High-performance liquid chromatographic determination of new 2,3-benzodiazepines

A simple high-performance liquid chromatographic assay with ultraviolet detection at 254 nm for simultaneous determination of 2,3-benzodiazepine derivatives (2,3-BZ2 and 2,3-BZ2Me) and their metabolites in rat plasma is described. The procedure involves a fast extraction of the drugs from the buffered sample using methanol. The extract is evaporated to dryness at 45 degrees C and the residue is redissolved in methanol (twice). A 20-microl aliquot is injected into the liquid chromatograph and eluted with methanol-water (65:35, v/v) on a C18 reversed-phase column. At a flow-rate of 1.5 ml/min the detection time was 3.1 min for 2,3-BZ2, 5.06 min for 2,3-BZ2Me and 10.9 min for prazepam, used as internal standard for the quantification of the studied compounds. The method has been used to investigate the steady-state concentrations of two 2,3-benzodiazepine derivatives in Sprague-Dawley rat plasma.