2-Methoxy-2, 4-diphenyl-3 (2H)-furanone as a Precolumn Fluorescence Derivatization Reagent for Amino Acids

Abstract

2-Methoxy-2, 4-diphenyl-3(2H)-furanone (MDPF), a fluorogenic reagent for amino cornpounds, has been evaluated as a precolumn fluorescence derivatization reagent for amino acids. The derivatization reaction was optimized by determining the reaction products formed under various conditions. α-Amino acids gave fluorescent MDPF derivatives which were directly determinable with a flow injection technique. On the other hand, N-alkyl-α-amino acids yielded nonfluorescent MDPF adducts. The adducts were first subjected to chromatography to remove interfering residual MDPF and its hydrolysis product and finally determined as fluorescent MDPF-2-aminoethanol after on-line postcolumn reaction with 2-aminoethanol. The established conditions involves the reaction of amino acids with 10 mmol·dm^-3 MDPF at pH 10 and 20°C for 40 min. The reaction mixture was separated on a reversed-phase C₁₈ column of TSK LS-410 K with gradient elution using 50 mmol·dm^-3 sodium phosphate buffer (pH 7.0)-methanol system. Although most of α-amino acids gave two fluorescent MDPF derivatives due probably to a pair of diastereomers, they were determined in quantities of 2-5 pmol. The relative standard deviations (n=5) of the method ranged from 2.9 to 5.3% for the analyses of 100 pmol of α-amino acids. Furthermore, in the preliminary examination MDPF was also found to be applicable to the precolumn fluorescence derivatization of peptides. Owing to high detectabilities and extreme chemical stabilities of its derivatives, MDPF seems to be promising as a precolumn fluorescence derivatization reagent for α-amino acids and peptides as well as primary amines.