Simultaneous determination of difloxacin and its primary metabolite sarafloxacin in rabbit plasma

An HPLC method with fluorescence detection is presented for the analysis of difloxacin (DIF) and sarafloxacin (SAR) in rabbit plasma using norfloxacin (NOR) as internal standard (Figure 1). Plasma sample preparations were carried out by adding phosphate buffer (pH 7.4, 0.1 M), followed by extraction with trichloromethane. Fluoroquinolones were separated on a reversed-phase column using an aqueous phosphate solution-acetonitrile (82:18) mobile phase. The concentrations of NOR, SAR and DIF eluting off the column, with retention times of 2.16, 5.60 and 6.20, respectively, were monitored by fluorescence detection at lambda(ex) 338 and lambda(em) 425 nm. The quantitation limit was 12 ng mL(-1) for SAR and DIE Standard curves were linearly related to concentration in the range from 1 to 1500 ng mL(-1) Recovery was determined as 76% and 70% for SAR and DIF, respectively. Inter-and intraassay coefficients of variation were less than 6% for all compounds.