Simultaneous Quantification and Identification of
Individual Chemicals in Metabolite Mixtures by Two-Dimensional Extrapolated
Time-Zero <sup>1</sup>H−<sup>13</sup>C HSQC (HSQC<sub>0</sub>)
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Abstract
Quantitative one-dimensional (1D) <sup>1</sup>H NMR spectroscopy
is a useful tool for determining metabolite concentrations because
of the direct proportionality of signal intensity to the quantity
of analyte. However, severe signal overlap in 1D <sup>1</sup>H NMR
spectra of complex metabolite mixtures hinders accurate quantification.
Extension of 1D <sup>1</sup>H to 2D <sup>1</sup>H−<sup>13</sup>C HSQC leads to the dispersion of peaks along the <sup>13</sup>C
dimension and greatly alleviates peak overlapping. Although peaks
are better resolved in 2D <sup>1</sup>H−<sup>13</sup>C HSQC
than in 1D <sup>1</sup>H NMR spectra, the simple proportionality of
cross peaks to the quantity of individual metabolites is lost by resonance-specific
signal attenuation during the coherence transfer periods. As a result,
peaks for individual metabolites usually are quantified by reference
to calibration data collected from samples of known concentration.
We show here that data from a series of HSQC spectra acquired with
incremented repetition times (the time between the end of the first <sup>1</sup>H excitation pulse to the beginning of data acquisition) can
be extrapolated back to zero time to yield a time-zero 2D <sup>1</sup>H−<sup>13</sup>C HSQC spectrum (HSQC<sub>0</sub>) in which
signal intensities are proportional to concentrations of individual
metabolites. Relative concentrations determined from cross peak intensities
can be converted to absolute concentrations by reference to an internal
standard of known concentration. Clustering of the HSQC<sub>0</sub> cross peaks by their normalized intensities identifies those corresponding
to metabolites present at a given concentration, and this information
can assist in assigning these peaks to specific compounds. The concentration
measurement for an individual metabolite can be improved by averaging
the intensities of multiple, nonoverlapping cross peaks assigned to
that metabolite