Extraction of NAD+ metabolites and nucleosides/nucleotides

Frozen PBMCs (5×10E6 cells total) were thawed on ice. 500 μl of ice cold 70:30 methanol:water was added along with 20 μl of internal standard and samples vortexed for 10 s. The resulting extract was centrifuged at 8000 × g for 5 min at 4 °C. The resulting supernatant was transferred to a new centrifuge tube and stored on ice. To the remaining pellet, 500 μl of ice cold methanol was added and the sample vortexed for 10 s to resuspend the pellet. The sample was then centrifuged at 8000 × g for 5 min at 4 °C. The entire supernatant was removed and combined with the 70% methanol supernatant. The resulting pellet was reserved and frozen at −70 °C for protein concentration analysis using the Bradford assay. The combined supernatants were centrifuged at 18,000 × g for 15 min and the resulting supernatant was transferred to a new tube and dried in a vacuum centrifuge at 55 °C. The dried samples were reconstituted in 100 μl of 1:1 methanol:water and centrifuged at 18,000 × g for 10 min at 4 °C. The supernatant was then transferred to a reduced surface activity autosampler vial for analysis.