The experimental design approach to the optimization of a simple quick easy cheap effective rugged and safe method for the analysis of phytoestrogens in complex soy-based food

The present paper deals with the multivariate optimization of an extraction-purification strategy for the determination of phytoestrogens (daidzein, genistein, coumestrol, formononetin, and biochanin A) in soy-based meat substitutes by high performance liquid chromatography with tandem mass spectrometry. For a reliable quantitation of these new concerning compounds in such a complex matrix, recovery and matrix effect must be carefully evaluated. Therefore, two sequential experimental designs were used to optimize the sample-pretreatment of soy-based burgers: the chosen technique was the quick, easy, cheap, effective, rugged and safe methodology, which does not require any particular facility or instrumentation. Thanks to the first screening design (Plackett-Burman), the significant factors influencing the studied responses were identified and further investigated through a response surface design (Box-Behnken). The optimal values of the variables (volume of extraction solvent mix/sample mass ratio and two clean-up sorbents) led to quantitative recoveries (97-104%) and low ion suppression (matrix effect 60-93%) for all analytes. This optimized method was characterized by low detection limits (0.2-1.5 ng/g) and excellent intraday precision (RSD 2-4%). It was applied to the determination of the considered compounds in several soy-burgers from the Italian market, detecting low ng/g levels (up to 40 ng/g) of coumestrol, formononetin, and biochanin A, and high concentrations (7.9-78 mu g/g) of genistein and daidzein.