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 f or the simultaneous determination of a fluoroquinolone, pefloxacin, an d its main active metabolite-norfloxacin (N-desmethyl metabolite) in s erum, Sample preparation involves protein precipitation with acetonitr ile. The drugs and the internal standard (acebutolol) were eluted from a 4-mu m Novapak C-18 cartridge at ambient temperature with an isocra tic mobile phase consisting of 14% acetonitrile in buffer solution, at a flow rate of 2.5 ml/min. The effluent was monitored on a fluorescen ce detector using excitation and emission wavelengths of 330 and 440 n m, respectively. Each analysis required no longer than 8 min. Quantifi cation was achieved by measurement of the peak-area ratio of the drugs to the internal standard, and the limit of quantification for both pe floxacin and norfloxacin in serum was 50 ng/ml. The intraday coefficie nt 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 concentration s. Relative recovery was 105.5 and 99.5% for pefloxacin and norfloxaci n, 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.