After oxidation of indole-3-acetic acid with ceric nitrate in the presence of hydroxylamine, a stable colored complex extractable to isoamyl alcohol is formed. This reaction is useful for the calorimetric estimation of indole-3-acetic acid in biological mixtures. The colored complex (whose chromophore probably has similar structure as the product of the well known Salkowski's reaction) is relativeiy stable in light. The specificity of the method is discussed.
Except for several modifications of laborious bioassays (l-3) or unspecific direct fluorometric assays (4,5) and except for some chromatographic methods (4,6,7), there are only a few useful calorimetric methods available for the estimation of indole-3-acetic acid. These methods are based on catalytic oxidation of indole-3-acetic acid with the formation of colored products. The same or similar colored products can be prepared from indole-3-acetic acid, for example, by oxidation with HNOs, FeC&, HClO, HBrO, HCIOs, H&rod, HzOz, CuSOa, HgC& and many others, especially in the presence of . All these colored products are usually unstable and/or do not obey Lambert-Beer's law in a wider range of concentrations.
Thus, several methods have been proposed to reduce the instability of the color, such as addition of gum arabic, HCIOd, arrangements of standard reaction time or light conditions, etc. (L2-17). But the methods stiI1 remained sensitive to reaction conditions and very sensitive to the interference of cysteine, ascorbic acid, polyphenols and other compounds ( 18).
The formation of orange-red indole-a-pyrone by the condensation of indole-3-acetic acid with acetic anhydride or glyoxyhc acid (19-21) was also used as a method for the calorimetric and fluorometric estimation of indole-3-acetic acid. This method is specific but is very laborious, very sensitive to reaction conditions and extractable impurities, and the final product under the conditions of the reaction is unstable. This makes it difficult to obtain accurate reproducible results.
I have found that indole-3-acetic acid is oxidized by ceric salts in the presence of hydroxylamine with the formation of a stable colored complex. The complex is quantitatively extractable to isoamyl alcohol and has sharp absorption maximum in visible light. The color complex 134 Copyright (Q I976 by Academic &es, inc. All rights of reproduction in any form reserved.