An update on the formation in tobacco, toxicity and carcinogenicity of N'-nitrosonornicotine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone.

The tobacco-specific nitrosamines N'-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) are considered 'carcinogenic to humans' by the International Agency for Research on Cancer (IARC) and are believed to be important in the carcinogenic effects of both smokeless tobacco and combusted tobacco products. This short review focuses on the results of recent studies on the formation of NNN and NNK in tobacco, and their carcinogenicity and toxicity in laboratory animals. New mechanistic insights are presented regarding the role of dissimilatory nitrate reductases in certain microorganisms involved in the conversion of nitrate to nitrite that leads to the formation of NNN and NNK during curing and processing of tobacco. Carcinogenicity studies of the enantiomers of the major NNK metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and the enantiomers of NNN are reviewed. Recent toxicity studies of inhaled NNK and co-administration studies of NNK with formaldehyde, acetaldehyde, acrolein and CO2, all of which occur in high concentrations in cigarette smoke, are discussed.

From cultivation to cancer: formation of N-nitrosamines and other carcinogens in smokeless tobacco and their mutagenic implications.

Tobacco use is a major cause of preventable morbidity and mortality globally. Tobacco products, including smokeless tobacco (ST), generally contain tobacco-specific N-nitrosamines (TSNAs), such as N'-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-butanone (NNK), which are potent carcinogens that cause mutations in critical genes in human DNA. This review covers the series of biochemical and chemical transformations, related to TSNAs, leading from tobacco cultivation to cancer initiation. A key aim of this review is to provide a greater understanding of TSNAs: their precursors, the microbial and chemical mechanisms that contribute to their formation in ST, their mutagenicity leading to cancer due to ST use, and potential means of lowering TSNA levels in tobacco products. TSNAs are not present in harvested tobacco but can form due to nitrosating agents reacting with tobacco alkaloids present in tobacco during certain types of curing. TSNAs can also form during or following ST production when certain microorganisms perform nitrate metabolism, with dissimilatory nitrate reductases converting nitrate to nitrite that is then released into tobacco and reacts chemically with tobacco alkaloids. When ST usage occurs, TSNAs are absorbed and metabolized to reactive compounds that form DNA adducts leading to mutations in critical target genes, including the RAS oncogenes and the p53 tumor suppressor gene. DNA repair mechanisms remove most adducts induced by carcinogens, thus preventing many but not all mutations. Lastly, because TSNAs and other agents cause cancer, previously documented strategies for lowering their levels in ST products are discussed, including using tobacco with lower nornicotine levels, pasteurization and other means of eliminating microorganisms, omitting fermentation and fire-curing, refrigerating ST products, and including nitrite scavenging chemicals as ST ingredients.

Associations between microbial communities and key chemical constituents in U.S. domestic moist snuff.

BACKGROUND: Smokeless tobacco (ST) products are widely used throughout the world and contribute to morbidity and mortality in users through an increased risk of cancers and oral diseases. Bacterial populations in ST contribute to taste, but their presence can also create carcinogenic, Tobacco-Specific N-nitrosamines (TSNAs). Previous studies of microbial communities in tobacco products lacked chemistry data (e.g. nicotine, TSNAs) to characterize the products and identify associations between carcinogen levels and taxonomic groups. This study uses statistical analysis to identify potential associations between microbial and chemical constituents in moist snuff products. METHODS: We quantitatively analyzed 38 smokeless tobacco products for TSNAs using liquid chromatography with tandem mass spectrometry (LC-MS/MS), and nicotine using gas chromatography with mass spectrometry (GC-MS). Moisture content determinations (by weight loss on drying), and pH measurements were also performed. We used 16S rRNA gene sequencing to characterize the microbial composition, and additionally measured total 16S bacterial counts using a quantitative PCR assay. RESULTS: Our findings link chemical constituents to their associated bacterial populations. We found core taxonomic groups often varied between manufacturers. When manufacturer and flavor were controlled for as confounding variables, the genus Lactobacillus was found to be positively associated with TSNAs. while the genera Enteractinococcus and Brevibacterium were negatively associated. Three genera (Corynebacterium, Brachybacterium, and Xanthomonas) were found to be negatively associated with nicotine concentrations. Associations were also investigated separately for products from each manufacturer. Products from one manufacturer had a positive association between TSNAs and bacteria in the genus Marinilactibacillus. Additionally, we found that TSNA levels in many products were lower compared with previously published chemical surveys. Finally, we observed consistent results when either relative or absolute abundance data were analyzed, while results from analyses of log-ratio-transformed abundances were divergent.

