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VGL uZ:W Assessment of Tobacco-specific N-Nitrosamines- in Tobacco Products' Dietrich Hotfrnann,2 John D. Adams, Klaus D: Brunnemann, and Stephen S. Hecht' + Division of Environmenta/ Carcinopenesis. Naylor Dana lnstitute 1or Disease Prevention. American HeatthFoundatiort, Valhalla, New York70595 Tobacco-specific nonvolatile N-nitrosamines in tobacco and in fresh mainstream and sidestream smoke of cigarettes and cigars were quantitatively determined with a thermal energy analyzer. The satoke was trapped in ascorbic acid solution buffered at pH 4.5 and extracted' with dichloromethane, and the organic phase was chromatographed and analyzed by high-performance liquid chromatography-thermal energy ana- lyzer methodology (sensitivity, 250 pg/injection). The nonvol- atile nitrosamines were further enriched by repeated chroma- tography and positively identified by gas-liquid chromatogra- phy-mass spectrometry. (2'-"C) N'-nitrosonornicotirte served as internal standard tor the quantitative analysis. The tobacco of five different cigarettes contained betweern t).22 and 7.0 ppm of the carcinogenic N'-nitrosonornicotine, 0.13 and 0.74 ppm of the carcinogenic 4-( N-methyl-N-ni- trosamino)-1-(3-pyridyl)-1-butanone, and 0:44 to 3.2 ppm of the newly identified N'-nitrosoanatabine. In unaged mainstream and sidestream smoke of the same cigarettes, vali,es ranged between 0.24 and 3.7 and-0.15 and' 6.1 ftg/cigarette for N'- nitrosonornicotine, between 0.11 and 0.42 and 0.19 and 0.66 µg/cigarette for 4-( N-methyl-N-nitrosamino)-1-(3-pyridyl)-1- butanone, and between 0.33 and 4.6 and 0.15 and 1.5 µg/ cigarette for N'-nitrosoanatabine, respectively: The relatively high concentrations of these carcinogenic N-nitrosamines in sidestream smoke are discusse6as possible tobacco-specific indicators for indoor pollution. INTRODUCTION The occurrence of NNH' and NNK (Chart 1) in tobacco and in cigarette smoke has been ascertained' during recent years (11). Tnese tobacco-specific N-nitrosamines are carcinogenic in mice, rats, and Syrian hamsters (2. 9. 12, 16, 24) and could conceivably contribute to the incidence of tobacco-related cancers. NNN, NNK. NAtB, and possibly other as yet uniden- tified N-nitcosamines are formed during curing and smokingg from nicotine, nornicotine, anatabine, and possibly from other minor tobacco alkaloids (11). Tobacco products contain these carcinogenic nitrosamines in the range of 0.1 to 100 ppm;, therefore, their detection and quantitative analysis required ' Supported by National Cancer Institute Contract NOT-CP-55666 and Amer- iean Cancer Society Grant BC56. The Alexander Ralston Peacock Memorial Gran1: This is PaDer 62 in the series, "Chemical Studies on~Tobacco Smoke." ° To whom.requests. (on reprints should be addressed, at Division of Environ- mertat Carcinogenesis. Naylor Dana Institute for Disease Prevention. Dana Road. American Health Foundation• Valhalla. N. Y. 10595. 3 Recipient ot National Cancerinslilute Research Career Development Award N0-5KO4CA00124. ' The abbreviations used are: NNN. N'-nitrosonornicotine; NNK, <-(N-methyl- N-nitrosamino)-1{3-pyridYl}1-butanone; NAtE. W'-nilrosoanatabine; TEA. ther, mat energy analyzer: HPLC• hiph-pertormance Jiquid chromatoyraphy; GLC-MS, pas-riouid chromatography-mass specttomelry; m (in spectral analysis). muttiptet: NDMA• nitrosodemetbytamine• Received January 11, 109; accepted April 3, 1979. lengthy enrichment procedures in earlier studies. The highly sensitive and specific TEA (7) simplifies the quantitative deter- mination of nitrosamines, in that these nonvolatile compounds need only be concentrated once prior to separaGon by HPLC an6detection. For an accurate measurement of these nitrosamines in chew- ing tobacco, tobacco leaves, and unaged smoke, extraction procedures as wellas trapping of the fseshly generated aerosol require the presence of ascorbic acid at pH.4•5 (15. 