Development of an online two-dimensional high-performance liquid chromatographic system in combination with tandem mass spectrometric detection for enantiomeric analysis of free amino acids in human physiological fluid

Chiharu Ishii, Takeyuki Akita, Masashi Mita, Tomomi Ide, Kenji Hamase

Research output: Contribution to journalArticle

13 Citations (Scopus)


An automated two-dimensional HPLC-MS/MS system was designed and developed for the highly selective determination of trace levels of D-amino acids. As the targets, frequently observed ones in mammalian physiological fluids, Ala, Asp, Glu, Leu, Pro and Ser, were selected because these D-amino acids are the potential biomarkers for the early and sensitive diagnoses of various diseases. The target analytes were derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), then isolated by a reversed-phase column in the first dimension. The NBD-amino acid fractions were automatically collected into the multi-loop device, and introduced into the enantioselective column in the second dimension to separate the D- and L-forms followed by detection by an MS/MS. The obtained resolution values of the enantiomers were 1.87–5.17 and the calibration lines, precision and accuracy were practically sufficient. By using the present 2D HPLC-MS/MS system, trace levels of D-amino acids in complex biological matrices were determined without disturbance by the intrinsic interfering compounds, and successfully applied to the analysis of the human clinical samples (plasma and urine).

Original languageEnglish
Pages (from-to)91-98
Number of pages8
JournalJournal of Chromatography A
Publication statusPublished - Oct 5 2018


All Science Journal Classification (ASJC) codes

  • Analytical Chemistry
  • Biochemistry
  • Organic Chemistry

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