Novel Nuclear Magnetic Resonance Method for Position-Specific Carbon Isotope Analysis of Organic Molecules with Significant Impurities

Abstract

We introduce a novel nuclear magnetic resonance (NMR) tool for determining position-specific carbon (13C/12C) isotope ratios within complex organic molecules. This analytical advancement allows us to measure position-specific isotope ratios of samples that contain impurities with NMR peaks that overlap with the signals of interest. The method involves collecting a series of alternating 13C-coupled and 13C-decoupled 1H NMR spectra using an NMR pulse sequence designed to optimize temperature stability, followed by a data reduction scheme that allows the signals of interest to be isolated from signals of impurities. The method was validated using glycine reference materials with known 13C/12C ratios from the US Geological Survey (USGS) into which impurities typically found in amino acid samples were intentionally introduced. Following validation, the method was used to determine position-specific 13C/12C ratios in a set of USGS l-valine materials (USGS73, -74, -75) that contain significant impurities associated with their biological origin. The l-valines were found to contain distinct intramolecular isotope variability, and the 13Cα isotope spikes in USGS74 and USGS75 were clearly detected, where they preserve carbon isotope ratios of -4.8 ± 0.9‰ and +11.5 ± 0.8‰, respectively. Carbon isotope abundance at the beta and gamma positions indicates that the USGS73 l-valine was obtained from a different source than USGS74 and -75. This analytical approach is a significant step forward in the field of position-specific isotope analysis at natural abundance via NMR because it enables the investigation of samples that contain impurities which are typically present in samples derived from natural sources.