Evaluation of fluorometric assay methods for serotonin in platelets. Plasma and whole blood samples by comparison with GC-MS-SIM Technique

Abstract

Different methods have been used for the determination of serotonin (5HT) in human blood platelets, whole blood and plasma samples, but there have been very few reports on inter-method correlations. We now report the evaluation of fluorometric method using o-phthalaldehyde (OPT), with 3H-5HT as internal standard and GC-MS method using deuterated (d4-) 5-HT as internal standard. Basic extracts (pH 9.5) of sonicated platelet samples, hemolyzed blood or plasma samples are converted to trifluoroacetyl (TFA) or pentafluoropropionyl (PFP) derivatives. An aliquot of this derivatized sample (10%) was used for 3H count for determining the percent recovery, a second aliquot for GC-MS, and the rest for quantitation of OPT fluorescence (Emax360 millimicrons and Fmax 470 millimicrons). In another series, duplicate samples are extracted, one for fluorometry and the second for GS-MS with d4-5-HT as internal standard. Ions m/e 351 and 354 are monitored for the TFA and m/e 451 and 454 for PFP derivatives. The correlation between OPT fluorescence and GS-MS methods was good (0.95) for the platelets, but was poor (0.63) for whole blood and plasma samples. Thin layer chromatography of whole blood and plasma extracts showed multiple spots which yield characteristic fluorescence with OPT spray having same Emax or high background fluorescence. Serotonin levels in platelet samples of normal controls, and patients with different diagnoses of psychiatric disorders are presented.