On-line metal chelate affinity chromatography clean-up for the high-performance liquid chromatographic determination of tetracycline antibiotics in animal tissues

An on-line high-performance liquid chromatographic (HPLC) method for the determination of tetracycline, oxytetracycline, chlortetracycline and demeclocycline using metal chelate affinity chromatography-reversed-phase HPLC has been developed. The drugs were extracted with succinate buffer and the extract diluted with EDTA-pentanesulphonate buffer. Diluted extract was then absorbed onto a C8 or XAD-2 solid-phase extraction (SPE) cartridge and eluted with methanol. The eluate was then injected onto a TSKgel chelate column which had been preloaded with copper(II). The tetracyclines were eluted from this column onto the analytical column (Polymer Labs. PLRP-S) with an EDTA-containing buffer. Elution of the analytical column was via a methanol-acetonitrile gradient and detection was by UV at 350 nm. Average recoveries at the 10, 20, 50 and 300 micrograms kg-1 levels were 50-80%. The limit of detection (LOD) was 10 micrograms kg-1 for oxytetracycline and tetracycline and 20 micrograms kg-1 for chlortetracycline and demeclocycline. The method was validated for sheep liver and cattle kidney.