Determination of substituted indole derivatives by ion suppression-reverse-phase high-performance liquid chromatography.

A method for the separation of substituted indole derivatives has been developed by the use of ion suppression-reversed-phase high-performance liquid chromatography (IS-HPLC). Signal response, selectivity (alpha), retention times (tR), and capacity factors (k') were monitored by varying the mobile phase with respect to methanol composition and pH. Chromatographic parameters including tR, k', K (distribution coefficients), alpha, number of theoretical plates, height equivalent to theoretical plates, and column resolution were calculated and assessed in regard to the suitability of four stationary phases in the analysis of 20 substituted indole derivatives. This work has established the chromatographic foundation needed to analyze over 10 specific enzyme reactions involved in the microbial and plant metabolism of auxins. Quantitative studies on the stability of auxins were possible by employing IS chromatography (ISC). The application of the developed IS chromatographic technique was employed to detect indole-3-acetic acid derived from L-tryptophan in a fluorescent pseudomonad culture.