Validation of a New High Performance Liquid Chromatography Assay for Glibenclamide in Human Plasma

A simple reversed phase high performance liquid chromatography assay for the determination of glibenclamide in human plasma was developed and validated. Glibenclamide and ketoconazole (internal standard) were extracted with dichloromethane/hexane (50:50, v : v), eluted on a Symmetry RP18 column with a retention factor of 4.6 and 2.1, respectively, and monitored fluorometrically at excitation and emission wavelengths of 235 and 354 nm, respectively. The mobile phase was a mixture of 0.05 M ammonium phosphate (pH 5.5) and acetonitrile (50:50, v : v) delivered at a flow rate of 1.0 ml/min. The limit of detection was 0.005 μg/ml. Calibration curves were linear (R2 ≥ 0.9993) over the range of 0.01 (lower limit of quantitation) to 0.60 μg/ml, and intra-and inter-day coefficients of variation were ≤ 5.9% and 6.5%, respectively. Mean extraction recovery of glibenclamide and ketoconazole were 91% and 93%, respectively. Glibenclamide was stable in plasma for 24 hours at room temperature (≥ 93%), 4 weeks at –20°C (≥ 97%), and after 3 cycles of freeze at –20°C and thaw at room temperature (≥ 97%). In extracted samples, glibenclamide was stable for 24 hours at room temperature (≥ 98%) and 48 hours at –20°C (≥ 102%). Glibenclamide stock solution (1 mg/ml in methanol) was stable for 48 hours at room temperature (98%) and 2 weeks at –20°C (88%).