Determination of Soy Isoflavones in Foods and Dietary Supplements by UPLC

Isoflavones are a class of plant-derived compounds, produced almost exclusively by members of the Fabaceae family, that have been shown to have estrogenic activity in mammals. The major source of isoflavones in the human diet comes from soybeans, in which genistein and daidzein are the predominant components. Isoflavones remain the subject of many scientific studies with accepted methods established by organizations such as AOAC1 and USP2 , utilizing traditional reversed-phase C18 columns. Recently, the National Institute of Standards and Technology (NIST) has developed a suite of soy-based candidate Standard Reference Materials (cSRMs) for the certification of soy isoflavones in foods and dietary supplements. For the analysis of those standards, they have developed a method utilizing a 60-minute gradient on a 5 µm cyano column which enables the separation of the soy isoflavones from other components not resolved using the standard C18 methods.3 Using these conditions, they have demonstrated resolution of the three main soy isoflavones and their glycosides (structures shown in Figure 1), as well as the acetyl and malonyl conjugates of the glycosides. Since the conjugates are somewhat unstable in solution, they have added an additional sample preparation step to hydrolyze these conjugates to their associated glycoside, providing a more accurate, reproducible determination of total isoflavone content. Optimization of this method utilizing the XSelect HSS Cyano XP 2.5 µm Column provides a significant reduction in analysis time by traditional HPLC. Further optimization of this method to UPLC® with the ACQUITY UPLC HSS Cyano 1.8 µm Column provides additional savings in time, while maintaining the resolution for accurate determination of soy isoflavones.