Validation of a high-performance liquid chromatography method for the determination of oxytetracycline, tetracycline, chlortetracycline and doxycycline in bovine milk and muscle

High-performance liquid chromatography with diode-array detection (HPLC–DAD) was optimised and validated for the determination of tetracyclines in bovine milk and tissues. Milk and tissue samples were extracted and purified using a solid-phase extraction HLB Oasis cartridge and analysed using HPLC–DAD set at 365 nm. The analyses were carried out using the mobile phase of 0.01 M oxalic acid–acetonitrile–methanol (60:25:15, v/v/v) on a C8 column (250×4.6 mm I.D., 5 μm). Recoveries of tetracyclines from spiked samples at the three concentrations (0.5, 1 and 1.5) of the maximum residues limits (corresponding to 100 μg/kg for milk and the muscle) were higher than 81.1% in milk and 83.2% in muscle. The method was successfully validated for bovine milk and muscle in compliance with requirements set by draft SANCO/1805/2000 European Decision. The decision limit (CCα) was in the range 113.2–127.2 μg/kg and 107.7–129.9 μg/kg for all compounds in milk and muscle, respectively. The detection capability (CCβ) was in the range 117.2–131.3 μg/kg and 114.9–133.1 μg/kg for all compounds in milk and muscle, respectively.