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graphed and analyzed, as described . under '"Tobacco An,a~lys'is" . "Y41-~colunnns 'of' 250 g silica gel each using ethyl Xace~ate, followed' ~ .. .. _ . , .. . .~ .. ._ . _J:fi~ ~,'s ~ .. M~i~.Y`zr-by an additional column of 50 g alumina, with 1'0!%methanol .- The fractions cantaining the tate as eluant ~~'~}~r t~i l . e y ace . ~i.-: . `Mass' Spectral ldentification, ~'~O1 R1 reference ' cig,arette, then enriched by two chromatographic ;.. . «-1,,,~~Js~-activity~ we~~re~ combined, =carefhlly~ ~ evaporated to" dryness and th~n redissolved in 100 p1l silylating agent. b) Mainstream Smoke Fraction t:' Fivehundred, 1iR1referencecigarettes were smoked. as~ ~ _ . . .,. , ``'`-~ "~,GC-MS using a capillary column,,30' m x 0i.25^mm i.d. SP -210'0, 1, Icribed!earlier. The enrichment, Ehromatographic~clean-up and silylation steps were the same,as those for.the ~tobacco~frac- )i Tobacco Fraction An extract was prepared from 2010 ,g of ~he tobacco of the "` ,, .. ;.., ..z tion. An aliqu~ot of the silylated concentrates ,was analyzed by . mil He/inin, splitless mode; the , GC program was 4't~min at 8'0`C, then 4°/min to 200'°C. -•Under these conditions, the silylated ~ derivative o1f' NDSLA had a retention time of 251.51 min. RESULTS ~ F~igure~~ 2, depicts typical chrom~atogramsof~ s~i~lylai~ed! NDS~LA~ from the concentrates of tobacco, cigarette smoke and MH-DELA

'~obtained ~uinder the `eonditions described~ under "Materials an . ., Occasionally, we observed_several larger peakss following the elution of' NDELA (TMS)., These were found to be ; ¢7.ue tci }tobaeco spebific nitrasamines NNN NNK and NAT (21For .,j« t1ass hie m spectral confirmation, we enriched NDELA from 200 g of •. . ' tobacco or from the mainstream smoke of 500 cigarettes (Figure -.- _ .~~ The precision'af the methodiwas' established by 5 analyses M , _ _ .. of 401 g Foff' ~tobacco of' the 1 R1 Kentucky cig,arette, and 5, analyses f tbesm~oke~ o1f~ 100 Kentucky~ IR1 cigarette~seach~:('~Table T)~. The standard deviations of 111.91V and 1I1'.7'% respectively, appear highr' but are likely due to the fact that we analyzed non- - "cominercialcigarettes and! that the MH-DELA residue is unevenly `,°distributed when -the tobaccolplants are sprayed. The recovery 'rate ©ff' the '14C-labeled NDELA which was used as internal stand- r ~i ardvared betwee~n~ 50! and, 7~01$ i tob ~ tmbacco~, smak d~ ~~n,acco!, ean MH-DELA samples. The addition of 5.5 mgi of DELA to a mixture ~~,~'~'!'). .. ... ~: .... ~.. : .~ .: . ,,... .. . . . . containing I g of~ asco~rb~ic~~ ac~id~, 40~ g of Kentucky 11R~~1 t~o~b~acco~, ,.~ ,. and 4010, ml of ethyl acetate did not lead! to an increase in the NDELA yield'; when 5.5 mg of DELA were added to the traps col- lecting the mainstream smoke of the DELA-free USDA cigarette (L-8' )I , the amount of NDELA detected' in the final concentrate was insignificant. This assures that under normal conditions, NDELA is not artifa'ctua'lly formedlduringi the analytical work- up. O This study demonstrates that silyl,ated nitrosamines p c~f CIt . ~ - 8 - ~

