Abstract: AimTo study the protein binding of glimepiride. MethodsAn HPLC-FA method is performed by using Pinkerton GFF II-S5-80 internal-surface reversed-phase silica support (150 mm×4.6 mm ID, 5 μm) at pH 7.4 in a 67 mmol·L^-1 isotonic sodium phosphate buffer at 37 ℃. Other conditions included flow rate of 0.2 mL·min^-1, UV detection at wavelength 230 nm and injection volume 900 μL. ResultsNonlinear regression parameter estimation was used for the association constant measurement of glimepiride to both primary and secondary sites, which were 5.1 (μmol·L^-1)^-1 and 1 for K₁ and n₁, and 0.017 (μmol·L^-1)^-1 and 7 for K₂ and n₂, respectively. ConclusionThe method is shown to be suitable for investigation of protein binding of glimepiride.