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

## TL;DR

A sustainable and sensitive HPLC method was developed to detect antibiotic residues in milk, ensuring food safety and reducing antimicrobial risks.

## Contribution

A new eco-friendly HPLC method for simultaneous detection of three antibiotics in milk with high accuracy and compliance to green chemistry principles.

## Key findings

- The method achieved high recoveries (97.67–98.89%) and low detection limits (0.01–0.015 µg/mL) for three antibiotics in milk.
- Validation confirmed the method's accuracy and precision compared to established techniques.
- The method is sustainable, with Eco-scale, AGREE, and RGB12 scores indicating compliance with green chemistry principles.

## Abstract

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.

The online version contains supplementary material available at 10.1038/s41598-025-32613-7.