Protocols

Current

Development and Validation of Methodologies for the Identification of Specialized Pro-Resolving Lipid Mediators and Classic Eicosanoids in Biological Matrices

Lipid mediators, including specialized pro-resolving mediators (SPMs) and classic eicosanoids, play critical roles in both initiating and resolving inflammation. However, our understanding of how these molecules are regulated across various inflammatory contexts is still limited. This gap hinders our comprehension of the mechanisms driving disease onset and progression. To address this, we developed highly sensitive methodologies for extracting, identifying, and quantifying these mediators. Using reverse-phase HPLC, we successfully separated and identified 37 lipid mediators, including isomers of Lipoxin A4, Protectin D1, and others. The method showed strong linearity (r² > 0.98), with low limits of quantitation (LLOQ) ranging from 0.01 to 8.5 pg. We also validated accuracy and precision, demonstrating reliable recovery in biological matrices (plasma and serum). The approach offers a robust platform for studying SPMs and eicosanoids in biological tissues, providing valuable insights into their roles in regulating inflammation. This will help advance our understanding of disease mechanisms and potential therapeutic targets.

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Methodologies and Procedures Employed in the Identification and Quantitation of Lipid Mediators via LC-MS/MS

Liquid chromatography tandem mass spectrometry (LC-MS/MS) is a sensitive and robust approach for the identification and quantitation of lipid mediators including the specialized pro-resolving mediators (resolvins, maresins, protectins and lipoxins), and the classic eicosanoids. Due to the relatively low concentration of these molecules in biological samples, this methodology is classically coupled with solid phase extraction (SPE) techniques that enhance the sensitivity of the assay. Herein, we provide a step-by-step description of instrumentation employed in the both SPE and LC-MS/MS and detailed methodologies employed in the extraction of these lipid mediators and their identification and quantitation using LC-MS/MS.

Lipid Mediator Metabolomics Via LC-MS/MS Profiling and Analysis

Solid-phase extraction coupled with liquid chromatography tandem mass spectrometry provides a robust and sensitive approach for the identification and quantitation of specialized pro-resolving mediators (lipoxins, resolvins, protectins, and maresins), their pathway markers and the classic eicosanoids. Here, we provide a detailed description of the methodologies employed for the extraction of these mediators from biological systems, setup of the instrumentation, sample processing, and then the procedures followed for their identification and quantitation.