Urinary metabolite profiling identifies novel colonic metabolites and conjugates of phenolics in healthy volunteers

Scope: The colonic metabolism of dietary flavonoids, phenolic acids and their phenolic metabolites is complex and many metabolites and conjugates have not yet been unambiguously identified in humans. Methods and results: Urine samples from nine healthy human volunteers obtained after the ingestion of a puree of five (poly)phenol-rich berry fruits were analysed using LC-Orbitrap MS to provide a preliminary indication of possible metabolites based on exact mass. In most cases, the identity of compounds was confirmed using standards produced either chemically or enzymically followed by analysis using LC-triple quadrupole MS. Sulphated, glucuronidated and methylated forms of catechol, pyrogallol and protocatechuic acid mostly appeared in urine after 8 h, suggesting colonic metabolism. Gallic acid and (-)-epicatechin conjugates appeared mainly before 4 h, indicative of absorption from the small intestine. Conjugates of ferulic, caffeic, and vanillic acid appeared at intermediate times. Conclusion: We have positively identified metabolites and conjugates, some novel, in the urine of healthy volunteers after intake of multiple phenolics from a mixed puree from berry fruits, with each being excreted at specific and signature times. Some of these compounds could potentially be used as biomarkers of fruit intake. The possible biological activities of these colonic metabolites require further assessment.