quantitative one - dimensional ( 1d ) 1h 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 1h nmr spectra of complex metabolite mixtures hinders accurate quantification . 
 extension of 1d 1h to 2d 1h13c hsqc leads to the dispersion of peaks along the 13c dimension and greatly alleviates peak overlapping . 
 although peaks are better resolved in 2d 1h13c hsqc than in 1d 1h 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 1h excitation pulse to the beginning of data acquisition ) can be extrapolated back to zero time to yield a time - zero 2d 1h13c hsqc spectrum ( hsqc0 ) 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 hsqc0 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 .