Full Collision Energy Ramp-MS<sup>2</sup> Spectrum in Structural Analysis Relying on MS/MS
Pengwei Guan, Wenjing Liu, Yan Cao, Huiting Tang, Hui-Xia Huo, Jian‐Bo Wan, Xue Qiao, Pengfei Tu, Jun Li, Yuelin Song
Abstract
Albeit frequently being overlooked, MS 2 spectrum variation against collision energy (CE) implies auxiliary structural clues for m / z values. Online energy-resolved MS (ER-MS) provides the opportunity to acquire the trajectory of ion intensity against CE for any fragment ion of interest, thus exactly offering the desired momentum to empower the conventional MS 2 spectrum at a certain CE forward to a full-CE ramp MS 2 spectrum (FCER-MS 2 ). Efforts were made here to construct an FCER-MS 2 spectrum and to evaluate its potential toward structural analysis. Flavonoids were employed as a proof of concept. MS 2 spectra of 76 compounds were recorded by LC–Q-Exactive-MS, and online ER-MS was subsequently programmed using LC–Qtrap-MS to build a breakdown graph for each obvious fragment ion. After defining the greatest value amongst all regressive apices as 100%, the normalized breakdown graphs comprised an FCER-MS 2 spectrum for each compound. The FCER-MS 2 spectrum contained the MS 2 spectrum at any CE as well as optimal CE (OCE) and maximal relative ion intensity (RII max ) of each fragment ion. Except the pronounced isomeric discrimination potential, either OCE or RII max reflected certain structural properties, such as aglycone, glycosidic bond, and hydroxy, methoxy, and glycosyl substituents. These rules were subsequently applied for flavonoid-focused characterization of a famous herbal medicine, namely Scutellariae Radix, and high-level structural annotation was accomplished for 75 flavonoids. Above all, the FCER-MS 2 spectrum includes m / z, OCEs, and RII max features, thus facilitating confidence-advanced structural analysis.