Derivatization of catecholamines in aqueous solution for quantitative analysis in biological fluids

Abstract

The reaction of methyl chloroformate with catecholamines at the nanogram level in aqueous solution has been estimated. Optimal pH conditions of the aqueous medium were established which provide a quantitative yield of the formate derivatives of primary and secondary catecholamines. First the catechol function was blocked by the reaction with methyl chloroformate under mild alkaline conditions (pH 7.2) and subsequently a pH shift was introduced (Δ pH = 2) to improve the reaction of the amine function. The formate derivatives were extracted effectively (≧ 99%) into ethyl acetate and subsequently converted to their O- tert.-butyldimethylsilyl, N-formate derivatives. These mixed derivatives appeared to be very suitable for quantitative determination of catecholamines and related compounds in biological fluids by gas chromatography—mass spectrometry. The coefficient of variation estimated in urine samples was 6% ( n = 6). The minimal detectable concentration in biological samples was 50 pg ml −1 with a signal-to-noise ratio of 5.