A Fluorogenic Reagent : 3-(4, 6-Difluorotriazinyl)amino-7-methoxycoumarin, for the Determination of Amantadine by High-Performance Liquid Chromatography

Abstract

A sensitive method for determination of amantadine by high-performance liquid chromatography (HPLC) has been developed. Amantadine was derivatized quantitatively into a fluorescent compound through the amino group by treatment with 3-(4, 6-difluorotriazinyl)amino-7-methoxycoumarin (FAMC) in benzene at 140°C for 15 min. The derivative was subjected to HPLC on TSK gel ODS-120T (250 × 4.6 mm i.d.) with acetonitrile-water (5 : 1) as the mobile phase and monitored by fluorescence detection (Ex. 345 nm; Em. 410 nm). The limit of detection was 250 fmol in 100μl of urine. This method was satisfactory with respect to recovery and precision to quantify amantadine spiked in urine.