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.