Phenolic profiling of rooibos using off-line comprehensive normal phase countercurrent chromatography×reversed phase...

Limited performance of one dimensional chromatographic methods provides the incentive for the development of multidimensional chromatographic techniques for the analysis of complex mixtures. Rooibos (Aspalathus linearis) represents such a complex sample, containing a range of phenolic compounds that cannot be separated and identified using a single chromatographic technique within a reasonable run time. By implementing NP high performance countercurrent chromatography (NP-HPCCC) in the first dimension (¹D) and RP ultra-high pressure LC (RP-UHPLC) in the second dimension (²D), a highly orthogonal (∼80%) off-line comprehensive two-dimensional separation of rooibos phenolic compounds was achieved in a total analysis time of 17h. The use of a gradient for the ¹D HPCCC separation ensured a good spread of relatively polar flavonoid di-C-glycosides and less polar mono- and di-O-glycosides, while the highly efficient UHPLC method was able to separate compounds eluting in the same ¹D fraction. Analysis of green ("unfermented") and traditional "fermented" rooibos samples enabled tentative identification of 39 phenolic compounds based on UV-vis and MS characteristics, of which 18 have not previously been reported in rooibos. Scolymoside (a flavone), hesperidin (a flavanone) and phloretin-3',5'-di-C-β-d-glucopyranoside (a dihydrochalcone) were identified for the first time in rooibos by comparison with authentic reference standards.