Optimization of a Ligand Exchange Chromatography Method for the Enantioselective Separation of Levofloxacin and Its Chiral Impurity

Abstract

Background Levofloxacin is a third-generation fluoroquinolone that has several advantages over its R-Ofloxacin isomer. It is used to treat different types of infections including urinary and prostatitis. Objective New HPLC method for the enantioselective separation of levofloxacin and its chiral impurity was developed and validated to improve the separation of the enantiomers of levofloxacin (impurity-R and active principle-S) by increasing the value of the resolution between the eutomer and the distomer. Methods Chromatographic separation was performed on Prodigy ODS -2, 5 µm 4.6 × 150 mm column, with a gradient of buffer solution and methanol, in the proportion of (80:20, v/v). The box-Behnken design was considered when optimizing the enantioseparation involving many factors effects such as the concentration of D-phenylalanine, the pH of the buffer, the percentage of organic modifier in the mobile phase, the flow rate, the temperature of the column, and the type column. Results Chiral separation was achieved with an optimal resolution of 3.8. The method was successfully validated following ICH Q2 (R1) guideline, fulfilling acceptance criteria for selectivity (no interference in the retention time of S-levofloxacin and R-levofloxacin), linearity [r > 0.999 in the ranges 1.25 to 3.75 µg/mL for all enantiomers], and precision (RSD < 2%). Accuracy was assessed by the application of the analytical method to an analyte of know purity, evidencing the usefulness of this monitoring system. Conclusions The method was successfully used for the determination of levofloxacin impurity in raw material and pharmaceutical dosage forms.