Separation of betacyanins from flowers of Amaranthus cruentus L. in a polar solvent system by high-speed counter-current chromatography.

Betacyanin extract of Amaranthus cruentus L. flowers was fractionated by semi-preparative high-speed counter-current chromatography in a highly polar solvent system: propan-1-ol/acetonitrile/(NH4 )2 SO4satd. soln /H2 O (1.0:0.5:1.2:1.0, v/v/v/v) in tail-to-head mode with 76% retention of the stationary phase. The crude extract as well as the fractions containing betacyanins were analyzed by liquid chromatography with tandem mass spectrometry as well as by high-resolution ion-trap time-of-flight mass spectrometry detection technique for the molecular formulae and multi-step fragmentation pattern elucidation. Four betacyanins; namely, amaranthin, betanin, 6'-O-formyl-amaranthin, and 6'-O-malonyl-amaranthin as well as their diastereomeric forms differing in the configuration of the C-15 carbon atom were identified in the fractions. Amaranthin was the dominant pigment in the extract and was additionally analyzed by nuclear magnetic resonance correlation techniques after the counter-current chromatographic and high-performance liquid chromatographic isolation. Betacyanins were highly enriched during a single high-speed counter-current chromatographic step; therefore, the tentative identification of new compounds for the whole Amaranthaceae family, 6'-O-formyl-amaranthin and 6'-O-malonyl-amaranthin was possible. Different elution profiles of the pigments observed in the counter-current chromatographic system in comparison to high-performance liquid chromatography system confirm a complementarity of both the techniques especially in the separation of diastereomeric pairs of betacyanins.