Modern Analytical Techniques for Flavonoid Determination

  • Mark A. Berhow
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 505)


The biological functionalities of plant natural products, both in plants and in the animals that use and consume them, is fueling new interest in phytochemical research (Cordell, 1995). A key component of this research is the ability to accurately identify and quantitate specific phytochemicals. Today analytical equipment is available that can rapidly separate, unequivocally identify, and accurately quantify phytochemicals from plant materials literally in a matter of minutes. The development of low-cost, high-powered computer systems allowed for the creation of computer-driven bench top chromatography instrumentation. These systems are able to perform complicated electronic functions, such as control of gas flow and liquid pumps, spectral detection, including optical and mass spectrometry and to accumulate, quantitate, and process large amounts of data. Previously, there may have only been one or two of these types of instruments at an institution. Now they are available at prices in the $20,000 to $200,000 range, which makes them affordable to individual researchers. These developments have made complex plant product analysis extremely practical and affordable.


Capillary Electrophoresis Flavonoid Glycoside Flavonoid Aglycone Plant Natural Product Modern Analytical Technique 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Andlauer, W., Martena, M. J., and Furst, P., 1999, Determination of selected phytochemicals by reverse-phase high-performance liquid chromatography combined with ultraviolet and mass spectrometric detection, J. Chromatog. A 849: 341–348.CrossRefGoogle Scholar
  2. Arnao, M. B., Casas, J. L., Del Rio, J. A., Acosta, M., and Garcia-Canovas, F., 1990, An enzymatic colorimetric method for measuring naringin using 2,2’-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) in the presence of peroxidase, Anal. Biochem. 185: 335–338.CrossRefGoogle Scholar
  3. Aussenac, T., Lacombe, S., and Dayde, J., 1998, Quantification of isoflavones by capillary zone electrophoresis in soybean seeds: effects of variety and environment, Am. J. Clin. Nutr. 68S:1480S–1485S.Google Scholar
  4. Barnes, S., Coward, L., Kirk, M., and Sfakianos, J., 1998, HPLC-mass spectrometry analysis of isoflavones, Proc. Soc. Exp. Biol. Med. 217: 254–262.Google Scholar
  5. Barnes, S., Kirk, M., and Coward, L., 1994, lsoflavones and their conjugates in soy foods: extraction conditions and analysis by HPLC-mass spectrometry, J. Agric. Food Chem. 42: 2466–2474.Google Scholar
  6. Barnes, S., Wang, C.-C., Smith-Johnson, A., and Kirk, M., 1999, Liquid chromatography: mass spectrometry of isoflavones, J. Medicinal Food 2: 1 11–117.CrossRefGoogle Scholar
  7. Berhow, M. A., and Vaughn, S. F., 1999, Higher plant tlavonoids: biosynthesis and chemical ecology, In: Principles and Practices in Plant Ecology: Allelochemical Interactions. lnderj it, Dakshini, K. M. M., and Foy, C., eds., CRC Press, Boca Raton, FL, pp. 423–438.Google Scholar
  8. Bocchini, P., Russo, M., and Galletti, G. C., 1998, Pyrolysis-gas chromatography/mass spectrometry used as a microanalytical technique for the characterization of Origanum heracleoticum from Calabria, southern Italy, Rapid Commun. Mass Spectrom. 12: 1555–1563.CrossRefGoogle Scholar
  9. Cancalon, P. F., 1999, Analytical monitoring of citrus juices by using capillary electrophoresis, J.AOAC Int. 82: 95–106.Google Scholar
  10. Careri, M., Elviri, L., and Mangia, A.,1999, Validation ofa liquid chromatography ion spray mass spectrometry method for the analysis of flavanones, flavones and flavonols, Rapid Commun. Mass Spec trom. 13: 2399–2405.Google Scholar
  11. Careri, M.. Mangia, A., and Musci, M., 1998, Overview of the applications of liquid chromatography-mass spectrometry interfacing systems in food analysis: naturally occurring substances in food, J. Chromatog. A 794: 263–297.CrossRefGoogle Scholar
