NNK) And N-nitrosonornicotine Research Articles

UHPLC-MS/MS method for the simultaneous determination of nicotine and tobacco-specific nitrosamines NNN and NNK for use in preclinical studies

A new method was developed and validated for the simultaneous determination of nicotine and tobacco-specific nitrosamines (TSNAs) 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N-nitrosonornicotine (NNN) in two different tests matrices: porcine buccal epithelium tissue and phosphate buffered saline (PBS) extracts of smokeless tobacco products. The novelty of this work is in the development of a liquid chromatography tandem mass spectrometry method that can provide simultaneous quantification of trace levels of TSNAs and high concentrations of nicotine in biological media. Precision, accuracy, and stability were evaluated during method validation to ensure the method was fit for purpose. Several sample preparation and extraction methods were evaluated to minimize matrix effects and maximize analyte recoveries. The method was accurate in the range of 81.1% – 117%; repeatability was estimated in the range of 1.5% – 13.6% across multiple concentrations. The linear regression correlation coefficient (R2) was greater than 0.9959 for all analytes, and the limit of detection (LOD) was determined for nicotine, NNK, and NNN at 1 ng/mL 0.005 ng/mL, and 0.006 ng/ mL, respectively. Our method was found to be appropriate for the analysis of nicotine, NNN, and NNK in the porcine buccal epithelium and PBS extracts of smokeless tobacco products.Graphical abstract

The potential role of nicotine in breast cancer initiation, development, angiogenesis, invasion, metastasis, and resistance to therapy.

A large body of research studying the relationship between tobacco and cancer has led to the knowledge that smoking cigarettes adversely affects cancer treatment while contributing to the development of various tobacco-related cancers. Nicotine is the main addictive component of tobacco smoke and promotes angiogenesis, proliferation, and epithelial-mesenchymal transition (EMT) while promoting growth and metastasis of tumors. Nicotine generally acts through the induction of the nicotinic acetylcholine receptors (nAChRs), although the contribution of other receptor subunits has also been reported. Nicotine contributes to the pathogenesis of a wide range of cancers including breast cancer through its carcinogens such as (4-methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N-nitrosonornicotine (NNN). Current study aims to review the mechanistic function of nicotine in the initiation, development, angiogenesis, invasion, metastasis, and apoptosis of breast cancer with the main focus on nicotine acetylcholine receptors (nAChRs) and nAChR-mediated signaling pathways as well as on its potential for the development of an effective treatment against breast cancer. Moreover, we will try to demonstrate how nicotine leads to poor treatment response in breast cancer by enhancing the population, proliferation, and self-renewal of cancer stem cells (CSCs) through the activation of α7-nAChR receptors.

CLCNt2 Mediates Nitrate Content in Tobacco Leaf, Impacting the Production of Tobacco-Specific Nitrosamines in Cured Leaves.

Nitrate accumulation in tobacco (Nicotiana tabacum L.) leaf, particularly in the burley (BU) type, is a reservoir for the generation of nitrosating agents responsible for the formation of tobacco-specific nitrosamines (TSNAs). TSNAs are mainly produced via the nitrosation of alkaloids occurring during the curing of tobacco leaves. Additional formation of TSNAs may also occur during tobacco storage, leaf processing and in some circumstances via pyrosynthesis during combustion. Two TSNA species, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N-nitrosonornicotine (NNN) are found in the tobacco products and have been documented to be animal carcinogens. A previous study showed that decreasing the accumulation of nitrate in tobacco leaf via the overexpression of a deregulated form of nitrate reductase is efficient to reduce the production of TSNAs. We pursue in finding another molecular genetic target to lower nitrate in BU tobacco. Suppressing expression or knocking-out CLCNt2 has a direct impact on leaf nitrate and TSNA reduction in cured leaves without altering biomass. This study provides now a straight path toward the development of new commercial tobacco varieties with reduced TSNA levels by breeding of variants deficient in active CLCNt2 copies.

Metabolites of Tobacco- and E-Cigarette-Related Nitrosamines Can Drive Cu2+-Mediated DNA Oxidation.