21). ["C]NNN serves as internal standard for the quantitative ana1- ysis. This study reports quantitative data for NNN, NNK, and the newly identified'NAtB in smoking and in chewing tobaccos and in the unaged mainstream smoke of cigarettes and cigars_ Sidest'ream smoke, which is generated during smoldering of tobacco in between puffs, trequently contains higher concen- trations of these tobacco-specific N-nitrosamines than does mainstream smoke. . 1. MATERIALS AND METHODS Apparatus. The generation of maissstream smoke of ciga- rettes•, little cigars, and cigars has been described'in a previous publication (15)i The smatt sideslrearzrn smoke collector used for cigarettes and little cigars (3, 4) and a large collector for cigars (23) required regutation of the.airaow to 25 tnl/sec. This airflow 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 (1). In this setting, the mainstream smoke was generated by a single-porLpiston- •type smoker (H'. Borgwatdt. Hamburg. Germany). ' • For the HPLC separation, we utilized a Model 6000A solvent delivery system (Waters Associates, tnc., Milford, Mass.) with a Model 70-10 Sample Injection Valve equipped with a Model 70,11 Loop Filler Port (Rheodyne, Berkeley, Calif.) and Corasit Il and µ-Porasil columns (Waters Associates, Inc.). The TEA with HPLC interface (Thermo Electron Corp., Waltham, Mass.) with a Hewlett-Packard Model 3380A recording integrator served as detector. Unkno%+n nitrosalnines were identified with a Hewlett-Packard Modef 5982A GLGMS instrument. A Nu- clear-Chicago Isocap 300 was used for scintillation counting in toluene with 0.5% PPO and 0.005% POPOP. Reagents. (2'-"CjNNN (14.1 mCi/mmol) was synthesized (17) and was pure accordrtg to HPLC with TEA and GLC-MS analysis (9). NNN and NNK were also synthesized by methods reported in the literature (9, 17). The initial NAtB was synthe- sized'from anatabine and purified by HPt-C: Synthesis of NALB. ou-Anatabine was prepared according to Ouan et al: (22) and was nitrosated to NAtB according to Hu et aL (17). The crude NAtS was purified by chromatography on preparative thin-layer chromatography plates (silica gel, CHCia_ methanol, 9:1.,. R, 0.59) and was diszitted~ at 0.5 mm Hg and 176° (oil bath). Gas chromatography on a 2-mm x 3.6-m glass

4`y cotumn with 10% UCW-98 on Gas-Chrom 0 at 200° showed' '~ a single peak. •.. Spectral Properties. IR (film): 3040, 2930, 1580, 1430, 1335, 1160, 978, 710 cm''. Mass spectrum m/e (relative intensity): 189 (M',15.1), 172 (20:5), 159 (100.0),157 (33.7), 144 (35.1), 130 (72.6). 117 (74.2), 105(58.9). 