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

Rationale Nitrogen isotopic compositions (delta N-15) 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 delta N-15 measurements of AAs (delta N-15-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 delta N-15 of the produced N2O via purge-and-trap continuous-flow isotope ratio mass spectrometry (PT/CF/IRMS). Results The cross-plots of delta N-15 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 parts per thousand-0.67 parts per thousand for Phe standards and 0.27 parts per thousand-0.35 parts per thousand for Glu standards. Our method and the GC/C/IRMS approach produced equivalent delta N-15 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 delta N-15-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 delta N-15 of N2O using PT/CF/IRMS. This method will help promote the use of delta N-15-AA in important studies of N cycling and trophic ecology in a wide range of research areas.

Automated summary

The study introduces a method for high-precision delta N-15 measurements of amino acids, optimized for certain sources and trophic amino acids. The method involves purification and separation via high-pressure ion-exchange chromatography, chemical conversion of amino acids to nitrous oxide, and determination of delta N-15 via continuous-flow isotope ratio mass spectrometry. The results show high accuracy and precision, making this method a reliable alternative for delta N-15-AA measurement.