Skip to main content
SpringerLink
Log in

Structural elucidation of low abundant metabolites in complex sample matrices

  • Review Article
  • Published:
Metabolomics Aims and scope Submit manuscript

Abstract

Identification of metabolites is a major challenge in biological studies and relies in principle on mass spectrometry (MS) and nuclear magnetic resonance (NMR) methods. The increased sensitivity and stability of both NMR and MS systems have made dereplication of complex biological samples feasible. Metabolic databases can be of help in the identification process. Nonetheless, there is still a lack of adequate spectral databases that contain high quality spectra, but new developments in this area will assist in the (semi-)automated identification process in the near future. Here, we discuss new developments for the structural elucidation of low abundant metabolites present in complex sample matrices. We describe how a recently developed combination of high resolution MS multistage fragmentation (MSn) and high resolution one dimensional (1D)-proton (1H)-NMR of liquid chromatography coupled to solid phase extraction (LC–SPE) purified metabolites can circumvent the need for isolating extensive amounts of the compounds of interest to elucidate their structures. The LC–MS–SPE–NMR hardware configuration in conjunction with high quality databases facilitates complete structural elucidation of metabolites even at sub-microgram levels of compound in crude extracts. However, progress is still required to optimally exploit the power of an integrated MS and NMR approach. Especially, there is a need to improve and expand both MSn and NMR spectral databases. Adequate and user-friendly software is required to assist in candidate selection based on the comparison of acquired MS and NMR spectral information with reference data. It is foreseen that these focal points will contribute to a better transfer and exploitation of structural information gained from diverse analytical platforms.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

Abbreviations

1D-1H-NMR:

One dimensional proton nuclear magnetic resonance (spectroscopy)

2D:

Two dimensional

3D:

Three dimensional

DAD:

Diode array detector

Da:

Dalton

HPLC:

High pressure/performance liquid chromatography

LC:

Liquid chromatography

MeOD:

Deuterated methanol

m/z :

Mass to charge

MS:

Mass spectrometry

MSn :

Multistage fragmentation mass spectrometry

MI:

Metabolite identification

MSI:

Metabolomics standard initiative

SPE:

Solid phase extraction

TOF:

Time of flight

Ref:

Reference

RT:

Retention time

UV:

Ultraviolet

References

  • Allwood, J. W., De Vos, R. C. H., Moing, A., Deborde, C., Erban, A., Kopka, J., et al. (2011). Plant metabolomics and its potential for systems biology research: Background concepts, technology, and methodology. Methods in Enzymology, 500, 299–336.

    Article  PubMed  CAS  Google Scholar 

  • Álvarez-Sánchez, B., Priego-Capote, F., & Castro, M. D. L. D. (2010). Metabolomics analysis II. Preparation of biological samples prior to detection. Trends in Analytical Chemistry, 29(2), 120–127.

    Article  Google Scholar 

  • Brand, W., Shao, J., Hoek-Van Den Hil, E. F., Van Elk, K. N., Spenkelink, B., De Haan, L. H. J., et al. (2010). Stereoselective conjugation, transport and bioactivity of S- and R-hesperetin enantiomers in vitro. Journal of Agriculture and Food Chemistry, 58(10), 6119–6125.

    Article  CAS  Google Scholar 

  • Brand, W., Oosterhuis, B., Krajcsi, P., Barron, D., Dionisi, F., Van Bladeren, P. J., et al. (2011). Interaction of hesperetin glucuronide conjugates with human BCRP, MRP2 and MRP3 as detected in membrane vesicles of overexpressing baculovirus-infected Sf9 cells. Biopharmaceutics & Drug Disposition, 32(9), 530–535.

    Article  CAS  Google Scholar 

  • Brown, M., Dunn, W. B., Dobson, P., Patel, Y., Winder, C. L., Francis-Mcintyre, S., et al. (2009). Mass spectrometry tools and metabolite-specific databases for molecular identification in metabolomics. Analyst, 134(7), 1322–1332.

    Article  PubMed  CAS  Google Scholar 

  • Chen, J., Zhao, X., Fritsche, J., Yin, P., Schmitt-Kopplin, P., Wang, W., et al. (2008). Practical approach for the identification and isomer elucidation of biomarkers detected in a metabonomic study for the discovery of individuals at risk for diabetes by integrating the chromatographic and mass spectrometric information. Analytical Chemistry, 80(4), 1280–1289.

