METHOD DEVELOPMENT AND VALIDATION FOR SIMULTANEOUS QUANTIFICATION OF OLEANOLIC ACID AND Β-SITOSTEROL FROM ACHYRANTHES ASPERA AND FROM MARKETED FORMULATION BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

Objective – To develop simple, accurate, precise and reproducible High Performance Liquid Chromatographic (HPLC) method for simultaneous quantification of Oleanolic acid and β-sitosterol in the whole plant extract of Achyranthes aspera . Methodology – The methanolic extract of the plant powder of A. aspera was separated on HPLC column (Phenomenox, Luna® 5 µm C18, LC Column 150 x 2 mm) with gradient mixture of HPLC grade Acetonitrile and Water as mobile phase. The separated components were detected using Photo Diode Array (PDA) detector at 210 nm. Results – Linear response was found in the concentration range of 10-300 μg/mL for β-sitosterol and 20-400 μg/mL for Oleanolic acid respectively. The relative standard deviation for inter-day and intra-day precision was found to be <2%. The validated method was successfully applied for quantification of the two components in a commercially available polyherbal formulation “Cystone” containing A.aspera extract. Conclusion – A precise, accurate and reproducible HPLC method is developed and validated for simultaneous quantification of two bio-active components from A.aspera . The method can be used as quality control tool in standardization of raw materials and marketed formulations.