Optimizing pre-treatment of alkaline hydrolysis RP-HPLC for enhancing accuracy of soy isoflavone determination

• Higher extraction and hydrolysis efficiency of improved alkaline hydrolysis. • Lower baseline noise and reduced co-elution risk of HPLC profile. • Total IFs content obtained by improved alkaline method increased 16.4%. Isoflavones (IFs) are important bioactive compounds which offers several benefits to human health. Despite the importance of IFs content, there are still some deficiencies in the existing methods. The objective of this study is to optimize key parameters of alkaline hydrolysis method for enhancing both reliability and accuracy of quantitative analysis of IFs contents in soybeans and soy protein products. Solvent type, extraction temperature, heating mode, alkaline concentration, hydrolysis temperature, hydrolysis time and some details are key points to target analytes which yield determining parameters. The biggest IFs content was obtained by extraction using methanol (MeOH)/water (80:20, v/v) in a 75 ℃ oven for 2h, and hydrolyzation with 3M NaOH solution at a room temperature for 10 mins. The total IFs content obtained by the improved method has increased by 16.4% compared with AOAC Official Method 2001.10. The accuracy of the method was evaluated using the relative standard deviation (RSD). Intraday accuracies in the total amount of isoflavones of four samples were 0.03%-0.63% (n=3). Interday accuracy in the total amount of isoflavones was 2.71% (n=6). LOD of IFs ranged from 0.1 μ g/mL for aglycones to 0.2 μ g/mL for glucosides. LOQ of IFs ranged from 0.3 μ g/mL for aglycones to 0.5 μ g/mL for glucosides. The improved method was proven to be a more accurate way to determine IFs contents in soybeans and soy protein products and thus making it useful for quality control and systematic in-depth study of soybeans and soy products; even to further assess the dietary soy exposure and the soy-health association.