High-performance liquid chromatographic determination of flurazepam and its metabolites in human blood plasma

A blood plasma sample was spiked with flurazepam and its metabolites and diluted with water (1 + 1); after protein precipitation, flurazepam and three of its metabolites were extracted into ethyl acetate at pH 9. After evaporation of the solvent, the residue was dissolved in the mobile phase for injection onto a reverse-phase LiChrosorb column; eluents were methanol-water (62:38) for separation of the metabolites and methanol-phosphate buffer (85:15) for flurazepam. The limit of detection varied from 0.1 to 1.6 ng. Recoveries from spiked plasma (1 μg ml^?1) were 94–95%. At 254 nm, the response was usually linear over the range 5–50 ng.