A reversed-phase high performance liquid chromatography coupled with resonance Rayleigh scattering detection for the determination of four tetracycline antibiotics Original Research Article

A new reversed-phase high performance liquid chromatography with resonance Rayleigh scattering detection (HPLC–RRS) was developed for simultaneous separation and determination of four tetracycline antibiotics (TCs). A good chromatographic separation among the compounds was achieved using a Synergi Fusion-RP column (150 mm × 4.6 mm; 4 μm) and a mobile phase consisting of methanol–acetonitrile–oxalic acid (5 mM) at the flow rate of 0.8 mL min−1. Column temperature was 30 °C. The RRS signal was detected at λex = λem = 370 nm. The recoveries of sample added standard ranged from 95.3% to 103.5%, and the relative standard deviation was below 2.79%. A detection limit of 2.12–5.12 μg mL−1 was reached and a linear range was found between peak height and concentration in the range of 10.36–518.0 μg mL−1 for oxytetracycline (OTC), 12.11–605.5 μg mL−1 for tetracycline (TC), 11.79–589.5 μg mL−1 for chlortetracycline (CTC) and 10.32–516.0 μg mL−1 for doxycycline (DC). The linear regression coefficients were all above 0.999. The method has been applied successfully to the determination of OTC, TC, CTC, DC in pharmaceutical formulations and in honey. The method was simple, rapid and showed a better linear relation and high repeatability.