ANALYSIS OF MIDAZOLAM AND METABOLITES IN PLASMA BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY - PROBE OF CYP3A

Hydroxylation of midazolam (MDZ) is mediated almost exclusively by CYP 3A isoforms. The authors describe a high-performance liquid chromatogr aphy assay involving MDZ, 1'-hydroxymidazolam, and 4-hydroxymidazolam in plasma. The compounds were eluted on an Ultrasphere ODS, 3-mu m par ticle size, 7.5 cm x 4.6 mm reversed-phase column and monitored by ult raviolet absorbance at 254 nm. The composition of the mobile phase was 35.2% acetonitrile:4.8% methanol:60% buffer acetate (vol/vol/vol), 0. 1 M, pH 4.7; the flow rate was 1 ml/minute. Calibration curves were li near (coefficients of correlation >0.99) within the range of concentra tions established (20 to 640 nM). Within- and between-day coefficients of variation were consistently better than 8%. The overall recovery w as >90% and the lowest detectable concentration was 8 nM. This approac h provides a simple, rapid, and sensitive assessment of MDZ and metabo lites in plasma, with a very good accuracy and precision, which enable s it as an in vivo marker of CYP3A activity in humans.