Nitrosamine metabolites resulting from cigarette smoking and E-cigarette (E-cig) vaping cause DNA damage that can lead to genotoxicity. While DNA adducts of metabolites of nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N-nitrosonornicotine (NNN) are well-known tobacco-related cancer biomarkers, only a few studies implicate NNN and NNK in DNA oxidation in humans. NNK and NNN were found in the urine of E-cigarette users who never smoked cigarettes. This paper proposes the first chemical pathways of DNA oxidation driven by NNK and NNN metabolites in redox reactions with Cu2+ and NADPH leading to reactive oxygen species (ROS). A microfluidic array with thin films of DNA and metabolic enzymes that make metabolites of NNN and NNK in the presence of Cu2+ and NADPH was used to estimate relative rates of DNA oxidation. Detection by electrochemiluminescence (ECL) employed a new ECL dye [Os(tpy-benz-COOH)2]2+ that is selective for and sensitive to the primary DNA oxidation product 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxodG) in DNA. Enzyme-DNA films on magnetic beads were used to produce nitrosamine metabolites that enter ROS-forming redox cycles with Cu2+ and NADPH, and liquid chromatography-mass spectrometry (LC-MS) was used to quantify 8-oxodG and identify metabolites. ROS were detected by optical sensors. Metabolites of NNK and NNN + Cu2+ + NADPH generated relatively high rates of DNA oxidation. Lung is the exposure route in smoking and vaping, human lung tissue contains Cu2+ and NADPH, and lung microsomal enzymes gave the highest rates of DNA oxidation in this study. Also, E-cigarette vapor contains 6-fold more copper than that in cigarette smoke, which could exacerbate DNA oxidation.

ECL Array and LC-MS Studies of Nitrosamine-Metabolite-Mediated DNA Oxidation

Metabolites of nitrosamines in cigarette smoke and E-cigarette (E-cig) vapor cause DNA damage that has been projected to lead to cancers in “genotoxic” pathways. DNA oxidation can also be mediated by reactive oxygen species (ROS) generated from redox cycling reactions involving nitrosamine metabolites, metal ions (Cu2+) and NADPH. DNA adducts of tobacco nitrosamines, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N-nitrosonornicotine (NNN) are well known tobacco-related biomarkers, but only a few reports implicate NNK and NNN in DNA oxidation. We used electrochemiluminescent (ECL) arrays and LC-MS to explore pathways of DNA oxidation driven by metabolites of NNK and NNN facilitated by Cu2+ and NADPH which are present in significant amounts in lung tissue. We used a 3-D printed microfluidic array that first makes reactive metabolites of test compounds using metabolic enzymes (mostly cyt P450s) and DNA in thin films. The assay measurement step detects DNA oxidation selectively by ECL using a new ECL dye, [Os(tpy-benz-COOH)2]2+ that is highly sensitive for primary DNA oxidation product 8-oxodG, which serves as a co-reactant in the ECL generation. Array results reveal that NNK and NNN oxidize DNA at relatively high rates. DNA/metabolic enzyme films on magnetic beads were utilized with nitrosamines, Cu2+, and NADPH for measurement of 8-oxodG in oxidized DNA by LC-MS/MS and specific ROS involved were identified by ROS-specific assays, that showed that nitrosamines metabolism in the presence of Cu2+ and NADPH produces hydroxyl radical (•OH), singlet oxygen (1O2), and hydrogen peroxide (H2O2). Possible reactive metabolites of NNK and NNN involved in ROS generation were identified by LC-MS/MS. Results provide insight into mechanistic pathways of cyt P450-mediated metabolism of NNK and NNN in ROS production and oxidation of DNA.

Detection and quantification of 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB) from smoker albumin and its potential as a surrogate biomarker of tobacco-specific nitrosamines exposure and bioactivation

