Stability and characteristics of the o-phthaldialdehyde/3-mercaptopropionic acid and o-phthaldialdehyde/ N-acetyl- l-cysteine reagents and their amino acid derivatives measured by high-performance liquid chromatography

Abstract

The quality and quantity of impurities present in o-phthaldialdehyde (OPA)/3-mercaptopropionic aicd (MPA) and OPA/ N-acetyl- l-cysteine (NAC) reagents have been measured and characterized performing fluorescence and photodiode array detection, simultaneously. The amounts of impurities determined are considerable. Consequently, they have to be deducted from the coeluting amino acid. Stability studies, carried out with 24 amino acids, including—as believed—the less stable OPA derivatives, such as glycine, γ-aminobutyric acid (GABA), β-alanine, histidine, lysine and ornithine, proved that up to 50 min their decomposition is not significant. After 6 h reaction time, the OPA/MPA amino acids manifest higher stability (≥93%) than the corresponding OPA/NAC ones (≥88%). In order to obtain quantitative interactions: (i) extended reaction time (7–28 min) is needed to achieve 100% yield for alanine, β-alanine, GABA, isoleucine, ornithine and lysine; (ii) those amino acids which furnish more than one derivative (glycine, GABA, β-alanine, histidine, lysine and ornithine) are to be quantitated on the basis of the total of their peaks; (iii) reproducibility investigations revealed that the mol ratios of the OPA reagent/amino acids should be at least 20 times larger than the total of amino acids to be determined. The probable composition of the double derivatives on the basis of their characteristic fluorescence intensities and UV absorbances were discussed: glycine, GABA, β-alanine and histidine might furnish the 1-thiosubstituted-2-alkyl- and the 1,3-dithiosubstituted-2-alkylisoindoles, while lysine and ornithine are eluting as their mono- and dimer isoindoles.