, Volume 8, Issue 3, pp 492–507 | Cite as

Metabolite profiling and fingerprinting of commercial cultivars of Humulus lupulus L. (hop): a comparison of MS and NMR methods in metabolomics

  • Mohamed A. Farag
  • Andrea Porzel
  • Jürgen Schmidt
  • Ludger A. Wessjohann
Original Article


Hop (Humulus lupulus L. Cannabaceae) is an economically important crop. In addition to its role in beer brewing, its pharmaceutical applications have been of increasing importance in recent years. Bitter acids (prenylated polyketides), prenylflavonoids and essential oils, are the primary phytochemical components that account for hop medicinal value. An integrated approach utilizing nuclear magnetic resonance (NMR) and mass spectrometry (MS) techniques was used for the first large-scale metabolite profiling in Humulus lupulus. Resins and extracts prepared from 13 hop cultivars were analysed using NMR, liquid chromatography (LC)-MS and fourier transform ion cyclotron resonance (FTICR)-MS in parallel and subjected to principal component analysis (PCA). A one pot extraction method, compatible with both MS and NMR measurement was developed to help rule out effects due to differences in extraction protocols. Under optimised conditions, we were able to simultaneously quantify and identify 46 metabolites including 18 bitter acids, 12 flavonoids, 3 terpenes, 3 fatty acids and 2 sugars. Cultivars segregation in PCA plots generated from both LC-MS and NMR data were found comparable and mostly influenced by differences in bitter acids composition among cultivars. FTICR-MS showed inconsistent PCA loading plot results which are likely due to preferential ionisation and also point to the presence of novel isoprenylated metabolites in hop. This comparative metabolomic approach provided new insights for the complementariness and coincidence for these different technology platform applications in hop and similar plant metabolomics projects.


Humulus lupulus L. Hop 1H NMR-based metabolomics ESI-FTICR MS LC-MS Humulones Lupulones Principal component analysis Flavonoids Isoprenoids Plant secondary metabolites 



Electrospray ionisation.


Fourier transform ion cyclotron resonance.


Gas chromatography.


Liquid chromatography.


Mass spectrometry


Tandem mass spectrometry


Nuclear magnetic resonance


Photodiode array detection


Principal component analysis



Dr. M. A. Farag thanks the Alexander von Humboldt-foundation, Germany for financial support. We are grateful to Hopsteiner Inc. (and here especially Dr. Martin Biendl, Hallertau, and Harald Schwarz) for hop resins, cones, and hop acid reference samples. We also thank Dr. Christoph Böttcher for assistance with the UPLC-MS, Prof. Barbara Seliger for support with HT29 measurements, and Orgentis Chemicals for reference samples of pure hop constituents. We are grateful to Dr. Steffen Neumann and Dr. Tilo Lübken for providing R scripts for NMR and MS data analysis.

Supplementary material

11306_2011_335_MOESM1_ESM.doc (511 kb)
Supplementary material 1 (DOC 511 kb)


