Quantitative determination of the macrolide antibiotics erythromycin, roxithromycin, azithromycin and clarithromycin in human serum by high-performance liquid chromatography using pre-column derivatization with 9-fluorenylmethyloxycarbonyl chloride and fluorescence detection

Abstract

A validated, highly sensitive and precise high-performance liquid chromatographic (HPLC) method for the determination of the macrolides erythromycin, azithromycin, clarithromycin and roxithromycin in human serum is described. A diethyl ether extract, obtained from serum using a saturated sodium carbonate solution, was treated with 9-fluorenylmethyloxycarbonyl chloride (FMOC–Cl) for 40 min at 40°C and chromatographed on a base-deactivated octadecyl column, maintained at 50°C during elution, using an eluent composed of acetonitrile–hydrogenphosphate buffer, pH 7.5, with 0.125% triethylamine (3:2, v/v). Fluorescence detection was used at an excitation wavelength of 255 nm and an emission wavelength of 315 nm. Erythromycin, clarithromycin, roxithromycin and azithromycin were found to have retention times of 8.8, 15.7, 17.1 and 20.7 min, respectively. Recoveries ranging from 93 to 104% were found with reproducibility coefficients of variation of 1.1–5%. Mean correlation coefficients of 0.9997, 0.9998, 0.9996 and 0.9994 were found for the linear calibration curves ( n=2) of erythromycin (0.320–16.1 mg/l), roxithromycin (3.24–19.4 mg/l), clarithromycin (0.190–19.4 mg/l) and azithromycin (0.0988–4.94 mg/l), respectively.