title: High-performance liquid chromatography-tandem mass spectrometry assay of fatty acid amide hydrolase (FAAH) in blood: FAAH inhibition as clinical biomarker.
abstract: Fatty acid amide hydrolase (FAAH) is one of the main enzymes responsible for the degradation of the endocannabinoid anandamide (N-arachidonoylethanolamine, AEA). FAAH inhibitors may be useful in treating many disorders involving inflammation and pain. Although brain FAAH may be the relevant target for inhibition, rat studies show a correlation between blood and brain FAAH inhibition, allowing blood FAAH activity to be used as a target biomarker. Building on experience with a rat leukocyte FAAH activity assay using [³H]AEA, we have developed a human leukocyte assay using stably labeled [²H₄]AEA as substrate. The deuterium-labeled ethanolamine reaction product ([²H₄]EA) was analyzed by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) in the positive electrospray ionization (ESI) mode. The response for [²H₄]EA was linear from 10 nM to 10 μM, and the analysis time was less than 6 min/sample. Results using the [²H₄]AEA and HPLC-MS/MS method agreed well with those obtained using the [³H]AEA radiometric assay. In addition to using a nonradioactive substrate, the HPLC-MS/MS method had increased sensitivity with lower background. Importantly, the assay preserved partial FAAH inhibition resulting from ex vivo treatment with a time-dependent irreversible inhibitor, suggesting its utility with clinical samples. The assay has been used to profile the successful inhibition of FAAH in recent clinical trials.
authors: Pfizer Global Research & Development, Groton, CT 06340, USA. udeni.yapa@pfizer.com
authors: Udeni
authors: U
authors: Yapa
authors: Jeffery J
authors: JJ
authors: Prusakiewicz
authors: Ann D
authors: AD
authors: Wrightstone
authors: Lori J
authors: LJ
authors: Christine
authors: Joe
authors: J
authors: Palandra
authors: Elizabeth
authors: E
authors: Groeber
authors: Arthur J
authors: AJ
authors: Wittwer
mesh_terms: D000581:Amidohydrolases / Q000037:antagonists & inhibitors / Q000097:blood*; D015415:Biomarkers / Q000097:blood; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D007962:Leukocytes / Q000201:enzymology*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*; D000099252:Fatty Acid Amide Hydrolases
chemical_list: D000581:Amidohydrolases; D015415:Biomarkers; D000099252:Fatty Acid Amide Hydrolases