Utilization of high resolution UPLC-Orbitrap-MS and quantitative UPLC-QqQ-MS/MS technique in proanthocyanidin analysis

Tannins are a group of large secondary metabolites produced by plants. Proanthocyanidins (PAs, i.e. condensed tannins) have the basic flavan-3-ol structure as the core monomeric unit [1,2]. Depending on the flavan-3-ol type, the oligomers and polymers formed are called procyanidins or prodelphinidins, or their mixtures (Fig. 1). The main difference in procyanidins and prodelphinidins is in the hydroxylation degree of the B-ring (Fig. 1). Oligomeric and further polymeric structures can be formed via interflavan bonds either by C–C bonds (B-type PAs) or by both C–C bonds and additional ether bonds (A-type PAs). These structures may contain only procyanidin units, but also variable numbers of prodelphinidin units. As a result of this structural diversity, proanthocyanidins usually occur as a complex mixture of complex structures in a plant cell.

Fig. 1: B-type proanthocyanidin dimer consisting of procyanidin (PC) and prodelphinidin (PD) units.

The fundamental trouble with PA analysis is the fact that all PA molecules cannot be effectively separated from each other by reversed phase liquid chromatography. Instead, PA polymers tend to produce a chromatographic hump that covers tens and hundreds of individual PA structures. In this study we have utilized a new approach in PA analysis by combining UPLC high resolution mass spectrometry and tandem mass spectrometry with a novel PA separation tool in an innovative way. With the new separation tool, we have the opportunity to separate distinguished PA fractions from the complete PA mixture and thus investigate the composition of the PA mixture more accurately than before.

[1] Waterman, P. G. and Mole, S, Oxford, Blackwell Scientific Publications; 1994: 1 – 35

[2] Haslam, E, Cambridge, UK: Cambridge University Press; 2005: 10 – 83.