, 2006; U.S. Federal Trade Commission, 2007), and the introduction of novel smokeless tobacco products that are marketed to smokers as a temporary Veliparib or permanent substitute for smoking (Hatsukami, Ebbert, Feuer, Stepanov, & Hecht, 2007; Rogers, Biener, & Clark, 2010). Moreover, there is no information available on the enantiomeric composition of NNN in cigarette smoke. In this study, we applied chiral gas chromatography (GC) analysis to determine (S)-NNN levels in three categories of U.S. products: conventional smokeless tobacco, novel tobacco products, and cigarettes. MATERIALS AND METHODS Chemicals and Standards 5-Methyl-N��-nitrosonornicotine (5-MeNNN) and NNN enantiomers were synthesized as previously described (Carmella et al., 2000). All other chemicals were obtained from Fisher Scientific.

Tobacco Samples Smokeless tobacco products were purchased between March 2010 and January 2012: conventional products were obtained from retailers in Minneapolis, and novel products were obtained as a part of the New Product Watch Project (Stepanov, Biener, et al., 2012). Cigarettes were purchased from retail stores in April 2010 (Stepanov, Knezevich, et al., 2012); all samples were king-size filtered cigarettes packaged in hard packs. Tobacco and Smoke Analyses NNN was analyzed in conventional and novel smokeless tobacco products by GC with detection by a thermal energy analyzer (TEA) (Thermedics Detection Inc.) as previously described (Stepanov, Biener, et al., 2012). The levels of NNN in the tobacco filler and smoke of cigarettes analyzed here have been previously published (Stepanov, Knezevich, et al.

, 2012). Sample purification for the analysis of NNN enantiomers was conducted as for the NNN quantitation. The prepared samples were analyzed by GC-TEA using a 30 m �� 0.25mm i.d., 0.25-��m film thickness Cyclosil-B chiral column (Agilent) supplied with a 2 m �� 0.53mm deactivated silica precolumn, as previously described (Carmella et al., 2000). The contribution of (S)-NNN to the measured NNN in each sample was calculated based on the peak areas of (S)-NNN and (R)-NNN. The calculated percentage contribution of (S)-NNN and the measured levels of NNN were further used to calculate the amount of (S)-NNN in the analyzed products. Moisture Content Conventional and novel smokeless tobacco products were analyzed for moisture content as previously described (Stepanov et al.

, 2008). The moisture content in the tobacco filler of cigarettes analyzed here was published Anacetrapib previously (Stepanov, Knezevich, et al., 2012). RESULTS The results of the analyses are summarized in Table 1. The levels of NNN in conventional smokeless products ranged from 1.21 to 4.25 ��g/g tobacco, and in the novel smokeless products, NNN levels ranged from 0.72 to 1.79 ��g/g product. Table 1. Levels of NNN and (S)-NNN in the Tobacco of U.S.