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

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.

Secondary or Specialized Metabolites, or Natural Products: A Case Study of Untargeted LC–QTOF Auto-MS/MS Analysis

“…Despite reports of 255 and 241 features [ 41 ], the observed 256 and 241 Da features can be regarded as congruent with a kaempferol-glu-rha feature. The additional glucose linked to the first glucose sugar could have caused this change.…”

“…The only detectable glycoside feature of 463 Da pointed to quercetin-3-glu or quercetin-7-glu. The 271 and 151 Da fragment ions have also been detected in quercetin-3-glu [ 41 ]. The finally assigned structure, quercetin-3-glu-rha-7-glu, belongs to the more commonly occurring flavonol glycosides [ 46 , 47 , 48 , 49 , 50 ].…”

“…Features at 283 Da—this one especially—and 271 Da, and 151 Da provided support for the first sugar being a rhamnose [ 41 ]. The feature at 446 Da provided further evidence that sugars 2 and 3 are both glucoses.…”

“…A feature at 593 Da indicated the loss of a glucose, and a feature at 447 Da the further loss of a rhamnose. The features 357 and 241 Da characterize a kaempferol-3-glu structure [ 41 ]. The proposed presents one of the more commonly occurring flavonol glycosides [ 46 , 49 , 52 , 53 ].…”

“…[ 40 ]. MS/MS fragmentation of flavonoid rings were compared to full collision energy ramp MS/MS spectra [ 41 ]. Trisaccharide fragmentation was compared to available CID studies [ 39 , 42 , 43 , 44 ].…”