Tobacco-specific Nitrosamines In Tobacco Research Articles

Determination of tobacco-specific nitrosamines in tobacco and mainstream cigarette smoke using one-step clean-up coupled with liquid chromatography- tandem mass spectrometry

A method for determining tobacco-specific nitrosamines (TSNAs) in tobacco and cigarette smoke using liquid chromatography-tandem mass spectrometry was established. The established method amended the deficiencies that exist in current mainstream methods. In this method, TSNAs in tobacco and cigarette smoke were extracted by water. The aqueous extract was then extracted by dichloromethane, and the extract could be analyzed by liquid chromatography-tandem mass spectrometry after a solvent replacement. This method was used to analyze flue-cured tobacco samples, and the response of the target compounds was about 10 times higher than that of the ammonium acetate extraction method. When analyzing cigarette smoke samples, the response strength and chromatographic peak purity of the target compounds were also significantly improved. The proposed method exhibited good linearities for both tobacco and cigarette smoke samples (r2 > 0.99). The limits of detection (LODs) for tobacco and cigarette smoke samples were 0.2–1.0 ng/g and 0.1–0.3 ng/cigarette, respectively. Additionally, this method exhibited desirable accuracy and precision. The TSNAs recovery values from tobacco and cigarette smoke samples ranged from 95.7 % to 107.7 % with inter- and intra-day relative standard deviations (RSDs) of less than 7.4 %. This method is simple, effective, and has wide adaptability. It is a useful upgrade to the existing methods for analyzing TSNAs in tobacco and cigarette smoke.

Trends in Analytical Methods for Analysis of Tobacco Products: An Overview

Abstract. Currently, due to the strong trends in policies to reduce smoking, several concerns related to quality control and regulation of tobacco products have been raised, as a great variety of products are available to consumers in the market. Considering that development of robust and selective analytical methods for tobacco and derivative products has been an important and necessary task over the years, however laborious, due to the complexity of the matrices. This work presents an overview of advances, innovation, and trends in analytical methods, focused on the chromatographic analysis of nicotine, tobacco-specific nitrosamines (TSNAs), humectants, pesticides, polycyclic aromatic hydrocarbons (PAHs) and sugars in different tobacco products. Gas chromatography (GC) coupled with different detectors is widely used to analyze nicotine, humectants and TSNAs in tobacco leaves and derivative products, while liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been frequently used to analyze TSNAs in mainstream cigarette smoke and smokeless tobacco products. Pesticides were mainly analyzed in tobacco using GC or LC coupled with mass detector (MS), while PAHs and sugars were generally analyzed in tobacco and smoke using LC-MS and GC-MS techniques, respectively. In addition to already established methods and despite the lack of a worldwide standardization of methods, significant efforts have been made to develop analytical procedures for a wide variety of tobacco products, with a broad range of innovative chromatographic methods available. In this sense, a potential trend is the possibility of simultaneous determination of multiple components aiming to reduce the analysis time. The present study examined the main works that developed or improved analytical methods for identifying and quantifying priority compounds in different tobacco matrices and aims to contribute to future research with this objective, in addition, to promoting standardization of technical terms used in this analytical area. It is noteworthy that some of the methods mentioned here have not been validated and further studies are needed in order to obtain reproducible analytical methods for regulatory purposes.
 Keywords: chromatography analyses; tobacco leaves; cigarette; cigarette smoke; smokeless tobacco products.

Cancer Progress and Priorities: Lung Cancer.

In the United States, lung cancer is the second most common diagnosed cancer and the leading cause of cancer-related death. Although tobacco smoking is the major risk factor accounting for 80% to 90% of all lung cancer diagnoses, there are numerous other risk factors that have been identified as

Simultaneous determination of alkaloids and their related tobacco-specific nitrosamines in tobacco leaves using LC-MS-MS.

Tobacco alkaloids (e.g., nicotine) and their metabolized tobacco-specific nitrosamines (TSNAs) are very important compounds for tobacco quality and safety. A simple and specific liquid chromatography-tandem mass spectrometry method was developed for the simultaneous determination of eight tobacco alkaloids and their related four TSNAs in tobacco leaves. The milled tobacco was extracted using 0.1 mol/L ammonium acetate solution and purified using methanol. Mass spectrometry parameters including declustering potential and collision energy were optimized to ensure that both the TSNAs and the tobacco alkaloids have suitable responses. Recoveries for accuracy were in the range of 80.2-105.2%. Intra-day and inter-day repeatability were in the range of 1.7-12.1% and 6.4-18.7%, respectively. Limit of detection and limit of quantitation were estimated in the range of 6 ng/g-45 μg/g and 24 ng/g-90 μg/g, respectively. The established method was applied to investigate the distribution of tobacco alkaloids and TSNAs in four kinds of tobacco. The result showed that the burley and the flue-cured have the highest (0.00047%) and the lowest (0.000024%) percentage of transformation from alkaloids to TSNAs, respectively. Thus, this method can be used for a wide range of samples.

