Assay Validation of Endogenous Fatty Acid Ethanolamides in Human Whole Blood

摘要

Fatty acid amide hydrolase (FAAH) is the major enzyme that catalyzes the hydrolysis of fatty acid ethanolamides (FAEs). FAEs act as lipid signaling molecules by binding to CB1 and CB2 cannabinoid receptors in the central nervous system (CNS) and in the periphery. Inhibition of FAAH is an attractive target for the treatment of pain by blocking the degradation of anandamide (AEA), the major FAE endocannabinoid. FAAH is also responsible for the catabolism of FAE noncannabinoids such as palmitylethanolamide (PEA) and oleylethanolamide (OEA). The purpose of this study was to develop a sensitive and robust LC/MS/MS method to measure AEA, PEA, and OEA in human whole blood (WB) as a potential pharmacodynamic (PD) biomarker for FAHH inhibitors for the treatment of inflammation and neuropathic pain. 0.5 mL of whole blood, spiked with 20 (mu)L of internal standards (AEA-d8, PEA-d4, and OEA-d2). Mixed with 2 mL of ACN, vortexed and incubated while rotating for 3 hours at 4 deg C. Centrifuged samples, transferred supernatant to a new tube and dried under nitrogen at room temperature. Resuspened in 30percent MeOH, then passed through Oasis HLB Solid Phase Extraction (SPE) sorbent using Rapid Trace. AEA, PEA and OEA were eluted with ACN. Samples dried under nitrogen at RT. Samples were reconstituted 50percent MeOH with 0.1percent formic acid with 2 mM NH_(4)OAc and assayed on LC/MS/MS. UFLC: (1) Shimadzu Prominence UFLC; (2) Buffer A: H_(2)O w/0.1percent formic acid with 2 mM NH_(4)OAc; (3) Buffer B: MeOH w/0.1percent formic acid with 2 mM NH_(4)OAc; (4) Gradient of 8 minutes (70percent to 82percent Buffer B); (5) Flow: 0.5 mL/min; (6) Column: Waters 2.5 (mu)m C18-BEH, 2.1X50 mm. Sciex API 4000. Positive mode – (AEA, PEA and OEA) Multiple Reaction Monitoring (MRM) AEA m/z 348.3/62.0, AEA-d8 m/z 356.3/63.0 PEA m/z 300.4/62.0, PEA-d4 m/z 304.4/62.0 OEA m/z 326.4/62.0, OEA-d2 m/z 328.4/62.0