The goal of this research was to develop a high-throughput, cost-effective method for metabolic profiling of
lipid mediators and
hormones involved in the regulation of
inflammation and energy metabolism, along with
polyunsaturated fatty acids and common over-the-counter non-steroidal anti-inflammatory drugs (
NSAIDs). We describe a 96-well plate
protein precipitation and filtration procedure for 50 μL of plasma or serum in the presence of 37 deuterated analogs and 2 instrument internal standards. Data is acquired in two back-to-back UPLC-MS/MS analyses using electrospray ionization with positive/negative switching and scheduled multiple reaction monitoring for the determination of 145 compounds, including
oxylipins,
endocannabinoids and like compounds,
bile acids,
glucocorticoids, sex
steroids,
polyunsaturated fatty acids, and 3
NSAIDs. Intra- and inter-batch variability was <25% for >70% of metabolites above the LOQ in both matrices, but higher inter-batch variability was observed for serum
oxylipins and some
bile acids. Results for NIST Standard Reference Material 1950, compared favorably with the 20 certified metabolite values covered by this assay, and we provide new data for
oxylipins, N-acylethanolamides,
glucocorticoids, and 17-hydroxy-progesterone in this material. Application to two independent cohorts of elderly men and women showed the routine detection of 86 metabolites, identified fasting state influences on essential
fatty acid-derived
oxylipins, N-acylethanolamides and conjugated
bile acids, identified rare presence of high and low
testosterone levels and the presence of
NSAIDs in ∼10% of these populations. The described method appears valuable for investigations in large cohort studies to provide insight into metabolic cross-talk between the array of mediators assessed here.