Objective: To establish an HPLC method for the simultaneous determination of eleven bufogenins, including pseudobufarenogin, gamabufotalin, arenobufagin, hellebrigenin, telocinobufagin, bufotaline, resibufagin, marinobufagenin, bufalin, resibufogenin and cinobufagin in Bufonis Venenum. Methods: The Agilent InfinityLab Poroshell 120 EC-C18 column (150 mm×3.0 mm, 2.7 μm) was used, and the mobile phase was water solution containing 0.3% acetic acid and acetonitrile with gradient elution, at the flow rate of 0.5 mL·min-1. The column temperature was at 30 ℃ and the detection wavelength was set at 296 nm. Results: The linear ranges of eleven bufogenins were 2.024-20.24 μg·mL-1 (r=0.999 8), 18.44-184.4 μg·mL-1 (r=0.999 9), 12.40-124.0 μg·mL-1 (r=0.999 5), 3.720-37.20 μg·mL-1 (r=0.999 5), 10.28-102.8 μg·mL-1 (r=0.999 8), 22.16-221.6 μg·mL-1 (r=0.999 7), 3.280-32.80 μg·mL-1 (r=0.999 4), 5.440-54.40 μg·mL-1 (r=0.999 7), 28.84-288.4 μg·mL-1 (r=0.999 8), 32.76-327.6 μg·mL-1 (r=0.999 7), 71.28-712.8 μg·mL-1 (r=0.999 6), respectively. Results of precision (RSD≤1.3%), repeatability (RSD≤4.4%) and stability (RSD≤3.3%) were good. The average recoveries were 95.6%-102.4% with RSDs of 0.98%-4.4%. The contents of pseudobufarenogin, gamabufotalin, arenobufagin, hellebrigenin, telocinobufagin, bufotaline, resibufagin, marinobufagenin, bufalin, resibufogenin and cinobufagin in 18 batches of samples were 0.285 0-1.018, 11.89-14.12, 9.246-10.98, 2.260-2.739, 6.813-8.093, 15.33-18.20, 0.582 0-1.178, 1.492-2.403, 17.79-26.17, 18.75-29.94 and 43.12-60.81 mg·g-1, respectively. The results of cluster analysis showed that 18 batches of samples could be divided into two categories. Conclusion: This method has the advantages of simple operation, good repeatability and high sensitivity, which can be used for quality control and quality evaluation of Bufonis Venenum.
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