Home Small Molecule HPLCHPLC determination of Besifloxacin in Ophthalmic Suspension using Chromolith^® SpeedROD RP-18e Column

HPLC determination of Besifloxacin in Ophthalmic Suspension using Chromolith^® SpeedROD RP-18e Column

Dr. Ajay Kaparwan, Dr. Sanjay Poman

, Mumbai Analytical Laboratory, Navi Mumbai, India

Scope

Here we demonstrate an application for Besifloxacin in ophthalmic suspension on RP-HPLC using Chromolith^® SpeedROD RP-18e column for system suitability, linearity, repeatability, percentage recovery, LOD and LOQ.

Introduction

Besifloxacin (Figure 1) is a bactericidal fluoroquinolone-type antibiotic, used for the treatment of bacterial conjunctivitis caused by susceptible bacteria. It was approved by US FDA in May 2009. Here, a solution is demonstrated for the determination of Besifloxacin in ophthalmic suspension dosage using HPLC with a monolith column and UV Detection at 289 nm. At present, there are no pharmacopeial monographs available for the assay of Besifloxacin in ophthalmic suspension dosage forms.

Figure 1.Chemical Structure of Besifloxacin

Instrumentation

Waters Alliance™ e2695 with Empower 3 Software
Milli-Q^® Integral 3 Water purification system
Ultrasonic bath from PCI analytics
Millex^® HV Durapore membrane filter (PVDF) 0.45 µm

Chromatographic Data of Besifloxacin Analysis

Chromatogram of Besifloxacin test solution

Chromatogram of blank solution (diluent)

Figure 2.Chromatograms of a) Besifloxacin reference STD solution; b) Besifloxacin test solution; and c) Blank solution (diluent)

Chromatograms showing peaks obtained for the LOQ and LOD estimation of besifloxacin on Chromolith® SpeedROD RP-18e column

Figure 3.LOQ and LOD chromatograms of Besifloxacin overlaid with blank

Linearity plot obtained for analysis of besifloxacin on Chromolith® SpeedROD RP-18e column

Figure 4.Linearity plot of Besifloxacin reference standard

Results and Discussion

An isocratic RP-HPLC method for the determination of Besifloxacin in ophthalmic suspension was developed using Chromolith^® SpeedROD RP-18 endcapped column with UV detection at 289 nm. The experimental data indicated an excellent linearity with an r² value of 0.999 (Figure 4) for the selected concentration range (0.005 – 75 µg/mL, Table 4). The LOD and LOQ values were estimated as 2.3 µg/mL and 7 µg/mL, respectively. The method precision for assay was below 1% RSD and the percentage recovery found to be ranging from 102.1 -104 % (Table 5).

In conclusion, the data for linearity, system suitability, repeatability of the method suggests its aptness for the assay of Besifloxacin in opthalmic suspension using Chromolith^® SpeedROD RP-18 endcapped column.

Materials

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