Soybean-derived isoflavones belong to the family of biologically active phytoestrogens. The purpose of this study was to develop a sensitive method, which permits quantification of the soybean isoflavonoids and equol in bovine rumen fluid and milk using LC-MS-(TOF). The samples of rumen fluid and milk were obtained from 12 lactating dairy cows ingesting 7,500-9,500 mg of total isoflavones daily. The validation of the developed method showed the limits of quantification to be in the range of 0.9-5.0 ng/mL. The precision was determined as relative standard deviation, which was lower than 25% in all cases. The recoveries of the most isoflavonoids were satisfactory. Lower recoveries of daidzin and glycitin can be solved by adding an internal standard. The presented method will be useful for kinetic studies of isoflavone metabolism in ruminants due to simultaneous quantification of free aglycones and glycosides in the rumen fluid.
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