Determination of three bufogenins in toad venom using reversed-phase high performance liquid chromatography

A reversed-phase high performance liquid chromatographic method for the quantitative determination of three bufogenins in toad venom was established. An Alltech Alltima C-18 column (250 mm x 4. 6 mill, 5 mu m) was used. The mobile phase was acetonitrile-0. 1 mol/L acetate buffer (pH 3.2) (50: 50, v/v), and the flow rate was 0. 8 mL/min. The detection wavelength was set at 299 nm. The method has good linearity in the ranges of 0. 25 - 0. 875 mu g for cinobufagin (r = 0. 999 0), 0. 25 - 0. 875 pLg for resibufogenin (r = 0. 999 1), and 0. 15 - 0. 525 mu g for bufalin (r = 0. 999 0). The average recoveries were 100. 3%, 100. 0% and 98. 0% for cinobufagin, resibufogenin, and bufalin, respectively. The results indicate that the method is simple, accurate, reproducible, and can be used for the quality control of bufogenins in toad venom.