Chromatographic Analysis of Naturally Fluorescing Compounds: I. Rapid Analysis of Nanogram Amounts of Indoles in Physiologic Fluids

Abstract

Abstract A chromatographic separation system has been developed for determination of various indole derivatives in physiologic fluids. A coupled-column configuration is used in which a 0.22 x 25 cm, jacketed, stainless-steel anion-exchange column is connected directly to a 0.22 x 50 cm, jacketed, stainless-steel cation-exchange column. The indole compounds are eluted with an ammonium acetate—acetic acid buffer that is 4 molar in ammonia and 5.8 molar in total acetate. An Aminco-Bowman spectrophotofluorometer, with an excitation setting of 292 nm and an emission setting of 330 nm, is used as the detector. The column temperature is 60°C. Effects of pH, temperature, and ionic strength on elution characteristics of the indole compounds have been investigated. The system can provide baseline separation of 50 to 100 ng of 5-hydroxytryptophan, tryptophan, 5-hydroxyindoleacetic acid, serotonin, indoleacetic acid, and tryptamine in 3 h. Use of the system in analyzing urine is illustrated.