are amenable to GC-TiEA detection with undiminished sensitivity. Table I'summariaes the findings for NDELA i~n tobacc© and .. . . v ,, . _ tobacco smoke of vari rv ,ous cigiarettes and other tobacco .4 ~: w z. i°t l the case of the hand-suckered tobaccoland the r1, k~ 1 e,~•... . ~:IDELA free tobacco, NDELA was not detected, (detection limit 0. . . ~ ...Y, ~ ppb in trobacco or 1,, ng~cigarettei a wery small~ge~k~obs ~rved at the retention time of NDELA was attributed to the internal w"::j~~:~1~I~Y~i~ , ~ ~ standard,, ~ N~DELA-1i4C'~)1.,_ A~ Frenchcigarette~ (purchased~ in ~1i975), ~„ y. ~eld i in the mainstream smoke did not i n the ~obacco nor NDELA which leads us to believe that the tobacco used for this „{ . .. ... ,. . . . . . ~ ., ,., .. .. • E. . . ..i. 5~ ir'. , product was not treated with MH-DELA. The two snuff samples .... 4 ,~ T . . .. ... . . ~ .. .. .. . .. . . .. „ . ,. ~ . . .. analyzed, showed NDELA values of 3.2 and 6.8 ppm, respectively. .111:,.~ For a popular 85 mm U.S. nonfilter cigarette we determined -43 ng of NDELA per cigarette in the sid'estream smoke, which is .{ ... .. . . . ..~ i., . . ..: =9. i• . , 7. .~. . . . ~i. ; i.+('.' T . ~.3.1~.' , Y L,.t.~ r.. generated in between puffs and'reSeased into the~y general en- t . , ' .- . : ., . , ....~. . . _ ~.... .„.~ ~..... ... ~ _~' > . ..: i• . .. . . . .k.:i , .wironment. ID!ISCLT!SSION Based on these findings one may expect that the MH-DELA Preparations sprayed a~rowth inhibitor onto the ~~~,,ssucker g~, tobacco, contain already NDELA. Table III shows that samples of. . . , . the MH-DELA preparations contained' between ©.6-1I.9 ppm of the nitrosamine. O Ch C!3 ~ od~ method developed ~ The~~ simp~~le~R r:e~pr~ucible~~ GC~-TEA~~ for the~ analysisiof N-nitrosodiethanollamine in tobacco products and' in. - 9 -

tobacco smoke demonstrated that~NDELAf in tobacco+ and its smoke derives from the treatment of tobacco with the sucker growth r :.y .- . . 4 i'~nhib~~itar male~ic~ hyd~raxide-di~ethanmlamine. ~ Alth~~o~ugh~.formula-~ ~ .NIC', -» ~~ a tlon~sf ' this widely used agricultural chemical contain~traces ' ' af NDELA, we expected~ thisnitrosamine to be primarily formed . . processing. The recommended effective dose of MH-DELA is 450! 1 JgtY A~ Sr ?3:3t~ tz i 6. n ` by nitrosatioof the amino alcohol residue duringtobacbo r mg per tmbacco plant of whiich 95 mg are DELA.''I'f one plant averages 160 g cured leaves the maximal residue on 1i kg tobacco ~:~~~ ~ thus, 1 kg cured tobacco may contain up, to 5.3'ug or 5.3 ppb of .. . ~ 1. 4 NDELA. We found in commercial U.S. tobaccae at least 108 8' ppb could be 59Omg DELA (20). The maximal amount of'NDELA found in the commercial MH-DELA preparation was 1' .9 ppm (Ta III )I ; b1c I Y ~'/^F'.~"y R £t.F_ ~y~r ~ . .t~ CVtX, ~ 6p ~-~ ;~ (Table II') , thus,, ma ximally 2.1$ of' the NDELA could derive from = the MEii-DELA preparation and most NDELA is formed during tobacco processing. This concept is also supported by the observation (3.2-6.8 ppm) than smoking tobacco does after curing (0.1-0!.4 . ~ that samples of~~ snuf~f~,~ ~which~~ went thr~ough, a~ long~ ~term; ..~ ., .. ...-.. . f~mentatio process,contai~n mueh higher amaunts0 f NDELA I ~:i F '-~t ~ . . ... ._. .... -.. .3 . . . _ . ,....,. y . _- . __. ~. ~ - - ppm).`Other corroborating evidence stems from the recent obr A, ,_ .} 4 '-+ , '" " " 'E;r servationn that levels of the carcinogenic, tobacco specific .N nitrosamines, NNN, NNK and NAT'are also significantly higher in snuf f [5. 5 to, 1:016 ppm, in 21 samples analyaed], than in 1P+ ~ cigarette tobac:co, [0.3 - 115 ppm in 151 sampl.es analyzed; 21114.Q N, .

