In-Syringe Automated Extraction of Selected Antibiotics in Surface Waters exploring Deep Eutectic Solvent coupled online to HPLC Analysis

• Lab-In-Syringe automated DLLME for antibiotics using natural deep eutectic solvent • Parallel operation of sample preparation and online coupled analyte separation • Throughput of 4.5/h using only 200 µL DES with preconcentration up to 8.5 • Excellent reproducibility with < 4.3% RSD and LOD values from 0.4 to 15 µg/L • Outstanding environmental friendliness with AGREE score of 0.80 Antibiotics are by design difficult to remove in the biological treatment of wastewater and are becoming ubiquitous in aquatic environments, which bears the risk for the formation of resistant bacterial strains. The new EU Urban Wastewater Treatment Directive demands fast, cheap, efficient, and environmentally-friendly methods for antibiotic determination. However, there is a general need for sample preparation and analyte preconcentration. For this purpose, liquid phase extractions remain reliable and often-used approaches, while they show drawbacks regarding procedural greenness. Herein, we applied dispersive liquid-liquid microextraction based on a hydrophobic natural deep eutectic solvent for the first time for the fully automated enrichment of the selected antibiotics tetracycline, oxytetracycline, doxycycline, chlortetracycline, sulfamethoxazole, and trimethoprim from surface waters. The methodology was based on the Lab-In-Syringe technique and was coupled online to high-performance liquid chromatography with spectrophotometric detection. Sample and solvent handling and extraction were carried out inside the void of a computer-controlled syringe with a multiposition head valve with an integrated magnetic stir bar for efficient solvent dispersion. The procedure required 200 µL solvent and was completed within 8 minutes, including all cleaning steps, and was operated in parallel to the chromatographic analysis of the previous extract. A greenness evaluation by the AGREE tool yielded a value of 0.8. Excellent linearity up to 1000 µg L −1 , detection limits ranging from 0.4 to 15 μg L⁻¹, and RSD values typically below 4.3% were achieved. The method was successfully applied to the determination of selected antibiotics in surface waters, demonstrating its practical application.