A stable isotope dilution approach to analyze ferulic acid oligomers in plant cell walls using liquid chromatography-tandem mass spectrometry

  • Martin Waterstraat
  • Mirko BunzelEmail author
Paper in Forefront


Diferulic (DFA) and triferulic acids (TriFA) acylate and cross-link plant cell wall polysaccharides, thereby being important structural elements within the cell wall, also affecting physicochemical properties of the isolated polysaccharides. Due to the large number of potential regio- and configurational isomers and due to the fact that oligoferulic acids are not commercially available as standard compounds, analysis of oligoferulic acids after alkaline hydrolysis is challenging. Eighteen di- and triferulic acids were synthesized both non-labeled as well as 13C-labeled. By using these standard compounds, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) (electrospray ionization, negative mode)-based stable isotope dilution approach was developed, fully validated and applied to plant materials. Whereas this stable isotope dilution approach is most useful to analyze plant materials with complex matrices (especially lignified tissues), less complicated matrices may not require this approach. Therefore, an alternative LC-MS/MS-based method that is based on using a single internal standard compound only was developed, too, validated, and compared to the stable isotope dilution approach. Although the stable isotope dilution approach appears to be superior, plant samples with simple matrices can also be screened by using the single internal standard method developed here.


Ferulate oligomers Phenolic cross-links Dietary fiber LC-MS/MS Isotopic labeling Synthetic strategies 



