Separation of 5-hydroxyindole-3-acetic acid and indole-3-acetic acid in urine by direct injection on a reversed-phase column containing a hydrogen-accepting stationary phase.

The separation of 5-hydroxyindole-3-acetic acid and indole-3-acetic acid from urine is demonstrated. The urine is injected directly on a liquid chromatographic column which is coupled to a flow-through fluorescence detector or a UV detector. A hydrogen-accepting stationary phase, tributyl phosphate (TBP), which retains carboxylic acid selectivity, was more selective than more common reversed-phase systems with alkyl-bonded stationary phases. The mobile phases were buffers, sometimes buffers containing methanol or acetonitrile. A simple method of removing late peaks in the chromatogram, in order to reduce analysis time, is also presented.