Targeted quantification of 25 tryptophan metabolites across three metabolic pathways in serum by ultra-high-performance liquid chromatography coupled with quadrupole orbitrap high-resolution mass spectrometry.

Tryptophan metabolism encompasses three interconnected pathways, namely the kynurenine, serotonin, and gut microbiota-mediated indole pathways, which contribute to immune regulation, neural signalling, and host-microbiota interactions. Quantitative analysis of metabolites spanning these pathways remains challenging because of their diverse physicochemical properties, broad concentration ranges, and matrix interference in biological samples. In this study, a targeted analytical method based on ultra-high-performance liquid chromatography coupled with quadrupole Orbitrap high-resolution mass spectrometry (UPLC-Q-Orbitrap HRMS) was developed for the quantification of 25 representative tryptophan metabolites in serum. Chromatographic and mass spectrometric conditions were systematically optimized, and the target metabolites were analysed using parallel reaction monitoring. All analytes showed linear responses within their respective calibration ranges, with coefficients of determination (R²) of at least 0.9990. The limits of detection and quantification ranged from 0.1 to 300 ng/mL and from 0.3 to 500 ng/mL, respectively. Intra-day and inter-day precision ranged from 0.2% to 6.9% and from 0.4% to 14.0%, respectively, while accuracy ranged from 85.2% to 117.5%. Extraction recoveries ranged from 80.6% to 108.2%, and matrix-effect values ranged from 72.9% to 119.9%, indicating analyte-dependent ion suppression or enhancement. The method was applied to serum samples from humans, rats, specific-pathogen-free mice, and germ-free mice. Comparative profiling revealed exploratory differences in the relative distributions of tryptophan metabolites among the four serum sources. Overall, the method provides broad analytical coverage of the three major tryptophan metabolic pathways and a useful approach for fit-for-purpose quantification and exploratory comparison of serum tryptophan metabolites.