High-Performance Liquid Chromatographic Method for Determination of Glibenclamide in Human Plasma

An HPLC method for quantitative determination of glibenclamide in human plasma is described. The methodology is based on simple one step extraction of glibenclamide and diazepam (internal standard) from human plasma with dichloromethane. The drugs were eluted from a resolve 5 u spherical C 18 column with a mobile phase consisting of 0.05 M ammonium dihydrogen phosphate:methanol (40:60%, v/v) adjusted to pH 4.0 with phosphoric acid. The drug and the internal standard were eluted at a flow rate of 1.2 ml per minute, and the optimum detector wavelength was 230 nm. The chromatography time was eleven minutes and the resolution between the drug and internal standard was excellent. Quantitation was achieved by the measurement of peak area ratios and the minimum detectable concentration was 5 ng/ml. The calibration curve of the assay was linear over the range of 10-400 ng/ml. The intraday coefficient of variation were ranged from 0.53% to 3.19%, whereas interday coefficient of variation were from 2.31% to 7.90%. The relative and absolute recoveries varied between 91.7% and 101.5%. Stability results showed that glibenclamide is stable for at least 10-weeks in plasma when freezed at -20°C. The utility of the analytical methodology for the quantitative determination of glibenclamide in pharmacokinetic studies in the human plasma was demonstrated.