De Novo StructuralElucidation of Acylglycerols bySupercritical Fluid Chromatography and Collision-Induced Dissociationof Electron-Deficient Precursor Ions

Various mass-spectrometric lipidomics approaches are described to resolve double bond (DB) positions, fatty acid attachments, and <i>sn</i>-position but require dedicated instrumentation or chemical derivatization. In this study, we demonstrate that dopant-assisted atmospheric-pressure photoionization (dAPPI) using chlorobenzene as a dopant generates radical cations (M^+.) and [M-H]⁺ cations of acylglycerols termed Electron Deficient Precursor Ions (EDP). Observed [M-H]⁺ ions are derived from radical hydrogen abstraction from M^+. ions. Collision-induced dissociation of EDP ions allows rule-based de novo annotation of DB positions, fatty acid attachments, and <i>sn</i>-position. Among 33 acylglycerol standards, selective ionization for acylglycerols with ≥1 DB was observed, where acylglycerols with ≤3 DB formed mainly [M-H]⁺ ions and those with ≥4 M^+. ions. EDP-CID generated characteristic intensity ratios of the fragment peaks for <i>sn</i>-positions and double-bond indicative elemental formula losses (EFL) originating from cleavages along the fatty acid alkyl chain with intensity maxima adjunct to DB positions. DB annotation was performed by following EFL series, such as M-CH₃(CH₂)<i>_n</i> for the first DB. For each DB lost, a Δ2H shift is observed (M-CH₃(CH₂)<i>_n</i> Δ2H_DB‑1) and used for the annotation of following DBs. Additional fragmentation series, indicative of DB positions, were observed after loss of hydroxyl groups or fatty acids (M-FA-CH₃(CH₂)<i>_n</i>). MsRadaR, an R package, was developed allowing to navigate through EDP-CID spectra and visualize relevant EFL series. Last, supercritical fluid chromatography coupled to dAPPI-EDP-CID was applied for the analysis of 57 acylglycerols in linseed oil with complete DB position characterization of 9 diglycerides and 24 triglycerides.