Integrative assessment of amino acid nitrogen isotopic composition in biological tissue samples determined by GC/C/IRMS, LC × EA/IRMS, and LC × GC/C/IRMS

Abstract

AbstractCompound‐specific isotope analysis of nitrogen (δ15N) in amino acids (CSIA‐AA) has significantly contributed to environmental sciences such as anthropology, biogeochemistry, and ecology. Several methods exist for determining δ15N 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 δ15N 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 × EA/IRMS), and LC separation followed by GC/C/IRMS (LC × GC/C/IRMS). The δ15N values of glutamic acid (δ15NGlu) and phenylalanine (δ15NPhe) 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 δ15NGlu values was found among the three analytical methods, their δ15NGlu and δ15NPhe values were fairly consistent between all pairs of methods (n = 8, R2 = 0.9968 for GC/C/IRMS vs. LC × GC/C/IRMS; 0.9936 for LC × EA/IRMS vs. LC × GC/C/IRMS; and 0.9912 for GC/C/IRMS vs. LC × 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.