HPLC Linked Electrospray Tandem Mass Spectrometry: A Rapid and Reliable Method to Analyse Indole-3-Acetic Acid Metabolism in Bacteria

Electrospray tandem mass spectrometry in combination with high-performance liquid chromatography can be used for the simultaneous detection and quantification of indole-3-acetic acid, indole-3-methanol, tryptophan, indole-3-acetamide, indole-3-ethanol, tryptamine, indole-3-acetaldehyde, indole-3-acetonitrile, indole-3-aldehyde, indole-3-lactic acid, indole-3-pyruvate and anthranilate on an individual basis. Although methylation is not an absolute prerequisite for analysis of most carboxyl-type indole compounds, samples were methylated before analysis and this improved the detection limits for tryptophan, indole-3-acetic acid, anthranilate and indole-3-lactate up to 1000-fold. Moreover, owing to this methylation no ion suppression conditions, necessary for chromatography of organic acids, have to be used and stability of indole-3-pyruvate was achieved. A detection limit of 100 fmol indole-3-acetic acid methyl ester injected on-column was obtained under multiple reaction monitoring conditions. In view of the analysis of a large number of samples, the chromatographic conditions selected are a compromise between speed of analysis and resolution.