Determination of the diastereomeric excesses of chiral flavonoids in organs of Citrus aurantium with two-dimensional supercritical fluid chromatography

Citrus aurantium (bitter orange), is widely used in culinary preparations, food supplements, cosmetics and traditional medicine. Its bioactivity is mainly associated to two major glycosylated flavanones, naringin and neohesperidin, each comprising two diastereoisomers having different bioactivities. To possibly relate bioactivity to stereoisomeric composition, an analytical method was necessary. A two-dimensional supercritical fluid chromatography (SFC) method was used to measure diastereomeric excesses of the flavonoids in bitter orange organs, including flowers, leaves and fruits at different stages of maturity. The extracts were prepared with supercritical fluid extraction (SFE) with CO 2 , ethanol and water. SFE conditions were defined following a 3-factor Box-Behnken design of experiments. While neohesperidin was highly stable, with circa 100 % stereomeric excess in all samples, naringin was prone to racemization, especially in mature fruits. This study also examined the importance of using a sufficient mass of sample to obtain accurate and meaningful measurements, particularly when stereoisomers are concerned. • CO 2 -ethanol-water provides efficient extraction of glycosylated flavonoids. • Two-dimensional supercritical fluid chromatography allows quick measurement of stereoisomeric excesses. • Sample mass should be larger when measuring stereoisomeric composition. • 2S-neohesperidin is highly stable while 2S-naringin racemizes when fruit matures. • Drying conditions may also affect enantiomeric stability.