Structural elucidation and quantification of phenolic conjugates present in human urine after tea intake

J.J.J. van der Hooft, R.C.H. de Vos, V. Mihaleva, R.J. Bino, L.O. Ridder, N. de Roo, D.M. Jacobs, J.P.M. van Duynhoven, J.J.M. Vervoort

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104 Citations (Scopus)


In dietary polyphenol exposure studies, annotation and identification of urinary metabolites present at low (micromolar) concentrations are major obstacles. In order 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-¿-valerolactone, three urolithin glucuronides, and indole-3-acetic acid glucuronide. We also obtained 26 hours of 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 low abundant phenolic conjugates down to nanomoles of trapped amounts of metabolite corresponding to micromolar metabolite concentrations in urine
Original languageEnglish
Pages (from-to)7263-7271
JournalAnalytical Chemistry
Issue number16
Publication statusPublished - 2012


  • tandem mass-spectrometry
  • green tea
  • black tea
  • metabolite identification
  • ellagic acid
  • metabolomics
  • polyphenols
  • nmr
  • ingestion
  • phytochemicals


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