Phenolics Profiling by Three-Phase Solvent System Step-Gradient Using High-Performance Countercurrent Chromatography with Off-Line Electrospray Mass-Spectrometry Detection.

The detailed metabolite profiling of was accomplished by high-performance countercurrent chromatography (HPCCC) using the three-phase system -hexane--butyl methyl ether-acetonitrile-water 2:3:3:2 (///) in step-gradient elution mode. The gradient elution was adjusted to the chemical complexity of the ethyl acetate partition and strongly improved the polarity range of chromatography. The three-phase solvent system was chosen for the gradient to avoid equilibrium problems when changing mobile phase compositions encountered between the gradient steps. The tentative recognition of metabolites including the identification of novel ones was possible due to the off-line injection of fractions to electrospray ionization mass spectrometry (ESI-MS/MS) in the sequence of recovery. The off-line hyphenation profiling experiment of HPCCC and ESI-MS projected the preparative elution by selected single ion traces in the negative ionization mode. Co-elution effects were monitored and MS/MS fragmentation data of more than 100 substances were used for structural characterization and identification. The metabolite profile in the extract comprised flavonoids, hydrolysable tannins, condensed tannins and low molecular weight polyphenols.