Plasma and serum oxylipin, endocannabinoid, bile acid, steroid, fatty acid and nonsteroidal anti-inflammatory drug quantification in a 96-well plate format

Authors: PEDERSEN, THERESA - University Of California, Davis; Gray, Ira; Newman, John

Submitted to: Analytica Chimica Acta
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/17/2020
Publication Date: 11/23/2020
Citation: Pedersen, T.L., Gray, I.J., Newman, J.W. 2020. Plasma and serum oxylipin, endocannabinoid, bile acid, steroid, fatty acid and nonsteroidal anti-inflammatory drug quantification in a 96-well plate format. Analytica Chimica Acta. 1143:189-200. https://doi.org/10.1016/j.aca.2020.11.019.
DOI: https://doi.org/10.1016/j.aca.2020.11.019

Interpretive Summary: The quantitative analysis of broad swaths of low abundance metabolites involved in the regulation of inflammation and energy metabolism can provides unique insights into normal biological processes associated with health and disease. However, current approaches are labor intensive, and not amenable for the study of large sample sets needed to understand the variability in the general population. 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 ultra performance liquid chromatography-tandem quadrupole mass spectrometry 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 limit of quantitation 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.

Technical Abstract: 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.