In silico–optimised RP-HPLC – FLD method coupled with MgSO ₄ -assisted salting-out extraction for ultra-trace detection of three Fluoroquinolone antibiotics in aquatic matrices

Fluoroquinolone (FQs) antibiotics are frequently detected as emergent contaminants in aquatic environments, necessitating sensitive and reliable analytical methods for their determination. In this study, an integrated in silico and in vivo strategy was developed for the trace-level detection of ciprofloxacin (CIP), norfloxacin (NOR), and ofloxacin (OFL) in diverse water matrices. Separation and quantification were achieved using reversed-phase high-performance liquid chromatography coupled with fluorescence detection (RP-HPLC-FLD). A linear mathematical model was employed to optimise chromatographic parameters, including mobile phase composition, pH, and flow rate, reducing reliance on conventional trial-and-error approaches without compromising chromatographic performance. The optimised excitation-emission wavelength pair (λex = 290 nm; λem = 460 nm) was used for all analytes, improving instrumental stability and analytical throughput. Under gradient elution with acetonitrile and methanol at a flow rate of 1 mL min−1, CIP, NOR, and OFL were resolved at 16.68, 15.27, and 14.51 min, respectively, demonstrating high selectivity and sensitivity. Sample preparation employed a magnesium sulphate-driven dual salting-out and drying (MgSO4-D2 S) strategy, providing efficient extraction. The method showed excellent linearity (R2 = 0.99) over 250–5 ng L−1, with recoveries above 90% and precision (RSD) of 0.59–3.87%. Limits of detection were 1.88, 0.79, and 1.57 ng L−1 for CIP, NOR, and OFL, while limits of quantification were 5.70, 2.39, and 4.77 ng L−1, respectively. The method was successfully applied to river, pond, wastewater, tap water, seawater, and microcosm samples, enabling reliable trace-level monitoring of FQs residues in aquatic environmental samples.