Sensitive determination of midazolam and 1′-hydroxymidazolam in plasma by liquid–liquid extraction and column-switching liquid chromatography with ultraviolet absorbance detection and its application for measuring CYP3A activity

This manuscript described a new sensitive determination of midazolam and its metabolite 1′-hydroxymidazolam by automated column-switching high-performance liquid chromatography. The test compounds were extracted from 2 ml of plasma using chloroform-hexane (30:70, v/v) and the extract was injected into a column I (TSK-PW precolumn, 10 μm, 35 mm × 4.6 mm i.d.) for clean-up and column II (C18 STR ODS-II analytical column (5 μm, 150 mm × 4.6 mm i.d.) for separation. The mobile phase for separation consisted of phosphate buffer (0.02 M, pH 4.6), perchloric acid (60%) and acetonitrile (57.9:0.1:42, v/v/v) and was delivered at a flow-rate of 0.6 ml/min. The peak was detected using a UV detector set at 254 nm. The method was validated for the concentration range 0.3–100 ng/ml, and good linearity (r > 0.998) was confirmed. Intra- and inter-day coefficient variations for midazolam and 1′-hydroxymidazolam were less than 8.5 and 6.1%, respectively, at the concentrations of 0.5, 5 and 50 ng/ml for the test compounds. Relative errors ranged from −14 to 6% and mean recoveries were 78–85%. The limit of quantification was 0.5 ng/ml for each compound. This method was sensitive enough for pharmacokinetic studies measuring CYP3A activity in human volunteers following an intravenous (1 mg) and a single-oral administration (2 mg) of a subtherapeutic midazolam dose.