Determination of levofloxacin in human plasma by HPLC: Method development, validation, stability evaluation and application to pharmacokinetics

This paper reported development, validation and stability evaluations of a simple, selective, sensitive, precise, accurate, and rapid high-performance liquid chromatographic method for the determination of levofloxacin in human plasma. The method was validated according to US FDA norms. Extrac-tion procedure employed a simple and rapid single-step protein precipitation to isolate levofloxacin from the plasma. Mobile phase consisted of 4 volumes of acetonitrile and 96 volumes of aqueous phase contain-ing 1.5% v/v tetrabutylammonium hydroxide, 0.3% v/v of triethylamine as ion-pairing agents, adjusted at 0.8 mL/min for isocratic separation. Levofloxacin and norfloxacin (internal standard) were separated on Zorbax SB-CN, a reversed-phase C18 HPLC column kept at 40 °C. The quantification was done by fluorescence detector set at an excitation and emission wavelengths of 290 and 456 nm, respectively. The method was found linear within 0.3-20.39 μg/mL of levofloxacin in plasma with a coefficient of correlation over 0.998. The overall precision and accuracy ranged between 1-7%. The stability of levofloxacin in plasma at different working conditions and long term storage condition were found ~ 98-103% and 93-95%, respectively. The utility of the method is reflected in its simplicity, rapidity, cost-effectiveness such as a high-throughput sample processing and analysis (a batch of 18 samples for a particular time period was processed within 20 min followed by a short run time of 12 min). The mean pharmacokinetic parameters of 18 subjects such as C_max, T_max & AUC were 5.50 ± 1.36 μg/mL, 1.306 ± 0.6836 h, and 40.38 ± 8.1751 μg-h/mL, respectively.