Application of a porous graphitic carbon column to carbon and nitrogen isotope analysis of underivatized individual amino acids using high-performance liquid chromatography coupled with elemental analyzer/isotope ratio mass spectrometry.

Rationale: Isolation of underivatized amino acids (AAs) using high-performance liquid chromatography (HPLC) is becoming a popular method for carbon (δ ¹³ C) and nitrogen isotope (δ ¹⁵ N) analyses of AAs because of the high analytical precision and for performing dual-isotope analysis. However, some AAs in natural samples, especially small, hydrophilic AAs, are not suitably separated using reversed-phase columns (e.g., C18) and ion-exchange columns (e.g., Primesep A).
Methods: We developed a new method for HPLC using a porous graphitic carbon column for the separation of nine hydrophilic AAs. After purification, δ ¹³ C and δ ¹⁵ N values of AAs were determined using elemental analyzer/isotope ratio mass spectrometry (EA/IRMS). We demonstrated the application of this method by determining δ ¹³ C and δ ¹⁵ N values of individual hydrophilic AAs in a biological sample, the muscle of blue mackerel (Scomber australasicus).
Results: Chromatographically, the baseline separation of hydrophilic AAs was achieved in both the standard mixture and the biological sample. We confirmed that δ ¹³ C and δ ¹⁵ N values of AA standards remained unchanged during the whole experimental procedure. The δ ¹³ C values of AAs in mackerel muscle are also in good agreement with the values obtained using another verified method for δ ¹³ C analysis.
Conclusions: The good separation performance of hydrophilic AAs and the reliability of δ ¹³ C and δ ¹⁵ N analyses of individual AAs using the porous graphite column offer a significant advantage over conventional settings. We suggest that, in the future, the HPLC × EA/IRMS method can be used for reliable δ ¹³ C and δ ¹⁵ N analyses of AAs in natural samples.
(© 2023 The Authors. Rapid Communications in Mass Spectrometry published by John Wiley & Sons Ltd.)