RP-HPLC with 9-fluorenylmethyl chloroformate fluorometric derivatization for the assay of amantadine hydrochloride in rats

A novel precolumn derivatization method is described for the quantitative determination of amantadine hydrochloride (AT) and vertilmicin sulfate as internal standard (IS) in rat plasma, which were both based on derivatization with 9-fluorenylmethyl chloroformate (FMOC-Cl) followed by reverse-phase HPLC-FLD, we also successfully applied this chromatography to actual pharmacokinetic study in rats. FMOC-Cl was introduced into the derivatization of AT, the amino group of AT and IS were trapped with FMOC-Cl to form AT-FMOC-Cl adduct and IS-FMOC-Cl adduct, which can be very sensitive for fluorescence detection. Effective chromatographic separation was achieved on a C 18 column with a mobile phase gradient consisting of acetonitrile and water at a flow-rate of 1.0 ml/min. The retention times of AT-FMOC-Cl and IS-FMOC-Cl adducts were 24.1 and 43.7 min, respectively. AT-FMOC-Cl and IS-FMOCCl were both detected at excitation 263 nm and emission 315 nm. Optimal conditions for the derivatization of AT were investigated, no endogenous interferences were found. The limit of detection (LOD) for AT-FMOC-Cl was 17.6 ng/ml in plasma, and the calibration curve showed linearity in the range 0.053-1.488 μg/ml with a regression coefficient corresponding to 0.9987. The pharmacokinetics of AT was studied in four male SD rats following intragastric administration, and pharmacokinetic parameters were estimated by use of the compartment model theory. The method was suitably validated and successfully applied to determination of AT in rat plasma samples and its oral pharmacokinetics.