The chemical composition of smokeless tobacco: a survey of products sold in the United States in 2006 and 2007.

Selected toxicant concentrations and other chemical measures have been determined for 43 U.S. smokeless tobacco products sold in 2006 and 2007. Products evaluated included moist snuff, dry snuff, loose leaf, plug, dissolvable and snus tobacco brands. Reference products available for scientific research purposes and eleven Swedish products were also evaluated and compared to the commercial products studied. Chemical endpoints determined included benzo[a]pyrene (B[a]P), N'-nitrosonornicotine (NNN), N'-nitrosoanatabine (NAT), N'-nitrosoanabasine (NAB), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), N-Nitrosodimethylamine (NDMA), nitrite, cadmium, lead, arsenic, nickel, chromium, chloride, water, pH and nicotine. Different toxicant profiles were observed for the products studied, with snus tobacco brands generally containing relatively low concentrations of B[a]P and tobacco specific nitrosamines (TSNAs) compared to other moist snuffs. Smokeless tobacco reference product toxicant profiles were similar to corresponding commercial products, with the exception of the TSNA content of the dry snuff reference material. TSNA concentrations observed for all commercial products were lower than historically reported values, likely reflecting changes in product shelf life, tobacco curing practices and, possibly, product blend formulations during the last 20-30 years. The survey results summarized provide a temporal point of comparison with future data anticipated from FDA "harmful and potentially harmful constituents in tobacco products" reporting.

Estimating tar and nicotine exposure: human smoking versus machine generated smoke yields.

OBJECTIVE: Determine human smoked (HS) cigarette yields of tar and nicotine for smokers using their own brand in their everyday environment. METHOD: A robust, filter analysis method was used to estimate the tar and nicotine yields for 784 subjects. Seventeen brands were chosen to represent a wide range of styles: 85 and 100 mm lengths; menthol and non-menthol; 17, 23, and 25 mm circumference; with tar yields [Federal Trade Commission (FTC) method] ranging from 1 to 18 mg. Tar bands chosen corresponded to yields of 1-3 mg, 4-6 mg, 7-12 mg, and 13+ mg. RESULTS: A significant difference (p<0.0001) in HS yields of tar and nicotine between tar bands was found. Machine-smoked yields were reasonable predictors of the HS yields for groups of subjects, but the relationship was neither exact nor linear. Neither the FTC, the Massachusetts (MA) nor the Canadian Intensive (CI) machine-smoking methods accurately reflect the HS yields across all brands. The FTC method was closest for the 7-12 mg and 13+ mg products and the MA method was closest for the 1-3mg products. The HS yields for the 4-6 mg products were approximately midway between the FTC and the MA yields. HS nicotine yields corresponded well with published urinary and plasma nicotine biomarker studies.

Assessment of dioxin and dioxin-like compounds in mainstream smoke from selected US cigarette brands and reference cigarettes.

Mainstream cigarette smoke (MSS) from 12 US cigarette brands and two reference cigarettes was evaluated to determine concentrations of dioxins (i.e., polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and dioxin-like polychlorinated biphenyls (PCBs)). The study included three 'tar' ranges based on Federal Trade Commission (FTC) determination: Low Yield (LY) < or = 5.5, Medium Yield (MY) 9.6-12.2, and High Yield (HY)> or = 14.5 mg/cig. Of the brands studied, the HY cigarettes yielded the greatest mean concentrations of 2005 World Health Organization Toxic Equivalents (WHO-TEQs) on a per cigarette basis. WHO-TEQ levels in LY cigarettes were significantly lower than for HY cigarettes (p=0.039) on a yield per cigarette basis and WHO-TEQ concentrations correlated with 'tar' yield (r=0.73, p=0.007), as did concentration on a WHO-TEQ per body mass per day basis (r=0.73, p=0.007). However, a statistically significant relationship was not observed between 'tar' yield levels and WHO-TEQ concentrations on a per mg Total Particulate Matter (TPM) basis. Concentrations for all brands tested ranged from 0.44 to 3.88 fg WHO-TEQ/mg TPM. Maximum daily exposure estimates calculated from this range (0.004-0.074 pg WHO-TEQ/kg bw/day) are below the current WHO Tolerable Daily Intake range of 1-5 pg/kg bw/day.

