Development of an Oxylipin Library Using Liquid Chromatography-Ion Mobility Quadrupole Time-of-Flight: Application to...

Oxylipins are bioactive lipid mediators derived from polyunsaturated fatty acids (PUFAs) that play crucial roles in physiological and pathological processes. The analysis and identification of oxylipins are challenging due to the numerous isomeric forms. Ion mobility (IM), which separates ions based on their spatial configuration, combined with liquid chromatography (LC) and mass spectrometry (MS), has been proven effective for separating isomeric compounds. In this study, we developed an extensive oxylipin library containing information on retention time (RT), m/z, and CCS values for 74 oxylipin standards using LC-IM-QTOF-MS in positive and negative ionization modes. The oxylipins in the library were grouped into 15 isomer categories to evaluate the efficacy of IM in isomeric separation. Various adducts were investigated, including protonated, deprotonated, and sodiated forms. The ΔCCS% for more than 1000 isomeric pairs was calculated, revealing that 30% of these exhibited a ΔCCS% greater than 2%. Positive ionization mode demonstrated superior separation capabilities, with 274 isomer pairs achieving baseline separation (ΔCCS% >  4%). Sodium adducts significantly improved isomer separation. With the inclusion of LC separation, only nine oxylipins coeluted, forming six different isomeric pairs. CCS values for the adducts [M+Na]⁺ and [M+2Na-H]⁺ separated three of these isomeric pairs. The CCS values were compared to experimental libraries, confirming the high reproducibility of CCS measurements, with average errors below 2%. Applying this library to mouse brain samples, 19 different oxylipins were identified by matching RT, m/z, and CCS values. Coeluting isomers, 9- and 13-HODE, 8- and 12-HETE, and 15-oxo-ETE and 14(15)-EpETrE, were successfully separated and identified using drift time separation.