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r I Anlage 8 . ~ INIL U) ASSESSMENT OF TOBACCO SPECIFIC N-NITROSAMINES IN TOBACCO PRODUCTSl Dietrich Hoffmann, John D. Adams, Klaus D. Brunnemann and Stephen S. Hecht Division of Environmental Carcinogenesis Naylor Dana Institute for Disease Prevention American Health Foundation Valhalla, New York 10595 1Supported by National Cancer Institute Contract NO1-CP-55666 and American Cancer Society Grant No. BC56 - The Alexander Ralston Peacock Memorial Grant. * G Stephen S. Hecht is recipient of National Cancer Institute Research Career Development Award NO-5K04CA00124. This is paper LXII in the series "Chemical Studies on Tobacco Smoke". The abbreviations used are: NNN - N'-Nitrosonornicotine NNK - 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-l-butanone ' NAB - N -Nitrosoanabasine NAtB - NDMA - N'-Nitrosoanatabine Nitrosodimethylamine ~' ~ NPYR - Nitrosopyrrolid'ine Q HPLC - High performance Liquid chromatography 0' GLC-MS - Gas-liquid chromatography-mass spectrometry )-.i TEA - Thermal Energy Analyzer r' Receivedi . Accepted (~ rr w

ABSTRACT Tobacco specific nonvolatile N-nitrosamines in tobacco and in fresh mainstream and sidestream smoke of cigarettes and cigars were quantitatively determined with a TEA. . The smoke was trapped in ascorbic acid solution buffered at pH 4.5, extracted with dichloromethane and the organic phase was chromatographed and analyzed by HPLC-TEA methodology (sensitivity, 250 pg per injection). The nonvolatile nitros- amines were further enriched by repeated chromatography and positively identified by GLC-MS. NNN--2' 14C served as internal standard for the quantitative analysis. The tobacco of 5 different cigarettes contained between 0.22 and 7.0 ppm of the carcinogenic NNN, 0.13 and 0.74 ppm of the carcinogenic NNK and 0,44-3.2 ppm of the newly identified NAtB. In unaged mainstream.smoke and sidestream smoke of the same cigarettes, values ranged between 0.24-3.7 Vg/cig and 0.15-6.1 Vg/cig for NNN, 0.11-0.42 Vg/cig and 0.19-0.66 ug/cig for NNK and 0.33-4.6 Vg/cig and 0.15-1.5 Vg/cig for NAtB, re- spectively. The relatively high concentrations of these carcinogenic N-nitrosamines in sidestream smoke are discussed as possible tobacco specific indicators for indoor pollution. ~ ~ - 1 -

/ INTRODUCTION The occurence of NNN and NNK (Chart 1) in tobacco and in cigarette smoke has been ascertained during recent years (11). ceivably contribute to the incidence of tobacco related cancers. NNN, NNK, NAtB and possibly other as yet unidentified N-nitros- amines are formed during curing and smoking from nicotine, nornicotine, anatabine and possibly, from other minor tobacco alka- loids (111. Tobacco products contain these carcinqgenic nitros- amines in the range of 0.1-100 ppm; therefore, their detection and quanti tat ive analysis require3 lengthy enrichrmnt pzooedures in earlier studies. The highly sensitive and specific thermal energy analyzer developed by Fine et al (8) simplifies the 'G quantitative determination of nitrosamines, in that these C nonvolatile compounds need only be concentrated once prior to C separation by IIPLC and detection with this new instrument. The ~ mechanism of detection of the nitrosamines involves pyrolytic Q decomposition to nitrogen oxide, separation from the solvents ~ G- in a cold trap, and oxidation of No by ozone to an excited state N02*. The latter decays to its ground state under emission of light in the near infrared region which is recorded after photomultiplication. These tobacco specific N-nitrosamines.are carcinoyenlc in mice, rats and Syrian hamsters (3,9,12,16,23) and could con- 2 t

