Analytical challenges and occurrence of antibiotics in biosolids from municipal sewage treatment plants in North Spain

• Multiclass, effective extraction of antibiotics from biosolids and sludge. • Sensitive LC-MS/MS determination after clean-up of ultrasound extracts. • Variable matrix effects as function of sludge treatments at sewage plants. • Ubiquity of fluoroquinolones, azithromycin and trimethoprim in biosolids. • Biosolids contain above 80% of fluoroquinolones emitted by sewage plants. Antibiotics are among the most concerning pharmaceutical contaminants released from municipal sewage treatment plants (STPs), occurring both in treated effluents and in dewatered biosolids. This study examines key analytical challenges during the determination of ten antibiotics in sewage wastes using liquid chromatography–tandem mass spectrometry (LC-MS/MS). Furthermore, results of their distribution in dewatered biosolids from different STPs are presented. Fluoroquinolones were identified as the most problematic compounds due to sorption on glassware, strong interaction with sample matrix, and signal suppression effects during LC-MS/MS analysis. Optimized extraction, based on sonication of freeze-dried samples with a buffered acetonitrile–water solution (pH 4.4, 1:1), yielded average recoveries between 74% and 108%, with moderate variability across sludge types. During simultaneous quantification of multiclass antibiotics, fluoroquinolones exhibited moderate to high signal attenuation depending on the matrix. For this group of compounds, signal suppression could be mitigated by fractionating extracts using mixed-mode (reversed phase and cation exchange) sorbents. Combined with isotopically labelled surrogate standards, solvent based calibration enabled accurate quantification of all targeted compounds, achieving limits of quantification below 5 ng g⁻¹. Azithromycin, clarithromycin, norfloxacin, ciprofloxacin and ofloxacin were ubiquitous in dewatered biosolids, with median concentrations ranging from 7 ng g -1 (clarithromycin) to 1761 ng g -1 (ofloxacin). A mass balance assessment of emissions through treated wastewater and biosolids highlighted azithromycin, ciprofloxacin, norfloxacin, and ofloxacin as the priority antibiotics for monitoring in final dewatered solid waste streams (biosolids) from STPs.