Separation of the enantiomers of underivatized amino acids by using serially connected dual column high-performance liquid chromatography-tandem mass spectrometry

In this article, a serially connected dual column liquid chromatography-tandem mass spectrometry (LC-MS/MS) method is described for the simultaneous separation and enantioseparation of proteinogenic amino acids. For this purpose, different achiral and chiral stationary phases (CSP) and mobile phase compositions have been tested. As a result of the optimization studies, the best enatioseparation for amino acids were achieved with a combination of zwitterionic and crown ether stationary phases using a gradient of two mobile phases: A (water:TFA 99.5:0.5, % v/v) and B (acetonitrile:ethanol:TFA 85:15:0.5, % v/v/v). The developed method provided simultaneous enantioseparation of all proteinogenic amino acids under this study including isomeric and isobaric ones except for proline. The method was successfully applied to human lung adenocarcinoma cells (A549) and healthy human lung epithelial cells (BEAS-2B) cultivated with D-amino acid containing cocktails in order to evaluate D-amino acids transfer rate in normal and cancer lines. TheD/L amino acid ratios were different in cancer and normal cell lines cultivated as mentioned above for aspartic acid, cysteine, methionine, phenylalanine, and serine.