Isolation and characterization of A- and B- type of cocoa proanthocyanidins by HPLC QTOF- MS/MS

Abstract

Cocoa powder has been described as the 4th richest dietary source of polyphenols, with increasing applications in food, pharmaceutical and cosmetic industries . In general, cocoa flavonoids consists of B-type procyanidins with a degree of polymerization (DP) up to 12 units. However, better elucidation is still a challenge due to the lack of separation of enantiomers and low sensitivity of traditional low-resolution MS. However, the application of the latest technological improvements in mass spectrometry may allow a deeper characterization of main cocoa flavonoids. Thereby, the aims of this work were: - Fractionation of cocoa procyanidins by preparative HPLC using a diol column. - Characterization and linkage elucidation of procyanidins by QTOF-MS/MS analysis. As expected, theobromine, caffeine, and theophylline -in less amount- were the main alkaloids detected in positive ionization mode. (-)-Epicatechin was the major flavan-3-ol followed by (+)- catechin. Regarding procyanidins, polymers up to decamers were identified. Overall, procyanidins mainly consisted of B-type species. Polymeric procyanidins between degree of polymerization (DP) 2 - 6 were detected as singly-charged ions, but those containing DP ≥ 7 were found as multiplycharged ions in the negative ESI mode. About 127 procyanidins were elucidated, including dimer, trimer, tetramer, pentamer, and hexamer procyanidins with m/z equal to 577.1358, 865.1993, 1153.2624, 1442.3301, 1730.3923, respectively. Fragment ions produced in MS/MS experiments followed well-known theoretical fragmentation pathway mechanisms such as quinone methide (QM), retro-Diels-Alder (RDA) as well as heterocyclic ring fissions (HRF). In general, B-type interflavan linkage were more frequent than A-type species, being identified by possessing 2 Da less than those of the B-type species. For example, trimer and tetramer species were detected at m/z 865.1918 and 863.1868 and 1153.2624 and 1149.3679 (-4 Da) for B- and A-types, respectively. Interestingly, the presence of one and two A-type linkages and glucose moiety (e.g., β- galactopyranose, α-arabinopyranose and glucopyranosyl) attached to procyanidins was also found. Therefore, dimer, trimer, tetramer with m/z 739.1888, 1025.2326, 1313.2987, respectively are reported. In addition, the presence of (epi)afzelechin-(epi)catechin (m/z 561.1358), (epi)afzelechin– (epi)catechin–(epi)catechin (m/z 849.2046) as well as (epi)afzelechin-(epi)afzelechin–(epi)catechin (m/z 833.1250), and novel dimer, trimer, pentamer, hexamer with m/z 575.1195, 849.2045, 1425.3268, 864.1901 ([M-2H]-2) in cocoa is reported for the first time. These observations highlight the importance of high resolution MS as useful tool for the characterization of cocoa proanthocyanidins as well as for further studies involving their detection in the organism upon metabolization.