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

Abstract

RationaleIsolation of underivatized amino acids (AAs) using high‐performance liquid chromatography (HPLC) is becoming a popular method for carbon (δ13C) and nitrogen isotope (δ15N) 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).MethodsWe developed a new method for HPLC using a porous graphitic carbon column for the separation of nine hydrophilic AAs. After purification, δ13C and δ15N values of AAs were determined using elemental analyzer/isotope ratio mass spectrometry (EA/IRMS). We demonstrated the application of this method by determining δ13C and δ15N values of individual hydrophilic AAs in a biological sample, the muscle of blue mackerel (Scomber australasicus).ResultsChromatographically, the baseline separation of hydrophilic AAs was achieved in both the standard mixture and the biological sample. We confirmed that δ13C and δ15N values of AA standards remained unchanged during the whole experimental procedure. The δ13C values of AAs in mackerel muscle are also in good agreement with the values obtained using another verified method for δ13C analysis.ConclusionsThe good separation performance of hydrophilic AAs and the reliability of δ13C and δ15N 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 δ13C and δ15N analyses of AAs in natural samples.