Sustainable HPLC method for simultaneous determination of Cefazolin, Sulfadimidine, and Marbofloxacin residues in milk.

Antibiotic residues in milk pose a food safety risk by promoting antimicrobial resistance and causing toxic effects in consumers. This study developed and optimized a sensitive chromatographic method for the simultaneous quantification of Cefazolin (CFZ), Sulfadimidine (SDD), and Marbofloxacin (MFC) residues in milk. These antibiotics are commonly used as a combination therapy for managing cattle metritis. Separation was performed on a C18 column with UV detection at 270 nm. The eluent consisted of a 0.05 M phosphate buffer (pH 3), acetonitrile, and methanol in a 60:35:5 ratio, flowing at 1.0 mL/min. Sample preparation included protein precipitation with acetonitrile, followed by dispersive solid-phase extraction (dSPE) using Enhanced Matrix Removal-Lipid (EMR-L) tubes to remove interfering lipids. Validation per ICH-Q2R1 showed a linearity range of 0.0125–0.5 µg/mL for CFZ and MFC and 0.025–0.5 µg/mL for SDD, with LOD/LOQ values between 0.01 and 0.015 µg/mL and % recoveries between 97.67% and 98.89%. The method effectively quantified the studied drug residues in real cattle milk samples after their withdrawal period, with no significant differences in accuracy or precision versus established methods. Additionally, an assessment of the method’s sustainability (Eco-scale: 71, AGREE: 0.69, RGB12: 88.8) confirmed its compliance with green and white analytical chemistry principles. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1038/s41598-025-32613-7.