A novel method for the determination of four tetracycline antibiotics (TCs) including tetracycline (TC), oxytetracycline (OTC), chlortetracycline (CTC) and doxycycline (DC) by means of high-performance liquid chromatography (HPLC) with chemiluminescence (CL) detection was proposed. The method was based on the enhancement effect of TCs on the chemiluminescence reaction between luminol and BrO^－, which was on-line electrogenerated by constant current electrolysis. The separation was carried out on Nucleosil RP-C₁₈ column (250 mm×4.6 mm i.d., 5 μm, pore size, 100 Å) at 25 ℃. The mobile phase consisted of a V(acetonitrile)∶V(0.05 mol•L^－1 potassium dihydrogen phosphate buffer, pH 2.5)＝30∶70 solution. At a flow-rate of 1.2 mL/min, the total run time was 11 min. The effects of several parameters on the HPLC resolution and CL emission were studied systematically. For the four TCs, the detection limits at a signal-to-noise of 3 ranged from 0.002 to 0.008 μg•mL^－1. The relative standard deviations for the determination of TCs ranged from 2.0% to 3.6% (n＝11, c＝0.01 μg•mL^－1). The method has been satis-factorily applied to the analysis of spiked raw milk samples.

Key words: tetracycline antibiotic, chemiluminescence detector, on-line electrogenerated BrO^－, high per-formance liquid chromatography, milk