Chromatographic resolution and reliable quantification of eleven endogenous and microbiota-derived tryptophan metabolites in human plasma by HPLC–MS/MS

Tryptophan metabolism plays a central role in host–microbiota interactions and immune regulation, yet the simultaneous quantification of metabolites from the kynurenine, serotonin, and indole pathways remains analytically challenging due to their diverse physicochemical properties and wide dynamic concentration ranges. Here, we report an efficient, derivatization-free HPLC–MS/MS workflow for the targeted quantification of eleven key tryptophan-derived metabolites in human plasma. The method employs a biphenyl stationary phase to achieve robust chromatographic resolution of structurally similar analytes, including compounds known to co-elute in C18 stationary phase, improving chromatographic selectivity and minimizing co-elution of structurally related analytes. Sample preparation requires only 50 µL of plasma and relies on a simple protein-precipitation step, allowing high throughput. The method demonstrated strong performance in terms of sensitivity, accuracy, and reproducibility, supported by extensive internal standardization. The combination of multi-pathway coverage, analytical cycle, and suitability for human plasma positions this workflow as a valuable tool for monitoring gut-related metabolic signatures in both research studies and potential clinical applications.