Untargeted Metabolomics Analytical Strategy Based on Liquid Chromatography/Electrospray Ionization Linear Ion Trap Quadrupole/Orbitrap Mass Spectrometry for Discovering New Polyphenol Metabolites in Human Biofluids after Acute Ingestion of Vaccinium myrtillus Berry Supplement

  • Claudia Ancillotti
  • Marynka Ulaszewska
  • Fulvio Mattivi
  • Massimo Del BubbaEmail author
Research Article


In this work, liquid chromatography, coupled with an electrospray ionization hybrid linear ion trap quadrupole/Orbitrap mass spectrometry, has been used to accurately identify polyphenol metabolites in human serum and urine after acute ingestion of a V. myrtillus berry supplement. The supplement was obtained by cryo-milling of bilberries, which were freeze-dried within 1 week after their harvesting, to maintain the berry native composition. Thirty-six derivatives of benzoic acids, hydroxyhippuric acids, cinnamic acids, phenylpropionic acids, phenylvaleric acids, phenylpentenoic acids and abscisic acid, together with two berry-native anthocyanins, one flavonol metabolite and two catechol derivatives were putatively identified in the investigated biofluids. The annotated compounds included 13 metabolites, among glucuronides and sulphates of phenylvaleric and phenylpentenoic acids, which have been identified for the first time in human biofluids after ingestion of V. myrtillus berries. It should be emphasized that the presence of phenylvaleric and phenylpentenoic acid derivatives is in agreement with their origin from fruit native flavanol monomers and oligomers, which are widely distributed in Vaccinium berries, but usually overlooked in metabolomics studies regarding bilberry. The identification of these compounds confirmed the key-role of untargeted metabolomics approach in the discovery of new metabolites which could be biologically active.

Graphical Abstract


Untargeted metabolomics High-resolution mass spectrometry Human serum Human urine Vaccinium myrtillus Polyphenol metabolites 



The authors wish to thank Prof. Riccardo Bartoletti from the Department of Translational Research on New Technologies in Medicine and Surgery of University of Pisa for his continuous and valuable support. We thank volunteers for participating in the study. We also thank Nicola La Monica (Sciex, Framingham, MA, USA) for XCMSplus license and Paul Benton (Scripps Center for Metabolomics, The Scripps Research Institute) for the support in data elaboration. CA and MDB acknowledge the support of the Regione Toscana and the private companies “Il Baggiolo S.r.l.,” Danti Giampiero & C. S.n.c.,” “Azienda Agricola Il Sottobosco,” and “Farmaceutica MEV S.r.l.,” through the “PRAF Misura 1.2. e)” grant. FM and MU acknowledge the support by the Autonomous Province of Trento, Italy, “ADP 2017” Project.

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflict of interest.

Supplementary material

13361_2018_2111_MOESM1_ESM.docx (593 kb)
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Copyright information

© American Society for Mass Spectrometry 2018

Authors and Affiliations

  • Claudia Ancillotti
    • 1
  • Marynka Ulaszewska
    • 2
  • Fulvio Mattivi
    • 2
    • 3
  • Massimo Del Bubba
    • 1
    Email author
  1. 1.Department of ChemistryUniversity of FlorenceFlorenceItaly
  2. 2.Department of Food Quality and Nutrition, Research and Innovation CentreFondazione Edmund Mach (FEM)TrentoItaly
  3. 3.Center for Agriculture Food and EnvironmentUniversity of TrentoSan Michele all’AdigeItaly

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