Determination of primary and secondary aliphatic amines by N-hydroxysuccinimidyl 4,3,2′-naphthapyrone-4-acetate and reversed-phase high-performance liquid chromatography

Abstract

A simple procedure for precolumn fluorescence derivatization of low molecular weight primary and secondary aliphatic amines with N-hydroxysuccinimidyl 4,3,2′-naphthapyrone-4-acetate is described. The derivatization reaction proceeds in aqueous solution at room temperature with a sodium borate buffer of pH 8.50 and gives corresponding stable fluorescent derivatives of primary amines and secondary amines within 5 and 15 min, respectively. Separation of eight-derivatized aliphatic amines has been optimized on a C 8 column with complete resolution in less than 15 min by isocratic elution with 56% aqueous methanol as the eluent. The derivatives were detected spectrofluorimetrically at 422 nm with excitation at 353 nm. The detection limits (signal-to-noise ratio=3) of primary amines and secondary amines can reach fmol level for an injection volume of 20 μl. As a preliminary application, the method was used to determine aliphatic amines in lake water.