Oasis prime hlb 1 cc cartridges

Solid phase extraction (SPE) was performed using OASIS PRiME HLB 1 cc cartridges (Waters Corporation, Milford, MA, USA, Part Number: 186008055), which utilize a reversed-phase Hydrophilic-Lipophilic Balance chemistry, and a positive pressure manifold (Biotage PRESSURE+48). Samples were diluted into an equal volume of 0.1% Trifluoroacetic acid (TFA) in water (75 μl each), vortexed for 30 s, and centrifuged at 10,000 rpm (RCF = 9632 × g) for 5 minutes. SPE cartridges were conditioned first with 1 ml acetonitrile (ACN) with 0.1% trifluoroacetic acid (TFA) and then 1 ml 0.1% TFA in water. The entire sample supernatant was applied to the cartridge. Each cartridge was then washed with 1 ml 10% ACN, 0.1% TFA. Finally, samples were eluted with 1 ml 30% ACN, 0.1% TFA. This elution is at a lower percent organic content than in previously published studies; for more information on the development of this method, see the appendix. The eluted samples were placed at −80°C overnight and lyophilized the next day using a centrivap (Labconco model #7810016) with the following settings: centrivap unheated, cold trap −80–85°C, vacuum 0.3–0.4 mbar. Samples were reconstituted in 250 μl assay buffer at the time of assay, resulting in a dilution factor of 1:3.33.