Determination of Nemonoxacin in Small Volumes of Rat Plasma and Bile by a Novel HPLC-Fluorescence Assay and Its Application to Disposition and Biliary Excretion Kinetics

Ruei-Lin Wu,1 Wei-Chun Wang,1 Ching-Ling Cheng,1,2 Cheng-Yuan Tsai,3 Chen-Hsi Chou1,4 1Institute of Clinical Pharmacy and Pharmaceutical Sciences, College of Medicine, National Cheng Kung University, Tainan City, Taiwan; 2School of Pharmacy, Tzu Chi University, Hualien City, Taiwan; 3TaiGen Biotechnology Co., Ltd., Taipei City, Taiwan; 4School of Pharmacy, National Cheng Kung University, Tainan City, TaiwanCorrespondence: Chen-Hsi Chou, Institute of Clinical Pharmacy and Pharmaceutical Sciences, College of Medicine, National Cheng Kung University, No. 1, University Road, East Dist, Tainan City, 701, Taiwan, Tel: +886 6 2353535 ext. 6207, Fax +886-6-2373149, Email [email protected]: Nemonoxacin is a novel non-fluorinated quinolone antibiotic for the treatment of community-acquired pneumonia. To investigate the pharmacokinetics (PK) of nemonoxacin, a simple and sensitive high-performance liquid chromatography assay (HPLC) was needed.Methods: An HPLC method with fluorescence (FL) detection was developed for the quantification of nemonoxacin in plasma and bile. Ultraviolet (UV) and FL characteristics were examined for the optimal detection conditions. Nemonoxacin and the internal standard gatifloxacin were extracted from plasma utilizing ethyl acetate-isopropanol (70/30, v/v). For bile sample preparation, direct dilution with the mobile phase buffer was used. Chromatographic separation was achieved on a C6-phenyl column (5 μm, 25 cm × 4.6 mm i.d.) at 30 °C with a flow rate of 1 mL/min. The mobile phase was composed of methanol and 50 mM potassium dihydrogen phosphate containing 0.5% (v/v) triethylamine (pH 7.5) (45/55 and 35/65 (v/v) for plasma and bile samples, respectively). FL was measured at an emission wavelength of 465 nm with excitation at 285 nm.Results: The calibration curves were linear with a lower limit of quantification of 5 and 100 ng/mL in a small volume of plasma (50 μL) and bile (10 μL). The intra- and inter-day precision was within 9.0% and the accuracy was within 7.6% deviation of the nominal concentration. Nemonoxacin was stable under various storage/handling conditions tested. The method was successfully employed to describe the plasma and biliary profiles of nemonoxacin in rats following a single intravenous dose of 1 mg/kg. Nemonoxacin displayed two-compartment disposition kinetics. The bile-to-plasma area under concentration–time curve ratio (AUCbile/plasma) estimated was 50.7, indicating that nemonoxacin was actively secreted into bile.Conclusion: A validated method was developed and found to be specific, precise and accurate. The applicability of this proposed method was substantiated in pharmacokinetic studies in rats. Keywords: nemonoxacin, fluorescence, biliary excretion, HPLC, pharmacokinetics