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.