A Low-Cost, High-Throughput Digital Image Analysis of Stain Patterns on Smoked Cigarette Filter Butts to Estimate Mainstream Smoke Exposure.

Standard machine smoking protocols provide useful information for examining the impact of design parameters, such as filter ventilation, on mainstream smoke delivery. Unfortunately, their results do not accurately reflect human smoke exposure. Clinical research and topography devices in human studies yield insights into how products are used, but a clinical setting or smoking a cigarette attached to such a device may alter smoking behavior. To better understand smokers' use of filtered cigarette products in a more natural environment, we developed a low-cost, high-throughput approach to estimate mainstream cigarette smoke exposure on a per-cigarette basis. This approach uses an inexpensive flatbed scanner to scan smoked cigarette filter butts and custom software to analyze tar-staining patterns. Total luminosity, or optical staining density, of the scanned images provides quantitative information proportional to mainstream smoke-constituent deliveries on a cigarette-by-cigarette basis. Duplicate sample analysis using this new approach and our laboratory's gold-standard liquid chromatography/tandem mass spectrometry (LC/MS/MS) solanesol method yielded comparable results (+7% bias) from the analysis of 20 commercial cigarettes brands (menthol and nonmentholated). The brands varied in design parameters such as length, filter ventilation, and diameter. Plots correlating the luminosity to mainstream smoked-nicotine deliveries on a per-cigarette basis for these cigarette brands were linear (average R2 > 0.91 for nicotine and R2 > 0.83 for the tobacco-specific nitrosamine NNK), on a per-brand basis, with linearity ranging from 0.15 to 3.00 mg nicotine/cigarette. Analysis of spent cigarette filters allows exposures to be characterized on a per-cigarette basis or a "daily dose" via summing across results from all filter butts collected over a 24 h period. This scanner method has a 100-fold lower initial capital cost for equipment than the LC/MS/MS solanesol method and provides high-throughput results (~200 samples per day). Thus, this new method is useful for characterizing exposure related to filtered tobacco-product use.

Characterization of Total and Unprotonated (Free) Nicotine Content of Nicotine Pouch Products.

INTRODUCTION: Nicotine pouch products, oral smokeless products that contain nicotine but no tobacco leaf material, have recently entered the US marketplace. Available data indicate sales of these products in the United States have increased since 2018; however, the extent of use among US youth and adults is uncertain. METHODS: To assay the chemistry of these emerging tobacco products, we analyzed 37 nicotine pouch brands from six total manufacturers. Almost all of the products had flavor descriptors (36 of 37), such as mint, licorice, coffee, cinnamon, and fruit. The amount of free nicotine, the form most easily absorbed, was calculated for each product using total nicotine, product pH, the appropriate pKa, and the Henderson-Hasselbalch equation. RESULTS: Nicotine pouch products varied in pouch content mass, moisture content (1.12%‒47.2%), alkalinity (pH 6.86‒10.1), and % free nicotine (7.7%‒99.2%). Total nicotine content ranged from 1.29 to 6.11 mg/pouch, whereas free nicotine ranged from 0.166 to 6.07 mg/pouch. These findings indicate that nicotine and pH levels found in some of these nicotine pouches are similar to conventional tobacco products, such as moist snuff and snus, and that most of these pouch products are flavored. CONCLUSIONS: Although these products likely lack many tobacco-related chemicals, each product analyzed contained nicotine, which is both addictive and can harm human health. Given that nicotine pouches may appeal to a spectrum of users, from novice to experienced users, it is important to include these emerging tobacco products in tobacco control research, policy, and practice. IMPLICATIONS: These "tobacco-free" nicotine pouches have similar pH and nicotine content to conventional tobacco products, such as moist snuff and snus. Although they lack many tobacco-related chemicals, most are highly flavored which could increase experimentation from new users. Given that nicotine pouches may appeal to a spectrum of users, from novice to experienced users, in terms of their flavors and nicotine content, it is important to examine and include these emerging tobacco products as they relate to tobacco control research, policy, and practice.