The mainstream~smoke of U.S. cigarettes treated'iwith the I sucker growth~~ i~nhibitor~ MH-DELA: cont~~a~ine~d2~0~-4a' n~g, of NDELA per~ " cigarette. ' ` It has not been determined~as yet how much of' NDELA in .the tobacco is transfetedi into the smoke and how much 'i's pyrosynthesia~~ea3 8uring smok~ing,~. Bioassavs on rats (51.6) andl on Syrian golden hamsters (7') M, ~~~ ~. ',! have clearly demonstrated that NDELA is a relatively strong animal carcinogen. This analytical study has shown that resi- d~ual amounts~ of the~sucker growth ~~ inhib~it~or~~male~ic hydraz~~id~e- n diethanolamine on tobacco leaves give rise to"NDELA in"tobacco ~ :?:i'. _ C:! 1 and, its smoke. Snuff, which is increasingly used as a smoke between 3'~-2~-6~.~8 ppm o~'f~ ND'ELA~.~ ~ Thus,~ this~~ N-nitros~anuine!addis to, oral cavity of' its long-term: users ( 22 )', was shown to contai'n: substitute by young people and which is a carcinoge in the the carcinogenic potential of the tobacco amines in snuf f[ 5.5 to 1I© 6 ppm; 21]. specifi'c N-n'itros- Both the bioassay data for NDELA and this analytical study strongly suggest a review of the use of maleic hydraaid'e-di- ethanolamine preparations for tobacco crops. It appears that certain' potassiumisalt preparations of' MH would be' equally effective asaisuicke~~r growth;in~h~ibito~~r and could thus be~ used • in place of the diethanolamine preparations o MH (121).

ACTCNOWLBDGEMRNTS ~ ~ <. We are grateful to T.C. Tso, U.S.D.A., Beltsvi,lle, 'MD. for j ~the experimental cigarettes and expert advice. We thank S. n.".'r•}.`'~7d t+`.I ", .Adams for his technical support and C. Lettre for the modifi- .. . . , ~. cations of the GC. This study was supported by Grant 1 P01 - CA-29'5801 from , ~ the National Cancer Institute. Parts of this paper were pre- sented' at the 34'th Tobacco Chemists' Research Conference, Rich- ..~c~ mond, VA, Oct. 27-29, 1980. . . . ., . . , . .. .. . «x..e .. . l. ... .~ ~...~ ..n. ~~~. ~ti.te ~r~~'.. .. A ~ . This is No. II,xIX of "Chemical S'tudies on Tobacco Smoke." ~ ~ - . . , . . . . .J W td . .. ~ ,X{I, .,,. ,d.wl~el.i , wi,.. r: .. ...~..... t4:% . . . . -.. , ... . . . • ,.. . . _. ~. . __.. . -. ~ : -.. L.~:. . . ~.. ... .. ' ..._i :ISZ"::~. ... . . . ~ .... .. .. ~ b~ . ... ~"J..~.~.. ~. !

, - IARC~~ Monographs ~©n~~ ~~th~e Eva~luation~ o~f~ Carcinogeni~c~ Risk o Chemistry to Mani (1'978') N-Ni~trosodiethanolamine, 17 i, ^i ni 1 . `REFERENCES 'Vita, giounds; a New Class of Nitrosating and r y t . . . .. . - . , . ~ ~~:K Fine,'D.e. (1978~) , C-Nitro Com- Agents, Tox,icol. Lett. ~=J"s.~S Y ± ,. ~ (1979')1, 2-Bromo-2-nitropro- for Diethanolamine: 4; r pane-1,3-diol as a Nitrosating Agent A Model Study. Food Cosmet. Toxicol. 17,`"105-109. 'Druckrey, H., Freussmann, R., Ivankovic, S.~and Schmaehl, D.'(11967), Organotrope'carcinQgene Wirkungen'bei 65 wer- ^'a,. ~ . . . ~ . , . . , ~ schIedenen N-Nitroso-VerbindUngen an -BD-Ratten. Z. Rrebs- Reuberp- M.D1.""and Manning,; „W.B. '(19'80) , Potent Carcinogenicity of Nitrosodietlianolamine in Rats. R. and' Schmaehl, D~. (198'1i ) Dose-Re- sponse Study on the Carcinogenicity of N-Nitro~sodiethanol- amine (NDELA) ini Male Sprague-Dawley Rats. J. Cancer Res. Clin. Oncol. 9'9, A27., Biilfrich, J., Schmeltz, I, andlHoffmann, D. ('1978). El~~fecta of N-ri1itrosodie~thiano~l~amine~ and 1,1-diethanolhydra- zine in Syrian golden hamsters. Cancer-'Lett. 4, 55-6101. Q