t For an accurate measurement of these nitros- amines in chewing tobacco, tobacco leaves and un- aged smoke, the extraction of these materials as well as the trapping of the freshly generated aerosol must be done with ascorbic acid solution at pH 4.5 (15,20). For the quantita- tive analysis 114CJNNN serves as internal standard. This study reports quantitative data for NNN,, NNK and the newly identified NAtB in smoking tobacco, in chewing tobacco and in unaged mainstream smoke of cigarettes and cigars. Sidastream smokA which is generated during smoulder- ing of tobacco in between puffs frequently contains higher concentrations of these tobacco-specific N-nitrosamines than mainstream smoke. MATERIALS AND METHODS Apparatus. In order to generate the mainstream smoke of cigarettes, little cigars and cigars we used a 2o-port auto- matic smoker (H. Borgwaldt, Hamburg, Germany) with a rotating head, of which.every second port was connected with a nitrogen 3

source, so that an exchange of air in the traps with nitrogen occurred every 2 seconds. (15). A small sidestream.. smoke collector was used for cigarettes and little cigars (4,5) and a large collector for cigars (22); in both cases the air-flow through these devices was regulated to be 25 ml/sec (4). This air flow rate renders total particulate matter and nicotine in the mainstream smoke at the same levels as•those produced by smoking a cigarette, or a cigar in the open air. In this setting the mainstream smoke was generated by a single-port piston-type smoker (H. Borgwaldt, Hamburg, Germany),. For the HPLC-separation we utilized a Model 60Q0A solvent delivery system 63ateis. Associates, I.nc, Milford, I4ass,1 with. a Model 70-10 Sample Injection Valve equipped wittt a Model 70-11 Loop Filler Port (Rheodyne, Berkeley, Californial and Corasil II and y-Porasil columns (Intaters Associates,Inc,). The thermal energy analyzer with HPLC interface •. (_Thermo Electron Corp., Walthamy Mass.) with a Hewiett-8ackard Model' 2230A recording integrator served, as detector. Unknown nitrosamines were identified with a Hewlett- Packard Model 5982A GLC-MS instrument. A Nuclear-Qiicago Isocap 300 was used for scintillation counting in toluene with 0.5% of PPO (2,5-diphenyloxazole) and 0.005$ POPOP (1,4-bis[2(5-pheny1- oxazolyl)7lbenzene). 4

Reagents. [2'-14C]NNN (14.1 mCi/mmole) was , and was pure, according to HPLC with TEA synthesized (17) and GLC-MS analysis (9). NNN and NNK were also synthesized by methods reported in the literature (9,17). The initial NAtB was synthesized from anatabine, which was kindly supplied by Dr. T.C. Tso, U.S.D.A., Beltsville, MD and was purified by HPLC. Synthesis of NAtB.- dl-Anatabine was prepared according to the method of Quan et al (21) and Was nitrosated to NAtB according to Hu et al (17:1- The crude NAtB was purified by chromatography on preparative TLC plates (silica gel; solvent CHC13/MeOH-9./1 Rf0_59) and by distillation under reduced pressure (0.5 mm Hg) at an oiL bath temperature of 176°. Gas chromatography on a 2 mm x 3.6 m 10€ UCCI-98 on Gas Chrom Q Ilass column Coven temperature 20VC) showed a single peak. Spectral properties_ IR (film) 3040, 2930, 1580, 1430, 1335, 1160,978, 710 cm 1; MS m/e (rel. intensity) 189 (M+, 15.1), 172 (20.5), (74.2), 8.9-8.2 6.7-3.9 (2H, m, 159 (100.0), 157 (33.7), 144 (35.1), 130 (72.6), 117 105 (58.91, 92 (46.0), 80 (30.4), 78 (53.7); NMR (CDC13) ppm (2H, m, pyridyl CH), 7. 8-7.1 ppm, (2H, m, pyridyl CH), ppm (5H, m, pyridyl-CH-N, N-CH2 -CH=CH), 3.7-2,6 ppm CH2-CH=CH); ' - 5

Elemental analysis (Calbraith Laboratories, Inc., Knoxville, Tennessee 37321): C10H11N30 Calculated: C 63.47, H 5.86, N 22.21 Found: . C 63.44, H 6.04, N 22.03 Tobacco Products. Commercial chewing tobacco, cigarettes, little cigars,and cigars were purchased on the open market in 1977 and 1478. *The experimental cigarettes were supplied by Dr. T.C. Tso, U.S.D.A., Beltsville{ MD. All tobacco products were stored for at least 24 hr in a humidity chamber at relative humidity of 60 + 3% and at 22° + 2°. The smoking laboratory was also kept at constant relative humidity of 60 + 5% and at 22° + 2°. The cigarettes and little cigars were weight selected Ci- 20 mg,of average weight of 200) and were smoked under standard conditions for cigarettes (1 puff/min; puff duration 2 sec; puff volume 35 ml; the butt length was determined by the filter plus overwrap plus 3 mm or was set at 23 mm, whichever was longer; 1). The cigars were selected by average wei~ght (± 5% average weight of 50 cigars) and were smoked under standard conditions (1 puff/ 40 sec; puff duration length 33 mm; 18). 1.5 sec; puff volume 20 ml; and butt 6

