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.

中文翻译：

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96 孔板形式的血浆和血清 oxylipin、内源性大麻素、胆汁酸、类固醇、脂肪酸和非甾体抗炎药的定量

本研究的目标是开发一种高通量、经济高效的方法，对参与炎症和能量代谢调节的脂质介质和激素，以及多不饱和脂肪酸和常见的非处方非甾体类药物进行代谢分析。抗炎药（NSAIDs）。我们描述了在 37 种氘代类似物和 2 种仪器内部标准存在的情况下对 50 μL 血浆或血清进行的 96 孔板蛋白质沉淀和过滤程序。在两次背靠背 UPLC-MS/MS 分析中采集数据，使用电喷雾电离和正/负切换和预定的多反应监测，测定 145 种化合物，包括氧脂、内源性大麻素和类似化合物、胆汁酸、糖皮质激素、性类固醇、多不饱和脂肪酸和 3 种非甾体抗炎药。两种基质中超过 70% 的代谢物的批次内和批次间变异性小于 25%，但观察到血清氧磷脂和一些胆汁酸的批次间变异性更高。NIST 标准参考材料 1950 的结果与该检测涵盖的 20 种认证代谢物值相比具有优势，我们提供了该材料中氧脂、N-酰基乙醇酰胺、糖皮质激素和 17-羟基-孕酮的新数据。对两个独立的老年男性和女性队列的应用表明，常规检测了 86 种代谢物，确定了禁食状态对必需脂肪酸衍生的氧脂、N-酰基乙醇酰胺和结合胆汁酸的影响，确定了罕见的高和低睾酮水平以及这些人群中约 10% 的非甾体抗炎药。