Simultaneous determination of asiaticoside and its novel metabolites in culture of Aspergilus niger by SPE-HPLC

When cultured with Aspergillus niger, asiaticoside, purified from Centella Asiatica (L.) Urban, was metabolized into four metabolites including one novel alkaloid. A simple and rapid SPE-HPLC method for simultaneous determination of asiaticoside and its four metabolites in culture of A. niger was developed. The chromatographic separation was achieved on a Dikma Diamonsi C₁₈ ODS column (200×4.6 mm i.d.) by gradient elution with 0.1% phosphoric acid in water and 0.1% phosphoric acid in methanol as the gradient mixtures. The flow rate was 1 mL/min, the detection wavelength was 204 nm and the column temperature was kept at 28°C. The retention times of asiaticoside, asiatic acid, M₁, M₂, and M₃ were 11.3, 9.4 and 17.1, 19.3 and 7.9 min, respectively. The mean recoveries of five analysts were all over 99% Quantification limits were 0.02 μg/mL for all tested compounds. The method was sucessively applied to the quantification of the biotransformation of asiaticoside.