92 (46.0), 80 (33:4), 78 (53.7). Nuclear magnetic resonance (CDCI3): 8.9 to 8.2 ppm (2H, m, pyridyt-CH), 7.8 to 7.1 ppm (2H, m, pyri+ dyi-CH), 6.7 to 3.9 ppm (5H; m, pyridyl-CH_-N, N--CH2-CH=CH), 3.7 to 2.6 ppm (2H, m, CK2-CH=CH). Elemental analysis (Gatbraith Laboratories, Inc., Knoxville, Tenn.): C,oH,1 Na0 Calcutated: C 63.47, H 5.86, N'22:21 Found: C 63.44, H 6.04, N22.03 Tobacco Products. Commercial chewing tobacco, ciga- rettes, little cigars, and cigars were purchased on the open market in 1977 and 1978. Experimental cigarettes were sup- plied by Dr. T. C. Tso (United States Department of Agriculture). Storage, conditioning, and smoking protocols for the various pro~ucts were according to standard procedures published eariier (5, 18, 23). . '° Tobacco Analysis. Fine-cut chewing tobacco and'the tobac- cos of cigarettes and little cigars were analyzed without grind- ing. Cigar tobaccos were ground in a blender before extraction: An aliquot was taken for water determination (25). For the actual analysis, about 25 g of tobacco were extracted for 24 hr tlty stirring at room temperature with 100 ml 5 mm ascorbic acid solution at pH' 4.5 (citric acid:sodium phosphate buffer) and 0.5 µg of, ['4C)NNN was added as internal standard. The exiract was filtered (5-µm nylon filter cup; Liquiflo, Inc., Plain- view; N. Y.) and adjusted to pH 5 by the addition of a few drops of 1' N sodium hydroxide. This adjustment facilitates the sepa- raticn of the nitroso compounds from nicotine in tobacco ex- trac:s (10). The aqueous phase was then extracted' 6 times with equal' volumes of ethyl acetate. The organic phase was driec (Na?SO.), concentrated to a few ml, and chrometo- gra„ned on 35 g of silica gel (J. T. Baker Chemical Co.; 40 to 140 mesh; 2- x 30-cm column). Ethyl acetate (200 ml) was used to elute impurities: acetone (150 ml) subsequently eluted the tobacco-specific nitrosamines. The acetone fraction was concentrated (1 to 5 ml) and was analyzed by HPLC:TEA (recovery rate of internal 'standard ["C]NNN, 75 to 90%). O O NA6 IO GH3 NNK \ O NAiB Cnal 1. ChemieaV structurps of tobacco-specific N-nitresamines. NAB, N'- nitrosoanabasine. 60 Crparettes. smokeo nto carate 1WIICr,/DN ~S) cOnt. 211' nuAascorbic acid r-----=. 1,,NNN-14 C 2. EcfraU .rdn,CM2Cl; iaueoas Uya L7i~t1J Ci1raC1 1. ory. Na2so. 2. Loncenuatt 3. Column cnrqq 9Dp AI?o3 Act il-nl 1. (NOo-VOalde tlutt wrttt 4:1 tM7Ct2 DMK 1 Narosarmnes) . NPLC• TEA elutt wnh a+,cl: (YotJlfrk NluosarmntsY. Char, 2. Analyticai procedure for the detetmination of nonvolatrl-e nitrosa- mines in cigarene smoke. Act. 11-I1J, Activitir o to Ilf: D/NK, OicNoror.nethane• cont., containing; chrom., chromatography. Mainstream Smoke Analysis. Sasty weight-sefected ciga- rettes or little cigars or 20 weight-seiected cigars were smoked with a 20-port automatic smoker under stanqard ssTloking conditions. The manner of collection of the mairtstrearra smoke particulates was described earlier (S)_ Each of the 2 cotlection vessels contained 100 ml of citrate SuHer (pH 4.5) with 20 mM ascorbic acid (15„ 21), and0.5 µg of j"C]NNN. After smoking, filter pads were extracted twice wittr.dichloromethane, and the buffer solutions from the collection vressets were extracted 5 times with 100 ml each of preequih-t)rated dichloromethane. The combined organic phases and fitter extracts were dried Wa?S0.), concentrated to 5 ml, and chromatocJraphed on 90 g of basic alumina (Woe)m: Activity 13 to 111) on a 2- x 30-cm column with dichloromethane (250 rrtl) and a 4:1 mix.-ure of dichloromethane with acetone (200 rasO. The latter eluates were concentrated to 1 to 5 ml depending on the amount of nonvol- atile nitrosamines present (Chart 2)- The recovery irate of ("C)NNN varied between 70 and 85%. Sidestream Smoke Anaiysis. Tlve smoke emitting: from~ a burning cigarette or cigar in betwee>wn puffs is defined' es side- stream smoke. Ten weight-selected cigarettes or tittlee cigars were smoked individually by a singfe-port, piston-type r^.achine as described earlier (3, 23). At an 2irflow of 25 rnl/sec, the sidestream smoke was 1ed' through two 250-ml gas wash bottles with 100 ml of 20 mM ascorbreacid'solution bur: Lered at pH 4.5 and containing 0.5 Fg of ["Cr2NN and, finally, t:hrough a filter holder with a 44-mm Cambridge CM-113 filter disa. The work-up of the trapped smoke was iOenticali to that of the mainstream smoke described above (recovery rate of ["C].