Validated Reverse Phase-High-Performance Liquid Chromatography Method for Simultaneous Determination of Vanillic acid and Glyburide in Amphiphilic Polymeric Micelles

Abstract

Background: A new sensitive and rapid reverse phase HPLC method was developed for the simultaneous determination of vanillic acid (VA) and glyburide in the formulation and validated according to (ICH) Q2 (R1) guidelines. Methods: The HPLC analysis was performed using the C-18 reverse phase column and a mobile phase consisting of acetonitrile and orthophosphoric acid (0.1% v/v) of ratio 70:30 v/v at 1 mL/min of flow rate. The detection was performed at the wavelength (λ) of 234 nm (isosbestic point), and the retention time of VA and glyburide was found around 2.6 and 5.4 min. The calibration plot gave a linear relationship over the concentration range of 2–10 μg/mL with a regression coefficient of 0.999. The LOD and LOQ for VA were 0.34 and 1.04 μg/mL, while for glyburide it was 0.38 μg/mL and 1.17 μg/mL respectively. The accuracy of the proposed method was determined by recovery studies and was in the range of 95% to 105%. The RSD% of the determination of precision was <2%. Results: The results of the robustness study were within the acceptable limits in response to changes in flow rate, ratio of mobile phase, and pH. The method was successfully applied for the determination of EE% and drug release from amphiphilic polymeric micelles. Conclusion: The EE% of both the drugs prepared in a liquid formulation of amphiphilic polymeric micelles was found to be greater than 90%. The results of drug release studies indicated almost 100% VA and 85.3% glyburide release using pH-gradient method within 4 and 48 h respectively.