,,,%e-- Tobacco Analysis. Fine-cut chewing tobacco and the tobaccos of cigarettes and little cigars were analyzed without grinding. Cigar tobaccos were ground in a blender before extraction. An aliquot was taken for water determination (2). For the actual analysis about 25 g tobacco was extracted for 24 hrs by stir- ring at room temperature with 1C0 ml 5mM ascorbic acid solution at pH 4.5 (citric acid-sodium phosphate buffer; .18) and 0.5 yg of (14CJNNN was added as internal standard. The extract was then filtered through a 5-micron nylon filter cup (Liquiflo Inc., Plainview, N.Y.), and adjusted to pH 5 by addition of a few drops of Msodium hydroxide. This aqueous phase was then extracted 6 times t;rith equal volumes of ethyl acetate. The ethyl acetate was dried (Nz2So4,), concentrated to a few milliliters,and chromatographed on 35g of silica gel (J.T. Baker Chemical Co., 40-140 mesh; column dimension 2cm x 30arr). Ethyl acetate (200 ml) was used to elute impurities; acetone (150 ml) subsequently eluted the tobacco specific nitrosamines. The acetone fraction was concentrated (1-5 ml) and analyzed by HPLC- Mainstream Smoke Analysis. Sixty weight-selected cigarettes or little cigars, or 20 weight selected cigars were smoked with a 2Q-port automatic smoker under standard smoking conditions ~~?QG11~~820 7

for cigarettes or cigars, respectively. The mainstream smoke was led through a series of two 250 ml gas wash bottles, each containing 100.ml of citrate buffer at pH 4.5, 20 mM of ascorbic acid C15,201 and 0.5 pg of [14C]NNN. A filter holder with a 44 mm Cambridge CM-113 filter disc was placed between the second gas wash bottle and the vacuum pump and discs were exchanged after smoking of 30 cigarettes, 30 - little cigars, or 10 cigars. The loaded filter pads were ex- tracted twice with 50 ml of dichloromethane. The smoke loaded buffer solution was extracted 5 times with 100 m1 each of pre- equilibrated dichloromethane. The combined organic phases and filter extracts were dried (NaZs04) and concentrated to 5 ml. This concentrate was chromatographed on 90 g of basic alumina (Woelm, activity II-lII) on a 2 cm x 30 cm column with dichloro- methane (250 mll and a 4:1 mixture of dichloromethane with ace- tone (•2000 ml), The latter fractions were concentrated to 1-5 mi depending on the amount of nonvolatile nitrosamines present (Chart 2). Sidestream~Smoke Analysis.. The smoke emitting from a burning cigarette, or cigar, in between puffs is defined as sidestream ~ smoke. Ten weight selected cigarettes or little cigars were ~ . . Q . C 0". 8

smoked individually in an apparatus described earlier (4,22) by a single-port piston-type machin?. At an airflow of 25 ml/sec the sidestream smoke was led throu;i two 250 ml gas wash bottles wxth 100 ml of 20 mM ascorbi,c acid'solution buffered at pH 4.5 and containing 0,5 pg of [14C) NN:; and, finally, through- a filter holder with a 44 mm Cambridge CM-113 filter disc. The workup of the trapped smoke was identical to that of the mainstream smoke, described above. HPLC with Thermal Energy Analyzer. Ten to 100 ul of the final concentrates from the tobacco mainstream or sidestream smoke extracts were injected into a 3 col,umn-BPLC-system. A Corasil II precolumn was followed by two y-Porasil columns. A solvent mixture of 68.6% chloroform, 30% cyclohexane and 1.4% methanol was used at a flow of 1 ml/min. Under these conditions the retention times were 13.4.min for :1At8, 13.4 min for NAB, 21.2 min for NNN, 20.8 min for the Z-NN{ isomer and 22.8 min for the E-NNK isomer (9), in this case, the E-•and Z-j,somers of NNN (10) and NAtB are not separated, Synthetic and isolated NNK are a mixture of E-(72.78) and Z-(27.3%) isomers (10). $ince the peak of the Z-NNR overlaps with the NNN peak, the contribution of the Z-NNK isomer was subtracted from the NNN value and the NNK values reported here are a total of the E- and Z-isomers. The detec- tion limits for NNN, NNK and NAtB were 250 pg per injection to chloroform as solvent; with "S .~. ~ ~ v. ~ other solvents, such as methanoL, lower responses were observed), h (detection limit applies only Mass Spectral Identification. One hundred grams of chewing; tobacco were extracted and 200 non-filter cigarettes (85 mm) were smoked for the identification of the unknown with the - 9 -

