The Composition of Cigarette Smoke I. Solanesyl Acetate

Alan Rodgman and Lawrence C. Cook Research Department, R. J. Reynolds Tobaeco Company Winston-Salem, North Carolina, U.S.A. Tobacco Science, 1959, 3-20, p. 86-88, ISSN.0082-4523.pdf

Solanesol, an unsaturated penta­ confirmation of the identity of the of solanesyl acetate from the smoke terpenoid alcohol, has been identified acid was provided by conversion of of Cigarettes B was an artifact due as a constituent of flue-cured tobacco the hydrated sodium acetate to to ester interchange occurring on the at various stages of treatment by p-pheny]phenacyl acetate (Shriner adsorbent between solanesol in the Rowland, Latimer and Giles (1956). and Fuson, 1948). These data firmly particular fraction investigated and Several solanesyl esters, namely, the establish the identity of solanesyl the ethyl acetate employed as eluent. caprate, caprylate, myristate, palmi­ acetate as a constituent of the ciga­ Mold and Booth (1957) reported tate, oleate, linoleate and linolenate, rette smoke. 130 mg. of sokmesol per 1000 ciga­ have also been reported in an extract Two separate lots of cigarettes of rettes smoked. We found 230 mg. of of flue-cured tobacco (Rowland and the same blend were smoked during this material per 1000 Cigarettes A Latimer, 1959). Recently, Rowland this investigation. One, Cigarettes A, and 370 mg. per 1000 Cigarettes B; (1959) has identified solanesyl ace­ consisting of 20,000 cigarettes, gave values approximately two or three tate in an extract of flue-cured to­ 0.35 g. (0.0025 per cent of tobacco times that of Mold and Booth. The bacrn. smoked) of solanesyl acetate in the difference in amounts appears to be Solanesol has also been identified smoke; the other, Cigarettes B, con­ related to the difference in smoking in cigarette smoke by Mold and sisting of 32,000 cigarettes, gave sol­ procedure. Mold and Booth (1957) Booth (1957). Wynder and Wright anesyl acetate at two stages of the employed a two-second puff per min­ (1957) originally reported geraniol chromatographic fractionation of the ute; whereas, in our studies, we em­ as a constituent of cigarette smoke; condensate. The first lot of solanesyl ployed a two-setond puff per 20 whereas, the compound isolated ac­ acetate from Cigarettes B appeared seconds. tually had an infrared spectrum iden­ at the same stage in the separation tical with that of solanesol (Wright, scheme as that obtained from the Experimental 1957). Recently, these authors (1958) smoke of Cigarettes A. The second All melting points were determined have reported solanesol in tobacco lot of solanesyl acetate (1.08 g,) ap­ using a Fisher-Johns melting point smoke. peared in a chromatographic fraction apparatus. The elemental analysis Solanesyl acetate has been isolated much later than anticipated. was performed by Huffman Micro­ from the condensable smoke of ciga­ This apparent discrepancy was analytical Laboratories, Wheatridge, rettes of a cased commercial blend of readily resolved by consideration of Colo. tobaccos. The melting point, infrared the following data: Isolatfon of Solanesyl Acetate absorption spectrum, and saponifica­ (1) The second lot of solanesyl from Cigarette Smoke. - Twenty tion products confirm the identity of acetate from the smoke of Cigarettes thousand filter tip ( 68-mm. tobacco this material. B was obtained from a chromato­ rod, 17-mm. filter tip) cigarettes of Saponification of the ester yielded graphic fraction rich in solanesol a cased commercial blend of tobaccos solanesol as the only alcohol frag­ and eluted from alumina by 1 :1 ( Cigarettes A) were smoked mechan­ ment and potassium acetate as the ethyl ether: ethyl