Simultaneous Quantification of Lupeol, Stigmasterol and β- Sitosterol in Extracts of Adhatoda vasica Nees Leaves and its Marketed Formulations by a Validated RP-HPLC Method

Background: Adhatoda vasica Nees (Acanthaceae) is a well-known medicinal plant used for the treatment of respiratory disorders such as asthma and bronchitis. Objective: To develop a simple and precise RP-HPLC method for the simultaneous assessment of lupeol, stigmasterol and β-sitosterol of various extracts of Adhatoda vasica Nees. Materials and Methods: The compounds were separated on RP-Phenomenex C₁₈ (250mm×4.6mm; 5μ) column with a mobile phase comprising of 0.1%v/v formic acid in water and methanol (28:82%v/v) splashed at a flow of 0.8mL/min with PDA detector at 208nm. Results: The retention time of lupeol, stigmasterol and β-sitosterol was found to be 16.89, 18.26 and 20.72 minutes respectively. The amount of lupeol was abundant in hexane extract (0.952%w/w) and formulation III (23.72ng/g) whereas, stigmasterol (0.285%w/w) and β-sitosterol (8.649%w/w) was highly abundant in chloroform extract and formulation I stigmasterol (2.57ng/g) and β-sitosterol (0.98ng/g). The optimized method was validated for different parameters and all the validated constraints were within the limits as per ICH guidelines. The proposed method was linear over the concentration range of 12.5-200μg/mL with correlation coefficients greater than 0.997. The LOD and LOQ values of lupeol, stigmasterol and β-sitosterol were found to be 0.66, 5.64 and 12.8μg/mL and 2.01, 17.10 and 36.62μg/mL respectively. Conclusion: To conclude, the developed method for the simultaneous estimation of lupeol, stigmasterol and β-sitosterol was simple, precise, accurate and thus reliable for the quality control investigations of crude drugs and its herbal formulations.