Analysis of L- and D-Amino Acids Using UPLC

Abstract

With the exception of glycine, α-amino acids are optically active, and two optical isomers (L- and D-) of each amino acid can be formed. Recent developments of analytical techniques have revealed that several free D-amino acids such as D-aspartate, D-serine and D-alanine exist in many kinds of organism including human and have biologically important roles. D-Aspartate regulates reproductive activity in animals and humans. D-Serine serves as a co-agonist of the N-methyl-D-aspartate receptor, which mediates glutamatergic neurotransmission. D-Alanine plays a role like osmolyte in crustaceans and mollusks. In this protocol, we describe a method for analysis of L- and D-amino acids using ultra-performance liquid chromatography (UPLC). To analyze D- and L-amino acids, the enantiomers are initially converted into diastereomers (diastereomers are stereoisomers that are not related as object and mirror image and are not enantiomers) using pre-column derivatization with o-phthaldialdehyde plus N-acylated cysteine (N-acethyl-L-cysteine or N-tert-butyloxycarbonyl-L-cysteine). The resultant derivatives are fluorescent diastereomers. This is followed by separation of the resultant fluorescent isoindol derivatives on an octadecylsilyl stationary phase using UPLC, and the fluorescence is detected by a fluorescence detector included in UPLC system. Using this method, 16 kinds of D-amino acid can be analyzed.