Tobacco-specific nitrosamine-hemoglobin adducts

Tobacco-specific nitrosamines are a group of carcinogens formed by nitrosation of nicotine and other tobacco alkaloids. Two of these compounds, N'-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), have been implicated in the etiology of cancers of the oral cavity, lung, pancreas, or esophagus caused by use of tobacco products. Based on the amounts of NNN and NNK in tobacco products, human exposure to these carcinogens is similar to the doses that cause tumors in laboratory animals. NNN and NNK are converted to highly electrophilic intermediates by enzymatic hydroxylation of the carbons α- to the N-nitroso group. Quantitation of hemoglobin adducts of NNN and NNK provide an index of their uptake and metabolic activation. The method consists of four major steps: (1) isolation of hemoglobin and treatment with base to release HPB, (2) solvent partitions to enrich HPB, (3) derivatization of HPB to its pentafluorobenzoate, which can be detected with great sensitivity and selectivity by capillary column gas chromatography–mass spectrometry with negative-ion chemical ionization detection and selected ion monitoring (GC–MS–NICI–SIM), and and (4) quantitation by GC–MS–NICI–SIM.