Abstract: A new rapid and high-throughput microbial chromogenic assay was developed to analyze antibiotic residues in animal-derived food samples. The positive samples were further confirmed by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method. Bacillus sterothermophilus was used as an indicator strain to produce a 96-well microplate for detecting a variety of antibiotics simultaneously. The results showed that the microbial assay was simple and cheap, and gave easy-to-interpret results. After extraction with phosphate buffer at pH 7.4, muscle samples were detected using a chromogenic assay system consisting of the reactant solution (150 μL), bacterial suspension (50 μL) with an initial optimal density (OD600 nm) of around 0.4, and the sample extract (100 μL). The limits of detection (LODs) of aminoglycosides, beta-lactam, macrolide, and tetracyclines antibiotics were 60, 20–40, 60–80, and 40–60 μg/kg, respectively, which reached the requirements for the determination of antibiotic residue levels in the country and abroad. Consistent results were obtained in examining antibiotic residue in 60 animal-derived food samples using the microbial chromogenic assay and HPLC-MS/MS, indicating that the proposed assay could be used to detect antibiotic residues in animal-derived food samples with reliable results.

Key words: high-throughput microbial chromogenic assay, antibiotic residues, high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), animal-derived food