Tandem mass spectrometry for sequencing proanthocyanidins

Proanthocyanidins (PAs) are a group of bioflavonoids consisting of oligomers based on catechin monomeric units. These polyphenolic compounds are widely distributed in higher plants and are an integral part of the human diet. A sensitive LC-tandem mass spectrometric (LC/ESI-MSn) method in the positive ion mode for sequencing these ubiquitous and highly beneficial antioxidants is described. The hydroxylation patterns and interflavanoid linkage for A- and B-type PAs were determined by fragment ions derived from a retro-Diels-Alder (RDA) fission, heterocyclic ring fission (HRF), a novel benzofuran-forming (BFF) fission described here for the first time, and a quinone methide (QM) fission. The subunit sequence of the PAs was determined by diagnostic ions derived from HRF/RDA fission, HRF/BFF fission, and RDA/HRF fission together with QM fission. A total of 26 PAs were reliably sequenced by the newly established tandem mass spectrometric protocol. It is shown that the protocol based on a combination of these different fragmentation patterns allows for uniquely identifying PA oligomers.