  12. Colegate, S. M., and Molyneux, R. J., 1993, Bioaclive Natural Products: Detection, Isolation, and Structural Determination, CRC Press, Boca Raton, FL.Google Scholar
  13. Cordell, G. A., 1995, Changing strategies in natural products chemistry, Phytochem. 40: 1585–1612.CrossRefGoogle Scholar
  14. Coward, L., Smith, M., Kirk, M., and Barnes, S., 1998, Chemical modification of isoflavones in soyfoods during cooking and processing, Ani. J. Clin. Nutr. 68S:1486S–1491S.Google Scholar
  15. Creaser, C. S., Koupai-Abyazani, M. R., and Stephenson, G. R., 1992, Gas chromatographic-mass spectromic characterization of flavanones in citrus and grape juices, Analyst 117: 1105–1109.CrossRefGoogle Scholar
  16. Dakora, F. D., 1995, Plant flavonoids: biological molecules for useful exploitation, Austral. J. Plant Physiol. 22: 87–99.CrossRefGoogle Scholar
  17. Davis, W. B., 1947, Determination of flavanones in citrus fruits, Anal. Chem. 19: 467–478.Google Scholar
  18. Deng, H., and Van Berkel, G. J., 1998, Electrospray mass spectrometry and UVNisible spectrophotometry studies of aluminum(III)-flavonoid complexes, J. Mass Spectrom. 33: 1080–1087.Google Scholar
  19. Dixon, R. A., 1999, lsoflavonoids: biochemistry, molecular biology. and biological functions, In: Comprehensive Natural Products Chemistry, Sankawa, U., ed., Elsevier, New York, NY, pp. 773–823.Google Scholar
  20. Dixon, R. A., and Paiva, N. L., 1995, Stress-induced phenylpropanoid metabolism, Plant Cell 7: 1085–1097.Google Scholar
  21. Garcia, M. C., Torre, M., Marina, M. L., and Laborda, F., 1997, Composition and characterization of soyabean and related products, Crit. Rev. Food Sci. Nutr. 34: 361–391.CrossRefGoogle Scholar
  22. Gengross, O., and Renda, N., 1966, Occurrence and quantitative estimation of naringin in citrus juices, Justus Liebigs Ann. Chem. 691: 186–189.CrossRefGoogle Scholar
  23. Hahlbrock, K., and Scheel, D., 1989, Physiology and biochemistry of phenylpropanoid metabolism, Ann Rev Plant Physiol. Plant Mol Biol. 40: 347–369.CrossRefGoogle Scholar
  24. Harbome, J. B., Mabry, T. J., and Mabry, H., eds., 1975, The Flavonoids,Chapman & Hall, New York, NY and London, UK.Google Scholar
  25. Harbome, J. B., and Mabry, T. J., eds., 1982, The Flavonoids: Advances in Research, Chapman & Hall, New York, NY and London, UK.Google Scholar
  26. Harbome, J. B., ed., 1988, The Flavonoids: Advances in Research since 1980, Chapman & Hall, New York, NY and London, UK.Google Scholar
  27. Harbome, J. B., ed., 1994, The Flavonoids: Advances in Research since 1986, Chapman & Hall, New York, NY and London, UK.Google Scholar
  28. Hasegawa, S., Berhow, M. A., and Fong, C. H., 1995, Analysis of bitter principles in Citrus, In: Modern Methods of Plant Analysis, Volume 18: Fruit Analysis, Linskens, H.F. and Jackson J. F., eds., Springer Verlag, Berlin; Heidelberg, pp. 59–80.Google Scholar
  29. Heinonen, S., Wahala, K., and Adlercreutz, H., 1999, Identification of isoflavone metabolites dihydrodaidzein, dihydrogenistein, 6-OH-O-DMA, and cis-4-OH-equol in human urine by gas chromatography-mass spectroscopy using authentic reference compounds, Anal. Biochem. 274: 211–9.CrossRefGoogle Scholar
  30. Heller, W., and Forkmann, G., 1994, Biosynthesis of flavonoids, in: The Flavonoids, Harbome, J. B., ed., Chapman & Hall, New York, NY, USA, pp. 499–536.Google Scholar
  31. Hendrickson, R., Kesterson, J. W., and Edwards, G. J., 1958, Ultraviolet absorption technique to determine the naringin content of grapefruit, Proc. Fla. State Hort. Soc. 71: 194–198.Google Scholar
  32. Horowitz, R. M., 1957, Detection of flavanones by reduction with sodium borohydride J.Org Chem. 22: 1733–1734.CrossRefGoogle Scholar
  33. Jourdan, P. S., Mansell, R. L., Oliver, D. G., and Weiler, E. W., 1984, Competitive solid phase enzyme-linked immunoassay for the quantification of limonin in citrus, Anal. Biochem. 138: 19–24.CrossRefGoogle Scholar
