Guidelines for the Use of Deuterium Oxide (D₂O) in ¹H NMR Metabolomics

In metabolomics, nuclear magnetic resonance (NMR) spectroscopy allows to identify and quantify compounds in biological samples. The sample preparation generally requires only few steps; however, an indispensable factor is the addition of a locking substance into the biofluid sample, such as deuterium oxide (D₂O). While creatinine loss in pure D₂O is well-described, the effects of different D₂O concentrations on the signal profile of biological samples are unknown. In this work, we investigated the effect of D₂O levels in the NMR buffer system in urine samples, in dependence on dwell time and temperature exposition. We reveal a decrease of the urinary creatinine peak area up to 35% after 24 h of dwell time at room temperature (RT) using 25% (v/v) D₂O, but only 4% loss using 2.5% D₂O. ¹H, inverse-gated (IG) ¹³C, DEPT-HSQC NMR, and mass spectrometry (MS) experiments confirmed a proton-deuterium (H/D) exchange at the CH₂. This leads to underestimation of creatinine levels and has an extensive effect when creatinine is used for normalization. This work offers a sample stability examination, depending on the D₂O concentration, dwell time, and temperature and enables a method to correct for the successive loss. We propose an equation to correct the creatinine loss for samples prepared with various D₂O concentrations and storage temperatures for dwell times up to 24 h. The correction function was validated against an external data set with n = 26 samples. To ensure sufficient creatinine stability in future studies, we suggest that a maximum of 10% D₂O should be used at 4 °C or 2.5% D₂O at RT, respectively.