ONLINE METAL CHELATE AFFINITY-CHROMATOGRAPHY CLEANUP FOR THE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC DETERMINATION OF TETRACYCLINE ANTIBIOTICS IN ANIMAL-TISSUES

An on-line high-performance liquid chromatographic (HPLC) method for t he determination of tetracycline, oxy tetracycline, chlortetracycline and demeclocycline using metal chelate affinity chromatography-reverse d-phase HPLC has been developed. The drugs were extracted with succina te buffer and the extract diluted with EDTA-pentanesulphonate buffer. Diluted extract was then absorbed onto a C-8 or XAD-2 solid-phase extr action (SPE) cartridge and eluted with methanol. The eluate was then i njected onto a TSKgel chelate column which had been preloaded with cop per(II). The tetracyclines were eluted from this column onto the analy tical column (Polymer Labs. PLRP-S) with an EDTA-containing buffer. El ution of the analytical column was via a methanol-acetonitrile gradien t and detection was by UV at 350 nm. Average recoveries at the 10, 20, 50 and 300 mu g kg(-1) levels were 50-80%. The limit of detection (LO D) was 10 mu g kg(-1) for oxytetracycline and tetracycline and 20 mu g kg(-1) for chlortetracycline and demeclocycline. The method was valid ated for sheep liver and cattle kidney.