Development of a methodology in an automated system for analysis of naproxen and bezafibrate in in-situ formation using a deep eutectic solvent by liquid chromatography.

This work presents an innovative analytical methodology that uniquely combines the in-situ formation of deep eutectic solvents (DESs) with hollow fiber microporous membrane liquid-liquid extraction (HF-MMLLE) in an automated 96-well plate system. Unlike conventional approaches where DESs are pre-prepared, in our method the eutectic phase is generated during the extraction step, establishing selective hydrogen-bonding interactions between octanoic acid and the functional groups of naproxen and bezafibrate. This in-situ supramolecular arrangement improves extraction efficiency while simplifying sample preparation and minimizing solvent consumption. The integration with a high-throughput 96-well platform allows the simultaneous processing of up to 96 samples, reducing the effective analysis time to less than one minute per sample, a feature not previously reported for DES-based extractions. Method optimization was performed through univariate and multivariate designs, and FTIR characterization confirmed the in-situ DES formation mechanism. The methodology presented detection limit of 7.6 μg L-1 and quantification limit of 25 μg L-1. The precision results were lower than 15.9% and recoveries ranged from 71 to 102%. The greenness of the methodology was confirmed using the AGREEprep tool, demonstrating that the proposed strategy offers a sustainable, selective, and high-throughput alternative for the determination of emerging pharmaceutical contaminants in aquatic environments.