4.8. Phytohormone Analysis

Quantitative analysis of phytohormones in leaf tissue was performed by a high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC–ESI–MS/MS) [10,44]. Brief, 50 mg of fresh leaves in a 2-mL tube was frozen in liquid nitrogen and ground using a Tissuelyser II (Qiagen). The ground sample was extracted with 500 μL of extraction solvent, 2-propanol/H₂O/concentrated HCl (2:1:0.002, v/v/v). Dichloromethane (1 mL) was added to the supernatant, and this was then centrifuged at 13,000× g for 5 min at 4 °C. The lower phase, which was poured into a clean screw-cap glass vial, was dried under nitrogen and dissolved in pure methanol. The completely dissolved extract, ensured by vortexing and sonicating, was transferred to a reduced volume liquid chromatography vial. Hormones were analyzed by a reverse phase C18 Gemini high-performance liquid chromatography (HPLC) column for HPLC–ESI–MS/MS analysis. The chromatographic separation of hormones and its internal standard from the plant extracts was performed on an Agilent 1100 HPLC (Agilent Technologies), Waters C18 column (15,092.1 mm, 5 l m), and API3000 MSMRM (Applied Biosystems), using a binary solvent system comprising 0.1% formic acid in water (Solvent A) and 0.1% formic acid in methanol (Solvent B) at a flow rate of 0.5 ml/min.