Sensitive spectrofluorimetric determination of tetracycline residues in bovine milk

A novel, sensitive, high-performance liquid chromatographic method for the residue determination of tetracyclines in bovine milk has been developed. Clean-up of the sample extracts is performed with metal chelate affinity chromatography. Detection is based upon the measurement of fluorescence induced by complexation of tetracyclines with the zirconium cation, which is added post-column to the high-performance liquid chromatographic eluate. The method has been validated for oxytetracycline, tetracycline and chlortetracycline. Calibration curves using 'blank' milk fortified at the 10-200 ng ml-1 level showed good linear correlation. Accuracy was measured at the 100 ng ml-1 level and was found to be within acceptable limits using a t-test (alpha = 0.05). At this level both within- and between-day precision, as measured by relative standard deviation (S(r)), were less than 16% for oxytetracycline and chlortetracycline, and less than 20% for tetracycline. The most striking advantages of the proposed method, over previously reported methods, are the higher sensitivity and specificity achieved. Using a signal-to-noise ratio of 4:1, the detection limits of the assay were 1 ng ml-1 for oxytetracycline, 2 ng ml-1 for tetracycline and 4 ng ml-1 for chlortetracycline.