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a CONFIDENTIAL CONCLUDING REPORT November 15, 1981 ON THE SELECTIVE REDUCTION OF TOBACCO-SPECIFIC N-NITROSAMINES IN CIGARETTE SMOKE Dietrich Hoffmann John D . Ad'ams Naylor Dana Institute for Disease Prevention American Health Foundation Valhalla, New York, 10595

ON THE SELECTIVE REDUCTION OF TOBACCO-SPECIFIC N-NITROSAMINES IN CIGARETTE SMOKE PURPOSE OF STUDY Page 1 1. GC-TEA METHOD FOR TSNA " 2 2. TRANSFER OF NNK " 7' 3. ON THE REDUCTION OF TSNA " 9 SIIMMARY " 19. Addendum 1 Progress Report 11-15-198'1 Addendum 2 1981 TCRC-Paper: Formation of NNK During Smokin g. ~ C

PURPOSE OF STUDY Four tobacco-specific N-nitrosamines (TSNA) have been identified in tobacco and tobacco smoke. These are N'-nitroso- nornicotime (NNN), 4-(m:ethylnitrosamino)-1-(3'-pyridyl)-1-buta- none (NNK), N'-nitrosoanatabine (NAT) and N'-nitrosoa.nabasine (NAB). NNN NNK NAT I NO N NAB ppm in tobacco 0.2-45 0.1 -35 0.6 -13 0.0-0.01 µg/cig. 0.2- 3.7 0.12- 0.44. 0.15- 4.6 0.0-0.15 These TSNA are formed during tobacco processing and smok- ing. They represent an important group of biologically active constituents of tobacco and its smoke. The aims of this study were: 1. To develop a rapid, reproducible analysis for TSNA in to- bacco products. 2. To determine the transfer rate of NNK - the biologically 1

most active TSNA - from tobacco into cigarette mainstream smoke and 3. to quantitate TSNA in the mainstream smoke (MS) of experi- mental cigarettes and explore the possibility of a select- ive red'uction of TSNA in MS by selective filtration. A progress report on this study was sent on April 6, 1981 (Addendum 1). 1. GC-TEA Method For Tobacco-Specific N-Nitrozamines A few years ago, we developed an analytical technique for the quantitative determInation of tobacco-specific N-nitros- amines (TSNA; 1) which yielded reproducible data, excluded artifactual TSNA formation and was highly sensitive (250 pg/compound). However, this method required improvements in efficiency in terms of time as well as in separating the Z-iso mer of NNK from NNN. Such improvements were the aim of this study. E%PERIMENTAL -0' ~ G Essential changes which led to a rapid, accurate and re- ~ producible method were as follows: The pyrolysis furnace of ~ the TEA was mounted at the column exit of a GC-unit (Figures 1C1 and 2). This direct connection of the GC-column and pyrolysis M 2

tube prevents [hermal degradation. A 3.3 x 2 mm (i.d'.) glass column was treated with a solution of dimethylchlorosilane in toluene. Subsequently, it was packed with 10% UCW-982 on Gas- Chrom Q 980/100 mesh) and was conditioned at 290°C for 3'0 mim. For the isothermic separation, the column was operated at 19-0°C (while the temperature of the TEA pyrolysis unit was 45-0:°C). Umder these conditons the retention times were: N"-nitroso- noxnicotin~e (NNN), 10 minutes; N'-nitrosoanatabine (NAT), 12 minutes; N'-nitrosoana:basine (NAB), 13 minutes; and 4-miethyL- nitrosamino)-1i-(3-pyridyl)-1-butanone (NNK), 18 minutes (Figure 3). The detection limit for an individual TSNA with this method is about 1100 pg per injection. The precision of the analysis for NNN in tobacco and in smoke was ± 8%. REFERENCE$ 1. Hoffmann, D., Adams, J'.D., Brunnemann, K.D., and Hecht, S.S. Assessment of tobacco-specific N-nitrosamines in tobacco products. Cancer Res. 39: 2505-2509, 1979. 3

Figure 1. Direct Interface Modification of GC-TEA System. 4

c Figure 2. Modification of the GC-TEA Interface. A: Catalytic Pyrolyzer; B: Heated Interface; C: 1/4" Graphite Ferrule; D: GC-Column. 5

GC - TEA TRACE OF TSNA IN CIGARETTE SMOKE NNN i NAT U NNK p \ 0 5 10 15 20 M I NUTES

2. Transfer of NNK from Cigarette Tobacco Into Mainstream Smoke. In~ previous studies we have shown that about 11% of the NNN which had been formed in cigarette tobacco during curing and processing transfers unichanige& into the mainstream smoke. Thlis occurs independent of influences of nitrate content of thie tobacco or pH of the smoke. Studies with 14'C-NNN have documented that 40-50% of the NNN in mainstream smoke originate by transfer from the NNN in tobacco and that the remainder is pyrosynthesize& from and nornicotine during the process of smoking (1,2). nicotinie It was the task of this project to determine the transfer rate of NNK into the smoke since this compound has even greater biological activity than NNN. For this stud~y, NNK-1-14C was synthesized, starting with nicotinic acid [carboxyl-1'4C]. Cigarettes were "spike&" individually with NNK-1-14'C with thie microsyringe metho&. Details of the method and analysis are summarized in a paper presented at the 35th Tobacco Chemists' Research Conference in Vinston Salem, N.C., 10-7 to 10-9, 1981, (Addendum 2). Table 1 summarizes our findings, showing that the transfer rate of NNK can vary between 7-11% an& that the NNK of the tobacco recovered in the MS canivary from 26 to 37%. `Spiking" of cigarettes with nicotine-methyl-14'C in&icated that about 0.002-0.003% of the nicotine give rise to NNK during smoking. 7