On-Line Three-Dimensional High-Performance Liquid Chromatographic System for the Comprehensive and Enantio/Stereoselective Determination of All Proteinogenic Amino Acids in Human Plasma and Urine

An online three-dimensional (3D) HPLC system was designed/developed for the comprehensive determination of all proteinogenic amino acid enantiomers, including allo-isoleucine, allo-threonine (aThr), and 3 cystine (Cys) stereoisomers (dd/dl/ll-forms), in real-world samples with one injection. d-Amino acids are candidates for physiologically active molecules and/or biomarkers, and the simultaneous determination of all the proteinogenic chiral amino acids is expected. However, the amounts of d-amino acids in biological samples are usually much lower than those of other intrinsic substances, including l-amino acids and peptides; thus, the analytical method for the determination of d-amino acids requires high selectivity. In the present study, a reversed-phase column (Singularity RP18) and a mixed-mode column (Singularity MX-103) were utilized to separate all of the proteinogenic amino acids as their scalemic mixtures, and a Pirkle-type enantioselective column (Singularity CSP-001S) was used for the chiral separations. By using the 3D-HPLC system, all of the proteinogenic amino acids and allo-forms were enantioselectively separated, and the determination of these chiral amino acids in human plasma and urine was carried out without interference from intrinsic substances. As a result, 5 d-amino acids (alanine, asparagine, aThr, proline, and serine) were clearly determined in human plasma, and the peaks of 19 d-amino acids (i.e., all the target d-amino acids except for d-isoleucine and dd-Cys) and dl-Cys were observed in human urine. The present 3D-HPLC system achieved the simultaneous and selective determination of all proteinogenic amino acid enantiomers, including 2 allo-forms and 3 Cys stereoisomers, in biological matrices for the first time, to the best of our knowledge.