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.

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. ©, This work was authored as part of the Contributor's official duties as an Employee of the United States Government and is therefore a work of the United States Government. In accordance with 17 U.S.C. 105, no copyright protection is available for such works under U.S. Law.

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.

The most alarming aspect of the Sudanese toombak smokeless tobacco is that it contains high levels of highly toxic Tobacco-Specific Nitrosamines (TSNAs). Understanding the microbiology of toombak is of relevance because TSNAs are an indirect result of microbial-mediated nitrate reductions. We conducted shotgun metagenomic sequencing on a toombak product for which relevant features are presented here. The microbiota was composed of over 99% Bacteria. The most abundant taxa included Actinobacteria, specifically genera Enteractinococcus and Corynebacterium, while Firmicutes were represented by Family Bacillaceae and the genus Staphylococcus. Selected gene targets were nitrate reduction and transport, antimicrobial resistance, and other genetic transference mechanisms. Canonical nitrate reduction and transport genes (i.e., nar) were found for Enteractinococcus and Corynebacterium while various species of Staphylococcus exhibited a notable number of antimicrobial resistance and genetic transference genes. The nitrate reduction activity of the microbiota in toombak is suspected to be a contributing factor to its high levels of TSNAs. Additionally, the presence of antimicrobial resistance and transference genes could contribute to deleterious effects on oral and gastrointestinal health of the end user. Overall, the high toxicity and increased incidences of cancer and oral disease of toombak users warrants further investigation into the microbiology of toombak.

Smokeless tobacco products (STP) contain bacteria, mold, and fungi due to exposure from surrounding environments and tobacco processing. This has been a cause for concern since the presence of microorganisms has been linked to the formation of highly carcinogenic tobacco-specific nitrosamines. These communities have also been reported to produce toxins and other pro-inflammatory molecules that can cause mouth lesions and elicit inflammatory responses in STP users. Moreover, microbial species in these products could transfer to the mouth and gastrointestinal tract, potentially altering the established respective microbiotas of the consumer. Here, we present the first metagenomic analysis of select smokeless tobacco products, specifically US domestic moist and dry snuff. Bacterial, eukaryotic, and viral species were found in all tobacco products where 68% of the total species was comprised of Bacteria with 3 dominant phyla but also included 32% Eukarya and 1% share abundance for Archaea and Viruses. Furthermore, 693,318 genes were found to be present and included nitrate and nitrite reduction and transport enzymes, antibiotic resistance genes associated with resistance to vancomycin, β-lactamases, their derivatives, and other antibiotics, as well as genes encoding multi-drug transporters and efflux pumps. Additional analyses showed the presence of endo- and exotoxin genes in addition to other molecules associated with inflammatory responses. Our results present a novel aspect of the smokeless tobacco microbiome and provide a better understanding of these products' microbiology. KEY POINTS: The findings presented will help understand microbial contributions to overall STP chemistries. Gene function categorization reveals harmful constituents outside canonical forms. Pathway genes for TSNA precursor activity may occur at early stages of production. Bacteria in STPs carry antibiotic resistance genes and gene transfer mechanisms.

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.

Smokeless tobacco (ST) products are used worldwide and are a major public health concern. In addition to harmful chemicals found in these products, microbes found in ST products are believed to be responsible for generating harmful tobacco-specific nitrosamines (TSNAs), the most abundant carcinogens in ST. These microbes also contribute endotoxins and other pro-inflammatory components. A greater understanding of the microbial constituents in these products is sought in order to potentially link select design aspects or manufacturing processes to avoidable increases in harmful constituents. Previous studies looked primarily at bacterial constituents and had not differentiated between viable vs nonviable organisms, so in this study, we sought to use a dual metatranscriptomic and metagenomic analysis to see if differences exist. Using high-throughput sequencing, we observed that there were differences in taxonomic abundances between the metagenome and metatranscriptome, and in the metatranscriptome, we also observed an abundance of plant virus RNA not previously reported in DNA-only studies. We also found in the product tested, that there were no viable bacteria capable of metabolizing nitrate to nitrite. Therefore, the product tested would not be likely to increase TSNAs during shelf storage. We tested only a single product to date using the strategy presented here, but succeeded in demonstrating the value of using of these methods in tobacco products. These results present novel findings from the first combined metagenome and metatranscriptome of a commercial tobacco product.

Certain South Asian smokeless tobacco, including ready-to-use products, are not well characterized. | | • | In this study, we measured pH and concentrations of nicotine, tobacco-specific nitrosamines, and flavor compounds. | | • | Products had a wide range of moisture (4.0–46.6%), pH (pH 4.99–10.0), total nicotine (0.39–35.2 mg/g) and unprotonated nicotine (<0.01–11.9 mg/g). | | • | Two khaini products analyzed contained higher levels of N′-Nitrosonornicotine, a compound associated with oral cancer. | | • | Understanding the constituents in these products is necessary for characterizing potential harm as these products become more available worldwide.

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.

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.

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 69 beats 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 ONDP use can increase plasma nicotine levels and heart rate and reduce cigarette cravings in abstinent smokers.

Rape, 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, rape 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 rape. Most rape 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 rapes (pH 9.75 - 10.2), which contain alkaline ashes. In tobacco-containing rapes, 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 rapes analyzed. Several TSNAs and PAHs identified in the products are known or probable carcinogens according to the International Agency for Research in Cancer. Milligram quantities of some non-tobacco constituents, such as camphor, coumarin, and eugenol, warrant additional evaluation.

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.

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 (CV, 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 g g-1), coumarin (439 g g-1), camphor (1060 g g-1) and diphenyl ether (4840 g g-1) 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.

