Structural Elucidation and Quantification of Phenolic Conjugates Present in Human Urine after Tea Intake

In dietary polyphenol exposure studies, annotation and
identification
of urinary metabolites present at low (micromolar) concentrations
are major obstacles. To determine the biological activity of specific
components, it is necessary to have the correct structures and the
quantification of the polyphenol-derived conjugates present in the
human body. We present a procedure for identification and quantification
of metabolites and conjugates excreted in human urine after single
bolus intake of black or green tea. A combination of a solid-phase
extraction (SPE) preparation step and two high pressure liquid chromatography
(HPLC)-based analytical platforms was used, namely, accurate mass
fragmentation (HPLC-FTMSn) and mass-guided SPE-trapping
of selected compounds for nuclear magnetic resonance spectroscopy
(NMR) measurements (HPLC-TOFMS-SPE-NMR). HPLC-FTMSn analysis
led to the annotation of 138 urinary metabolites, including 48 valerolactone
and valeric acid conjugates. By combining the results from MSn fragmentation with the one-dimensional (1D)-1H
NMR spectra of HPLC-TOFMS-SPE-trapped compounds, we elucidated the
structures of 36 phenolic conjugates, including the glucuronides of
3′,4′-di- and 3′,4′,5′-trihydroxyphenyl-γ-valerolactone,
three urolithin glucuronides, and indole-3-acetic acid glucuronide.
We also obtained 26 h-quantitative excretion profiles for specific
valerolactone conjugates. The combination of the HPLC-FTMSn and HPLC-TOFMS-SPE-NMR platforms results in the efficient identification
and quantification of less abundant phenolic conjugates down to nanomoles
of trapped amounts of metabolite corresponding to micromolar metabolite
concentrations in urine.