Development and validation of a rapid, selective, and sensitive LC-MS/MS method for simultaneous determination of d and l-amino acids in human serum: application to the study of hepatocellular carcinoma

A validated liquid chromatography-tandem mass spectrometry method was developed for the simultaneous determination of d- and l-amino acids in human serum. Under the optimum conditions, except for dl-proline, l-glutamine, and d-lysine, the enantioseparation of the other 19 enantiomeric pairs of proteinogenic amino acids and nonchiral glycine was achieved with a CROWNPAK CR-I(+) chiral column within 13 min. The lower limits of quantitation for l-amino acids (including glycine) and d-amino acids were 5-56.25 mu M and 0.625-500 nM, respectively, in human serum. The intraday precision and interday precision for all the analytes were less than 15%, and the accuracy ranged from -12.84% to 12.37% at three quality control levels. The proposed method, exhibiting high rapidity, enantioresolution, and sensitivity, was successfully applied to the quantification of d- and l-amino acid levels in serum from hepatocellular carcinoma patients and healthy individuals. The serum concentrations of l-arginine, l-isoleucine, l-aspartate, l-tryptophan, l-alanine, l-methionine, l-serine, glycine, l-valine, l-leucine, l-phenylalanine, l-threonine, d-isoleucine, d-alanine, d-glutamate, d-glutamine, d-methionine, and d-threonine were significantly reduced in the hepatocellular carcinoma patients compared with the healthy individuals (P < 0.01). d-Glutamate and d-glutamine were identified as the most downregulated serum markers (fold change greater than 1.5), which deserves further attention in hepatocellular carcinoma research.