Optimization of a liquid chromatographic method for determination of oxytetracycline, tetracycline, and chlortetracycline in milk.

A liquid chromatographic (LC) method was developed for the simultaneous identification and quantitation of oxytetracycline, tetracycline, and chlortetracycline in milk. Milk samples (5 mL) were deproteinized by adding 1 mL 1N HCl and 24 mL acetonitrile, and filtering. Dichloromethane and hexane were added to 15 mL filtrate to separate the water layer. The organic layer was washed with 1 mL deionized water, and the combined water layers were diluted to 4 mL. Sample aliquots of 1000 microL were then injected directly and analyzed on an LC system. The sensitivity limit of the method is 5 ppb for each antibiotic; no interferences are present at their retention times. Mean recoveries from milk spiked at 0.01-1 ppm ranged from 87 to 99%, and precision was good.