Trace level analysis of indole-3-acetic acid from light- grown, highly pigmented leaf tissue

Abstract

A new chromatographic method has been developed for the isolation and quantitation of trace levels of the plant hormone, indole-3-acetic acid (IAA). The individuals steps in the method were selected and adjusted specifically to remove the large amounts of interfering substances present in highly pigmented, light-grown plant tissue. This method employs a rapid extraction and preparative cleanup by open column chromatography, followed by preparative reversed-phase ion-pair high- performance liquid chromatography to remove the bulk of the interfering substances. The latter step maximizes the resolution of the IAA from its native matrix by interaction with the tetrabutylammonium (TBA) acetate ion-pair at the ideal pH for the quantitative formation of the TBA—IAA ion-pair. Two additional high-performance liquid chromatographic separations ultimately produce a baseline resolved IAA peak, which is quantified by fluorescence and amperometric detection. Identity of the IAA peak was verified by capillary gas—liquid chromatography with nitrongen—phosphorus detection and by gas chromatography—mass spectrometry analysis. The method is illustrated here by the quantitation of IAA from 1 g fresh weight of highly-pigmented, light-grown cotton leaf tissue. The limit of detection is as low as 1 ng of IAA from 1 g fresh weight with average recoveries of aroung 50% at the quantitation step. Although the method was designed to quantitate IAA, the extraction and high-performance liquid chromatographic steps can be used to prepare purified IAA samples for esterification and gas chromatography—mass spectrometry analysis.