Analysis of tetracycline antibiotics using HPLC with pulsed amperometric detection

The analysis of tetracycline, oxytetracyline, chlortetracycline and doxycycline by high-performance liquid chromatography with pulsed amperometric detection using an anodized boron-doped diamond thin film (BDD) electrode is originally reported. The analyses were carried out using the mobile phase, phosphate buffer (0.01 M, pH 2.5)-acetonitrile (80:20; v/v), on a C-18 column (250 x 4.6 mm i.d., 5 mu m) at a flow rate of 1.0 mL/min. The optimal PAD waveform parameters at the anodized BDD were 1.5 V (versus Ag/AgCl) detection potential (E-det) for 290 ms (200 ms delay time and 90 ms integration time), 2.0 V (versus Ag/AgCl) oxidation potential (E-oxd) for 200 ms oxidation time (t(oxd)) and 0.4 V (versus Ag/AgCl) reduction potential (E-red) for 200 ms reduction time (t(red)). The proposed method showed the simultaneous determination of tetracycline, oxytetracyline, chlortetracycline and doxycycline with a linear range of 0.1-100 mu g/mL, detection limits of 0.05-0.1 mu g/mL and recoveries of 70.8 - 96.0%. The application of this method to real samples was demonstrated and validated using a shrimp sample.