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

Nuclear magnetic resonance (NMR) based C 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 H-C/C is introduced that uses "in-phase/opposite-phase" encoding to simultaneously detect and discriminate both protons attached to C and C at full H sensitivity in every scan. Unlike traditional approaches that focus on the C/C satellite ratios in a H spectrum, this approach creates separate sub-spectra for the C and C 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 C dipole, faster relaxation of the H-C pairs results in slight underestimation compared to the H-C pairs. However, this is easily compensated for, by collecting an additional reference spectrum, from which the absolute percentage of C can be calculated by difference. When combined with the result, C and C percent enrichment in both H-C and H-C fractions are obtained. As the approach uses isotope filtered H NMR for detection, it retains nearly the same sensitivity as a standard H spectrum. Here, a proof-of-concept is performed using simple mixtures of C and C glucose, followed by suspended algal cells with varying C /C ratios representing a complex mixture. The results consistently return C/C ratios that deviate less than 1 % on average from the expected. Finally, the sequence was used to monitor and quantify C% enrichment in Daphnia magna neonates which were fed a C diet over 1 week. The approach helped reveal how the organisms utilized the C lipids they are born with vs. the C lipids they assimilate from their diet during growth. Given the experiments simplicity, versatility, and sensitivity, we anticipate it should find broad application in a wide range of tracer studies, such as fluxomics, with applications spanning various disciplines.