Development and Validation of an RP‐HPLC Method for Simultaneous Quantification of β‐Sitosterol and Stigmasterol in <i>Limonia acidissima</i> Leaves

Abstract Limonia acidissima (LA) is a medicinal plant recognized for its therapeutic potential, particularly due to phytosterols like β‐sitosterol and stigmasterol. This study aimed to develop and validate a reverse phase‐high performance liquid chromatography (RP‐HPLC) method for simultaneously quantifying these compounds in LA leaves. Chromatographic separation was achieved using a Waters xBridges C18 column with detection at 210 nm. The mobile phase consisted of acetonitrile and water (80:20 v/v), delivered at a flow rate of 0.8 mL/min, with the pH adjusted to 3.8 using 0.04% glacial acetic acid. Validation was conducted following ICH Q2 (R1) guidelines, assessing system suitability, specificity, linearity, accuracy, and robustness. Linear responses for β‐sitosterol and stigmasterol were observed in the range of 10–80 µg/mL, with correlation coefficients of 0.9993 and 0.9988, respectively. The limits of detection were 0.012 µg/mL for β‐sitosterol and 0.088 µg/mL for stigmasterol, while limits of quantification were 0.038 and 0.269 µg/mL, respectively. Recovery rates ranged from 99% to 101%, confirming the method's accuracy and precision. This is the first RP‐HPLC method developed for the simultaneous quantification of these phytosterols in LA leaves, showcasing excellent specificity, accuracy, and robustness for food and biological sample analysis.