retention time of NAtB and for NNN and NNK. After the first enrichment of the nonvolatile nitrosamines, as described above, the concentrate was evaporated to dryness, dissolved in dichloro- methane and chromatographed on 500 g of basic alumina (Woelm, activitiy II-III). Those fractions which gave positive responses with the HFLC-TEA at the retention times for NAtB, NNN and NNK, were combined and concentrated to =0.3 ml. For the identification of NAtB, NNN and NNK in the tobacco extract, about 10 u1 of this concentrate was injected into the GLC-MS system. For the unambiguous identi- fication of NAtB, NNN and NNK in smoke, it was necessary to rechromatograph the final concentrate (=0.3 ml) on thin layer chromatography plates (silica gel, 50 p; 10% methanol in chloro- form) and to extract the respective bands and analyze the concentrated extracts by GLC-MS. The GLC separation of NNN and NNK was achieved at 220° on a 1.8 m glass column filled with 10% Carbowax 20M-TPA on Gas-Chrom Q (injection port 250°), and that of NAtB was achieved at 200° on a 3.5 m glass column filled with 10% UCW-98 on Gas-Chrom 0 (injection port 250°). RESULTS AND DISCUSSION, Chart 3 represents a high performance liquid chromatogram of concentrates with tobacco specific nitrosamines detected.by W - 10 -

/ the TEA. The peaks were identified by comparison with the retention times of reference compounds and by mass spectral analyses of concentrates from 100 g of chewing tobacco or from the mainstream smoke of 200 cigarettes with- out filter tips. As in earlier studies (1L), we did not detect NAB, likely because the precursor anabasine is only a minor alkaloid, whereas, anatabine is the most abundant secondary alkaloid in the major tobacco varieties (7,19). ' 2.7% by anatabine, 0.96% by nornicotine and 0.35% by Recently it was reported that about.94.5% of the alkaloid fraction of cigarette tobacco was represented by nicotine, anabasine (7). Chart 4 compares mass spectra of synthetic NAtB and NAtB isolated from chewing tobacco and from cigarette smoke. The presence of Niz1tB was suggested earlier by Klus and Kuhn who found in a thin layer chromatogram a compound with a- retention time of this nitrosamine (25). Evaluation of the carcino- genic activity of NAtB is currently in progress in bioassays with mice and rats. The quantitative values obtained for nonvolatile nitros- amines with a HPLC-thermal energy analyzer system could possibly be inflated, or reduced by the presence of unknowns.in the con- centrates. One must consider such interferences since tobacco smoke contains more than 2,000 known compounds and since merely a short enrichment procedure was used for the nonvolatile nitros- amines. The presence of unknowns in the concentrate may affect the signal originating from the excited 110 2 which derived from nitrosamimes by cleavage and ozoni~zation. In order to clarify this point, we analyzed NNN fron the mainstream smoke of one 14 type of cigarette four times with [ C]\".V as a; standaxl. After (v V 1 1 '' 4 - 11 - ~'

