DETERMINATION OF ALPRAZOLAM AND ALPHA-HYDROXYALPRAZOLAM IN HUMAN PLASMA BY GAS-CHROMATOGRAPHY NEGATIVE-ION CHEMICAL-IONIZATION MASS-SPECTROMETRY

A sensitive and specific method was developed for the determination of alprazolam and its major metabolite alpha-hydroxyalprazolam in plasma . After the addition of deuterium-labeled internal standards, plasma s amples were buffered to pH 9 with 1 ml of saturated sodium berate buff er, extracted with toluene-methylene chloride (7:3) and evaporated to dryness. The residues were treated with N,O-bis(trimethylsilyl)trifluo roacetamide containing 1% of trimethylchlorosilane and analyzed on a F innigan-MAT mass spectrometer operated in the negative-ion chemical io nization mode with methane as the reagent gas. Chromatographic separat ion was achieved on a Restek-200 capillary column using hydrogen as th e carrier gas. The assay was linear from 0.25 to 50 ng ml(-1) for both compounds. The intra-assay precision for alprazolam was 16.1% at 0.5 ng ml(-1) and 4.6% at 50 ng ml(-1) and that for alpha-hydroxyalprazola m was 15.8% at 0.5 ng ml(-1) and 4.2% at 50 ng ml(-1). The method was used to determine alprazolam and alpha-hydroxyalprazolam in human plas ma samples collected after a single 2 mg oral dose of alprazolam. A pe ak concentration of 32.9 ng ml(-1) of alprazolam was detected at 1 h f ollowing the dose.