Development of a Simple and Rapid HPLC-UV Method for Simultaneous Determination of Ciprofloxacin Hydrochloride and Oxytetracycline Hydrochloride in Aqueous Matrix

Background Reliable and effective methods capable of simultaneously determining ciprofloxacin hydrochloride (CIP) and oxytetracycline hydrochloride (OTC) can be useful when concentrations fall within the validated working range. Objective This study aimed to develop and validate a simple and rapid reversed-phase high-performance liquid chromatography with ultraviolet detection (RP-HPLC-UV) method for simultaneous determination of CIP and OTC in aqueous matrices. Methods The separation was performed using an Agilent InfinityLab Poroshell 120 EC-C18 column (150 × 4.6 mm, 4 μm) with a mobile phase consisting of 0.1% formic acid and acetonitrile at a flow rate of 1.0 mL/min. The method was validated for linearity, precision, selectivity, recovery, sensitivity, robustness and system suitability. Matrix performance was assessed using fortified lake-water samples. Results Linearity over the concentration range of 10–25 mg/L, with R² values of 0.9947 for OTC and 0.9923 for CIP was achieved. The LOD values were 1.40 mg/L for OTC and 1.69 mg/L for CIP, while the corresponding LOQ values were 4.24 and 5.11 mg/L, respectively. Peak-area RSDs were below 1% in both intraday and interday precision studies. Recoveries in fortified lake water ranged from 91.12–109.13% for OTC and 82.88–107.72% for CIP and from 95.84–104.91% and 89.56–112.88%, respectively, in interday analysis. Robustness testing showed that satisfactory chromatographic separation was maintained under the investigated variations in flow rate, column temperature, and detection wavelength. Conclusion The HPLC-UV approach was applicable for the simultaneous determination of CIP and OTC in aqueous samples containing relatively elevated concentrations of the target antibiotics. The method is not intended for direct trace-level determination in typical environmental waters without an appropriate preconcentration step. Highlights A simple RP-HPLC-UV method was developed for the simultaneous determination of CIP and OTC. The method is applicable to aqueous samples containing relatively elevated antibiotic concentrations.