Measurement by HPLC of midazolam and its major metabolite, 1-hydroxymidazolam in plasma of very premature neonates.

A simple, selective high-performance liquid chromatographic method with ultraviolet detection at 220 nm is described for quantitation of midazolam and its primary metabolite 1-hydroxymidazolam in 100 microL plasma samples from premature infants. A mobile phase of acetonitrile:tetrahydrofuran:phosphate buffer (0.01 M, pH 6.7) (35:5:60 v/v) was pumped at 1 mL/min through a C8 Symmetry (150 x 3.9 mm) column. Plasma (100 microL) was extracted with 10% v/v isopropyl alcohol in dichloromethane containing 25 ng/mL climazolam (internal standard, IS) followed by back extraction into phosphoric acid (0.02 M). 1-Hydroxymidazolam, midazolam, and climazolam (IS) were eluted at 4.9, 7.4, 8.4 min, respectively. Recoveries were > 70%. Calibration curves in blank plasma were linear (r > 0.999) from 12.5 to 800 ng/mL. Within-day and between-day imprecision (CV%) was 1.8-6.5%, and 4.1-8.8%, respectively. Inaccuracy was 12.3%. Application of the method was demonstrated by a pharmacokinetic analysis of midazolam and 1-hydroxymidazolam in plasma drawn from 15 premature neonates after a single intravenous dose of midazolam (0.1 mg/kg).