Chemical analysis of snus products from the United States and northern Europe.

INTRODUCTION: Snus is an oral tobacco product that originated in Sweden. Snus products are available as fine-cut loose tobacco or in pre-portioned porous "pouches." Some snus products undergo tobacco pasteurization during manufacturing, a process that removes or reduces nitrite-forming microbes, resulting in less tobacco-specific nitrosamine content in the product. Some tobacco companies and researchers have suggested that snus is potentially less harmful than traditional tobacco and thus a potential smoking cessation aid or an alternative to continued cigarette consumption. Although snus is available in various countries, limited information exists on snus variants from different manufacturers. METHODS: Moisture, pH, nicotine, and tobacco-specific N'-nitrosamines (TSNAs) were quantified in 64 snus products made by 10 manufacturers in the United States and Northern Europe (NE). Reported means, standard errors, and differences are least-square (LS) estimates from bootstrapped mixed effects models, which accounted for correlation among repeated measurements. Minor alkaloids and select flavors were also measured. RESULTS: Among all product types, moisture (27.4%-59.5%), pH (pH 5.87-9.10), total nicotine (6.81-20.6 mg/g, wet), unprotonated nicotine (0.083-15.7 mg/g), and total TSNAs (390-4,910 ng/g) varied widely. The LS-mean unprotonated nicotine concentration of NE portion (7.72 mg/g, SE = 0.963) and NE loose (5.06 mg/g, SE = 1.26) snus were each significantly higher than US portion snus (1.00 mg/g, SE = 1.56). Concentrations of minor alkaloids varied most among products with the highest total nicotine levels. The LS-mean NNN+NNK were higher in snus sold in the US (1360 ng/g, SE = 207) than in NE (836 ng/g, SE = 132) countries. The most abundant flavor compounds detected were pulegone, eucalyptol, and menthol. CONCLUSION: Physical and chemical characteristics of US and NE products labeled as snus can vary considerably and should not be considered "equivalent". Our findings could inform public health and policy decisions pertaining to snus exposure and potential adverse health effects associated with snus.

Monoamine oxidase inhibitory activity of flavoured e-cigarette liquids.

BACKGROUND AND AIMS: Monoamine oxidase inhibitors have been hypothesised to be important in tobacco dependence, reinforcing the brain's response to nicotine by delaying the degradation of neurotransmitters by monoamine oxidases. The development of electronic cigarettes has provided an alternative nicotine delivery system, which is widely viewed as less toxic than tobacco smoke. However, significant data gaps remain. This paper reports the results of measurements of monoamine oxidase inhibitory activity in a small sample of commercially available, flavoured e-liquids. METHODS: Twelve e-liquids were tested for monoamine oxidase inhibitory activity, using the kynuramine assay and monoamine oxidase enzymes (human, recombinant). Control samples of carrier liquids, propylene glycol and glycerol, and nicotine were also tested. RESULTS: Four e-liquids contained high levels of inhibitory activity, four more were moderately inhibitory. The remaining four e-liquids were mildly inhibitory, while the carrier liquids, and nicotine were inactive at relevant concentrations. The active compounds in the e-liquids were subsequently identified as vanillin and ethyl vanillin. Under some conditions of use, the sampled e-liquids with the highest concentrations of monoamine oxidase inhibitory activity have the potential to expose consumers to physiologically significant levels of MAO inhibitory activity. CONCLUSIONS: While only a small sample of e-liquids was tested, the findings suggest that some flavours have pharmacological actions, with potential to enhance the response to nicotine or to other drugs. The public health implications of these preliminary findings on addiction and smoking cessation warrant exploration and further research.

LC-MS/MS Analysis of Sugars, Alditols, and Humectants in Smokeless Tobacco Products.

