Development of the quantitative determination of asiaticoside, madecassoside, asiatic acid and madecassic acid in centella asiatica (Linn.) urban by high-performance liquid chromatography

Abstract

A H PLC m ethod was d eveloped t o d etemiine madecassoside (MS), a siaticoside (AS), madecassic acid (MA) and asiatic acid (AA) simultaneously. The system comprises of a Hi-Q sil column (C₁₈, 4.6x150 mm, 5micron) as stationary phase, a 29:71 mixture of acetonitnle : phosphate buffer (l0mM, pH 7.1) as mobile phase (flow rate 1.0 ml/min), prednisolone (PL) as an internal standard with a photodiode array detector at wavelength 210 nm. Each analyte sample from Centella asiatica (CA) extract was cleaned up by using the solid phase extraction techniques prior to inject to the system. Furthermore, thin-layer chromatographic (TLC) method was developed to determine MS and AS by using silica gel plate GF₂₅₄ as stationary phase and chloroform : methanol : water ( 30:15:2) as developing solvent. The detection of the TLC spot was developed by spraying with anthrone reagent and scanned with densitometer at wavelength 525 nm. These methods were validated following ICH guideline. The percentage recovery were in range 96-104 and percent of RSD were not more than 3. The developed method was applied to analyze the 22 plant samples collected every month throughout the year from two gardens, five extract powder and two plant extract during the accelerated stability program.