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 chromatography on a narrow-bore C-18 column, has been developed for the simultaneous determination of genistein, daidzein, formononetin, and biochanin A. The 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 concern at trace levels. Good linearity was demonstrated over three orders of magnitude of concentration for each analyte (r(2) = 0.998 - 1.000). The intra-day repeatability was evaluated in terms of RSD (%) at two concentration levels for each analyte (RSD % < 1.8%). Good inter-day reproducibility of data was proved by performing homoscedasticity and ANOVA tests (P > 0.05 at the 95% confidence level). The method was applied to the determination of genistein and daidzein in yellow soybeans, after optimization of the method for extraction of isoflavonoid aglycones from soybeans by experimental design, i.e. central composite design. Extraction recoveries up to 87 +/- 4% were obtained when the corresponding glycosidic forms (genistin and daidzin) were added to soybean samples.