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

Abstract

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 (RIImax) 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.