High-performance liquid chromatographic assay with fluorescence detection for the evaluation of inhibitors against fatty acid amide hydrolase.

A fluorescent assay for the evaluation of inhibitors of fatty acid amide hydrolase (FAAH) is described. Microsomes from rat brain served as enzyme source. N-(2-Hydroxyethyl)-4-pyren-1-ylbutanamide was designed and synthesized as novel fluorogenic substrate. For substrate solubilization, Triton X-100 was employed. The FAAH activity was determined directly without further sample clean-up by measuring the amount of 4-pyren-1-ylbutanoic acid released by the enzyme with reversed-phase HPLC and fluorescence detection. The known FAAH inhibitors URB597, phenyl hexanoyl oxazolopyridine (PHOP) and [6-(2-methyl-4,5-diphenyl-1H-imidazol-1-yl)hexyl]carbamic acid phenyl ester were used to validate the test assay.