Abstract P180: Quantification of Substance P in Human Blood by Mass Spectroscopy

Abstract

Background: Substance P (SP), a tachykinin, may contribute to the effects of ACE inhibitors. Measurement of SP has been limited by its short half-life and cross-reactivity with metabolites. Here we quantify SP in human blood using mass spectroscopy (MS). Methods: Venous blood was obtained from healthy subjects before and during arterial infusion of SP (2, 4, and 8 pmol/min). Venous blood (3 mL) was immediately added to 9 mL chilled ethanol to denature proteases. After 30 min at 4°C, samples were centrifuged and the supernatant stored at -70°C. 13 C 6 , 15 N 4 SP internal standard was then added to the ethanolic supernatant. Samples were purified with Nexus and C 18 Sep-Pak cartridges. Sample was further resolved by UPLC (40°C) on a C18 column using solvent mixture of 5% acetonitrile/0.1% formic acid at a flow rate 0.14 mL/min to 30% acetonitrile/0.1% formic acid in 7 min with a linear gradient, and quantified by positive ESI-MS/MS on a “Vantage” triple quadrupole MS with SRM. Results: The lower limit of detection of SP was 0.1 pg. Linearity was confirmed over a 100-fold concentration range. Precision was measured by analyzing five 2-mL aliquots of pooled ethanolic supernatant from 3 volunteers collected during SP 8 pmol/min. The coefficient of variation was 2.2%. The mean SP concentration was 2.3 pg/mL at baseline and increased with intra-arterial dose (Figure). Conclusion: SP can be measured in blood using MS. This assay can be used to study the effects of ACE, DPP4, and neprilysin inhibitors on SP.