Detection of Antibiotic Residues of Tetracycline, Oxytetracycline and Chlortetracycline in Animals' Raw Milk in Hamedan Province, Iran in 2022 Using the Four-Plate Test, ELISA, and HPLC Techniques.

Antibiotic resistance has become a health concern as it is associated with the death of numerous people worldwide. Milk safety is one factor that guarantees the quality of dairy products. This study was designed to determine Tetracycline (TC), Oxytetracycline (OTC), and Chlortetracycline (CTC) residues in raw milk from animals in Hamedan, Iran using a four-plate test (FPT), enzyme-linked immunosorbent assay (ELISA) and high-performance liquid chromatography (HPLC) techniques. Over a two -year Period a cross-sectional sampling was conducted, during which 246 unprocessed raw milk samples were collected from dairy farms and milk collection centers in different regions of Hamedan, the western part of Iran. FPT was used as the initial screening tool for the presence of antibiotics. Positive samples were subsequentlyanalyzed for antibiotic residue using ELISA. Finally, the HPLC method was applied to identify the type and quantity of Tetracycline residues. In the primary evaluation, forty-seven samples (19.11 %) tested positive for antimicrobial residues using FPT. ELISA analysis indicated that 29.79 % (14/47) of these samples contained TCs residues higher than the maximum residue limit (MRL) suggested by EU (100 µg/L). The average TCs residue in positive samples was 98.43±6.86 µg/Land the lowest and highest levels were 100.59 µg/L and 129.56 µg/L, respectively. Having used HPLC, the overall average of TCs level was calculated as 105.73±7.25 µg/L (TC=100.67, OTC=103.38, and CTC=107.11 μg/L). The detection of antibiotic residues in animal products highlights the need for monitoring these residues in milk and other animal-derived food products. Training farmers on the correct use of drugs, especially antibiotics, is recommended. Additionally, acomprehensive protocol for regularly evaluating livestock products is necessary to prevent high-contamination products from entering the production cycle.