Validation of a high-performance liquid chromatographic method for determination of isoflavonoids in soybeans. Study of the extraction procedure by experimental design

An improved analytical method, reversed-phase high-performance liquid chrom atography on a narrow-bore C-18 column, has been developed for the simultan eous determination of genistein, daidzein, formononetin, and biochanin A. T he method was validated in terms of detection limits, quantitation limits ( LOG), linearity, and precision. LOO in the 0.04 - 0.1 mug mL(-1) range were calculated, enabling determination of these compounds of nutritional conce rn at trace levels. Good linearity was demonstrated over three orders of ma gnitude of concentration for each analyte (r(2) = 0.998 - 1.000). The intra -day repeatability was evaluated in terms of RSD (%) at two concentration l evels for each analyte (RSD % < 1.8%). Good inter-day reproducibility of da ta was proved by performing homoscedasticity and ANOVA tests (P > 0.05 at t he 95% confidence level). The method was applied to the determination of ge nistein and daidzein in yellow soybeans, after optimization of the method f or extraction of isoflavonoid aglycones from soybeans by experimental desig n, i.e. central composite design. Extraction recoveries up to 87 +/- 4% wer e obtained when the corresponding glycosidic forms (genistin and daidzin) w ere added to soybean samples.