Elucidation of (-)-epicatechin metabolites after ingestion of chocolate by healthy humans

Lucas Actis-Goretta, Antoine Lévèques, Francesca Giuffrida, Fedor Romanov-Michailidis, Florian Viton, Denis Barron, Montserrat Duenas-Paton, Susana Gonzalez-Manzano, Celestino Santos-Buelga, Gary Williamson, Fabiola Dionisi

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113 Citations (Scopus)


After absorption in the gastrointestinal tract, (-)-epicatechin is extensively transformed into various conjugated metabolites. These metabolites, chemically different from the aglycone forms found in foods, are the compounds that reach the circulatory system and the target organs. Therefore, it is imperative to identify and quantify these circulating metabolites to investigate their roles in the biological effects associated with (-)-epicatechin intake. Using authentic synthetic standards of (-)-epicatechin sulfates, glucuronides, and O-methyl sulfates, a novel LC-MS/MS-MRM analytical methodology to quantify (-)-epicatechin metabolites in biological matrices was developed and validated. The optimized method was subsequently applied to the analysis of plasma and urine metabolites after consumption of dark chocolate, an (-)-epicatechin-rich food, by humans. (-)-Epicatechin-3′-β-d-glucuronide (Cmax 290±49 nM), (-)-epicatechin 3′-sulfate (Cmax 233±60 nM), and 3′-O-methyl epicatechin sulfates substituted in the 4′, 5, and 7 positions were the most relevant (-)-epicatechin metabolites in plasma. When plasmatic metabolites were divided into their substituent groups, it was revealed that (-)-epicatechin glucuronides, sulfates, and O-methyl sulfates represented 33±4, 28±5, and 33±4% of total metabolites (AUC0-24 h), respectively, after dark chocolate consumption. Similar metabolites were found in urine samples collected over 24 h. The total urine excretion of (-)-epicatechin was 20±2% of the amount ingested. In conclusion, we describe the entire metabolite profile and its degree of elimination after administration of (-)-epicatechin-containing food. These results will help us understand more precisely the mechanisms and the main metabolites involved in the beneficial physiological effects of flavanols.

Original languageEnglish
Pages (from-to)787-795
Number of pages9
JournalFree Radical Biology and Medicine
Issue number4
Publication statusPublished - 15 Aug 2012
Externally publishedYes


  • (-)-Epicatechin
  • Chocolate
  • Cocoa
  • Free radicals
  • Humans
  • Metabolites

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