The relative influence of derivatization and normalization procedures on the compound-specific stable isotope analysis of nitrogen in amino acids

Abstract

Rationale: Within the last decade, applications of compound-specific stable isotope analysis of nitrogen (δ¹⁵ N values) in amino acids (CSIA-AA) have developed rapidly, particularly within organismal ecology. Unlike with bulk stable isotope analysis (BSIA), the reproducibility of δ¹⁵ N-AA measurements has not been critically assessed. Two primary concerns include the diversity of techniques available for the derivatization of amino acids prior to analysis by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) and the myriad of standardization practices and quality assurance procedures used across studies.

Methods: We examined the relative effect of three normalization procedures, (1) internal reference calibration, (2) compound-specific calibration, and (3) scale-normalization, on the accuracy and precision of δ¹⁵ N-AA measurements by GC/C/IRMS and the comparability of δ¹⁵ N-AA measurements by two derivatization techniques, methoxycarbonylation-esterification and acetylation-esterification, across a range of organisms.

Results: The overall accuracy and precision of δ¹⁵ N-AA measurements were improved following both compound-specific calibration and scale-normalization, as was the comparability of δ¹⁵ N-AA measurements of individual amino acids between derivatization techniques across organisms. The mean difference of scale-normalized δ¹⁵ N-AA values across all organisms between the two derivatization techniques was 0.19‰, much less than the typical analytical error associated with δ¹⁵ N-AA measurements (±1‰)_. CONCLUSIONS: Adoption of standardized calibration procedures will be important to establishing reproducibility in δ¹⁵ N-AA measurements, particularly across derivatization techniques. It is both technically practical and desirable for users of CSIA-AA to adopt practices in quality control and assessment similar to those outlined for BSIA, including the compound-specific calibration of δ¹⁵ N-AA values, followed by scale-normalization. Copyright © 2017 John Wiley & Sons, Ltd.