Endogenous cytosine methylation and the formation of carcinogen carcinogen-DNA adducts

All CG dinucleotides along exons 5-8 of the p53 tumor suppressor gene contain endogenous 5-methylcytosine ((Me)C, X = Me in Scheme 1). The same sites (e.g. p53 codons 157, 158, 245, 248, and 273) are mutational hotspots in smoking induced lung cancer, suggesting that methylated CG dinucleotides may be preferentially targeted by the reactive metabolites of tobacco carcinogens. We employed a stable isotope labeling HPLC-ESI-MS/MS approach to demonstrate that methylated CG dinucleotides of the p53 gene are the preferred binding sites for the diolepoxide metabolites of bay region polycyclic aromatic hydrocarbons, e.g. benzo[a]pyrene diol epoxide (BPDE). In contrast, cytosine methylation was protective against O(6)-guanine alkylation by tobacco tobacco-specific nitrosamines, e.g. 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), To investigate the mechanisms behind these effects, a series of structural analogs of (Me)C were prepared, and their effects on reactivity of base the paired dG towards BPDE was examined. We found that the presence of the C-5 substituent on cytosine influences the reactivity of its partner guanine towards BPDE and modifies the stereoisomeric composition of the resulting N(2)-BPDE-dG adducts.

Tobacco-specific nitrosamines in tobacco from U.S. brand and non-U.S. brand cigarettes

Tobacco-specific nitrosamines in tobacco from U.S. brand and non-U.S. brand cigarettes

Relationship between Tobacco-Specific Nitrosamines and Nitrite from Different Air-Cured Tobacco Varieties

Three tobacco varieties of differing agronomic characteristics were grown and air-cured during the 1988-1990 growing seasons. The leaves were separated into lamina and midvein from three stalk positions. The replicated samples were analyzed for individual alkaloids, nitrate, nitrite, N'-nitrosonornicotine, N'-nitrosoanatabine, N'-nitrosoanabasine, and 4-[(methylnitroso)amino]-1-(3'-pyridyl)-1-butanone. The air-cured flue-type tobacco contained consistently lower quantities of the individual tobacco-specific nitrosamines (TSNA). There were no statistical differences in TSNA and stalk position or TSNA and curing season. Correlation coefficients were determined between nitrite and individual TSNA. Their coefficients showed there was a positive relationship between nitrite levels and individual TSNA concentrations in the tobacco tissue. From this 3-year study there was no relationship between alkaloids or nitrate and individual TSNAs. This study indicates that accumulation of TSNA in tobacco is limited by the amount of nitrite accumulated during air-curing.

Tobacco-specific nitrosamines in Canadian cigarettes

Twenty-five brands of Canadian commercial cigarettes were analyzed for tobacco-specific nitrosamines (TSNA) in tobacco and in mainstream smoke as well as for nitrate in tobacco. Preformed N'-nitrosonor-nicotine (NNN) in the tobacco ranged from 265 ng to 979 ng/cigarette, preformed 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) ranged from 465 ng to 878 ng/cigarette. The mainstream smoke concentration for NNN was between 5 ng and 39 ng/cigarette and for NNK between 5 ng and 97 Ng/cigarette. The nitrate levels were between 0.3 mg and 3.4 mg/cigarette. The NNK levels in tobacco and in mainstream smoke were higher than the NNN levels, which is typical for Virginia-type cigarettes. Based upon the average mainstream smoke concentration of the three most popular Canadian cigarette brands, an average TSNA delivery for 20 cigarettes of 0.7 micrograms NNN and 1.7 micrograms NNK can be calculated, which is less than the average for West German cigarettes. The results of this investigation demonstrate that there seems to be a good correlation between the TSNA and tar deliveries in mainstream smoke. However, no correlation between the level of preformed TSNA in tobacco and the tar delivery in mainstream smoke could be observed. It is demonstrated that the good correlation between the tar and TSNA deliveries in mainstream smoke can only be attributed to the unusual good correlation between the tar delivery and the ventilation ratio. For the cigarettes investigated, which seemed to be Virginia-type cigarettes, with few exceptions, the ventilation ratio had a much higher influence on the mainstream smoke concentration than the level of preformed TSNA in tobacco.

