Reverse Phase High Performance Liquid Chromatography Method for the Estimation of Mangiferin in Nano Formulations: Development and Optimization by Analytical Quality-by-Design.

A novel reverse-phase high-performance liquid chromatography (RP-HPLC) method was developed and optimized for the estimation of Mangiferin (MGF) by employing a Quality by Design (QbD) approach. Preliminary method screening was done by the Taguchi OA, followed by using a Box-Behnken Design (BBD), enabling systematic evaluation of critical factors with a reduced number of experimental runs. The model efficiently established significant correlations between selected variables and analytical responses, thereby enhancing the method's robustness and reliability. Chromatographic separation was achieved on a Hyperclone C18 column (4.6 × 250 mm, 5 μm) using an isocratic mobile phase consisting of phosphate buffer (pH 3.2) and acetonitrile (30%) + methanol (70%) in a 78:22% v/v ratio, at a flow rate of 1.0 mL/min. Detection was performed using a photodiode array detector at 258 nm. The method was validated in accordance with ICH Q2-(R2) guidelines. Validation parameters, including system suitability, linearity, accuracy, precision, robustness, sensitivity, and solution stability, were found within acceptable limits. The proposed method was successfully applied for the analysis of the stability study of MGF and to analyze the release kinetics of MGF SLNs, formulated via high-pressure homogenization and the sonication technique. The results confirm the applicability of the developed RP-HPLC method for routine quality control of combination nanocarrier systems.