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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 -