Simultaneous determination of stigmasterol and β-sitosterol in <i>Justicia adhatoda</i> leaves using reverse-phase high-performance liquid chromatography (RP-HPLC)

The study developed and validated an RP-HPLC method for the simultaneous estimation of β-sitosterol and stigmasterol in <i>Justicia adhatoda</i> leaves. Separation was accomplished chromatographically using a Waters xBridges C₁₈ column, with detection at 210 nm. The separation employed an isocratic mobile phase composed of acetonitrile and water in a 70:30 volume ratio, adjusted to pH 3.5, and delivered at a flow rate of 0.5 mL per minute. Validation assessed system appropriateness, specificity, linearity, accuracy, and robustness per ICH Q2 (R1). The approach demonstrated linearity for both substances (10–80 μg/mL), with correlation values of 0.9992 for β-sitosterol and 0.9988 for stigmasterol. For β-sitosterol, LOD and LOQ were 0.057 and 0.172 μg/mL, while for stigmasterol, they were 0.063 and 0.193 μg/mL. Recovery was 99%–104%. The first RP-HPLC technique for these chemicals in Justicia adhatoda leaves is highly selective and has food and biological uses.