...QS. the short standard enrichment procedure, we recorded the average value from`four runs at 0.21 + 0.025 ug NNN/cigarette. After this analysis, which required less than 38 of the sample we enriched the NNN from the concentrate by 2 successive coluan chromatographies on basic alumina. The average NNN value from 4 runs, after one column (enrichment step 1) was 0.21 + 0.020 pg/cigarette and after two columns (enrichment step 2) 0.20 + 0.024 pg/cigarette (Table 1).Thus we concluded that the quantita- tive values for NNN and, most likely also,for the two other tobacco specific nitrosamines obtained after the short enrich- ment step and by the HPLC-thermal energy analyzer, do in fact represent the actual am'ount present in the smoke concentrate. In Table 11, we have summarized the findings for tobacco specific N-nitrosamines in tobacco, mainstream smoke and side- stream smoke. These data indicate that tobacco and smoke of little cigars and cigars, as well as of cigarettes made entirely of Burley tobacco are richer in tobacco-specific nitrosamines than in tobacco and in smoke from cigarettes made of Bright and blended tobaccos. As was shown earlier, Burley and cigar tobaccos are rich in nitrate (23') and thus generate during smok- ing relatively high amounts of nitrogen oxides. The smoke of 1000121525 - 12 -

these tobacco products also has pH values of 6.5 and above, especially for the last puffs and thus contains •unprotonated . nicotine and other tobacco alkaloids (4). The unprotonated form of tobacco alkaloids is at least partially present in the gas phase and not just in the particulate phase as in the smoke of Bright or blended tobaccos(pH'<61. High levels of. nitrate in tobacco and nitrogen oxides in the smoke and elevated pH appear to favor the formation of nonvolatile N-nitros- amines in the smoke as was previously observed for volatile nitros- amines (6). presently,we are exploring this ooncept experimentally. Whereas most of the volatile nitrosamines are formed during smoking by pyrosynthesis*(6), some tobacco-specific nitrosamines transfer directly from tobacco into smoke (13). The influence of smoke pH, and the concentrations of nitrogen oxides and tobacco alkaloids on the formation of these tobacco specific carcinogens must be fully studied in order to permit approaches towards the reduction of these carcinogenic nitrosamines in the smoke. It is important to note that in some cases the sidestream smoke contains higher concentrations of nonvolatile nitrosamines,than mainstream smoke. Similar to volatile

nitrosamines,nonvolatile nitrosamines appeared to be formed in higher amounts during smouldering (sidestream smoke forma- tion) than during puffing (mainstream smoke formation). The sidestream smoke of one popular nonfilter cigarette ~ contains 1,7_00 nq NNN, 410 ng NNK and 270 ng NAtB compared ~ to 680 ng NDMA and 300 ng NPYR. For the determination of the a volatile and nonvolatile nitrosamines in sidestream smoke an air flow of 25 m1/sec was drawn through the apparatus. This flow rate was chosen in order to reproduce values for the mainstream smoke corresponding to open air smoking. It is known from our previous study however, that the levels of nitrosamines in sidestream smoke are influenced by the air flow rate thYough-the smoke apparatus (6). With the aid of the TEA, we detected volatile 2iDMA in smoke polluted rooms (10-130 ng/m3). In highly polluted rooms the amount of NDMA in the air inhaled during one hour is equivalent to the amount of NDMA present in the mainstream smoke of 1-35 cigarettes (5). However, origin of NDMA in room air is not necessarily limited to tobacco products. Based on the data for sidestream smoke from this study, we may be able to detect tobacco-specific nitrosamines in polluted in- door environments and thus define more clearly the contribution of tobacco smoke to the carcinogenic potential of a given polluted indoor environment.

w rv 4 xiFEREr:CLs 2. Bethmann, M. von, Lipp, G. and Nooy', H. van, Feuchtigkeits- Bates, Griffith, R.B., Harlo:v, H.S., Senkus, W. and tsakehamy H. Determination and Reporting of Total, Particulate Matter, Water in Total. Par ti culate Matter ... and Nicotine. Tobacco Sci.,12: 192-196, 1968- bestim,-nung im Tabak. Beitr. iabakforsch.,1: 19-29, 1961.- 3. Boyland, E., Roe, F.J. and Gorrod, J. W. Induction of Pulmonary Tumors in Mice by Nitrosonornicotine, a p©ssible Constituent of Tobacco Smoke. Nature 202: 1126, 1964. 4. Brunnemann, K.D. and Hoffmann, D. Chemical Studies on Tobacco Smoke XXV. The pH of Tobacco Smoke.Food Cosmet. Toxicol., 12: 115-124, 1974'. 5. Brunnemann, K.D. and Hoffmann, D. Analysis'of Volatile Nitrosam_ines.in Tobacco Smoke and Polluted Indoor Environ-~ ments. Intern. Agency Ies. Cancer Sci..Publ: 19: 343-356, 1978. 0 C- 6. Brunnemann, K.D., Yu, L., and Hoffmann, D. Assessm°nt of ~ Carcinogenic Volatile N-Nitrosaaiines in Tobacco and in OA Mainstream and Sidestream Smoke from Cigarettes. ~ •Cancer Res., 37: 3218-3222, 1977 Q~ 7. Elmenhorst,• H. Gas Chromatographic Analysis of Nicotine and Minor Alkaloids. Coresta znform. Bull. 1978 Special,120.

