Comparison of Anthocyanin Content in Seed Coats of Black Soybean [Glycine max(L.) Merr.] Cultivars Using Liquid Chromatography Coupled to Tandem Mass Spectrometry

  • Shin, Sung-Chul (Department of Chemistry and Research Institute of Life Science, Gyeongsang National University) ;
  • Lee, Soo-Jung (Department of Food and Nutrition, Institute of Agriculture and Life Science, Gyeongsang National University) ;
  • Lee, Sung-Joong (Department of Chemistry and Research Institute of Life Science, Gyeongsang National University) ;
  • Chung, Jong-Il (Department of Agriculture and Life Science Research Institute, Gyeongsang National University) ;
  • Bae, Dong-Won (Central Laboratory, Gyeongsang National University) ;
  • Kim, Soo-Taek (Department of Informational Statistics and Research Institute of Natural Science, Gyeongsang National University) ;
  • Sung, Nak-Ju (Department of Food and Nutrition, Institute of Agriculture and Life Science, Gyeongsang National University)
  • Published : 2009.12.31


The seed coat of the black soybean contains 3 main anthocyanins such as delphinidin-3-O-$\beta$-glucoside, cyanidin-3-O-$\beta$-glucoside, and petunidin-3-O-$\beta$-glucoside. As a part of our effort on discovering and breeding new black soybean cultivars which possesses specific anthocyanin component rich, we determined the anthocyanin profiles of the 2 cultivars recently developed soybean cv. Gaechuck #1 and cv. Gyeongsang #1, using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and compared their content and identity with those of previously known 10 cultivar controls. The Cosmosil-$5C_{18}$-AR-II column were selected for the analysis because of the best peak separation. The column temperature was set up at $35^{\circ}C$. The mobile phase consisting of water containing 0.5%(v/v) formic acid and methanol gave good separation between the 3 anthocyanin analytes and internal standard (quercetin 3-O-$\beta$-rutinoside) and peaks with suppressed tail. The MS/MS spectra of each individual anthocyanin standard were detected in positive electron spray ionization (ESI) modes. It was disclosed that the anthocyanin contents of the soybean cv. Gaechuck#1 and cv. Gyeongsang#1 are roughly higher than those of the 10 controls.


