Conditions to obtain precise and true measurements of the intramolecular C distribution in organic molecules by isotopic C nuclear magnetic resonance spectrometry

Intramolecular ¹³C composition gives access to new information on the (bio) synthetic history of a given molecule. Isotopic ¹³C NMR spectrometry provides a general tool for measuring the position-specific ¹³C content. As an emerging technique, some aspects of its performance are not yet fully delineated. This paper reports on (i) the conditions required to obtain satisfactory trueness and precision for the determination of the internal ¹³C distribution, and (ii) an approach to determining the “absolute” position-specific ¹³C content. In relation to (i), a precision of <1% can be obtained whatever the molecule on any spectrometer, once quantitative conditions are met, in particular appropriate proton decoupling efficiency. This performance is a prerequisite to the measurement of isotope fractionation either on the transformed or residual compound when a chemical reaction or process is being studied. The study of the trueness has revealed that the response of the spectrometer depends on the ¹³C frequency range of the studied molecule, i.e. the chemical shift range. The “absolute value” and, therefore, the trueness of the ¹³C NMR measurements has been assessed on acetic acid and by comparison to the results obtained on the fragments from COOH and CH₃ by isotopic mass spectrometry coupled to a pyrolysis device (GC-Py–irm-MS), this technique being the reference method for acetic acid. Of the two NMR spectrometers used in this work, one gave values that corresponded to those obtained by GC-Py–irm-MS (thus, the “true” value) while the other showed a bias, which was dependent to the range covered by the resonance frequencies of the molecule. Therefore, the former can be used directly for studying isotope affiliations, while the latter can only be used directly for comparative data, for example in authenticity studies, but can also be used to obtain the true values by applying appropriate correction factors. The present study assesses several key protocol steps required to enable the determination of position-specific ¹³C content by isotopic ¹³C NMR, irrespective of the NMR spectrometer: parameters to be adjusted, performance test using [1,2-¹³C₂]acetic acid, generation of correction factors.