Validation of a levofloxacin HPLC assay in plasma and dialysate for pharmacokinetic studies

An HPLC method with fluorescence detection suitable for routine determination of levofloxacin in plasma and dialysate has been validated. Sample preparation was assured by one-step protein precipitation for plasma or direct injection of the dialysate solution, respectively. Separation occurred on an YMC Pro C18 RP column (150 mm x 2 mm) with an acidic binary gradient mobile phase and detection at excitation and emission wavelengths of 296 and 504 nm. The assay was linear between 0.1 and 6 microg/ml for plasma and 0.1 and 5 microg/ml for dialysate with intra- and inter-day precision and accuracy lower than 10%. No degradation of levofloxacin was observed under the applied conditions for both matrices. The method was successfully applied to an in vitro pharmacokinetic study and patient samples as well.