Establishment and Application of an Automated Chiral Two-dimensional High Performance Liquid Chromatography Method for Bio-analysis of D-Acidic Amino Acids

The physiological and bio-marker function of D-acidic amino acids is now becoming the focus in metabolomics study and new drug discovery. A fully automated two-dimensional high performance liquid chromatography (2D-HPLC) was established by using silica-based monolithic ODS column as the first dimension column with acetonitrile-trifluoro acetic acid-water (9:0.05:91, V/V) as the mobile phase, micro Chiralpak QD-1-AX column as the enantiomer separation column with 10 mM citric acid in methanol- acetonitrile (50:50, V/V) as the mobile phase for the second dimension separation, and 4-fluoro-7-nitro-2,1,3- benzoxadiazole (NBD-F) as the fluorometrical derivative reagent. The method has a higher separation efficiency (Rs > 2.5) and a higher detection sensitivity (LOD = 1 fmol) than that of existing methods in the determination of acidic amino acids enantiomers, meanwhile, an online confirmation of the enantiomers amounts were achieved by this method. The recoveries were around 97%–104%, and the RSDs of intra-day and inter-day were less than 5%. Furthermore, by analyzing the aging model senescence accelerated mouse prone 1 (SAMP1) mice which have low immunocompetence, the amounts of D-aspartic acid in thymus and spleen were determined as (206 ± 18) nmol g−1 and (264 ± 21) nmol g−1, respectively. In the experiment, it was found for the first time that there was an obvious increasing trend of D-aspartic acid (p < 0.01) in thymus and spleen of SAMP1 mice compared to senescence accelerated mouse resistant 1 (SAMR1) mice.

In this 2D-HPLC system, NBD-Asp and NBD-Glu were firstly separated in a reversed-phase model, their fractions were collected in the different loops of multi-loop valve, respectively, then switched to the QD-1-AX column immediately for the enantiomeric separation at different time. NBD-Asp and NBD-Glu enantiomers were completely separated (Rs > 2.5) without interference.Figure optionsDownload as PowerPoint slide