Assay of chlortetracycline in animal feeds by liquid chromatography with fluorescence detection

A liquid chromatographic (LC) method is described to assay chlortetracycline (CTC) at gram-per-ton and gram-per-pound levels. Feeds and premixes are extracted with acid-acetone following AOAC Method 957.23B(h). Extracts are filtered and quantitated by LC using a C18 column and fluorescence detection at 390 nm excitation and 512 nm emission. The mobile phase (1.5 mL/min) uses a methanol gradient with an initial hold time of 5 min at 35% methanol followed by an increase to 60% methanol over 8 min. The aqueous component contains CaCl2, disodium EDTA, and sodium acetate buffered to pH 6.5. The method provides significant improvement in selectivity and limit of quantitation compared to LC with UV detection. Recovery of spiked samples averaged 98.1% and assay of samples correlated closely with the results obtained with AOAC microbiological Method 967.39. CTC and epi-CTC eluted at approximately 12.5 and 4.0 min, respectively. No interference was observed from several drugs and other antibiotics often used in combination with CTC in feeds.