Contents of this article first appeared in M. Waterstraat’s doctoral thesis [45]. We thank Ina Dix and Emine Sager for conducting and interpreting X-ray crystallography measurements.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. 1.
    Harris PJ, Trethewey JAK. The distribution of ester-linked ferulic acid in the cell walls of angiosperms. Phytochem Rev. 2010;9:19–33.CrossRefGoogle Scholar
  2. 2.
    Ishii T. Structure and functions of feruloylated polysaccharides. Plant Sci. 1997;127:111–27.CrossRefGoogle Scholar
  3. 3.
    Allerdings E, Ralph J, Steinhart H, Bunzel M. Isolation and structural identification of complex feruloylated heteroxylan side-chains from maize bran. Phytochemistry. 2006;67:1276–86.CrossRefGoogle Scholar
  4. 4.
    Bunzel M, Ralph J, Steinhart H. Association of non-starch polysaccharides and ferulic acid in grain amaranth (Amaranthus caudatus L.) dietary fiber. Mol Nutr Food Res. 2005;49:551–9.CrossRefGoogle Scholar
  5. 5.
    Colquhoun IJ, Ralet MC, Thibault JF, Faulds CB, Williamson G. Structure identification of feruloylated oligosaccharides from sugar-beet pulp by NMR-spectroscopy. Carbohydr Res. 1994;263:243–56.CrossRefGoogle Scholar
  6. 6.
    Ishii T, Tobita T. Structural characterization of feruloyl oligosaccharides from spinach-leaf cell walls. Carbohydr Res. 1993;248:179–90.CrossRefGoogle Scholar
  7. 7.
    Wefers D, Gmeiner BM, Tyl CE, Bunzel M. Characterization of diferuloylated pectic polysaccharides from quinoa (Chenopodium quinoa WILLD.). Phytochemistry. 2015;116:320–8.CrossRefGoogle Scholar
  8. 8.
    Bunzel M. Chemistry and occurrence of hydroxycinnamate oligomers. Phytochem Rev. 2010;9:47–64.CrossRefGoogle Scholar
  9. 9.
    Nino-Medina G, Carvajal-Millan E, Rascon-Chu A, Marquez-Escalante JA, Guerrero V, Salas-Munoz E. Feruloylated arabinoxylans and arabinoxylan gels: structure, sources and applications. Phytochem Rev. 2010;9:111–20.CrossRefGoogle Scholar
  10. 10.
    Micard V, Thibault J-F. Oxidative gelation of sugar-beet pectins: use of laccases and hydration properties of the cross-linked pectins. Carbohydr Polym. 1999;39:265–73.CrossRefGoogle Scholar
  11. 11.
    Waldron KW, Parker ML, Smith AC. Plant cell walls and food quality. Comp Rev Food Sci Food Safety. 2003;2:128–46.CrossRefGoogle Scholar
  12. 12.
    Vogel B, Gallaher DD, Bunzel M. Influence of cross-linked arabinoxylans on the postprandial blood glucose response in rats. J Agric Food Chem. 2012;60:3847–52.CrossRefGoogle Scholar
  13. 13.
    Ford CW, Hartley RD. GC/MS characterisation of cyclodimers from p-coumaric and ferulic acids by photodimerisation - a possible factor influencing cell wall biodegradability. J Sci Food Agric. 1989;46:301–10.CrossRefGoogle Scholar
  14. 14.
    Ford CW, Hartley RD. Cyclodimers of p-coumaric and ferulic acids in the cell walls of tropical grasses. J Sci Food Agric. 1990;50:29–43.CrossRefGoogle Scholar
  15. 15.
    Dobberstein D, Bunzel M. Identification of ferulate oligomers from corn stover. J Sci Food Agric. 2010;90:1802–10.Google Scholar
  16. 16.
    Geissmann T, Neukom H. Vernetzung von Phenolcarbonsäureestern von Polysacchariden durch oxydative phenolische Kupplung. Helv Chim Acta. 1971;54:1108–12.CrossRefGoogle Scholar
  17. 17.
    Ralph J, Quideau S, Grabber JH, Hatfield RD. Identification and synthesis of new ferulic acid dehydrodimers present in grass cell walls. J Chem Soc Perkin Trans. 1994;1(1):3485–98.CrossRefGoogle Scholar
  18. 18.
    Schatz PF, Ralph J, Lu F, Guzei IA, Bunzel M. Synthesis and identification of 2,5-bis-(4-hydroxy-3-methoxyphenyl)-tetrahydrofuran-3,4-dicarboxylic acid, an unanticipated ferulate 8-8-coupling product acylating cereal plant cell walls. Org Biomol Chem. 2006;4:2801–6.CrossRefGoogle Scholar
  19. 19.
    Bunzel M, Ralph J, Funk C, Steinhart H. Isolation and identification of a ferulic acid dehydrotrimer from saponified maize bran insoluble fiber. Eur Food Res Technol. 2003;217:128–33.CrossRefGoogle Scholar
  20. 20.
    Bunzel M, Ralph J, Funk C, Steinhart H. Structural elucidation of new ferulic acid-containing phenolic dimers and trimers isolated from maize bran. Tetrahedron Lett. 2005;46:5845–50.CrossRefGoogle Scholar
  21. 21.
    Funk C, Ralph J, Steinhart H, Bunzel M. Isolation and structural characterisation of 8–O–4/8–O–4- and 8–8/8–O–4-coupled dehydrotriferulic acids from maize bran. Phytochemistry. 2005;66:363–71.CrossRefGoogle Scholar
  22. 22.
    Bunzel M, Ralph J, Brüning P, Steinhart H. Structural identification of dehydrotriferulic and dehydrotetraferulic acids from insoluble maize bran fiber. J Agric Food Chem. 2006;54:6409–18.CrossRefGoogle Scholar
  23. 23.
    Waterstraat M, Bunzel D, Bunzel M. Identification of 8-O-4/8-5(cyclic)- and 8-8(cyclic)/5-5-coupled dehydrotriferulic acids, naturally occurring in cell walls of mono- and dicotyledonous plants. J Agric Food Chem. 2016;64:7244–50.CrossRefGoogle Scholar
  24. 24.
