High‐Performance Liquid Chromatography–Based Standardization of Stigmasterol in Moringa oleifera: Method Development and Validation Through Design of Experiment Approach

Abstract

The primary objective of this investigation was to develop and validate a robust reversed‐phase high‐performance liquid chromatography (RP‐HPLC) method for quantifying stigmasterol in both Moringa oleifera powder and tablets. The analysis was conducted using a Box–Behnken design. Stigmasterol separation was achieved with a Phenomenex Luna C‐18 column (4.6 × 150 mm2, 5 µm) and a mobile phase consisting of methanol and 0.1% formic acid in an 80:20% v/v ratio. The separation was performed at a flow rate of 1 mL/min, and stigmasterol was detected at a wavelength of 210 nm using a photodiode array detector. Method validation followed the guidelines set forth by the International Council for Harmonisation (ICH) Q2 (R1). This rigorous validation process evaluated critical parameters, including linearity, accuracy, system suitability, precision, and robustness. The results for each parameter were well within the acceptable range, confirming the reliability and efficacy of the established RP‐HPLC method. The findings of this study highlight the potential of the RP‐HPLC method as a dependable tool for the routine analysis of stigmasterol in herbal formulations.