Sep pak vac 3 cc 200 mg c18 cartridges

Here, 20 µL of serum was transferred to a 1.5 mL Eppendorf tube. Then, the sample was extracted twice with ethanol. In each extraction step, 200 µL of ethanol was mixed with serum, and the mixture was sonicated for 10 min before centrifugation (14,000× g, for 15 min). The supernatant of the two extraction steps was mixed and ultimately evaporated. The residue was reconstituted with 0.5 mL of 60% methanol for solid-phase extraction using Sep-Pak Vac 3 cc (200 mg) C18 cartridges (Waters Corp. Milford, MA, USA). The sample was loaded into the cartridge, which was activated using methanol (6 mL) and conditioned by 60% of methanol (6 mL). The cartridge was washed with 6 mL of methanol 60%. Then, inotodiol was eluted with 100% of methanol (3 mL) in the glass tube. The sample was then evaporated. The residue was resolved in methanol before HPLC-MS/MS analysis.

For plasma samples, 3 mL methanol were added to an aliquot of 600 μL plasma, and vortexed for 5 min. The mixture was centrifuged at 12,000 rpm for 10 min, and then the supernatant was separated and evaporated to dryness under a stream of nitrogen gas at 35 °C. Then the residue was reconstituted with 100 μL of methanol, vortexed for 1 min, then centrifuged at 12,000 rpm for 10 min, and the supernatant was separated. An aliquot of 4 μL was injected into the UHPLC-ESI(electrospray ionization) -Q-TOF system for analysis.
SPE (solid phase extraction) procedures (Waters Sep-Pak^® Vac 3cc, 200 mg C₁₈ Cartridges) were used for urine and bile sample pretreatment, and each sample was extracted independently. A total of 5 mL methanol and 5 mL distilled water was used to precondition the SPE cartridge. Each sample was vortexed for 30 s and centrifuged at 12,000 rpm for 10 min before loaded into the SPE cartridge. Then, the SPE cartridge was washed with 2 mL water and eluted with 2 mL methanol. The eluent was evaporated to dryness under a stream of nitrogen gas at 35 °C. Then the residue was reconstituted with 500 μL of methanol, vortexed for 1 min, then centrifuged at 12,000 rpm for 10 min, and the supernatant was separated. An aliquot of 4 μL was injected into the UHPLC-ESI-Q-TOF system for analysis.