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 highperformance liquid chromatography with tandem mass spectrometry. For a reliablequantitation of these new concerning compounds in such a complex matrix, recov-ery and matrix effect must be carefully evaluated. Therefore, two sequential experi-mental designs were used to optimize the sample-pretreatment of soy-based burgers:the chosen technique was the quick, easy, cheap, effective, rugged and safe method-ology, which does not require any particular facility or instrumentation. Thanks tothe first screening design (Plackett-Burman), the significant factors influencing thestudied responses were identified and further investigated through a response surfacedesign (Box-Behnken). The optimal values of the variables (volume of extraction sol-vent 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 opti-mized method was characterized by low detection limits (0.2–1.5 ng/g) and excellentintraday precision (RSD 2–4%). It was applied to the determination of the consideredcompounds 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 concentra-tions (7.9–78μg/g) of genistein and daidzein.