Combining near-complete characterization with quantitation for lipid analysis in matrix using electron activated dissociation

EAD has become a valuable tool for comprehensively characterizing glycerophospholipids and neutral lipids—from lipid class to double bond configuration—on the ZenoTOF 7600 system. However, lipid mediators remain to be fully characterized using EAD analysis. This dissociation tool utilizes electron capture and excitation to provide richer molecular structure information compared to conventional CID. Limited information is often provided by CID, conversely the EAD cell produces diagnostic fragmentation that can be used to quantify, identify, and characterize lipid mediators. Lipid mediators are diverse in terms of the number of possible double bond and functional group positions and geometries; however, the number of distinct parent masses and fragment ion masses is relatively small due to these compounds being derived from a limited set of fatty acid precursors and having many shared structural features (e.g., conjugated dienes, trienes, and tetrataenes; usually 1-3 hydroxyl groups). As a result, liquid chromatography methods for lipid mediator profiling generally demands a longer run time on the order of 15 – 20 minutes (or longer) to achieve sufficient separation of the hundreds of distinct mediators and their numerous epimers and other isobaric species. IDA was initially used with an inclusion list to trigger and collect spectra for a library of lipid mediator standards. Then, using scheduled MRMhr analysis, diagnostic fragments were monitored for PGE2 and PGD2 to quantify these two isoelemental compounds at the same chromatographic retention time. This newly described methodology for the characterization and quantitation of lipid mediators uses EAD on a ZenoTOF 7600 System. The rich MS/MS data produced by EAD contains unique fragment ions with patterns and intensities that can be used for complete lipid structural  confirmation. Additionally, the scheduled MRMhr EAD experiment is sensitive, reproducible, and fast, enabling quantitation from diagnostic fragments from complex mixtures during rapid LC separation.