On the accurate determination of serotonin in human plasma

Serotonin (5-hydroxytryptamine, 5-HT) in blood is stored in platelets and has vascular and platelet stimulating effects when released into plasma. Accurate measurements of 5-HT in plasma are complicated by inadvertent platelet activation causing sampling artifacts and by analytical problems when determining trace levels. We developed an assay for plasma 5-HT based on solid-phase extraction (Sep-Pak C18), aqueous acetylation, pentafluoropropionylation, and negative ion chemical ionization gas chromatography-mass spectrometry. The method was able to recover 5-HT from plasma by > 90% and to quantitate with a precision of 7.5% at a level of 0.5 nmol/l. It was used to define blood sampling and sample handling procedures giving low and consistent values for 5-HT. A good blood sampling technique, adequate platelet stabilization in the test tube, and rapid high speed centrifugation of the blood resulted in low plasma levels of both 5-HT and beta-thromboglobulin (a platelet release product). Using these procedures plasma 5-HT levels in healthy volunteers were found to be 0.77 +/- 0.38 (mean +/- S.D.; range 0.27-1.49) nmol/l (n = 18), which is 4-100-fold lower than previously reported values.