4.4. Gibberellin Content Measurement by Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry

The sample preparation and analysis of GAs were performed according to the method described in [48] with some modifications. Briefly, tissue samples of 26–60 mg DW (three independent technical replicates of each of the tree biological samples) were ground to fine consistency using 3-mm zirconium oxide beads (Retsch GmbH & Co. KG, Haan, Germany) and an MM 301 vibration mill at a frequency of 30 Hz for 3 min (Retsch GmbH & Co. KG, Haan, Germany), with 1 mL of ice-cold 80% acetonitrile, containing 5% formic acid as extraction solution. The samples were then extracted overnight at 4 °C using a benchtop laboratory rotator Stuart SB3 (Bibby Scientific Ltd., Staffordshire, UK), after adding 17 internal GA standards ([2H2]GA1, [2H2] GA₃, [2H2]GA4, [2H2]GA5, [2H2]GA6, [2H2]GA7) purchased from OlChemIm, Olomouc, Czech Republic. The homogenates were centrifuged at 36,670× g and 4 °C for 10 min; corresponding supernatants further purified using reversed-phase and mixed mode SPE cartridges (Waters, Milford, MA, USA) and analysed by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS; Micromass, Manchester, UK). GAs were detected using multiple-reaction monitoring mode of the transition of the ion [M–H]- to the appropriate product ion. Masslynx 4.1 software (Waters, Milford, MA, USA) was used to analyse the data and the standard isotope dilution method [49] was used to quantify the GAs levels.