Development and Validation of RP-HPLC Method for Simultaneous Estimation of Glibenclamide and Thymoquinone in Rat Plasma and Its Application to Pharmacokinetics

Abstract

A rapid, accurate, and sensitive reverse phase high-performance liquid chromatographic method was developed and validated for the simultaneous determination and quantification of glibenclamide and thymoquinone in rat plasma in the presence of internal standard (thymol). Chromatograms were developed with methanol, acetonitrile, and buffer (50:20:30, v/v/v) solvent system on a Symmetry® C18 (5 μm, 3.9 × 150 mm) column, and pH was adjusted to 4.5 with orthophosphoric acid. Mobile phase was pumped at a flow rate of 1.5 mL min−1 with 254 nm ultraviolet (UV) detection. Validation of the method was performed in order to demonstrate its selectivity, linearity, precision, accuracy, limits of detection, and quantification (LOD and LOQ). Standard curves were linear (r2 = 0.996 and 0.999 for glibenclamide and thymoquinone) over the concentration range 0.5–50 μg mL−1. The coefficient of variation (CV) of < 6% and accurate recovery of 87.54–105.19% for glibenclamide and CV of <5% and accurate recovery of 86.08–103.19% ...