Systematic evaluation of pre-HPLC sample processing methods on total and individual isoflavones in soybeans and soy products Suqin Shao a,b , Alison M. Duncan b , Raymond Yang a , Massimo F. Marcone c , Istvan Rajcan d , Rong Tsao a, ⁎ a Guelph Food Research Centre, Agriculture & Agri-Food Canada, 93 Stone Road West, Guelph, Ontario, Canada N1G 5C9 b Department of Human Health and Nutritional Sciences, University of Guelph, 50 Stone Road East, Guelph, Ontario, Canada N1G 2W1 c Department of Food Science, University of Guelph, 50 Stone Road East, Guelph, Ontario, Canada N1G 2W1 d Department of Plant Agriculture, University of Guelph, 50 Stone Road East, Guelph, Ontario, Canada N1G 2W1 abstract article info Article history: Received 1 October 2010 Accepted 21 December 2010 Keywords: Soybean Isoflavone Glucosides Acetylglucosides HPLC Hydrolysis Direct analysis There are mainly two protocols in isoflavone analysis, one that involves hydrolysis prior to HPLC analysis and the other direct HPLC analysis. In this study, three different hydrolysis methods were systematically re- evaluated, and compared with direct HPLC analysis. Acidic hydrolysis (1.2–3 M HCl in ethanol at 80 °C) showed a maximum conversion of ca. 92% from glucosides to aglycones in 2 h; however, longer reaction caused degradation of genistein. Alkaline hydrolysis using 2 M NaOH converted acetylglucosides and malonylglucosides to their respective glucosides within 10 min. Glucuronidase from H. pomatia effectively converted isoflavone glucosides and acetylglucosides to aglycones. Quantification of the total isoflavones in various soy food products showed no significant difference among direct injection and the three hydrolysis methods (P b 0.05). We conclude that direct analysis of isoflavones in crude extracts is a rapid and accurate method to obtain isoflavone profiles and compositions in soybean, soy foods and beverages. © 2011 Elsevier Ltd. All rights reserved. 1. Introduction Interest in introducing soybean and soy food products to the Western diet has been growing steadily owing to the health promoting effects of soy protein and isoflavones over the past several years. Soy and its constituent protein and/or isoflavones have been associated with reduced risk of cardiovascular disease (Clarkson, 2002), prostate (Yan & Spitznagel, 2005), breast cancers (Wu, Yu, Tseng, & Pike, 2008), and improved bone health (Messina, Ho, & Alekel, 2004). There are 12 major known isoflavones in soybean and soy products, three aglycones genistein, daidzein and glycitein, their respective 7-O-β-D-glucosides (genistin, daidzin and glycitin), 6″-O- acetyl-7-O-β-D-glucosides (acetylgenistin, acetyldaidzin and acetyl- glycitin) and 6″-O-malonyl-7-O-β-D-glucosides (malonylgenistin, malonyldaidzin and malonylglycitin) (Fig. 1). The concentrations of these forms vary in soybean and soy products depending on cultivar, geographic region, agronomical practice, environmental conditions of plant growth, and processing conditions (Kim, Jung, Ahn, & Chung, 2005; Kirakosyan et al., 2006; Mebrahtu, Mohamed, Wang & Andebrhan, 2004; Pinto, Lajolo, & Genovese, 2005). Quantification of all of these isoflavone forms is not only important in quality control of soy products, but also in understanding the effect of processing conditions on isoflavone content of foods, and isoflavone bioavailability (Delmonte & Rader, 2006; Jackson et al., 2002; Song, Barua, Buseman, & Murphy, 1998). A chromatographic separation is therefore necessary before all isoflavones can be accurately quanti- fied. Different chromatographic techniques have been developed to separate soy isoflavones in all forms; however, selection of a particular technique will vary depending upon the objectives of the investigation. Among commonly used chromatographic techniques, high- performance liquid chromatography (HPLC), is currently the most widely used technique for isoflavone analysis. Great advances have been made to obtain complete profiles of all 12 isoflavones in soybean and soy products (Collison, 2008; Griffith & Collison, 2001; Hsieh, Kao & Chen, 2004; Pinto et al., 2005; Zhang, Hettiarachchy, Chen, Horax, Cornelious, & Zhu, 2006). This is largely due to the availability of the 12 isoflavone standards, and to the technological advances in separation chemistry. Such effort has resulted in good HPLC separa- tions of all soy-derived isoflavones such as that done by Collison (2008). Meanwhile, analysis of the aglycones after acidic, alkaline or enzymatic hydrolysis of the extracted native isoflavones (i.e. aglycones, glucosides, acetylglucosides and malonylglucosides) is still employed in many methods (Kano, Takayanagi, Harada, Sawada, & Ishikawa, 2006; Klump, Allred, MacDonald, & Ballam, 2001; Seguin, Zheng, Smith, & Deng, 2004). Hydrolysis reduces the number of analytes for the HPLC analysis (Kano et al., 2006; Klump et al., 2001; Seguin et al., 2004), which not only significantly simplifies the Food Research International 44 (2011) 2425–2434 ⁎ Corresponding author. Guelph Food Research Centre, Agriculture & Agri-Food Canada, 93 Stone Road West, Guelph, Ontario, Canada N1G 5C9. Tel.: +1 519 780 8062. E-mail address: caor@agr.gc.ca (R. Tsao). 0963-9969/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2010.12.041 Contents lists available at ScienceDirect Food Research International journal homepage: www.elsevier.com/locate/foodres