Stereoisomeric Separation of Flavonoids by Two-Dimensional Supercritical Fluid Chromatography: Identification of Adequate Chiral Columns and Application to Honey Analysis

Abstract

Chiral flavonoids are frequently encountered in natural products consumed as food or therapeutic products. However, the taste and other bioactivities of stereoisomers are known to differ. Hence, stereoseparation methods are necessary to resolve these isomers, whether to measure stereoisomeric ratios or to purify individual stereoisomers for activity testing. In this study, we looked for the best conditions to resolve different families of chiral flavonoids, including auronols, flavanonols, aglycon, and glycosylated flavanones. Furthermore, we aimed to include this stereoseparation as the second-dimension method in a two-dimensional supercritical fluid chromatography system (SFC-SFC), to achieve the resolution of chiral flavonoids in complex samples. First, a set of 10 standards of chiral flavonoids was examined on 5 immobilized polysaccharide chiral stationary phases. With the help of Derringer desirability functions, the columns offering the best options for rapid resolution were identified. Then, a sample of honey was analyzed in an SFC-SFC system in heart-cutting mode. The first-dimension achiral separation resolved the complex mixture of honey and isolated the fraction of pinocembrin enantiomers, which were re-analyzed in the second-dimension chiral separation to measure the enantiomeric excess, and resolve co-eluting isobaric species. This experiment demonstrates the relevance of this strategy in the resolution of flavonoid enantiomers from complex samples.