Analysis of Plasma Amino Acids Using RP-HPLC and Pre-Column Derivatization with OPA/3-MPA

ABSTRACT        Background and Objective: Measurement of amino acids is an important tool for metabolic studies and evaluation of patients’ clinical condition. The aim of this study was to analyze the plasma amino acids using reverse phase high performance liquid chromatography techniques (RP-HPLC) with pre-column derivatization by o-phthalaldehyde (OPA) in combination with 3- mercaptopropionic acid (3-MPA).        Methods: Overall, 107 neonates and babies suspected of having metabolic disorder were enrolled in this study. The level of amino acids in plasma samples was analyzed within 65 minutes by HPLC with pre-column derivatization by OPA/3-MPA. This was a gradient RP-HPLC method that was performed using two solvents with a ratio of methanol and sodium acetate. L-norvaline internal standard was used as the reference peak for amino acids. Standard mixture of amino acids was used to determine the concentrations of amino acids.         Results: According to the values of coefficient of variation obtained for each amino acid, the results indicated a good chromatographic separation of amino acids by this method. The use of OPA/3-MPA derivative reagent increased the efficiency and resolution of amino acids chromatographic separation.       Conclusion: Due to simple preparation and accurate assessment, determination of plasma amino acids using OPA/3-MPA derivatives and RP-HPLC is a suitable method in many clinical samples.