Optimized Quantitative Method for Determining Isoflavones and Equol in Bovine Digestive Fluids and Feces

ABSTRACTMany studies have been conducted on the impact of animal feed on isoflavones and their metabolite concentrations in bovine milk, but few studies have focused on the development and validation of analytical protocols for quantifying these compounds in biological matrices other than milk and plants. The purpose of this study was to develop a method that would enable four isoflavones and equol in cows’ feces and digestive fluids to be quantified simultaneously. The method is based on aglycones released by methanolic ultrasound-assisted extraction, followed by enzymatic hydrolysis and high-performance liquid chromatography tandem–mass spectrometry analysis. The sample preparation was optimized using the Box–Behnken design. The selected extraction conditions were 80°C, 10 min, and 50% methanol for digestive fluids and 70°C, 35 min, and 60% methanol for feces. For hydrolysis, the selected conditions were 37°C, 1 h, and a pH of 6 for both matrices. The analytical method showed a good linear regression mo...