title: Quantitation of alprazolam and alpha-hydroxyalprazolam in human plasma using liquid chromatography electrospray ionization MS-MS.
abstract: A sensitive and specific electrospray ionization high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS-MS) method has been developed for the quantitative determination of alprazolam (AL) and alpha-hydroxyalprazolam (OH-AL) in plasma. After the addition of deuterium labeled internal standards of AL and OH-AL, plasma samples were buffered to alkaline pH and extracted with toluene/methylene chloride (7:3). Dried extract residues were reconstituted in HPLC mobile phase and injected onto a reversed-phase C18 HPLC column. The analytes were eluted isocratically at a flow rate of 250 microL/min using a solvent composed of methanol and water (60:40) containing 0.1% formic acid. The analyses were performed using selected reaction monitoring. The assay was sensitive to 0.05 ng/mL for both the parent drug and metabolite and linear to 50 ng/mL. The intra-assay percent coefficients of variation (%CV) for AL at 2, 5, and 20 ng/mL were all < or = 5.6. At these concentrations, and all OH-AL intra-assay %CVs were < or = 8.4. The interassay variabilities for AL were 11.8%CV, 8.7%CV, and 8.7%CV at 2.0, 5.0, and 20.0 ng/mL, respectively. The OH-AL interassay variabilities were 9.6%CV, 9.2%CV, and 7.8%CV at the same concentrations, respectively. The assay accuracy was less than or equal to +/- 6.6% for both analytes at the three concentrations. The method was used to quantitate AL and OH-AL in plasma samples collected from 10 subjects who were administered a 1-mg oral dose of AL. The mean AL concentration peaked at 11.5 ng/mL 1 h after the dose and AL was detectable for 48 h. The mean OH-AL concentration peaked at 0.18 ng/mL 4 h after the dose and was undetectable by 36 h. Hydrolysis of the plasma samples had little effect on the detected AL concentrations but increased OH-AL concentrations substantially. Plasma/blood ratios for AL and OH-AL exceeded 1 in the study samples.
authors.affiliation: Center for Human Toxicology, University of Utah, Department of Pharmacology and Toxicology, Salt Lake City 84112, USA.
authors.forename: D J
authors.initials: DJ
authors.lastname: Crouch
authors.forename: D E
authors.initials: DE
authors.lastname: Rollins
authors.forename: D V
authors.initials: DV
authors.lastname: Canfield
authors.forename: D M
authors.initials: DM
authors.lastname: Andrenyak
authors.forename: J E
authors.initials: JE
authors.lastname: Schulties
mesh_terms: D000525:Alprazolam / Q000031:analogs & derivatives* / Q000097:blood*; D014151:Anti-Anxiety Agents / Q000097:blood*; D002138:Calibration; D005260:Female; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D006868:Hydrolysis; D008297:Male; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D015813:Substance Abuse Detection / Q000379:methods*
chemical_list: D014151:Anti-Anxiety Agents; C032318:alpha-hydroxyalprazolam; D000525:Alprazolam