89: Development of a Novel Micro LC-MS/MS Method for Assay of Agonist-Induced Release of Endogenous Serotonin from Human Platelets

Abstract

Objective: Assessment of platelet function testing is often limited when platelet counts are low, as typically found in pediatric and thrombocytopenic populations. We aim to develop a high sensitive method for measuring the release of endogenous serotonin by mass spectrometry to address this limitation. Methods: 100 μL citrated blood was diluted 10-fold to 1.0 mL with Modified HEPES/Tyrode Buffer (MTB). 80 μL of the diluted blood was then incubated with a platelet agonist, such as ADP, unstirred, in a total volume of 100 μL for 10 minutes at 37°C. Following incubation, the sample was centrifuged to obtain platelet releasate (supernatant fraction) for further analysis. An unstimulated blood sample was lysed with perchloric acid for determination of total platelet serotonin content. Deuterated serotonin (C/D/N isotope, Quebec) was used as an internal standard (IS). Calibrators were prepared by spiking serotonin standards (Sigma) into MTB. IS and perchloric acid were added to the calibrators and samples, and were extracted with reverse phase cartridges. The eluents were dried under nitrogen, and the residues were dissolved in 50µL of 1:9 methanol/water (v/v) prior to an injection into the MicroLC-6500 QTrap (SCIEX, MA). Separation on a micro C18 column (SCIEX, MA) with a gradient of 2-35% methanol (0.1% formic acid) and H 2 O (0.1% formic acid) at a flow rate of 25 µL/min. Results: Serotonin and IS were detected in positive mode using the following transitions: serotonin: 177/160 (quantifier), 177/132 (qualifier); IS: 184/164. Our method showed good linearity over a concentration range of 9-869 pg/mL (r 2 > 0.999). Recovery study using known standards showed excellent recovery (90%-110%). Within-day precision study showed CVs < 15% for the 3 level QCs. ADP-induced serotonin release from platelets typically reached a maximal level of approximately 50-150 pg/10 6 platelets at a concentration of 10 μmol/L ADP. Conclusions: The approach we have developed permits extensive study of platelet-dense granule secretion from extremely small blood samples. Virtually all the platelet-stimulating agents routinely used in classic platelet aggregation studies can be employed in parallel, and even in multiple concentrations, from just 100 μL of citrated blood. Moreover, by the approach of assaying endogenous platelet serotonin, radioactivity can be completely avoided. Finally, we anticipate that the high sensitivity of this method should significantly extend the opportunity to assess platelet-dense granule secretion in thrombocytopenic patients and in the pediatric population.