Full Collision Energy Ramp-MS² Spectrum in Structural Analysis Relying on MS/MS

Albeit frequently being overlooked, MS² 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 MS² spectrum at a certain CE forward to a full-CE ramp MS² spectrum (FCER-MS²). Efforts were made here to construct an FCER-MS² spectrum and to evaluate its potential toward structural analysis. Flavonoids were employed as a proof of concept. MS² 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-MS² spectrum for each compound. The FCER-MS² spectrum contained the MS² 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-MS² spectrum includes m/z, OCEs, and RII_max features, thus facilitating confidence-advanced structural analysis.