Development and validation of HPLC method for the determination of glibenclamide in rat serum.

Abstract

A rapid, sensitive, accurate and specific HPLC assay with UV-Visible detection (253nm) for the determination of glibenclamide in rat serum was developed and validated. Glipizide was used as an internal standard (IS). The serum proteins were precipitated by a single step liquid-liquid extraction using methanol. Chromatographic separation was achieved with a combination of acetonitrile and 25mM monobasic potassium dihydrogen orthophosphate (pH 3.5 adjusted with phosphoric acid) at 60:40 v/v ratios was run isocratically through a C18 (250mmX4.6mm, 5µm) reverse phase analytical column.  Analytical run time was less than 10 min. Mean recovery was 97.12% for 0.1-10 µg/ml concentrations. The assay exhibited good linear relationship. Quantification limit was at 50ng/ml of glibenclamide and accuracy and precision were over the concentration range of 0.1-10 µg/ml. The method was validated with excellent sensitivity, accuracy, precision and recovery. The assay has been applied successfully to pharmacokinetic studies.