Method for Determining Molar Concentrations of Metabolites in Complex Solutions from Two-Dimensional 1H−13C NMR Spectra

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Abstract
One-dimensional (1D) 1H nuclear magnetic resonance (NMR) spectroscopy is used extensively for high-throughput analysis of metabolites in biological fluids and tissue extracts. Typically, such spectra are treated as multivariate statistical objects rather than as collections of quantifiable metabolites. We report here a two-dimensional (2D) 1H−13C NMR strategy (fast metabolite quantification, FMQ, by NMR) for identifying and quantifying the ∼40 most abundant metabolites in biological samples. To validate this technique, we prepared mixtures of synthetic compounds and extracts from Arabidopsis thaliana, Saccharomyces cerevisiae, and Medicago sativa. We show that accurate (technical error 2.7%) molar concentrations can be determined in 12 min using our quantitative 2D 1H−13C NMR strategy. In contrast, traditional 1D 1H NMR analysis resulted in 16.2% technical error under nearly ideal conditions. We propose FMQ by NMR as a practical alternative to 1D 1H NMR for metabolomics studies in which 50-mg (extract dry weight) samples can be obtained.