Characteristics and stability of the OPA/3-mercaptopropionic acid and OPA/N-acetyl-L-cysteine derivatives of amino acids

Abstract

The behavior of o-phthaldialdehyde (OPA)/3-mercaptopropionic acid (MPA) and OPA/N-acetyl-L-cysteine (NAC) derivatives of amino acids (AAs) have been studied from analytical view point, based on their UV and fluorescence responses, simultaneously. The particular aim was to clarify the background of the believed instability of AAs, that give more than one OPA-derivative, such as glycine, γ-amino butyric acid (GABA), β-alanine, histidine, ornithine and lysine. To confirm the stoichiometric correlation and to improve reproducibility of interaction the mode ratios of OPA to the SH-group-containing additive, the concentration of reactants, pH of injected analyte, temperature of interaction and reactivity of substituted AAs toward the OPA/MPA reagent have been investigated. Varying the mole ratios of [OPA] to [MPA] and [OPA] to [NAC], it is shown that the higher the concentration of SH-group-containing additive the lower the transformation rate of the initially formed OPA-derivative to those forthcoming. Performing all reactions with the same mole ratios, at higher concentrations resulted in increased amounts of the succesively-formed OPA-derivatives. Decreasing the pH of OPA-AAs from 9.3 to 7.2 before injecting them onto the column, lead to considerable loss of response. Increasing the temperature of reactions from 4°C to 30°C resulted in increased decomposition rate of derivatives. The stability, and hence reproducibility of reactions can be improved (i) by applying the the same mole ratios of OPA and SH-group-containing additive, (ii) by injecting the analyte of derivatives at the derivatization pH, and, (iii) performing reactions with refrigerated stock solutions and maintained before injection possible at 4°C.