Development and Validation of a Reversed-Phase HPLC Method for Estimation of Glimepiride in Rabbit Serum and Its Application to Pharmacokinetic Studies

Abstract

A reversed-phase high-performance liquid chromatography (RP-HPLC) method reported for glimepiride was modified and standardized for its estimation in rabbit serum, using pioglitazone as an internal standard (IS), to support pharmacokinetic evaluation of the drug in a rabbit model. Chromatographic separation was achieved on an RP C18 column (250 × 4.6 mm, 5 μm) using acetonitrile and phosphate buffer (pH 4.3) in a 75:25 (v/v) ratio as the mobile phase at a flow rate of 1 mL/min, with UV detection at 258 nm. Glimepiride and the internal standard were extracted from rabbit serum by liquid–liquid extraction with dichloromethane, and the ratio of the peak area (AUC) of glimepiride to that of pioglitazone was used for quantification. The method was validated for linearity, system suitability, precision, and recovery/accuracy. The calibration curve was linear over 20–1000 ng/mL (y = 0.0089x − 0.0078, r² = 0.9980). The limit of detection (LOD) and limit of quantification (LOQ) were 18.55 and 20.00 ng/mL, respectively. The column gave 14,352 theoretical plates and a tailing factor of 1.08 for glimepiride, with retention times of 6.8–8.02 min for glimepiride and 4.2–5.05 min for the internal standard. Intra-day and inter-day precision (%RSD) were below 3% at all concentrations tested, and absolute recovery ranged from 98.85% to 99.82%. The validated RP-HPLC method is simple, sensitive, precise, and accurate, requires only 200 μL of serum, and is suitable for pharmacokinetic, bioavailability, and drug-interaction studies of glimepiride in rabbits.