Open Access Open Badges Methodology

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

Gerd Ulrich Balcke1, Vinzenz Handrick12, Nick Bergau1, Mandy Fichtner1, Anja Henning1, Hagen Stellmach1, Alain Tissier1, Bettina Hause1 and Andrej Frolov13*

Author Affiliations

1 Department of Cell and Metabolic Biology, Leibniz Institute of Plant Biochemistry, Weinberg 3, Halle (Saale), 06120, Germany

2 Present address: Max Planck Institute for Chemical Ecology, Department of Biochemistry, Hans-Knoell-Str. 8, Jena, 07745, Germany

3 Faculty of Chemistry and Mineralogy, Institute of Bioanalytical Chemistry, Centre for Biotechnology and Biomedicine, Leipzig University, Deutscher Platz 5, Leipzig, 04103, Germany

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Plant Methods 2012, 8:47  doi:10.1186/1746-4811-8-47

Published: 22 November 2012



Phytohormones 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.


Here 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.


The 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.

Phytohormones; Jasmonates; LC-MS/MS; Solid phase extraction (SPE); Quantification; Electrospray ionization; Plant stress response; Wounding