Simultaneous determination of moxifloxacin and ofloxacin in physiological fluids using high performance liquid chromatography with ultraviolet detection

•Simultaneous determination of moxifloxacin and ofloxacin in physiological fluids using HPLC–UV detection is carried out for the first time.•This method is more sensitive compared to reported methods.•This method is applicable for analysis of both drugs in plasma, aqueous humor and pharmaceutical dosage forms.•Extraction is carried out using simple protein precipitation method.

A novel, sensitive and validated RP-HPLC–UV method was developed for simultaneous determination of moxifloxacin and ofloxacin using timolol maleate as internal standard in physiological fluids. Different experimental parameters were optimized and validated according to international guidelines. Complete separation of the analytes was achieved with Kromasil 100-5C18 analytical column (250 mm × 4.6 mm × 5 μm), methanol and 0.05% trifloroacetic acid (TFA) (38:62 v/v) were used as mobile phase, pumped at flow rate of 1.1 ml/min in isocratic phase, column oven temperature maintained at 45 °C and detection wavelength of 290 nm. Protein precipitation method was applied to extract the drugs from human plasma and bovine aqueous humor samples using methanol as precipitating solvent. This method is linear in concentration range of 0.018–100 μg/ml for moxifloxacin and 0.014–20 μg/ml for ofloxacin. The recoveries of the method were 97.52 and 97.39% in human plasma for MX and OFN respectively, while in aqueous humor 94.48% for MX. The LOD values in plasma were found to be 10.0 and 8.00 ng/ml for MX and OFN respectively, while their respective LOQ values were 18.0 and 14 ng/ml. In aqueous humor the LOD and LOQ for MX were 16.0 and 24 ng/ml respectively. In future, this method will be used to study the pharmacokinetic profile of moxifloxacin and ofloxacin in biological fluids and pharmaceutical products.