ml. A small amount of triazolam was detected only in bile (176 ng/ml) and liver (23 ng/g). The cause of death was respiratory paralysis produced by the additive effects of methomyl and nicotine shortly after consumption.

Postmortem changes in tissue concentrations of triazolam and diazepam in rats.

The aim of this study was to investigate the postmortem changes of concentrations of triazolam and diazepam in the rat model and to confirm the factor causing the phenomenon. We administered triazolam or diazepam orally to rats and then sacrificed them 1 h after administration. Abdominal tissues including stomach content and small intestine, thigh muscle and heart blood were collected at 0, 12, 24 h after death, and postmortem changes of the two drug concentrations were compared. Drug concentrations in the samples were analyzed by a selected ion monitoring of gas chromatography/mass spectrometry. Gastrointestinal postmortem concentrations of triazolam and diazepam decreased. On the other hand, the drug concentrations in the liver and kidney increased markedly, and those in lung and heart blood increased slightly during postmortem periods by diffusion from the gastrointestinal tract. The patterns of both drug concentration changes were similar. However, the extent of the triazolam increase tended to be larger than that of diazepam. This difference may be accounted for by lower triazolam concentrations before death. The postmortem drug concentrations of thigh muscle did not change when administered with triazolam and diazepam. For both triazolam and diazepam, a significant correlation was observed in the drug concentration between the small intestine and the kidney at 24 h after death, indicating that major cause of the change in the drug concentration in the kidney was diffusion from the small intestine up to 24 h after death. The results in this study indicate that diazepam as well as triazolam diffuses from the gastrointestinal tract into the surrounding tissues after death and suggest that the diffusion is influenced by their pharmaceutical properties before death.

A case of fatal triazolam overdose.

A 57-year-old man was found dead lying down in a bamboo thicket. Moderate to severe petechiae were present on his conjunctivae, buccal mucosa, and laryngeal mucosa at autopsy. Cardiac chambers contained a normal volume of fluid blood. Moderate atherosclerosis and fatty liver were observed. No remarkable changes, other than congestion in other organs, were observed. Gas chromatographic screening of the stomach contents, blood and urine was positive for triazolam and alpha-hydroxytriazolam that were confirmed by gas chromatography-mass spectrometry. Blood concentrations of triazolam and free alpha-hydroxytriazolam were 62-251 and 10-66 ng/ml, respectively. A substantial amount of triazolam was detected in bile (1130 ng/ml), but not in urine. Free and total alpha-hydroxytriazolam concentrations were 3920 and 7050 ng/ml, respectively, in the bile and 3710 and 9670 ng/ml, respectively, in urine. Organs contained 216-583 ng/g triazolam. The concentration of free alpha-hydroxytriazolam in the kidney (246 ng/g) was higher than in any other organ. Free alpha-hydroxytriazolam was not detected in the liver. The concentrations of total alpha-hydroxytriazolam in the liver and kidney were 784 and 381 ng/g, respectively. Free to total ratios of alpha-hydroxytriazolam were 0.14-0.56 in fluid samples and 0.56-0.92 in tissue samples, except for the liver. A large quantity of triazolam (8.4 mg) remained in the stomach. The victim probably died of postural asphyxia caused by triazolam poisoning.

Distribution of triazolam and alpha-hydroxytriazolam in a fatal intoxication case.

The case of a 77-year-old woman who was found dead in her bathtub with her head clearly above the water line is presented. The decedent had a medical history of depression, liver disease, spinal stenosis, and diabetes mellitus. An empty medication bottle of triazolam was found in the trashcan. At autopsy, no injury or evidence of drowning was found. Toxicological analysis identified triazolam at a concentration of 0.12 mg/L in the heart blood. Triazolam and alpha-hydroxytriazolam were quantitated in the specimens received. The medical examiner ruled that the cause of death was triazolam intoxication and the manner of death was suicide.

Determination of triazolam involving its hydroxy metabolites in hair shaft and hair root by reversed-phase liquid chromatography with electrospray ionization mass spectrometry and application to human hair analysis.

