Accurate quantitative 13C NMR spectroscopy:: Repeatability over time of site-specific 13C isotope ratio determination

The stability over time (repeatability) for the determination of site-specific C-13/C-12 ratios at natural abundance by quantitative C-13 NMR spectroscopy has been tested on three probes: enriched bilabeled [1,2-C-13(2)]ethanol; ethanol at natural abundance; and vanillin at natural abundance. It is shown in all three cases that the standard deviation for a series of measurements taken every 2-3 months over periods between 9 and 13 months is equal to or smaller than the standard deviation calculated from 5-10 replicate measurements made on a single sample. The precision which can be achieved using the present analytical 13C NMR protocol is higher than the prerequisite value of 1-2 parts per thousand for the determination of site-specific C-13/C-12 ratios at natural abundance (C-13-SNIF-NMR). Hence, this technique permits the discrimination of very small variations in C-13/C-12 ratios between carbon positions, as found in biogenic natural products. This observed stability over time in C-13 NMR spectroscopy indicates that further improvements in precision will depend primarily on improved signal-to-noise ratio.