A rapid and simple high‐performance liquid chromatography method for the determination of human plasma levofloxacin concentration and its application to bioequivalence studies

A high‐performance liquid chromatography method with fluorescence detection (HPLC‐FLD) for the determination of levofloxacin in human plasma is described. Neutralized with phosphate buffer (pH 7.0), the sample (0.1 mL) was extracted with dichlormethane (1 mL). After voltex‐mixing and centrifuged at 3000 g for 6 min at 4°C, the upper aqueous layer was aspirated using a micro vacuum pump and the organic layer was directly transferred to a clean test tube without pipetting. The organic solvent was evaporated and the residues were reconstituted with the mobile phase. Levofloxacin and terazosin (internal standard, IS) were chromatographically separated on a C 18 column with a mobile phase containing phosphate buffer (pH 3.0, 10 m m ), acetonitrile and triethylamine (76:24:0.076, v/v/v) at a flow rate of 1 mL/min. The analytes were detected using fluorescence detection at an excitation and emission wavelength of 295 and 440 nm, respectively. The linear range of the calibration curves was 0.0521–5.213 µg/mL for levofloxacin with a lower limit of quantitation (0.0521 µg/mL). The retention times of levofloxacin and terazosin were 2.5 and 3.1 min, respectively. Within‐ and between‐run precision was less than 12 and 11%, respectively. Accuracy ranged from −6.3 to 4.5%. The recovery ranged from 86 to 89% at the concentrations of 0.0521, 0.5213 and 5.213 µg/mL. The present HPLC‐FLD method is sensitive, efficient and reliable. The method described herein has been successfully used for the pharmacokinetic and bioequivalence studies of a levofloxacin formulation product after oral administration to healthy Chinese volunteers. Copyright © 2007 John Wiley & Sons, Ltd.