Use of LC-MS for the quantitative analysis of (poly)phenol metabolites does not necessarily yield accurate results: Implications for assessing existing data and conducting future research

Plant-derived, dietary (poly)phenols have potential effects on disease-risk reduction and primary disease prevention. The characterization of (poly)phenol absorption, distribution, metabolism and excretion (ADME) is recognized as crucial step to further advance nutritional and biomedical research of these compounds; and given that (poly)phenols are extensively metabolized after ingestion, accurate assessments of their in vivo metabolites is required. It has become common practice to use unmetabolized parent compounds as reference standards when quantifying (poly)phenol metabolites by LC-MS, although little is known about the accuracy of this approach. To investigate this situation with routinely used LC-MS conditions, the signal yielded by the flavan-3-ol (−)-epicatechin was compared to those of authentic standards of its phase II and microbiota-derived metabolites. The results obtained revealed underestimations up to 94% and overestimations up to 113% of individual epicatechin metabolites. Inaccurate quantitative estimates were also obtained when phase II metabolites of other (poly)phenols were quantified by reference to their unmetabolized parent compounds. This demonstrates the importance of using structurally-identical authentic metabolites as reference compounds when quantifying (poly)phenol metabolites by LC-MS. This is of importance, not just to the accuracy of ADME studies, but for the identification and validation of (poly)phenol metabolites as biomarkers of intake in epidemiological studies. [Display omitted] •The use of quantitative LC-MS-based (poly)phenol metabolite analysis is increasing.•Utilization of unmetabolized parent compounds as analytical standards is common.•We show that this approach carries high risk of generating inaccurate results.•Authentic metabolites should be used as analytical reference standards.•This is of high importance to bioavailability studies and epidemiological research.