Comprehensive two-dimensional liquid chromatographic analysis of anthocyanins

• The first application of LC × LC for anthocyanin analysis is reported.
• Improved resolution is provided by off-line HILIC × RP-LC analyses.
• Practical peak capacities of up to 6950 were obtained.
• Group-type separation facilitated compound identification by means of contour plots.

Anthocyanins are naturally occurring plant pigments whose accurate analysis is hampered by their complexity and unique chromatographic behaviour associated with on-column conversion reactions. This paper reports the evaluation of off-line comprehensive two-dimensional liquid chromatography (LC × LC) for the analysis of anthocyanins. Hydrophilic interaction chromatography (HILIC) was used in the first dimension in combination with reversed phase liquid chromatography (RP-LC) in the second dimension. For the selective detection of anthocyanins, diode array detection was used, while high resolution quadrupole-time-of-flight mass spectrometry (Q-TOF) was used for compound identification. As application, the HILIC × RP-LC separation of diverse anthocyanins in blueberries, red radish, black beans, red grape skins and red cabbage is demonstrated. Off-line HILIC × RP-LC revealed information which could not be obtained by one-dimensional HPLC methods, while the structured elution order for the anthocyanins simplifies compound identification and facilitates the comparison of anthocyanin content of natural products by means of contour plots.