Pharmacokinetics of fluoroquinolones in critical care patients: A bio-analytical HPLC method for the simultaneous quantification of ofloxacin, ciprofloxacin and moxifloxacin in human plasma

A high-performance liquid chromatographic (HPLC) method with fluorescence detection was developed and validated for the simultaneous quantification of ofloxacin, ciprofloxacin and moxifloxacin in human plasma. Sarafloxacin was used as internal standard. Chromatography was carried out using a Waters XBridge™ C18 HPLC column and a gradient mobile phase consisting of CH3CN/MeOH/0.025 M TBA·Cl/TFA (eluent A at 75/25/899/1 (v/v); eluent B at 150/50/799/1 (v/v); both at pH 3.5). Excitation/emission wavelengths were 279/442 nm for ciprofloxacin and 290/500 nm for ofloxacin, moxifloxacin and internal standard. Prior to chromatography, plasma samples were treated with acetonitrile for protein precipitation, followed by evaporation of the liquid layer and reconstitution in eluent A. The method was validated for the three fluoroquinolones over the clinically relevant concentration range from 0.02 to 7.50 μg/ml. The method showed acceptable linearity with correlation coefficients, r2 > 0.995, as well as high precision (RSD% <7% in each case), accuracy (90.4–105.4%) and selectivity. The limit of quantification for the three fluoroquinolones was established at 0.02 μg/ml. Ofloxacin, ciprofloxacin and moxifloxacin were extracted from plasma with a mean recovery of 95%, 86.4% and 94.2%, respectively. During validation, the concentration of the three fluoroquinolones was found to be stable after 3 freeze–thaw cycles and for at least 15 h after extraction. This bio-analytical method was finally applied to the analysis of samples which have been obtained from patients, participating in a pharmacokinetic study on moxifloxacin.