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
Yuchen Sun, Nanako O. Ogawa, Naoto F. Ishikawa, Thomas M. Blattmann, Yoshinori Takano, Naohiko Ohkouchi
Abstract
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.