  34. Jourdan, P.S., Mansell, R. L., and Weiler, E. W., 1982, Radioimmunoassay for the citrus bitter principle, naringin, and related flavonoid-7-O-neohesperidosides, J. Medicinal Plant Res. 44: 82–86.CrossRefGoogle Scholar
  35. Jourdan, P. S., McIntosh, C. A., and Mansell, R. L., 1985a, Naringin levels in citrus tissues. Il. Quantitative distribution of naringin in Citrus paradisi Macfad., Plant Physiol. 77: 903–908.CrossRefGoogle Scholar
  36. Jourdan, P. S., Weiler, E. W., and Mansell, R. L., 19856, Naringin levels in citrus tissues. I. Comparison of different antibodies and tracers for the radioimmunoassay of naringin, Plant Physiol. 77: 896–902.Google Scholar
  37. Junghuth, G., and Ternes, W., 2000, HPLC separation of flavanols, flavones and oxidized tlavanols with UV-, DAD-, electrochemical and ESL-ion trap MS detection, Fresenius J. Anal. Chem. 367: 661–666.CrossRefGoogle Scholar
  38. Kirchner, J. G., ed., 1978, Thin-Layer Chromatography, Second Edition, Techniques of Chemistry Series Vol. XIV, John Wiley & Sons, New York, NY.Google Scholar
  39. Kohen, F., Lichter, S., Gayer, B., DeBoever, J., and Lu, L.. J., 1998, The measurement of the isoflavone daidzein by time resolved fluorescent immunoassay: a method for assessment of dietary soya exposure, J. Steroid Biochem. Mol Biol. 64: 217–222.CrossRefGoogle Scholar
  40. Kudou, S., Fleury, Y., Wetli, D., Magnolato, D., Uchida, T., Kitamura, K. and Okubo, K., 1991, Malonyl isoflavone glycosides in soybean (Glycine max Merrill) Agric. Biot. Chem. 55:2227–2233.Google Scholar
  41. Kwierty, A., and Braverman, J. B., 1959, Critical evaluation of the cyanidin reaction for flavonoid compounds, Bull. Res. Council Israel Sect. C 7: 187–196.Google Scholar
  42. Lin, L.-Z., He, X.-G., Lindenmaier, M., Yang, J., Cleary, M., Qiu, S.-X., and Cordell, G. A., 2000, LC-ESI-MS study of the flavonoid glycoside malonates of red clover (Trifolium pratense). J. Agric. Food Chem. 48: 354–365.Google Scholar
  43. Luthria, D. L., Jones, A. D., Donovan, J. L., and Waterhouse, A. L., 1998, GC-MS determination ofcatechin and epicatechin levels in human plasma, Book of Abstracts, 215th ACS National Meeting, Dallas, March 29–April 2, 1998. AGFD 008.Google Scholar
  44. Mabry, T. J., Markham, K. R., and Thomas, M. B., 1970, The Systematic Identification of Faavonoids, Springer Verlag, New York, NY.CrossRefGoogle Scholar
  45. Mann, J., R. Davidson, S., Hobbs, J. B., Banthorpe, D. V., and Harborne, J. B., 1994, Natural Products: Their Chemistry and Significance, Addison Wesley Longmann Ltd., Edinburgh Gate, Harlow.Google Scholar
  46. Matsumoto, R. and Okudai, N., 1991, Early evaluation of citrus bitter component, flavanone neohesperidosides by enzyme immunoassay using anti-naringin antibody, J. Japanese Soc. Horticult. Sci. 60: 191–200.CrossRefGoogle Scholar
  47. Messina M. J., 1999, Legumes and soybeans: overview of their nutritional profiles and health effects, Am. J. Clin. Nutr. 70S: 439S–450S.Google Scholar
  48. Novotny, L., Vachalkova, A., Al-Nakib, T., Mohanna, N., Vesela, D., and Suchy, V., 1999, Separation of structurally related flavonoids by GC/MS technique and determination of their polarographic parameters and potential carcinogenicity, Neoplasma 46: 231–236.Google Scholar
  49. Pietta, P. G., Mauri, P. L., Rava, A., and Sabbatini, G., 1991, Application of micellar electrokinetic capillary chromatography to the determination of flavonoid drugs, J. Chromatog. A 549: 367–373.CrossRefGoogle Scholar
  50. Runkel, M., Muehlau, A., Duecker, D., Tegtmeier, M., and Legrum, W., 1998, Capillary electrophoresis (CE): An efficient tool for the quality control of fruits as shown for the constituents ofgrapefruit, Fruit Process. 8: 102–104.Google Scholar
  51. Seigler, D. S.. 1981, Secondary metabolites and plant systematics, In: Secondary Plant Products, Stumpf, P. K., and Conn, E. E., eds., Academic Press. New York, pp. 139–175.Google Scholar