    Article  PubMed  CAS  Google Scholar 

  • Clarkson, C., Staerk, D., Honoré Hansen, S., & Jaroszewski, J. W. (2005). Hyphenation of solid-phase extraction with liquid chromatography and nuclear magnetic resonance: Application of HPLC-DAD-SPE-NMR to identification of constituents of Kanahia laniflora. Analytical Chemistry, 77(11), 3547–3553.

    Article  PubMed  CAS  Google Scholar 

  • de Rijke, E., Out, P., Niessen, W. M. A., Ariese, F., Gooijer, C., & Brinkman, U. A. T. (2006). Analytical separation and detection methods for flavonoids. Journal of Chromatography A, 1112(1), 31–63.

    Article  PubMed  Google Scholar 

  • Elyashberg, M. E., Williams, A. J., & Martin, G. E. (2008). Computer-assisted structure verification and elucidation tools in NMR-based structure elucidation. Progress in Nuclear Magnetic Resonance Spectroscopy, 53(1–2), 1–104.

    Article  CAS  Google Scholar 

  • Exarchou, V., Godejohann, M., Beek, T. A. V., Gerothanassis, I. P., & Vervoort, J. (2003). LC-UV-solid-phase extraction-NMR-MS combined with a cryogenic flow probe and its application to the identification of compounds present in Greek Oregano. Analytical Chemistry, 75, 6288–6294.

    Article  PubMed  CAS  Google Scholar 

  • Exarchou, V., Krucker, M., Beek, T. A. V., Vervoort, J., Gerothanassis, I. P., & Albert, K. (2005). LC–NMR coupling technology: Recent advancements and applications in natural products analysis. Magnetic Resonance in Chemistry, 43, 681–687.

    Article  PubMed  CAS  Google Scholar 

  • Harborne, J. B. (1967). Comparative biochemistry of the flavonoids. London: Academic Press.

    Google Scholar 

  • Heinonen, M., Rantanen, A., Mielikäinen, T., Kokkonen, J., Kiuru, J., Ketola, R. A., et al. (2008). FiD: A software for ab initio structural identification of product ions from tandem mass spectrometric data. Rapid Communications in Mass Spectrometry, 22(19), 3043–3052.

    Article  PubMed  CAS  Google Scholar 

  • Hill, A. W., & Mortishire-Smith, R. J. (2005). Automated assignment of high-resolution collisionally activated dissociation mass spectra using a systematic bond disconnection approach. Rapid Communications in Mass Spectrometry, 19(21), 3111–3118.

    Article  CAS  Google Scholar 

  • Jaroszewski, J. W. (2005). Hyphenated NMR methods in natural products research, part 2: HPLC-SPE-NMR and other new trends in NMR hyphenation. Planta Medica, 71(9), 795–802.

    Article  PubMed  CAS  Google Scholar 

  • Jewison, T., Knox, C., Neveu, V., Djoumbou, Y., Guo, A. C., Lee, J., et al. (2012). YMDB: The yeast metabolome database. Nucleic Acids Research, 40(D1), D815–D820.

    Article  PubMed  CAS  Google Scholar 

  • Kesting, J. R., Olsen, L., Staerk, D., Tejesvi, M. V., Kini, K. R., Prakash, H. S., et al. (2011). Production of unusual dispiro metabolites in Pestalotiopsis virgatula endophyte cultures: HPLC-SPE-NMR, electronic circular dichroism, and time-dependent density-functional computation study. Journal of Natural Products, 74(10), 2206–2215.

    Article  PubMed  CAS  Google Scholar 

  • Kind, T., & Fiehn, O. (2007). Seven golden rules for heuristic filtering of molecular formulas obtained by accurate mass spectrometry. Bmc Bioinformatics, 8, 105.

    Article  PubMed  Google Scholar 

  • Kind, T., & Fiehn, O. (2010). Advances in structure elucidation of small molecules using mass spectrometry. Bioanalytical Reviews, 2(1), 23–60.

    Article  PubMed  Google Scholar 

  • Kueger, S., Steinhauser, D., Willmitzer, L., & Giavalisco, P. (2012). High-resolution plant metabolomics: From mass spectral features to metabolites and from whole-cell analysis to subcellular metabolite distributions. Plant Journal, 70(1), 39–50.

    Article  PubMed  CAS  Google Scholar 

  • Laatikainen, R., Niemitz, M., Weber, U., Sundeun, J., Hassinen, T., & Vepsäläinen, J. (1996). General strategies for total-lineshape-type spectral analysis of NMR spectra using integral-transform iterator. Journal of Magnetic Resonance, Series A, 120(1), 1–10.

