Development and validation of a fast isocratic liquid chromatography method for the simultaneous determination of norfloxacin, lomefloxacin and ciprofloxacin in human plasma

Abstract

A simple and fast liquid chromatographic method coupled with fluorescence detection (LC-FD) is reported, for the first time, for the simultaneous quantification of norfloxacin (NOR), ciprofloxacin (CIP) and lomefloxacin (LOM) in human plasma, using levofloxacin as internal standard (IS). Sample preparation consists of a single-step precipitation of plasma proteins followed by vortex-mixing and centrifugation. Chromatographic separation was achieved within 7 min on a reversed-phase C(18) column with a mobile phase consisting of 0.1% aqueous formic acid (pH = 3.0, triethylamine)-methanol (82:18, v/v) pumped isocratically at 1.2 mL/min. The detector was set at excitation/emission wavelengths of 278/450 nm. Calibration curves were linear (r(2) ≥ 0.994) in the range of 0.02-5.0 µg/mL, and the limit of quantification was established at 0.02 µg/mL for all analytes (NOR, CIP and LOM). The overall precision did not exceed 8.19% and accuracy was within ±10.91%. NOR, CIP and LOM were extracted from human plasma with an overall mean recovery ranged from 90.1 to 111.5%. No interferences were observed at the retention times of the analytes and IS. This novel LC-FD method enables the reliable determination of NOR, CIP and LOM in a single chromatographic run, which may be suitable to support human pharmacokinetic-based studies with those antimicrobial agents.