Simultaneous determination of multiple tetracycline residues in milk using metal chelate affinity chromatography

A method was developed for the determination of 7 tetracyclines in milk. Raw milk samples are defatted, acidified, and centrifuged to remove proteins, and the tetracyclines are specifically absorbed from the milk by chelation with metal ions bound to small Chelating Sepharose Fast Flow columns. The tetracyclines are removed from these columns with EDTA-containing buffer, and the extracts are further cleaned up by centrifugal ultrafiltration. Finally, the extracts are concentrated and analyzed simultaneously by on-line concentration. This method has limits of detection for individual tetracyclines of < 5 ng/mL and was validated with fortified milk samples at 15, 30, and 60 ng/mL. Recoveries exceeded 60% for all tetracyclines at all levels, with good precision. The method was also tested on milk from cows dosed with each of the tetracyclines. Advantages of this method over existing methods include its sensitivity, minimal use of organic solvents, and speed; with an autosampler, at least 14 samples can be processed and analyzed in 1 day.