RAPID HPLC DETERMINATION OF TETRACYCLINE ANTIBIOTICS IN MILK

A previously described method using on-line concentration and gradient elution was modified for more rapid isocratic analysis. Milk (5 mL) was extracted/deproteinized with 1 mL of 1 N HCl and 15 mL of acetonitrile. The resulting filtrate (12 mL) was evaporated directly, or the water layer resulting from addition of hexane and methylene chloride was evaporated. The extract could be concentrated to about 1 mL without significant degradation of tetracyclines. The concentrates were filtered. For analysis, a Polymer Laboratories PLRP-S column was used with a mobile phase of 0.02 M H3PO4 and 0.01 M sodium decanesulfonate-acetonitrile, 72 + 28 for oxytetracycline and tetracycline and 68 + 32 for chlortetracycline. The injection volume was 200 mu L with UV detection at 380 nm. Average recoveries were greater than 80% with detection limits of 2-4 ppb.