Whole Blood Preparation for 1D NMR

1. Blood samples are first aliquoted

2. Proteins are precipitated by the addition of twice the amount of cold methanol purged of oxygen for 30 minutes by nitrogen gas.

3. Antibacterial NaN3 is added 2.0% by volume to avoid any bacterial growth

4. The 1:2 mixture of serum: solvent is then vortexed for 10 seconds

5. Samples are then sonicated in a water bath for 10 min at 4°C, followed by incubation at -20°C for 20 minutes

6. Samples are diluted with 600 μL H2O and thoroughly mixed by pipette followed by centrifugation at 14,000 rpm for 30 min at 4°C to pellet cell debris

7. Supernatant is collected and divided for NMR and MS analysis, 80/20% respectively. Samples are snap frozen in Liquid Nitrogen and freeze dried to remove methanol by speed vacuum centrifugation (SpeedVac R Plus, Savant) and water by lyophilization using FreeZone™ (Labconco, Kansas City, MO) for 24 hours and stored at -20°C until NMR analysis.