DEVELOPMENT AND VALIDATION OF RP-HPLC METHOD FOR QUANTIFICATION OF BETULINIC ACID IN VITEX NEGUNDO L. AND ITS POLYHERBAL FORMULATIONS

The Vitex negundo, which is traditional Indian medicine, has been used for treatment of various diseases. A simple, sensitive, precise and accurate RP-HPLC method has been developed for the analysis of Vitex negundo L. samples and its polyherbal formulations for their Betulinic acid content. The extraction was checked using different solvents like methanol, chloroform, 95% ethanol, acetone and dichloromethane. It was found chloroform was good extraction solvent that allowed extraction of Betulinic acid with highest content. The method involves RP C18 column with methanol-acetonitrile-water (90:5:5 % v/v/v) pH 2.5 adjusted with ortho phosphoric acid as mobile phase and UV detection at 270 nm. The flow rate was 1.3 ml/min. The retention time was 2.29 min. The linearity range was 5-30 mu g/ml. The method was validated for linearity range, precision, LOD, LOQ, robustness and accuracy. Mean recovery was 99.16-100.76 %. The method was successfully applied for the quantification of Betulinic acid in leaves, seeds of Vitex negundo L. and its polyherbal formulations. The results demonstrated that the content of Betulinic acid was depending on different extraction solvents and ensure its clinical benefits. The content of total triterpenoids was also determined by colorimetry method.