Quantifying Valeric Acid in Cerebral Cortex

The amount of valeric acid in the cerebral cortex harvested 24 h after the last intraperitoneal injection of valeric acid was determined by a method similar to that reported before [51 , 52 (link)]. Briefly, 0.1 g cerebral cortex was homogenized in 500 µl aqueous acetonitrile. Supernatant was extracted with 8 ml extraction buffer (hexane:diethyl ether = 1:1) and centrifuged at 1800 g for 5 min. 7.5 ml supernatant was collected, mixed with 93 µl 20 mM KOH in methanol and dried at 40 °C under nitrogen gas. Dried residue was reconstituted in 50 µl 2.5% 18-Crown-6 in acetonitrile and was further derivatized with 9-chloromethylanthracene in acetonitrile with addition of tetramethylammonium hydroxide. Finally, 30 µl derivatization solution was loaded to Acclaim C18 column (3 µm, 4.6 × 100 mm, Thermo Scientific) and separated in Ultimate 3000 high performance liquid chromatograph (HPLC, Thermo Scientific) equipped with UV-visible detector. The peak of derivatized valeric acid was detected at a wavelength of 254 nm. 2-Ethylbutyric acid (2-EA) was added as an internal reference control. The peak area of valeric acid was measured as mAU*min and its peak area of each sample was normalized with that of 2-EA in the sample. Final results were presented as ratios of valeric acid group to NS group.