Evaluation of a Method to Quantify Isoflavones in Soybean by Single and Multi-laboratory Validation Studies

Abstract

The recovery of isoflavones spiked into a soy sample at two different concentrations ranged from 94% to 105%. The multilaboratory validation study with 7 participating laboratories showed satisfactory interlaboratory precision; all HorRat values were < 2.Keywords: soy isoflavone, multilaboratory validation study, AOAC guideline IntroductionSoy contains isoflavone aglycones such as genistein, daidzein and glycitein, and their glycosides such as genistin, daidzin and glycitin (Munro et al., 2003). Recently, the antioxidant, cancergrowth inhibition, and other health promotion effects of soy isoflavones have been elucidated (Perabo et al., 2008, Cuevas et al., 2003, Nagata et al., 2002. A microbiota metabolite of isoflavone glycoside showed growth inhibitory activity for cancer cells and anti-inflammatory activity (Atkinson et al., 2005, Hirata et al., 2013, Shimoda and Hamada et al., 2011. Moreover, soy isoflavones are recognized to be plant estrogens, binding estrogen receptors to induce physiological effects. Japanese people consume isoflavones mainly from soy in everyday life; no other Japanese food contains such a high amount of isoflavones. The daily consumption of soy isoflavones is limited to 70~75 mg aglycone equivalent, as defined by the Food Safety Commission of Japan, because isoflavones are endocrine disruptors of human reproductive systems (Clarke et al., 2003). Therefore, it is important to analyze the isoflavone concentration in common foods and "food for specific health uses" rapidly and with high precision (Cederroth et al., 2012). However, the quantitative analysis of isoflavones by the official methods of AOAC international (OMA) 2001.10 and OMA 2008.03 is both time-consuming and requires a large amount of soy sample (i, Collison et al., 2008). With the OMA 2001.10 and OMA T. Ogita et al. 474 2008.03 methods, isoflavone content in soy samples can be quantified using 2 to 10 g of sample, and the analysis time by HPLC is 44.5 min and 63 min, respectively. Thus, the conventional method is not suitable for the analysis of large numbers of samples. Accordingly, the development of a better analysis method for quantification of isoflavones in soy samples is required.In this study, we developed a quantification method for soy isoflavones requiring shorter analysis time and less sample than the conventional method. ExperimentalChemicals Daidzin, daidzein, genistin, genistein, glycitin and glycitein were purchased from Nacalai Tesque (Kyoto, Japan) and used as the standards. All the chemicals were > 97% purity based on HPLC analysis. Soybeans (30 g) were frozen overnight at _ 80 ℃, and then ground using a Rotor-Speed MillP14 (Fritsch GmbH, Idar-Oberstein, Germany), passed through a 0.5 mm filter, and freeze-dried using a freeze dryer. Powdered soybean samples were thoroughly mixed, divided into 8 portions, and placed in plastic bags. Freeze-drying and mixing steps were repeated twice. Finally, soy powder samples were stored in aluminum laminated bags at _ 80℃. The soy powder particles were analyzed u...

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