Salicylic acid quantification

Salicylic acids were extracted and quantified from freeze-dried tomato samples according to the protocol described by Seskar, Shulaev & Raskin (1998). The freeze-dried whole plant tissues (0.2 g) were accordingly extracted with 90% and 100% methanol. The samples were than centrifuged at 10,000×g and the combined methanol extract was vacuum-dried. Dried samples were resuspended in 2.5 mL of 5% trichloroacetic acid (TCA) and further partitioned with ethyl acetate, cyclopentane, and isopropanol (ratio of 100:99:1, v/v). The upper organic layer containing free SA was transferred to a 4-mL vial and dried with nitrogen gas. The dry SA was again suspended in 1 mL of 70% methanol and was subjected to HPLC, using a Shimadzu device outfitted with a fluorescence indicator (Shimadzu RF-10AxL) with excitation at 305 nm and emission at 365 nm, filled with a C18 reverse-phase HPLC column (HP Hypersil ODS, particle size 5 μm, pore size 120 Å, Waters). Flow rates of 1.0 mL/min were used.