Detection, characterization and quantification of salicylic acid conjugates in plant extracts by ESI tandem mass spectrometric techniques

摘要

An approach for the detection and characterization of SA derivatives in plant samples is presented basedon liquid chromatography coupled to electrospray ionization (ESI) tandem mass spectrometric techniques. Precursor ion scan methods using an ESI triple quadrupole spectrometer for samples from plantschallenged with the virulent Pseudomonas syringae pv tomato DC3000 allowed us to detect two potentialSA derivatives. The criterion used to consider a potential SA derivative is based on the detection ofanalytes in the precursor ion scan chromatogram upon selecting m/z 137 and m/z 93 that correspond tothe salicylate and its main product ion, respectively. Product ion spectra of the newly-detected analytesas well as accurate m/z determinations using an ESI Q-time-of-flight instrument were registered asmeans of characterization and strongly suggest that glucosylated forms of SA at the carboxylic and at thephenol functional groups are present in plant samples. The specific synthesis and subsequent chromatography of salicylic glucosyl ester (SGE) and glucosyl salicylate (SAG) standards confirmed the chemicalidentity of both peaks that were obtained applying different tandem mass spectrometric techniques andaccurate m/z determinations. A multiple reaction monitoring method has been developed and applied toplant samples. The advantages of this LCeESIeMS/MS methods with respect to the traditional analysis ofglucosyl conjugates are also discussed. Preliminary results revealed that SA and the glucosyl conjugatesare accumulated in Arabidopsis thaliana in a time dependent manner, accordingly to the up-regulation ofSA-dependent defenses following P. syringae infection. This technique applied to plant hormones orfragment ions may be useful to obtain chemical family members of plant metabolites and help identifytheir contribution in the signaling of plant defenses