Validated method for phytohormone quantification in plants

Phytohormones are long time known as important components of signaling cascades in plant development and plant responses to various abiotic and biotic challenges. Quantifications of phytohormone levels in plants are typically carried out using GC or LC-MS/MS systems, due to their high sensitivity, specificity, and the fact that not much sample preparation is needed. However, mass spectrometer-based analyses are often affected by the particular sample type (different matrices), extraction procedure, and experimental setups, i.e., the chromatographic separation system and/or mass spectrometer analyser (Triple-quadrupole, Iontrap, TOF, Orbitrap). For these reasons, a validated method is required in order to enable comparison of data that are generated in different laboratories, under different experimental set-ups, and in different matrices. So far, many phytohormone quantification studies were done using either QTRAP or Triple-quadrupole mass spectrometers. None of them was performed under the regime of a fully-validated method. Therefore, we developed and established such validated method for quantification of stress-related phytohormones such as jasmonates, abscisic acid, salicylic acid, IAA, in the model plant Arabidopsis thaliana and the fruit crop Citrus sinensis, using an Iontrap mass spectrometer. All parameters recommended by FDA (US Food and Drug Administration) or EMEA (European Medicines Evaluation Agency) for validation of analytical methods were evaluated: sensitivity, selectivity, repeatability and reproducibility (accuracy and precision).