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.
Compound-specific isotope analysis of nitrogen (δ 15 N) in amino acids (CSIA-AA) has significantly contributed to environmental sciences such as anthropology, biogeochemistry, and ecology. Several methods exist for determining δ 15 N of amino acids (AAs). Although these methods have their own strength and weakness, they have not been intercalibrated yet, especially for biological samples with matrices. To address this issue, we systematically compared AA δ 15 N values among three methods using gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS), preparative liquid chromatography (LC) separation followed by elemental analyzer/ IRMS (LC Â EA/IRMS), and LC separation followed by GC/C/IRMS (LC Â GC/C/IRMS). The δ 15 N values of glutamic acid (δ 15 N Glu ) and phenylalanine (δ 15 N Phe ) in fish muscle, two crucial AAs for estimating the trophic positions (TPs) of organisms, were compared among methods. Although a significant difference in fish muscle δ 15 N Glu values was found among the three analytical methods, their δ 15 N Glu and δ 15 N Phe values were fairly consistent between all pairs of methods (n = 8, R 2 = 0.9968 for GC/C/IRMS vs. LC Â GC/C/IRMS; 0.9936 for LC Â EA/IRMS vs. LC Â GC/C/IRMS; and 0.9912 for GC/C/IRMS vs. LC Â EA/IRMS), which resulted in similar TP estimates among the methods. Thus, the results provide empirical validation that the CSIA-AA is comparable among different methods in interdisciplinary research fields. We also highlighted some critical features of each of the three analytical methods that can be used as a guideline for future CSIA-AA research.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.