Background: Adenoon indicum Dalz, a member of the Asteraceae family, is a substantial source of phytosterol chemicals, including stigmasterol. The plant traditionally used in Maharashtra, India, for ulcers, gastrointestinal irritations, and wounds, lacks a documented phytochemical profile. Objectives: The absence of standardization for Adenoon indicum Dalz leaves and extracts complicates quality monitoring. This study aims to develop a straight forward, novel, dependable, and precise HPTLC technique for quantifying stigmasterol in Adenoon indicum Dalz leaf extract. Materials and Methods: The isolation and measurement of stigmasterol were performed using HPTLC plates covered with Silica gel 60 F254 as the stationary phase. Toluene, ethyl acetate, methanol, and formic acid were combined in the mobile phase in a 3:3:2:1 volumetric ratio. The detection and quantification were performed by densitometric scanning at 265 nm. Results: An HPTLC technique was established using toluene, ethyl acetate, methanol, and formic acid in a ratio of 3:3:2:1 (v/v/v/v) as the optimal mobile phase. TLC plates at 490 nm revealed a singular phytochemical with an Rf value of 0.894±2.21. The approach demonstrated selectivity for stigmasterol in the extract of Adenoon indicum Dalz, as shown by the overlay of UV spectra. The technique was verified for linearity, precision, accuracy, Limit of Detection (LOD), and Limit of Quantification (LOQ). It exhibited linear calibration curves (400-1200 ng/band, r²>0.995), % RSD < 2%, LOD and LOQ of 889.823 and 2696.433 ng/band, respectively, and a recovery rate of 98.2-100%. The methodology demonstrated robustness, with a relative standard deviation percentage of under 5%. Conclusion: A straightforward and sensitive HPTLC approach was validated for the detection of stigmasterol in Adenoon indicum Dalz, providing advantages in terms of time and cost-effectiveness.
