Analytical Sciences

Analysis of Tetracycline Antibiotics Using HPLC with Pulsed Amperometric Detection

Thiraporn CHAROENRAKS,* Suchada CHUANUWATANAKUL,* Kensuke HONDA,** Yoko YAMAGUCHI,** and Orawon CHAILAPAKUL*

*Department of Chemistry, Faculty of Science, Chulalongkorn University, Patumwan, Bangkok 10330, Thailand
**Department of Chemistry and Earth Sciences, Faculty of Science, Yamaguchi University, 1677-1, Yoshida, Yamaguchi-shi, Yamaguchi 753-8512, Japan

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 C₁₈ column (250 × 4.6 mm i.d., 5 µm) 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 µg/mL, detection limits of 0.05 - 0.1 µg/mL and recoveries of 70.8 - 96.0%. The application of this method to real samples was demonstrated and validated using a shrimp sample.

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