Optimization and validation of a two-step extraction dispersive liquid-liquid microextraction (UA-DLLME) method for...

A rapid and efficient method based on two-step dispersive liquid-liquid microextraction (DLLME) was developed for the simultaneous determination of antibiotics belonging to six different subclasses (fluoroquinolones, macrolides, sulfonamides, lincosamides, nitroimidazoles and trimethoprim) in influent wastewater samples. Analysis was performed using ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS). During method optimization, several parameters affecting DLLME performance were evaluated: the type and volume of extraction and dispersive solvents, sample pH, sample volume, salt content (sodium chloride) for the salting-out effect, and ultrasonication time. Under optimized conditions, the method's performance characteristics exhibited linearity between 100 and 4000 ng/L (R² ≥ 0.98). The matrix effect (ME) ranged from 32.5% to 93.3%. Recovery values were satisfactory, mostly ranging between 80% and 120%. Relative standard deviation (RSD) values indicated good repeatability and intermediate precision (< 20%), while limits of quantification (LOQs) ranged from 10.7 to 369 ng/L. Finally, the validated method was applied to the analysis of influent wastewater samples collected from the Psyttalia Wastewater Treatment Plant.