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 13 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 13 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 13 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 13 C values of AAs in H. bleekeri varied by as much as 30‰ with glycine being most enriched in 13 C. Conclusions The consistency between the δ 13 C values of reference and processed AAs demonstrates that the experimental procedure generates accurate δ 13 C values unaffected by fractionation effects and contamination. This method is therefore suitable for δ 13 C analysis of biological samples with higher precision than conventional approaches. We propose this new method as a tool to measure δ 13 C values of AAs in biological, ecological and biogeochemical studies.