Ultrahigh-Performance Liquid Chromatography TandemMass Spectrometry with Electrospray Ionization Quantification of TryptophanMetabolites and Markers of Gut Health in Serum and Plasma—Applicationto Clinical and Epidemiology Cohorts

摘要

A targeted ultrahigh-performance liquid chromatography tandem mass spectrometry with electrospray ionization (UHPLC-ESI-MS/MS) method has been developed for the quantification of tryptophan and its downstream metabolites from the kynurenine and serotonin pathways. The assay coverage also includes markers of gut health and inflammation, including citrulline and neopterin. The method was designed in 96-well plate format for application in multiday, multiplate clinical and epidemiology population studies. A chromatographic cycle time of 7 min enables the analysis of two 96-well plates in 24 h. To protect chromatographic column lifespan, samples underwent a two-step extraction, using solvent protein precipitation followed by delipidation via solid-phase extraction (SPE). Analytical validation reported accuracy of each analyte <20% for the lowest limit of quantification and <15% for all other quality control (QC) levels. The analytical precision for each analyte was 2.1–12.9%. To test the applicability of the method to multiplate and multiday preparations, a serum pool underwent periodic repeatanalysis during a run consisting of 18 plates. The % CV (coefficientof variation) values obtained for each analyte were <15%. Additionalbiological testing applied the assay to samples collected from healthycontrol participants and two groups diagnosed with inflammatory boweldisease (IBD) (one group treated with the anti-inflammatory 5-aminosalicylicacid (5-ASA) and one group untreated), with results showing significantdifferences in the concentrations of picolinic acid, kynurenine, andxanthurenic acid. The short analysis time and 96-well plate formatof the assay makes it suitable for high-throughput targeted UHPLC-ESI-MS/MSmetabolomic analysis in large-scale clinical and epidemiological populationstudies.