Simultaneous analysis of five triterpenes in Centella asiatica by high performance liquid chromatography with...

Triterpenes are considered the major active components in Centella asiatica (L.) Urb. (C. asiatica), such as asiatic acid, madecassic acid, asiaticoside, madecassoside and asiaticoside B. It is difficult to simultaneously determine five triterpenes because of madecassoside isomers (madecassoside and asiaticoside B), and the great polarity difference between triterpene acid and triterpene glycoside. In this study, a simple high performance liquid chromatography method with isocratic elution employing cyclodextrins (CDs) as the mobile phase additives was developed to determine five triterpenes in C. asiatica. Various factors affecting triterpenes retention in the C18 column, such as the nature of CDs, γ-CD concentration, acetonitrile percentage and temperature, were studied. Experimental results showed that γ-CD, as an effective mobile phase additive, could markedly reduce the retention of triterpenes (especially asiatic acid and madecassic acid), and improve the separation for madecassoside and asiaticoside B. The elution of five triterpenes could be achieved on an ODS C18 column within 30 min using the acetonitrile-0.2% phosphoric acid contained 4.0 mM γ-CD (20:80, v/v) mixture as the mobile phase. The retention modification of triterpenes may be attributed to the formation of the triterpenes-γ-CD inclusion complexes. The optimized method was successfully applied for simultaneous determination of five triterpenes in C. asiatica.