Development and validation of liquid chromatographic method for the simultaneous determination of some fluoroquinolone antibiotics in biological fluids using new generation columns

In this study, a liquid chromatography method for the determination of levofloxacin, ciprofloxacin, moxifloxacin and gemifloxacin which are the fluoroquinolones used in the treatment of infectious diseases was developed for the first time. Efficiency of 5 different columns in the separation of fluoroquinolones was compared. Experimental parameters that affect the separation such as percentage of organic solvent, pH, temperature, gradient shape and detector wavelength were optimized by employing a step-by-step approach. Phosphate buffer (pH 2, 20 mM) and acetonitrile mixture was employed as mobile phase in the gradient elution mode. Using a pentafluorophenyl core-shell column (100 mm x 4.6 mm, 2.7 μm), the separation of four analytes was accomplished in less than 7.5 min. Total analysis time was 22 min, including column clean-up and column conditioning steps. Fluorescence detector was operated simultaneously on two different channels. The diode array detector was set to 280 nm. Flow rate was 1.0 mL/min and the column temperature was 25 °C. The developed method was validated for determination of analytes in both urine and plasma with respect to sensitivity, specificity, linearity (r≥0.9989), recovery (%79.46-102.69), accuracy (%89.55-111.94), precision (%0.35-8.05), and stability (%85.79-111.07). Afterwards, feasibility of method was demonstrated by analysing urine and plasma samples of patients receiving orally levofloxacin, ciprofloxacin or moxifloxacin. Developed method is suitable for therapeutic drug monitoring of these fluoroquinolones and can be applied to pharmacokinetic and toxicological studies.