+ YECS~iC .. 1 ,.r0 5 .,iC`SS Fine, D.H-, Rufeh, F., Lieb,"D_ and Rour.behler, D.. Description of the Thermal Energy Analyzer (TEA) for Trace Determination of Volatile and i.'on-volatiLe NLNitroso Compounds. Anal. Chem., 47: 1188-1190, 1975 9. Hecht, S.S., Chen, C.B., Hirota, N. Ornaf, R.M., Tso, T.C. .and Hoffmann, D. Tobacco Specfie•Nitrosamines: Formation from Nicotine in Vitro and During Tobacco 10. Curring and Carcinogenicity in Strain A Mice. J. • Natl. Cancer. Inst., 60 : 819-824, 1978. . Hecht, S.S., Chen, C.B., Dong, tL,, Ornaf, Hoffmann, D. and Tso, T.C. Chemical Studies on Tobacco Smoke LI Studies on Non-Volatile Nitrosa.mines in Tobacco. Beitr. Tabakforsch.. 4: 1-6, 1977. 11. . Hecht, S.S., Schmeltz, I. and Hoffmann, D. Nitrogenous Compounds in Cigarette Smoke and their Possible Precursors. Recent Adv. Tobacco Sci., 3: 59-93, 1977. 12. Hilfrich, J., Hecht, S.S. and Hoffmann, D. Effects of N'-i>sitrosonornicotine and'N' -'Mitrosoanabasine in Syrian Golden Hamsters. Cancer t,etters, 2: 169-176, 1977• 13. . Floffmann, D., Dong, M. and Hecht, S.S. Origin in Tobacco Smoke of N'-Nitrosonornicotine, a Tobacco-Specific Carcinogen: Brief Communication. J. Natl. Cancer Inst.,58: 1841-1844, 1977. 14.' • HoffrtZnn, D., Flecht, S.S., Ornaf, R.M. and Wynder, E.L. N'-N.itrosonornicotine in Tobacco•. Science,186: 265-267, 1974. Hoffmann, D., Hecht, S.S., Schneltz, I,,Brunnemann, R.D. and Wynder, E.L. New Separation Techniques for Classes of Smoke Compounds. Recent. Adv. Tobacco Sci., 1: 97-122, 1975-

a Hoffmann, D., Raineri, P.., Hecht, S.S., Maronpot, R_ and Wynder, E.L. A Study of Tobacco Carcinogenesis XIV Effects of N'-Nitrosonorr.icotine and N'-Nitroso- anabasine in Rats. J. Natl. Cancer Inst..,55: 977-981, 1975. 17. Hu, M.W. , Bondinell, I•7.E. and Eo:fmann, D. Chemical Studies on Tobacco Smoke XXIII. Synthesis of Carbon-14 • Labelled Myosmine, Nornico tine and N'-Nitrosnornicotine J. Labelled Comp.,10: 79-88, 1974. 13. . International Committee for Cigar Smoke Study. Machine Smoking of Cigars. Coresta Inf. Bulletin 1974-1, 31-34. 19. Kuhn, H, Die Tabakalkaloide und ihre Pyrolyseprodukte im Tabakra•uch. Mitt, Oesterr. Tabakregie,5: 73-82, 1964. Mirvish, S.S., WalLcave, L., Eagen, Ei_ and Shubik, P. Ascorbate-Nitrate Reaction: Possible 2leans of Blocking the Formation of Carcinogenic %I-Nitroso Compounds. Science,177: 65-68, 1972. 21. Quan, P.M., Karns, T.K.B. and Quin, L.D. The Synthesis of 20. 22. 23. Anatabine and Related Compounds. J. Org_ Chem.,30: 2769-2772, 1')65 Schmeltz, I., Brunnemann, K.D., Hoffmann, D. and Cornell, A. Chenical Studies on Tobacco Smcke XLV. On the Chemistry of Cigar Smoke: Comparison beto-:een Experimental Little andl Large Cigars. Beitr. Tabakforsch.;8: 367-377, 1976. Singer, G.M. and Taylor, H.W. Carcinogenicity of N'-Nitroso- nornicotine in Sprague-Dat:ley Rats. J. Natl. Cancer Inst., C 57: 1275-1276, 1976. ~ t•7yn.der, E.L. and Hoffmann, D. Tobacco and To3acco Smoke. ~ Studies in Experimental Carcincgenesis. Academic Press, ~ New York, New York, 1967, pp 453-458. ~ ~ . O

