Separation of isomeric triglycerides by cyclic ion mobility mass spectrometry

Triacylglycerols (TAGs) are the most abundant lipid class in foods. They consist of three fatty acid chains esterified to a glycerol backbone and are often present in various isomeric forms. These include sn-positional, cis-trans configuration, acyl chain length, double bond positional, and mixed type isomers. Elucidating these complex isomeric mixtures is of great interest as different isomers can have distinct properties, such as digestibility, oxidative stability or lipid metabolism. However, TAG isomer separation remains challenging. In this study, we developed a method with cyclic ion mobility mass spectrometry (cIMS-MS), for the separation and identification of all types of TAG isomers. The influence of different adducts (Li+, NH4+, Na+, and K+) on the cIMS separation of TAG isomers was studied. Overall, it was concluded that the sodium adduct is the best all-round choice to efficiently separate TAG isomers. In addition, trends were found in the influence of specific structural features on the drift time order. An order of relative influence was established, from most to least influential: (1) degree of unsaturation on the exterior position, (2) acyl chain length on the exterior position, (3) cis/trans configuration, and (4) double bond position. Finally, various specific cIMS-MS strategies were developed for separation of complex mixtures of four or more isomers. To conclude, this method can be used for separation of complex mixtures of TAG isomers and has great potential to be expanded to other similar type of lipids such as di- and monoacylglycerol isomers.