Enantioselective and multi-dimensional HPLC analysis of amino acids as their free/protein bound forms for the screening of novel biomarkers

Kenji Hamase, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan (hamase@phar.kyushu-u.ac.jp)

Enantioselective analysis of amino acids is gathering high attention recently for the screening of new physiologically active substances and/or biomarkers in mammals.  However, the amounts of these chiral amino acids, especially the minor D-forms are extremely low, and the sensitive and selective analytical methods are essential.  In the present study, therefore, multi-dimensional chiral HPLC systems combining a reversed-phase column, an anion-exchange/mixed-mode column and an enantioselective column have been designed and applied to the analyses of biological samples including human clinical samples.

Prior to the HPLC analysis, amino acids were derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) and detected by a fluorescence detector and also by a tandem mass spectrometer.  Following the derivatization, NBD-amino acids were injected into the three-dimensional (3D) HPLC system and separated by a reversed-phase column (KSAARP) as a first dimension.  The target fractions were collected to a multi-loop device, and introduced into the second dimension (KSAAAX or KSAAMX columns), and the target amino acids were separated again as their scalemic mixtures.  The target fractions were transferred into the third dimension where D and L amino acids were separated by the enantioselective columns (KSAACSP).  The second dimension could be omitted when the selectivity is sufficient or the tandem mass spectrometer is used as a detector.

By using the present multi-dimensional HPLC concept, chiral amino acids in a variety of biological samples including human serum/plasma and urine were analyzed.  In the human serum/plasma, small amounts of D-Ala, D-Asn, D-Pro and D-Ser were observed, and the amounts of these D-amino acids, especially D-Asn and D-Ser have clear correlation to the kidney function.  In the urine, the presence of non-negligible amounts of various D-amino acids (D-Ala, D-Arg, D-Asn, D-Asp, D-Glu, D-allo-Ile, D-Ser and D-allo-Thr) were shown.  Concerning the protein bound type D-amino acid residues, proteins were hydrolyzed in the presence of deuterium oxide plus deuterium chloride.  The resultant amino acids were derivatized with NBD-F, and analyzed by a 2D HPLC-MS/MS system.  In a variety of proteins, several D-amino acid residues were observed.  In ovalbumin, small but clear peak of D-Ser was detected, and D-Asn/Asp residue was found in mouse lysozyme.  The present multi-dimensional HPLC concept is useful for the determination of trace levels of D-amino acids in biological samples, and further applications are in progress.

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Abstract Reference & Short Personal Biography of Presenting Author

Kenji HAMASE graduated from The University of Tokyo in 1991, and received a Master of Pharmacy in 1993.  He then obtained his Ph.D. degree in Pharmaceutical Sciences from The University of Tokyo in 1996.  Throughout the doctoral course, he obtained research fellowships from the Japan Society for the Promotion of Science for Young Scientists (1993-1996).  Subsequently, he began his academic carrier as an Assistant Professor at Kyushu University, and was promoted to an Associate Professor in 2001, and to the full Professor in 2016.  He is a Program Officer of Research Center for Science Systems, Japan Society for the Promotion of Science (2018-2020).  He received The Japan Society for Analytical Chemistry Award for Young Scientists in 2003, and The Pharmaceutical Society of Japan Award for Young Scientists in 2006.  His current research interests focus on the development of analytical methods for chiral amino acids and the study on their physiological functions, diagnostic values and the design of functional foods.