acetate. ically on a manifold-type smoking acid fragment. The infrared absorp­ (2) Chromatography of pure sol­ machine. Thirty cigarettes were tion spectrum of the potassium ace­ anesol on alumina using 3: 1 ethyl puffed simultaneously. Thirty-five to tate was somewhat masked by that ether :ethyl acetate as eluent gave 40-ml. puffs, each of two-second dura­ of the excesg potassium hydroxide solanesyl acetate in the eluate. tion, were taken at 20-second inter­ present. The potassium hydroxide­ The solanesyl acetate from the vals for each cigarette. The observed potassium acetate mixture was acidi­ smoke of Cigarettes A and which ap­ temperature of combustion (860° C.) fied, steam distilled and the distillate peared early in the separation of the cigarettes was in agreement neutralized with dilute sodium hy­ scheme for the smoke condensate of with the values reported by Touey droxide. The acid fragment was thus Cigarettes B was obtained from frac­ and Mumpower (1957) employing recovered as sodium acetate trihy­ tions of the condensate which were their method of measurement. A 50- droxide. The acid fragment was thus never exposed to ethyl acetate. Thus, mm. length of each cigarette was infrared absorption data. Additional it was concluded that the second lot smoked. Thus, the tobacco smoked in

(Tobacco Science 86) this lot of cigarettes weighed 14.0 kg. The smoke was condensed in a Table 1: Chromatography of Methanol-eluted Fraction from series of all-glass traps at Dry Ice Hexane Soluble Neutral-acidic Fraction (Cigareffes A) temperature. The smoke condensate from 1200 Eluent to 1250 cigarettes was rinsed from Fraction wt., the traps and manifold arms using Fraction Vol. per Chromatogram Total in 400 to 500 ml. of methanol. The No. Solvent in liters grams methanol solution was acidified with 1 benzene 1.0 14.9 650 ml. of 0.2 N hydrochloric acid 2 1 :1 benzene:ether 1.0 19.6 and extracted with a total of 1500 3 ether 1.0 9.5 ml. of hexane. The resulting hexane 4 1 : 1 ether-ethyl acetate 1.0 32.5 solution was dried (anhydrous sod­ 5 ethyl acetate 1.0 9.3 ium sulfate), filtered and concen­ ° 6 9 : 1 ethyl acetate :methanol 1.0 12.3 trated at 40 C. using a water as­ 7 methanol 1.0 1.3 pirator. Repetition of this procedure for the 20,000 cigarettes gave a total

of 248 g. of the so-called hexane Tobacco Science, 1959, 3-20, p. 86-88, ISSN.0082-4523.pdf soluble neutral-acidic fraction. Preliminary chromatography of tion with 9: 1 hexane: benzene gave Fr. 4' ( 46.2 g.) was chromato­ this material on Aluminum Oxide 0.35 g. of the ester, m.p. 30.0-31.0" graphed on Aluminum Oxide Merck Merck (Merck Cat. No. 71707) [45 C. (reported as 32-33° C. by Rowland to give solanesyl acetate (1.08 g.), mm. (diam.) x 250 mm.] in 16 ali­ et al., 1956). The infrared absorp­ m.p. 31.0-31.5 ° C., with an infrared quots gave three main fractions; the tion spectrum of this material was absorption spectrum identical with first eluted by hexane (600 ml.), the identical with that of an authentic that of an authentic sample (Row­ second by 15 percent benzene-hex­ sample (Rowland et al., 1956), i.e., land et al., 1956) and with that of ane (1200 ml.) and the third by me­ absorption due to ester carbonyl at the sample isolated from Fr. 1 of the thanol (600 ml.). Combination of the 5.77 µ,, absorption due to carbon-car­ smoke from Cigarettes A. A mixed methanol-eluted fractions from the bon double bonds at 6.05 µ, and ab­ melting point of this material with 16 chromatograms gave 113 g. of a sorption due to acetate at 8 :15 p,. A an authentic sample gave no depres­ chocolate-brown, viscous oil. mixed melting point of the isolated sion. material with authentic solanesyl Saponification of the Solanesyl The methanol-eluted fraction ° (113 g.) was chromatographed in 10- acetate, m.p. 31.5-32.0 C., gave no Acetate.