Plasma Biomarker Quantification Protocol

Blood was collected by retro-orbital bleeding into EDTA-coated tubes (450532; Greiner Bio-One, Kremsmünster, Austria) and kept on ice. Plasma was separated by centrifugation and 5 mg reduced glutathione (G4251; Sigma–Aldrich, St. Louis, MO, USA) was added per mL of plasma after centrifugation to prevent degradation of epinephrine and norepinephrine. Plasma was snap-frozen and stored at −80 °C until further use. Corticosterone, epinephrine, and norepinephrine were measured by online solid phase extraction (SPE) in combination with isotope dilution liquid chromatography tandem mass spectrometry (LC-MS/MS). Corticosterone measurement was performed essentially as described by Hawley et al. [28 (link)], using corticosterone-D4 as a stable isotope-labeled internal standard on an online SPE manager in combination with a XEVO TQ mass spectrometer (Waters, Milford, MA, USA). Epinephrine and norepinephrine were analyzed as described by Van Faassen et al. [29 (link)], using epinephrine-D3 and norepinephrine-D6 as stable isotope-labeled internal standard on a Symbiosis Pharma online SPE system (Spark Holland, Emmen, The Netherlands) in combination with a XEVO TQ mass spectrometer (Waters, Milford, MA, USA).