4-(Methylnitrosamino)-l-(3-pyridyl)-1-butanone (NNK) and N-nitrosonornicotine (NNN), two tobacco specific nitrosamine carcinogens, can form adducts with DNA and proteins via pyridyloxobutylation upon phase I enzyme-mediated bioactivation. Such DNA modifications have been proposed as the root cause to initiate carcinogenesis. Upon hydrolysis, both DNA and protein modifications would release 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB). The released HPB, being tobacco carcinogen specific, has the potential to serve as a surrogate biomarker for both tobacco exposure and carcinogen bioactivation. Because of its easy access, blood is a great source of such investigations with the potential in epidemiological application. HPB quantification from haemoglobin (Hb), however, has been demonstrated with limited success. To further explore this potentially paradigm-shift opportunity, we reported, for the first time, the detection and quantification of HPB from albumin (Alb) adducts formed by the tobacco-specific nitrosamines in mice and in human smokers. The time-course quantitative analysis of HPB from mouse Alb upon NNK exposure suggests that such an Alb adduct is stable. The amounts of HPB from Alb adducts in smoker plasma averaged 1.82 ± 0.19 pg/mg Alb (0.42 to 3.11 pg/mg Alb), which was 36 times the value in nonsmokers (0.05 ± 0.01 pg/mg Alb). Importantly, HPB level from Alb correlated positively with the level of human tobacco exposure estimated by urinary total nicotine equivalent (TNE) (R2 = 0.6170). For comparison, HPB level from Alb was 16.5 times that of Hb (0.12 ± 0.02 pg/mg Hb) in the plasma and red blood cell (RBC) samples of the same smokers. In addition, there was no significant correlation between HPB levels from Hb and TNE (R2 = 0.0719). These data overall suggest that HPB from Alb adducts can serve as a surrogate biomarker to monitor the level of tobacco exposure and carcinogenic nitrosamine bioactivation.

Expression of a constitutively active nitrate reductase variant in tobacco reduces tobacco-specific nitrosamine accumulation in cured leaves and cigarette smoke.

SummaryBurley tobaccos (Nicotiana tabacum) display a nitrogen‐use‐deficiency phenotype that is associated with the accumulation of high levels of nitrate within the leaf, a trait correlated with production of a class of compounds referred to as tobacco‐specific nitrosamines (TSNAs). Two TSNA species, 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanone (NNK) and N‐nitrosonornicotine (NNN), have been shown to be strong carcinogens in numerous animal studies. We investigated the potential of molecular genetic strategies to lower nitrate levels in burley tobaccos by overexpressing genes encoding key enzymes of the nitrogen‐assimilation pathway. Of the various constructs tested, only the expression of a constitutively active nitrate reductase (NR) dramatically decreased free nitrate levels in the leaves. Field‐grown tobacco plants expressing this NR variant exhibited greatly reduced levels of TSNAs in both cured leaves and mainstream smoke of cigarettes made from these materials. Decreasing leaf nitrate levels via expression of a constitutively active NR enzyme represents an exceptionally promising means for reducing the production of NNN and NNK, two of the most well‐documented animal carcinogens found in tobacco products.

Adolescents' and Young Adults' Knowledge and Beliefs About Constituents in Novel Tobacco Products.

Novel tobacco products, such as little cigars, cigarillos, hookah, and e-cigarettes, and their smoke or aerosol contain chemicals which the FDA has determined to be Harmful or Potentially Harmful Constituents. We explored adolescents' and young adults' knowledge and beliefs about constituents in novel tobacco products and their smoke or aerosol, in order to inform risk communication messages. Seventy-seven adolescents and young adults (ages 13-25) participated in 10 focus groups, including 47 novel tobacco product users and 30 susceptible nonusers. Participants were asked to discuss 10 pre-selected constituents found in novel tobacco products and their smoke or aerosol. The first author analyzed the discussion for emergent themes. Participants were generally familiar with arsenic, carbon monoxide, formaldehyde, and nicotine, but unfamiliar with acetaldehyde, acrolein, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanon (NNK), and N-nitrosonornicotine (NNN). All participants had negative beliefs about most constituents, although users had positive beliefs about nicotine. "Unfamiliar" constituents were associated with similarly-sounding words (eg, acetaldehyde sounds like acetaminophen), and some participants recognized words in the chemical names of NNK/NNN (eg, "nitro"). "Familiar" constituents were associated with negative health effects and other common products the constituents are found in. All participants wanted more information about the constituents' health effects, toxicity, and other common products. Most participants were unaware the constituents discussed are in novel tobacco products and their smoke or aerosol. Risk communication messages could capitalize on negative associations with familiar constituents, or attempt to educate about unfamiliar constituents, to discourage novel tobacco product use among adolescents and young adults. The results of this study have implications for how the FDA and other agencies can communicate about the risks of novel tobacco products to the general public, which will be particularly important once the Deeming Rule is finalized. Our findings suggest it may be effective to capitalize on the public's negative beliefs about and associations with familiar constituents, or to educate about unfamiliar constituents and their health effects, their concentration and toxicity in novel tobacco products and their smoke or aerosol, and other products they are found in.

