Revisiting fatty acid fragmentation: New insights into structural identification by high-resolution and multi-stage mass spectrometry

• Characteristic ions were utilized to confirm the presence of a carboxylic group • C-6 acts as a boundary between charge-remote and charge-driven fragmentation • A novel conjugated structure was proposed for [M-H-H 2 O]⁻ of fatty acids • A characteristic collision energy index was designed to differentiate fatty acids High-resolution, multi-stage mass spectrometry data acquired at multiple collision energies for 41 fatty acids were obtained from the mzCloud database, and their fragmentation patterns were reanalyzed. Characteristic fragment ions, including [(M-H)-2H]⁻, [(M-H)-H 2 O]⁻, [(M-H)-H 2 O-2H]⁻, [(M-H)-2H 2 O]⁻, and [(M-H)-2H 2 O-2H]⁻ in MS 2 spectra, as well as [(M-H-H 2 O)-2H]⁻, [(M-H-H 2 O)-H 2 O]⁻, and [(M-H-H 2 O)-H 2 O-2H]⁻ in MS 3 spectra, were identified as diagnostic markers for carboxylic acid groups. A novel structural configuration was proposed for the [(M-H)-H 2 O]⁻, in which the C=C double bond between C-2 and C-3 forms a conjugated system with the carbonyl (C=O) group. A new parameter, characteristic collision energy, was introduced and utilized to discriminate fatty acids with various chain lengths and unsaturation degrees. It also demonstrated potential for distinguishing isomers with different double-bond positions. This work enhances the interpretability of fatty acid mass spectral data and facilitates more confident structural determination.