Quantitation of Alprazolam and a-Hydroxyalprazolam in Human Plasma Using Liquid Chromatography Electrospray Ionization MSÐMS

Published: Journal of Analytical Toxicology, Volume 23, Number 6, October 1999, pp.479-485

Quantitation of Alprazolam and a-Hydroxyalprazolam in Human Plasma Using Liquid Chromatography Electrospray Ionization MS–MS
Dennis J. Crouch, Douglas E. Rollins, Dennis V. Canfield, David M. Andrenyak, and Janie E. Schulties

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 a-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 µL/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 ² 5.6. At these concentrations, and all OH-AL intra-assay %CVs were ² 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.

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