Quantification of alprazolam in serum or plasma by liquid chromatography

A reversed-phase "high-performance" liquid-chromatographic assay for the quantification of alprazolam in serum or plasma is described. Serum or plasma is extracted with toluene/isoamyl alcohol (99/1 by vol), evaporated, and reconstituted in the mobile phase. The latter is washed with hexane, then subjected to reversed-phase liquid chromatography and ultraviolet detection at 202 nm. Either U-31485, an alprazolam analog, or lorazepam, a 3-hydroxybenzodiazepine, is satisfactory as internal standards. Major alprazolam metabolites and various other commonly used drugs do not interfere. The useful lower limit of sensitivity for quantification is 2.5 micrograms/L. Peak height and alprazolam concentration are linearly related from 2.5 to 100 micrograms/L. For 10 and 20 micrograms/L concentrations, within-run CVs were 1.4% and 0.9% and the between-runs CVs 4.8% and 3.2%. Steady-state serum concentrations ranged from 25 to 55 micrograms/L in patients taking 1.5 to 6.0 mg per day, orally. Preliminary data suggest the method is also suitable for analysis of the structurally similar triazolobenzodiazepine, triazolam.