Improvement of Pharmacopeial High-Performance Liquid Chromatography Method for Determination of Enantiomeric Purity of Moxifloxacin Drug Substance.

New chiral isocratic HPLC conditions were proposed for investigation of moxifloxacin hydrochloride and its potential isomeric impurity (R,R)-isomer (impurity G) using the chiral additive copper (II)-sulfate and isoleucine in the mobile phase using an achiral C18 chromatographic column, 150 × 4.6 mm; 3 µm, as a significant improvement of the official European Pharmacopeia method concerning economy and baseline resolution. The mechanism of separation of enantiomers of moxifloxacin using the chiral additive copper (II)-sulfate is also elucidated. In the optimization of the experimental conditions of the chiral HPLC method, the central composite design was used, and for multiobjective optimization the desirability function with its desired value 1.00 was used. After optimization and method validation the best conditions concerning the concentration of chiral agents (0.01 M), organic solvent content in the mobile phase (30%, v/v methanol), pH value of mobile phase (3.5), and column temperature (23°C) with 10 µL injection volume, were defined. Compared to the pharmacopeial HPLC method, the analysis time was shorter and significantly lower amount of chiral agents in mobile phase was required. The optimized method was validated with respect to linearity, accuracy, precision, and robustness. The linearity of the method has been demonstrated in the range of 0.30-2.50 µg/mL (R2 > 0.998). Limit of detection and limit of quantification for moxifloxacin chiral impurity G determination were 0.098 and 0.298 µg/mL, respectively. The method was found to be suitable for quantification of moxifloxacin chiral impurity G in moxifloxacin drug substance.