Measurement of 15N enrichment of glutamine and urea cycle amino acids derivatized with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate using liquid chromatography–tandem quadrupole mass spectrometry

Abstract

6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) is an amino acid-specific derivatizing reagent that has been used for sensitive amino acid quantification by liquid chromatography–tandem quadrupole mass spectrometry (LC–MS/MS). In this study, we aimed to evaluate the ability of this method to measure the isotopic enrichment of amino acids and to determine the positional 15N enrichment of urea cycle amino acids (i.e., arginine, ornithine, and citrulline) and glutamine. The distribution of the M and M+1 isotopomers of each natural AQC–amino acid was nearly identical to the theoretical distribution. The standard deviation of the (M+1)/M ratio for each amino acid in repeated measurements was approximately 0.1%, and the ratios were stable regardless of the injected amounts. Linearity in the measurements of 15N enrichment was confirmed by measuring a series of 15N-labeled arginine standards. The positional 15N enrichment of urea cycle amino acids and glutamine was estimated from the isotopic distribution of unique fragment ions generated at different collision energies. This method was able to identify their positional 15N enrichment in the plasma of rats fed 15N-labeled glutamine. These results suggest the utility of LC–MS/MS detection of AQC–amino acids for the measurement of isotopic enrichment in 15N-labeled amino acids and indicate that this method is useful for the study of nitrogen metabolism in living organisms.