A comparison of the mainstream smoke chemistry and mutagenicity of a representative sample of the US cigarette market with two Kentucky reference cigarettes (K1R4F and K1R5F).

The incorporation of technologies into cigarettes such as filters, filter ventilation, porous cigarette papers, expanded tobacco and reconstituted tobacco sheet has resulted in cigarettes with a wide range of "tar" yields. The objectives of this study were to characterize the US cigarette market according to "tar" category (i.e. full flavor, FF; full flavor low tar, FFLT; or ultra low tar, ULT) and to determine whether the Kentucky reference cigarettes K1R4F and K1R5F are representative of FFLT and ULT cigarettes, respectively. As a means of characterization and comparison, the mainstream smoke from a representative sample of commercially available cigarettes from each market segment and the K1R4F and K1R5F Kentucky reference cigarettes was analyzed for the presence and level of 18 selected chemical constituents. In addition, a measure of the mutagenic activity of the mainstream smoke condensate from these cigarettes was determined using an Ames Salmonella mutagenicity assay. All cigarettes were smoked according to US Federal Trade Commission (FTC) guidelines. Results indicated that, overall, mainstream smoke constituent levels are well predicted by FTC "tar" yield--constituent levels increased as "tar" delivery increased. Based on the selected analytes measured in mainstream smoke, the K1R4F reference cigarette was generally representative of the FFLT segment of the US cigarette market. The K1R5F reference cigarette was representative of the ULT segment of the US cigarette market for cigarettes with "tar" deliveries approximate to it. In terms of mutagenic activity, a direct relationship was also demonstrated on a per cigarette basis-revertants per cigarette increased with increasing "tar" delivery. There was a weak tendency (R-square = 0.12, P = 0.08) for specific activity (revertants/mg "tar") to increase with decreasing "tar" yield-lower "tar" products had a slightly higher specific activity. No significant differences (P > 0.05) were observed when the specific activities of the condensates from the K1R4F and K1R5F reference cigarettes were compared to the market segments that they were designed to represent, FFLT and ULT, respectively. Overall, these results support the use of the K1R4F and the K1R5F as acceptable reference cigarettes for comparative mutagenicity and smoke chemistry studies of cigarettes available on the US market.

GC-AED Studies of Nicotine Fate in a Burning Cigarette.

An atomic emission detector (AED) for a gas chromatograph (GC) can be used to selectively detect compounds labeled with stable isotopes, such as D, (13)C, and (15)N. This capability can be used to determine precursor-fate relationships within complex matrixes, using stable isotopes rather than radiolabeled isotopes. Employing stable isotopes removes the safety consideration associated with radiolabeling studies. Two previous reports have employed GC-AED in fate studies for (13)C-labeled precursors. The goal of this study was to evaluate the utility of GC-AED for precursor-fate determinations in tobacco science. In this work, GC-AED was used to determine the fate of nicotine-d(4) in a burning cigarette. GC-MSD was then employed to identify the compounds that the AED indicated contained D. Spectral confirmation of the presence of D was performed on each chromatographic peak of interest with both AED and MSD to ensure that the identification of the products was correct. Nicotine and nicotine-d(4) standards were used to evaluate the effect of coelution of unlabeled compounds with the labeled compounds on the AED response for D. It was shown that the AED response for D at λ = 308 nm decreases with increased concentration of unlabeled compound. Detection at λ = 656 nm, however, is unaffected by the presence of the unlabeled compound. Compound-independent calibration is also possible at this wavelength. GC-AED studies with nicotine-d(4) core injected into cigarettes demonstrated that most of the nicotine (79%) is distilled, unchanged, into the mainstream (MS) and sidestream (SS) smoke. The degradation products that do occur include 3-substituted pyridines and nicotine-oxidation products. These are found only in the SS smoke.

