Review for "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"

Abstract

Rationale To achieve better precision and accuracy for delta C-13 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 delta C-13 values of AAs were measured by EA/IRMS. Results The complete isolation of all 15 AAs in the standard mixture was achieved. The delta C-13 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 delta C-13 values of AAs inH. bleekerivaried by as much as 30 parts per thousand with glycine being most enriched in(13)C. Conclusions The consistency between the delta C-13 values of reference and processed AAs demonstrates that the experimental procedure generates accurate delta C-13 values unaffected by fractionation effects and contamination. This method is therefore suitable for delta C-13 analysis of biological samples with higher precision than conventional approaches. We propose this new method as a tool to measure delta C-13 values of AAs in biological, ecological and biogeochemical studies.