- NNN, 70 to 85%). HPLC with TEA. Ten to 100 µl of V-K-- final concentratm from the tobacco mainstream or sidestre=n smoke extracts were

~. injected into an HPLC system consisting~of a Corasil Ii precol- . a:a umn followed by 2 µPorasil columns. A solvent mixture of 68.65 chloroform„309'o cyclohexane, and 1.4% methanol was used' at a flow of 1 mI/min. Under these conditions, the reten- tion times were 13.4 min for NAtB as well!as for N'-nitrosoan- abasine, 21.2 min for NNN. 20.8 min tor the Z NNK isomer, and 22.8 min for the E-NNK isomer (9); in this case, the E and Z isomers of NNN (8) and NAtB are not separated. Synthetic and isolated NNK are a mixture of E(72.7°.0) and' Z(27.39a) .isomers (8): Since the peak of the Z-NNK overlaps the NNN peak, the contribution of the Z-NNK isomer was subtracted from the NNN value, and the NNK values reported represent the sum total of E: and Z isomers. The detection limits for NNN, NNK, and' Ni4iB in chloroform were 250 pg/injection (with other solvents, such as methanol,, the responses were lower). Mass Spectral Identification. One hundred g of chewing tobacco were extracted, and 200 nonfilter cigarettes (85 mm) were smoked for the identification of the unknown; with the retention time of NAtB and for NNN and NNK. After the first enrichment step for the nonvolatile nitrosamines, the concen- trate was evaporated to d'ryness, dissolved in dichloromethane, and ehromatographed on 500 g of basic alumina (Woelm; Activity Il to III). Fractions which oave positive responses at the retention times for NAtB. NNN, an&NNK were combined and concentrated~ to =0.3 ml. About 10 pl of this concentrate were injected into the GLC-MS system. For the unambiguous iden- tification of NAtB, NNN, and NNK in smoke, it was necessary NAtB C) iO x 11 ko r: N N'N w cn z 0 C_ cn W Q G NNK ~~'~ i l `~, 0 IO 20 30 .,. M,INUTES " Char1 3. Hiqh.performance liouid chromatogram of nonvotatile nitrosamines from tobacso. Nvnvula,iit rvlt,(~,a, r,ri„s,u /vi,ac.i„c,, rurvui-i~ .~ 0J N, NO REPERENCE 20 40 60 ao 100 m/c RO 140 160, IW 200 Chart 4. Mass spectra of NAS and of unknowns isolated trom. tobacco and from tobacco smoke. to rechromatograph, the finati concentrate (=0.3 rsrd) on thin- layer chromatography plates (silica: gel. 50 µ; 10% rnethanoti in chloroform), and to extract the respective bands and analyze the concentrated', extracts by GLC-MS. The gas-tiquid chro- matography separation of NNN and NNK was achieved at 220° on a 1.8-m glass column fille6 with 10°ro Carbo%wax 20M- terephthalic acid' on Gas-Chrom 0 (injection port, 250°), and' NAtB was separated at 200° on a 3.5-m glass cofumn filled' with 10% UCVJ-98 on Gas-Chrorn Q(injection port, 250P). RESULTS AND DISCUSSION' Chart 3 represents a: high-performance Iiquid chromatopram of concentrates with tobacco-specific nitrosamines. detected bythe TEA. The peaks were identified by comaarisom 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 without filter tips. As in, earlier studies (1'11 we did noU detect Nr-nitrasoanaba- sine„probably because the precursor anabasine is orxy a minor alkaloid, whereas anatabine is the most abundant secondary alkaloid in.the major tobacco varieties (6, 20). Recer.;Tly, it was reported that about 94.5% of the alkaloid fraction of cigarette tobacco was represented by nicotine, 2.7% by anatabine. 