Optimization of (15)n detection with an atomic emission detector.

Gas chromatography with atomic emission detection is a useful tool for the detection of stable isotope labels in complex samples. While papers involving the analysis of D and (13)C are numerous, little work has been done in the area of (15)N detection. For (15)N isotope detection, three reagent gases are used: H(2), O(2), and CH(4). In this work, the reagent gas flows were varied to optimize the sensitivity of (15)N detection without sacrificing isotope selectivity. The optimal gas flows determined in this work produce the following ratios of the spectral peak areas: O 725 area/He 728 area = 0.039 with only O(2) flowing; H 486 area/He 492 area = 12 with only H(2) flowing; C 496 area/He 502 area = 0.41 with O(2), H(2), and CH(4) flowing for C and no gases flowing for He. When using these gas settings, the (15)N sensitivity is increased by nearly 2 orders of magnitude relative to the manufacturer-recommended settings. It was also demonstrated that the presence of a compound in both the labeled and unlabeled forms in the same sample does not affect the response. The ratios of (15)N to (14)N in standards, calculated from calibration plots (which are linear for both isotopes), agree well with the actual values. A tobacco smoke sample containing various (15)N-labeled compounds was used to show the utility of the GC-AED for indicating which compounds in a complex sample contain the label. This sample also demonstrates the necessity for optimal sensitivity when dealing with samples containing small amounts of compounds with low incorporation levels.

Chemical and biological studies of a new cigarette that primarily heats tobacco. Part 1. Chemical composition of mainstream smoke.

A new-technology cigarette has been developed. While the new cigarette burns some tobacco, it does not use tobacco as the fuel to sustain combustion and provide heat to the cigarette. Rather, the new cigarette primarily heats tobacco thereby reducing products of smoke formation mechanisms such as tobacco combustion, tobacco pyrolysis and pyrosynthesis. The mainstream smoke composition from a cigarette based on the new design (TOB-HT) has been characterized in comparative chemical testing with two reference cigarettes using the FTC puffing regimen. Thermal properties, UV absorption characteristics, elemental composition and materials balance studies all suggest a simplified smoke aerosol. Twenty-five smoke constituents ("target compounds") identified by the scientific community as compounds that may contribute to the diseases statistically associated with smoking have also been measured. Mainstream smoke concentrations of most target compounds are significantly lower with the TOB-HT cigarette when compared with reference cigarettes in the ultra-light "tar" and light "tar" categories. Taken together, chemical analysis results suggest simplified TOB-HT smoke chemistry with marked reductions in specific chemicals reported to be biologically active.

Chemical and biological studies of a new cigarette that primarily heats tobacco. Part 1. Chemical composition of mainstream smoke.

A new-technology cigarette has been developed. While the new cigarette burns some tobacco, it does not use tobacco as the fuel to sustain combustion and provide heat to the cigarette. Rather, the new cigarette primarily heats tobacco thereby reducing products of smoke formation mechanisms such as tobacco combustion, tobacco pyrolysis and pyrosynthesis. The mainstream smoke composition from a cigarette based on the new design (TOB-HT) has been characterized in comparative chemical testing with two reference cigarettes using the FTC puffing regimen. Thermal properties, UV absorption characteristics, elemental composition and materials balance studies all suggest a simplified smoke aerosol. Twenty-five smoke constituents ("target compounds") identified by the scientific community as compounds that may contribute to the diseases statistically associated with smoking have also been measured. Mainstream smoke concentrations of most target compounds are significantly lower with the TOB-HT cigarette when compared with reference cigarettes in the ultra-light "tar" and light "tar" categories. Taken together, chemical analysis results suggest simplified TOB-HT smoke chemistry with marked reductions in specific chemicals reported to be biologically active.

Evaluation of the genotoxic and cytotoxic potential of mainstream whole smoke and smoke condensate from a cigarette containing a novel carbon filter.

