A method for stable carbon isotope measurement of underivatized individual amino acids by multi-dimensional high-performance liquid chromatography and elemental analyzer/isotope ratio mass spectrometry.

Rationale: To achieve better precision and accuracy for δ C analysis of individual amino acids (AAs), we have developed a new analytical method based on multi-dimensional high-performance liquid chromatography (HPLC) and elemental analyzer/isotope ratio mass spectrometry (EA/IRMS). Unlike conventional methods using gas chromatography, this approach omits pre-column chemical derivatization, thus reducing systematic errors associated with the isotopic measurement.

Methods: The separation and isolation of individual AAs in a standard mixture containing 15 AAs and a biological sample, spear squid (Heterololigo bleekeri) were performed. AAs were isolated using an HPLC system equipped with a reversed-phase column and a mixed-mode column and collected using a fraction collector. After the chromatographic separation and further post-HPLC purification, the δ C values of AAs were measured by EA/IRMS.

Results: The complete isolation of all 15 AAs in the standard mixture was achieved. The δ C values of these AAs before and after the experiment were in good agreement. Also, 15 AAs in the biological sample, H. bleekeri, were successfully measured. The δ C values of AAs in H. bleekeri varied by as much as 30‰ with glycine being most enriched in C.

Conclusions: The consistency between the δ C values of reference and processed AAs demonstrates that the experimental procedure generates accurate δ C values unaffected by fractionation effects and contamination. This method is therefore suitable for δ C analysis of biological samples with higher precision than conventional approaches. We propose this new method as a tool to measure δ C values of AAs in biological, ecological and biogeochemical studies.