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

Background: Levofloxacin is a third-generation fluoroquinolone that has several advantages over its (R) ofloxacin isomer. It is used to treat different types of infection, including urinary infection and prostatitis.

Objective: A 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.

Method: Chromatographic separation was performed on a Prodigy ODS -2, 5 µm 4.6 × 150 mm column, with a gradient of buffer solution and methanol (80:20, v/v). A Box-Behnken design was considered when optimizing the enantioseparation involving the effects of many factors 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 of column.

Results: Chiral separation was achieved with an optimal resolution of 3.8. The method was successfully validated following the International Conference on Harmonization Q2 (R1) guideline, fulfilling the acceptance criteria for selectivity [no interference in the retention time of (S) levofloxacin and (R) levofloxacin], linearity (r ≥0.999 in the range 1.25-3.75 µg/mL for all enantiomers), and precision (RSD <2%). Accuracy was assessed by the application of the analytical method to an analyte of known purity, providing evidence for 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.

Highlights: The following method is accurate and robust to quantify and characterize the presence of levofloxacin impurity in raw material for pharmaceutical compounds.