Development of pH-Switchable Hydrophobic Deep Eutectic Solvents for the Extraction and Preconcentration of Aflatoxin M1 in Milk-Based Athlete Sports Supplements

A novel, eco-friendly, and efficient dispersive liquid–liquid microextraction method was developed, utilizing pH-switchable hydrophobic deep eutectic solvents. This method, which combines with liquid chromatography and fluorescence detection, allows for the extraction and analysis of aflatoxin M1 in milk-based sports supplements. At first, twelve deep eutectic solvents were prepared and their pH switchability was investigated. pH-Switchable solvents were used for extraction and preconcentration of aflatoxin M1. The strength of the proposed method is that the dispersion of extraction solvent in the aqueous phase and the separation of phases occurred only by pH change and do not require the addition of dispersing solvent, ultrasonic, vortex, and centrifugation. In this study, the key factors that affect the extraction process, including the type and volume of DES (deep eutectic solvent), KOH concentration, volume of hydrochloric acid, the effect of adding salt, and the duration of extraction, were investigated, and the optimal conditions for each of these parameters were determined to maximize the extraction efficiency. Under optimum conditions, the relative standard deviations (RSDs) including intra- and inter-day precision of the method were determined by analyzing 5 μg kg⁻¹ of aflatoxin M1 in target samples seven times. The RSDs were 2.6% for intra-day and 4.5% for inter-day precision. The calibration curves showed linearity in the range of 0.015 to 50 μg kg⁻¹. The limit of detection (LOD) for this method was 0.005 μg kg⁻¹. When real milk-based sports supplements were spiked with varying concentrations of aflatoxin M1, the relative recoveries ranged from 92.0 to 105.0%.