Development of a highly selective two-dimensional HPLC-MS/MS system for the screening of intrinsic D-amino acids in human clinical samples

D-Amino acids, minor stereoisomers of chiral amino acids in living beings, have recently been discovered in mammals and several D-forms (especially, D-aspartate (Asp) and D-serine (Ser)) have been clarified to have physiological functions.  These D-amino acids have also been paid attention as new biomarkers for diseases since the significant alterations of their intrinsic levels were reported.  However, the amounts of D-enantiomers are extremely trace, and the determination of chiral amino acids is frequently interfered by unknown compounds even using the high selective two-dimensional (2D) HPLC or LC-MS/MS methods.  Accordingly, development of the method having higher selectivity has been required for the precise screening of various intrinsic D-amino acids.  Therefore, in the present study, a highly selective 2D HPLC-MS/MS system combining reversed-phase separation, enantioselective separation and detection by an MS/MS was developed to analyze a wide variety of intrinsic amino acids in human physiological fluids.

Human plasma and urine were appropriately deproteinized or diluted prior to the pre-column derivatization of the amino acids with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F).  As the target analytes, alanine (Ala), Asp, glutamate, leucine, lysine, methionine, phenylalanine, proline (Pro), Ser and valine were selected considering the presence of D-forms in mammals.  For the development of the 2D HPLC-MS/MS system, the conditions of reversed-phase separation (1^st dimension), enantioselective separation (2^nd dimension) and also the MS/MS conditions were thoroughly investigated.  As a result, all of the target NBD-amino acids were separated by a C18 column (KSAARP, 1.0 x 500 mm) within 450 min using aqueous solutions containing 10-28% acetonitrile (MeCN) and 0.05% trifluoroacetic acid.  The amino acid fractions were automatically collected and introduced to the next dimension.  All of the target enantiomers were further separated into their D and L-forms by an original enantioselective column (KSAACSP-001S, 1.5 x 250 mm) within about 30 min using the mixtures of methanol and MeCN containing formic acid as mobile phases.  Using the developed system, the intrinsic amounts of chiral amino acids in human clinical samples were successfully determined without interferences.  As a result, trace levels of D-Ala, D-Pro and D-Ser were observed in the plasma, and the ratios of D-enantiomers (D/(D+L) x 100) were 0.6, 0.2 and 1.6%, respectively.  In the urine, all of the target D-enantiomers were detected and the ratios of D-forms were 0.7-36.7%.  The %D values of target analytes were confirmed using the mobile phases of different compositions, and approximately equal values were observed.  Further clinical applications are ongoing.