Determination of midazolam and its α-hydroxy metabolite in human plasma and urine by high-performance liquid chromatography

We describe a new high-performance liquid chromatography (HPLC) method for measurement of midazolam and its major metabolite, alpha-hydroxymidazolam, in clinical samples. Plasma or urine was mixed with 100 ng internal standard Ro 05-6669 and borate buffer, 0.1 M, pH 9. Midazolam and its related compounds were extracted into diethylether. The organic phase was evaporated to dryness. The residue was dissolved in HPLC mobile phase [methanol-isopropyl alcohol-perchloric acid, 0.5 microM (57:25:18)] and injected into the chromatograph. The separation of substances was performed on an Spherisorb S5CN 250 x 4.6 mm HPLC column maintained at 45 degrees C. The detection was performed by absorption measurement at 245 nm. At a flow rate of 1.7 ml/min, the retention times of Ro 05-6669, 1,4 dihydroxymidazolam, alpha-hydroxymidazolam, 4-hydroxymidazolam and midazolam were 4.0, 6.7, 7.8, 9.6, and 10.8 min, respectively. In the concentration range of 5-1,000 ng/ml, the calibration graphs for both compounds were linear. The coefficients of variation of the between-day and within-day assay were < 14% for the concentration range 5-10 and < 7% for the range 10-600 ng/ml. The limits of detection for midazolam and alpha-hydroxymidazolam were 2 and 4 ng/ml, respectively. This assay is more sensitive than earlier methods; it is simple and rapid, and it enables the quantification of midazolam and its alpha-hydroxy metabolite with very good precision and accuracy in human plasma and urine.