Understanding the variability of compound quantification from targeted profiling metabolomics of 1D- 1 H-NMR spectra in synthetic mixtures and urine with additional insights on choice of pulse sequences and robotic sampling

The growing use of ‘targeted profiling’ approaches for the deconvolution of 1D-1H-NMR spectra by comparison to a pure compound library has created a need for an in-depth characterization of quantification variability that is beyond what is currently available in the literature. In this study, we explore the underlying source of quantification variability (tube insertion, spectra acquisition, and profiling) as well as a number of other factors, such as temporal consistency of repeated NMR scans, human consistency in repeated profiles, and human versus machine sampling. We also look at the effect of different pulse sequences on the differences between acquired spectra and the peak reference library. Two sample types were considered for this work—a synthetic five compound mixture as well as human urine. The result is a comprehensive examination of 1D-1H-NMR quantification error. Our investigation into variability sources revealed that apart from profiling, sample insertion and/or shimming can play a significant role in final quantification, a finding that is equally applicable to all integration-based methods of quantification. Both sources of error were also found to have temporal relationships, with bias identified as a function of both scan and profiling order, reinforcing the need for randomization in scanning and profiling. As well as presenting a practical estimate of variability in human urine samples, we have uncovered a considerable amount of complexity in underlying NMR variability that will hopefully serve as impetus for future exploration in this area.