A Validated Rp-hplc Method for Simultaneous Determination of Betulin, Lupeol and Stigmasterol in Asteracantha Longifolia Nees

Objective: The aim of the present study was to develop a reversed phase high-performance liquid chromatography (RP-HPLC) method for simultaneous estimation of betulin, lupeol and stigmasterol in Asteracantha longifolia extract. Methods: The chromatographic separation was achieved using Luna C18 column (5 μm, 250 x 4.6 mm) under isocratic elution of acetonitrile and 0.1% acetic acid in water (94: 6, v/v) with a flow rate of 1.0 mL min-1 and the detection wavelength was set at 215 nm. The column temperature was maintained at 25°C and the run time was set at 30 min. The method was validated for linearity, specificity, system suitability, accuracy, limit of detection, limit of quantification, precision and ruggedness. Results: Calibration curves showed a good linearity relationship in the concentration range of 5 200 μg mL-1 for betulin (r2 = 0.9994) and 10 800 μg mL-1 for lupeol (r2 = 0.9995) and 10 800 μg mL-1 for stigmasterol (r2 = 0.9986). Limits of detection for betulin, lupeol and stigmasterol were found to be 0.33, 1.0 and 3.0 μg mL-1 and limits of quantification were 2.0, 5.0 and 8.0 μg mL-1, respectively. Mean recovery values were in the range of 98.92 100.11%. The %RSD values of intraand inter-day precision analysis were lower than 2%. System suitability parameters were also found to be satisfactory. Conclusion: The developed method allows rapid and reliable simultaneous determination of betulin, lupeol and stigmasterol in A. longifolia extract.