Characterization and application of acridine-9-N-acetyl-N-hydroxysuccinimide as a pre-column derivatization agent for fluorimetric detection of amino acids in liquid chromatography

Abstract

A simple and sensitive LC method that rapidly labels amino compounds including amino acids, using acridine-9-N-acetyl-N-hydroxysuccinimide (AAHS) which was synthesized by the reaction of acridine-9-N-acetic acid with benzenedisulfonyl-N-hydroxysuccinimide, was developed. A mixture of amines is treated with AAHS in the presence of triethylamine in non-aqueous acetonitrile or in 0.2 mol l^–1 borate buffer at pH 8.0–9.0 in 40% v/v acetonitrile solution to give quantitative yields of amides. The emission maximum for the derivatized amines is 435 nm (λ_ex = 404 nm). The labeled derivatives are very stable; no significant decomposition is observed after heating in 50% acetonitrile at 40 °C for 24 h. Studies on the derivatization conditions indicate that amines or amino acids react very rapidly with AAHS under the proposed conditions. The method, in conjunction with a multi-step gradient, offers baseline resolution of common amine or amino acid derivatives on a reversed-phase C₁₈ column. This method is more convenient and more efficient than previous methods which require prior conversion of carboxylic acids to acyl chlorides, which are unstable to moisture. The LC separation of amine or amino acid derivatives has good reproducibility. The established method is also suitable for the determination of other amine compounds in various biological fluids.