Investigations on the origin of tobacco-specific nitrosamines n mainstream smoke of cigarettes

The origin of tobacco-specific nitrosamines (TSNA) in mainstream smoke and the possible contribution of synthesis during the smoking procedure was investigated. Addition of the nitrosamine precursors nitrate and nicotine to the tobacco prior to smoking did not change the mainstream smoke concentrations of N'-nitrosonornicotine (NNN) and 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK), whereas the mainstream smoke concentration of N'-nitrosoanabasine (NAB) and N'-nitrosoanatabine (NAT) increased after spiking the cigarettes with nitrate. Data for TSNA in tobacco and in mainstream smoke and for nitrate in tobacco of commercial cigarettes of the West German market, taken from previous investigations, were used to calculate the mainstream smoke/tobacco ratios for NNN and NNK. These ratios were corrected for ventilation and cigarette length. It is shown that the ratios are constant and neither depend on the nicotine level nor on the nitrate level of the tobacco except for NNK in the nitrate rich dark tobacco type cigarettes. For nonfilter cigarettes the transfer rates of NNN and NNK which had been corrected for ventilation and cigarette length amounted to 23 or 34% respectively. For filter cigarettes a transfer rate of 13% for NNN and 23% for NNK was calculated. Furthermore it is shown that the mainstream smoke/tobacco ratios for NNN and NNK are constant over the whole length of the cigarettes except for NNK in dark tobacco type cigarettes. The results of this investigation indicate that pyrosynthesis of NNN does not occur and that it is very unlikely for NNK at least for lower nitrate levels. Thus with few exceptions the TSNA burden of smokers is predominantly influenced by the amount of preformed NNN and NNK in tobacco.

Tobacco-specific nitrosamines, an important group of carcinogens in tobacco and tobacco smoke.

Tobacco-specific nitrosamines are a group of carcinogens that are present in tobacco and tobacco smoke. They are formed from nicotine and related tobacco alkaloids. Two of the nicotine-derived nitrosamines, NNK and NNN, are strong carcinogens in laboratory animals. They can induce tumors both locally and systemically. The induction of oral cavity tumors by a mixture of NNK and NNN, and the organospecificity of NNK for the lung are particularly noteworthy. The amounts of NNK and NNN in tobacco and tobacco smoke are high enough that their total estimated doses to long-term snuff-dippers or smokers are similar in magnitude to the total doses required to produce cancer in laboratory animals. These exposures thus represent an unacceptable risk to tobacco consumers, and possibly to non-smokers exposed for years to environmental tobacco smoke. The permission of such high levels of carcinogens in consumer products used by millions of people represents a major legislative failure. Indeed, the levels of tobacco-specific nitrosamines in tobacco are thousands of times higher than the amounts of other nitrosamines in consumer products that are regulated by government authorities. Although the role of tobacco-specific nitrosamines as causative factors in tobacco-related human cancers cannot be assessed with certainty because of the complexity of tobacco and tobacco smoke, several lines of evidence strongly indicate that they have a major role, especially in the causation of oral cancer in snuff-dippers. Epidemiologic studies have demonstrated that snuff-dipping causes oral cancer. NNK and NNN are quantitatively the most prevalent known carcinogens in snuff, and they induce oral tumors when applied to the rat oral cavity. A role for NNK in the induction of lung cancer by tobacco smoke is likely because of its organospecificity for the lung. Tobacco-specific nitrosamines may also be involved in the etiology of tobacco-related cancers of the esophagus, nasal cavity, and pancreas. Because they are derived from nicotine, and therefore should be associated only with tobacco, tobacco smoke and other nicotine-containing products, tobacco-specific nitrosamines as well as their metabolites and macromolecular adducts should be ideal markers for assessing human exposure to, and metabolic activation of, tobacco smoke carcinogens. Ongoing research has demonstrated the formation of globin and DNA adducts of NNK and NNN in experimental animals. Sensitive methods for the detection and quantitation of these adducts in humans would provide an approach to assessing individual risk for tobacco-related cancers.(ABSTRACT TRUNCATED AT 400 WORDS)