Relation of pH to fluorescence of serotonin, melatonin, and other indole compounds reacted with o-phthaldialdehyde.

We distinguished serotonin, malatonin, and other indole compounds by their markedly different fluorescence behavior when heated with o-phthaldialdehyde in different concentration of HCl, or after the subsequent addition of alkali. Fluorescence may increase, disappear, or change from blue to orange-red. Only melatonin developed significant fluorescence (at less than 0.5 mug/liter) when heated with the reagent in 50 mmol/liter HCl. Serotonin and 5-hydroxyindole compounds with a C-3 aliphatic chain lost their blue fluorescence when brought from strong to weakly acid and alkaline pH. Intense reddish fluorescence developed in strongly alkaline solution. C-5 methoxy derivatives, such as melatonin, maintained their blue fluorescence in alkaline solution. Differences in fluorescence color (and RF) also characterized these compounds on silica gel plates. The use of toluene decreased blank fluorescence, while sample preparation in dim light, as often recommended, resulted in diminished and less-stable fluorescence.