An improved UPLC method for rapid analysis of levofloxacin in human plasma

A rapid, specific, and sensitive ultra-performance liquid chromatographic method for analysis of levofloxacin in human plasma has been developed and validated. Plasma samples were spiked with the internal standard (enoxacin) and extracted with 10:1 (v/v) ethyl acetate-isopropanol. UPLC was performed on a 100 × 2.1 mm i.d., 1.7 μm particle, C₁₈ column with 88:12 (v/v) 0.4% triethylamine buffer (pH 3)-acetonitrile as mobile phase, pumped isocratically at a pressure of 11,000 psi (758 bar) and a flow-rate of 0.3 mL min^-1. Ultraviolet detection was performed at 300 nm. The retention times of levofloxacin and enoxacin were 3.4 and 2.8 min, respectively, and the run-time was 5 min. Calibration showed that response was a linear function of concentration over the range 0.05-10 μg mL^-1 (r² ≥ 0.99) and the method was validated over this range for both precision and accuracy. The relative standard deviation was <15% for both intra-day and inter-day assay (n = 5). Levofloxacin and enoxacin were stable in plasma; there was no evidence of degradation during three freeze-thaw cycles, post-preparative stability at 20°C was ≥24 h, short-term stability at room temperature was ≥6 h, and long-term stability at -70°C was ≥30 days. The method was successfully used in a study of the bioequivalence of two levofloxacin tablet formulations in healthy volunteers.