Globally, smokeless tobacco (ST) includes a wide array of chemically diverse products generally used in the oral cavity. Although ST has been widely investigated, this study was undertaken to determine the levels of sugars (mono- and di-saccharides), alditols, and humectants present in major ST categories/subcategories by using high performance liquid chromatography coupled with a triple quadrupole mass spectrometer (HPLC-MS/MS). The products studied included chewing tobacco (loose leaf, plug, twist), US moist snuff, Swedish snus, creamy snuff, dry snuff, dissolvable tobacco products, and tobacco-coated toothpicks. The highest mean sugar level was detected in chewing tobacco (9.3-27.5%, w/w), followed by dissolvable tobacco (2.1%); all other products were lower than 1%. Creamy snuff had the highest mean alditol levels (22.6%), followed by dissolvable tobacco (15.4%); all others had levels lower than 1%. The detected mean humectant levels ranged from non-detectable to 5.9%. This study demonstrates the broad chemical diversity among ST. This research may aid researchers and public health advocates investigating the exposures and risks of ST. [Beitr. Tabakforsch. Int. 28 (2019) 203-213].

Dans l'ensemble, le terme de tabac sans fumée (TSF) recouvre une large gamme de diverses substances chimiques consommées, en règle générale, par la cavité buccale. Malgré les vastes analyses déjà consacrées au TSF, la présente étude fut entamée dans le but de déterminer les niveaux de sucres (mono- et disaccharides), de sucres-alcools et d'humectants présents dans les principales catégories/sous-catégories de TSF à l'aide de la chromato-graphie en phase liquide à haute performance couplée à une spectrométrie de masse à triple quadripôle (HPLC-MS/MS). Les produits analysés inclurent le tabac à chiquer (feuilles en vrac, tablette, corde), du tabac à priser humide américain, du snus suédois, du creamy snuff, du tabac à priser sec, des produits de tabac soluble et des cure-dents à la nicotine. Le niveau médian de sucres le plus élevé fut détecté dans le tabac à chiquer (9,3­27,5%, p/p), suivi du tabac soluble (2,1%); tous les autres produits présentèrent des niveaux inférieurs à 1%. Le creamy snuff présenta les niveaux médians de sucre-alcool les plus élevés (22,6%), suivi du tabac soluble (15,4%); tous les autres produits affichèrent des niveaux inférieurs à 1%. Les niveaux médians d'humectants détectés oscillèrent entre non-détectable et 5,9%. La présente étude démontre la grande diversité chimique parmi les formes de TSF et peut aider les chercheurs et les intervenants en faveur de la santé publique à explorer les degrés d'exposition et les risques du TSF. [Beitr. Tabakforsch. Int. 28 (2019) 203­213].

Surveillance of Nicotine and pH in Cigarette and Cigar Filler.

OBJECTIVE: We examined differences between nicotine concentrations and pH in cigarette and cigar tobacco filler. METHODS: Nicotine and pH levels for 50 cigarette and 75 cigar brands were measured. Non-mentholated and mentholated cigarette products were included in the analysis along with several cigar types as identified by the manufacturer: large cigars, pipe tobacco cigars, cigarillos, mini cigarillos, and little cigars. RESULTS: There were significant differences found between pH and nicotine for cigarette and cigar tobacco products. Mean nicotine concentrations in cigarettes (19.2 mg/g) and large cigars (15.4 mg/g) were higher than the other cigars types, especially the pipe tobacco cigars (8.79 mg/g). The mean pH for cigarettes was pH 5.46. Large cigars had the highest mean pH value (pH 6.10) and pipe tobacco cigars had the lowest (pH 5.05). CONCLUSIONS: Although cigarettes are the most common combustible tobacco product used worldwide, cigar use remains popular. Our research provides a means to investigate the possibility of distinguishing the 2 tobacco product types and offers information on nicotine and pH across a wide range of cigarette and cigar varieties that may be beneficial to help establish tobacco policies and regulations across product types.

Smokeless tobacco products harbor diverse bacterial microbiota that differ across products and brands.

Smokeless tobacco products contain numerous chemical compounds, including known human carcinogens. Other smokeless tobacco constituents, including bacteria, may also contribute to adverse health effects among smokeless tobacco users. However, there is a lack of data regarding the microbial constituents of smokeless tobacco. Our goal was to characterize the bacterial microbiota of different smokeless tobacco products and evaluate differences across product types and brands. DNA was extracted from 15 brands of smokeless tobacco products (including dry snuff, moist snuff, snus, and Swedish snus) and 6 handmade products (e.g., toombak) using an enzymatic and mechanical lysis approach. Bacterial community profiling was performed using PCR amplification of the V1-V2 hypervariable region of the 16S rRNA gene, followed by 454 pyrosequencing of the resulting amplicons and sequence analysis using the QIIME package. Total viable counts were also determined to estimate the number of viable bacteria present in each product. Average total viable counts ranged from 0 to 9.35 × 107 CFU g-1. Analysis of the 16S rRNA gene sequences revealed high bacterial diversity across the majority of products tested: dry snuff products where characterized by the highest diversity indices compared to other products. The most dominant bacterial phyla across all products were Firmicutes, Proteobacteria, Actinobacteria, and Bacteroidetes. Significant differences in both bacterial community composition and in silico predicted gene content were observed between smokeless tobacco product types and between brands of specific smokeless tobacco products. These data are useful in order to comprehensively address potential health risks associated with the use of smokeless tobacco products.

