Long-term stability of various drugs and metabolites in urine, and preventive measures against their decomposition with special attention to filtration sterilization.

The long-term stability of drugs and metabolites of forensic interest in urine, and preventive measures against their decomposition have been investigated, with special attention to filtration sterilization. An aseptic urine collection kit, which was recently developed based on filtration sterilization, was utilized for the aseptic collection and storage of urine samples. For evaluating preservation measures, methamphetamine (MA), amphetamine (AP), nitrazepam (NZ), estazolam (EZ), 7-aminoflunitrazepam (7AF), cocaine (COC), and 6-acetylmorphine (6AM) were spiked into urine at 500 ng/mL each, and were monitored for 6 months at 25, 4, and -20 degrees C, after the addition of NaN(3) and/or filtration sterilization using the aseptic collection kit. In severely contaminated urine with bacteria, there were significant losses of 7AF and NZ, and slight decomposition of MA and AP at 25 degrees C. However, such degradation was successfully suppressed by the use of the kit, though the use of the kit and NaN(3) were preferred for 7AF. The kit was also effective in preventing the hydrolyses of COC and 6AM, while it was suggested that the common preservative NaN(3) can accelerate the hydrolysis of such ester-type drugs and metabolites.

[Identification of estazolam, alprazolam and triazolam in human urine by LC/MSn].

AIM: To investigate the fragmentation behavior of triazolobenzodiazepines and to develop a specific, sensitive and rapid LC/MSn assay for simultaneous determination of estazolam, alprazolam and triazolam in human urine. METHODS: After oral administration of a single 4 mg dose of the drugs to each of three healthy volunteers, urine samples were purified by solid-phase extraction, and then injected into an ODS column (150 mm x 4.6 mm) with a mobile phase of methanol-water (8:2) for LC/MSn analysis. The structures of estazolam, alprazolam and triazolam in human urine were identified by direct comparison of the observed mass spectra and the chromatographic retention time with those of the reference substance. The mass spectrometer (Finnigan LCQ) was operated in positive mode and in two scan modes including SIM and full scan MS/MS mode. The obtained mass spectra was analyzed assisted with the software Mass Frontier 1.0 for their fragmentation pathways. RESULTS: The full scan MS/MS spectra of each compound gave characteristic fragment ions of [M + H - N2]+ and [M + H - Cl]+. The detection limit was below 0.5 ng.mL-1 for estazolam, alprazolam and triazolam in human urine. CONCLUSION: The method is useful in forensic and clinical toxicology in which unequivocal identification of eatazolam, alprazolam and triazolam is desired.

Urinary screening for adinazolam and its major metabolites by the Emit d.a.u. and FPIA benzodiazepine assays with confirmation by HPLC.

Adinazolam is a triazolobenzodiazepine, currently under clinical investigation, that possesses antidepressant and anxiolytic activity. It has a short half-life (less than 3 h), and less than 2% of an oral dose is excreted unchanged. The major urinary metabolite is N-desmethyladinazolam, and minor metabolites are estazolam and alpha-OH-alprazolam. The objective of this study was to characterize the reactivity of adinazolam, N-desmethyladinazolam, and estazolam in the Emit d.a.u. benzodiazepine assay and the Abbott TDx urine (FPIA) benzodiazepine assay. N-desmethyladinazolam and estazolam gave an equivalent response to the Emit cutoff calibrator (300 ng/mL) at 100-200 ng/mL, and adinazolam gave an equivalent response at 200 ng/mL. By FPIA, N-desmethyladinazolam and adinazolam had equivalent net polarization values as the 300-ng/mL low control at 500-1000 ng/mL, and estazolam gave a positive response at 300 ng/mL. Six volunteers received single oral doses of 10, 30, and 50 mg of adinazolam. Urine specimens (N = 7) were collected from 0 to 36 h post-administration. By Emit, all urine specimens at all doses were positive from 2 to 36 h, and all FPIA analyzed specimens were positive from 2 to 24 h. Confirmation testing was performed by HPLC by analyzing for N-desmethyladinazolam. All urine specimens were confirmed positive for N-desmethyladinazolam (greater than 200 ng/mL) except for the blank specimens (time = 0) and 7 of 18 specimens collected 36 h post-administration. In conclusion, both immunoassay screening assays are acceptable for detecting the presence of adinazolam in human urine for up to 24 h after a single oral dose of 10-50 mg.

A new method for the determination in blood and urine of a novel triazolobenzodiazepine (estazolam) by HPLC.

In this study the authors report a new method to determine estazolam in blood and urine by high pressure liquid chromatography (HPLC). This compound is a triazolobenzodiazepine and shows an interesting psychopharmacological activity. The extraction from biological fluids was carried out using a mixture of ethylenechloride, methylenechloride, and ethylacetate (1:1:8). The HPLC analysis was carried out with a C18 column, using methanol:phosphate buffer (pH 7.5, 0.011M):acetonitrile (65:33:2) as mobile phase. The detector was set at lambda = 240 nm. The method shows good repeatability and a linear response in the range of 0.6 to 10 micrograms estazolam/mL both in serum and urine. A case of non-lethal acute intoxication in which the method was applied is also reported.

Metabolism of 14C-estazolam in dogs and humans.

14C-Estazolam (2 mg) administered orally to dogs and human subjects was rapidly and completely absorbed with peak plasma levels occurring within one hour. In humans, plasma levels peaked at 103 +/- 18 ng/ml and declined monoexponentially with a half-life of 14 h. The mean concn. of estazolam in dog plasma at 0.5 h was 186 ng/ml. Six metabolites were found in dog plasma at 0.5 and 8 h, whereas only two metabolites were detected in human plasma up to 18 h. Metabolites common to both species were 1-oxo-estazolam (I) and 4-hydroxy-estazolam (IV). Major metabolites in dog and human plasma were free and conjugated 4-hydroxy-estazolam; the concn. were higher in dogs. After five days, 79% and 87% of the administered radioactivity was excreted in dog and human urine, respectively. Faecal excretion accounted for 19% of the dose in dog and 4% in man. Eleven metabolites were found in the 0-72 h urine of dogs and humans; less than 4% dose was excreted unchanged. Four metabolites were identified as: 1-oxo-estazolam (I), 4'-hydroxy-estazolam (II), 4-hydroxy-estazolam (IV) and the benzophenone (VII), as free metabolites and glucuronides. The major metabolite in dog urine was 4-hydroxy-estazolam (20% of the dose), while the predominant metabolite in human urine (17%) has not been identified, but is likely to be a metabolite of 4-hydroxy-estazolam. The metabolism of estazolam is similar in dog and man.