A novel carbon filter has been developed which primarily reduces the amount of certain vapor phase constituents of tobacco smoke with greater efficiency than the charcoal filters of cigarettes currently in the market. In vitro indicators of genotoxic and cytotoxic potential were used to compare the cigarette smoke condensate (particulate phase) or whole cigarette smoke (vapor phase and particulate phase) from cigarettes containing the novel carbon filter with smoke condensate or whole smoke from commercial or prototype cigarettes not containing the novel carbon filter. Ames bacterial mutagenicity, sister chromatid exchange (SCE) in Chinese hamster ovary (CHO) cells, and neutral red cytotoxicity assays in CHO cells were utilized to assess the genotoxic and cytotoxic potential of the cigarette smoke condensates. SCE and neutral red cytotoxicity assays were utilized to assess the genotoxic and cytotoxic potential of the whole smoke. As expected, the novel carbon filter did not significantly affect the genotoxic or cytotoxic activity of the smoke condensate, although we did observe that the use of low-nitrogen tobacco reduced the mutagenicity of the condensate in Salmonella typhimurium strain TA98. However, the whole smoke from cigarettes containing the novel carbon filter demonstrated significant reductions in genotoxic and cytotoxic potential compared to cigarettes without the novel carbon filter. The toxicity of the smoke was correlated (r = 0.7662 for cytotoxicity and r = 0.7562 for SCE induction) to the aggregate mass of several vapor phase components (acetone, acetaldehyde, acrolein, acrylonitrile, 1,3-butadiene, ammonia, NOx, HCN, benzene, isoprene, and formaldehyde) in the smoke of the cigarettes utilized in this study. In conclusion, this novel carbon filter, which significantly reduced the amount of carbonyls and other volatiles in mainstream cigarette smoke, resulted in significant reductions in the genotoxic and cytotoxic activity of the smoke as measured by these assays.

Inhibitory activity of cigarette-smoke condensate on the mutagenicity of heterocyclic amines.

Cigarette-smoke condensate (CSC) is a complex mixture containing over 3800 identified chemicals including nicotine, water, mutagens, antimutagens, cytotoxins and inert chemicals. Although CSC is mutagenic in the Ames test, its effect on the activity of other mutagens has not been characterized. Using the Ames Salmonella bacterial mutagenesis assay, we found CSC exerts a significant inhibitory effect on mutagens requiring bioactivation. Those studied included heterocyclic amines (Glu-P-1, Glu-P-2, IQ, MeIQ, Trp-P-1 and Trp-P-2), benzo[a]pyrene (B[a]P) and aflatoxin B1. However, CSC had no effect on the activity of direct-acting mutagens (2-nitrofluorene, sodium azide, 4-nitro-1,2-phenylenediamine, 4-nitroquinoline N-oxide and methyl methanesulfonate). With indirect-acting mutagens, the reduced number of revertants observed in the presence of CSC was not attributable to cytotoxicity. CSC exhibited a potent inhibitory effect on the cytochrome P-450 dependent monooxygenases, ethoxyresorufin O-deethylase and B[a]P hydroxylase. This suggests inhibition of the cytochrome P-450 isozymes as one possible mechanism for the antimutagenicity of CSC. Fractionation studies of CSC revealed that the neutral, weakly acidic (phenolic) and basic fractions are all effective as antimutagens against Glu-P-1, a representative heterocyclic amine. This indicates that several classes of chemicals contribute to the inhibitory effect of CSC on the mutagenicity of the heterocyclic amines.

Quantitative determination of selected compounds in a Kentucky 1R4F reference cigarette smoke by multidimensional gas chromatography and selected ion monitoring-mass spectrometry.

