N^α-(5-Fluoro-2,4-dinitrophenyl)-L-leucinamide-Derivatized LC/MS/MS Analysis of Amino Acid Enantiomers in HepG2 Cells

Abstract

D-Amino acids have recently been found to be present in mammals, and their synthetic pathways require investigation. We analyzed D- and L-amino acids in HepG2 cells using the N^α-(5-fluoro-2,4-dinitrophenyl)-L-leucinamide (FDLA) chiral derivatization method for liquid chromatography-tandem mass spectrometry (LC/MS/MS). D-Asp and D-Ser were detected in HepG2 cells and their culture medium, and the concentrations of these amino acids increased in a time-dependent manner during culture. In addition, in the presence of the D-aspartate oxidase (DDO) inhibitor, 5-aminopyridine-3-carboxylic acid, the concentrations of D-Asp and D-Ser increased compared to those in the control cells. These results suggest that D-Asp and D-Ser are biosynthesized in HepG2 cells and that DDO is involved in the regulation of D-Asp and D-Ser concentration in these cells.