Targeted metabolic profiling of phenolics in urine and plasma after regular consumption of cocoa by liquid chromatography–tandem mass spectrometry

The biological properties of cocoa (Theobroma cacao L.) polyphenols are strictly dependent on their bioavailability. A long-term cocoa feeding trial was performed with subjects at high risk for cardiovascular disease. Subjects (n = 42) received two sachets of 20 g of cocoa powder/day with 250 mL of skimmed milk each, or only 500 mL/day of skimmed milk, both for two 4-week periods. The phenolic metabolic profile including phase II conjugated metabolites and phenolic acids derived from the intestinal microbiota was determined by LC-MS/MS in both 24-h urine and fasting plasma. The analysis of 24-h urine revealed significant increases of phase II metabolites, including glucuronides and sulfate conjugates of (−)-epicatechin, O-methyl-epicatechin, 5-(3′,4′-dihydroxyphenyl)-γ-valerolactone and 5-(3′-methoxy-4′-hydroxyphenyl)-γ-valerolactone, after regular cocoa intake. In the case of plasma, only glucuronide conjugates of dihydroxyphenylvalerolactones increased. Regular consumption of cocoa also resulted in a significant increase in the urinary excretion of colonic microbial-derived phenolic metabolites, including vanillic, 3,4-dihydroxyphenylacetic and 3-hydroxyphenylacetic acids, and particularly 5-(3′,4′-dihydroxyphenyl)-γ-valerolactone, whereas only the two latter metabolites showed a significant increase in fasting plasma. The results found herein indicate that 5-(3′,4′-dihydroxyphenyl)-γ-valerolactone and hydroxyphenylacetic acids could be good biomarkers of the regular consumption of cocoa and therefore, of flavanol-rich foods.