N-hydroxysuccinimidyl fluorescein- O-acetate as a highly fluorescent derivatizing reagent for aliphatic amines in liquid chromatography

Abstract

A sensitive fluorescent reagent, N-hydroxysuccinimidyl fluorescein- O-acetate (SIFA), has been developed as a precolumn derivatizing reagent for aliphatic amines in liquid chromatography (LC). SIFA was shown to be a successful reagent with advantages including high fluorescent quantum yield in the visible region, specificity to aliphatic amines, mild derivatization conditions and few by-products. Methylamine, ethylamine, ethylene diamine, isopropylamine, isobutylamine, n-butylamine, 1,4-butylene diamine and 1,5-amylene diamine were used to investigate the separation and derivatization conditions. In a pH 8.5 H 3BO 3–Na 2BO 7 buffer solution, SIFA reacted with amines at 45°C for 30 min to form stable derivatives. The derivatives could be separated on a C 18 column in 18 min with methanol–water (46/54, v/v) containing 10 mmol l −1 pH 5.40 citric acid–Na 2HPO 4 buffer as a mobile phase. The detection limits of these amines were in the range 85–520 amol (signal-to-noise ratio=3), which were better than those using the other derivatizing reagents for amine analysis by LC.