-The isolated ester ( 0.93 to 10.5-g. aliquots on Aluminum depression. The amount isolated rep­ g.) was refluxed with 40 ml. of 40 resents a yield of 0.0025 percent of per cent methanolic potassium hy­ Oxide Merck [45 mm. (diam.) x 100 Anal. mm.] to yield seven main fractions. the cigarette tobacco smoked. droxide (w/v) for one hour. The These fractions were eluted by the Calcd. for C02H.,O,: C, 84.25; H, cooled reaction mixture was diluted amounts of solvent per chromato­ 11.42. Found: C, 83.58 H, 11.20. ,vith 40 ml. of water and extracted In a similar experiment in which with a total of 100 ml. of ethyl ether. gram, shown in Table 1, to give the B, total weights indicated. 32,000 cigarettes (Cigarettes 22.5 kg. of tobacco smoked), similar to The ether extract was washed with Fr. 3 (9.5 g, l was rich in solane­ Cigarettes A above, were smoked, water, dilute potassium hydroxide, and again with water, dried over sol. one modification was introduced. The Fr. 1 (14.9 g.) was chromato­ hexane solution obtained by extrac­ anhydrous sodium sulfate, filtered graphed on Florisil (60/100 mesh) tion of the acidic methanol fraction and concentrated to give 0.89 g. (100 using 9: 1 to 4: 1 hexane:benzene of whole condensate from 1200 to per cent) of solanesol, m.p. 37.0- solutions aR eluent to yield a mixture 1250 cigarettes was extracted with a 40.0" C. Purification of this material of high molecular weight esters total of 680 ml. of a solution of 7 .5: 1 by chromatography on Florisil gave (3.82 g.) in addition to other com­ N 0.70 g. (80 per cent) of solianesol, 0.2 sodium hydroxide:methanol. ° pounds. This ester mixture was The so-called hexane soluble neutral m.p. 41.0-42.0 C., the infrared ab­ eluted with 9.1 hexane: benzene. fraction was obtained after concen­ sorption spectrum of which was Chromotography of this fraction identical with that reported (Row­ tration of the resulting hexane solu­ et al., (3.82 g.) on silicic acid (Mallinck­ tion. land 1956). rodt Chemical Works) using 9 :1 The methanol-eluted fraction (168 The combined aqueous residue hexane:benzene solution as eluent g.) obtained by preliminary chroma­ and washings from the ether extrac­ permitted separation of four main tography of this hexane soluble neu­ tion were concentrated to dryness. groups of compounds. These com­ tral fraction was chromatographed Infrared absorption studies indicated pounds or mixture of compounds in on Aluminum Oxide Merck to yield a mixture of potassium acetate and order of elution were as follows: (a) seven main fractions. potassium hydroxide but the hydrox­ 0.86 g. of an ester fraction contain­ Fr. 1' (5.02 g.) was eluted by ide seriously masked the absorption ing an aliphatic saturated ester, m.p. benzene and contained solanesyl ace­ of the acetate. This mixture was dis­ 77 ° C., designated by Rowland and tate and other solanesyl esters. Fr. solved in water, acidified with 10 Latimer (1959) as Ester X, (b) a 4' (46.2 g.) was eluted by 1 :1 ethyl per cent sulfuric acid and steam dis­ mixture of solanesy) esters (1.48 g.) ether:ethyl acetate •and was rich in tilled. The acidic aqueous distillate similar to that identified by Rowland solanesol. Fr. 4' also contained some was neutralized with 4.5 ml. of 0.2 N and Latimer (1959) in flue-cured to­ solanesyl acetate. Subsequently, it sodium hydroxide (phenolphthalein) bacco, (c) crude solanesyl acetate was demonstrated that the solanesyl and concentrated to dryness to give (0.78 g.), and (d) a high molecular acetate in Fr. 4' was an artifact aris­ 0.12 g. (77 per cent) of