Limited impact of presaturation time on the accuracy of 1H qNMR using a NOESY with high-power presaturation

A one-dimensional NOESY sequence with presaturation (NOESY-presat) is widely used for water suppression in ¹ H NMR-based metabolomics. While presaturation strengths below approximately 100 Hz are generally recommended to maintain accuracy of quantitative ¹ H NMR ( ¹ H qNMR), higher presaturation strengths may be employed in practice to suppress large H ₂ O signals and improve signal-to-noise ratios of ¹ H NMR spectra. However, the accuracy of ¹ H qNMR using NOESY-presat under such non-ideal conditions has not been systematically evaluated, particularly with respect to presaturation time. In this study, the effect of presaturation time on analyte concentration biases was examined under high presaturation strength of 356 to 370 Hz. Two H ₂ O/D ₂ O (90/10 vol %) solutions containing glycine (Gly), maleic acid (MA), and 3-(trimethylsilyl)-1-propanesulfonic acid- d ₆ (DSS- d ₆ ), or L-phenylalanine (Phe), L-valine (Val), MA, and DSS- d ₆ were measured by ¹ H qNMR using NOESY-presat. The results showed that large concentration biases of − 49% to − 8% for analyte signals located near the presaturation frequency corresponding to the bulk H ₂ O signal were not reduced by altering the presaturation time over a wide range of 50 to 60,000 ms. These findings indicate that presaturation time is not an effective parameter for reducing concentration biases under high presaturation strengths. Therefore, presaturation strength is the dominant factor affecting accuracy of ¹ H qNMR using NOESY-presat experiments. When high presaturation strengths are unavoidable, the resulting biases should be recognized as systematic and evaluated for each analyte signal, consistent with the established understanding for low presaturation strengths in ¹ H qNMR.