High-Resolution Metabolomics of 50 Neurotransmitters and Tryptophan Metabolites in Feces, Serum, and Brain Tissues Using UHPLC-ESI-Q Exactive Mass Spectrometry

Recent evidence indicates that tryptophan metabolites and neurotransmitters are potential mediators of the microbiome-gut-brain interaction. Here, a high-resolution ultra-high performance liquid chromatography-electrospray ionization tandem mass spectrometry (UHPLC-ESI-MS/MS) assay was developed and validated for quantifying 50 neurotransmitters, tryptophan metabolites, and bacterial indole derivatives in mouse serum, feces, and brain. The lower limit of quantitation for the 50 compounds ranged from 0.5 to 100 nmol/L, and sample preparation procedures were adapted for individual compounds to allow quantitation within linearity of the assay with a correlation coefficient >0.99. Reproducibility was tested by intra- and interday precision and accuracy of analysis: intra- and interday precision at the lower limit of quantitation was less than 20% for all compounds, with over two-thirds of the compounds achieving an interday precision below 10%, while the interday accuracy at the lower limit of quantitation ranged from 82.3 to 128.0% for all compounds. The analyte recovery was assessed based on sample-spiked stable-isotope-labeling standards, illustrating a need to consider matrix-specific recovery discrepancies when performing interorgan comparison. Carryover was evaluated by intermittent solvent blank injection. The assay was successfully applied to determining the concentration profiles of neurotransmitter and tryptophan metabolites in serum, feces, and brain of conventionally raised specific pathogen-free (SPF) C57BL/6 mice. Our method may serve as a useful analytical resource for investigating the roles of tryptophan metabolism and neurotransmitter signaling in host-microbiota interaction.

Automated summary

Recent evidence suggests that tryptophan metabolites and neurotransmitters play potential roles in the microbiome-gut-brain interaction. A high-resolution UHPLC-ESI-MS/MS assay for quantifying 50 compounds in mouse serum, feces, and brain was developed and validated, demonstrating good reproducibility and accuracy. This method provides a useful analytical resource for investigating the roles of tryptophan metabolism and neurotransmitter signaling in host-microbiota interaction.