Restoring the Duality between Principal Components of a Distance Matrix and Linear Combinations of Predictors, with Application to Studies of the Microbiome.

Appreciation of the importance of the microbiome is increasing, as sequencing technology has made it possible to ascertain the microbial content of a variety of samples. Studies that sequence the 16S rRNA gene, ubiquitous in and nearly exclusive to bacteria, have proliferated in the medical literature. After sequences are binned into operational taxonomic units (OTUs) or species, data from these studies are summarized in a data matrix with the observed counts from each OTU for each sample. Analysis often reduces these data further to a matrix of pairwise distances or dissimilarities; plotting the first two or three principal components (PCs) of this distance matrix often reveals meaningful groupings in the data. However, once the distance matrix is calculated, it is no longer clear which OTUs or species are important to the observed clustering; further, the PCs are hard to interpret and cannot be calculated for subsequent observations. We show how to construct approximate decompositions of the data matrix that pair PCs with linear combinations of OTU or species frequencies, and show how these decompositions can be used to construct biplots, select important OTUs and partition the variability in the data matrix into contributions corresponding to PCs of an arbitrary distance or dissimilarity matrix. To illustrate our approach, we conduct an analysis of the bacteria found in 45 smokeless tobacco samples.

Characterization of Bacterial Communities in Selected Smokeless Tobacco Products Using 16S rDNA Analysis.

The bacterial communities present in smokeless tobacco (ST) products have not previously reported. In this study, we used Next Generation Sequencing to study the bacteria present in U.S.-made dry snuff, moist snuff and Sudanese toombak. Sample diversity and taxonomic abundances were investigated in these products. A total of 33 bacterial families from four phyla, Actinobacteria, Firmicutes, Proteobacteria and Bacteroidetes, were identified. U.S.-produced dry snuff products contained a diverse distribution of all four phyla. Moist snuff products were dominated by Firmicutes. Toombak samples contained mainly Actinobacteria and Firmicutes (Aerococcaceae, Enterococcaceae, and Staphylococcaceae). The program PICRUSt (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) was used to impute the prevalence of genes encoding selected bacterial toxins, antibiotic resistance genes and other pro-inflammatory molecules. PICRUSt also predicted the presence of specific nitrate reductase genes, whose products can contribute to the formation of carcinogenic nitrosamines. Characterization of microbial community abundances and their associated genomes gives us an indication of the presence or absence of pathways of interest and can be used as a foundation for further investigation into the unique microbiological and chemical environments of smokeless tobacco products.

Nicotine delivery and pharmacologic response from Verve, an oral nicotine delivery product.

Verve, an oral nicotine delivery product (ONDP), was introduced by Nu Mark (Altria Client Group, Richmond VA) for smokers to use in places where smoking is prohibited. This study assessed the effect of this ONDP on plasma nicotine levels, heart rate, product satisfaction, and ability to suppress smoking urge and cigarette cravings. Thirteen daily cigarette smokers [8 men and 5 women; average age 33.4years] attended two laboratory sessions, one occurred after overnight tobacco abstinence. Plasma samples were collected before and after ONDP use and measured for nicotine. In non-abstinent smokers, mean plasma nicotine levels increased from 18.3 to 21.0ng/mL. In abstinent smokers, average nicotine levels increased from 3.1 to 4.5ng/mL. After overnight tobacco abstinence, ONDP use significantly (p<0.01) increased heart rate from 69beats per minute (bpm) to 75bpm; while urge to smoke decreased significantly (p<0.01) from a score of 8.6 to 4.9. Participants indicated moderate product satisfaction that was not changed by tobacco abstinence. Analysis of unused ONDP revealed total nicotine levels of 1.68±0.09mg/disc. Spent ONDP discs were also analyzed to determine % nicotine liberated during chewing; results were 80% in the non-abstinent and 82% in the abstinent conditions (ns). Our study results indicate that ONDP use can increase plasma nicotine levels and heart rate and reduce cigarette cravings in abstinent smokers.

