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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.<\/jats:p>","DOI":"10.1021\/acs.analchem.6c03014","type":"journal-article","created":{"date-parts":[[2026,8,22]],"date-time":"2026-08-22T09:57:17Z","timestamp":1787392637000},"page":"25166-25173","source":"Crossref","is-referenced-by-count":1,"title":["On-Line Three-Dimensional\nHigh-Performance Liquid\nChromatographic System for the Comprehensive and Enantio\/Stereoselective\nDetermination of All Proteinogenic Amino Acids in Human Plasma and\nUrine"],"prefix":"10.1021","volume":"98","author":[{"given":"Yuri","family":"Nagata","sequence":"first","affiliation":[{"name":"Kyushu University , , 3-1-1 Maidashi , Higashi-ku, ,","place":["Fukuoka, Japan, 812-8582"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Takeyuki","family":"Akita","sequence":"additional","affiliation":[{"name":"Kyushu University , , 3-1-1 Maidashi , Higashi-ku, ,","place":["Fukuoka, Japan, 812-8582"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chiharu","family":"Ishii","sequence":"additional","affiliation":[{"name":"Kyushu University , , 3-1-1 Maidashi , Higashi-ku, ,","place":["Fukuoka, Japan, 812-8582"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Masataka","family":"Ikeda","sequence":"additional","affiliation":[{"name":"Kyushu University , , 3-1-1 Maidashi , Higashi-ku, ,","place":["Fukuoka, Japan, 812-8582"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Masashi","family":"Mita","sequence":"additional","affiliation":[{"name":"KAGAMI, Inc. , 7-7-15 Saito-asagi , , ,","place":["Ibaraki, Osaka, Japan, 567-0085"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tomomi","family":"Ide","sequence":"additional","affiliation":[{"name":"Kyushu University , , 3-1-1 Maidashi , Higashi-ku, ,","place":["Fukuoka, Japan, 812-8582"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6140-4787","authenticated-orcid":true,"given":"Kenji","family":"Hamase","sequence":"additional","affiliation":[{"name":"Kyushu University , , 3-1-1 Maidashi , Higashi-ku, ,","place":["Fukuoka, Japan, 812-8582"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,8,21]]},"reference":[{"key":"2026090306464521800_cit1","doi-asserted-by":"publisher","first-page":"11","DOI":"10.15583\/jpchrom.2024.001","article-title":"Development of Two-Dimensional LC-MS\/MS Methods for\nHighly-Selective Analysis of Chiral Amino Acids and the Evaluation\nof Their Intrinsic Amounts in Mammals and Stereoinversion in Proteins","volume":"45","author":"Ishii","year":"2024","journal-title":"Chromatography"},{"key":"2026090306464521800_cit2","doi-asserted-by":"publisher","first-page":"103","DOI":"10.1023\/A:1016031014871","article-title":"d-Amino Acids in Living Higher Organisms","volume":"32","author":"Fujii","year":"2002","journal-title":"Orig.\nLife Evol. 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