Ultimate 3000 high performance liquid chromatograph

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.

Hormone analysis was carried out on four samples, each of which contained all seedlings from six of the 24 wells or from the four-week-old A. thaliana three leaf discs from every single plant were sampled, three individual plants were sampled as one sample. Plant hormone levels were determined as described by^{40 (link)}. Briefly, samples were homogenized in tubes with 1.3 mm silica beads using a FastPrep-24 instrument (MP Biomedicals, USA). The samples were then extracted with a methanol/H₂O/formic acid (15:4:1, v:v:v) mixture, which was supplemented with stable isotope-labeled phytohormone internal standards (10 pmol per sample) in order to check recovery during purification and validate the quantification. The clarified supernatants were subjected to solid phase extraction using Oasis MCX cartridges (Waters Co., USA). The eluates were evaporated to dryness and the generated solids dissolved in 30 μl of 15% (v/v) acetonitrile in water. Quantification was performed on an Ultimate 3000 high-performance liquid chromatograph (Dionex, USA) coupled to a 3200 Q TRAP hybrid triple quadrupole/linear ion trap mass spectrometer (Applied Biosystems, USA) as described by^{41 (link)}. Metabolite levels were expressed in pmol/g fresh weight (FW).