Quantitative determination of azithromycin in plasma, blood and isolated neutrophils by liquid chromatography using pre-column derivatization with 9-fluorenylmethyloxycarbonyl-chloride and fluorescence detection

In this study, a high-performance liquid chromatographic method with pre-column derivatization and fluorescence detection was optimised and validated for the quantification of azithromycin (AZM) in plasma. Clarithromycin (CLM) was used as an internal standard. Pre-column derivatization was done with 9-fluorenylmethyloxycarbonyl-chloride. Recovery from blood and polymorphonuclear neutrophils (PMNNs) isolated by a gravity separation procedure was also assessed. Analytical separation was carried out using a C18 column as stationary phase and acetonitril–phosphatebuffer as mobile phase. Peak quantification was carried out by excitation at 267 nm and detection at 317 nm. A lower limit of quantitation of 0.042 ± 0.017 mg/l in plasma, 0.119 ± 0.065 mg/l in blood and 0.072 ± 0.036 in water was achieved. Linearity was assessed from 0 to 1.5 mg/l in plasma and blood and from 0–9 mg/l in water. The analytical method proved to be applicable in a pharmacokinetic study of AZM in a Cystic Fibrosis patient.