    Article  CAS  Google Scholar 

  • Lin, L.-Z., Chen, P., & Harnly, J. M. (2008). New phenolic components and chromatographic profiles of green and fermented teas. Journal of Agricultural and Food Chemistry, 56(17), 8130–8140.

    Article  PubMed  CAS  Google Scholar 

  • Ludwig, C., Easton, J. M., Lodi, A., Tiziani, S., Manzoor, S. E., Southam, A. D., et al. (2012). Birmingham metabolite library: A publicly accessible database of 1-D 1H and 2-D 1H J-resolved NMR spectra of authentic metabolite standards (BML-NMR). Metabolomics, 8(1), 8–18.

    Article  CAS  Google Scholar 

  • Moco, S., & Vervoort, J. (2012). Chemical identification strategies using liquid chromatography-photodiode array-solid-phase extraction-nuclear magnetic resonance/mass spectrometry. In N. W. Hardy, & R. D. Hall (Eds.). Methods in Molecular Biology, 860, 287–316.

    Article  PubMed  CAS  Google Scholar 

  • Moco, S., Bino, R. J., Vorst, O., Verhoeven, H. A., de Groot, J., van Beek, T. A., et al. (2006). A liquid chromatography-mass spectrometry-based metabolome database for tomato. Plant Physiology, 141(4), 1205–1218.

    Article  PubMed  CAS  Google Scholar 

  • Moco, S., Bino, R. J., De Vos, R. C. H., & Vervoort, J. (2007). Metabolomics technologies and metabolite identification. Trends in Analytical Chemistry, 26(9), 855–866.

    Article  CAS  Google Scholar 

  • Molinski, T. F. (2010). NMR of natural products at the ‘nanomole-scale’. Natural Products Reports, 27(3), 321–329.

    Article  CAS  Google Scholar 

  • Muñoz, M. A., Perez-Hernandez, N., Pertino, M. W., Schmeda-Hirschmann, G., & Joseph-Nathan, P. (2012). Absolute Configuration and 1H NMR characterization of rosmaridiphenol diacetate. Journal of Natural Products, 75(4), 779–783.

    Article  PubMed  Google Scholar 

  • Nicoli, R., Martel, S., Rudaz, S., Wolfender, J. L., Veuthey, J. L., Carrupt, P. A., et al. (2010). Advances in LC platforms for drug discovery. Expert Opinion Drug Discovery, 5(5), 475–489.

    Article  CAS  Google Scholar 

  • Olsen, H., Aaby, K., & Borge, G. I. A. (2009). Characterization and quantification of flavonoids and hydroxycinnamic acids in curly kale (Brassica oleracea L. convar. acephala var. sabellica) by HPLC-DAD-ESI-MSn. Journal of Agriculture and Food Chemistry, 57(7), 2816–2825.

    Article  CAS  Google Scholar 

  • Ottaviani, J. I., Momma, T. Y., Heiss, C., Kwik-Uribe, C., Schroeter, H., & Keen, C. L. (2011). The stereochemical configuration of flavanols influences the level and metabolism of flavanols in humans and their biological activity in vivo. Free Radical Biology and Medicine, 50(2), 237–244.

    Article  PubMed  CAS  Google Scholar 

  • Peironcely, J. E., Reijmers, T., Coulier, L., Bender, A., & Hankemeier, T. (2011). Understanding and classifying metabolite space and metabolite-likeness. PLoS One, 6(12), e28966.

    Article  PubMed  CAS  Google Scholar 

  • Psychogios, N., Hau, D. D., Peng, J., Guo, A. C., Mandal, R., Bouatra, S., et al. (2012). The human serum metabolome. PLoS One, 6(2), e16957.

    Article  Google Scholar 

  • Puiggròs, F., Solà, R., Bladé, C., Salvadó, M. J., & Arola, L. (2011). Nutritional biomarkers and foodomic methodologies for qualitative and quantitative analysis of bioactive ingredients in dietary intervention studies. Journal of Chromatography A, 1218(42), 7399–7414.

    Article  PubMed  Google Scholar 

  • Qiu, F., Imai, A., McAlpine, J. B., Lankin, D. C., Burton, I., Karakach, T., et al. (2012). Dereplication, residual complexity, and rational naming: The case of the actaea triterpenes. Journal of Natural Products, 75(3), 432–443.

