Authors:A. Ahmad, R. M. A. Khan and K. M. Alkharfy
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% for thymoquinone were found to be in the selected concentration range of 0.5–50 μg mL−1. The lower limits of detection and quantitation of the method were 0.109 and 0.332 μg mL−1 for glibenclamide and 0.119 and 0.361 μg mL−1 for thymoquinone, respectively. The within and between-day coefficients of variation were less than 7%. The validated method has been successfully applied to measure the plasma concentrations in a drug interaction study of glibenclamide with thymoquinone in an animal model to illustrate the scope and application of the method.