Precise measurement for the purity of amino acid and peptide using quantitative nuclear magnetic resonance

Precise measurement for the purity of organic compounds will fundamentally improve the capabilities and measurement services of the organic chemical analysis. Quantitative nuclear magnetic resonance (qNMR) is an important method to assess the purity of organic compounds. We presented a precise measurement method for the purity of small molecule with identification of impurities. In addition, the qNMR was rarely applied to purity of large compounds such as peptide, for which qNMR peaks are too crowded. Other than general idea of qNMR, we removed unwanted exchangeable peaks by proton exchange, as a new approach for qNMR, to make the quantitative protons of peptide isolated, which can ensure precise measurement. Moreover, a suitable internal standard, acesulfame potassium, was applied. The analytes were valine and peptide T5, due to their importance for protein analysis. For valine, the intraday CV was 0.052%, and the interday CV during 8 months was 0.071%. For peptide T5, simpler operation, shorter analytical time (1 h vs. 3 days) and smaller CV (0.36% vs. 0.93%) were achieved by qNMR, compared with a traditional method (amino acid based isotope labeled mass spectrometry) via a hydrolysis reaction. This method has greatly increased the quantitative precision of qNMR for small compounds, and extended application scope of qNMR from small compounds to peptides.