Optimization and validation of the direct HPLC method for the determination of moxifloxacin in plasma

Moxifloxacin (1-cyclopropyl-6-fluoro-1,4-dihydro-8-methoxy-7-[(4aS,7aS)-octahydro-6H-pyrrolo-[3,4-b]pyridin-6-yl]-4-oxo-3-quinolinecarboxylic acid hydrochloride) is new, fourth generation fluoroquinolone with broaden spectrum of antibacterial activity. In the present work simple and rapid RP-HPLC method for the direct determination of moxifloxacin in human plasma is described. Separation of moxifloxacin from plasma components was achieved on Supelco LC-Hisep shielded hydrophobic phase column. The mobile phase consisted of acetonitrile and 0.25 mol/dm3 Na3PO4 (pH 3) in a volume percent ratio (5:95, v/v) and was delivered at a rate of 1 mL/min. Fluorescence detection was employed with excitation at 290 nm and emission at 500 nm. Ofloxacin was used as internal standard and sodium dodecylsulfate solution was used as a displacing agent. Sample preparation was simplified and involved only addition of displacing agent and internal standard and dilution with water. The separation conditions were optimized by the response surface method in two factor space, i.e. the dependence of the retention time on volume percent of acetonitrile and on pH of aqueous phase was optimized. The method was fully validated and validation parameters were: linearity range 3–1300 μg/L; correlation coefficient, 0.99986; mean recovery, 92.5%; limit of quantification, 3.0 μg/L and limit of detection, 1.0 μg/L. Method was applied for the determination of moxifloxacin in human plasma after single or repeated oral doses of 400 mg Avelox® tablets. The proposed method proved to be rapid and accurate and can be successfully used in pharmacokinetic studies and routine clinical practice.