The relative influence of derivatization and normalization procedures on the compound-specific stable isotope analysis of nitrogen in amino acids.
BACKGROUND
Within the last decade, applications of compound-specific stable isotope analysis of nitrogen (δ15N values) in amino acids (CSIA-AA) have developed rapidly, particularly within organismal ecology. Unlike with bulk stable isotope analysis (BSIA), the reproducibility of δ15N-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 δ15N-AA measurements by GC/C/IRMS and the comparability of δ15N-AA measurements by two derivatization techniques, methoxycarbonylation-esterification and acetylation-esterification, across a range of organisms.


RESULTS
The overall accuracy and precision of δ15N-AA measurements were improved following both compound-specific calibration and scale-normalization, as was the comparability of δ15N-AA measurements of individual amino acids between derivatization techniques across organisms. The mean difference of scale-normalized δ15N-AA values across all organisms between the two derivatization techniques was 0.19‰, much less than the typical analytical error associated with δ15N-AA measurements (±1‰).CONCLUSIONS: Adoption of standardized calibration procedures will be important to establishing reproducibility in δ15N-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 δ15N-AA values, followed by scale-normalization. Copyright © 2017 John Wiley & Sons, Ltd.
Stable Isotope Facility, University of California, Davis, 1 Shields Avenue, Davis, CA, 95616, USA.
Christopher T
http://orcid.org/0000-0003-4559-0465
CT
Yarnes
Stable Isotope Facility, University of California, Davis, 1 Shields Avenue, Davis, CA, 95616, USA.
Julian
J
Herszage
D000596:Amino Acids / Q000737:chemistry*; D000818:Animals; D002138:Calibration; D003094:Collagen / Q000737:chemistry; D004285:Dogs; D005399:Fishes; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D006868:Hydrolysis; D016014:Linear Models; D009587:Nitrogen Isotopes / Q000032:analysis*; D010944:Plants; D015203:Reproducibility of Results
D000596:Amino Acids; D009587:Nitrogen Isotopes; D003094:Collagen