Eight compounds from a Kentucky 1R4F reference cigarette smoke condensate have been determined by selected ion monitoring-mass spectrometry (SIM-MS) to confirm the validity of multidimensional gas chromatography (MDGC) as a quantitative tool in complex mixture analyses. Four electrostatically precipitated smoke condensate samples of 100 cigarettes each are dissolved individually in 25 mL of 2-propanol. The 2-propanol contains two methyl esters (C8 and C14) and seven deuterium-labeled compounds used as internal standards (IS). Analysis of the compounds of interest, pyridine; acetamide; acrylamide; phenol; o-, m-, and p-cresol; and quinoline, is accomplished by using two heartcuts. Heartcut times of the MDGC analysis are selected such that at least one IS is transferred with each group of compounds being analyzed. This study shows that the MDGC technique previously developed and described can be used for quantitative analyses. A comparison is made between the two types of internal standards. The results obtained for both types of internal standards agree within 20% of each other, on the average, with higher standard deviations for approximately 60% of the compounds where methyl esters are used as internal standards.

Separation and quantitation of monovalent anionic and cationic species in mainstream cigarette smoke aerosols by high-performance ion chromatography.

A simple method has been developed to separate and quantitate monovalent ionic species in mainstream cigarette smoke aerosols based on ion chromatography (IC) with conductivity detection. The method entails collecting the smoke aerosol particulate phase by electrostatic precipitation, dissolving the smoke condensate in methanol (MeOH), and separating the ionic species on either a cation- or anion-exchange column. The method has been applied to the analysis of smoke aerosols from two cigarettes, 1R4F Kentucky Reference cigarettes and a new cigarette that heats but does not burn tobacco. The predominant cations in smoke aerosols from 1R4F Kentucky Reference and the new cigarettes are sodium (Na+), ammonium (NH4+), and potassium (K+) ions; the predominant anions are acetate (AcO-) and formate (HCOO-). Trace amounts of chloride (Cl-), nitrite (NO2-), and nitrate (NO3-) ions are also present.

Chemical and biological studies of a cigarette that heats rather than burns tobacco.

Cigarettes can be developed that heat rather than burn tobacco. Such products would be expected to have less "tar" and other combustion products than cigarettes that burn tobacco. With one product of this type, benzo(a)pyrene, N-nitrosamines, phenolic compounds, acetaldehyde, acrolein, hydrogen cyanide, and N-heterocyclic compounds have been reduced 10- to 100-fold compared to the Kentucky reference (1R4F) cigarette, a representative low-tar cigarette. The yields of nicotine and carbon monoxide from this new cigarette are less than the yields of 95% and 75%, respectively, of the cigarettes sold in the United States during 1988. Nicotine absorption from smoking this new cigarette is not significantly different from that of tobacco-burning cigarettes yielding equivalent levels of nicotine. The urine mutagenicity of smokers of new cigarettes is significantly less (P less than .05) than that of smokers of tobacco-burning cigarettes and is not significantly different (P greater than .10) from that of nonsmokers. We conclude that cigarettes which heat rather than burn tobacco can reduce the yield of tobacco combustion products. This simplification of smoke chemistry had no effect on nicotine absorption in smokers and resulted in a reduction of biological activity in smokers as measured by urine mutagenicity.

Cigarette smoke composition. Part 2. Method for determining major components in smoke of cigarettes that heat instead of burn tobacco.

A method is described for determining major constituents in the smoke of a cigarette that heats, but does not burn, tobacco. Dual, simultaneous separations are performed in a single gas chromatographic oven to determine water, glycerol, nicotine, and propylene glycol in a rapid and cost-effective manner. A materials balance of new cigarette smoke total particulate matter was attempted from both Cambridge filter and electrostatic precipitation smoke collection data. Serious deficiencies were found when Cambridge filter smoke collection was applied for this purpose. Electrostatic precipitation smoke collection eliminated these problems. The data obtained by electrostatic precipitation smoke collection indicate that water, glycerol, nicotine, and propylene glycol make up about 94% of new cigarette smoke total particulate matter.

Cigarette smoke composition. Part 1. Limitations of FTC method when applied to cigarettes that heat instead of burn tobacco.

