The ABA and proline extraction was performed on 10mg of freeze-dried leaf tissue as described by Forcat et al. (2008 (link)). The samples were analyzed for ABA and proline using LCMS/MS, and were filtered through a 0.45μm cellulose acetate syringe. The phytohormones separation was done using a C18 column (ZORBAX Eclipse Plus). An injection of 2 μl was loaded onto the C18 column (1.8μm particle size, 2.1mm inner diameter, and 50mm long) at a flow rate of 0.2 ml/min and the column temperature was kept at 35°C. The liquid chromatography was connected to an Agilent Technologies Mass Spectrometry (6420 Triple Quad detector). For elution, solvent A consists of formic acid (0.1%) with distilled water and solvent B consists of an LCMS grade acetonitrile were used. The analytical procedure was as follows.
Solvent A was used (5min), then the gradient from 0 to 100% solvent B was used (5–20min), after the solvent B was kept constant (5min) and at 25.1min solvent A was 100% was used for 30min. During the analysis with LC–MSMS only negative polarity mode was used for ABA and Proline analysis. For fragmentation, nitrogen gas was used. The capillary voltage was 4,000V, the gas flow was 8 L/min, the gas temperature was 300°C and the nebulizer pressure was 45 psi.
Solvent A was used (5min), then the gradient from 0 to 100% solvent B was used (5–20min), after the solvent B was kept constant (5min) and at 25.1min solvent A was 100% was used for 30min. During the analysis with LC–MSMS only negative polarity mode was used for ABA and Proline analysis. For fragmentation, nitrogen gas was used. The capillary voltage was 4,000V, the gas flow was 8 L/min, the gas temperature was 300°C and the nebulizer pressure was 45 psi.
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