Sensitive assay for midazolam and its metabolite 1′-hydroxymidazolam in human plasma by capillary high-performance liquid chromatography

A sensitive high-performance liquid chromatographic method is described for the quantification of midazolam and 1'-hydroxymidazolam in human plasma. Sample (1 mi plasma) preparation involved a simple solvent extraction step with a recovery of approximately 90% for both compounds. An aliquot of the dissolved residue was injected onto a 3 mu m capillary C-18 column (150 mmx0.8 mm I.D.). A gradient elution was used. The initial mobile phase composition (phosphate buffer-acetonitrile, 65:35) was maintained during 16 min and was then changed linearly during a 1-min period to phosphate buffer-acetonitrile, 40.60. The flow-rate of the mobile phase was 16 mu l/min and the eluate was monitored by UV detection. The limits of quantification for midazolam and 1'-hydroxymidazolam were 1 ng/ml and 0.5 ng/ml, respectively. The applicability of the method was demonstrated by studying the pharmacokinetics, of midazolam, and its major metabolite l'-hydroxymidazolam, in human volunteers following i.v. bolus administration of a subtherapeutic midazolam dose (40 mu g/kg). (C) 1998 Elsevier Science B.V. All rights reserved.