Development of an enantioselective three-dimensional HPLC system for the determination of alanine, valine, isoleucine, allo-isoleucine and leucine in human plasma and urine

A three-dimensional (3D) HPLC system was designed/developed for the discriminative determination of aliphatic chiral amino acids, namely, alanine (Ala), valine (Val), isoleucine (Ile), allo-Ile (aIle) and leucine (Leu), in human physiological fluids. These aliphatic amino acid enantiomers are expected to be new physiologically active molecules and/or biomarkers in mammals. Among these aliphatic amino acids, the structural chain isomers of Leu (Ile/aIle/Leu) have similar chemical-physical properties, and the analytical method for these aliphatic amino acids is required to be highly enantio- and chemo-selective. In the present study, a reversed-phase column (Singularity RP18, first dimension) and a mixed-mode column (Singularity MX-103, second dimension) were utilized to separate these aliphatic amino acids as their scalemic mixtures, then chiral separations were performed using a Pirkle-type enantioselective column (Singularity CSP-001S) in the third dimension. By using the 3D-HPLC system, these aliphatic amino acid enantiomers were completely discriminated, and the analysis of these aliphatic chiral amino acids in the human plasma and urine was successfully carried out. The obtained amounts of the aliphatic amino acids (and %D value, the percentage of D-form to D + L forms) in the human plasma were 2.1 µM (0.5%) for D-Ala, and trace/not-detected for the other D-forms. In human urine, the values were 102.0 µM (24.7%) for D-Ala, 2.0 µM (3.4%) for D-Val, 2.3 µM (10.8%) for D-aIle (%D value of D-aIle was calculated using D-aIle and L-Ile) and 3.3 µM (5.5%) for D-Leu. The present 3D-HPLC system is a powerful and well validated tool for the simultaneous determination of aliphatic amino acid enantiomers, and further biological and clinical studies are expected.