CHROMATOGRAPHY, Vol. 35 (2014), No. 1, pp. -49

D-Amino acids are now increasingly recognized as physiologically functional molecules and biomarkers in mammals. However, the amounts of D-amino acids are extremely low in most cases, thus highly sensitive and selective analytical methods are practically essential. In the present review article, enantioselective two-dimensional high-performance liquid chromatographic (2D-HPLC) methods are introduced and their applications to the determination of D-amino acids in mammalian samples are described. For the sensitive determination, amino acid enantiomers are derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), and are determined by a fluorescence detector. The 2D-HPLC system consists of the reversed-phase non-enantioselective, but chemoselective separation of NBD-amino acids in the first dimension, and the sequential chiral separation in the second dimension. By using the system, neuroactive D-amino acids (D-Ser and D-Ala) were determined in the brain of various mammalian species and the regulation of their intrinsic amounts are also demonstrated. The physiological and diagnostic meanings of the trace amounts of D-Ser are also discussed. The 2D-HPLC systems for the acidic D-amino acids (D-Asp and D-Glu), proline analogues (D-Pro, trans- and cis-4-hydroxy-D-Pro) and hydrophilic D-amino acids (D-Arg, D-Asn, D-Asp, D-Gln, D-Glu, D-His, D-Ser, D-allo-Thr and D-Thr) are also shown.