Separation of Madecassoside and Madecassic Acid Isomers by High Performance Liquid Chromatography Using β -Cyclodextrin as Mobile Phase Additive

Abstract

An improved HPLC method for the separation of madecassoside isomers (madecassoside and asiaticoside-B) has been developed. The isomers can be separated with high resolution from extracts of Centella asiatica by HPLC using <TEX>$\beta$</TEX>-cyclodextrin as a mobile phase added on a <TEX>$C_{18}$</TEX> column. The result shows that the isomers can be separated with a mobile phase consisting of methanol:water (50:50, v/v) with 4 mmol/L <TEX>$\beta$</TEX>-CD. To elucidate the mechanism of the separation of madecassoside and asiaticoside-B, this paper studied the separation of their aglycon parts (madecassic acid and terminolic acid), another pair of isomers. The isomers can also be separated with high resolution with a mobile phase consisting of methanol:water (65:35, v/v) with 4 mmol/L <TEX>$\beta$</TEX>-CD and the pH of the mobile phase was adjusted to 4. The paper also studied the separation of the two isomers by HPLC using <TEX>$\alpha$</TEX>-CD and Glucosyl-<TEX>$\beta$</TEX>-CD as a mobile phase additive in order to elucidate the mechanism of the separation process.