BBD-driven optimization of an RP-HPLC method for simultaneous analysis of two major ısoflavone aglycones in Tofu

Tofu, one of soy food products widely consumed in Indonesia, has been reported as a source of natural protein. It is important to evaluate the contents of genistein and daidzein to provide information according to the nutrient information on soy food products. A reversed-phase high performance liquid chromatography (RP-HPLC) was developed in this study in order to analyze genistein and daidzein content in tofu samples simultaneously. Employing a Box-Behnken design (BBD) for designing the experiment, the response surface methodology (RSM) was then applied to optimize chromatographic conditions including methanol composition, flowrate, and column temperature. Separation response such as retention time, resolution, and tailing factor were evaluated to build an optimization model followed by analyzing the desirability. It was found that the model predicted a composite desirability of 0.9778 can be obtained by applying the methanol composition of 60%, flowrate of 0.80 mL.min-1, and column temperature of 50ºC. The optimized HPLC conditions met the acceptance criteria for retention time and area deviations, resolution, tailing factor, and theoretical plates number.