Quantitative ABA Analysis via HPLC

Seed ABA was extracted following the methods described by Tombesi et al.^{57 (link)}. Analyses were performed on an Agilent 1260 HPLC (Agilent Ltd., California, USA) equipped with an Agilent C18 ZORBAX (5 mm × 150 mm × 4.6 mm) column at a flow rate of 0.013 ml s⁻¹. The injection volume was 10 μL and the detection was made at 265 nm. The mobile phase of acetonitrile/methanol/water (8:40:52 v/v/v, 0.6% acetic acid) was previously filtered and degassed. ABA was identified by comparing the retention times with those of standard ABA, and the peak area quantified by an external standard method. Stock solutions of ABA standards was prepared by diluting a solution (0.1 mg mL⁻¹ in acetonitrile) to obtain a range of concentrations from 0.1 to 10 μg mL⁻¹.