Comprehensive chemical characterization of Rapé tobacco products: Nicotine, un-ionized nicotine, tobacco-specific N'-nitrosamines, polycyclic aromatic hydrocarbons, and flavor constituents.

Rapé, a diverse group of smokeless tobacco products indigenous to South America, is generally used as a nasal snuff and contains substantial amount of plant material with or without tobacco. Previously uncharacterized, rapé contains addictive and harmful chemicals that may have public health implications for users. Here we report % moisture, pH, and the levels of total nicotine, un-ionized nicotine, flavor-related compounds, tobacco-specific N-nitrosamines (TSNAs) and polycyclic aromatic hydrocarbons (PAHs) for manufactured and hand-made rapé. Most rapé products were mildly acidic (pH 5.17-6.23) with total nicotine ranging from 6.32 to 47.6 milligram per gram of sample (mg/g). Calculated un-ionized nicotine ranged from 0.03 to 18.5 mg/g with the highest values associated with hand-made rapés (pH 9.75-10.2), which contain alkaline ashes. In tobacco-containing rapés, minor alkaloid levels and Fourier transform infrared spectra were used to confirm the presence of Nicotiana rustica, a high nicotine tobacco species. There was a wide concentration range of TSNAs and PAHs among the rapés analyzed. Several TSNAs and PAHs identified in the products are known or probable carcinogens according to the International Agency for Research on Cancer. Milligram quantities of some non-tobacco constituents, such as camphor, coumarin, and eugenol, warrant additional evaluation.

Chemical Composition and Evaluation of Nicotine, Tobacco Alkaloids, pH, and Selected Flavors in E-Cigarette Cartridges and Refill Solutions.

INTRODUCTION: Electronic cigarette (e-cigarette) use is increasing dramatically in developed countries, but little is known about these rapidly evolving products. This study analyzed and evaluated the chemical composition including nicotine, tobacco alkaloids, pH, and flavors in 36 e-liquids brands from 4 manufacturers. METHODS: We determined the concentrations of nicotine, alkaloids, and select flavors and measured pH in solutions used in e-cigarettes. E-cigarette products were chosen based upon favorable consumer approval ratings from online review websites. Quantitative analyses were performed using strict quality assurance/quality control validated methods previously established by our lab for the measurement of nicotine, alkaloids, pH, and flavors. RESULTS: Three-quarters of the products contained lower measured nicotine levels than the stated label values (6%-42% by concentration). The pH for e-liquids ranged from 5.1-9.1. Minor tobacco alkaloids were found in all samples containing nicotine, and their relative concentrations varied widely among manufacturers. A number of common flavor compounds were analyzed in all e-liquids. CONCLUSIONS: Free nicotine levels calculated from the measurement of pH correlated with total nicotine content. The direct correlation between the total nicotine concentration and pH suggests that the alkalinity of nicotine drives the pH of e-cigarette solutions. A higher percentage of nicotine exists in the more absorbable free form as total nicotine concentration increases. A number of products contained tobacco alkaloids at concentrations that exceed U.S. pharmacopeia limits for impurities in nicotine used in pharmaceutical and food products.

Custom Mentholation of Commercial Cigarettes for Research Purposes.

In the U.S. menthol remains the sole permitted characterizing cigarette flavor additive in part because efforts to link menthol cigarette use to increased tobacco-related disease risk have been inconclusive. To perform definitive studies, cigarettes that differ only in menthol content are required, yet these are not commercially available. We prepared research cigarettes differing only in menthol content by deposition of L-menthol vapor directly onto commercial nonmenthol cigarettes, and developed a method to measure a cigarette's menthol and nicotine content. With our custom-mentholation technique we achieved the desired moderately high menthol content (as compared to commercial brands) of 6.7 ± 1.0 mg/g (n = 25) without perturbing the cigarettes' nicotine content (17.7 ± 0.7 mg/g [n = 25]). We also characterized other pertinent attributes of our custom-mentholated cigarettes, including percent transmission of menthol and nicotine to mainstream smoke and the rate of loss of menthol over time during storage at room temperature. We are currently using this simple mentholation technique to investigate the differences in human exposure to selected chemicals in cigarette smoke due only to the presence of the added menthol. Our cigarettes will also aid in the elucidation of the effects of menthol on the toxicity of tobacco smoke.

