Determination of the agonist-antagonist anxiolytic agent 6-(2-chlorophenyl)-4-hydroxy-4H-imidazo[1,5-a]-[1,4]benzodiazepine- 3-carboxamide and its benzophenone carboxylic acid metabolite in plasma by high-performance liquid chromatography.

A rapid, sensitive and selective high-performance liquid chromatographic (HPLC) assay was developed for the determination of 6-(2-chlorophenyl)-4-hydroxy-4H-imidazo[1,5-a]-[1,4]benzodiazepine- 3-carboxamide (I) and its benzophenone carboxylic acid metabolite, 1-[2-(2-chlorobenzoyl)phenyl]-4-(aminocarbonyl)-1H-imidazole-5-car boxylic acid (II) in plasma. The assay involves the extraction of both compounds into benzene from buffered plasma (pH 5.4) and subsequent analysis by reversed-phase HPLC. The overall recovery of I and II is 98.3 +/- 9.4 and 59.7 +/- 15.7% for dog plasma, 86.0 +/- 14.7 and 52.8 +/- 15.1% for rat plasma and 98.1 +/- 9.3 and 66.9 +/- 18.0% for human plasma, respectively. The sensitivity limit of the assay for I and II is 20.0 and 40.0 ng/ml of plasma using ultraviolet detection at 254 nm. The assay was used in studies in dog and rat.