The design of a new cigarette that heats rather than burns tobacco calls for modifications to the Federal Trade Commission (FTC) method for analytical smoking. These changes include eliminating sample conditioning at 75 degrees F and 60% RH, exercising greater care in lighting cigarettes, and smoking cigarettes to self-extinguishment rather than to a predetermined butt length as a measure of complete consumption. By several gross analytical measures, smoke condensate from the new cigarette differs substantially from that of tobacco-burning cigarettes. This is inferred from the lack of coloration of smoke condensate collected on Cambridge filters. Elemental analysis demonstrates reduced carbon and nitrogen content concurrent with increased hydrogen. Thermogravimetric analysis shows almost quantitative weight loss at Tmax = 220 degrees C. Ultraviolet (UV) spectrophotometric analysis shows greatly reduced levels of tobacco-derived smoke components and qualitative differences in chemical entities being measured. By design, the heat required for smoke formation is supplied by a carbon heat source embedded in the cigarette tip. Tobacco contained in the cigarette is not burned and is exposed to temperature less than 300 degrees C. Thus, it is apparent (1) that smoke from the new cigarette contains little or no "tar" as tar is classically defined, and (2) that the FTC method even as modified to account for cigarette design differences is appropriate only for determination of nicotine and carbon monoxide yielded from this cigarette.

Separation and quantitation of phenolic compounds in mainstream cigarette smoke by capillary gas chromatography with mass spectrometry in the selected-ion mode.

Cigarette smoke condensate is a complex chemical matrix and determination of phenolic compounds in it frequently requires extensive and laborious sample preparation. By utilizing derivatization techniques and capillary column gas chromatography with mass spectrometry in the selected-ion mode, separation and quantitation of selected phenolic compounds found in mainstream cigarette smoke can be accomplished with minimal sample preparation. This method has been used to determine concentrations of phenol, o-cresol, m-cresol, p-cresol, catechol, resorcinol and hydroquinone in cigarette smoke condensate from a number of commercially available cigarettes and a new cigarette which heats, but does not burn, tobacco. Unlike tobacco-burning cigarettes, levels of the phenolic compounds in the new cigarette smoke are at or below the detection limits for most of the compounds. This result is attributed to the unique design of the new cigarette.

Isotope dilution gas chromatography-mass spectrometry in the determination of benzene, toluene, styrene and acrylonitrile in mainstream cigarette smoke.

A cryogenic trapping method with isotope dilution gas chromatography-mass spectrometry analysis has been developed for the determination of benzene, toluene, styrene and acrylonitrile in mainstream vapor phase cigarette smoke. The method is simple, direct, and quantitative. Vapor phase samples are collected cryogenically in a series of four traps following removal of the particulate phase with a Cambridge filter pad. For all four analytes, 75-85% of the total amounts recovered were found in the initial trap and less than 1% in the final trap. Assessment of instrumental precision by multiple injections of a sample gave relative standard deviations of less than 2%. Linear calibration for all analytes over the analysis range gave an r2 value greater than 0.99 with average relative standard deviations at the mean ranging from 1.4 to 8.2%. The cigarettes analyzed include a reference cigarette (Kentucky 1R4F), a commercial ultra-low "tar" mentholated cigarette, and two cigarettes that heat but do not burn tobacco. The values determined for the four analytes in the 1R4F samples are comparable to reported values of similar cigarettes. The cigarettes which heat rather than burn tobacco yield less of all four analytes compared to the other cigarettes in the study.

Analysis of flue-cured tobacco essential oil by hyphenated analytical techniques.

The major components of an alkaloid-free, flue-cured, tobacco essential oil sample are isolated and identified. This is accomplished by utilizing modern hyphenated analytical methods. The instrumentation developed to accomplish this are an automated multidimensional gas chromatograph/mass spectrometer/flame ionization detector (MDGC/MS/FID) and a multidimensional gas chromatograph/matrix isolation/Fourier transform infrared spectrometer (MDGC/MI/FTIR). A total of 306 compounds is identified in the essential oil, of which 80 are found as tobacco constituents for the first time.