High-level structural analysis of proanthocyanidins using full collision energy ramp-MS² spectrum

Proanthocyanidins (PACs) refer to a group of polyphenols consisting of flavan-3-ol units, and are ubiquitously distributed in fruits, vegetables, nuts and grains. PACs possess high-level structural diversity because of the fickle linkage manners amongst units, the polymerization degree and stereoisomeric forms, thus leading to a great challenge for structural analysis. Although LC-MS/MS currently serves as the workhorse to profile PACs in complicated matrices, it's still challenging to achieve confirmatively structural annotation even employing the cutting-edged high-resolution MS/MS techniques, and the key technical obstacle lies at isomeric discrimination. To pursue as many auxiliary structural clues as possible, full collision energy ramp-MS² (FCER-MS²) spectrum was conceptually designed here to involve all mass fragmentation behaviors of a given compound, such as m/z, optimal collision energy (OCE) and the maximal relative ion intensity (RII_max) aiming to advance the structural annotation confidences of PACs through reliably differentiating isomers. Thirteen authentic compounds were collected to mine relationships between chemical structures and FCER-MS² spectra that were correlated by three progressive steps: (1) recording MS/MS spectrum by LC-Q-TOF-MS; (2) proposing mass fragmentation pathways to assign those obvious fragment ion species; and (3) acquiring breakdown graph for each concerned fragment ion species by programming online energy-resolved mass spectrometry to compose FCER-MS² spectrum. Afterwards, the rules were applied for PACs-focused chemical characterization of a medicinal herb namely Indigofera stachyodes (Chinese name: Xuerenshen), and as a result, 22 PACs were captured and more importantly, isomerically identified by deciphering FCER-MS² spectra. Therefore, FCER-MS² spectrum provides a promising way to achieve in-depth isomeric discrimination of, but not limited to, PACs.