In‐source fragmentation and accurate mass analysis of multiclass flavonoid conjugates by electrospray ionization time‐of‐flight mass spectrometry

Abstract

In-source collision-induced dissociation (CID) fragmentation features of multiclass flavonoid glycoconjugates were examined using liquid chromatography electrospray time-of-flight mass spectrometry. Systematic experiments were performed to search for optimal conditions for in-source fragmentation in both positive and negative ion modes. The objective of the study was to attain uniformly appropriate conditions for a wide range of analytes independently of the aglycone, the attached sugar part and the type of bond between the aglycone and the glycan moieties (O- or C-glycosides). Studied substances included representatives of flavonols, flavones, flavanones and anthocyanins and, regarding their glycan parts, mono-, di- and triglycosides with varying distribution of carbohydrate moieties (di-O-glycosides, O-diglycosides, O,C-diglycosides). The breakdown properties of the analytes along with the abundances of the characteristic diagnostic ions required for structural elucidation of complex flavonoid derivatives were evaluated. An optimized value was found for the instrument parameter (fragmentor voltage) affecting the in-source CID fragmentation of the analytes [230 V (ESI+) and 330 V (ESI-)]. Thus, appropriate performance in terms of both highly sensitive full-scan acquisition and fragmentation information was obtained for all the investigated flavonoids. In addition, singularities in the abundance of selected diagnostic ions (e.g. Y(0), Y(1) and Y*) due to variations in the interglycosidic linkage (rutinoside-neohesperidoside) in the glycan part were found and are also evaluated and discussed in detail. The combination of in-source CID fragmentation with high mass accuracy MS detection establishes a working basis for the development of versatile and useful LC-MS methods for wide-scope screening, non-targeted detection and tentative identification of flavonoid derivatives.

Systematic bottom-up approach for flavonoid derivative screening in plant material using liquid chromatography high-resolution mass spectrometry

“…When a selected diagnostic ion is extracted, its corresponding sodium adduct can be extracted in parallel, to find out whether the extracted ion is just a fragment ion of the sought flavonoid derivative or, on the contrary, actually the intact [M+H] + ion. According to preliminary experiments [25,30], intact flavonoid derivatives mass spectra usually includes [M+Na] + adducts, even without the addition of any Na-containing reagent to foster this process while fragment ions are not able to form sodium adducts under the same conditions. Once the sodiated ion occurs together with the protonated ion, there is no need to search for more complex flavonoid derivatives.…”

“…1). Based on the results of our preliminary studies, independently of the glycan parts of the flavonoid compounds, 0,2 X ion was the most intense one among the studied i,j X ions in positive ion mode [25]. Consequently, in this study, 0,2 X ion was tested as an Y 0 + substitute in the case of C-glycosides.…”

“…Optimization of fragmentor voltage is described elsewhere [25], LC-MS accurate mass spectra were recorded across the range of 50-1,000m/z. The instrument performed the internal mass calibration automatically, using a dual-nebulizer electrospray source with an automated calibrant delivery system, which introduces the flow from the outlet of the chromatograph together with a low flow (approximately 10 μL min −1 ) of a calibrating solution.…”

“…In this technique, the ions generated in the ion source and entering the vacuum region can be accelerated by applying voltages, prompting collisions with surrounding species that can produce sufficient energy to yield diagnostic fragment ions. In-source fragmentation usually generates fragmentation patterns consistent with those attained in dedicated collision cells, in which an auxiliary collision gas is used for that purpose, and after precursor ion selection and isolation [25]. In-source fragmentation offers the advantage that fragmentation information is obtained simultaneously on an unlimited number of compounds without any compromise on the analysis speed.…”

Chiral Differentiation of Amino Acids by In-Source Collision-Induced Dissociation Mass Spectrometry

“…e technique is usually called as in-source CID. [21][22][23] It is also referred with di erent names, such as: nozzle-skimmer fragmentation, 24) skimmer-CID or source-CID. 25) It is not a real MS/MS method since the precursor ions cannot be selected and isolated, and the experiments can be performed in single-stage MS instruments.…”