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Characterization of indole-3-acetic acid in cambial zone tissue by ultra-performance liquid chromatography-tandem mass spectrometry

Renan Marcel Bonilha Dezena, Renata Mauri, Fabio Alessandro Proença Barros, Paulo César Pires Rosa.

Abstract
The developmental plasticity of plants according to changes in their growth conditions is mediated by signaling molecules called hormones. The major classes of plant hormones are auxin, gibberellins, abscisic acid, and ethylene. Among these, the most important is indole-3-acetic acid (IAA). Quantification of IAA in an extract of approximately 10 mg obtained from the cambial zone of Eucalyptus leaves was performed by ultra-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (UPLC/ESI-MS/MS). The leaves were extracted using acetone, and the extract was analyzed using a reverse-phase column (Acquity UPLC BEH C18, 2.1 × 100 mm, 1.7 µm) at a flow rate of 0.2 mL min-1 with gradient elution of an aqueous mobile phase (containing 0.1% formic acid) with methanol. This gradient elution provided an excellent performance in terms of specificity/selectivity, linearity, precision, and ruggedness/stability. Additionally, the run time was short (6 min), with excellent linearity (R2 > 0.99) in the range of 10–70 ng mL-1. The structure of IAA was elucidated using UPLC/ESI-MS/MS, operating and quantifying in multiple reaction monitoring (MRM) mode.

Key words: Ultra-performance liquid chromatography, mass spectrometry, indole-3-acetic acid (IAA), Eucalyptus, hormone analysis.


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