Advances in the o-phthalaldehyde derivatizations: Comeback to the o-phthalaldehyde–ethanethiol reagent

The main aims of this work were (a) to present the characteristics and stability of the o-phthalaldehyde (OPA)–ethanethiol (ET) derivatives of 22 amino acids, including the believed-to-be less stable OPA derivatives providing glycine, γ-aminobutyric acid, β-alanine, histidine, ornithine, lysine and the C1–C5 aliphatic amines; (b) to compare the stability properties of the most common amino acids and amines as OPA–ET-fluorenylmethyl chloroformate (FMOC) derivatives to the corresponding ones obtained from OPA reagents containing various (SH)-additives; (c) to show the molar responses of alanine and lysine depending on the OPA reagentʹs composition; as well as (d) to prove the practical utility of these basic researches, by the simultaneous HPLC separation of 22 amino acids and 15 amines as their OPA–ET–FMOC derivatives. Investigations have been carried out by varying the composition of the reagents, the molar ratios of reactants and the reaction time, applying diode array and fluorescence detections simultaneously. Average reproducibility of quantitations, characterized with the relative standard deviations (RSDs) based on the fluorescence intensities of derivatives, in the order of listing, proved to be 1.2–5.9% for amino acids and 1.1–8.7% for amines. The practical utility of the method is demonstrated by the analysis of the amino acid and amine contents of mouse tissues, with an average reproducibility of 3.5%.