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

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 orthophthalaldehyde-3-mercaptopropionic acid (OPA-3-MPA) and filtered through a 0.20 mu m syringe filter. The excitation and emission wavelengths of 240-450 nm (.ex-.em) were used for the detection of amino acids. Chromatographic separation was achieved by gradient RP-HPLC-FLD through C18 symmetry column (150 x 4.6 mm, particle size 3.5 mu m). 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.