SIMULTANEOUS DETERMINATION OF SELECTED VETERINARY ANTIBIOTICS AND THEIR MAIN METABOLITES IN FISH AND MUSSEL SAMPLES BY HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY WITH DIODE ARRAY-FLUORESCENCE (HPLC-DAD-FLD) DETECTION

A reversed-phase high-performance liquid chromatography (RP-HPLC) method for the quantitative determination of 11 antibiotics (drugs) and the main metabolites of five of them in fish tissue and mussel samples were developed, optimized, and validated. The analytes belong to four different classes of antibiotics (sulfonamides, tetracyclines, penicillin, and amphenicols). The analyzed compounds were sulfadiazine and its N-4-acetylsulfadiazine metabolite; sulfamethazine and its N-4-acetylsulfamethazine; sulfamerazine and its N-4-acetylsulfamerazine; sulfamethoxazole; trimetroprim; amoxicillin and its main metabolite amoxicilloic acid; ampicillin and its main metabolite ampicilloic acid; chloramphenicol; thiamphenicol; oxytetracycline; and chlortetracycline. For HPLC analysis, diode array and fluorescence detectors were used. The separation of the analyzed compounds was conducted by means of a C-18 (150mm x 4.6mm I. D., particle size 5 mu m) analytical column with LiChrospher (R) C-18 (4mm x 4 mm, particle size 5 mu m) guard-column. Analyzed drugs were determined within 35 minutes using formic acid 0.1% in water and acetonitrile in gradient elution mode as the mobile phase. The method was applied to the determination of the analytes in tissue of hake (Merluccius merluccius), anchovy (Engraulis encrasicolus), mussel (Mytltus sp.), and wedge sole (Solea solea). The proposed method was also evaluated by a laboratory assay consisting of the determination of the targeted analytes in samples of Cyprinus carpio that were previously administered controlled doses of the antibiotics.