Determination of serotonin and 5-hydroxyindoleacetic acid in urine by reversed-phase ion-pair partition chromatography with fluorimetric detection.
The determination of serotonin and 5-hydroxyindoleacetic acid in urine is described. The compounds are separated by means of reversed-phase chromatography using tri-n-butylphosphate as the stationary phase and a mobile phase containing perchlorate as an ion-pairing agent. Detection is performed fluorimetrically. The compounds can be assayed directly, using two mobile phases with different pH values. Alternatively, both compounds can be determined with the same mobile phase after urine extraction on a small XAD-2 column for clean-up of serotonin. Two different approaches were evaluated for this XAD-2 extraction, viz.: (i) desorption of serotonin with a methanol-rich solvent after a washing step with an aqueous solvent; (ii) direct desorption of serotonin in the washing step using a solvent with a small percentage of methanol. Only the latter approach proved to be successful. The linearity of the method was investigated by standard additions to urine. The method was applied to the determination of both compounds in urine. The stability of the compounds in standard solutions and in urine was investigated under various storage conditions.
D002852:Chromatography, Ion Exchange; D006801:Humans; D006897:Hydroxyindoleacetic Acid / Q000652:urine*; D011137:Polystyrenes; D012701:Serotonin / Q000652:urine*; D013050:Spectrometry, Fluorescence
D011137:Polystyrenes; D012701:Serotonin; D006897:Hydroxyindoleacetic Acid; C003771:Amberlite XAD-2 resin