Exploring the Fragmentation of Sodiated Species Involving Covalent-Bond Cleavages for Metabolite Characterization.

Rationale: Electrospray (ESI), the most popular desorption/ionization technique used in mass spectrometry-based metabolomics, generates both protonated and deprotonated molecules, as well as adduct ions, sodium being the most frequent monoatomic cation entering their composition. With the spread and generalization of untargeted data-dependent and independent tandem mass spectrometry experiments, considering product ion spectra of sodium-containing entities appears relevant to complement fragmentation information of their protonated and deprotonated counterparts.

Methods: Solutions of pure standards, mainly amino and organic acids, were prepared at 1 μg/mL and injected either by direct infusion or by flow-injection prior to ESI-MS/MS analysis. Product ion spectra of (de)protonated and sodiated molecules were recorded both in positive and negative modes on Orbitrap instruments under both non-resonant and resonant excitation conditions. Various normalized collision energies (NCE) were applied and the resulting collisional spectra were analyzed.

Results: Examination of the resulting collisional spectra clearly revealed that fragmentation of sodiated ion species may produce spectra significantly different from [M + H]⁺ or [M - H]^-. They can be highly informative and result from specific fragmentation mechanisms based on covalent bond cleavages (CBCs) compared to protonated or deprotonated molecules. These specific CBCs involving sodium retention either in product ions or in neutral losses have been investigated and seem to occur when the sodium cation is involved in an ion-ion type interaction within the structure.

Conclusions: Overall, we show, using representative examples of biologically relevant metabolites, the benefits of considering MS/MS data generated from sodiated entities, in addition to [M + H]⁺ and [M - H]^- collisional data, to improve metabolite identification. The differentiation of four positional isomers is a striking illustration of the power of fragmentation information obtained with species of the [M - 2H + Na]^- form. Considering the number of metabolites featuring chemical groups capable of interacting with Na⁺, systematic integration of these data into annotation workflows should be considered.