During the metabolic characterization of compound I, 2-{6-cyano-3-[(2,2-difluoro-2-pyridin-2-ylethyl)amino]-2-oxopyrazin-1(2H)-yl]-N-[(3-fluoropyridin-2-yl)methyl]acetamide, evidence was obtained for extensive oxidative bioactivation of the pyrazinone ring system and some of the resulting metabolites were apparently devoid of the cyano moiety. Two assays, a spectrophotometric and a high-pressure liquid chromatography (HPLC) pre-column derivatization method, were evaluated for their ability to detect and quantify cyanide that is metabolically generated from liver microsomal incubations. When I was incubated (45 microM) in the presence of NADPH-fortified human liver microsomes for 2h, 7.5 microM of cyanide was detected using the spectrophotometric assay and 8.9 microM was measured using the HPLC methodology. Overall, the results from the two assays appeared to agree reasonably well with each other. However, the HPLC assay was the preferred method for the evaluation of cyanide formation in vitro due to its sensitivity, reliability, and ease of use.