The use of deuterium-labeled precursors allowed us to specifically identify NNN as formed from the precursors added to saliva prior to incubation, obviously thus eliminating concerns that NNN measured in saliva of our nonsmoking volunteers could have other sources, for example, exposure to secondhand smoke. We also took particular precautions to ensure that artifactual nitrosation did not take place after the incubation during sample preparation for LC-MS/MS analysis. The method used in this study is practically identical to the one that has been previously shown not to result in artifactual NNN formation (Stepanov & Hecht, 2005; Stepanov, Carmella, Briggs, et al., 2009). Nitrosation of [pyridine-D4]nicotine during saliva incuba tion experiments was minimal, which is in agreement with kinetic studies showing that nornicotine is nitrosated to form NNN much more efficiently than nicotine (Mirvish, Sams, & Hecht, 1977).

These results also suggest that nornicotine, and not nicotine, is the major precursor of endogenously synthesized NNN in some users of oral NRT products. There was significant interindividual variation in the amount of [pyridine-D4]NNN formed upon incubation with [pyridine-D4]nornicotine of saliva collected from nonsmoking volunteers (Table 1). Endogenous formation of N-nitrosamines in humans can be greatly affected by various dietary and host factors. For example, ascorbic acid and vitamin E inhibit endogenous nitrosation in humans, whereas some phenolic compounds can both inhibit and catalyze N-nitroso compound formation, depending on a variety of factors (Bartsch, Ohshima, & Pignatelli, 1988).

The time of nicotine or nornicotine contact with nitrosating agents relative to the time of food consumption is also critical: under fasting conditions, the concentration of nitrite in saliva varies between 10 and 1000 ��mol/L, increasing 2- to 5-fold for several hours after the ingestion of nitrate-containing food (McColl, 2005). In addition, certain oral microflora can catalyze nitrosation reactions at neutral pH (Jiebarth et al., 1997), and the use of antiseptic mouthwash reduces endogenous nitrosamine formation by 62%�C74% (Shapiro, Hotchkill, & Roe, 1991). The potential role of oral microflora AV-951 in the formation of [pyridine-D4]NNN in this study is supported by the previous finding that NNN formation does not occur upon urine incubation with nornicotine (Stepanov, Carmella, Briggs, et al., 2009). Although the effect of diet, oral health status, and other factors on the salivary synthesis of [pyridine-D4]NNN is outside the scope of this report, the observed interindividual variation in the capacity for oral nitrosation is in agreement with our previous observation that only some oral NRT users form NNN endogenously. About 0.4%�C0.