Development of a Nonradioactive Platelet Serotonin Uptake and Release Assay by Micro-Liquid Chromatography Tandem Mass Spectrometry Using Minimal Blood Volume

Abstract

Abstract Objectives Analysis of platelet functional responses to stimuli is presently quite limited with respect to measurement of dense granule secretion. We sought to develop a nonradioactive assay of stimulated serotonin release using liquid chromatography tandem mass spectrometry (LC-MS/MS). Methods Citrated whole blood (200 μL) was incubated with deuterated serotonin (d45-HT). Following uptake by platelets, blood was diluted 10-fold and aliquots were incubated with platelet stimuli. Following stimulation, blood was further diluted, centrifuged, and supernatant was assayed for released d45-HT by micro-LC–MS/MS. Results This study demonstrated a broad linear range of 50 to 2,000 pg/mL d45-HT, with a total precision of less than 15.0% coefficient of variation at all quality control levels and a limit of quantitation of 50 pg/mL. Conclusions Quantification of d45-HT by micro-LC–MS/MS assay offers a highly sensitive, nonradioactive methodology for quantitating platelet serotonin uptake and dense granule secretion, requiring only small volumes of patient blood.