Determination Of Triazolam Research Articles

LC-MS determination of triazolam and its hydroxy metabolites in mouse dried blood spots: application to transgenic mouse pharmacokinetic studies.

The objective of this study was to develop a LC-MS/MS method for the simultaneous determination of triazolam (TRZ) and its two hydroxy metabolites in transgenic mouse dried blood spots (DBS) using BALB/c mouse blood as a surrogate biomatrix. The DBS method involved spotting volume of 10 μl using Ahlstrom 226 sample collection cards. A 'whole spot' analysis (6-mm punch) involved extraction of analytes using water and acetonitrile containing an internal standard. DBS samples were analyzed by a validated LC-MS/MS method with a run time of 4 min. This validated LC-MS/MS method using DBS extraction was applied to quantitation of TRZ, α-hydroxytriazolam and 4-hydroxytriazolam in a CYP3A4 transgenic mouse oral pharmacokinetic study of TRZ.

Simultaneous Quantification of Triazolam and its Metabolites in Human Urine by Liquid Chromatography-Tandem Mass Spectrometry

A simple, simultaneous, sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS-MS) method for the determination of triazolam and its metabolites, α-hydroxytriazolam (α-OHTRZ) and 4-hydroxytriazolam (4-OHTRZ), in human urine was developed and validated. Triazolam-d4 was used as the internal standard (IS). This analysis was carried out on a Thermo(®) C(18) column, and the mobile phase was composed of acetonitrile/H(2)O/formic acid (35:65:0.2, v/v/v). Detection was performed on a triple-quadrupole tandem MS using positive ion mode electrospray ionization, and quantification was performed by multiple reaction monitoring mode. The MS-MS ion transitions monitored were m/z 343.1 → 308.3, 359.0 → 331.0, 359.0 → 111.2, and 347.0 → 312.0 for triazolam, α-OHTRZ, 4-OHTRZ, and triazolam-d(4), respectively. The lower limits of quantification of the analytical method were 0.5 ng/mL for triazolam, 5 ng/mL for α-OHTRZ, and 0.5 ng/mL for 4-OHTRZ. The within- and between-run precisions were less than 15%, and accuracy was -12.33% to 9.76%. The method was proved to be accurate and specific, and it was applied to a urinary excretion study of triazolam in healthy Chinese volunteers.

Determination of triazolam and its metabolites 1-hydroxymethyltriazolam and 4-hydroxytriazolam in eight autopsy cases by GC-MS

Triazolam (Trz) concentrations in body fluids and organ tissues of eight autopsy cases were determined by gas chromatography-mass spectrometry (GC-MS). Blood Trz concentrations in seven out of eight cases ranged from less than 2 to 274 ng/g. The distribution of Trz in various organ tissues could be demonstrated in four cases. These distribution data are useful for cadavers that lack blood in forensic autopsy cases. In addition, urinary levels of 1-hydroxymethyltriazolam and 4-hydroxytriazolam, the main metabolites of Trz, were also determined with and without β-glucuronidase treatment by GC-MS for the eight cases. Using the ratios of urine Trz concentration to blood Trz concentration and the ratios of urine 1-hydroxymethyltriazolam concentration to urine 4-hydrozytriazolam concentration, we proposed a method for estimation of intervals between Trz intake and death, although further studies are needed to corroborate it.

Disposition of triazolam in the rat by brain microdialysis and semi-micro column high-performance liquid chromatography with UV absorbance detection.

A semi-micro column high-performance liquid chromatography with ultraviolet detection for the determination of triazolam is described. The method was applied to determine plasma and brain microdialysate concentrations of triazolam after single intravenous bolus of 2.5 mg/kg to rat. The separation was achieved on a 250 x 1.5 mm i.d. C(18) column and the column effluent was monitored at 222 nm. The detection limits at a signal-to-noise ratio of 3 obtained using spiked plasma and artificial cerebrospinal fluid were 2.1 and 0.7 ng/mL, respectively. The intra- and inter-day reproducibility of the present method were satisfactory with the highest relative standard deviation of 9.1 (n > or = 5). The present method was successfully applied to study the disposition of triazolam in rat (n = 5) by analyzing plasma and brain microdialysate samples.

Determination of triazolam by GC-MS in two autopsy cases: distribution in body fluids and organs

A detailed procedure for analysis of triazolam by GC-MS was constructed in our laboratory. At the concentration of 100 ng/ml, recoveries of triazolam in plasma and urine were 84.9 and 91.0%, respectively. The coefficients of variation in terms of its recovery were 11.5 (plasma) and 10.2% (urine). The detection limit for quantitation by the method was approximately 5 ng/g. This method was applied to two autopsy cases, giving triazolam distribution in body fluids and organs. In one case (33-year-old woman), concentrations of triazolam in the heart blood, urine, brain, lung, liver, kidney, skeletal muscles and stomach contents were 83.9, 741, 106, 165, 507, 293, 125 and 343 ng/g, respectively. From these toxicological data together with autopsy findings, her cause of death was diagnosed as triazolam poisoning. In the other case (45-year-old man), triazolam concentrations in the urine and stomach contents were 7.81 and 41.1 ng/g, respectively, but it could not be detected in the pleural blood; his cause of death was judged to be a traumatic shock, based on autopsy findings.

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.

Determination of triazolam in a drug tablet by thermal desorption gas chromatography

Thermal desorption gas chromatography using a vertical microfurnace pyrolyzer was applied to determine the amount of triazolam in a tablet sample. About 1 mg of ground tablet was directly subjected to analysis without any preconcentration. When the sample was heated at 300°C, triazolam desorbed from the powder was observed as a completely isolated peak in a chromatogram together with some additional peaks formed from the main components of the tablet through pyrolysis and/or thermal desorption. The correlation coefficient of the calibration curve obtained by use of pure triazolam ranging from 2.4 to 10.5 μg was 0.999. Triazolam in the tablet sample (about 3 μg/mg) could be determined using the calibration curve. The relative standard deviation of this method was 2.4% for four runs.

Determination of triazolam in serum by deactivated metal capillary gas chromatography with electron-capture detection

A method for the determination of trace amounts of triazolam in serum by deactivated metal capillary gas chromatography with electron-capture detection was established. The column used exhibits excellent thermostability in high-temperature analysis and easy handling and a long lifetime of the column and well shaped peaks on the chromatograms are obtained. With the metal capillary column, it was found to be easier to maintain suitable analytical conditions for the routine assay of triazolam than with a fused-silica column. With this method, 0.5 ng/ml of triazolam in serum can be determined. The method is useful for pharmacokinetic and therapeutic purposes.

Methods for Assay, Related Substances, and Organic Volatile Impurities in Triazolam Raw Materials and Formulations

Abstract Liquid chromatographic methods have been developed for determination of triazolam and related compounds in drug raw materials and tablets. The methods resolve 9 available related compounds from the drug with quantitation limits of 0.1 % or less. Eight raw materials and 16 formulations were examined for related compounds. Total impurities in raw materials ranged up to 0.6% and up to 1% in formulations, not including a compound in some tablets that is believed to be excipient related. Assay values were between 99.9 and 101.3% for raw materials and between 89.4 and 105.5% for tablets. A gas chromatographic method was developed for organic volatile Impurities in drug raw materials. The major solvents found were methyl ethyl ketone (100-750 ppm), ethyl acetate (510 ppm), and n-propanol (80-300 ppm), with a trace of benzene (<30 ppm) in several samples.