Separation and determination of closely related triterpenic acids by high performance thin-layer chromatography after iodine derivatization.

A method based on high performance thin-layer chromatography combined with densitometry for the simultaneous determination of oleanolic and ursolic acids is described. Because of the similarity of chemical structure the prechromatographic derivatization was necessary to separate these triterpenic acids. The samples were treated by 1% iodine solution in chloroform directly on the chromatographic plate and developed with the mobile phase consisting of A (petroleum ether), B (ethyl acetate) and C (acetone) (8.2:1.8:0.1, v/v/v). After drying, the plates were sprayed with 10% (v/v) ethanol solution of sulfuric acid(VI) and heated to 120 degrees C for 3 min. Quantification was performed in absorbance/transmittance mode at a wavelength of 530 nm by using a computer-controlled densitometer Desaga CD 60. The presented method was validated for linearity, precision and accuracy. Correlation coefficient (r(2)>0.99), R.S.D. values (1.4-3.5%), detection limits as well as recovery values (98.4-103.1%) were found to be satisfactory. The method has been successfully applied in the analysis of both triterpenic acids in plant extract.