Ultra-high performance liquid chromatography coupled to high resolution Orbitrap mass spectrometry for metabolomic profiling of the endogenous phytohormonal status of the tomato plant

Phytohormones are key signalling biomolecules and are of particular interest because of their regulating role in numerous physiological and developmental plant processes. Since the plant response to a given stimulus results amongst others from the complex interaction between phytohormones, there is a mounting interest for multiple phytohormone analysis. Therefore, with the primary aim of profiling the hormonal status of the tomato plant, a generic extraction protocol and an U-HPLC–Orbitrap-MS analysis were developed and validated for both tomato fruit and leaf tissue. To this end, eight phytohormones were considered, i.e. gibberellic acid, indol-3-acetic acid, abscisic acid, jasmonic acid, salicylic acid, zeatin, N6-benzyladenine and epibrassinolide, representing the major hormonal classes. The sample pre-treatment involved liquid extraction with a buffer of methanol, ultrapure water and formic acid (75:20:5, v/v/v), after which the extract was purified by means of an Amicon® Ultra centrifugal unit. Subsequently, analytes were chromatographically separated on a sub-2 μm particles Nucleodur Gravity C18 column and detected by an Exactive™ high-resolution mass spectrometer. Validation of the analytical method demonstrated that linearity (≥0.99), precision (CV ≤ 15%) and mean corrected recovery (between 80% and 110%) performed well for the majority of the eight targeted phytohormones. In addition, the generic nature of the extraction protocol and the full scan approach of the Orbitrap mass spectrometer allowed metabolomic profiling of the hormonal status of the tomato plant.