Synthesis of 4,5,6,7 and 2,4,5,6,7 Deuterium-labeled Indole-3-Acetic Acid for Use in Mass Spectrometric Assays

Abstract

Syntheses are described for tetra and pentadeutero indole-3-acetic acid (IAA) labeled in positions 4, 5, 6, 7 or 2, 4, 5, 6, 7 of the indole moiety. Polydeuterated IAA is proposed as an internal standard for gas chromatographic-mass spectrometric analysis of IAA by selected ion monitoring. Nanogram amounts of IAA may be assayed by monitoring the base peak of IAA at m/z = 130 (134 for d₄-IAA) and the molecular ion of the methyl ester of IAA at 189 (193 for d₄-IAA). Deuterium in positions 4, 5, 6, and 7 and, to only a slightly lesser extent, that in position 2 of IAA is retained during alkali treatment, thus permitting use of these compounds as internal standards for assay of IAA released by alkaline hydrolysis of ester and amide conjugates. The use of polydeutero internal standards separates the standards from the “isotope cluster” caused by the normal abundance of heavy isotopes and also permits use of reduced mass resolution, thus leading to a 10-fold increase in sensitivity. Tetradeutero IAA was used as an internal standard for determining free plus ester IAA in alkaline hydrolysates of Zea mays, and showed exact agreement between estimates based on the molecular ion of the methyl ester and those based upon base peak. Application of the method to measuring free IAA in the upper and lower halves of geotropically stimulated Zea shoots showed 61 ± 4% of the free IAA to be on the lower side.