Analysis of human gut model metabolites by GCxGC-TOF

Dietary phenolic compounds are plant derived secondary metabolites, which can be divided into flavonoids, phenolic acids, stilbenes, tannins and lignans. Catechins can be found as monomers e.g. in tea or as condensed forms, proanthocyanidins (PA), in bilberries, apples, grapes and beverages derived from those fruits. PAs are formed from (+)-catechin and (-)-epicatechin units. Phenolic compounds are ubiquitous metabolites that are metabolised extensively during uptake. It is possible that a major proportion of the metabolites are formed in the colon by microbiota. For example lignans are converted to enterodiol and enterolactone, flavonols are converted to phenylacetic acids and flavanols and flavanones to phenylpropionic acid derivatives. These metabolites, predominantly small phenolic acids, have been identified in human plasma and urine. The residence time of the colonic metabolites is longer than the original structures from plant foods. Epidemiological studies have shown that high concentration of enterolactone is associated to lowered risk of chronic diseases like cancer and cardiovascular disease. Similar evidence for flavanoids and other phenolic compounds is lacking. Identification of diverse flavonoid metabolites is needed. This work aims to compare the in vitro microbial metabolism of (+)-catechin and (-)-epicatechin units using human faecal microbiota as an inoculum to identify their main degradation products. Pure (+)-catechin and (-)-epicatechin were fermented with pooled human faecal microbiota in strictly anaerobic conditions. The sample preparation steps include extraction with ethyl acetate and silylation with MSTFA. GCxGC-TOF analytical method was developed for the analysis of the microbial metabolites. We will describe the method including data processing steps and the preceding sample preparation steps for the analysis of microbial metabolites of (+)-catechin and (-)-epicatechin. The compounds include e.g. hydroxyphenyl propionic and acetic acid derivatives. Financial support of the project STREP-FLAVO (Food-CT-2004-513960) is gratefully acknowledged.