CYP2A13 Genetic Polymorphisms in Relation to the Risk of Bladder Cancer in Japanese Smokers.

Tobacco-specific nitrosamines including 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N-nitrosonornicotine (NNN), which can be activated by the metabolic enzyme CYP2A13, are potent procarcinogens. Smoking plays a role in carcinogenesis in the human bladder, which expresses CYP2A13 at a relatively high level. Numerous genetic polymorphisms of CYP2A13 causing amino acid substitution might reduce CYP2A13 metabolic activity toward NNK and NNN, resulting in decreased susceptibility to bladder cancer. The aim of this study was to reveal any association between bladder cancer development and CYP2A13 genetic polymorphisms in Japanese smokers. The CYP2A13 genotype of each subject (163 bladder cancer patients and 161 controls) was determined by next-generation sequencing (NGS) of the full CYP2A13 gene. All samples were genotyped for five CYP2A13 variant alleles (CYP2A13*2, *3, *4, *6, *7). Based on biological logistic regression, the odds ratio (95% confidence interval) for the CYP2A13*1/*2 genotype was 0.34 (0.17-0.69). Thus, CYP2A13 genetic polymorphisms might play important roles in the development of bladder cancer in Japanese smokers.

Influence of usage time on exposure of snus users to nicotine, NNN and NNK from snus pouches

Poster POS1-23 presented at 2012 SRNT 18th Annual Meeting, March 13-16, Houston, Texas, USA. This study was entirely funded by British American Tobacco INTRODUCTION Measuring the amount of constituents extracted by snus consumers during use is a valuable step in estimating exposure to tobacco constituents. A factor potentially influencing the extent of exposure is the length of time that individual consumers keep snus pouches in their mouths during use. A 2007/2008 survey of snus use in Sweden established that, on average, use of pouched snus extends from approximately 30 minutes to just less than 120 minutes1. The objective of this study therefore was to quantify the importance of use duration on constituent transfer from pouched snus to the consumer over this timescale. The constituents selected for evaluation were nicotine and the two tobacco-specific nitrosamines (TSNAs) classified by IARC as Group 1 (sufficient evidence of carcinogenicity in humans)2, 4-(N-Methylnitrosamino)-1-(3-pyridinyl)-1-butanone (NNK) and N-Nitrosonornicotine (NNN).

TSNA levels in machine-generated mainstream cigarette smoke: 35 years of data

This paper characterizes historical and current tobacco specific nitrosamine (TSNA) levels in mainstream (MS) cigarette smoke of US commercial cigarettes. To conduct this analysis, we gathered 35years of published data of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N-nitrosonornicotine (NNN) levels in MS cigarette smoke. We also assessed internal data of MS smoke NNK and NNN levels generated from various market monitoring initiatives and from control cigarettes used in a multi-year program for testing cigarette ingredients. In all, we analyzed machine smoking data from 401 cigarette samples representing a wide range of products and design characteristics from multiple manufacturers and market leaders. There was no indication that TSNA levels systematically increased in cigarette MS smoke over the 35-year analysis period. In particular, TSNA levels expressed as either per cigarette or normalized for tar suggest a downward trend in MS smoke over the past 10years. The apparent downward trend in TSNA levels in MS smoke may reflect industry and agricultural community efforts to reduce levels of TSNAs in tobacco and cigarette smoke.

