Quantitative Determination of Levofloxacin in Ophthalmic Solution by High-Performance Liquid Chromatography

Abstract

Background: Levofloxacin, as a prototypical agent of the third generation of fluoroquinolones, is an antimicrobial agent routinely administered for treating bacterial keratitis. Levofloxacin is available under different trade names as liquid pharmaceutical formulations, such as infusions and eye drops. Objectives: This paper reports a fast, simple, accurate, and precise high-performance liquid chromatography (HPLC) technique for levofloxacin determination in liquid pharmaceutical formulations. Methods: The HPLC method was applied using a photodiode array detector (DAD), and measurements were conducted at a 294 nm UV-Vis wavelength. The technique was developed to enable the immediate estimation of levofloxacin in the Oftaquix (5 mg/mL) ophthalmic formulation. The ODS-phenyl column was used and maintained at 30 ± 2°C and 294 nm λmax conditions. A mixture of acetonitrile: 0.1% trifluoroacetic acid (18:82 v/v) was used as the mobile phase, with a flow rate of 1.5 mL/min. The method was validated in relation to system suitability, accuracy, linearity, and precision in agreement with International Conference on Harmonization (ICH) and Food and Drug Administration (FDA) guidelines. Results: The levofloxacin peak was eluted at 9.5 minutes with good resolution. The calibration curve was linear within the 50 - 150 μg/mL range with a linearity coefficient higher than 0.9999. The limit of detection (LOD) and limit of quantification (LOQ) of the developed method were 2.13 and 6.47 μg/mL, respectively, and the lowest concentration from the calibration curve is obtained as 50.19 ± 0.24 μg/mL. The average inter- and intraday precision was in the range of 96.465 ± 0.0080 - 97.060 ± 0.0034%. Analyzing the amount of drug in Oftaquix 5 mg/mL ophthalmic solution revealed a 4.96 mg/mL levofloxacin in this formulation, with 99.3% accuracy and 0.84% relative standard deviation (RSD) value. Conclusions: The HPLC-DAD method developed in this study can be applied for routine analysis of levofloxacin amounts in pharmaceutical formulations and bioequivalence studies in quality control departments and the pharmaceutical industry.