Validation of a High Performance Liquid Chromatography (HPLC) Method for the Determination of Oxytetracycline Residues in Milk

Abstract

Abstract | Tetracyclines are antibacterial agents widely used in preventing and treating infectious diseases in food producing animals. This extensive use can lead to an increase in their residues levels in food of animal origins. These residues will pass through the food chain to the consumers. In addition, the residues in milk may have many consequences on consumers’ health: allergic reactions in human’s antibiotics give rise to an increase in the antibiotic resistance of pathogenic bacteria, which may result in direct toxity, allergy and resistance to antibiotics. In this way, authors look to detect and quantify Oxytetracycline residues in milk by High Performance Liquid Chromatography (HPLC). They have, at first, proceeded to the optimization of various analysis parameters [stationary phase, mobile phase, absorption spectrum (wavelengths), flow rate and injected volume], then they have continued with their validation using adequate statistical analysis methods (Repetitiveness reproducibility, Linearity of the calibration curve) and the adaptation of an extraction technique of this antibiotic from cow’s milk. The results of the validation process led to the adaptation of following parameters: Sample concentration: 0, 4 mg/mL (diluted in methanol), stationary phase: column C18 reversed phase (Grafted silica gel), mobile phase: acetonitrile, injected volume: 11 µl, wavelength (UV detector): 325 nm and flow rate: 1, 5 mL/min. The drug was extracted with mixture acetonitrile/HPLC water and simply cleaned up on Whatman filter, then on a nylon membrane filter of 45 µm/ 47 mm