Isotope In-Phase/Opposite-Phase-Edited 2D 1H-1H (12C/13C) TOCSY and Complementary 1D 1H Selective NMR Experiments for Carbon Tracing in Complex Samples.

Nuclear magnetic resonance (NMR)-based 13C tracing is widely used in medicine, metabolomics, and environmental research. Here, we introduce a 2D 1H-1H (12C/13C) "in-phase/opposite-phase" (IP/OP) TOCSY experiment that uniquely generates 2D subspectra discriminating 13C-13C, 12C-13C, and 12C-12C connectivity. The sequence was demonstrated first on a standard mixture of 50/50 13C-phenylalanine and 1-13C-glucose, followed by an in vivo ethanol fermentation using brewer's yeast (Saccharomyces cerevisiae) with 1-13C-glucose. Finally, incorporation of 13C in marine copepods (Tigriopus californicus) was monitored ex vivo. Copepods were analyzed at natural abundance and again on 7 days of feeding >98% 13C-enriched green algae. The sequence successfully identified three carbon pools: intact fragments or molecules from the 13C diet (13C-13C), intact fragments from pre-existing biomass (12C-12C), and new bond formation between 12C and 13C molecules during metabolite turnover (12C-13C). Molecules involved in osmotic regulation, alanine, proline, glycine, choline, betaine, and TMAO, were particularly abundant in the 12C-13C pool, suggesting rapid turnover by combining 13C food with existing 12C biomass. This was supported by a quantitative 1D 1H-(12C/13C) IP/OP experiment, measuring average enrichment of the six osmolytes at 22.8 ± 0.1% 13C on day 7. A complementary "singlet-only" experiment quantified glycine at 27.4 ± 0.2% 13C and betaine at 3.7 ± 0.4% 13C, enabling detection of molecules without scalar couplings. In summary, the 2D 1H-1H IP/OP TOCSY simultaneously identified pre-existing and newly synthesized molecules, while 1D experiments provide quantitative support, offering a sensitive framework to study carbon dynamics, preservation, and transformation in complex in vivo and ex vivo processes.