An LC-MS method to determine concentrations of isoflavones and their sulfate and glucuronide conjugates in urine.

Most methods for detecting isoflavones in biological samples do not measure the concentration of sulfate conjugates. An LC-MS method is reported here to estimate urinary concentrations of genistein and daidzein, their sulfate and glucuronide conjugates and other major metabolites. Human and rat urine samples were extracted with diethyl ether, or pre-digested with sulfatase and/or beta-glucuronidase followed by extraction. The isoflavones were separated using gradient LC methods and detected by negative single ion monitoring on an MS system using a heated nebulizer atmospheric pressure chemical ionization interface. CVs for inter- and intra-assay variability were generally < 20 and 10%, respectively. Preliminary studies using these procedures demonstrate 52+/-4 and 26+/-4% of genistein in rat urine was found as the aglycone and sulfate conjugates, respectively, compared to 0.36 and 4%, respectively, in human urine. This method is suitable for the study of isoflavone sulfate conjugates in biological fluids.