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 car ried 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 pha se. The concentrations of NOR, SAR and DIF eluting off the column, with ret ention times of 2.16, 5.60 and 6.20, respectively, were monitored by fluore scence 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 relate d to concentration in the range from 1 to 1500 ng mL(-1) Recovery was deter mined as 76% and 70% for SAR and DIF, respectively. Inter-and intraassay co efficients of variation were less than 6% for all compounds.