Enantiomer-Specific Stable Carbon and Nitrogen Isotopic Analyses of Underivatized Individual l - and d -Amino Acids by HPLC + HPLC Separation and Nano-EA/IRMS

作者：Yuchen Sun, Thomas M. Blattmann, Yoshinori Takano, Nanako O. Ogawa, Yuta Isaji, Naoto F. Ishikawa, Naohiko Ohkouchi · 发表于：Analytical Chemistry · 年份：2024 · DOI：10.1021/acs.analchem.4c02851 · 被引用次数：9 · 研究领域：Amino Acid Enzymes and Metabolism、Isotope Analysis in Ecology、Mass Spectrometry Techniques and Applications

We developed a new method for stable carbon and nitrogen isotopic (δ 13 C and δ 15 N) analysis of underivatized amino acid (AA) enantiomers simultaneously, based on high-performance liquid chromatography (HPLC) separation and off-line isotopic measurement. l - and d -Enantiomers of each AA were isolated using a ReproSil Chiral-AA column, purified by wet chemical procedure, and analyzed for δ 13 C and δ 15 N values with a nanomol-scale elemental analyzer/isotope-ratio mass spectrometry (nano-EA/IRMS) system. We successfully achieved the separation of l - and d -enantiomers of 15 proteinogenous AAs, with all l -enantiomers eluting before respective d -enantiomers. The δ 13 C and δ 15 N values of AA enantiomers were consistent before and after HPLC separation, demonstrating that this analytical method conserves isotopic information. By coupling this column with a multidimensional HPLC system for isolating individual AAs, we analyzed l - and d -AAs in a natural sample, peptidoglycan isolated from Gram-positive bacterium Bacillus subtilis . Results show a surprisingly large 15 N-depletion, up to 20‰, in d -glutamic acid relative to its l -counterpart. The first example, to our knowledge, of δ 13 C and δ 15 N analyses of underivatized AA enantiomers is expected to contribute to various research areas in the future.