Simultaneous Determination of Indigo, Indirubin and Glycyrrhizic Acid in Wufu Huadu Pill by HPLC

OBJECTIVE To develop an HPLC method for simultaneous determination of indigo, indirubin and glycyrrhizic acid in Wufu Huadu pill. METHODS An Eclipse XDB-C₁₈ column(250 mm×4.6 mm, 5 μm) was adopted. The mobile phase was methanol-0.2 mol·L^-1 ammonium acetate-glacial acetic acid(65∶35∶1) at the flow rate of 1.0 mL·min^-1. The UV-detection wavelength was at 250, 288 nm with the column temperature of 25 ℃. RESULTS Calibration curves were found to be linear in the ranges of 2.501-25.01 μg·mL^-1 for indigo, 0.633-6.330 μg·mL^-1 for indirubin and 5.10-51.02 μg·mL^-1 for glycyrrhizic acid. The correlation coefficients were 1.000 0, 0.999 5, 1.000 0. The average recoveries(n=9) of indigo, indirubin and glycyrrhizic acid were 98.3%, 98.4% and 98.2%. CONCLUSION The method is simple, accurate and reproducible. It could be reliable for the quality control of Wufu Huadu Pill.