Multiscale workflow for the profiling and identification of urinary food bioactives metabolites Part I: Optimizing...

The relationship between diet, human health, and disease prevention is well-established, with food bioactive compounds (FBs) widely recognized for their beneficial effects. Metabolism is key in transforming precursor FBs molecules and facilitating their circulation in the human body. Urine has proven to be a valuable biofluid for monitoring dietary exposure. However, the low concentrations of FBs metabolites, their chemical variability, and the lack of appropriate reference standards present challenges in metabolite identification. To address these challenges, developing urine preparation methods for scalable metabolite isolation and unambiguous structure elucidation could significantly improve the coverage and accurate annotation of urine metabolites.

Urine samples were collected from a healthy volunteer after hydroxytyrosol (HT) supplementation. Traditional urine pretreatment protocols, such as liquid-liquid extraction (LLE) and solid-phase extraction (SPE), were tested alongside enrichment methods using resins (XAD-4, XAD-7, ion-exchange). Extracts were analyzed in parallel using HPLC-DAD/ELSD, UPLC-HRMS, and NMR to assess profiles and annotate metabolites. Methods were evaluated based on extraction yield, metabolite chemical and biochemical diversity, metabolite coverage, selectivity, as well as cost, ease and time. The most promising protocols were further tested on a larger scale. Among the methods evaluated, XAD-7 resin and LLE (Urine/EtOAc 1:3) showed the best performance. Furthermore, detailed identification of metabolites (endogenous and exogenous) per protocol was performed using LC-HRMS/MS and NMR. Additionally, investigation of each protocol performance in respect to the biochemical pathway in which metabolites are implicated was assessed.

The suggested workflow is compatible with both profiling and isolation set-ups and could provide essential insights into urine metabolome and FBs biotransformation. It ensures confident identification and high coverage of metabolites, providing more complete and accurate interpretation of metabolism studies' results and, therefore, valuable input in profiling approaches towards the role of diet on human health.