An UPLC-MS/MS method for highly sensitive high-throughput analysis of phytohormones in plant tissues

Abstract

BackgroundPhytohormones are the key metabolites participating in the regulation of multiple functions of plant organism. Among them, jasmonates, as well as abscisic and salicylic acids are responsible for triggering and modulating plant reactions targeted against pathogens and herbivores, as well as resistance to abiotic stress (drought, UV-irradiation and mechanical wounding). These factors induce dramatic changes in phytohormone biosynthesis and transport leading to rapid local and systemic stress responses. Understanding of underlying mechanisms is of principle interest for scientists working in various areas of plant biology. However, highly sensitive, precise and high-throughput methods for quantification of these phytohormones in small samples of plant tissues are still missing.ResultsHere we present an LC-MS/MS method for fast and highly sensitive determination of jasmonates, abscisic and salicylic acids. A single-step sample preparation procedure based on mixed-mode solid phase extraction was efficiently combined with essential improvements in mobile phase composition yielding higher efficiency of chromatographic separation and MS-sensitivity. This strategy resulted in dramatic increase in overall sensitivity, allowing successful determination of phytohormones in small (less than 50 mg of fresh weight) tissue samples. The method was completely validated in terms of analyte recovery, sensitivity, linearity and precision. Additionally, it was cross-validated with a well-established GC-MS-based procedure and its applicability to a variety of plant species and organs was verified.ConclusionThe method can be applied for the analyses of target phytohormones in small tissue samples obtained from any plant species and/or plant part relying on any commercially available (even less sensitive) tandem mass spectrometry instrumentation.

Development of an Ultrahigh-Performance Liquid Chromatography–Electrospray Ionization–Tandem Mass Spectrometry Method for the Simultaneous Determination of Salicylic Acid, Jasmonic Acid, and Abscisic Acid in Rose Leaves

“…Our findings are in agreement with these results and the choice of the MCX cartridge as the best SPE cartridge for the purification of the phytohormones 1, 2, and 3 from plant material. The method currently presented offers a more effective extraction protocol when compared to the method by Balcke et al 30 The amount of leaf material used was reduced to 20 mg and combined with an easier analysis procedure. Furthermore, the method was tested on a woody plant.…”

“…Using matrix-matched calibration curves for quantitation, we worked with a woody plant species. We assume that the method is also applicable and improves the analysis of phytohormones proposed earlier 30 in other plant material (e.g., tomato, rice, etc.) as well.…”

“…In contrast, cation-exchange cartridges MCX and WCX and the generic cartridge HLB retained all three phytohormones examined. Balcke et al 30 developed a purification method similar to the method presented in this paper. They tested 11 types of SPE devices and found strong cation-exchange solid-phase material to give the best recovery results.…”

“…Instead, methanol is the preferred solvent, allowing for efficient plant cell penetration during extraction. 31,32 Because methanol produced the best recoveries in preliminary experiments (data not shown), experiments were performed using methanol alone 30 and with different proportions of ultrapure water [methanol/water, 75:25; 20 methanol/water/formic acid, 75:20:5; 29 and methanol/water/ acetic acid, 10:89:1 33 ). The results obtained are summarized in Table 2.…”

“…Many procedures and solvents have been developed and used for plant hormone extraction. 29,30 The polarity of the extraction solvent is chosen to closely match that of the target compounds; thus, the ratio of organic solvent/water is defined according to the polarity of hormones. Nonpolar solvents, such as ether, are rarely used to extract plant hormones.…”