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

A validated, highly sensitive and precise high-performance liquid chromatog raphic (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 carb onate solution, was treated with 9-fluorenylmethyloxycarbonyl chloride (FMO C-CI) for 40 min at 40 degrees C and chromatographed on a base-deactivated octadecyl column, maintained at 50 degrees C during elution, using an eluen t composed of acetonitrile-hydrogenphosphate buffer, pH 7.5, with 0.125% tr iethylamine (3:2, v/v). Fluorescence detection was used at an excitation wa velength of 255 nm and an emission wavelength of 315 nm. Erythromycin, clar ithromycin, roxithromycin and azithromycin were found to have retention tim es of 8.8, 15.7, 17.1 and 20.7 min, respectively. Recoveries ranging from 9 3 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. (C) 1998 Elsevier Science B .V. All rights reserved.