  52. Seitz, U., Oefner, P. J., Nathakarnkitkool, S., Popp, A., and Bonn, G. K., 1992, Capillary electrophoretic analysis of flavonoids, Electrophoresis 13: 35–38.CrossRefGoogle Scholar
  53. Shelnutt, S. R., Cimino, C. O., Wiggins, P. A., and Badger, T. M., 2000. Urinary pharmacokinetics of the glucuronide and sulfate conjugates of genistein and daidzein, Cancer Epidemiol. Biomarkers Prey. 9: 413–419.Google Scholar
  54. Shihabi, Z. K., Kute, T., Garcia, L. L., and Hinsdale, M.,1994, Analysis of isoflavones by capillary electrophoresis. J. Chromatog. A 680: 181–185.Google Scholar
  55. Song, T., Barua, K., Buseman, G., and Murphy, P. A., 1998, Soy isoflavone analysis: quality control and a new internal standard, Am. J. Gin. Num. 68S: 1474S–1479S.Google Scholar
  56. Stobiecki, M., 2000, Application of mass spectrometry for identification and structural studies of flavonoid glycosides. Phytochem. 54: 237–256.CrossRefGoogle Scholar
  57. Stobiecki, M., Malosse, C., Kerhoas, L., Wojlaszek, P., and Einhorn, J., 1999, Detection of isoflavonoids and their glycosides by liquid chromatography/electrospray ionization mass spectrometry in root extracts of lupin (Lupinus albus), Phytochem. Anal. 10: 198–207.CrossRefGoogle Scholar
  58. Swantsitang, P., Tucker, G., Robards, K., and Jardine, D.. 2000, Isolation and identification of phenolic compounds in Citrus sinensis, Anal. Chini Acta 417: 231–240.CrossRefGoogle Scholar
  59. Tsukamoto, C., Shimada, S., Igata, K., Kudou, S., Kokubun, M., Okubo, K., and Kitamura, K., 1995, Factors affecting isoflavone content in soybean seeds: changes in isoflavones, saponins, and composition of fatty acids at different temperatures during seed development, J. Agric. Food Chem. 43: 1184–1192.CrossRefGoogle Scholar
  60. Voirin, B., Sportouch, M., Raymond, O., Jay, M., Bayet, C., Dangles, O., and El Hajji, H., 2000, Separation offlavone C-glycosides and qualitative analysis of Pass fora incarnata L. by capillary zone electrophoresis, Phytochem. Anal. 11: 90–98.CrossRefGoogle Scholar
  61. Wang, C. Y., Ma, Q., Pagadala, S., Sherrard, M. S., and Krishnan, P. G., 1998, Changes of isoflavones during processing of soy protein isolates, J. Amer Oil Chem. Soc. 75: 337–342.CrossRefGoogle Scholar
  62. Wang, C. Y., Sherrard, M., Pagadala, S., Wixon, R., and Scott, R. A., 2000, Isoflavone content among maturity group 0 to 11 soybeans, J. Amer Oil Chem. Soc. 77: 483–487.CrossRefGoogle Scholar
  63. Wang, H., and Murphy, P. A., 1994, Isoflavone content in commercial soybean foods, J. Agric. Food Chem. 42: 1666–1673.CrossRefGoogle Scholar
  64. Wang, H. J., and Murphy, P. A., 1996, Mass balance study of isoflavones during soybean processing, J. Agric. Food Chem. 44: 2377–2383.CrossRefGoogle Scholar
  65. Watson, D. G., and Pitt, A. R., 1998, Analysis of flavonoids in tablets and urine by gas chromatography/mass spectrometry and liquid chromatography/mass spectrometry, Rapid Commun. Mass Spectrom. 12: 153–156.CrossRefGoogle Scholar
  66. Wolfender, J. L., Rodriguez, S.. Hostettmann, K., and Wagner-Redecker, W., 1995, Comparison of liquid chromatography/electrospray, atmospheric pressure chemical ionization, thermospray and continous flow fast atom bombardment mass spectrometry for the determination of secondary metabolites in crude plant extracts, J. Mass Spectrom. and Rapid Commun. Mass Spectrom. Special: S35–S46.Google Scholar
  67. Zhou, S,. and Hamberger, M., 1996, Application of liquid chromatography atmospheric pressure ionization mass spectrometry in natural products analysis: Evaluation and optimization of electrospray and heated nebulizer interfaces, J. Chromatog. A 755: 189–204.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2002

Authors and Affiliations

  • Mark A. Berhow
    • 1
  1. 1.U. S. Department of Agriculture, Agricultural Research ServiceNational Center for Agricultural Utilization ResearchPeoriaUSA

Personalised recommendations