Simultaneous separation and determination of four bioactive constituents in traditional Chinese medicinal tablet Xinkeshu by HPLC-DAD

A reversed-phase high performance liquid chromatographic method for simultaneous determination of four bioactive constituents was established for the quality control of traditional Chinese medicinal tablet Xinkeshu. Danshensu, protocatechualdehyde, puerarin and daidzein were successfully separated on a Diamonsil C-18 column (150 x 4.6 mm i.d., 5 mum) with a guard column (12.5 x 2.1 mm i.d., 5 mum) packed with the same material at 25 degreesC. The mobile phase was a mixture of acetonitrile and 0.02 mol/L potassium dihydrogen phosphate employing gradient elution at a flow rate of 0.8 mL/min. Detection was accomplished with a diode-array detector and detection wavelength was set at 280 nm before 22 min, then it was varied to 250 nm. The four constituents were identified by comparing their retention time and UV spectra in the range of 190-400 nm with those of authentic standards. The linear calibration ranges were 15.4-123.2, 3.11-25.22, 21.6-172.8, and 0.26-3.47 mug/mL for danshensu, protocatechualdehyde, puerarin and daidzein, respectively. The detection limits were 0.03, 0.04, 0.10, 0.03 mug/mL for danshensu, protocatechualdehyde, puerarin, and daidzein, respectively. The contents of these four bioactive constituents in Xinkeshu tablet were successfully determined by the proposed method.