Sample preparation for NMR metabolomics analysis

Each serum sample was thawed for 1 h and passed through a centrifugal filter with a 5 kDa cut-off to remove macromolecules shown to interfere with the NMR metabolite signals. First, the centrifugal filters were washed four times with 100 mM NaCl and twice with MiliQ water, prior to sample treatment to remove glycerol. Filtered serum was transferred to NMR tubes and proportionally added to phosphate buffer prepared in D₂O containing an internal standard (4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS) 0.25 mM). The solution in the NMR tube was homogenized by repeated inversions avoiding the formation of air bubbles. This protocol was established after optimization of several steps, namely serum thawing time, sample stability and ultrafiltration conditions with the main goal of improving reproducibility and minimizing time dependent sample degradation. To evaluate sample stability NMR spectra were acquired immediately after sample preparation and after 4, 9, 13, 16 and 27 h. Comparison of NMR spectra profiles has shown deviations in chemical shifts, in particular for histidine, due to changes in sample pH, which was minimized using a phosphate buffer (50 mM pH 7.0) and keeping 3 h timing between sample preparation and NMR data acquisition.