Determination of the stereoisomeric impurities of sitafloxacin by capillary electrophoresis with dual chiral additives.

Because of the bioactivity against the human topoisomerase II, the stereoisomeric impurities of sitafloxacin should be controlled in the process of manufactory. In the present work, a capillary electrophoresis (CE) method was developed and validated for simultaneous determination of three stereoisomeric impurities of sitafloxacin. The separation with high resolution not only for the separation of enantiomers, but also for the separation of diastereoisomers was achieved by using a background electrolyte composed of dual chiral selectors, namely γ-cyclodextrin (γ-CD) and Cu(2+)-d-phenylalanine (D-Phe) complex. The combination of two chiral selectors is indispensable to gain a high separation selectivity due to the cooperativity effect of different chiral discrimination modes: inclusion complexation (γ-CD) and ligand exchange (Cu(2+)-d-Phe). The concentrations of γ-CD, Cu(2+) and D-Phe were found to be critical to the separation. Because two chiral selectors were involved in the enantiomer separation system, multiple factors and their interaction should be simultaneously optimized by using the response surface methodology (RSM) with a face centred central composite design (FCCD). The obtained optimized separation conditions were as follows: 15mmol/L dipotassium hydrogenphosphate solution (pH 4.5) containing 15mmol/L D-Phe, 20mmol/L CuSO4 and 20mmol/L γ-CD, separation voltage 15kV. The method was then validated and the robustness of the method was tested. Under the optimized conditions, as low as 0.1% (m/m) stereoisomeric impurities of sitafloxacin can be determined by the method.