4.6. NMR Sample Preparation

Aliquots were thawed and 500 μL of serum was diluted to a final volume containing 50% (v/v) serum, 40% (v/v) dd ¹H₂O (18.2 MΩ), 10% (v/v) 1 M PO₄³⁻ pH 7.4 buffer (Na₂HPO₄, VWR International Ltd., Radnor, PA, USA and NaH₂PO₄, Sigma-Aldrich, Gillingham, UK) in deuterium oxide (²H₂O, Sigma-Aldrich) and 1.2 mM sodium azide (NaN₃, Sigma-Aldrich). Samples were vortexed for 1 min, centrifuged at 13,000× g at 4 °C for 2 min and 600 μL transferred into 5 mm outer diameter NMR tubes (Bruker, Coventry, UK).

Urine & saliva samples were thawed at room temperature before addition of 500 μL to 500 μL of 1 M phosphate buffer (Na₂HPO₄ and NaH₂PO₄) at pH7.4 with 20% ²H₂O, 200 μM TSP and 2.4 mM sodium azide. The samples were vortexed for 30 s prior to 5 min centrifugation at 21,500× g and 4 °C before transferring 600 μL of sample to Bruker SampleJet 5 mm (outer diameter) NMR tubes. The final concentration in the NMR tube was 50% urine or saliva, 10% ²H₂O, 1.2 mM sodium azide and 100 μM TSP.