Development and validation of a modified QuEChERS-LC-MS/MS method for multi-residue antibiotic determination: Application to Milk from southern Mozambique

Antibiotic residues in milk may pose significant public health risks, including allergic reactions, microbiota disruption, and the development of antimicrobial resistance development. Monitoring these residues requires highly sensitive analytical methods; however, traditional sample preparation techniques typically consume large volumes of organic solvents (10–20 mL per sample), generating substantial chemical waste and increasing operational costs. These challenges are particularly critical in resource-limited settings and developing countries. This study presents the comprehensive development and validation of a solvent-minimized LC-MS/MS method for the simultaneous determination of 20 antibiotics from six different classes in milk and its application to samples from Mozambique. A modified QuEChERS method was optimized to minimize organic solvent consumption while maintaining high sensitivity and selectivity. The method was validated according to VICH guidelines, achieving limits of detection (LOD) and quantification (LOQ) of 3.3–8.3 μg/Kg and 10–25 μg/Kg, respectively, all well below the established maximum residue limits (MRLs). Accuracy (recovery) ranged from 74.4% to 117.2%, with intra-day and inter-day precision (coefficient of variation) values of 1.0–14.7% and 3.7–19.81%, respectively, meeting all acceptance criteria. The method demonstrated excellent linearity (r ≥ 0.994) across all analytes. Analysis of 96 milk samples from the southern region of Mozambique, including both family and commercial farming sectors, revealed that no samples contained antibiotic residues above the LOD for all 20 analytes analyzed. This work demonstrates the feasibility of developing sensitive, selective, and sustainable analytical methods for antibiotic residue monitoring in developing countries, reducing solvent consumption by 80–90% compared to traditional approaches while maintaining regulatory compliance standards.