Identification and analysis of a nicotine-derived N-nitrosamino acid and other nitrosamino acids in tobacco

Research on carcinogenic, tobacco-specific N-nitrosamines (TSNA) led to the identification and analysis of 4-(methylnitrosamino)-4-(3-pyridyl)butyric acid (iso-NNAC) in tobacco and tobacco smoke. In order to isolate iso-NNAC, an aqueous tobacco extract at pH 4 was partitioned with ethyl acetate after the Other N-nitrosamino acids and TSNA were removed at pH 2 and pH 9 respectively. The structure of iso-NNAC was confirmed by GC-MS after enrichment of the methylated pH 4 fraction by chromatography on an alumina column. Iso-NNAC, 3-(methylnitrosamino)propionic acid, 4-(methylnitrosamino)butyric acid, N-nitrosoproline and TSNA were determined by GC-TEA in various smokeless tobaccos as well as in reference cigarettes. The levels of iso-NNAC in tobacco products ranged from 0.01 p.p.m. in chewing tobacco to 0.95 p.p.m. in dry snuff. The transfer rate of unchanged iso-NNAC into the mainstream smoke of a non-filter cigarette amounted to 0.85%. Iso-NNAC does not induce DNA repair in primary rat hepatocytes and is inactive as a tumorigenic agent in strain A mice.