MEASUREMENT BY HPLC OF MIDAZOLAM AND MAJOR METABOLITE, 1-HYDROXYMIDAZOLAM IN PLASMA OF VERY PREMATURE NEONATES

A simple, selective high-performance liquid chromatographic method wit h ultraviolet detection at 220 mn is described for quantitation of mid azolam and its primary metabolite 1-hydroxymidazolam in 100 mu L plasm a samples from premature infants. A mobile phase of acetonitrile: tetr ahydrofuran :phosphate buffer (0.01 M, pH 6.7) (35:5:60 v/v) was pumpe d at 1 mL/min through a C-8 Symmetry(TM) (150 x 3.9 mm) column. Plasma (100 mu L) was extracted with 10% v/v isopropyl alcohol in dichlorome thane 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, and 8.4 min, r espectively. 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. Inacc uracy was 12.3%. Application of the method was demonstrated by a pharm acokinetic analysis of midazolam and 1-hydroxymidazolam in plasma draw n from 15 premature neonates after a single intravenous dose of midazo lam (0.1 mg/kg).