DESENVOLVER E VALIDAR UMA METODOLOGIA ANALÍTICA UTILIZANDO CLAE/DAD PARA DETERMINAÇÃO QUANTITATIVA DE RESÍDUOS DE TETRACICLINAS EM CARNE BOVINA, EMPREGANDO A TÉCNICA DE EXTRAÇÃO POR QUECHERS

Abstract

Tetracyclines (TCs) are part of a class of antibiotics widely used to treat infections in both humans and large and small animals, and are one of the most widely consumed in the world. They have a broad spectrum of action, acting against Gram-positive and Gram-negative bacteria. Some of the routes of interaction between humans and TCs can be through direct administration (through treatment with the drug) or indirect, such as the ingestion of contaminated food. There are reports in the literature of the presence of antibiotics in foods such as milk, eggs, meat, water, vegetables, and plants, among others. Human exposure to antibiotic residues contributes to the development of microorganisms resistant to the therapeutic action of these drugs, causing microbial resistance to antibiotics, which has become increasingly evident among public health problems. Thus, the present study aims to evaluate the presence of the antibiotic tetracycline, oxytetracycline and chlortetracycline in a beef sample from the region of Mato Grosso do Sul. For this purpose, the optimization and validation of the QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) method is proposed. The detection and identification of the analytes were performed by highperformance liquid chromatography with diode array detection. The validation of the chromatographic system demonstrated the following analysis conditions: mobile phase in isocratic mode using the solvents oxalic acid 0.01 mol L-1, acetonitrile, and methanol, in the proportions of 70%, 22% and 8%, respectively. Flow rate of 1 mL min-1, wavelength of 355 nm, oven temperature at 30 ºC and 25 cm C18 column (5 µm particle diameter). The meat sample was obtained in natura from a butcher shop in Campo Grande, Mato Grosso do Sul. It was cut into small pieces, freeze-dried, and ground. In the analysis of the parameters for the QuEChERS method, a 26-2 fractional factorial design with central point and Doehlert matrix was applied. The variables analyzed per stage were: sample quantity (extraction stage), mass of magnesium sulfate (MgSO4) and sodium chloride (NaCl) salts (partition stage) and mass of C18, mass of primary secondary amine (PSA) and MgSO4 (cleaning stage). The evaluation of the design was performed through analysis of variance (ANOVA). The optimization of the method with a 95% confidence interval demonstrated that the best conditions for analyzing the mentioned TC analytes are: Sample 0.77 g; MgSO4 0.50 g; PSA 91 mg. The parameters maintained were: NaCl 0.10 g, centrifugation from 1 to 5000 rpm. The following values were obtained as limits of detection (LD) and quantification (LQ), respectively: 46.53 and 141.01 µg L-1 for tetracycline (TC), 24.60 and 74.55 µg L-1 for oxytetracycline (OT), and 38.88 and 117.81 µg L-1 for chlortetracycline (CT). The matrix effect values were within the expected range, being -5.83% for TC, -6.71% for OT, and - 9.18% for CT. The lowest recovery value was 73.05% for OT at a concentration of 100 µg L-1, and the maximum recovery was 110.67% for TC, also at this concentration. The highest relative standard deviation value was 8.59. The coefficient of determination values for the optimized QuEChERS were 0.9906 for TC, 0.9988 for OT and 0.9952 for CT. The results were satisfactory according to the statistical analysis methods used and the competent bodies and agencies (National Health Surveillance Agency - ANVISA and National Institute of Metrology, Quality and Technology - INMETRO) and were adequate for the validation of the proposed method.