Abstract
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 μl of mobile phase [0.02 M ammonium phosphate monobasic buffer—methanol—acetonitrile (60:35:5, v/v) (300 ml), 600 μl 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 × 4.6 mm I.D.) maintained at 30°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.
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