Determination of roxithromycin in human plasma by HPLC with fluorescence and UV absorbance detection: application to a pharmacokinetic study

A selective HPLC method with fluorescence detection for the determination of roxithromycin (ROX) in human plasma was described. After solid-phase extraction (SPE), ROX and erythromycin (internal standard, I.S.) were derivatized by treatment with 9-fluorenylmethyl chloroformate (FMOC-Cl). Optimal resolution of fluorescence derivatives of ROX and I.S. was obtained during one analytical run using reversed phase, C(18) column. The mobile phase was composed of potassium dihydrogenphosphate solution, pH 7.5 and acetonitrile. Fluorescence of the compounds was measured at the maximum excitation, 255 nm and emission, 313 nm, of ROX derivatives. Validation parameters of the method were also established. After SPE, differences in recoveries of ROX and erythromycin from human plasma were observed. The linear range of the standard curve of ROX in plasma was 0.5-10.0 mg/l. The validated method was successfully applied for pharmacokinetic studies of ROX after administration of a single tablet of ROX.