  1. Agnolet, S., Jaroszewski, J. W., Verpoorte, R., & Staerk, D. (2010). 1H NMR-based metabolomics combined with HPLC-PDA-MS-SPE-NMR for investigation of standardized Ginkgo biloba preparations. Metabolomics, 6, 292–302.PubMedCrossRefGoogle Scholar
  2. Aharoni, A., De Vos, C., Verhoeven, H. A., Maliepaard, C. A., Kruppa, G., & Bino, R. (2002). Nontargeted metabolome analysis by use of fourier transform ion cyclotron mass spectrometry. OMICS A Journal of Integrative Biology, 6, 217–234.PubMedCrossRefGoogle Scholar
  3. Arraez-Roman, D., Cortacero-Ramirez, S., Segura-Carretero, A., Contreras, J. A. M. L., & Fernandez-Gutierrez, A. (2006). Characterization of the methanolic extract of hops using capillary electrophoresis-electrospray ionization-mass spectrometry. Electrophoresis, 27, 2197–2207.PubMedCrossRefGoogle Scholar
  4. Biais, B., Allwood, J. W., Deborde, C., Xu, Y., Maucourt, M., Beauvoit, B., et al. (2009). 1H NMR, GC-EI-TOFMS, and data set correlation for fruit metabolomics: Application to spatial metabolite analysis in melon. Analytical Chemistry, 81, 2884–2894.PubMedCrossRefGoogle Scholar
  5. Biendl, M., & Pinzl, C. (2008). Hops and health. uses, effects, history. Wolznach: German Hop Museum Wolznach.Google Scholar
  6. Catchpole, G. S., Beckmann, M., Enot, D. P., Mondhe, M., Zywicki, B., Taylor, J., et al. (2005). Hierarchical metabolomics demonstrates substantial compositional similarity between genetically modified and conventional potato crops. Proceedings of the National Academy of Sciences of the United States of America, 102, 14458–14462.Google Scholar
  7. Chadwick, L. R., Nikolic, D., Burdette, J. E., Overk, C. R., Bolton, J. L., van Breemen, R. B., et al. (2004). Estrogens and congeners from spent hops (Humulus lupulus). Journal of Natural Products, 67, 2024–2032.PubMedCrossRefGoogle Scholar
  8. Chadwick, L. R., Pauli, G. F., & Farnsworth, N. R. (2006). The pharmacognosy of Humulus lupulus L. (hops) with an emphasis on estrogenic properties. Phytomedicine, 13, 119–131.PubMedCrossRefGoogle Scholar
  9. Culik, J., Jurkova, M., Horak, T., Čejka, P., Kellner, V., Dvořák, J., et al. (2009). Extraction of bitter acids from hops and hop products using Pressurized Solvent Extraction (PSE). Journal of the Institute of Brewing, 115, 220–225.Google Scholar
  10. de Rijke, E., Zappey, H., Ariese, F., Gooijer, C., & Brinkman, U. A. T. (2003). Liquid chromatography with atmospheric pressure chemical ionization and electrospray ionization mass spectrometry of flavonoids with triple-quadrupole and ion-trap instruments. Journal of Chromatography A, 984, 45–58.PubMedCrossRefGoogle Scholar
  11. Dumas, M. E., Maibaum, E. C., Teague, C., Ueshima, H., Zhou, B., Lindon, J. C., et al. (2006). Assessment of analytical reproducibility of H-1 NMR spectroscopy based metabonomics for large-scale epidemiological research: The INTERMAP study. Analytical Chemistry, 78, 2199–2208.PubMedCrossRefGoogle Scholar
  12. Fiehn, O., Kopka, J., Dörmann, P., Altmann, T., Trethewey, R. N., & Willmitzer, L. (2000). Metabolite profiling for plant functional genomics. Nature Biotechnology, 18, 1157–1161.PubMedCrossRefGoogle Scholar
  13. Fortes, A., Santos, F., Choi, Y., Silva, M. S., Figueiredo, A., Sousa, L., et al. (2008). Organogenic nodule development in hop (Humulus lupulus L.): Transcript and metabolic responses. BMC Genomics, 9, 445.PubMedCrossRefGoogle Scholar
  14. Garcia-Viguera, C., Zafrilla, P., & Tomas-Barberan, F. A. (1998). The use of acetone as an extraction solvent for anthocyanins from strawberry fruit. Phytochemical Analysis, 9, 274–277.CrossRefGoogle Scholar
  15. Gerhauser, C. (2005). Beer constituents as potential cancer chemopreventive agents. European Journal of Cancer, 41, 1941–1954.PubMedCrossRefGoogle Scholar