    Article  PubMed  CAS  Google Scholar 

  • Rasche, F., Scheubert, K., Hufsky, F., Zichner, T., Kai, M., Svatoš, A., et al. (2012). Identifying the unknowns by aligning fragmentation trees. Analytical Chemistry, 84(12), 3417–3426.

    Article  PubMed  CAS  Google Scholar 

  • Ridder, L., van der Hooft, J. J. J., Verhoeven, S., De Vos, R. C. H., Van Schaik, R., & Vervoort, J. (2012). Substructure-based annotation of high-resolution multistage MSn spectral trees. Rapid Communication in Mass Spectrometry, 26(20), 2461–2471.

    Article  CAS  Google Scholar 

  • Rojas-Cherto, M., Peironcely, J. E., Kasper, P. T., van der Hooft, J. J. J., De Vos, R. C. H., Vreeken, R. J., et al. (2012). Metabolite identification using automated comparison of high resolution MSn spectral trees. Analytical Chemistry, 84(13), 5524–5534.

    Article  PubMed  CAS  Google Scholar 

  • Rojas-Chertó, M., van Vliet, M., Peironcely, J. E., van Doorn, R., Kooyman, M., Beek, T. T., et al. (2012). MetiTree: A web application to organize and process high resolution multi-stage mass spectrometry metabolomics data. Bioinformatics, 28(20), 2707–2709.

    Article  PubMed  Google Scholar 

  • Scalbert, A., Andres-Lacueva, C., Arita, M., Kroon, P., Manach, C., Urpi-Sarda, M., et al. (2011). Databases on food phytochemicals and their health-promoting effects. Journal of Agricultural and Food Chemistry, 59(9), 4331–4348.

    Article  PubMed  CAS  Google Scholar 

  • Sprogøe, K., Staerk, D., Ziegler, H. L., Jensen, T. H., Holm-Møller, S. B., & Jaroszewski, J. W. (2008). Combining HPLC-PDA-MS-SPE-NMR with circular dichroism for complete natural product characterization in crude extracts: Levorotatory gossypol in Thespesia danis. Journal of Natural Products, 71(4), 516–519.

    Article  PubMed  Google Scholar 

  • Sturm, S., & Seger, C. (2012). Liquid chromatography-nuclear magnetic resonance coupling as alternative to liquid chromatography-mass spectrometry hyphenations: Curious option or powerful and complementary routine tool? Journal of Chromatography A, 1259, 50–61.

    Article  PubMed  CAS  Google Scholar 

  • Sumner, L. W., Amberg, A., Barrett, D., Beale, M. H., Beger, R., Daykin, C. A., et al. (2007). Proposed minimum reporting standards for chemical analysis. Metabolomics, 3(3), 211–221.

    Article  CAS  Google Scholar 

  • Tang, H., Xia, C., & Wang, Y. (2009). Important roles of the hyphenated HPLC-DAD-MS-SPE-NMR technique in metabonomics. Magnetic Resonance in Chemistry, 47, S157–S162.

    Article  PubMed  CAS  Google Scholar 

  • Tohge, T., & Fernie, A. R. (2009). Web-based resources for mass-spectrometry-based metabolomics: A user’s guide. Phytochemistry, 70(4), 450–456.

    Article  PubMed  CAS  Google Scholar 

  • van der Hooft, J. J. J., Mihaleva, V., Bino, R. J., de Vos, R. C. H., & Vervoort, J. (2011a). A strategy for fast structural elucidation of metabolites in small volume plant extracts using automated MS-guided LC-MS-SPE-NMR. Magnetic Resonance in Chemistry, 49(S1), S55–S60.

    Article  PubMed  CAS  Google Scholar 

  • van der Hooft, J. J. J., Vervoort, J., Bino, R. J., Beekwilder, J., & De Vos, R. C. H. (2011b). Polyphenol identification based on systematic and robust high-resolution accurate mass spectrometry fragmentation. Analytical Chemistry, 83(1), 409–416.

    Article  PubMed  Google Scholar 

  • van der Hooft, J. J. J., Akermi, M., Ünlü, F. Y., Mihaleva, V., Roldan, V. G., Bino, R. J., et al. (2012a). Structural annotation and elucidation of conjugated phenolic compounds in black, green, and white tea extracts. Journal of Agricultural and Food Chemistry, 60, 8841–8850.

