A direct analysis in real-time (DART) ion source coupled to a high-resolution orbitrap mass spectrometer was used for the quantitative analysis of isoflavones isolated from soybeans. For the isolation of genistein, daidzein, glycitein, and their respective acetyl, malonyl, and glucoside forms, an extraction employing 80% aqueous MeOH enhanced by sonication was used. As far as the total isoflavones (expressed as aglycones) were to be determined, an acid hydrolysis with 80% aqueous EtOH and refluxing had to be employed, while in the latter case a good agreement of the results with the data generated by the UHPLC-orbitrap MS method was achieved, in the case of the analysis of non-hydrolyzed extracts, some overestimation of the results as compared with those generated by UHPLC-orbitrap MS was observed. A careful investigation of this phenomenon showed that the free aglycones originated from the conjugated forms of isoflavones in the DART ion source, thus contributing significantly to the “free” genistein/daidzein/glycitein signals during the DART analysis. Good recoveries (95–102%) and repeatabilities (RSD: 7–15%) were obtained at the spiking levels of 0.5, 1, and 0.05 g/kg, for daidzein, genistein, and glycitein, respectively. The limits of detection estimated for the respective analytes were 5 mg/kg.
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