Abstract: Objective:High performance liquid chromatography(HPLC)was used for separation and determination of four isoflavone compounds(daidzein,genistein,formononetin,and biochanin A)in Trifolium pretense L.Methods:HPLC quantitated the quanitity of the four main isoflavones in Trifolium pretense L.The qualification of HPLC:C18 column(250 mm ×4.6 mm):the mobile phase MeOH-CH3CN-H2O(3.7:1.3:5,V:V:V),the flow rate 1.0 ml/min,the filling amount 20μl,the column temperature room temperature,UV-detection performed at 260 nm.Methods about treating with the sample:the sample was prepared by dissolving in 40 ml solution of the HCl methanol(42:500,V:V)at 85 ℃ hot-water for 2 hours.After the sample was cool,it was added methanol to reach the volume 50 ml in all.After the 0.45μm membrane was used to filtrate the sample and the filtrate was analyzed by HPLC.Results:According HPLC analysis,in Trifolium pretense L.(1#),the quantity of the four main isoflavones:formononetin(4.79%),and biochanin A(1.32%),daidzein(0.43%),genistein(0.35%);in Trifolium pretense L.(2#),the quantity of the four main isoflavones:daidzein(0.60%),genistein(0.64%),formononetin(9.29%),and biochanin A(2.63%).

Key words: Trifoloum pretense L., isoflavones, HPLC