  16. Goodacre, R., Shann, B., Gilbert, R. J., Timmins, E. M., McGovern, A. C., Alsberg, B. K., et al. (2000). Detection of the dipicolinic acid biomarker in Bacillus spores using Curie-point pyrolysis mass spectrometry and fourier transform infrared spectroscopy. Analytical Chemistry, 72, 119–127.PubMedCrossRefGoogle Scholar
  17. Hoek, A. C., Hermans-Lokkerbol, A. C. J., & Verpoorte, R. (2001). An improved NMR method for the quantification of alpha-acids in hops and hop products. Phytochemical Analysis, 12, 53–57.PubMedCrossRefGoogle Scholar
  18. Holtzel, A., Schlotterbeck, G., Albert, K., & Bayer, E. (1996). Separation and characterisation of hop bitter acids by HPLC-H-1 NMR coupling. Chromatographia, 42, 499–505.CrossRefGoogle Scholar
  19. Junot, C., Madalinski, G., Tabet, J. C., & Ezan, E. (2010). Fourier transform mass spectrometry for metabolome analysis. Analyst, 135, 2203–2219.PubMedCrossRefGoogle Scholar
  20. Lamy, V., Roussi, S., Chaabi, M., Gossé, F., Schall, N., Lobstein, A., et al. (2007). Chemopreventive effects of lupulone, a hop β-acid, on human colon cancer-derived metastatic SW620 cells and in a rat model of colon carcinogenesis. Carcinogenesis, 28, 1575–1581.PubMedCrossRefGoogle Scholar
  21. Le Gall, G., Colquhoun, I. J., Davis, A. L., Collins, G. J., & Verhoeyen, M. E. (2003). Metabolite profiling of tomato (Lycopersicon esculentum) using H-1 NMR spectroscopy as a tool to detect potential unintended effects following a genetic modification. Journal of Agricultural and Food Chemistry, 51, 2447–2456.PubMedCrossRefGoogle Scholar
  22. Leon, C., Rodriguez-Meizoso, I., Lucio, M., Garcia-Cañas, V., Ibañez, E., Schmitt-Kopplin, P., et al. (2009). Metabolomics of transgenic maize combining Fourier transform-ion cyclotron resonance-mass spectrometry, capillary electrophoresis-mass spectrometry and pressurized liquid extraction. Journal of Chromatography A, 1216, 7314–7323.PubMedCrossRefGoogle Scholar
  23. Li, H. J., & Deinzer, M. L. (2006). Structural identification and distribution of proanthocyanidins in 13 different hops. Journal of Agricultural and Food Chemistry, 54, 4048–4056.PubMedCrossRefGoogle Scholar
  24. Magalhaes, P. J., Guido, L. F., Cruz, J. M., & Barros, A. A. (2007). Analysis of xanthohumol and isoxanthohumol in different hop products by liquid chromatography-diode array detection-electrospray ionization tandem mass spectrometry. Journal of Chromatography A, 1150, 295–301.PubMedCrossRefGoogle Scholar
  25. Moco, S., Bino, R. J., De Vos, R. C. H., & Vervoort, J. (2007). Metabolomics technologies and metabolite identification. Trac-Trends in Analytical Chemistry, 26, 855–866.CrossRefGoogle Scholar
  26. Moco, S., Forshed, J., De Vos, R. C. H., Bino, R. J., & Vervoort, J. (2008). Intra- and inter-metabolite correlation spectroscopy of tomato metabolomics data obtained by liquid chromatography-mass spectrometry and nuclear magnetic resonance. Metabolomics, 4, 202–215.CrossRefGoogle Scholar
  27. Simons, R., Vincken, J. P., Bakx, E. J., Verbruggen, M. A., & Gruppen, H. (2009). A rapid screening method for prenylated flavonoids with ultra-high-performance liquid chromatography/electrospray ionisation mass spectrometry in licorice root extracts. Rapid Communications in Mass Spectrometry, 23, 3083–3093.PubMedCrossRefGoogle Scholar
  28. Smith, C. A., Want, E. J., O’Maille, G., Abagyan, R., & Siuzdak, G. (2006). XCMS: Processing mass spectrometry data for metabolite profiling using Nonlinear peak alignment, matching, and identification. Analytical Chemistry, 78, 779–787.PubMedCrossRefGoogle Scholar
  29. Sterner, J. L., Johnston, M. V., Nicol, G. R., & Ridge, D. P. (2000). Signal suppression in electrospray ionization Fourier transform mass spectrometry of multi-component samples. Journal of Mass Spectrometry, 35, 385–391.PubMedCrossRefGoogle Scholar