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 40.1mg/g. The most readily absorbable form of nicotine (unprotonated nicotine) varied more than 350-fold, ranging from 0.01 to 3.7mg/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 76,500ng/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.

INTRODUCTION: Iq'mik, a form of smokeless tobacco (ST), is traditionally used by Cup'ik and Yup'ik Eskimo people of western Alaska. Iq'mik is sometimes incorrectly considered to be a healthier alternative to smoking because its ingredients are perceived as "natural." Our chemical characterization of iq'mik shows that iq'mik is not a safe alternative to smoking or other ST use. METHODS: We measured nicotine and pH levels of tobacco and ash used to prepare iq'mik. We also characterized levels of toxins which are known to be present in ST including tobacco-specific nitrosamines (TSNAs) and polycyclic aromatic hydrocarbons (PAHs) using chromatographic separations coupled with isotope dilution mass spectrometry. RESULTS: Nicotine content in the iq'mik tobacco was very high, ranging from 35 to 43mg/g, with a mean of 39mg/g. The pH of the iq'mik tobacco-ash mixture was 11, an extremely high level compared with most ST products. High levels of PAHs were seen in the fire-cured tobacco samples with a benzo[a]pyrene level of 87ng/g. Average TSNA levels in the tobacco were 34, 2,700, and 340ng/g for 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), N'-nitrosonornicotine (NNN), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), respectively. CONCLUSIONS: Iq'mik contains high levels of the more easily absorbed unionized nicotine as well as known carcinogenic TSNAs and PAHs. The perception that iq'mik is less hazardous than other tobacco products due to the use of "natural" ingredients is not warranted. This chemical characterization of iq'mik gives a better understanding of the risk of possible adverse health effects of its use.

OBJECTIVE: Oral tobacco products contain nicotine and carcinogenic tobacco-specific N-nitrosamines (TSNAs) that can be absorbed through the oral mucosa. The aim of this study was to determine typical pH ranges and concentrations of total nicotine, unionised nicotine (the most readily absorbed form) and five TSNAs in selected oral tobacco products distributed globally. METHODS: A total of 53 oral tobacco products from 5 World Health Organisation (WHO) regions were analysed for total nicotine and TSNAs, including 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), using gas chromatography or liquid chromatography with mass spectrometric detection. Unionised nicotine concentrations were calculated using product pH and total nicotine concentrations. Fourier transform infrared spectroscopy was used to help categorise or characterise some products. RESULTS: Total nicotine content varied from 0.16 to 34.1 mg/g product, whereas, the calculated unionised nicotine ranged from 0.05 to 31.0 mg/g product; a 620-fold range of variation. Products ranged from pH 5.2 to 10.1, which translates to 0.2% to 99.1% of nicotine being in the unionised form. Some products have very high pH and correspondingly high unionised nicotine (eg, gul powder, chimo, toombak) and/or high TSNA (eg, toombak, zarda, khaini) concentrations. The concentrations of TSNAs spanned five orders of magnitude with concentrations of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) ranging from 4.5 to 516 000 ng/g product. CONCLUSIONS: These data have important implications for risk assessment because they show that very different exposure risks may be posed through the use of these chemically diverse oral tobacco products. Because of the wide chemical variation, oral tobacco products should not be categorised together when considering the public health implications of their use.

OBJECTIVE: This study examines child poisonings resulting from ingestion of tobacco products throughout the nation and assesses the potential toxicity of novel smokeless tobacco products, which are of concern with their discreet form, candy-like appearance, and added flavorings that may be attractive to young children. METHODS: Data representing all single-substance, accidental poisonings resulting from ingestion of tobacco products by children <6 years of age, reported to poison control centers, were examined. Age association with ingestion of smokeless tobacco versus other tobacco products was tested through logistic regression. Total nicotine content, pH, and un-ionized nicotine level were determined, and the latter was compared with values for moist snuff and cigarettes. RESULTS: A total of 13,705 tobacco product ingestion cases were reported, >70% of which involved infants <1 year of age. Smokeless tobacco products were the second most common tobacco products ingested by children, after cigarettes, and represented an increasing proportion of tobacco ingestions with each year of age from 0 to 5 years (odds ratio: 1.94 [95% confidence interval: 1.86-2.03]). A novel, dissolvable, smokeless tobacco product with discreet form, candy-like appearance, and added flavorings was found to contain an average of 0.83 mg of nicotine per pellet, with an average pH of 7.9, which resulted in an average of 42% of the nicotine in the un-ionized form. CONCLUSION: In light of the novelty and potential harm of dissolvable nicotine products, public health authorities are advised to study these products to determine the appropriate regulatory approach.

In recent years, there has been a rapid proliferation of smokeless products with a wide range of nicotine content and flavoring formulations that may appeal to new users and existing cigarette smokers. The CDC nicotine method, which employs gas chromatography-flame ionization detection (GC-FID), provides a robust means for measuring nicotine in smokeless tobacco. However, several compounds, identified in a few flavored smokeless products, interfere with nicotine quantification using GC-FID. In response, the standard nicotine method (26.7 min run time) was modified to use faster GC ramping (3.7 min run time) and detection with mass spectrometry (GC-MS) in selected ion-monitoring mode to reduce signal interferences that can bias nicotine values. Seven conventional smokeless samples (n = 12) and blank tobacco samples spiked at three nicotine concentration levels (n = 5) were analyzed using the GC-FID and GC-MS methods and found to be in excellent agreement. However, only the GC-MS method provided confirmation of chromatographic peak purity in certain highly flavored products. The GC-MS method is not intended to replace the GC-FID method but to provide a method versatile enough to analyze a wide range of nicotine values in domestic and international samples of varying complexity. Accurate nicotine quantification is important for determining total nicotine content in tobacco and in subsequent calculations of un-protonated nicotine content.