[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 C18 column (250 mm x 4.6 mm, 5 microm) 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 microg for cinobufagin (r = 0.999 0), 0.25 -0.875 microg for resibufogenin (r = 0.999 1), and 0.15 - 0.525 microg 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.