Quantitative analysis of ofloxacin in human urine by using HPLC-UV detector: Application to a pharmacokinetic study in Iranian healthy volunteer

Ofloxacin (OFL), (±)-9-fluoro-2,3-dihydro-3-methyl-10-(4-methyl-1-piperazinyl)-7-oxo-7H-pyrido[1,2,3-de]-1,4-benzoxazine-6-carboxylic acid, a synthetic broad-spectrum antimicrobial fluoroquinolone (FQ) that exhibits wide spectrum of bactericidal activity against both Gram-positive and negative bacteria, is used for the treatment of urinary tract infections, respiratory tract infections, and osteomyelitis [1,2]. This compound belongs to a class of FQs that possess similar chromophore core units that are responsible for their UV activities [3].A simple, selective, and sensitive high performance liquid chromatography (HPLC) procedure has been developed for determination of Ofloxacin in human urine. Sample preparation involved ultrasound-assisted solvent dispersive liquid-liquid microextraction by chloroform. The HPLC separation was performed on a ZORBAX Eclipse Plus C18 column (100 mm × 4.6 mm, 3.5Micron, Agilent, USA) with a mobile phase of methanol/2-propanol/0.04 % phosphoric acid (pH adjusted to 3.0) (18:2:80, v/v) at a flow rate of 1.0 mL/min. The peaks were detected by using UV/Vis detector at 294 nm. The extraction recovery was 72.6–88.3% and the method was over the concentration range of 10.0–1486 ng/mL with a lower limit of quantitation (LLOQ) of 23.0 ng/mL using 300 L of urine. The intra- and inter-day accuracy of the method at three concentrations ranged from 96.7% to 104.2% for ofloxacin with precision of 2.9–9.7%. This validated method was successfully applied to a pharmacokinetic study enrolling one Iranian volunteers administered a single oral ofloxacin extended-release tablet of 300 mg.