Quantitative analysis of metabolic mixtures by two-dimensional 13C constant-time TOCSY NMR spectroscopy.

An increasing number of organisms can be fully (13)C-labeled, which has the advantage that their metabolomes can be studied by high-resolution two-dimensional (2D) NMR (13)C-(13)C constant-time (CT) total correlation spectroscopy (TOCSY) experiments. Individual metabolites can be identified via database searching or, in the case of novel compounds, through the reconstruction of their backbone-carbon topology. Determination of quantitative metabolite concentrations is another key task. Because strong peak overlaps in one-dimensional (1D) NMR spectra prevent straightforward quantification through 1D peak integrals, we demonstrate here the direct use of (13)C-(13)C CT-TOCSY spectra for metabolite quantification. This is accomplished through the quantum mechanical treatment of the TOCSY magnetization transfer at short and long-mixing times or by the use of analytical approximations, which are solely based on the knowledge of the carbon-backbone topologies. The methods are demonstrated for carbohydrate and amino acid mixtures.