Optimization of High-Performance Liquid Chromatography (HPLC) Conditions for Isoflavones Using Deep Eutectic Solvents (DESs) as Mobile Phase Additives by the HCI Program

Abstract

Isoflavone exhibits estrogen-like structure and activity and has various physiological and pharmacological functions, such as anticancer, antioxidant, and anti-inflammatory. In this study, several deep eutectic solvents (DESs) were synthesized and used as mobile phase additives to optimize the analytical conditions of five isoflavones. The DES composed of choline chloride:citric acid (molar ratio 1:1) was the most effective mobile phase additive with reduced peak tailing, improved peak shape, and increased number of theoretical plates. The effectiveness of the DES highlights its potential as a mobile phase additive. The optimal high-performance liquid chromatography (HPLC) conditions for the isoflavones were determined by the Intelligent HPLC simulator. The optimal conditions for gradient elution were water (A): acetonitrile (B) for 0 min, 25% B; 15 min, 25% B; 17 min, 35% B; and 50 min, 35% B. This approach provided excellent linearity (0.5 to 500 μg/mL, R2 ≥ 0.9991), limits of detection (0.024 to 0.109 μg/mL), limits of quantification (0.079 to 0.363 μg/mL), and precision (1.74 to 4.80%).