Direct determination of ofloxacin enantiomers in human urine by ligand exchange chromatography

A sensitive and simple method for the determination of ofloxacin enantiomers in human urine was developed by ligand exchange high performance liquid chromatography. Chiral separation was performed on a C-18 column, where the mobile phase consisted of a methanol-water solution (containing1.2 mmol L-1 L-phenylalanine and 1.0 mmol L-1 copper sulphate) (15:85, v/v) and its flow rate was set at 1.0 mL min(-1). After centrifugation, the human urine was injected into a C18 column directly. Baseline separation of ofloxacin enantiomers in human urine were obtained with a resolution of 3.24 in less than 25 min, and no interference by the protein or endogenous compounds were observed. The effects of different separation conditions were investigated and the concentration of ligand and pH of the mobile phase play a critical role in the enantioseparation. The standard curves showed excellent linearity over the concentration range from 0.8 to 400 mu g mL(-1) for ofloxacin enantionters. The linear correlation equations are: Y = 171.11 X + 504.13 (r = 0.9995) and Y = 169.01X + 631.59 (r = 0.9994) for (S)-ofloxacin and (R)-ofloxacin, respectively. The average recover), of ofloxacin enantiomers from human urine samples was more than 96%. The procedure developed was successfully applied to investigate the stereoselectivity and pharmacokinetics of ofloxacin enantiomers in the human body.