0.96% by nornicotine, and 0.35% by anabasine (6)- Chart 4 compares mass spectra of synthetic NAtS; and NAtB isolated from chewing tobacco and from cigarette smoke. Evaluation of the carcinogenic activity of NAtB is cztrrentty in progress in bioassays with mice and rats. The possible pres- ence of nitrosoanatabine in cigarette smoke had earlier been suggested' oy Klus and' Kuhn (19). The quantitative values obtained for nonvolatile nitr•.osamines with an HPLC:TEA system could possibly be inflated or reduced by the presence of unknowns in the concentrates. One must~ consider such interferences since tobacco smoke contains more than 2000 knowmm compounds and since mere=ty a short enrichment procedure was used for the nonvolatile nitrosa- mines. The presence of unknowns in the concen:rate may affect the signal originating trom the excited N07, which derives

v. Hotlrnann et al. . . ~ from nitrosamines by cleavage and ozonization. To clarify this poin;, we analyzed NNN from the mainstream smoke of one ~ 1 type of cigarette 4 times with ("C)NNN as internal standardL After the short standard enrichment procedure, we recorded the average value from 4 runs at 0:21 ± 0.025 (S.D.) µg NNN per cigarette. This analysis required less than 3% o.f, the sam- ple. The NNN from the concentrate was then enriched by 2 successive column chromatographies on basic alumina. The ave°age NNN value from 4 runs, after 1 column (Enrichment Step 1) was 0.21 --4- 0.020 µg/cigarette and after 2 columns (Enrichment Step 2) was 0.20 ± 0.024 µg/cigarette (Table 1). Thus, we concluded that the values for NNN and probably also for the 2 other tobacco-specific nitrosamines determined with the !-iPLC:TEA, after the short enrichment step do; in fact, represent the actual amount present in the smoke concentrate. In Table 2, we have summarized the findings for tobacco- specific 1V-nitrosamines in tobacco, mainstream, and' side- stream smoke. Tobacco and smoke of little cigars and of cigars as well as of cigarettes made entirely of Burley tobacco are richer in tobacco-specific nitrosamines than tobacco and smoke from cigarettes made of Bright and blended tobaccos. As was shown earlier, Burley and cigar tobaccos are rich in nitra:e (26) and thus generate relatively high amounts of nitro- gen oxides during smoking. The smoke of these t'obacco pro:ucts also has pH' values of 6.5 and' above, especially for the iast pufts, and thus contains unprotonated nicotine and other tobacco alkaloids (3). In the smoke of Burley and 'cigar tobaccos, the unprotonated form of tobacco alkaloids is at least partiatty present in the gas phase and not just in the paaic::Iate phase as in the smoke of Bright or blended tobaccos (pH s 6): High levels of nitrate in tobacco, nitrogen oxides in the smoke, and elevated pH appear to favor the formation of nonvolatile N-nitrosamines in the smoke, as was previously obse-ved for volatile nitrosamines (5). Presently, we are ex- plorir;y mechanisms for this concept experimentally. Table 1 Ouantitative analysis forNNN'in cigarerte mainstream smoke Ei;-;y-five-rnm cigarettes without filter tips, were used for analyses. =or ceails see "Results and Discussion." (yQ/tigarette): Whereas most of the volatfile nitrosamines are tormed during smoking by pyrosynthesis M), some