Multistep Analysis of Diol-LC-ESI-HRMS Data Reveals Proanthocyanidin Composition of Complex Plant Extracts (PAComics)

Proanthocyanidins (PACs) are complex oligomeric or polymeric phenolic biopolymers composed of flavan-3-ol building blocks. PACs exert manifold functional bioactivities and are assessed as bioactive ingredients in a variety of food products, beverages, medicinal plants, and phytopharmaceuticals. Although analytical methods for PACs with low degree of polymerization (DP) are well established, a lack of methods for the detailed analysis of higher oligomers and polymers from complex plant extracts is obvious. For this, the present study investigated PAC-enriched extracts from four different plants, traditionally used for medical purpose (Lime flower, Hawthorn leaf and flower, Japanese Wisteria fruits without seeds, and Common Sorrel herb). PACs were separated on diol stationary-phase high-performance liquid chromatography according to the respective DP and detected by fluorescence and quadrupole time-of-flight mass spectrometry (qTOF-MS). The qTOF-MS contour plots [t_R → m/z] provided a sufficient overview on the respective PAC distribution. Subsequently, high-resolution mass spectrometry data were used for Kendrick mass defect (KMD) analysis, with (epi)catechin, the main flavan-3-ol unit in PACs, as the reference unit. The resulting KMD plots enabled an elucidation of the general polymer chain composition with regard to DP, building blocks, and potential secondary modifications (e.g., galloylation). Subsequently, analysis of MS² fragmentation patterns of PAC oligomers confirmed the structural features obtained from the KMD plots. While Lime flower contained oligomeric A- and B-type PACs, composed of (epi)catechin and (epi)afzelechin, Japanese Wisteria fruit contained PACs consisting of three different hydroxylated flavan-3-ols. Cinchonains, A-type PACs, and B-type PACs were detected in the Hawthorn plant material. Galloylated oligo- and polymeric PACs were detected in Common Sorrel herb. This multistep analysis reveals collective insights into the PAC composition of the extracts. The protocol offers a fast and reliable methodology to be used in a standard laboratory. On the other hand, this methodology reaches its limits for higher oligomeric PACs, and further optimization is necessary for a better detection of the polymers, as the optimal DP cluster detection depends on the resolution of diol stationary-phase chromatography and is therefore limited.