DETERMINATION OF MIDAZOLAM AND 2 OF ITS METABOLITES IN HUMAN PLASMA BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

A high-performance liquid chromatographic assay coupled with UV detection (254 nm) has been developed for the determination of midazolam and two of its hydroxylated metabolites, 1-hydroxymidazolam (1-OH) and 4-hydroxymidazolam (4-OH), in human plasma. Following a novel solid-phase extraction procedure, midazolam and its metabolites are well recovered from plasma. The analytes were extracted with C-1 cartridges and the extracts were evaporated to dryness. The dry residues were dissolved in 200 mu l of mobile phase [0.02 M ammonium phosphate monobasic buffer-methanol-acetonitrile (60:35:5, v/v) (300 ml), 600 mu l of 0.2 M tetrabutylammonium bromide solution, adjusted to a pH' (apparent pH) of 4.10]. The separation of the analytes was performed on a Spherisorb C-8 column (10 cm x 4.6 mm I.D.) maintained at 30 degrees C. The mobile phase was pumped at a flow-rate of 1.5 ml/min. The method has a lower limit of quantitation of 15 ng/ml of plasma for midazolam and proved to be reproducible (inter-assay precision 5.4%) and accurate (94 +/- 5%) over the therapeutic range of concentrations.