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Formononetin is an isoflavonoid phytoestrogen found in animal fodder and in certain human foodstuffs. Since high consumption of formononetin has been implicated in infertility among ruminants and may relate to human breast cancer, we developed a radioimmunoassay method and applied it to quantitate formononetin in murine plasma and mammary glandular tissue for animal model studies.The radioimmunoassay utilized an antiserum raised in rabbits following immunization with formononetin-7-O-(carboxymethyl)ether coupled to bovine serum albumin. The tracer was an [3H]leucine derivative of formononetin synthesized by mixed anhydride reaction between formononetin-7-O-(carboxymethyl)ether and [3H]leucine. The bound and free forms of formononetin were separated by adding dextran-coated charcoal. This radioimmunoassay procedure enabled the quantification of 4 ng/ml of plasma or 50 pg/mg of mammary tissue, and the antiserum showed no marked cross-reaction with the reactants tested. The reliability and reproducibility of the assay were demonstrated by intra- and inter-assay variation that was 6.5% and 11.9%, respectively. This radioimmunoassay was compared with a high-performance liquid chromatographic method by determining concentrations of formononetin in ethanol extracts of red clover. Good correlation existed between the radioimmunoassay and high-performance liquid chromatographic method (r = 0.980), but the radioimmunoassay values were on the average 5% higher than high-performance liquid chromatographic values. Furthermore, we used the radioimmunoassay to assess the pharmacokinetics of formononetin in castrated female BALB/c mice after a single subcutaneous injection at 40 mg/kg dose. The sensitivity of the radioimmunoassay permitted the detection of a prolonged elimination phase for formononetin in both plasma and mammary glandular tissues, with mean elimination half-lives of 2 and 2.5 hr, respectively.A specific, sensitive, and fast radioimmunoassay procedure has been developed for the determination of formononetin in murine plasma and mammary glandular tissue. We conclude that the presented radioimmunoassay is a valid alternative method to the quantification of formononetin in biological fluids and/or plants.