3.2. Deuterium Labeled Internal Standards Addition Procedures

For deuterium labeled internal standards recovery study, five samples were prepared as pre-spikes and five samples were prepared as post-spikes, using each extraction procedure. Preliminary experiments (not shown) have indicated that UltimateSPLASH™ One Internal Standard can be detected at the dilution level of 1:10 in plasma robustly and reliably (2.5–15 μg/mL depending on the lipid). To prepare a pre-spiked plasma sample at this level without affecting the solvent characteristics for the different sample preparations techniques, 100 μL aliquots UltimateSPLASH™ One Internal Standard was initially dried down, and reconstituted in 1000 μL SRM 1950 plasma. This mixture was vortexed and ultrasonicated for 10 min on ice and stored at −80 °C until use. To prepare the post-spike samples, 100 μL aliquot of UltimateSPLASH™ One Internal Standard was initially dried down and then reconstituted in 1 mL 1:1 BuOH/MeOH. This solution was vortexed and ultrasonicated for and was used to reconstitute dried post-spike plasma samples.

For the lipids profiling studies, all human plasma samples were extracted following the methods procedure, and then reconstituted in a solution containing EquiSPLASH^® Internal Standard in 1:1 BuOH/MeOH. To prepare the reconstituted solution, an aliquot of 100 μL EquiSPLASH^® Internal Standard was initially dried down and then reconstituted in 1 mL 1:1 BuOH/MeOH. This mixture was vortexed and ultrasonicated for 10 min on ice.