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A novel metabolomics approach for NMR‐based stable isotope tracer studies called PEPA is presented, and its performance validated using human cancer cells. PEPA detects the position of carbon label in isotopically enriched metabolites and quantifies fractional enrichment by indirect determination of  13 C‐satellite peaks using 1D‐ 1 H‐NMR spectra. In comparison with  13 C‐NMR, TOCSY and HSQC, PEPA improves sensitivity, accelerates the elucidation of  13 C positions in labeled metabolites and the quantification of the percentage of stable isotope enrichment. Altogether, PEPA provides a novel framework for extending the high‐throughput of  1 H‐NMR metabolic profiling to stable isotope tracing in metabolomics, facilitating and complementing the information derived from 2D‐NMR experiments and expanding the range of isotopically enriched metabolites detected in cellular extracts.

Positional Enrichment by Proton Analysis (PEPA): A One‐Dimensional 1 H‐NMR Approach for 13 C Stable Isotope Tracer Studies in Metabolomics