Position specific ¹⁵N isotope analysis at natural abundance by ¹H NMR

Stable isotope analysis of nitrogen (N) at natural abundance provides key insights into biochemical processes. However, position-specific isotope analysis (PSIA) of ¹⁵N remains technically challenging due to the low sensitivity of conventional approaches. The approach we present here relies on resolving the ¹H-¹⁵N doublets embedded within dominant ¹H-¹⁴N triplets and quantifying them via spectral deconvolution under rigorously optimized quantitative NMR conditions. Using ammonium chloride as a model, we established the experimental and processing parameters required for precision (≤2‰) and trueness (<3‰), validated against isotope ratio monitoring Mass Spectrometry (irm-MS). The method was successfully extended to structurally more complex compounds (aniline, indole, urea, and N-acetylated tryptophan methyl ester), achieving accurate position-specific δ¹⁵N determinations. Importantly, we demonstrate the feasibility of applying this strategy to a human urine sample, enabling direct δ¹⁵N measurement of endogenous urea in a complex biological matrix at natural abundance. This non-destructive method requires minimal sample amounts and offers a robust complement to irm-MS, expanding the analytical toolbox for nitrogen isotope research in environmental, biochemical, and biomedical contexts.