Liquid-chromatographic assay of diazepam and its major metabolites in plasma

We describe a method for simultaneous quantitation of diazepam and its metabolites, N-desmethyldiazepam and oxazepam, by reversed-phase liquid chromatography with ultraviolet detection at 254 nm. After extraction with chloroform at pH 11.0, the organic extract is evaporated, and the residue is reconstituted in a small volume of mobile-phase and chromatographed. Concentrations as low as 12 to 20 microgram/L can be detected after extraction of only 250 microL of plasma. The maximum within-day and day-to-day coefficients of variation for each compound at concentrations of 50, 250, and 1000 microgram/L are 8.0 and 10.3%, respectively. Of 14 commonly used anticonvulsant, sedative, and psychotherapeutic drugs, only carbamazepine and lorazepam may interfere with analysis for oxazepam or N-desmethyldiazepam, or both. Total chromatography time for a single sample is less than 10 min. Applicability of the method to diazepam disposition studies in children is illustrated.