Simultaneous determination of tetracycline, oxytetracycline, and 4-epitetracycline in milk by high-performance liquid chromatography

A reversed-phase high-performance liquid chromatography with photodiode-array detection (HPLC–PAD) was optimized and validated for the simultaneous determination of tetracycline (TC), 4-epitetracycline (4-epiTC) and oxytetracycline (OTC) in milk. Milk samples were extracted and cleaned-up using solid-phase extraction Discovery SPE DSC-18 tubes. The separation were accomplished in less than 8 min in a Waters Symmetry C18 column at ambient temperature with a mobile phase consisted of 0.010 M aqueous oxalic acid:acetonitrile:methanol (150:20:20 by volume). Quantitation was carried out by the peak area method, with detection limits of 2.0 μg/l of each tetracycline. Average recoveries of TC, 4-epiTC and OTC from spiked samples at the four concentrations (0.25, 0.5, 1.0 and 1.5 μg/ml) were 91.5, 71.5 and 83.1, respectively, with their standard deviations less than 4% within a day and 7% between days. This method was applied for the simultaneous determination of TC, 4-epiTC and OTC in market milk samples purchased from local supermarkets. Oxytetracycline was found being present in all samples in a concentration range of 13–106 μg/l, 4-epiTC in most samples at 18–65 μg/l, TC in one sample at 44 μg/l.