title: Determination of diazepam and its metabolites in human urine by liquid chromatography/tandem mass spectrometry using a hydrophilic polymer column.
abstract: Diazepam and its major metabolites, nordazepam, temazepam and oxazepam, in human urine samples, were analyzed by liquid chromatography (LC)/tandem mass spectrometry (MS/MS) using a hydrophilic polymer column (MSpak GF-310 4B), which enables direct injection of crude biological samples. Matrix compounds in urine were eluted first from the column, while the target compounds were retained on the polymer stationary phase. The analytes retained on the column were then eluted into an acetonitrile-rich mobile phase using a gradient separation technique. All compounds showed base-peak ions due to [M+H]+ ions on LC/MS with positive ion electrospray ionization, and product ions were produced from each [M+H]+ ion by LC/MS/MS. Quantification was performed by selected reaction monitoring. All compounds spiked into urine showed method recoveries of 50.1-82.0%. The regression equations for all compounds showed excellent linearity in the range of 0.5-500 ng/mL of urine. The limits of detection and quantification for each compound were 0.1 and 0.5 ng/mL of urine, respectively. The intra- and inter-day coefficients of variation for all compounds in urine were not greater than 9.6%. The data obtained from actual determination of diazepam and its three metabolites, oxazepam, nordazepam and temazepam, in human urine after oral administration of diazepam, are also presented.
authors.affiliation: Department of Legal Medicine, Showa University School of Medicine, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan.
authors.forename: Hironobu
authors.initials: H
authors.lastname: Umezawa
authors.forename: Xiao-Pen
authors.initials: XP
authors.lastname: Lee
authors.forename: Yoshiko
authors.initials: Y
authors.lastname: Arima
authors.forename: Chika
authors.initials: C
authors.lastname: Hasegawa
authors.forename: Akemi
authors.initials: A
authors.lastname: Marumo
authors.forename: Takeshi
authors.initials: T
authors.lastname: Kumazawa
authors.forename: Keizo
authors.initials: K
authors.lastname: Sato
mesh_terms: D000284:Administration, Oral; D000328:Adult; D014151:Anti-Anxiety Agents / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism* / Q000652:urine*; D002853:Chromatography, Liquid / Q000379:methods*; D003975:Diazepam / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism* / Q000652:urine*; D004355:Drug Stability; D005615:Freezing; D006801:Humans; D008875:Middle Aged; D015394:Molecular Structure; D011786:Quality Control; D012015:Reference Standards; D012044:Regression Analysis; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry / Q000379:methods*; D013997:Time Factors
chemical_list: D014151:Anti-Anxiety Agents; D003975:Diazepam