According to the on-line DAD spectra of the analytes from 190400 nm, the detection wavelength was chosen at 210 nm, which provided an optimum S/N for simultaneous quantitative analysis of these three compounds. Studies showed that 0.1% formic acid added into the mobile phase (water) remarkably improved the symmetries of chromatographic peaks, and 0.06% formic acid added into the mobile phase (methanol) conduced to avoiding baseline drift at 210 nm in gradient elution. Besides, in order to prevent IS from being interfered by other peaks existing in rhubarb decoction, the temperature of column was optimized systematically from 25 to 40 °C. Eventually, the column temperature of 30 °C was preferred to separate them. The chromatographic separation of these analytes is shown in Fig. 2. Chromatographic peaks in the samples were identified by comparing their retention time and UV spectra with those of the reference standards.