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

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.