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

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 % on average from the expected. Finally, the sequence was used to monitor and quantify ¹³C% enrichment in Daphnia magna neonates which were fed a ¹³C diet over 1 week. The approach helped reveal how the organisms utilized the ¹²C lipids they are born with vs. the ¹³C 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.