Development and validation of a reversed-phase liquid chromatography method for the simultaneous determination of indole-3-acetic acid, indole-3-pyruvic acid, and abscisic acid in Barley (Hordeum vulgare L.)

摘要

A simple, sensitive, precise, and specific reverse HPLC method was developed and validated for the determination of plant hormones in barley (Hordeum vulgare L.). The method includes extraction in aqueous organic solvent followed by solid-phase extraction, sample evaporation, and reversed-phase HPLC analysis in a general purpose UV-visible (abscisic acid (ABA)) and fluorescence detection (indole-3-acetic acid (IAA) and indole-3-pyruvic acid (IPA)), high-performance liquid chromatography system. The separation was carried out on Zorbax Eclipse XDB C8 column (150 × 4.6 mm I.D) with a mobile phase composed of methanol and 1% acetic acid (60:40v/v) in isocratic mode at a flow rate of 1mlmin ~(-1). The detection was monitored at 270nm (ABA) and at 282nm (Ex) and 360nm (Em) (IAA, IPA). The developed method was validated in terms of accuracy, precision, linearity, limit of detection, limit of quantification, and robustness. The determined validation parameters are in the commonly acceptable ranges for that kind of analysis.