Analysis of Tobacco-Specific Nitrosamines in Tobacco and Tobacco

Chapter 45

Analysis of Tobacco-Specific Nitrosamines in Tobacco and Tobacco Smoke Klaus D. Brunnemann and Dietrich Hoffmann

Downloaded by AUBURN UNIV on December 2, 2016 | http://pubs.acs.org Publication Date: March 28, 1994 | doi: 10.1021/bk-1994-0553.ch045

American Health Foundation, 1 Dana Road, Valhalla, NY 10595

Several groups of nitrosamines have been identified in tobacco and to bacco smoke; these include volatile nitrosamines (mainly nitroso dimethylamine and nitrosopyrrolidine), non-volatile nitrosamines (such as nitrosodiethanolamine), nitrosamino acids (see abstract by Djordjevic et al. in this volume) and tobacco-specific nitrosamines (TSNA). The latter derive from the Nicotiana alkaloids. The major TSNA found in tobacco and tobacco smoke are Ν'-nitrosonornicotine (NNN), Ν'-nitrosoanatabine (NAT), Ν'-nitrosoanabasine (NAB), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-buta none (NNK). Of these, NNN and NNK are strong carcinogens in mice, rats and hamsters where they induce benign and malignant tumors of the lung, nasal cavity, mouth, esophagus, and/or pancreas. For the analysis of TSNA, 5-10 g of tobacco were extracted with citrate buffer pH 4.5 containing 20 mM ascorbic acid; N-nitrosopentylpicolylamine (NPePicA) served as internal standard. The buffer was then partitioned with dichloromethane (DCM) and the DCM fraction was chromatographed on 65 g Al O using 250 ml DCM (to yield the VNA fraction) followed by 250 ml DCM-acetone (4:1 to yield the TSNA fraction). The latter was analyzed by GC-TEA We analyzed 16 samples of moist snuff and found 0.8-64 μg NNN/g, 0.2-215 μg NAT/g, 0.016.7 μg NAB/g, and 0.08-8.3 μg NNK/g (expressed per dry tobacco weight). In six samples of dry snuff, we found 9.4-55 μg NNN/g, 11-40 μg NAT/g, 0.5-1.2 μg NAB/g, and 0.88-14 μg NNK/g. These levels of TSNA are at least three orders of magnitude higher than those N-nitrosamines found in any other consumer product. A newly developed method for the extraction of TSNA in tobacco is based on the extraction with methanol-modified supercritical carbon dioxide (1). This method yielded up to 7 times more NNK than did conventional solvent extraction, while the yields of other TSNA were similar among the different procedures. For the analysis of TSNA in cigarette smoke, 10-40 cigarettes (for mainstream smoke collection) or 5-10 cigarettes (for sidestream smoke collec tion) were smoked into two gas wash bottles containing citrate buffer p H 4.5 with 20 mM ascorbic acid as scavenger and NPePicA as internal standard, followed by a 90-mm Cambridge filter. Others claimed that this method may lead to artefact formation (2), however, in our hands there was no difference 2

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0097-6156/94/0553-0369$08.00/0 © 1994 American Chemical Society

Loeppky and Michejda; Nitrosamines and Related N-Nitroso Compounds ACS Symposium Series; American Chemical Society: Washington, DC, 1994.

NITROSAMINES AND RELATED JV-NITROSO COMPOUNDS

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whether the smoke was collected this way or onto a Cambridge filter treated with ascorbic acid or onto an untreated Cambridge filter directly (3,4). The buffer and the filter were extracted with D C M and analyzed analogously to tobacco. Table I lists the major TSNA found in the mainstream smoke of some domestic cigarettes. Some foreign brands (Russian and French) yielded much higher TSNA data than domestic brands, while Japanese brands clearly had lower TSNA levels. It is noteworthy that the brand yielding the highest TSNA levels in mainstream smoke yielded the lowest TSNA levels in the sidestream smoke and vice versa. Moreover, the sidestream/mainstream ratio for NNN ranged from 0.6-8.3 while that for N N K ranged from 1.5-21, a two to three fold increase. This result means that during smoldering of the cigarette more NNK is pyrosynthesized in the reducing zone of the burning cone. We observed a similar trend earlier (5). Table I. TSNA in the Mainstream Smoke of Domestic Cigarettes (ng/cig.) Total NNK NAB NAT Cigarette Type NNN A D G I J

NF F F, M F, L F, U L

278 209 250 138 40

236 172 192 114 37

30 21 20 14 4

156 156 173 87 17

700 558 635 353 98

NF=non-filter; F=filter; M=menthol; L=light; UL=ultra light We also analyzed the TSNA in the sidestream smoke of domestic cigarettes (Table II). Table II. TSNA in the Sidestream Smoke of Domestic Cigarettes (ng/cig.) Total NNK NAB Type NNN NAT Cigarette A D G I J

NF F F, M F, L F, U L

170 191 238 214 330

105 119 132 132 221

20 19 24 18 41

241 312 303 216 352

536 641 697 580 944

For abbreviations see Table I. We also applied supercritical fluid extraction (SFE) to the analysis of TSNA The mainstream smoke of only one cigarette was collected on a 44mm Cambridge filter and then extracted with supercritical C O 2 containing 5% methanol. 2-(2-Methylnitrosaminoethyl)pyridine served as internal standard. The recoveries of TSNA standards ranged from 60-68%. In contrast to tobacco, the SFE method yielded only 60-90% of the TSNA obtained with conventional solvent extraction. Efforts are underway to improve the efficiency by using different modifiers. ACKNOWLEDGEMENTS Supported by Grant CA-29580 from the National Cancer Institute.


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REFERENCES 1. 2. 3. 4.


5.

Prokopczyk, B., Hoffmann, D., Cox, J.E., Djordjevic, M.V., and Brunnemann, K.D. Chem. Res. Toxicol. 5: 336-340, 1992. Caldwell, W.S. and Conner, J.M. 43rd Tobacco Chemists' Research Conf., Richmond, VA, Oct. 2-5, 1989; abstr. 45. Djordjevic, M.V., Sigountos, C.W., Brunnemann, K.D., and Hoffmann, D. J. Agric. Food Chem. 39: 209-213, 1991. Brunnemann, K.D., Cox, J.E., Liu, Y. and Hoffmann, D. 46th Tobacco Chemists' Research Conf., Montreal, Canada, Sept 27-30, 1992; abstr. 22. Adams, J.D., Ο Mara-Adams, K.J. and Hoffmann, D. Carcinogenesis 8: 729-731, 1987.

RECEIVED January 26, 1994


Analysis of Tobacco-Specific Nitrosamines in Tobacco and Tobacco

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