Quantitative profiling of aromatic amino acids and their host-microbial co-metabolites in human urine via UPLC-MS/MS

The aromatic amino acids tryptophan, tyrosine and phenylalanine are involved in many biochemical pathways and their metabolism and co-metabolism by the human and the gut microbiota results in the production of a number of metabolites. Many of these are phenols which are excreted in the urine after either sulfation or glucuronidation by the host. These metabolic processes can be dysregulated due to factors such as inflammation, disease, dietary and/or pharmaceutical interventions. Validated, quantitative methods for the analysis of aromatic amino acids and their metabolites may therefore provide insights into host-gut microbiota symbiosis and its association with pathological conditions. As sulfation is often the favored form of conjugation for phenolic compounds, the development of analytical methods would benefit from access to the sulfate conjugates as reference standards, which unfortunately are scarce. To overcome this limitation nine sulfate conjugates were synthesized using standard chemical routes and were subsequently purified by preparative HPLC. Following structure confirmation (¹H NMR and MS/MS analysis) the standards were used along with 24 other analytes for the development and validation of a quantitative LC-MS/MS-based assay. The method used a CSH Phenyl-Hexyl column to separate and analyze 33 aromatic amino acids and their metabolites in urine. The method was validated and subsequently applied to the analysis of urine obtained from 20 healthy individuals to obtain information on the relevant concentrations in human urine.