2.5. Solid-phase extraction for enrichment of FAHFAs

SPE was performed according to the previously reported method with slightly modified [7]. Briefly, SPE was conducted with a HyperSep silica SPE cartridge (200 mg bed weight, 3 mL, Thermo Scientific, cat. no. 60108–410) at room temperature. The SPE cartridge was firstly conditioned with 15 mL of hexane. Individual aforementioned lipid extract dried with N₂ stream was reconstituted in 200 μL of chloroform, and then loaded onto the cartridge. Neutral lipids (i.e., triglyceride, and cholesterol and its esters) were eluted with 15 mL of 5% ethyl acetate in hexane, followed by elution of FAHFAs with 10 mL of ethyl acetate. Afterward, the FAHFAs eluent collected in a disposable conical tube was totally dried under a stream of N₂, and stored at −80 °C prior to use.