Skip to content
BY-NC-ND 3.0 license Open Access Published by De Gruyter Open Access September 27, 2011

Use of multivariate statistical techniques to optimize the separation of isoflavones by liquid chromatography

Irena Baranowska, Sylwia Magiera and Jacek Baranowski
From the journal Open Chemistry


The aim of this paper is to optimize and validate a high performance liquid chromatography (HPLC) method for separation and quantification of five isoflavones. A statistical central composite design was used to separate all peaks. These multivariate procedures were efficient in determining the optimal separation condition using resolution, capacity factor, asymmetry and number of theoretical plates. The effective separation of the examined compounds was applied on a Develosil RP Aqueous AR 5 RP-30 column with a gradient mobile phase system and a DAD detector. The isolation and preconcentration of the isoflavones from urine and plasma samples were conducted by means of the solid-phase extraction (SPE). For optimize SPE conditions various sorbents were tested. Furthermore, high recoveries and good relative standard deviations were obtained when the samples were passed through the Oasis HLB column. The developed method was validated and successfully applied for determination of isoflavones in urine and plasma.

[1] D.F. Birt, S. Hendrich, W. Wang, Pharm. Ther. 90, 157 (2001) in Google Scholar

[2] B.H. Havsteen, Pharmacol. Ther. 96, 67 (2002) in Google Scholar

[3] T. Walle, Free Rad. Biol. Med. 36, 829 (2004) in Google Scholar

[4] L.W. Lu, L.D. Broemeling, M.V. Marshall, V.S. Ramanujam, Cancer Epidemiol. Biomark. Prev. 4, 497 (1995) Search in Google Scholar

[5] K. Setchell, M. Faughnan, T. Avades, L. Zimmer-Nechemias, N. Brown, B. Wolfe, W. Brashear, P. Desai, M. Oldfield, N. Botting, A. Cassid, Am. J. Clin. Nutr. 77, 411 (2003) 10.1093/ajcn/77.2.411Search in Google Scholar

[6] A.A. Franke, L.J. Custer, Clin. Chem. 42, 955 (1996) 10.1093/clinchem/42.6.955Search in Google Scholar

[7] S. Yamamoto, T. Sobue, S. Sasaki, M. Kobayashi, Y. Arai, M. Uehara, H. Adlercreutz, S. Watanabe, T. Takahashi, Y. Iitoi, Y. Iwase, M. Akabane, S. Tsugane, J. Nutr. 131, 2741 (2001) 10.1093/jn/131.10.2741Search in Google Scholar

[8] J. Maubach, M.E. Bracke, A. Heyerick, H.T. Depypere, R.F. Serreyn, M.M. Mareel, D. De Keukeleire, J. Chromatogr. B 784, 137 (2003) in Google Scholar

[9] Z. Ma, Q. Wu, D.Y.W. Lee, M. Tracy, S.E. Lukas, J. Chromatogr. B 823, 108 (2005) in Google Scholar PubMed

[10] B. Yan, D. Xing, Y. Ding, J. Tao, L. Du, J. Pharm. Biomed. Anal. 37, 297 (2005) in Google Scholar PubMed

[11] S. Vega-López, K.J. Yeum, J.L. Lecker, L.M. Ausman, E.J. Johnson, S. Devaraj, I. Jialal, A.H. Lichtenstein, Am. J. Clin. Nutr. 81, 43 (2005) 10.1093/ajcn/81.1.43Search in Google Scholar PubMed

[12] C.L. Holder, M.I. Churchwell, D.R. Doerge, J. Agric. Food Chem. 47, 3764 (1999) in Google Scholar

[13] N. Fang, S. Yu, T.M. Badger, J. Agric. Food Chem. 50, 2700 (2002) in Google Scholar

[14] P.B. Grace, J.I. Taylor, N.P. Botting, T. Fryatt, M.F. Oldfield, S.A. Bingham, Anal. Biochem. 315, 114 (2003) in Google Scholar

[15] P.B. Grace, J.I. Taylor, N.P. Botting, T. Fryatt, M.F. Oldfield, N. Al-Maharik, S.A. Bingham, Rapid Commun. Mass Spectrom. 17, 1350 (2003) in Google Scholar

[16] P.B. Grace, N.S. Mistry, M.H. Carter, A.J.C. Leathem, P. Teale, J. Chromatogr. B 853, 138 (2007) in Google Scholar

[17] L. Gu, M. Laly, H.C. Chang, R.L. Prior, N. Fang, M.J.J. Ronis, T.M. Badger, J. Agric. Food Chem. 53, 6858 (2005) in Google Scholar

[18] J.K. Prasain, K. Jones, N. Brissie, R. Moore, J.M. Wyss, S. Barnes, J. Agric. Food Chem. 52, 3708 (2004) in Google Scholar

[19] B. Klejdus, J. Vacek, V. Adam, J. Zehnálek, R. Kizek, L. Trnková, V. Kubáň, J. Chromatogr. B 806, 101 (2004) in Google Scholar

[20] T. Nurmi, H. Adlercreutz, Anal. Biochem. 274, 110 (1999) in Google Scholar

[21] J.A. Starkey, Y. Mechref, C.K. Byun, R. Steinmetz, J.S. Fuqua, O.H. Pescovitz, M.V. Novotny, Anal. Chem. 74, 5998 (2002) in Google Scholar

[22] F. Kohen, S. Lichter, B. Gayer, J. De Boever, L.J.W. Lu, J. Steroid Biochem. Molec. Biol. 64, 217 (1998) in Google Scholar

[23] M. Uehara, O. Lapcík, R. Hampl, N. Al-Maharik, T. Mäkelä, K. Wähälä, H. Mikola, H. Adlercreutz, J. Steroid. Biochem. Molecular. Biol. 72, 273 (2000) in Google Scholar

[24] I. Baranowska, S. Magiera, Anal. Bioanal. Chem. 399, 3211 (2011) in Google Scholar

[25] I. Baranowska, S. Magiera, J. Baranowski, J. Chromatogr. B 879, 615 (2011) in Google Scholar

[26] A.R. Lomasney, C. Guillo, A.M. Sidebottom, M.G. Roper, Anal. Bioanal. Chem. 394, 313 (2009) in Google Scholar

[27] E. Nemutlu, S. Kır, D. Katlan, M.S. Beksaç, Talanta 80, 117 (2009) in Google Scholar

[28] A. Müller, D. Flottmann, W. Schulz, W. Seitz, W.H. Weber, Anal. Bioanal. Chem. 390, 1317 (2008) in Google Scholar

[29] C. Hartmann, J. Smeyers-Verbeke, D.L. Massart, R.D. McDowall, J. Pharm. Biomed. Anal. 17, 193 (1998) in Google Scholar

[30] H.F. Cueto-Rojas, N.O. Pérez, G. Pérez-Sánchez, I. Ocampo-Juárez, E. Medina-Rivero, J. Chromatogr. B 878, 1019 (2010) in Google Scholar PubMed

Published Online: 2011-9-27
Published in Print: 2011-12-1

© 2011 Versita Warsaw

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Scroll Up Arrow