Addition of Porphyrins to Cigarette Filters To Reduce the Levels of Benzo[a]pyrene (B[a]P) and Tobacco-Specific N-Nitrosamines (TSNAs) in Mainstream Cigarette Smoke

Tobacco-specific N-nitrosamines (TSNAs) and benzo[a]pyrene (B[a]P) in mainstream cigarette smoke (MSS) cause smoking-related diseases and environmental pollution. Porphyrins were added to cigarette filters to reduce B[a]P (porphyrins A-E) and TSNAs (porphyrin F) in MSS. The porphyrin-B[a]P and porphyrin F-TSNAs (N'-nitrosoanabasine (NAB), N'-nitrosoanatabine (NAT), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and N-nitrosonornicotine (NNN)) interactions were investigated by fluorescence quenching and UV-visible spectroscopy. The correlation coefficients were 0.987-0.997 (B[a]P) and 0.994-0.999 (TSNAs), and the binding constants were (1.67-5.02) × 10(5) (B[a]P) and 3.42 × 10(3)-1.40 × 10(4) (TSNAs). Up to 36.72% of B[a]P and 46.67% of the TSNAs were eliminated from MSS, with greater reductions when more porphyrin was included in the filter. With the same mass of porphyrin in the filter, the reduction trend for B[a]P by porphyrins A-E was A > B > C > D > E. The reduction trend for TSNAs by porphyrin F was NNN > NAB > NNK > NAT. The porphyrin mode of action is possibly through strong π-π interactions.

Quantification of four tobacco-specific nitrosamines in cigarette filter tips using liquid chromatography-tandem mass spectrometry

Tobacco-specific N-nitrosamines (TSNA) have been suggested by some scientists to play an important role in tobacco smoke carcinogenesis. We have developed and validated an LC-MS/MS method for the determination of TSNA, notably N-nitrosoanabasine (NAB), N-nitrosoanatabine (NAT), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N-nitrosonornicotine (NNN), extracted from smoked cigarette filter tips. Reporting limits of 0.44, 0.89, 0.91 and 0.91ng mL−1 for NAB, NAT, NNK and NNN respectively were achieved. The newly developed method may find application in the filter analysis methodology for estimating the mouth-level exposure to NAB, NAT, NNK and NNN for cigarette smokers. TSNA levels were determined in mainstream smoke collected on industry standard Cambridge Filter Pads following smoking on a smoking machine. TSNA yields were compared with TSNA levels extracted from cigarette filter tips. However, the observation of the progressive post-smoking accumulation of TSNA during ambient storage of smoked cigarette filter tips potentially compromises use of this technique as an estimate of mouth-level exposure. Storage of smoked cigarette filter tips at sub-ambient temperatures reduced substantially the post-smoking synthesis of TSNA.

CYP2A6 Polymorphisms and Risk for Tobacco-Related Cancers

Tobacco consumption is the main identifiable risk to cancer, contributing to the majority of tumors in upper aerodigestive tissues. The psychoactive compound responsible for tobacco addiction, nicotine and the potent carcinogens present at high concentrations either in cigarette mainstream smoke or in smokeless tobacco products, 4-(methylnitrosamino)-1-(3-pyridyl)-butanone (NNK) and N-nitrosonornicotine (NNN) can be metabolized by CYP2A6. CYP2A6 is expressed in many aerodigestive tissues with high interindividual variability. The CYP2A6 gene is highly polymorphic and CYP2A6 alleles coding for enzymes with altered expression or metabolic capacity produce alterations in nicotine metabolism in vivo and seem to influence smoking behavior. These polymorphisms may change the rate of NNK and NNN activation and, therefore, may influence cancer risk associated with tobacco consumption. However, to date only a few and inconclusive studies have addressed the risk that a given CYP2A6 polymorphism presents for the development of tobacco-related tumors. Most, but not all, show a reduced risk associated with alleles that result in decreased enzyme activity. The overlapping substrate specificity and tissue expression between CYP2A6 and the highly similar CYP2A13 may add to the conflicting results observed. The intricate regulation of CYP2A6 and the variation of structurally different chemical compounds capable of inhibiting CYP2A enzymes also add to the complexity. Finally, the interaction between polymorphisms of genes that code for CYP2A6, CYP2A13 and other potent carcinogen-metabolizing CYP enzymes may help to determine individuals that are at higher risk of developing tumors associated with tobacco consumption.

