Determination of midazolam and 1′-hydroxymidazolam by liquid chromatography–mass spectrometry in plasma of patients undergoing methadone maintenance treatment

A rapid, sensitive and selective LC–MS method was developed for the simultaneous determination of midazolam (MDZ) and 1′-hydroxymidazolam (1′-OHMDZ) in plasma taken from 54 patients undergoing methadone maintenance therapy, most of whom were multidrug users. Samples spiked with prazepam, the internal standard, and were extracted into diethyl ether. Compounds were separated on a Phenomenex Luna C18 column and a mobile phase of acetonitrile–ammonium acetate buffer (10 mM, pH 4.7) (52:48, v/v) at a flow-rate of 1 ml/min. The limit of detection was 0.65 and 0.68 (ng/ml) for MDZ and 1′-OHMDZ, respectively. Within-day relative standard deviations were less than 8%.