Alkaline–Enzymatic Hydrolysis for Improved Quantitation of Total Isoflavones in Soy Protein-Based Infant Food by UPLC–MS/MS

Abstract

Early life exposure to isoflavones may have detrimental effects on the reproductive system. However, determining the total 12 isoflavones found in soybeans is challenging due to the unavailability of reference standards for all isoflavones. In this study, a method was developed using only three aglycone isoflavones to quantitate all isoflavones. Three different β-glucosidases were evaluated to hydrolyze all glucoside isoflavones, but resistance was observed for malonyl and acetyl glucosides. To address this issue, 80% methanol containing 0.3% NaOH was used as the extraction solution and to facilitate the hydrolysis, allowing 0.5 U of βglucosidase from Thermotoga maritima to efficiently hydrolyze all glucoside isoflavones with the corresponding aglycones obtained within 10 min. Combined with ultraperformance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS), efficient quantitation of all isoflavones was achieved with low limits of quantitation (0.010−0.23 μg/g) and good recoveries (97.0−98.6%) for soy protein-based infant food. This developed method is expected to normalize total isoflavones to aglycone equivalents to avoid overestimating isoflavone contents caused by the presence of glucoside forms.