Determination of midazolam (Versed) and its metabolites in plasma by high-performance liquid chromatography

A rapid, sensitive and selective high-performance liquid chromatographic (HPLC) assay was developed for the determination of midazolam, 8-chloro-6-(2-fluorophenyl)-1-methyl-4H-imidazo[1,5-a][1,4]- benzodiazepine (I), and its four metabolites in plasma. The assay involves extraction into diethyl ether--methylene chloride (7:3) from plasma buffered to pH 9 and subsequent analysis by reversed-phase HPLC with ultraviolet detection at 254 nm. The overall recovery of I from dog plasma was 94.5 +/- 7.1% and greater than 89.0% for its metabolites. The sensitivity limit of the assay was 50 ng/ml of plasma for all compounds. The HPLC assay was used to determine plasma concentrations of I and its metabolites from selected samples taken from an oral toxicity study in the dog.