In this study,the high performance liquid chromatography (HPLC) was used to establish fingerprint and multi-component determination method of Bufonis Venenum.The analysis was performed on Agilent EC-C₁₈ column (150 mm×3 mm，2.7 μm) with 0.1% acetic acid water (A) - acetonitrile (B) as mobile phase in a gradient elution mode.The flow rate was 0.5 mL/min,the column temperature was 30 ℃,the detection wavelength was set at 296 nm and the injection volume was 5 μL.The similarity of 40 batches of Bufonis Venenum ranged from 0.779 to 0.991,indicating that there was difference between batches of Bufonis Venenum.There were 21 common peaks in the HPLC chromatogram of 40 batches of Bufonis Venenum, among which the percentages of bufarenogin,gamabufotalin,arenobufagin,bufotalidin, telocinobufagin,bufotalin, resibufagin,marinobufagin,bufalin,resibufogenin and cinobufagin were as follows:0.01%-0.163%, 0.528%-1.608%, 0.943%-5.413%,0.204%-0.815%,0.474%-1.825%,1.115%-2.019%,0.030%-0.249%,0.148%-1.661%,1.206%-2.752%,0.345%-3.287%,1.426%-5.875%.According to the cluster analysis,the 40 batches of Bufonis Venenum were classified into three categories;principal component analysis screened out four principal components,with the cumulative variance contribution rate of 91.122%,indicating that the principal components contained most information of original data.Partial least squares discriminant analysis marked 12 differential components in the medicinal materials.This method can provide reference for the quality control and evaluation of Bufonis Venenum.

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