First liquid chromatography method for the simultaneous determination of levofloxacin, pazufloxacin, gatifloxacin, moxifloxacin and trovafloxacin in human plasma

For the first time a simple, selective and sensitive liquid chromatography method was developed and validated for the simultaneous determination of levofloxacin (LEV), pazufloxacin (PAZ), gatifloxacin (GAT), moxifloxacin (MOX) and trovafloxacin (TRO) in human plasma. Samples were pre-treated with acetonitrile for precipitation of plasma proteins followed by evaporation and reconstitution steps. Chromatographic separation of the analytes and norfloxacin, used as internal standard (IS), was performed under gradient elution on a LiChroCART® Purospher Star C18 column (55 mm × 4 mm, 3 μm). The mobile phase comprised a mixture of 0.1% aqueous formic acid adjusted to pH 3.0 with triethylamine, acetonitrile and methanol pumped at a flow rate of 1.0 mL/min. The detector was set at excitation/emission wavelengths of 260/455 nm. Calibration curves were linear (r2 ≥ 0.9923) in the ranges of 0.005-5 μg/mL for GAT, 0.02-5 μg/mL for LEV, PAZ and MOX and 0.04-5 μg/mL for TRO. The intra and interday precision did not exceed 7.32% and the intra and interday accuracy ranged from −11.73 to 8.92%. The limits of quantification were established at 0.005 μg/mL for GAT, 0.02 μg/mL for LEV, PAZ and MOX and 0.04 μg/mL for TRO. No endogenous or tested exogenous compounds were found to interfere at the retention times of the analytes and IS. Since the proposed method proved to be reliable for the quantitative determination of LEV, PAZ, GAT, MOX and TRO it may be a useful tool for routine analysis and to support clinical pharmacokinetic and toxicological studies involving these antibiotics.