*2.6. Gadolinium Effect on Plasma Metabolites*

We next assessed the effects of Gd on our ability to detect and quantify metabolites using a commercially available standard pooled human plasma sample. We recorded a 25% NUS 1H-13C HSQC with or without the addition of Gd, and with a relaxation delay of 0.8 s, a constant scan number of 72, and an acquisition time of ~1 h. The addition of Gd led to a 1.12-fold increase in overall mean peak intensities. This increase was slightly less pronounced than the fold change of 1.25 observed with the model mixture and could be due to the presence of additional anions and salts, which are known to influence the impact of PRE agents [22,24]. Nonetheless, as noted for the model mixtures, we observed that the largest increase in fold change was associated with low-intensity resonances. These results further suggest that the addition of Gd could improve our ability to detect low abundant metabolites (Figure 5c). Furthermore, the % CV was lowered from 15% to 10% for the pooled human plasma sample in the presence of Gd (Figure 5d). Collectively, these results suggest that the addition of Gd to plasma samples increases the S/N for metabolite NMR resonances, especially for low abundant metabolites, and increases the reproducibility of intensity measurements. Overall, the addition of Gd to a metabolomics sample could facilitate an increase in the confidence and reliability in the detection and quantification of metabolite NMR resonances.


**Table 2.** Limit of detection (LOD) for measured metabolite resonances.


**Table 3.** Limit of quantification (LOQ) for measured metabolite resonances.

**Figure 4.** Gadolinium maintains the reproducibility of NMR intensity measurements. The percent coefficient of variation (%CV) in the peak intensities measured from NUS 1H-13C HSQC spectra. The %CV decreased for metabolite resonances in the presence of Gd (orange) compared to no Gd (blue).

**Figure 5.** Gadolinium improves S/N and the mean intensity of plasma metabolite resonances. 1H-13C HSQC spectra (**a**) without and (**b**) with the addition of gadolinium. (**c**) Fold change of median normalized intensity in the presence of gadolinium. (**d**) Percent coefficient of variation (% CV) measured intensity decreases for metabolite resonances in the presence of Gd (orange) compared to no Gd (blue).
