Sensitive and specific determination of midazolam and 1-hydroxymidazolam in human serum by liquid chromatography–electrospray mass spectrometry

Abstract

A liquid chromatographic–mass spectrometric technique was designed for the determination of the anaesthetic benzodiazepine midazolam (MID) and its active metabolite 1-hydroxymidazolam (1-OHM), with the aim of conducting pharmacokinetic/pharmacodynamic studies. MID and 1-OHM were extracted from alkalinised (pH 9.5) spiked and clinical serum samples using a single step, liquid–liquid extraction procedure with diethyl ether–2-propanol (98:2, v/v). The chromatographic separation was performed on a Nucleosil C 18, 5 μm (150×1 mm I.D.) column, using a gradient of acetonitrile in 5 m M ammonium formate, pH 3.0 as the mobile phase, delivered at a flow-rate of 50 μl/min. The compounds were ionised in the ionspray source of an atmospheric pressure mass spectrometer, fragmented by in-source collisions and the pseudomolecular and fragment ions detected in the selected ion monitoring mode. The recovery was between 79 and 87% for MID, between 83 and 87% for 1-OHM and 81.5% for methylclonazepam. The limit of detection was 0.2 μg/l for MID and 0.5 μg/l for 1-OHM, the limit of quantitation (LOQ) was 0.5 μg/l for both. Linearity was verified from these LOQs up to 2000 μg/l and the method was found accurate and precise over this range. It was successfully applied to a preliminary study to establish the concentration versus time curve of MID and 1-OHM in a patient administered midazolam by continuous infusion.