Título da Dissertação: Determinação de resíduos de oxitetraciclina, tetraciclina, clortetraciclina e doxiciclina em leite bovino por cromatografia líquida de alta eficiência.

Orientador: Prof. Dr. Miguel Machinski Junior

Data da Defesa: 03/04/2012



 INTRODUCTION. Tetracyclines are a large spectrum class of antibiotic, widely used in prevention and treatment of infection in dairy cattle. Though its indiscriminate useand the disrespect to the withdraw period may result residues in milk, which can cause bacteria resistance, allergies, and economic losses to producers of milk and dairy products. Therefore, surveillance and monitoring of the presence of antibiotics in milk are needed to ensure consumer safety and avoid financial losses. For that, several methods have been used, and the high performance liquid chromatography is preferred by presenting analysis speed, selectivity, accuracy and reproducibility. Moreover, laws of several countries have established safe limits of residues in food (LMR).
OBJECTIVES. (I) Provide information about the methods used in the determination of tetracyclines in milk reported in the literature. (II) Validate a multiresidue method for the determination of tetracyclines (oxytetracycline, tetracycline, chlortetracycline and doxycycline) in bovine milk by high performance liquid chromatography with UV-VIS detector according to analytical criteria defined by European Commission 2002/657/EC. (III) Investigate the occurrence of tetracyclines residue in bovine pasteurized milk by high performance liquid chromatography detection system with UV/VIS. (IV) Assessment the population exposure to antimicrobial residues in milk commercialized in the State of Parana, Brazil.
MATERIAL AND METHODS. A review of analythical methods for determination of tetracyclines reported in the literature was performed. Validation of an extraction method, wherein the milk samples were deproteinated with Mcllvaine buffer and 20% trichloroacetic acid and cleanup carried out in C18 columns. Tetracyclines have been separated into a high efficiency liquid chromatograph with UV/VIS detector set at 365 nm by a gradient composed of oxalic acid 0.01 mol/L, acetonitrile and triethylamine in C8 column. To investigate the occurrence of tetracyclines, 100 samples were collected in the State of Parana, between March/2010 and October/2011, and analyzed by the validated method. To evaluate the exposure to the tetracyclines residue, it was calculated the Estimated Daily Intake (EDI) according to the JECFA. The EDI was compared to the Acceptable Daily Intake (ADI) from 0 to 0.03 mg/kg body weight (bw).
RESULTS AND DISCUSSION. The steps for determining antibiotic residues in milk are: extraction, cleanup, identification/quantification and confirmation. The main problem with this analysis due the matrix effect that may interfere with the determination of these residues. Therefore, we have investigated methods of extraction and cleanup witch consist of deproteinization and removal of these components, but that are simple and quick. Thus, many researchers have used to solutions and acidic buffers, and solid phase columns. For identification and quantification, the most used techniques are high performance liquid chromatography and capillary electrophoresis, which show precise results and the confirmation is generally made by mass spectrometry. The validated method in this work presented the limits of detection and quantification from 42.3 to 75.8 and 43.2 to 76.4 μg/kg, respectively. The decision limit (CCα) and detection capability (CCβ) varied from 114.2 to 143.7 and 129.3 to 188.7 μg/kg, respectively. Recoveries of tetracyclines in fortified samples were above 82.5%. Of 100 samples analyzed, three were contaminated with values of 121.8 μg/kg for oxytetracycline, 93.5 μg/kg for tetracycline and 134.6 μg/kg for chlortetracycline (61.6μg/kg) and doxycycline (73.0 μg/kg). The median of residues of tetracyclines found in the samples was 42.3 μg/kg and EDI was 0.05 μg/kg bw/day for Brazil, 0.08 μg/kg bw/day for the Southern region and 0.07 μg/kg bw/day for the State of Parana.
CONCLUSIONS. Among the analytical techniques for the determination of tetracyclines residue in milk, the most commonly used is the high performance liquid chromatography (HPLC) with ultraviolet detection system (UV) or diode array. Although there are techniques which have good results, but with lower sensitivity such as capillary electrophoresis, but allows a rapid and less polluting analysis. The method validated in this study for determination of tetracyclines in pasteurized bovine milk, according to regulation 2002/657/EC of the European Union, presented linearity, specificity, selectivity, precision and accuracy. Therefore, this method showed efficiency enough for the monitoring of tetracyclines in milk. Results of analysis of the collected samples showed that the occurrence of tetracylines in milk was low (3%), but had samples above the MRL (2%), and the risk to the population to these residues in the milk was regarded as negligible (<1% ADI).
Keywords: Bovine milk, Veterinary drug residues, Tetracyclines, HPLC, Food safety,

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