High‐performance liquid chromatography‐based assay optimization for the detection of plasma amino acids for applications in metabolic disorders in developing countries

Abstract

Plasma amino acids are generally analyzed through ion exchange chromatography, a reproducible but time‐consuming method. Here, we report the optimization of a reverse‐phase‐high‐performance liquid chromatography with fluorescence detector (RP‐HPLC‐FLD) assay for the detection and quantification of plasma amino acids for potential applications in metabolic disorders (e.g., aminoacidopathies, a rare group of Inborn Errors of Metabolism). For assay development, initially standard amino acids were derivatized with ortho‐phthalaldehyde‐3‐mercaptopropionic acid (OPA‐3‐MPA) and filtered through a 0.20 μm syringe filter. The excitation and emission wavelengths of 240–450 nm (λex—λem) were used for the detection of amino acids. Chromatographic separation was achieved by gradient RP‐HPLC‐FLD through C18 symmetry column (150 × 4.6 mm, particle size 3.5 μm). HPLC assay was successfully optimized and was able to detect amino acids in the range of 10–400 ng/mL and good linearity (R2 > 0.98) was achieved in the mixture for each standard amino acid. Moreover, the current assay showed great efficiency with two additional advantages: the use of low‐cost mobile phases, and the detection and quantification of amino acids at low level (ng/mL) concentration in biofluids. This assay could be applied for the analysis of human plasma to identify aminoacidopathies in newborn screening programs, and other metabolic disorders.