Method for the determination of indole-3-acetic acid and related compounds of L-tryptophan catabolism in soils.

An optimized method for the determination of substances occurring in auxin metabolism and L-tryptophan (TRP) catabolism was developed. It is based on solid-phase extraction (SPE), two isocratic reversed-phase high-performance liquid chromatographic (HPLC) separations at different liquid phase conditions and the simultaneous detection of fluorescence and UV absorbance at different wavelengths. Advantages of the proposed method are: the solvent (ethanol) and liquid phase (containing 2-propanol) provide optimum stability and selectivity; almost no toxic wastes are produced; no time-consuming liquid-liquid extractions (LLE), derivatization procedures or column re-equilibration (obligatory for gradient systems) are necessary, no need for antioxidants, ion-pair or derivatization reagents; recovery rates of the SPE system are superior to LLE effeciencies; high sensitivity, selectivity and identification capacity are provided by the proposed HPLC and detection system. By measuring various chromatographic and spectral parameters simultaneously, the determination reliability is improved. The characteristic chromatographic and spectral data for selected indole derivatives and TRP catabolites are presented. In samples from two different soils that were tested with the proposed method, the actual contents of TRP were 1.4 and 5.8 μg/g dry soil. In addition, traces of indole-3-acetic acid (IAA) could be detected. When TRP was added, IAA was the predominant catabolite in both soils, and reached values of 2.9 and 8.0 μg/g dry soil. In addition to IAA, indole-3-ethanol, indole-3-aldehyde, indole-3-carboxylic acid, indole-3-lactic acid, anthranilic acid and traces of indole-3-acetamide were identified and determined