Mass Spectrometric Analysis of Tobacco-specific Nitrosamine Hemoglobin Adducts in Snuff Dippers, Smokers, and Nonsmokers

Hemoglobin adducts of the carcinogenic tobacco-specific nitrosamines 4-(methylnitrosaminoH-(3-pyridyl)-l-butanone and N’-nitrosono:nico-tine were quantified in blood samples collected from snuff dippers, smokers, and nonsmokers. Mild base treatment of hemoglobin adducted by4-(ethylnitrosamino)-1-(3-idyH-butanoneor'sonorni-cotinereleases4-hydroxy-l-(3-pyridyl)-l-butanone (HPB). HPB was enriched by solvent partitioning and derivatized to its pentafluoroben-zoate. After purification by high performance liquid chromatography, HPB-pentafluorobenzoate was analyzed by capillary column gas chromatography with detection by negative ion chemical ionization mass spectrometry and selected ion monitoring. [4,4-D2]HPB was used as internal standard. The detection limit for HPB-pentafluorobenzoate was approximately 100 amol/injection or 5 fmol/g hemoglobin. Mean adduct levels (fmol HPB/g hemoglobin) were 517 ²153 in snuff dippers, 79.6 ±smokers, and 293 ± nonsmokers. Adduct levels in snuff dippers and in a subgroup of smokers were higher than would have been predicted solely based on estimates of exposure to tobacco-specific nitrosamines. The results of this study provide the first measurements of tobacco-specific nitrosamine hemoglobin adducts in humans and suggest new approaches to understanding the metabolic activation of 4-(methyl-nitrosamino-1--pyridyl)-l-butanone and N'nornicotine in humans.