Determination of tetracyclines in meat using two phases freezing extraction method and HPLC-DAD

The protein binding of tetracyclines may cause the common extraction methods are not able to completely remove them from the proteins. To overcome this problem, we introduced a simple, reliable, sensitive, and reproducible method for simultaneous clean-up and preconcentration of 4 tetracyclines in chicken meats based on two phase freezing method. The tetracyclines were extracted by solid liquid extraction using Britton-Robinson buffer (0.008 mol L-1, pH = 10) from chicken breast, thigh and liver samples. Then simultaneous separation, clean up, protein-drug bonds breaking and preconcentration of tetracyclines from extract solution was performed by two phase freezing method. In this method the acetonitrile was mixed with aqueous extractant solution, the mixture solution was frozen and the organic phase was removed by centrifuge at -20 A degrees C. For separation of tetracyclines a reversed-phase high-performance liquid chromatography was used. In two phase freezing method several parameters such as pH of extract solution, the type of miscible organic solvent, salting out, the ratio of organic/aqueous phases, and extraction cycles were investigated. The recoveries of tetracyclines spiked at three levels of concentration (50, 100, and 150 mu g Kg(-1)) were changed from 76.5 % to 95.5 % with the relative standard deviation values less than 11 %. This method is a reliable tool and could be applied to analyze tetracyclines residues in chicken meats.