LC-MS/MS based molecular networking approach for the identification of cocoa phenolic metabolites in human urine

Dietary phenolic compounds are often transformed by gut microbiota prior to absorption. This transformation may modify their structures, producing novel gut flora metabolites associated with numerous health benefits. Traditional mass spectrometry (MS) based approaches for assessing dietary exposure of cocotea (cocoa, coffee and tea) products provided very little information about the modification and fate of dietary phenolics after ingestion, mainly due to limitation of complex sample nature and their data analyses. Mass spectrometry techniques are well-suited to a high-throughput characterization of natural products, however, analyzing MS based data of complex biological matrix is still considered a challenge. In order to overcome such limitations and simplify the analysis of complex MS data, a cocotea based human trial was conducted where MS based molecular networking approach was implemented. To demonstrate the utility of this approach in one of the specific cocotea diets, we have applied it to a diverse collection of human (n = 15) urine samples, who consumed cocoa rich in polyphenols over a 48-h period. This approach illustrated the power of the new strategy, allowing the rapid identification of new analogues of cocoa metabolites after human consumption. Analysis of human urine samples after cocoa consumption revealed (by assignment of unknown metabolites based on the network similarities) that monomeric flavanols are mainly absorbed and transformed directly into their glucuronide and sulfated moieties. Subsequently, the hydroxy and methoxy phenyl-g-velerolactone as well as their smaller metabolites (such as hydroxyphenyl valeric acids, hydroxy and methoxy phenyl propionic acids and their derivates) are indicative of bacterial metabolism of cocoa major flavanols. For the first time, our study exemplifies and highlight the implementation of MS based molecular networking approach in illustrating the tracking of various structural motifs of ingested cocoa phenolics in human based study.