Determination of Pefloxacin and Its Main Active Metabolite in Human Serum by High-Performance Liquid Chromatography

A high-performance liquid chromatographic (HPLC) method is described for the simultaneous determination of a fluoroquinolone, pefloxacin, and its main active metabolite norfloxacin (N-desmethyl metabolite) in serum. Sample preparation involves protein precipitation with acetonitrile. The drugs and the internal standard (acebutolol) were eluted from a 4-microns Novapak C-18 cartridge at ambient temperature with an isocratic mobile phase consisting of 14% acetonitrile in buffer solution, at a flow rate of 2.5 ml/min. The effluent was monitored on a fluorescence detector using excitation and emission wave-lengths of 330 and 440 nm, respectively. Each analysis required no longer than 8 min. Quantification was achieved by measurement of the peak-area ratio of the drugs to the internal standard, and the limit of quantification for both pefloxacin and norfloxacin in serum was 50 ng/ml. The intraday coefficient of variation (CV) ranged from 1.3 to 4.4% and from 2.2 to 7.5% for pefloxacin and norfloxacin, respectively, at the concentration ranges evaluated. The interday CV ranged from 1.1 to 5.9% and from 2.3 to 5.6% for pefloxacin and norfloxacin, respectively, at three concentrations. Relative recovery was 105.5 and 99.5% for pefloxacin and norfloxacin, respectively. Stability tests show that pefloxacin and norfloxacin are stable in serum for at least 3 weeks when stored at -20 degrees C. This method has been used successfully in pharmacokinetic studies in humans.