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Background and Aim: Food of animal origin such as milk is vital for nutritional security and should be free of any antibiotic residues due to its public health significance. We designed a study aiming to determine the occurrence of antibiotic residues and in further levels of oxytetracycline residues in pooled raw milk samples from Palakkad district, Kerala. Materials and Methods: We collected pooled raw milk samples were collected from Alathur, Chittoor, and Palakkad blocks of Palakkad district, Kerala. A total of 215 samples were screened for antibiotic residues by microbial inhibition assay (MIA) and the positive samples were subjected to enzyme-linked immunosorbent assay (ELISA) to determine oxytetracycline residues, this was further confirmed using high-performance liquid chromatography (HPLC). Results: We found that out of the 215 pooled raw milk samples screened for antibiotic residues using MIA, 22 samples (10.23%) were positive for antibiotic residues from Palakkad, Kerala. Out of these 22 samples, five (2.33%) were positive for oxytetracycline residues. We further calculated the mean concentration of oxytetracycline residues in these five samples and estimated it to be 201.00±41.25 ng/mL and 272.11±53.21 ng/mL using ELISA and HPLC, respectively. On analyzing these five samples, we found that four samples (1.86%) exceeded the maximum residue limits level of 100 ng/mL for oxytetracycline residues in milk as specified by Codex Alimentarius Commission/Food Safety and Standards Authority of India (FSSAI). Conclusion: This study revealed that the occurrence of oxytetracycline residues in pooled raw milk samples in the Palakkad district of Kerala. Hence, there is a need for surveillance and monitoring of antibiotic residues in milk due to its impact on public health to ensure consumer safety.

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