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 δ13C 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 δ13C values of AAs were measured by EA/IRMS. Results The complete isolation of all 15 AAs in the standard mixture was achieved. The δ13C 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 δ13C values of AAs in H. bleekeri varied by as much as 30‰ with glycine being most enriched in13C. Conclusions The consistency between the δ13C values of reference and processed AAs demonstrates that the experimental procedure generates accurate δ13C values unaffected by fractionation effects and contamination. This method is therefore suitable for δ13C analysis of biological samples with higher precision than conventional approaches. We propose this new method as a tool to measure δ13C values of AAs in biological, ecological and biogeochemical studies.