Effects of Smokeless Tobacco and Tumor Promoters on Cell Population Growth and Apoptosis of B Lymphocytes Infected with Epstein–Barr Virus Types 1 and 2

The effects of smokeless tobacco purified products 4-(N-methyl-N-nitrosamine)-1-3-pyridinyl)-1-butanone (NNK) and N-nitrosonornicotine (NNN), smokeless tobacco extracts (dry snuff, moist snuff, and loose leaf), and the tumor promoters 12-O-tetradecanoyl phorbol-13-acetate (TPA) and n-butyrate on cell population growth, cell death, and apoptosis were studied in B lymphocyte cell lines harboring Epstein–Barr virus (EBV) type 1 (Raji and X50-7) or type 2 (HR-1K and AG876) and in an EBV-uninfected control lymphocyte cell line (Ramos). Spontaneous apoptosis was present in all EBV-infected cell lines, but at varying levels. Spontaneous and induced apoptosis were generally greater by Student–Newman–Keuls tests in cells harboring EBV type 2 compared to EBV type 1. The greatest effects on cell population growth, cell death, and apoptosis on cells harboring lytic EBV type 1 (X50-7) was with each of the three smokeless tobacco extracts. The greatest effects on cells harboring EBV type 2 was with TPA and n-butyrate. There were no effects of smokeless tobacco extracts on the Raji cell line that harbors EBV type 1 incapable of lytic replication. Smokeless tobacco purified products, NNN and NNK, had no discernible effects. At the concentrations used in these experiments, there appears to be an EBV type-specific response to chemical induction, with greater susceptibility of lytic EBV type 1 to smokeless tobacco extracts and lytic EBV type 2 to TPA and n-butyrate. This EBV type-specific susceptibility to the effects of smokeless tobacco extracts is another phenotypic difference between EBV types. The use of smokeless tobacco products may affect B lymphocytes infected with replication-capable EBV in the oropharynx. The absence of significant effects with NNK and NNN suggests that these properties reside with other compounds present in tobacco extracts.

Inhibition of mutagenicity of N-nitrosamines by tobacco smoke and its constituents

Tobacco smoke is a complex chemical mixture including pyridine alkaloids and N-nitrosamines, with the concentration of the former several orders of magnitude higher than that of the N-nitrosamines. The major biologically important N-nitrosamines present in tobacco smoke are N-nitrosodimethylamine (NDMA), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and N-nitrosonornicotine (NNN). These nitrosamines require metabolic activation by cytochrome P-450s for the expression of mutagenicity. Although nicotine, the major pyridine alkaloid in tobacco, has been shown to inhibit the metabolic activation of NNK, its effect on the mutagenicity of NNK and other N-nitrosamines has not been reported. In the present study, the ability of three pyridine alkaloids (nicotine, cotinine, nornicotine) and aqueous cigarette smoke condensate extract (ACE) to inhibit the mutagenicity of tobacco-related N-nitrosamines was tested on Salmonella typhimurium strain TA1535 in the presence of a metabolic activation system (S9). All three of the pyridine alkaloids tested, as well as ACE, inhibited the mutagenicity of NDMA and NNK, but not NNN, in a concentration-dependent manner. The induction of SCEs in mammalian cells (CHO) by NNK in the presence of metabolic activation was also significantly reduced by nicotine and cotinine. None of the observed reductions in mutagenicity could be explained by cytotoxicity. These results demonstrate that tobacco smoke contains chemicals, pyridine alkaloids and other unidentified constituent(s), which inhibit the mutagenicity of N-nitrosamines.

The Application of Solid Phase Extraction to the Analysis of Tobacco-Specific Nitrosamines

A new isolation and separation method has been developed for the alkaloid-related nitrosamines in tobacco, called tobacco-specific nitrosamines (TSNA). The improved isolation procedure involves the extraction by sonication of TSNA from cured tobacco with toluene and an aqueous citrate buffer solution, while purification is achieved by solid phase extraction on small silica cartridges. Interfering extract materials are eluted from the silica cartridges with the toluene and a chloroform-methylene chloride solvent, while subsequent elution with chloroform yields the TSNA in a purified fraction. Gas chromatographic analyses on an SE-54-coated glass capillary column and detection by a nitrogen-phosphorous detector produce very good data on the four TSNAs, which include N-nitrosanatabine (NATB), N-nitrosoanabasine (NAB), 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (called NNK), and N-nitrosonornicotine (NNN). The method is shown to be quantitative, reproducible, and applicable to the determination of TSNA in various tobacco samples, such as stems and lamina of cured tobacco leaves, as well as to smokeless tobacco.