Rapid, sensitive analysis method for determining the nitrogen stable isotope ratio of total free amino acids in soil

The amino acid-nitrogen (AA-N) isotope analysis of naturally abundant or isotope-labeled samples is indispensable for tracing nitrogen transfer in soil nitrogen biogeochemical cycling processes. Despite the usefulness of AA-N isotope analysis, the preparation methods are complex and time-consuming, and necessitate the use of toxic reagents.

We present an improved, rapid method for AA-N isotope analysis with high precision. At a high pH, AA-N was released and oxidized to N₂ O using ClO^- under vacuum. Additionally, purge-and-trap isotope ratio mass spectrometry was used to analyze N₂ O. Moreover, we investigated the effect of various factors on the N₂ O conversion process with glycine and applied the results to seven representative single-N AAs (alanine, serine, cysteine, aspartic acid, glutamic acid, leucine, and phenylalanine) and five poly-N AAs (lysine, arginine, histidine, tryptophan, and asparagine), as well as side-chain analogs, blank reagent, and other N forms.

The concentration of ClO^- and the pH were determined to be crucial factors for achieving desirable AA-N to N₂ O conversion efficiencies. Glycine-N had the highest N₂ O yield of 70%, with isotopic results consistent with those of the reference values at a high precision (within 0.5‰ for natural abundance and 0.01 atom% for ¹⁵ N-enrichment) at the nanomolar N level. Additionally, the α-NH₂ AAs were labile, and the single-N AAs were more easily converted to N₂ O than poly-N AAs. With the exception of γ-aminobutyric acid, the N₂ O conversion efficiencies of the side-chain N analogs were very low (below 5%). This method was also applicable to the ¹⁵ N analysis of the total free AAs in complex soil samples without interference from analytical blanks and other forms of N.

Our method is highly selective for the α-NH₂ groups of an amino acid, and the oxidation of the side chain is difficult. In addition, the method is sensitive, rapid, and convenient, and does not require toxic reagents.