sodium ace­ weight aliphatic ketone (0.68 g.). ing by elution of solanesol from tate trihydrate, identified on the The crude solanesyl acetate was alumina with solutions rich in ethyl basis of its infrared absorption spec­ chromatographed on silicic acid. Elu- acetate. trnr.1. (Toba:cco Science 87} The sodium acetate trihydrate with an infrared absorption spec­ Esters. Anal. Chem., 18, 31-32 (0.12 g.) was converted to p-phenyl­ trum identical with that reported (1946). phenacyl acetate by the method of (Rowland et al., 1956). This repre­ Mold, J. D. and Booth, J. S., Isola­ Shriner and Fuson (1948) and puri­ sents a yield of 0.032 per cent based tion of Solanesol from Cigarette fied chromatographically (Kirchner on the weight of the tobacco smoked Smoke. Tobacco Science, l, 38-39 et al., 1946) to give 0.18 g. of or 230 mg. per 1000 cigarettes (1957). p-phenylphenacyl acetate, m.p. 109.5- smoked. Rowland, R. L., Private communica­ 0 111.0 C • A mixed melting point with Fr. 4' (46.2 g.), the solanesol-rich tion, 1959. au authentic sample gave no depres­ fraction from the smoke of Ciga­ Rowland, R. L. and Latimer, P. H., sion. The infrared absorption spec­ rettes B, gave 11.9 g. of solanesol, Isolation of Solanesyl Esters from trum was identical with that of an m.p. 40.5-41.5° C. This represents a Flue-cured Tobacco. Tobacco authentic sample. The over-all yield yield of 0.053 per cent of the ciga­ Science, 3, 1-3 (1959). of p-phenylphenacyl acetate based on rette tobacco smoked or 370 mg. per Rowland, R. L., Latimer, P. H. and the solanesyl acetate s,aponified was 1000 cigarettes smoked. Giles, J. A., Flue-cured Tobacco. 57 per cent. IV. Isolation of Solanesyl Esters. Summary J. Am. Chem. Soc., 78, 4680-4683 Preparation of Solanesyl Acetate (1956). by Ester Interchange. Solanesol Solanesyl acetate has been identi­ Tobacco Science, 1959, 3-20, p. 86-88, ISSN.0082-4523.pdf ° fied in the smoke of a commercial Shriner, R. L. and Fuson, R. C., (1.31 g.), m.p. 41.0-42.0 C. was "Identification of Organic Com­ chromatographed on Aluminum Ox­ blend of cigarette tobaccos. This ma­ terial in the smoke comprises 0.0025 pounds." John Wiley and Sons, ide Merck [24 mm. (diam.) x 100 Inc., 3rd Ed., 1948, p. 157. mm.] using 3: 1 ethyl ether :ethyl per cent of the tobacco consumed during the smoking process. Touey, G. P. and Mumpower II, R. acetate as the eluent. The initial 50- C., Measurement of the Combus­ in vacuo, ml. fraction, concentrated tion-Zone Temperature of Ciga­ contained 0.5;� g. This material was Acknowledgments rette:!. Tobacco Science, l, 33-37 shown to be predominately solanesyl We are indebted to Mr. Bruce W. (1957). acetate admixed with some solanesol Woosley and Mr. Max A. Wagoner Wright, G. F, Private communica­ on infrared absorption study. Subse­ for technical assistance. We are also tion, 1957. quent 50-ml. fraetions contained un­ indebted to Mr. John J. Whalen and A ehanged solanesol, m.p. 40-41.0° C. Wynder, E. L. and Wright, G. F, .:\fr. John L. Stewart for the infrared Study of Tobacco Carcinogenesis. Solanesol.---Fr. ;{ (9.53 g.), the absorption spectra. I. The Primary Fractions. Can­ solanesol-rich frattion from the cer, 10, 255-271 (1957). smoke of Cigarettes A, was purified Literature Cited Wynder, E. L., Wright, G. F, and by chromatography on alumina and Kirchner, J. C., Prate1·, A. N., and Lam, J., A Study of Tobacco Car­ then Florisil after the method of Haagen-Smit, A. J., Separation of cinogenesis. V. The Role of Py­ Rowland et al. ( 1956) to give 4.54 Acids by Chromatographic Ad­ rolysis. Cance1·, 11, 1140-1148 I!. of solane:sol, m.p. 41.0-42.0° C., sorption of their p-Phenylphenacyl (1958).

(Tobacco Science 88)