Unraveling the Double Bond Position of Fatty Acids by GC-MS Using Electron Capture APCI and In-Source Fragmentation Patterns

The position of double bonds in unsaturated fatty acids is strongly connected to their biological effects, but their analytical characterization is still challenging. However, the ionization of unsaturated fatty acids by a GC-APCI leads to regiospecific in-source fragment ions, which can be used to identify the double bond position. The fragment ions are oxidized species that occur mostly at the double bond closest to the carboxylic acid group. This effect can be further promoted by using benzaldehyde as a gas-phase reactant. This allows the identification of the Δ-notation of the fatty acid, and based on additional information such as m/z and retention time, it is possible to annotate the corresponding fatty acid. The developed method also enables the quantification of fatty acids in one step with high selectivity and sensitivity. Moreover, rare fatty acids can be identified in suspected target approaches that are often not available as standards. This was demonstrated by analyzing fish oil samples that provide a complex mixture of highly unsaturated fatty acids and by identifying rare fatty acids such as hexadecatetraenoic acid (FA 16:4 Δ6).