Analytical considerations for quantitative determination of serotonin and its metabolically related products in biological matrices

We discuss the use of 'high-performance' liquid chromatography with electrochemical detection for the quantification of serotonin (5-hydroxytryptamine) and metabolically related products (i.e., 5-hydroxyindoleacetic acid and 5-hydroxytryptophan) in biological matrices. Two methods are described: one for routine quantification of nanogram amounts or more; the other a two-column purification-separation ('trace enrichment') technique for determining subnanogram amounts of these compounds. Various factors affecting the liquid-chromatographic separation and quantification of 5-hydroxyindoles are detailed, including selection of appropriate internal standard, sample purification, and effects of changes in mobile phase composition, e.g., ion-pair reagent, organic modifier content, and pH. We describe in detail the trace-enrichment method for determinations in the range of 25 pg to 25 ng, and discuss the general factors to consider when developing or modifying an assay for specific applications.