Practical considerations for improved reliability and precision during determination of δ¹⁵N values in amino acids using a single combined oxidation–reduction reactor

Abstract

Rationale There has been increased interest in the measurement of δ15N values in amino acids (AAs) to gain simultaneous insight into both trophic relationships and the composition of biogeochemical sources used by producers at the base of the food web. A new combustion reactor design in gas chromatography/combustion isotope ratio mass spectrometry (GC/C‐irMS) equipment has brought to light variable outcomes in performance, highlighting the need for better information about best practices for new systems. Methods Precision for δ15N values in amino acids using the single combined oxidation–reduction reactor is improved across a sequence of analyses if the reactor is oxidized for a substantial period (2 h) and subsequently maintained throughout the sequence with 12–17 s seed oxidation before each run during GC/C‐irMS. A five‐point calibration curve using amino acids with a range of δ15N values from −2.4‰ to +61.5‰ was used in combination with a 13–15 amino acid mixture to consistently normalize measurements to internationally calibrated reference materials. Results Combining this oxidation method with normalization techniques using both internal and external standards provided a reliable throughput of ~25 samples per week. It allowed for a reproducible level of precision of <±0.5‰, n = 10 within a derivatized standard mixture across each sequence and an average sample precision of ±0.27‰ n = 3, which is lower than the analytical precision typically associated with δ15N values for amino acid analysis (<±1‰). Conclusions A few practical considerations regarding oxidation and conditioning of the combustion reactor allow for increased sequence capacity with the single combined oxidation–reduction reactor. These considerations combined with normalization techniques result in a higher throughput and reduced analytical error during the measurement of δ15N values in amino acids.

Reconstructing the diet, trophic level and migration pattern of mysticete whales based on baleen isotopic composition

“…High δ 15 N Gly values observed for the humpback (approx. 0‰; figure 4b) also mirror δ 15 N Gly values found in small fish migrating from estuarine into coastal waters where elevated δ 15 N Gly values have been previously observed, such as herring [45].…”

“…The method is a modified version of the amino acid analysis method by Chikaraishi et al . [ 37 ] as described in Riekenberg et al [ 45 ]. In short, at the Royal Netherlands Institute for Sea Research (NIOZ) samples were hydrolysed, derivatized into N-pivaloyl/isopropyl (NPiP) derivatives and analysed in duplicate with a Trace 1310 gas chromatograph coupled to a Delta V Advantage isotope ratio mass spectrometer (Thermo Scientific, Bremen) via an IsoLink II and Conflo IV.…”

“…In short, at the Royal Netherlands Institute for Sea Research (NIOZ) samples were hydrolysed, derivatized into N-pivaloyl/isopropyl (NPiP) derivatives and analysed in duplicate with a Trace 1310 gas chromatograph coupled to a Delta V Advantage isotope ratio mass spectrometer (Thermo Scientific, Bremen) via an IsoLink II and Conflo IV. Details about the temperature ramp, programme settings and normalization procedures are provided in Riekenberg et al [ 45 ]. We report δ 15 N values for 12 amino acids including alanine (Ala), aspartic acid (Asp), Glu, Gly, isoleucine, leucine, lysine, Phe, serine, Thr, tyrosine and valine.…”

Reconstructing the diet, trophic level, and migration pattern of Mysticete whales based on baleen isotopic composition

“…Since baleen grows continuously throughout a whale's life the material closest to the gingiva reflects the most recently produced layer with the material farther away from the gums reflecting increasingly older periods of the whale's foraging history. Subsampled material (3-5 mg) for analysis of δ 15 N of amino acids were selected based on the variation within the bulk δ 15 (42). We report δ 15 N values for 12 amino acids including alanine, aspartic acid, Glu, Gly, isoleucine, leucine, lysine, Phe, serine, Thr, tyrosine, and valine.…”

Stable nitrogen isotope analysis of amino acids as a new tool to clarify complex parasite-host interactions within marine food webs

“…For CSIA-AA analysis, dried and homogenized tissue (2-5 mg) was lipid extracted, acid hydrolyzed, derivatized to pivaloyl-isopropyl esters and analysed for δ 15 N of AAs via gas chromatography-combustion isotope mass spectrometry using a Thermo Trace 1310 GC attached to Delta V Advantage isotope ratio mass spectrometer via an Isolink 2 following the method presented in Riekenberg et al (2020). δ 15 N values for 14 AAs are reported: alanine (Ala), aspartic acid (Asp), glutamic acid (Glu), glycine (Gly), leucine (Leu), lysine (Lys), isoleucine (Ile), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tyrosine (Tyr), and valine (Val).…”