Identification of Glycosylated and Prenylated (Iso)flavonoids Using cIMS-MS

Separation and identification of (iso)flavonoids, including their isomers, is of great interest due to their differential bioactivity. Yet, identification of substituted (iso)flavonoids and differentiation of their isomers in complex plant extracts by LC-MSⁿ is complicated due to the large diversity of these compounds, along with the subtle differences in their structures that result in highly similar retention times and fragmentation patterns. In this study, a cyclic ion mobility-MS (cIMS-MS) method was developed to strengthen the identification of prenylated and glycosylated (iso)flavonoids using collisional cross section (^cIMSCCS_N2) values. Among the ions investigated, the lithium adducts were most stable and offered high-resolution separation. We created a library containing the ^cIMSCCS_N2 values of lithium adducts of 51 (iso)flavonoids. For 47 of the 50 isomer pairs, the ^cIMSCCS_N2 values of the two isomers within the pair were statistically significantly different, confirming that the ^cIMSCCS_N2 values in the library can be used in combination with retention times, m/z values, and fragmentation patterns to achieve identification of these compounds. Furthermore, pre-cIMS trap fragmentation of (iso)flavonoid glycosides can be used to determine ^cIMSCCS_N2 values of the corresponding aglycones for their identification. In addition, a modeling approach was explored, revealing the potential of integrating molecular dynamics simulations for CCS prediction and identification of unknowns. The combined LC-MSⁿ and cIMS-MS approach was able to strengthen identification of several prenylated and glycosylated (iso)flavonoids in a proof of concept analysis of a licorice root extract, thereby demonstrating the power of this approach for the identification of substituted (iso)flavonoids in complex extracts.