Development of an HPLC method for the simultaneous determination of azithromycin, clarithromycin, and erythromycin in...

The widespread occurrence of macrolide antibiotics in aquatic environments poses serious ecological and public health concerns, highlighting the need for reliable, accessible, and validated analytical methods. In this study, a rapid and cost-effective high-performance liquid chromatography method with photodiode array detection (HPLC-PDA) was developed and validated for the simultaneous determination of azithromycin (AZM), clarithromycin (CLR), and erythromycin (ERY) in wastewater. Chromatographic separation was achieved on an Agilent Zorbax Eclipse C18 column (250 × 4.6 mm, 5 µm) at 35 °C using a mobile phase of acetonitrile and 10 mM NaH₂PO₄ buffer (60 : 40, v/v, pH 6.0), flow rate 0.8 mL min^-1, and injection volume 10 µL. Retention times were 3.76, 4.91, and 6.23 min for AZM, CLR, and ERY, respectively, with baseline resolution (R _s > 1.5). The method exhibited excellent linearity over 2-15 µg mL^-1 for all analytes (R ² = 0.997-0.999). Limits of detection (LOD) were 5-6 µg L^-1 and limits of quantification (LOQ) were 17-20 µg L^-1, meeting AOAC (Association of Official Analytical Chemists) performance standards. Intra- and inter-day precision (%RSD) were below 2.0%, and mean recoveries from spiked wastewater samples ranged from 99.1% to 104.5%. Minor deliberate changes in pH (±0.2) or flow rate (±0.1 mL min^-1) produced negligible effects (<2% deviation), confirming the robustness of the method. Matrix effects were minimal (<3%), demonstrating that the wastewater matrix did not interfere with analyte detection. Application of the validated method to hospital and livestock wastewater samples revealed macrolide concentrations up to 13.36 mg L^-1 (CLR), 11.27 mg L^-1 (ERY), and 7.80 mg L^-1 (AZM). The developed HPLC-PDA method is therefore suitable for routine environmental monitoring, providing a simple and reliable tool for assessing macrolide antibiotic contamination in wastewater.