Analytical and Bioanalytical Chemistry

, Volume 409, Issue 9, pp 2477–2488 | Cite as

Enumeration of carboxyl groups carried on individual components of humic systems using deuteromethylation and Fourier transform mass spectrometry

  • Alexander Zherebker
  • Yury Kostyukevich
  • Alexey Kononikhin
  • Oleg Kharybin
  • Andrey I. Konstantinov
  • Kirill V. Zaitsev
  • Eugene Nikolaev
  • Irina V. Perminova
Research Paper


Here, we report a novel approach to enumeration of carboxylic groups carried by individual molecules of humic substances using selective chemical modification and isotopic labeling (deuteromethylation) and high-resolution electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI FTICR MS). Esterification was conducted with a use of thionyl chloride–deuteromethanol reagent under mild conditions to avoid transesterification. The deuteromethylated products were subjected to solid phase extraction using PPL Bond Elute cartridges prior to FTICR MS analysis. An amount of carboxyl groups in the individual molecular component was estimated from the length of identified deuteromethylation series. The method allowed for discerning between compounds with close elemental compositions possessing different protolytic properties. We found that different carboxylic moieties occupy distinct regions in molecular space of humic substances (HS) projected onto Van Krevelen diagram. These locations do not depend on the source of the humic material and can be assigned to carboxyl-rich alicyclic molecules (5 to 6 COOH), hydrolyzable tannins (3-4 COOH), lignins (1 to 2 COOH), condensed tannins and lignans (0 to 1 COOH), and carbohydrates (0 COOH). At the same time, the alignment pattern of these carboxylated species along the structural evolution lines in Van Krevelen diagrams was characteristic to the specific transformation processes undergone by the humic materials in the different environments. The obtained data enable mapping of molecular ensemble of HS with regards to their specific acidic compartments and might be used for directed fractionation of HS.

Graphical abstract

Selective isotopic labeling followed by FTICR MS enables discerning between humic molecules with close elemental compositions carrying different numbers of carboxylic groups.


Humic substances FTICR MS Isotopic labeling Modification Carboxylic group 1H NMR spectroscopy 



We would like to acknowledge the Interdisciplinary Research Centre for Magnetic Tomography and Spectroscopy of the Lomonosov MSU and personally Dr. Borisova N.E. for the assistance in 1H NMR analysis.

This work was partially supported by the Russian Foundation for Basic Research (grants 16-04-01753-a and 16-33-00914-mol-a). 1H NMR spectroscopy studies were supported by the Russian Science Foundation (grant 16-14-00167), and the high-resolution mass spectrometry study was funded by the Russian Science Foundation grant no. 14-24-00114.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.

Supplementary material

216_2017_197_MOESM1_ESM.pdf (1.7 mb)
ESM 1 (PDF 1684 kb)


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Copyright information

© Springer-Verlag Berlin Heidelberg 2017

Authors and Affiliations

  • Alexander Zherebker
    • 1
  • Yury Kostyukevich
    • 2
    • 3
    • 4
    • 5
  • Alexey Kononikhin
    • 3
    • 4
    • 5
  • Oleg Kharybin
    • 2
    • 5
  • Andrey I. Konstantinov
    • 1
  • Kirill V. Zaitsev
    • 1
  • Eugene Nikolaev
    • 2
    • 3
    • 4
    • 5
  • Irina V. Perminova
    • 1
  1. 1.Department of ChemistryLomonosov Moscow State UniversityMoscowRussia
  2. 2.Skolkovo Institute of Science and TechnologySkolkovoRussia
  3. 3.Orekhovich Institute of Biomedical ChemistryRussian Academy of Medical SciencesMoscowRussia
  4. 4.Institute for Energy Problems of Chemical Physics of RASMoscowRussia
  5. 5.Moscow Institute of Physics and TechnologyDolgoprudnyiRussia

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