Sample preparation for phytohormone analysis

Fresh samples (50 mg) were frozen in liquid nitrogen and ground into a powder, which was extracted with 1 ml of isopropanol:H₂O:HCl (2:1:0.002, v/v/v) using a vortex for 20 min. Dichloromethane (1 ml) was then added, and the mixture was centrifuged at 15,000 rpm for 10 min for fractionation. After fractionation, two phases were obtained, where the dichloromethane fraction was eluted through a C18 cartridge using methanol. The filtered sample was concentrated and re-dissolved in 25 µl of methanol.