HPLC separation and quantification of ofloxacin enantiomers in rabbit plasma. Application to pharmacokinetic studies

A sensitive HPLC method with marbofloxacin (MAR) as internal standard and fluorescence detection is described for the analysis of ofloxacin (OFL) enantiomers in plasma samples. Plasma samples were prepared by adding phosphate buffer (pH 74, 0.1 M), then extracted with trichloromethane. S-OFL R-OFL, and the internal standard were separated on a reversed-phase column with water-methanol, 85.5:14.5, as mobile phase. The concentrations of S-OFL and R-OFL eluting from the column (retention times 75 and 8.7 min, respectively) were monitored by fluorescence detection with), lambda(ex) = 331 and lambda(em) = 488 nm. The detection and quantitation limits were 10 and 20 ng mL(-1), respectively, for S-OFL and 11 and 21 ng mL(-1) for R-OFL. Response was linearly related to concentration in the range 10 to 2500 ng mL(-1) Recovery was close to 93% for both compounds. The method was applied to determination of the enantiomers of OFL in plasma samples collected during pharmacokinetic studies.