Determination of 5-hydroxytryptamine and 5-hydroxyindoleacetic acid in plasma by direct injection in coupled-column liquid chromatography with electrochemical detection.

A method is described that allows determination of 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) in platelet-poor human blood plasma after direct injection of 50-100 microliters of plasma into a coupled-column liquid chromatographic system. The chromatographic system comprised an enrichment column and two separation columns with different selectivity properties. The samples were injected into the C18 enrichment column, which was eluted with a buffer solution as the mobile phase. 5-HT, 5-HIAA and the internal standard 5-hydroxy-N-methyl-tryptamine (5-HMT) were then desorbed by a stronger mobile phase and flushed into a cation exchanger, which separated 5-HT and 5-HMT. 5-HIAA passed straight through and was switched to a C18 column where it was retarded. After 5-HT and 5-HMT had been eluted from the cation exchanger and detected, the eluate from the C18 column was directed to the detector and 5-HIAA was determined. Basic plasma levels, about 4 nM for 5-HT and 30 nM for 5-HIAA, were measured with a relative standard deviation of about 5%.