A simple and rapid gradient RP-HPLC method for simultaneous separation and determination of related ginsenosides during the process of biotransformation of ginsenoside Rb1 has been developed. As many as four process ginsenosides have been separated and identified on an Eclipse XDB C18 column (4.6 mm×150 mm, 5 μm) with gradient elution using water and ACN as a mobile phase. The column was maintained at 30°C and the eluents were monitored with diode array detection at 203 nm. The method was validated in terms of linearity, sensitivity, precision, and accuracy. The correlation coefficients (r) for calibration curves of ginsenosides were in the range of 0.9996–1.0000. The proposed RP-HPLC method was successfully applied to the analysis of fermentation broth and the recoveries of ginsenosides were in the range of 94.4–103.1% with RSD <2.87%. The method could be of use for rapid and routine evaluation of the quantity of ginsenosides during the biotransformation process of ginsenoside Rb1.
