After acquiring serum from the mouse, 80 μL serum was added to 240 μL cold methanol/acetonitrile (2:1, v/v). 10 μL internal tagging standard (L-2-chlorine-phenylalanine, 0.3 mg/mL, dissolved in methanol) was added, the samples were vortexed for 2 min, and then ultrasonic extraction was performed for 5 min. The samples were allowed to stand at -20° C for 20 min, and then centrifuged for 10 min (14,000 RPM, 4° C). 200 μL of the supernatant was loaded into a sample bottle with a lining tube for LC/MS analysis. A Waters UPLC I-class system equipped with a binary solvent delivery manager and sample manager, coupled to a Waters VION IMS Q-TOF Mass Spectrometer equipped with an electrospray interface (Waters Corporation, Milford, MA, USA) was used for LC/MS analysis. An Acquity BEH C18 column (100 mm × 2.1 mm; i.d., 1.7 μm; Waters, Milford, USA) was used for LC separation. Information on the peak picking, alignment, deconvolution, and further processing of raw LC-MS data can be found in previously published protocols [35 (link), 40 (link)] (Sah et al. 2017; Sonnerburg et al., 2016).
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