A sensitive method using reversed-phase liquid chromatography coupled with electrospray ionization mass spectrometry has been developed for simultaneous determination of triazolam and its hydroxy metabolites in hair. After the addition of deuterium-labeled 1-hydroxymethyltriazolam as an internal standard, the analytes in hair shaft and hair root samples were extracted with a basic medium, CH(2)Cl(2):MeOH:28% NH(4)OH (20:80:2) at room temperature overnight. The chromatographic separation of the analytes was achieved using a semimicro HPLC column (3-microm particle size; 100 x 2.0-mm i.d.) by gradient elution with acetonitrile in water containing 1% acetic acid as eluent. The mass spectrometer was operated in selected-ion monitoring mode at quasi-molecular ions [M+H](+) of triazolam and its metabolites. The method has been applied to determine the incorporation of triazolam and its metabolites into the hair shafts and hair roots of Dark Agouti rats administered 3 or 6 mg/kg triazolam intraperitoneally twice a day for 5 days. Triazolam, 1-hydroxymethyltriazolam, and 4-hydroxytriazolam were incorporated into the hair shafts and the hair roots. The concentration of 4-hydroxytriazolam was the highest of all compounds detected. An unknown substance considered to be 1,4-dihydroxytriazolam also appeared in the hair samples. The structural elucidation was performed with online HPLC-MS after acetylation of the substance with acetic anhydride and pyridine. The time course studies of triazolam and the metabolites in both rat hair roots and plasma were carried out after single intraperitoneal administration of triazolam. The concentrations of triazolam and the metabolites in the hair roots reflected those in the plasma. The proposed method using selected-reaction monitoring was applied to the determination of triazolam and the metabolites in human hairs of a triazolam addict. Triazolam, 1-hydroxymethyltriazolam, and 4-hydroxytriazolam were identified in the black hair shafts, whereas only triazolam was detected in the hair roots and the white hair shafts. This is the first report on the detection of triazolam and its metabolites in human hairs.

Detection of triazolam and its hydroxy metabolites in rat hair by reversed-phase liquid chromatography with electrospray ionization mass spectrometry.

A sensitive method using reversed-phase liquid chromatography coupled with electrospray ionization mass spectrometry (LC-ESI-MS) for simultaneous determination of triazolam (TZ) and its hydroxy metabolites in hair has been developed. After the addition of deuterium-labeled 1 -hydroxymethyltriazolam (1-HT-d4) as an internal standard, analytes in hair shaft and hair root samples were extracted with a basic medium, CH2Cl2/MeOH/28% NH4OH (20:80:2), at room temperature overnight. The chromatographic separation of the analytes was achieved using a 3-microm micro HPLC column (100 x 2.0-mm i.d.) with a gradient of acetonitrile in water containing 1% acetic acid as the mobile phase at a flow rate of 0.15 mL/min. The mass spectrometer was operated in selected-ion monitoring mode at quasi molecular ions [M+H]+ of TZ and its metabolites. Under the proposed conditions, the ranges of quantitation of TZ, 1-HT, and 4-HT were 0.1-10 ng/0.2 mL. The method has been applied to determine the hair shaft and hair root incorporation of TZ and its metabolites into Dark Agouti rats administered with 3 mg/kg or 6 mg/kg intraperitoneally twice a day for five days. Judging from the retention behavior by the chromatography and the mass spectra of the peaks detected, TZ, 1-HT, and 4-HT were incorporated in the hair shaft and the hair root. The concentration of 4-HT was the highest of all compounds detected. An unknown substance thought to be 1,4-diHT also appeared in both hair shaft and hair root samples. This substance was obtained from in vitro metabolic studies of TZ using rat liver microsome fraction and was accompanied by the other two metabolites, 1-HT and 4-HT. Structural elucidation was performed with online high-performance liquid chromatography-MS after acetylation of the substance with acetic anhydride and pyridine. This is the first report of the detection of the hydroxy metabolites of TZ in hair. The method has been found to be useful as a screening procedure of TZ intake in humans.

Use of high-performance liquid chromatography as an extraction procedure for analysis of triazolam in decomposed human muscle by gas chromatography-mass spectrometry.

A reliable and sensitive method for the determination of triazolam in human muscle using gas chromatography-mass spectrometry (GC-MS) is described. The drug was extracted from decomposed human muscle using three-step liquid-liquid extraction and HPLC which was performed isocratically on a conventional ODS column with a mobile phase of 0.01 M phosphate buffer (pH 6.5)-acetonitrile (7:3). Estazolam was used as an internal standard. GC-MS analysis was performed on a DB-5 capillary column. Excellent linearity was obtained over the concentration range 1-200 ng/g. The lower limit of detection was approximately 0.5 ng/g. Forensic application of the present method was also described.

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 husband's 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.