A simple 1H (12C/13C) filtered experiment to quantify and trace isotope enrichment in complex environmental and biological samples

[Display omitted] •The 1D 1H-(12C/13C) in-phase/opposite-phase experiment can simultaneously detect and discriminate both protons attached to 12C and 13C at full 1H sensitivity in every scan.•Unlike traditional approaches that focus on the 12C/13C satellite ratios in a 1H spectrum, the in-phase/opposite-phase experiment creates separate sub-spectra for the 12C and 13C bound protons.•The sub-spectra can be used for both quantitative and qualitative analysis of complex environmental and biological samples with significant spectral overlap. Quantitative analysis requires an additional equivalent reference spectrum.•The in-phase/opposite-phase experiment is useful for process monitoring as it can provide a simple overview of the components in each 12C and 13C fraction and how they change over time.•The in-phase/opposite-phase experiment has broad application in a wide range of carbon tracer studies spanning different disciplines (i.e., fluxomics). Nuclear magnetic resonance (NMR) based 13C tracing has broad applications across medical and environmental research. As many biological and environmental samples are heterogeneous, they experience considerable spectral overlap and relatively low signal. Here a 1D 1H–12C/13C is introduced that uses “in-phase/opposite-phase” encoding to simultaneously detect and discriminate both protons attached to 12C and 13C at full 1H sensitivity in every scan. Unlike traditional approaches that focus on the 12C/13C satellite ratios in a 1H spectrum, this approach creates separate sub-spectra for the 12C and 13C bound protons. These spectra can be used for both quantitative and qualitative analysis of complex samples with significant spectral overlap. Due to the presence of the 13C dipole, faster relaxation of the 1H–13C pairs results in slight underestimation compared to the 1H–12C pairs. However, this is easily compensated for, by collecting an additional reference spectrum, from which the absolute percentage of 13C can be calculated by difference. When combined with the result, 12C and 13C percent enrichment in both 1H–12C and 1H–13C fractions are obtained. As the approach uses isotope filtered 1H NMR for detection, it retains nearly the same sensitivity as a standard 1H spectrum. Here, a proof-of-concept is performed using simple mixtures of 12C and 13C glucose, followed by suspended algal cells with varying 12C /13C ratios representing a complex mixture. The results consistently return 12C/13C ratios that deviate less than 1 %