Improving Resolution of Isomeric Flavonoids and Their Glycosides Using Two-Dimensional Liquid Chromatography Coupled With High-Resolution Mass Spectrometry

The separation of isomers is one of the key issues in analysing complex natural matrices, thus commanding a higher separating power from the applied analytical system. In this study, 42 common flavonoid glycosides from three key subclasses (flavanones, flavones, flavonols) were analysed in three steps to maximise chromatographic and spectrometric resolution via: (1) C18 liquid chromatography (LC) coupled to a quadrupole time-of-flight mass spectrometer (QTOF/MS), (2) tandem mass spectrometry (MS/MS), and (3) two-dimensional LC with heart-cutting for specific regions of co-elution. While the LC-QTOF/MS step was found to separate the majority of flavonoid glycoside isomers, some isomers with identical accurate mass were not resolved. Following this, increased spectrometric resolution was achieved with MS/MS; however, this technique was not suitable for particularly challenging isomers that were also identical in aglycone and saccharide structure. Furthermore, a heart-cutting method was developed using a three-level full-factorial design to separate such challenging isomers, and satisfactory resolution was obtained for the targeted isomers ( R > 2.00) with reasonable efficiency (total run time < 25 min) and repeatability (RSD < 2%). In conclusion, the developed strategy highlights the significance of combining high-resolution mass spectrometry with chromatographic separation for added confidence in the identification of isomers.