25. Klus, H. and Kuhn, H. Untersuchungen t7ber die nichtflilchtigen N-Nitrosamine der Tabakalkaloide. Fach. Mitt. Austria Tabakwerke A.G. 307-317, 1975.

Table 1. Quantitative Analysis for NNN in Cigarette Nainstream Smokea,b ug/cig. Analysis 1 2. 3 4 Average Standard Method 0.21 0.18 0.24 0.20, 0.21 Enrichment Step 1 .0.21 0.19 0.23 0.19 0.21 Enrichment Step 2 0.21 0.18 0.23 0.18 0.20 a85 mm cigarette without filter tip. bDetails see "Results and Discussion"

Table 2. Tobacco Specific Nitrosamines In Tobacco and Tobacco Smoke* T?P~myo MIu9/~yaTm S~(V9~ci j)m Tobacco Producta NAtB NNN NNK NAtB NNN NNK NAtB NNN NNK Burley cigarette 3.2 7.0 N.D. without filter Bright cigarette 0.44 0.22 0.37 without filter Commercial cigarette 1.64 1.7 •'0.74 without filter Commercial cigarette 1.3 1.4 0.70 with filter Kentucky IRT 0.62 0.63 0.13 Little Cigar with 13.4 45.3 35.4 filter 4.6 3.7 0.32 1.5 6.1 0.66 0.41 0:62 0.42 0.39 1.7 0.50 0.33 0.24 0.11 0.27 1.7 0.41 0.37 0.31 0.15 0.15 0.15 0.19 0.53 0.39 0.16 0.19 0.21 0.24 1.7 5.5 4.2 0.57 0.88 0.81 Columbia Cigar (5..7g) 3.3 10.7 1.1 1.9 3.2 1.9 Nnt. 16.6 15.7 Fine-cut Chewing Tobacco 44.1 39.3 2.4 aAll cigarettes and the little cigar were 85 mm long. * NNN values are quantitative (14C-NNN used an internal standard), NAtB and NNK are isolated amounts. ND= not detected. NDt= not determined (interference by unknown(s)). /U:&TZAI0000 P 0

Chart 1. Chemical structures of tobacco specific N-nitrosamines. Chart 2. Analytical procedure for the determination of nonvolatile nitrosamines in cigarette smoke. Act. II-III, activity II-III. Chart 3. High performance liquid chromatogram of non- volatile nitrosamines from tobacco. Chart 4. Mass spectra of N'-nitrosoanatabine and of unknowns isolated from tobacco and from tobacco smoke. -

I r NNN NAB CHART 1. NNx NAtB

60 Gg.uettes. smoked into citrate bultrr (pH 4 5) conL 20 mM ascorbw acid; L,NNN114 C 2. ExtracCwith CHZCIt Aqueouslayer CH2CI2 extract 1. Dry. Na2S0a 2. Concentrate 3- Column,chrom. 90g, A1203 Act. II-111 1 elule with 4:1 CHZCI2:,DMK I (Nun-Vatatite Nitrosa,niiiesl t HPLC- TEA Chart 2. elute with CH;CI _ (Vulatile Nitrosaminesl!

0 .. -f,,~y+s' . Z c. y ` e UU~U~L 0 10 20 30 _ MINUTES Chart 3.

r I ~ I00r ISOLATED; Q 80}• TOBACCO Lu 60 > ;z 40 W 20 ~ 20 Chart 4. 40 60 80 100 120 140 160 180 200 m/e

`t rt q