Quantification of nebivolol hydrochloride in human plasma by liquid chromatography using fluorescence detection: Use in pharmacokinetic study.

A simple, rapid, and sensitive method of reversed-phase high-performance liquid chromatography with fluorescence detection has been developed and validated for use in determining levels of nebivolol•HCl in human plasma. Sample preparation involves a simple single-step protein precipitation procedure and extraction of nebivolol in acetonitrile. The separation was performed on a Kromasil® RP-C18 column (Ф 4.6 mm × 250 mm, 5 μm) with a mobile phase consisting of 0.05 M potassium dihydrogen phosphate buffer/acetonitrile (40:60, v/v) adjusted to pH 3 using orthophosphoric acid. Analysis was carried out under isocratic conditions at a flow rate of 1.5 mL/min and at room temperature using a fluorescence detector with excitation at 288 nm and emission at 310 nm. The chromatographic run was 4 min. The calibration curve was linear over the concentration range 0.2-20 ng/mL. The method was validated in terms of its accuracy, precision, and specificity. The assay enabled the measurement of nebivolol with a minimum quantification limit of 0.16 ng/mL. The average recovery of nebivolol from spiked human plasma was 98.4 ± 3.3%. This method was successfully used in a pharmacokinetic study of oral administration of 5-mg tablets to healthy human volunteers.