    Article  PubMed  Google Scholar 

  • van der Hooft, J. J. J., De Vos, R. C. H., Mihaleva, V., Bino, R. J., Ridder, L., de Roo, N., et al. (2012b). Structural elucidation and quantification of phenolic conjugates present in human urine after tea intake. Analytical Chemistry, 84(16), 7263–7271.

    Article  PubMed  Google Scholar 

  • van der Hooft, J. J. J., Vervoort, J., Bino, R. J., & de Vos, R. C. H. (2012c). Spectral trees as a robust annotation tool in LC-MS based metabolomics. Metabolomics, 8(4), 691–703.

    Article  Google Scholar 

  • Watson, J. T., & Sparkman, O. D. (2007). Introduction to mass spectrometry (4th ed.). West Sussex: Wiley.

    Book  Google Scholar 

  • Wishart, D. S. (2011). Advances in metabolite identification. Bioanalysis, 3(15), 1769–1782.

    Article  PubMed  CAS  Google Scholar 

  • Wishart, D. S., Knox, C., Guo, A. C., Eisner, R., Young, N., Gautam, B., et al. (2009). HMDB: A knowledgebase for the human metabolome. Nucleic Acids Research, 37(Suppl 1), D603–D610.

    Article  PubMed  CAS  Google Scholar 

  • Wolf, S., Schmidt, S., Möller-Hannemann, M., & Neumann, S. (2010). In silico fragmentation for computer assisted identification of metabolite mass spectra. BMC Bioinformatics, 11, 148.

    Article  PubMed  Google Scholar 

  • Wolfender, J. L., Marti, G., & Queiroz, E. F. (2010). Advances in techniques for profiling crude extracts and for the rapid identification of natural products: Dereplication, quality control and metabolomics. Current Organic Chemistry, 14(16), 1808–1832.

    Article  CAS  Google Scholar 

  • Wubshet, S. G., Johansen, K. T., Nyberg, N. T., & Jaroszewski, J. W. (2012). Direct 13C NMR detection in HPLC hyphenation mode: Analysis of Ganoderma lucidum terpenoids. Journal of Natural Products, 75(5), 876–882.

    Article  PubMed  CAS  Google Scholar 

  • Xiao, J. F., Zhou, B., & Ressom, H. W. (2012). Metabolite identification and quantitation in LC-MS/MS-based metabolomics. Trends in Analytical Chemistry, 32, 1–14.

    Article  PubMed  Google Scholar 

Download references

Acknowledgments

The authors thank Prof. Dr. Alan Crozier for his time to read and comment on the manuscript. This research was funded by the Netherlands Metabolomics Centre (JJJvdH, JV, RCHdV) and the Centre for BioSystems Genomics (RJB, RCHdV), both of which are part of the Netherlands Genomics Initiative/Netherlands Organization for Scientific Research.

Author information

Author notes

  1. Justin J. J. van der Hooft

    Present address: University of Glasgow, Joseph Black building, Glasgow, G12 8QQ, UK

Authors and Affiliations

  1. Laboratory of Biochemistry, Wageningen University, Dreijenlaan 3, 6703 HA, Wageningen, The Netherlands

    Justin J. J. van der Hooft, Lars Ridder & Jacques Vervoort

  2. BU Bioscience, Plant Research International, POB 16, 6700 AA, Wageningen, The Netherlands

    Justin J. J. van der Hooft & Ric C. H. de Vos

  3. Netherlands Metabolomics Centre, Einsteinweg 55, 2333 CC, Leiden, The Netherlands

    Justin J. J. van der Hooft, Ric C. H. de Vos & Jacques Vervoort

  4. Centre for Biosystems Genomics, POB 98, 6700 AB, Wageningen, The Netherlands

    Ric C. H. de Vos

  5. Netherlands eScience Center, Amsterdam, The Netherlands

    Lars Ridder

  6. Laboratory of Plant Physiology, Wageningen University, P.O. Box 658, 6700 AR, Wageningen, The Netherlands

    Raoul J. Bino

Authors
  1. Justin J. J. van der Hooft
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ric C. H. de Vos
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Lars Ridder
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jacques Vervoort
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Raoul J. Bino
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Justin J. J. van der Hooft.

Rights and permissions

Reprints and permissions

About this article

Cite this article

van der Hooft, J.J.J., de Vos, R.C.H., Ridder, L. et al. Structural elucidation of low abundant metabolites in complex sample matrices. Metabolomics 9, 1009–1018 (2013). https://doi.org/10.1007/s11306-013-0519-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11306-013-0519-8

Keywords

Search

Navigation