Targeted quantification of lipid mediators in skeletal muscles using restricted access media-based trap-and-elute...

Lipid mediators (LMs) are a class of bioactive metabolites of the essential polyunsaturated fatty acids (PUFA), which are involved in many physiological processes. Their quantification in biological samples is critical for understanding their functions in lifestyle and chronic diseases, such as diabetes, as well allergies, cancers, and in aging processes. We developed a rapid, and sensitive LC-MS/MS method to quantify the concentrations of 14 lipid mediators of interest in mouse skeletal muscle tissue without time-consuming liquid-liquid or solid-phase extractions. A restricted-access media (RAM) based trap was used prior to LC-MS as cleanup process to prevent the analytical column from clogging and deterioration. The system enabled automatic removal of residual proteins and other biological interferences presented in the tissue extracts; the target analytes were retained in the trap and then eluted to an analytical column for separation. Matrix evaluation tests demonstrated that the use of the combined RAM trap and chromatographic separation efficiently eliminated the biological or chemical matrix interferences typically encountered in bioanalytical analysis. Using 14 LM standards and 12 corresponding deuterated compounds as internal standards, the five-point calibration curves, established over the concentration range of 0.031-320 ng mL^-1, demonstrated good linearity of r² > 0.9903 (0.9903-0.9983). The lower detection limits obtained were 0.016, 0.031, 0.062, and 0.31 ng mL^-1 (0.5, 1, 2, and 10 pg on column), respectively, depending on the specific compounds. Good accuracy (87.1-114.5%) and precision (<13.4%) of the method were observed for low, medium, and high concentration quality control samples. The method was applied to measure the amount of 14 target LMs in mouse skeletal muscle tissues. All 14 analytes in this study were successfully detected and quantified in the gastrocnemius muscle samples, which provided crucial information for both age and gender-related aspects of LMs signaling in skeletal muscles previously unknown. This method could be applied to advance the understanding of skeletal muscle pathophysiology to study the role of LMs in health and disease. Furthermore, we will expand the application of this methodology to humans and other tissues/matrices in the near future.