Track: Bioanalytics - Chemical - Biomarker Quantification - Mass Spectrometry (LC-MS) Methods
Category: Late Breaking Poster Abstract
Sensitive and Comprehensive Lipid Mediator Profiling using Advanced Scheduled MRM with Polarity Switching and QTRAP Enhanced Product Ion Scanning
Purpose: Lipid mediators regulate diverse physiological processes and play critical roles in modulating human inflammation-resolution responses. Understanding the complex roles and regulation of lipid mediators in human health relies on sensitive and comprehensive methods to capture complete pathway profiles. In biological samples, these bioactive lipids are often present at or below nanomolar concentrations and require a highly sensitive LC-MS/MS platform for identification and quantitation. In this work, a quantitative and qualitative workflow is described for the analysis of different lipid mediator species including specialized pro-resolving mediators (SPM), leukotrienes, prostaglandins, hydroxy-eicosatetraenoic acids (HETEs) and epoxy-eicosatrienoic acids (EETs), endocannabinoids (e.g., anandamide and 2-AG), as well as metabolites and markers of reactive oxygen and nitrogen species. Methods: Using a QTRAP® 7500 system coupled with an ExionLC™ System with SciexOS software, we developed and expanded a targeted panel of now 108 compounds for comprehensive profiling of lipid mediators and pathway markers. A Kinetex® Polar C18 column was used for LC based separation of lipid mediators, epimers and other isoelemental structures within a 20 min run time. Advanced scheduled sMRM was used to optimize scanning windows and dwell weighting. The negative mode lipid mediator panel includes pro-inflammatory prostaglandins, leukotrienes and their primary metabolites, specialized pro-resolving mediators (SPM) including resolvins, protectins, maresins, and lipoxins, biomarkers of ROS and NOS, and fatty acid and mono-hydroxy fatty acid precursors. The positive mode panel includes cysteinyl leukotrienes, PAF, and maresin and protectin conjugates in tissue regeneration (MCTRs and PCTRs). Using standards, MRM transitions were developed and optimized for 88 different lipid mediators, including 16 deuterium labeled internal standards. Next a number of columns were evaluated. The Kinetex Polar C18 column was selected because it provided both polar and non-polar retention that facilitates the separation of lipid mediator epimers and isobaric compounds which would improve assay performance statistics and minimize false positives. As an example, baseline separation of LXA4 and 15-epi-LXA4 epimers, as well as RvD1 and 17-epi-RvD1 epimers was achieved which enables the differentiation of enzymatic pathway utilization for pro-resolving lipid mediator production. Results: Concentration curves were generated by injecting 3 replicate injections of 8 different concentrations of each lipid mediator standard. Excellent linearity is observed with R2 of at least 0.99 for all analytes. Excellent sensitivity was obtained for all lipid mediator species with LLOQ as low as 40 fg on column with CV < 20%. Finally, the unique qualitative / quantitative QTRAP platform also enabled MRM triggered EPI experiments which provided high sensitivity MS/MS data for confirmation of low level analytes. Conclusion: The polar C18 LC-MS/MS method provides a unique qualitative / quantitative platform for rapid and sensitive profiling of lipid mediators with sensitivity levels capable of measuring lipid mediators in different matrix types.