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

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.