Lij',insky, W. , Losikoff, A.M. and' Sansone, E.B. .(,1!9'S'1). Penetration ©f Rat Skin by Ni-Nitrosodiethanolamine and' u.N-Nitrosomorphoiline. ,,7'. Natl., Cancer. Inst. 66, 125-127. ,-Edwards, G.S., Peng, M., SAiegelhalder, B. and Kann, ~.. <,...,.: : :... . . . .. . . .,~,.. . ~ ~ .:. s,.' ' ,a, , . ~ ....'. -. s ..: ".;.~.~.,: i.. ! (',1i9791) Detection of N-Nitrosodiethanolamine in Humann clr:;Urine Followring, Application of a Contaminated Cosmetic. ;~~::halder, B. .09178).. yUrinar~y EXcretion of~ N-N:~itrosodli..- , ~ S ~ 2'17-222. ,~,0 . r . Preussmann, R., Wuertele, G., Eisenbrand, G. . . .. . .... . . ~.(: ethanolamine Administered Orally to Rats.Cance.r Lett. (1!981) ,,Effects of Maleic Hydrazide on Cig,arette Smoke Inhalation Toxicity in Syrian Golden Ham- United States Department of Agriculture (1979) ',"'The logic and Economic Assessment off'Maleic,Hydrazide." U.S.D.A. Techn. Bull. No. 1634, p. 106. 3 . , . . .. ::~ ~.~ PA. , p. 393'. Tsol, T.C'. (1972) '"Physiology and Biochemistry of Tobacco Plants.` Dowd~en, Hutchinson and Ross, Inc., Stroudsburg,. 14'. Steffens, G.L. (!1979) Influence of Growth Regulators and Herbicides on the Chemistry of Tobacco. Recent Adwan. To- bacco Sci.. 5, 133-1'6'3'. ~'

genic Agents in Unburned Processed Tobacco: I . .' Brunnemann, 1C «D!. ''and Wynder, E. L. .'(11975 ) Chemical studies on tobacco ~;. . ,., ..~.~. , _ smoke. XLIV. New separation techniques for classes smoke compounds. Recent Advan. Tobacco . Sci. 1: 97-122. 17. ..Brunnemann, K.D. and Hoffmann, D. (1974) The pH of Tobac- . . co Smoke. Food Cosmet. To:cicol« 12, 115-1,24 18. Bates, W.W., Griffith,R.B., Harlow, H.S., Senkus, W., and' Wakehami, H. (1968) Determination and Reporting of Total Particuliate Matter, Water in Total Particulate Matter and Nicotine. Tobacco Sci. 12, 192-196. - „ • . , ~'~`.~'-' V 19. International Comunittee for Cigar Smoke Study (1974) Oliver,,J.E..` _,(1981i)1. Agricultural Environmental Quality -,--'Machine Smoking of Cigars. Coresta Inf. Bull. 1, 31-34. .~ _, . . . . <>,. Institute, U.S'.D.A., Personal Communication. 21. Hoffmann, D. and Adams, J.D. ('11981) Carcinogenic Tobacco Specific N-Nitrosamines in Snuff and in the Saliva of Snuff Dippers.Submitted~. 22« Winn, Di.M., Blot, W.J., Shy, C. M. , PickTe, L.W., Toledo, M.A., and Fraumeni, Jr., J.F. ( 1'9'81i) Snuff Dipping and Oral Cancer Among Women in the Southern United!States. N=Nitrosodi- ~ ;: .: , ..,, -, -,:. , .. ,..~ W~ it T ,,.. . . . -Dimethylhydraaine« Cancer Lett.

ample # , ~ Tbbacco (ppb,) Mainstream Smoke . ._r :. 1us 5 . 7 . 3'0'• 1 2 ~,~: 74',°' 28 . 1 (i "e T Table I 1 J PRECISION Q OF NDELA ANALYSES* ~ 86' 8~ 2~8 3 ' : r, s1 rMean : Sigma « . y,. 1OI.6 ,Rel. Stdi. Dev. , ._. , __. 1'1!.9% tX S z; ~ . . • ... T~. . . } ... ... ...~ ..~...~~.5~ . ,~ ~ . . A . 4 Cw~ _a x: i' Y ii i n i]YC *Performed on the Kentucky IR1 Eteference cigarette. .,.. . 98.5' 36.3 99.8 0 28, .