tobacco-specific nitros- amines transfer directly frorm tobacco into smoke (13). The influence of smoke pH and. •of the concentrations of nitrogen oxides and tobacco alkaloids on the formation of these to- bacco-specific carcinogens must be fully studied to permit approaches toward the redutrtion of these carcinogenic nitros- amines in the smoke. It is important to note that in some cases the sidestream smoke contains higher corzcentratic,ns of nonvolatile nitros- amines than does the mairastrearn smoke. Similar to volatile nitrosamines, nonvolatile nitrnosarnines appeared to be formed in higher amounts during srmouldering (sidestream smoke for- mation) than during puffing (vmainstream smoke formation). The sidestream smoke m1f one popular nonfilter cigarette contains 1700 ng NNN; 4::f 0 rsg ' NNK, and 270 ng NAtB compared to 680 ng NDMA and 300 ng nitrosopyrrolidine. It must be kept in mind that tta-ese values were obtained' with an airflow of, 25 ml;/sec in the snrJestr.eam apparatus to reproduce values for the mainstream sr.noke most closely approximating those obtained' during operr. air snoking. It is known from our previous study that the leveits of nitrosamines in sidestream smoke are influenced by the airrow rate through the smoke apparatus (5). With the aid of the TEA, we:detected volatile NDMA in smoke- polluted rooms (10 to 130 tag%m'7_ In highly polluted'environ- ments, the amount of NDMA in the air inhaled during 1 hr is equivalent to the amount of. tA1Dt~'A present in the mainstream smoke of 1 to 35 cigarettes;t(4). However, origin of NDMA in room air is not necessarily lirmited lo tobacco prod'ucts, Based on the data for sidestream ssnoke :rom this study, we may be able to detect tobacco-speciiiic nitrosamines in polluted indoor environments and thus define mor.=_ clearly the contribution of tobacco smoke to the carcirn3senic potentiallof a givenpotluted indoor environment. ;, ACKNOWLEDGMENTS The authors are gratefut to Dr_ -r. C. '.'so, United States CDepar•ment or Agriculture. Seltsville, Md.• for supGfwmg aar•~ aJine for the NA:5 analysis. - Analysis Analysis Analys;s Anatys;s 1 2 3 e Av. REEERENCES Stancvc method ' 0:21 • 0.18 0.2<' 0.20 0.21 Ennc'neat Step 1 0:21 0.19 0.23 0.19 0.21 1. Enrichment Step 2 0:21 0.18 0.23 0.18 0.20 ' Table 2 Eates. W. W.. Gritfitn, R. S.. Haraow, Yi. 5.• Senkus• W, and Y•lakeham, H. Determination and reporting of :M:al Cxnicutate matter„waten in total par6ic- ubate matter and nicotine. Tob_5¢i.. T= 192-196• 1968. ~. A 7obacco-specific nirrosamines in tobacco and tobacco smoke NN'I values are quantitative (("CJNNN used aniinternal standard). NAtB and r.NK are isolated amounts 0 ~ Tobacco (ppm) r.'tamstream (jrg/cigarette) S;oestream (i,9tc;gzrettey ~ Tobacco producfa NAt6 NNN NNK' NAIS NNN NNK NAtB NNN' NNK ~ _urle) c:;,arene ~.•ithout filter 3.2 7.0 NDP 4.6 3.7 0.32 1.5 6.1 0.66 Q _righ- c:garette witnout finer 0.44 0.22 0.37 0:4 1 0.62 0.42 0.39 1.7 0.50 : fjn ; omrrre~ciat cigarette without filter 1.6 11.7 0.74 0.33 0.24 0.11 a'J.27 1.7 0.41 ~ =cm^e-aat cigarette with fitter 1.3 1.4 0.70 0:37 ._ 0.31 - 0.15 '0.15 0.16 0.19 -;entuc-y IRI 0X2 0.63 0.13 0.53 0.39 0.16 a.19 0.21 0.24 -nte c,yanwrih f;her 130 45.0 35.0 1.7 5.5 4.2 0.57 0.68 0.81 ;ctun:.•a cigar (5.7 g) 3.3' t0.7 ' 1.1 1.9 3.2 1.9 tNDt° 16.6 15.7 chewing tobacco 44.0 39.0 2.4 A; s parer,es and tne titfle cigar were 85 mm long. 6 N:I not oetected; r•Dt; not determined (interterence by unknown(s))J

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