State of the Art Oxylipin Analysis and New Developments

Eicosanoids and other oxylipins are oxygenation products from polyunsaturated fatty acids. Several oxylipins are highly potent lipid mediators, and many are discussed as biomarkers for diseases. Oxylipin analysis is analytically challenging due to their low abundance and rapid metabolism. Both chromatography and MS/MS are needed because of the presence of closely eluting isomers and similar fragmentation patterns of the molecules. The quantitation is based on external calibration using authentic (isotope-labeled) standards and a sound sample preparation strategy.

The webinar explains how to start method development for oxylipin analysis on an LC coupled to a triple quadrupole (QqQ) MS instrument and a high-resolution mass spectrometer (QTof). What is needed, which parameters can be chosen as a starting point, and where can resources/information, e.g., an SOP for sample preparation or calibration, be found?

The second part will focus on what is required to characterize the method. This is particularly important when the method is used in publications. Examples of “reporting criteria” recently published as “good practice” by the lipidomics community (https://doi.org/10.5281/zenodo.15044739) will be discussed.

The accurate and precise quantitation of oxylipins is then shown for human blood and cells following supplementation with long-chain n3-PUFA. Using these examples, a major limitation of current LC-MS analysis is illustrated: The lack of separation of configuration isomers, i.e. enantiomers, by reversed-phase chromatography. This can be addressed with two-dimensional LC separation: By multiple heart-cutting, peaks are collected following reversed-phase separation and transferred onto a chiral column. Based on the analysis of blood samples, we highlight that the stereo configuration of many oxylipins should only be stated when an enantioselective separation was carried out.

Key learnings for attendees:

• Method development for oxylipin analysis by LC-MS/MS
• Good practices for method characterization in lipidomics
• Overcoming challenges in oxylipin quantitation

Who should attend:

• Life science researchers looking to enhance their understanding of oxylipin analysis