目的研究建立双波长同时检测人尿中大豆异黄酮代谢产物雌马酚等6种化学成分含量的高效液相色谱分析方法。方法尿样品用β-葡萄糖苷酶水解,乙酸乙酯萃取;采用乙酸乙酯-甲醇-0.05%磷酸盐缓冲液作为流动相,梯度洗脱;程序波长紫外检测器检测,检测波长为254nm、280nm。结果方法的线性范围为0.05～10μg/ml;检出限为0.03～0.05μg/ml,定量限为0.1～0.17μg/ml;6种成分的平均回收率为71.4%～102.1%;相对标准偏差为1.0%～7.9%。应用新建立的方法测定了10份人尿样品中6种活性化学成分的含量。结论该方法可靠,适用于人尿中大豆异黄酮代谢产物的分析检测。 OBJECTIVE To establish a dual-wavelength high performance liquid chromatographic method for the simultaneous determination of six chemical constituents, such as equol, of the metabolite of soybean isoflavone in human urine. Methods Urine samples were hydrolyzed with β-glucosidase and extracted with ethyl acetate. The mobile phase consisted of gradient elution with ethyl acetate-methanol-0.05% phosphate buffer. The detection wavelength was 254 nm at 280 nm . Results The linear range of the method was 0.05 ~ 10μg / ml. The detection limit was 0.03 ~ 0.05μg / ml, the limit of quantification was 0.1 ~ 0.17μg / ml. The average recoveries of the six components were 71.4% ~ 102.1% 1.0% ~ 7.9%. The newly established method was used to determine the contents of six active chemical constituents in 10 human urine samples. Conclusion The method is reliable and suitable for the analysis and detection of soybean isoflavone metabolites in human urine.