  1. da Costa CT, Horton D, Margolis SA. Analysis of anthocyanins in foods by liquid chromatography, liquid chromatography–.mass spectrometry and capillary electrophoresis. J. Chromatogr. A 881: 403-410 (2000)
  2. Torskangerpoll K, Andersen OM. Colour stability of anthocyanins in aqueous solutions at various pH values. Food Chem. 89: 427-440 (2005)
  3. Sullivan J. Anthocyanin. Available from: http://www.charlies-web. com/specialtopics/anthocyanin.html. Accessed July 15, 2009
  4. Hedin PA, Jenkins JN, Collum DH, White WH, Parrot WL. Multiple factors in cotton contributing to resistance to the tobacco budworm, Heliothis virescens (F.). pp. 347-365. In: Plant Resistance to Insects. ACS Symposium Series. Hedin PA (ed). American Chemical Society, Washington, DC, USA (1983)
  5. Gao L, Mazza G. Quantitation and distribution of simple and acylated anthocyanins and other phenolics in blueberries. J. Food Sci. 59: 1057-1059 (1994)
  6. Ortega-Regules A, Romero-Cascales I, Lopez-Roca J, Ros-Garcia JM, Gomez-Plaza E. Anthocyanin fingerprint of grapes: Environmental and genetic variations. J. Sci. Food Agr. 86: 1460-1467 (2006)
  7. Nam SH, Choi SP, Kang MY, Koh HJ, Kozukue N, Friedman M. Antioxidative activities of bran extracts from twenty one pigmented rice cultivars. Food Chem. 94: 613-620 (2006)
  8. Philpott M, Gould KS, Lim C, Ferguson LR. In situ and in vitro antioxidant activity of sweetpotato anthocyanins. J. Agr. Food Chem. 52: 1511-1513 (2006)
  9. Tsuda T, Horio F, Osawa T. Cyanidin 3-O-$\beta$-D-glucoside suppresses nitric oxide production during a zymosan treatment in rats. J. Nutr. Sci. Vitaminol. 48: 305-310 (2002)
  10. Kim HJ, Tsoy I, Park JM, Chung JI, Shin SC, Chang KC. Anthocyanins from soybean seed coat inhibit the expression of TNF-$\alpha$-induced genes associated with ischemia/reperfusion in endothelial cell by NF-(B-dependent pathway and reduce rat myocardial damages incurred by ischemia and reperfusion in vivo. FEBS Lett. 580: 1391-1397 (2006)
  11. Hyun JW, Chung HS. Cyanidin and malvidin from Oryza sativa cv. Heugjinjubyeo mediate cytotoxicity against human monocytic leukemia cells by arrest of $G_2$/M phase and induction of apoptosis. J. Agr. Food Chem. 52: 2213-2217 (2004)
  12. Zhao C, Giusti MM, Malik M, Moyer MP, Magnuson BA. Effects of commercial anthocyanin-rich extracts on colonic cancer and nontumorigenic colonic cell growth. J. Agr. Food Chem. 52: 6122-6128 (2004)
  13. Tsuda T, Horio F, Uchida K, Aoki H, Osawa T. Dietary cyanidin 3-O-$\beta$-D-glucoside-rich purple corn color prevents obesity and ameliorates hyperglycemia in mice. J. Nutr. 133: 2125-2130 (2003)
  14. Choi Y, Jeong HS, Lee J. Antioxidant activity of methanolic extracts from some grains consumed in Korea. Food Chem. 103: 130-138 (2007)
  15. USDA. Evaluation/characterization data queries. Available from: Accessed July 14, 2009
  16. Todd JJ, Vodkin LO. Pigmented soybean (Glycine max) seed coats accumulate proanthocyanidins during development. Plant Physiol. 102: 663-670 (1993)
  17. Choung MG, Baek IY, Kang ST, Han WY, Shin DC, Moon HP, Kang KH. Isolation and determination of anthocyanins in seed coats of black soybean (Glycine max (L.) Merr.). J. Agr. Food Chem. 49: 5848-5851 (2001)
  18. Kuroda C, Wada M. The colouring matter of 'Kuro-mame'. Vol. 9, pp. 17-18. In: Proceedings of the Imperial Academy. Majima R, Jan MIA (eds). Institute of Physical and Chemical Research, Tokyo, Japan (1933)
  19. Yoshida K, Sato Y, Okuno R, Kameda K, Isobe M, Kondo T. Structural analysis and measurement of anthocyanin from colored seed coats of Vigna, Phaseolus, and Glycine legumes. Biosci. Biotech. Bioch. 60: 589-593 (1996)
  20. Chung JI. Gaechuck #1 (patent 2831; 2006), Gyenogsnag #1 (patent 2338; 2008). Korea Seed and Variety Service, Seoul, Korea
  21. Flamini R. Mass spectrometry in grape and wine chemistry. Part I: Polyphenols. Mass Spectrom. Rev. 22: 218-250 (2003)
  22. Stobiecki M. Application of mass spectrometry for identification and structural studies of flavonoid glycosides. Phytochemistry 54: 237-256 (2000)
  23. Cooke DN, Thomasset S, Boocock DJ, Schwarz M, Winterhalter P, Steward WP, Gescher AJ, Marczylo TH. Development of analyses by high-performance liquid chromatography and liquid chromatography/tandem mass spectrometry of bilberry (Vaccinium myrtilus) anthocyanins in human plasma and urine. J. Agr. Food Chem. 54: 7009-7013 (2006)
  24. Tian Q, Giusti MM, Stoner GD, Schwartz SJ. Characterization of a new anthocyanin in black raspberries (Rubus occidentalis) by liquid chromatography electrospray ionization tandem mass spectrometry. Food Chem. 94: 465-468 (2006)
  25. Ju ZY, Howard LR. Effects of solvent and temperature on pressurized liquid extraction of anthocyanins and total phenolics from dried red grape skin. J. Agr. Food Chem. 51: 5207-5213 (2003)
  26. Hosseinian FS, Muir AD, Westcott ND, Krol ES. AAPH-mediated antioxidant reactions of secoisolariciresinol and SDG. Org. Biomol. Chem. 5: 644-654 (2007)
  27. Kang J, Sun JH, Zhou L, Ye M, Han J, Wang BR, Guo DA. Characterization of compounds from the roots of Saposhnikovia divaricata by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry. Rapid Commun. Mass Sp. 22: 1899-1911 (2008)
  28. Hosseinian FS, Li W, Beta T. Measurement of anthocyanins and other phytochemicals in purple wheat. Food Chem. 109: 916-924 (2008)
  29. Oliveira MC, Esperanca P, Almoster Ferreira MA. Characterisation of anthocyanidins by electrospray ionization and collision-induced dissociation tandem mass spectrometry. Rapid Commun. Mass Sp. 15: 1525-1532 (2001)