    Waterstraat M, Bunzel M. A multi-step chromatographic approach to purify radically generated ferulate oligomers reveals naturally occurring 5-5/8-8(cyclic)-, 8-8(noncyclic)/8-O-4-, and 5-5/8-8(noncyclic)-coupled dehydrotriferulic acids. Front Chem. 2018;6:190.CrossRefGoogle Scholar
  25. 25.
    Waldron KW, Parr AJ, Ng A, Ralph J. Cell wall esterified phenolic dimers: identification and quantification by reverse phase high performance liquid chromatography and diode array detection. Phytochem Anal. 1996;7:305–12.CrossRefGoogle Scholar
  26. 26.
    Vismeh R, Lu F, Chundawat SPS, Humpula JF, Azarpira A, Balan V, et al. Profiling of diferulates (plant cell wall cross-linkers) using ultrahigh-performance liquid chromatography-tandem mass spectrometry. Analyst. 2013;138:6683–92.CrossRefGoogle Scholar
  27. 27.
    Bunzel M, Ralph J, Marita JM, Hatfield RD, Steinhart H. Diferulates as structural components in soluble and insoluble cereal dietary fibre. J Sci Food Agric. 2001;81:653–60.CrossRefGoogle Scholar
  28. 28.
    Dobberstein D, Bunzel M. Separation and detection of cell wall-bound ferulic acid dehydrodimers and dehydrotrimers in cereals and other plant materials by reversed phase high-performance liquid chromatography with ultraviolet detection. J Agric Food Chem. 2010;58:8927–35.CrossRefGoogle Scholar
  29. 29.
    Jilek ML, Bunzel M. Dehydrotriferulic and dehydrodiferulic acid profiles of cereal and pseudocereal flours. Cereal Chem. 2013;90:507–14.CrossRefGoogle Scholar
  30. 30.
    Fieser LF, Fieser M. Reagents for organic synthesis, vol. 1. New York: John Wiley & Sons; 1967.Google Scholar
  31. 31.
    Lu F, Wei L, Azarpira A, Ralph J. Rapid syntheses of dehydrodiferulates via biomimetic radical coupling reactions of ethyl ferulate. J Agric Food Chem. 2012;60:8272–7.CrossRefGoogle Scholar
  32. 32.
    Bunzel M, Funk C, Steinhart H. Semipreparative isolation of dehydrodiferulic and dehydrotriferulic acids as standard substances from maize bran. J Sep Sci. 2004;27:1080–6.CrossRefGoogle Scholar
  33. 33.
    Packert M, Steinhart H. Separation and identification of some monomeric and dimeric phenolic acids by a simple gas chromatographic method using a capillary column and FID-MSD. J Chromatogr Sci. 1995;33:631–9.CrossRefGoogle Scholar
  34. 34.
    Zeller WE, Schatz PF. Synthesis of monomethyl 5,5′-dehydrodiferulic acid. Tetrahedron Lett. 2015;56:1076–9.CrossRefGoogle Scholar
  35. 35.
    Ralph J, Garcia-Conesa MT, Williamson G. Simple preparation of 8-5-coupled diferulate. J Agric Food Chem. 1998;46:2531–2.CrossRefGoogle Scholar
  36. 36.
    Bunzel M, Heuermann B, Kim H, Ralph J. Peroxidase-catalyzed oligomerization of ferulic acid esters. J Agric Food Chem. 2008;56:10368–75.CrossRefGoogle Scholar
  37. 37.
    Lu F, Marita JM, Lapierre C, Jouanin L, Morreel K, Boerjan W, et al. Sequencing around 5-hydroxyconiferyl alcohol-derived units in caffeic acid O-methyltransferase-deficient poplar lignins. Plant Physiol. 2010;153:569–79.CrossRefGoogle Scholar
  38. 38.
    Hu K, Jeong J-H. A convenient synthesis of an anti-Helicobacter pylori agent, dehydrodiconiferyl alcohol. Arch Pharm Res. 2006;29:563–5.CrossRefGoogle Scholar
  39. 39.
    Ralph J, Bunzel M, Marita JM, Hatfield RD, Lu F, Kim H, et al. Peroxidase-dependent cross-linking reactions of p-hydroxycinnamates in plant cell walls. Phytochem Rev. 2004;3:79–96.CrossRefGoogle Scholar
  40. 40.
    Ichikawa M, Takahashi M, Aoyagi S, Kibayashi C. Total synthesis of (−)-incarvilline, (+)-incarvine C, and (−)-incarvillateine. J Am Chem Soc. 2004;126:16553–8.CrossRefGoogle Scholar
  41. 41.
    Carpita N, McCann M. The cell wall. In: Buchanan BB, Gruissem W, Jones RL, editors. Biochemistry and molecular biology of plants. Waldorf, MD, USA: American Society of Plant Physiologists; 2000. p. 52–108.Google Scholar
  42. 42.
    Brueggemann L, Quapp W, Wennrich R. Test for non-linearity concerning linear calibrated chemical measurements. Accred Qual Assur. 2006;11:625–31.CrossRefGoogle Scholar
  43. 43.
    Gu H, Liu G, Wang J, Aubry A-F, Arnold ME. Selecting the correct weighting factors for linear and quadratic calibration curves with least-squares regression algorithm in bioanalytical LC-MS/MS assays and impacts of using incorrect weighting factors on curve stability, data quality, and assay performance. Anal Chem. 2014;86:8959–66.CrossRefGoogle Scholar
  44. 44.
    Barberousse H, Roiseux O, Robert C, Paquot M, Deroanne C, Blecker C. Analytical methodologies for quantification of ferulic acid and its oligomers. J Sci Food Agric. 2008;88:1494–511.CrossRefGoogle Scholar
  45. 45.
    Waterstraat M (2017) Entwicklung von Massenspektrometrie-basierten Multimethoden zur Bestimmung von Ferulasäure, Oligoferulasäuren und Ferulasäure-Metaboliten. Doctoral Thesis. 2017, Karlsruhe Institute of Technology (KIT).Google Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Food Chemistry and PhytochemistryInstitute of Applied Biosciences, Karlsruhe Institute of Technology (KIT)KarlsruheGermany

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