Simultaneous determination of oxytetracycline, tetracycline and chlortetracycline in animal tissues using liquid chromatography, post-column derivatization with aluminium, and fluorescence detection

Abstract

A method is described for the simultaneous determination of the antibiotics tetracycline, oxytetracycline and chlortetracycline in liver, kidney and muscle using liquid chromatography, post-column derivatization with Al³⁺, and fluorescence detection. Samples are extracted by homogenizing in glycine–HCl buffer, and are then applied to cyclohexyl Bond Elut cartridges. These are washed with water and tetracylines eluted with methanol. The extracts are evaporated to dryness under nitrogen and the residues dissolved in an oxalic acid–acetonitrile mobile phase and injected onto the HPLC system. A gradient system is used to control sample elution. Following elution, tetracyclines are derivatized with Al³⁺ ions at 60 °C to produce a highly fluorescent derivative. The fluorescence detector is set with an excitation wavelength of 390 nm and an emission wavelength of 490 nm. The assay is linear up to 2500 ng g^–1, and has a limit of determination of 20 to 230 ng g^–1, depending on tissue and compound. The assay is used in this laboratory for determination of residual concentrations of tetracyclines in statutory testing schemes.