Regioselective solvent-phase deuteration of polyphenolic compounds informs their identification by mass spectrometry

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Liquid chromatography-mass spectrometry (LC-MS) is a highly sensitive tool for the analysis of polyphenolic compounds in complex food and beverage matrices. However, the high degree of isomerism among polyphenols in general often complicates this approach, especially for identification of novel compounds. Here, we explore the utility of mild acid-catalyzed deuterium (MACD) labeling via electrophilic aromatic substitution as a complementary method for informing polyphenolic compound structure elucidation. To prevent hydrolysis of acid-labile glycosidic linkages, optimal reaction conditions that maximize regioselective hydrogen/deuterium (H/D) exchange of aromatic protons while preserving compound integrity were characterized (60°C, pH 3.0, 72 h). Under these conditions, standard compounds varying in the number and position of hydroxyl, glycosyl, and methyl groups about their aromatic core structure produced distinguishable H/D exchange patterns. The applicability of this method for the analysis of complex mixtures was demonstrated in red wine where the extent of deuterium exchange, together with accurate mass information, led to the putative identification of an unknown compound. The identification was further supported by tandem MS (MS/MS) data, which matched conclusively to the same compound in the Metlin LC-MS/MS library. With the capacity to discriminate between select isomeric forms, MACD labeling provides structural information that complements accurate mass and tandem mass spectral measurements for informing the identification of polyphenolics by MS.

Original languageEnglish (US)
Pages (from-to)76-85
Number of pages10
JournalAnalytical Biochemistry
Issue number1
StatePublished - May 1 2014

Bibliographical note

Funding Information:
This work was funded by National Science Foundation (NSF) Plant Genome Research Program grants IOS-0923960 and IOS-1238812 . We thank the University of Minnesota Office of the Vice President for Research and the Grant in Aid program for salary support (M.R.R.). We further acknowledge supplemental support from the Gordon and Margaret Bailey Endowment for Environmental Horticulture and the Minnesota Agricultural Experiment Station. We also thank Gary Gardner (University of Minnesota) for helpful discussions throughout the project.


  • Compound identification
  • Deuterium labeling
  • Flavonoid
  • Mass spectrometry
  • Metabolomics
  • Polyphenolics
  • Stilbenoid


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