Off-line comprehensive 2-dimensional hydrophilic interaction × reversed phase liquid chromatography analysis of procyanidins

The development of an off-line comprehensive 2-dimensional liquid chromatography (2-D-LC) method for the analysis of procyanidins is reported. In the first dimension, oligomeric procyanidins were separated according to molecular weight by hydrophilic interaction chromatography (HILIC), while reversed phase LC was employed in the second dimension to separate oligomers based on hydrophobicity. Fluorescence, UV and electrospray ionisation mass spectrometry (ESI-MS) were employed for identification purposes. The combination of these orthogonal separation methods is shown to represent a significant improvement compared to 1-dimensional methods for the analysis of complex high molecular weight procyanidin fractions, by simultaneously providing isomeric and molecular weight information. The low correlation (r2 < 0.2100) between the two LC modes afforded a practical peak capacity in excess of 2300 for the optimal off-line method. The applicability of the method is demonstrated for the analysis of phenolic extracts of apple and cocoa.