Quantitation of Ten Flavor Compounds in Unburned Tobacco Products.

Most research on unburned tobacco has focused on the harmful chemicals associated with the tobacco itself. However, certain flavor additives in tobacco products can pose additional health risks. Flavors like camphor, coumarin, pulegone, eugenol, methyl salicylate, menthol and diphenyl ether have exhibited biological activity and/or toxicity in both lab animals and humans. This publication presents a new GC/MS method for the quantitation of ten flavor compounds (eucalyptol, camphor, menthol, pulegone, ethyl salicylate, methyl salicylate, cinnamaldehyde, eugenol, diphenyl ether and coumarin) in a variety of tobacco products, including smokeless products and cigar filler. Excellent linearity (>0.997), accuracy (93.9% - 106.6%) and precision (C.V., 0.5% - 3.0%) were achieved for all flavor analytes measured. A summary of the concentrations of these flavors in selected international smokeless tobacco (SLT) products including zarda, quiwam, gutkha, and khaini varieties from Southeast Asia and snuff, clove cigarette filler and flavored cigar filler from the United States is reported. High concentrations of eugenol (2110 µg/g), coumarin (439 µg/g), camphor (1060 µg/g) and diphenyl ether (4840 µg/g) were found in selected products. Accurate identification and quantitation of potentially hazardous flavor compounds is important because they can exist in relatively high levels in some tobacco products, including international SLT products. We outline a versatile method which can be used to quantitate flavor compounds in multiple types of tobacco products.

Chemical characterization of domestic oral tobacco products: total nicotine, pH, unprotonated nicotine and tobacco-specific N-nitrosamines.

In the United States, moist snuff has been studied more widely than other distinct categories of oral tobacco. In this study, we measured pH, moisture, nicotine (total and unprotonated), and tobacco-specific N-nitrosamines (TSNAs) for other established (twist, loose leaf, plug, and dry snuff without pouch) and emerging oral tobacco products (dry snuff pouch, US-made snus, and dissolvable tobacco). Among the seven product categories, product pH ranged from 4.7 to 7.9, and total nicotine concentration spanned from 3.9 to 40.1 mg/g. The most readily absorbable form of nicotine (unprotonated nicotine) varied more than 350-fold, ranging from 0.01 to 3.7 mg/g. While the highest total nicotine concentrations were observed in twist products, snus and dissolvable tobacco had the highest unprotonated nicotine levels. Among all products, total TSNA concentrations ranged from 313 to 76,500 ng/g with dry snuff having the highest total TSNA concentrations. This study demonstrates the diversity among oral tobacco products and highlights the potential of these products to deliver a wide range of nicotine and carcinogenic TSNAs. Characterizing the chemical content of these products may be helpful in further understanding the risk of marketing these products to oral tobacco users and smokers as an alternative and discrete form of tobacco.

Application of GC-MS/MS for the analysis of tobacco alkaloids in cigarette filler and various tobacco species.

This publication reports the first known use of gas chromatography-tandem mass spectrometry for the quantitation of five minor tobacco alkaloids (nornicotine, myosmine, anabasine, anatabine, and isonicoteine) in various tobacco samples. A summary of the concentrations of these minor alkaloid levels in the filler from 50 popular cigarette brands were found to be 659-986 µg/g nornicotine, 8.64-17.3 µg/g myosmine, 127-185 µg/g anabasine, 927-1390 µg/g anatabine, and 23.4-45.5 µg/g isonicoteine. Levels of minor alkaloids found in reference cigarettes (1R5F, 2R4F, 3R4F, CM4, and CM6) as well as burley, flue-cured, oriental, reconstituted, and Nicotiana rustica and Nicotiana glauca tobacco types are also reported. Quantitation of the minor tobacco alkaloids is important because the alkaloids have been shown to be precursors of carcinogenic tobacco specific N'-nitrosamines.