High‐precision measurement of phenylalanine and glutamic acid δ¹⁵N by coupling ion‐exchange chromatography and purge‐and‐trap continuous‐flow isotope ratio mass spectrometry

Abstract

Rationale Nitrogen isotopic compositions (δ15N) of source and trophic amino acids (AAs) are crucial tracers of N sources and trophic enrichments in diverse fields, including archeology, astrobiochemistry, ecology, oceanography, and paleo‐sciences. The current analytical technique using gas chromatography‐combustion‐isotope ratio mass spectrometry (GC/C/IRMS) requires derivatization, which is not compatible with some key AAs. Another approach using high‐performance liquid chromatography‐elemental analyzer‐IRMS (HPLC/EA/IRMS) may experience coelution issues with other compounds in certain types of samples, and the highly sensitive nano‐EA/IRMS instrumentations are not widely available. Methods We present a method for high‐precision δ15N measurements of AAs (δ15N‐AA) optimized for canonical source AA‐phenylalanine (Phe) and trophic AA‐glutamic acid (Glu). This offline approach entails purification and separation via high‐pressure ion‐exchange chromatography (IC) with automated fraction collection, the sequential chemical conversion of AA to nitrite and then to nitrous oxide (N2O), and the final determination of δ15N of the produced N2O via purge‐and‐trap continuous‐flow isotope ratio mass spectrometry (PT/CF/IRMS). Results The cross‐plots of δ15N of Glu and Phe standards (four different natural‐abundance levels) generated by this method and their accepted values have a linear regression slope of 1 and small intercepts demonstrating high accuracy. The precisions were 0.36‰–0.67‰ for Phe standards and 0.27‰–0.35‰ for Glu standards. Our method and the GC/C/IRMS approach produced equivalent δ15N values for two lab standards (McCarthy Lab AA mixture and cyanobacteria) within error. We further tested our method on a wide range of natural sample matrices and obtained reasonable results. Conclusions Our method provides a reliable alternative to the current methods for δ15N‐AA measurement as IC or HPLC‐based techniques that can collect underivatized AAs are widely available. Our chemical approach that converts AA to N2O can be easily implemented in laboratories currently analyzing δ15N of N2O using PT/CF/IRMS. This method will help promote the use of δ15N‐AA in important studies of N cycling and trophic ecology in a wide range of research areas.

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

“…Recently, Riekenberg et al (2020) have examined a combined oxidation-reduction reactor on GC/C/IRMS for CSIA-AA. Furthermore, Zhang et al (2021) have established a method using purge-and-trap continuous-flow isotope ratio mass spectrometry. In their method, the target AAs can be converted to nitrous oxide (N 2 O) rather than dinitrogen (N 2 ) to determine their δ 15 N values.…”

Different Nitrogen Sources Fuel Symbiotic Mussels at Cold Seeps

“…The δ 15 N of AAs was measured by coupling instrumental analysis (ion chromatography and Precon-GC-IRMS) and oxidationreduction of AAs (Zhang et al, 2021;Wang et al, 2022). Briefly, AAs were separated using an IC system (Dionex, ICS-5000 + , Thermo Scientific, USA) equipped with an autosampler (Dionex, AS-AP) with an adjustable injection volume and coupled to an automated fraction collector (Dionex, UltiMate 3000).…”

“…For the separation of AAs, the IC was equipped with an IC column (Dionex™ AminoPac™PA10, Thermo Scientific, USA), and a solvent ramp programme was used. At the end of each sample, 1 M sodium acetate was used to wash the column to remove any residue (Zhang et al, 2021). IC procedural blanks were collected after AA fractions, and were re-injected in IC system to ensure that all AAs has been collected completely.…”

“…Meanwhile, the solvent blanks were also collected between Phe and Glu. These blanks were used to correct the isotopic interference from any nitrogenous compounds introduced by the analytical procedure up to this point that could be potentially converted to nitrite by NaClO [more details showed in Zhang et al (2021)] Purified AAs were oxidized to NO 2 − by Strecker degradation (Schonberg and Moubacher, 1952;Zhang and Altabet, 2008). The next step was the conversion of NO 2 − to N 2 O; here, NH 2 OH•HCl was used as an alternative reductant (Bothner-By and Friedman, 1952;Liu et al, 2014).…”

“…Standard AAs (Merck, USA; Glu, USGS40, Gly, USGS64, the Reston Stable Isotope Laboratory of the U.S. Geological Survey) were treated with the same protocol as the samples and were used to calibrate the δ 15 N value of the AAs. The results from this method were compared with the general GC-IRMS analysis with two lab standards (McCarthy Lab AA mixture and cyanobacteria) and only 0.6‰ deviations were found (Zhang et al, 2021).…”