A HPLC Method for Determination of Ursolic Acid and Betulinic Acids from their Methanolic Extracts of Vitex Negundo Linn

Ursolic and betulinic acids are very useful nutraceuticals available in herbs. Their quantitative estimation from the solvent extracts of the herbs is a great challenge. So far all the chromatographic methods available for identification and quantification of these acids are distinctly different, meaning thereby, each acid would require a separate method. Reverse phase (RP-HPLC) method is developed for determination of ursolic acid and betulinic acid from their methanol extract of Vitex negundo Linn leaves. Analysis was carried out using Waters’ symmetry C-18 column with acetonitrile: methanol (80:20) as isocratic elution mode with UV detection (λ=210 nm). The method is pretty linear for ursolic acid in the range of 0.01-0.1 mg/ml (R2 = 0.9961) and for betulinic acid in the range of 0.003-0.018 mg/ml (R2 = 0.999). The peaks of ursolic acid and betulinic acid were confirmed by LC-MS. The method was validated by mixing these acids standards in methanol and found that it is accurate, sensitive and has a good reproducibility.