  30. Stevens, J. F., & Page, J. E. (2004). Xanthohumol and related prenylflavonoids from hops and beer: To your good health!. Phytochemistry, 65(10), 1317–1330.PubMedCrossRefGoogle Scholar
  31. Stevens, J. F., Taylor, A. W., Nickerson, G. B., Ivancic, M., Henning, J., Haunold, A., et al. (2000). Prenylflavonoid variation in Humulus lupulus: Distribution and taxonomic significance of xanthogalenol and 4’-O-methylxanthohumol. Phytochemistry, 53, 759–775.PubMedCrossRefGoogle Scholar
  32. Takahashi, H., Kai, K., Shinbo, Y., Tanaka, K., Ohta, D., Oshima, T., et al. (2008). Metabolomics approach for determining growth-specific metabolites based on Fourier transform ion cyclotron resonance mass spectrometry. Analytical and Bioanalytical Chemistry, 391, 2769–2782.PubMedCrossRefGoogle Scholar
  33. Tolstikov, V. V., & Fiehn, O. (2002). Analysis of highly polar compounds of plant origin: Combination of hydrophilic interaction chromatography and electrospray ion trap mass spectrometry. Analytical Biochemistry, 301, 298–307.PubMedCrossRefGoogle Scholar
  34. Van Cleemput, M., Cattoor, K., De Bosscher, K., Haegeman, G., De Keukeleire, D., & Heyerick, A. (2009). Hop (Humulus lupulus)—Derived bitter acids as multipotent bioactive compounds. Journal of Natural Products, 72, 1220–1230.PubMedCrossRefGoogle Scholar
  35. van den Berg, R. A., Hoefsloot, H. C. J., Westerhuis, J. A., Smilde, A. K., & van der Werf, M. J. (2006). Centering, scaling, and transformations: Improving the biological information content of metabolomics data. BMC Genomics, 7, 142. doi: 10.1186/1471-2164-7-142.
  36. van der Kooy, F., Maltese, F., Choi, Y., Kim, H., & Verpoorte, R. (2009). Quality control of herbal material and phytopharmaceuticals with MS and NMR based metabolic fingerprinting. Planta Medica, 75, 763–775.PubMedCrossRefGoogle Scholar
  37. Vanhoenacker, G., Dermaux, A., De Keukeleire, D., & Sandra, P. (2001). Single-run capillary electrochromatographic analysis of hop acids and prenylated hop flavonoids. Journal of Separation Science, 24, 55–58.CrossRefGoogle Scholar
  38. von Roepenack-Lahaye, E., Degenkolb, T., Zerjeski, M., Franz, M., Roth, U., Wessjohann, L. A., et al. (2004). Profiling of Arabidopsis secondary metabolites by capillary liquid chromatography coupled to electrospray ionization quadrupole time-of-flight mass spectrometry. Plant Physiology, 134, 548–559.CrossRefGoogle Scholar
  39. Wang, G. D., Tian, L., Aziz, N., Broun, P., Dai, X., He, J., et al. (2008). Terpene biosynthesis in glandular trichomes of hop. Plant Physiology, 148, 1254–1266.PubMedCrossRefGoogle Scholar
  40. Wessjohann, L., Wilhelm, H., & Biendl, M. Verfahren zur Herstellung von Naringeninderivaten aus Xanthohumol. [DE 10 2005 013 258.8-44]. 22-3-2005. Germany.Google Scholar
  41. Wilhelm, H., & Wessjohann, L. A. (2006). An efficient synthesis of the phytoestrogen 8-prenylnaringenin from xanthohumol by a novel demethylation process. Tetrahedron, 62, 6961–6966.CrossRefGoogle Scholar
  42. Zhang, X. Z., Liang, X. M., Xiao, H. B., & Xu, Q. (2004). Direct characterization of bitter acids in a crude hop extract by liquid chromatography-atmospheric pressure chemical ionization mass spectrometry. Journal of the American Society for Mass Spectrometry, 15, 180–187.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Mohamed A. Farag
    • 1
    • 2
  • Andrea Porzel
    • 1
  • Jürgen Schmidt
    • 1
  • Ludger A. Wessjohann
    • 1
  1. 1.Department of Bioorganic ChemistryLeibniz Institute of Plant BiochemistryHalle (Saale)Germany
  2. 2.Pharmacognosy Department, College of PharmacyCairo UniversityCairoEgypt

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