Development of a Highly-Sensitive Two-Dimensional HPLC System with Narrowbore Reversed-Phase and Microbore Enantioselective Columns and Application to the Chiral Amino Acid Analysis of the Mammalian Brain

Aogu Furusho, Reiko Koga, Takeyuki Akita, Yurika Miyoshi, Masashi Mita, Kenji Hamase

Research output: Contribution to journalArticlepeer-review

Abstract

<p>A highly-sensitive two-dimensional high-performance liquid chromatographic (2D-HPLC) system with narrowbore reversed-phase and microbore enantioselective columns has been developed for the determination of alanine (Ala) and serine (Ser) enantiomers. The amounts of D-amino acids in biological samples are extremely low in most cases and improvement in the sensitivity is still required. For a highly-sensitive analysis, a narrowbore reversed-phase column, KSAARP (1.5 mm x 500 mm, having a wider ID than the microbore column) was used in the first dimension, and a microbore enantioselective column, KSAACSP-001S (1.0 mm x 250 mm, having a narrower ID than the frequently reported enantioselective columns) was used in the second dimension. The amino acids were derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole and detected by the fluorescence detectors. The 2D-HPLC system was validated using a mouse cerebrum in addition to the standard amino acids, and satisfactory calibration lines and precision results were obtained. In the mouse cerebrum, a large amount of D-Ser and a small amount of D-Ala were clearly observed. These results indicated that the present method is applicable to the determination of the trace amounts of D-amino acids in tissue samples.</p>
Original languageEnglish
Pages (from-to)83-90
Number of pages8
JournalCHROMATOGRAPHY
Volume39
Issue number2
DOIs
Publication statusPublished - 2018

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