The regions (and the corresponding states) are as follows: Northwest (Alaska), West (Colorado), Midwest (Indiana, more Nebraska, Minnesota), Northeast (New Hampshire, New York, Massachusetts), Mid-Atlantic/Appalachian (West Virginia), and South (Arkansas, Georgia). In order to obtain representative averages for constituent levels, we sought to acquire a sample of all flavors of each product from three different locations within each region. The monitors were provided with prepaid envelopes and asked to ship samples to our laboratory the day of purchase via overnight delivery. To provide a reference level of analytes, we also purchased Ariva and Stonewall��lozenges that contain compressed powdered tobacco and have been marketed as an alternative to smokers since 2001.
We previously demonstrated that these products contain very low levels of TSNA (Stepanov, Jensen, Hatsukami, & Hecht, 2006). Four flavors of Ariva and two flavors of Stonewall were purchased online. Upon receipt, each sample was labeled with a unique identification number. Detailed information on each sample, including but not limited to the brand, flavor, and date and place of purchase, was recorded. Storage conditions��refrigerated or not upon purchase, between purchase and shipment, and after the sample was received in our laboratory��were also recorded to monitor for potential variations in constituent levels due to different storage conditions. After the information was recorded, the samples were sealed in plastic sleeves and stored at 4 ��C until analysis.
Tobacco Analysis Approach to Sample Analyses Sample preparation for analysis of TSNA, nicotine, moisture content, and pH started after all the samples were obtained in the laboratory. For the measurement of moisture content and pH, all received samples were treated as one large set, the measurements being performed in a single run. Sample preparation for TSNA and nicotine analysis was carried out in three subsets, and analyses of the prepared samples for each analyte were performed in a single run on the corresponding instrument as described below. During sample preparation, one blank sample (corresponding extraction solvent without addition of tobacco) and one positive control sample (Copenhagen Snuff for which nicotine and TSNA contents were previously established) were included in order to monitor for potential contamination and day-to-day analytical variation.
Tobacco-Specific Batimastat N-Nitrosamines Analysis of TSNA in smokeless tobacco was performed essentially as previously described (Stepanov et al., 2006). Briefly, tobacco samples were extracted with citrate�Cphosphate buffer; the extracts were loaded on ChemElut cartridges (Varian, Harbor City, CA) and extracted with CH2Cl2. This was followed by solid-phase extraction on Sep-Pak Plus silica cartridges (Waters Corp.