On the accurate measurement of serotonin in whole blood.

Levels of serotonin (5-hydroxytryptamine; 5-HT) in whole blood reflect the levels in the platelets, but there are problems with instability of 5-HT in measurement of whole blood extracts. We therefore developed and validated an assay for whole blood 5-HT using HPLC with fluorometric detection. The procedure involved collecting blood in EDTA vacutainer tubes and aliquoting it with internal standard, alpha-methyl-5-HT, before freezing. The aliquots were thawed on ice and proteins precipitated with perchloric acid containing EDTA and ascorbic acid. The 5-HT was stable in fresh whole blood for 24 h after sampling when stored at room temperature, refrigerated, or ice-cooled. Measurements of the levels of 5-HT in blood collected in heparin, citrate or EDTA were similar, but in blood collected in ACD-buffer the levels were approximately 25% lower. Cell lysis by sonication decreased the 5-HT levels, but this was compensated for by the internal standard. Determinations of 5-HT by HPLC were in good agreement with those by gas chromatography - mass spectrometry. The intra-individual variability between days was 3.6% (n=6). However, single and repeated ingestion of bananas, which are rich in 5-HT, elevated 5-HT in whole blood, indicating dietary influences on platelet 5-HT.