Flow-Batch Sample Preparation for Fractionation of the Stress Signaling Phytohormone Salicylic Acid in Fresh Leaves

Salicylic acid (SA) is an important stress signaling phytohormone and plays an essential role in physiological processes in plants. SA fractionation has been carried out batchwise, which is not compatible with the high analytical demand in agronomical studies and increases susceptibility to analytical errors. In this context, a novel flow-batch sample preparation system for SA fractionation on fresh plant leaves was developed. It was based on microwave-assisted extraction with water and conversion of the conjugated species to free SA by alkaline hydrolysis. Free and total SA were quantified by fluorimetry after separation by sequential injection chromatography in a C18 monolithic column. The proposed procedure is directly applicable to plant leaves containing up 16 mg kg^-1 SA, with a limit of detection of 0.1 mg kg^-1 of SA, coefficient of variation of 3.0% (n = 10), and sampling rate of 4 samples h^-1. The flow-batch sample preparation system was successfully applied to SA fractionation in sugarcane, corn, and soybean leaves without clogging or increasing in backpressure. The proposed approach is simple, less time-consuming, and more environmentally friendly in comparison to batchwise procedures.