High-precision measurement of phenylalanine and glutamic acid δ¹⁵ N by coupling ion-exchange chromatography and purge-and-trap continuous-flow isotope ratio mass spectrometry

Rationale: Nitrogen isotopic compositions (δ¹⁵ N) 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 δ¹⁵ N measurements of AAs (δ¹⁵ N-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 (N₂ O), and the final determination of δ¹⁵ N of the produced N₂ O via purge-and-trap continuous-flow isotope ratio mass spectrometry (PT/CF/IRMS).

Results: The cross-plots of δ¹⁵ N 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 δ¹⁵ N 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 δ¹⁵ N-AA measurement as IC or HPLC-based techniques that can collect underivatized AAs are widely available. Our chemical approach that converts AA to N₂ O can be easily implemented in laboratories currently analyzing δ¹⁵ N of N₂ O using PT/CF/IRMS. This method will help promote the use of δ¹⁵ N-AA in important studies of N cycling and trophic ecology in a wide range of research areas.