13C-Satellite Decoupling Strategies for Improving Accuracy in Quantitative Nuclear Magnetic Resonance.

Abstract

Quantitative 1H nuclear magnetic resonance (qHNMR) with an appropriate internal standard is a well-established quantitation method for assigning purity to organic molecules. For accurate measurements, the premise of qHNMR relies on the careful selection of integrals, for both the analyte and the standard, in such a way that the selected integrals are free from interferences. The 13C-satellite signals of adjacent integrals, low-level impurities, and tautomer signals are among the common integral interferences that are typically encountered. One of the simplest ways to identify and avoid these interferences is to decouple the 13C-satellites. Two decoupling schemes were explored to illustrate the benefits of 13C-decoupling for qHNMR or qH{13C}NMR: GARP and bilevel adiabatic broadband decoupling. Unwanted sample heating and nuclear Overhauser effect (NOE) enhancements are the two main drawbacks of decoupling schemes. We show that with careful optimization of acquisition parameters and decoupling power, no excessive sample heating occurred during acquisition at 400 MHz. At 900 MHz, only bilevel adiabatic decoupling could be safely implemented. Furthermore, any undesirable NOE enhancements were completely avoided if acquisition was executed with an inverse-gated pulse sequence. We explored and confirmed the benefits of qH{13C}NMR through the quantitation of a diverse set of compounds, namely, small molecules (dimethyl terephthalate and zearalenone), a 13C-labeled compound (13C6-ochratoxin A), and an octapeptide (angiotensin II). Statistical comparisons confirmed that qH{13C}NMR produced comparable data to qHNMR. However, with qH{13C}NMR data providing added clarity about the presence of overlapping 13C-satellites, impurities, and tautomers, it has an edge over qHNMR for accurate measurements.