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

We developed a new method for stable carbon and nitrogen isotopic (δ¹³C and δ¹⁵N) 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 δ¹³C and δ¹⁵N 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 δ¹³C and δ¹⁵N 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 ¹⁵N-depletion, up to 20‰, in d-glutamic acid relative to its l-counterpart. The first example, to our knowledge, of δ¹³C and δ¹⁵N analyses of underivatized AA enantiomers is expected to contribute to various research areas in the future.