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)-gamma-valerolactone and 5-(3'-methoxy-4'-hydroxyphenyl)-gamma-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)-gamma-valerolactone, whereas only the two latter metabolites showed a significant increase in fasting plasma. The results found herein indicate that 5-(3',4'-dihydroxyphenyl)-gamma-valerolactone and hydroxyphenylacetic acids could be good biomarkers of the regular consumption of cocoa and therefore, of flavanol-rich foods.