Litt Scopoletin in Commercial p hanc 1 ed I Lorn Tobacco Products Ha!J wor1 Rale Bri_g SnuJ Chao-Hwa Yang. Yasushi Nakagawa and Simon H. Wender Scot Mild The Chemistry Department, University of Oklahoma Bull Norman, Oklahoma, U.S.A. N. 1 Skoa Beec Cl. bers­ Following the discovery by Yang

Tobacco Science, 1958, 2-24, p. 111-114, ISSN.0082-4523.pdf used Table 1.-Determination of scopoletin in tobacco et al. (1958) that the tobacco and been the mainstream smoke from cigar­ Red Tobacco product Volume (ml) Scopoletin Scopoletin Scopoletin ettes commonly used in the U. S. shee1 type and brand aliquot streaked (µ,g) per (µ,g) per I g (%) in contained scopoletin (6-methoxy-7- notec on paper aliquot tobacco* tobacco hydroxycoumarin), these workers imat, (1) (2) (3) (4) (5) devised quantitative methods for its cendi Cigars determination in cigarette tobacco tial and smoke. Recently, they reported lC jars quantitative results obtained for sco­ Sample 1 0.0092 steel 10 18.4 92.0 poletin in 24 brands of cigarettes Sample 2 0.0088 inets 10 17.6 88.0 (Yang et al.; in press). These find­ 2C 193/s' ings have suggested the extension empl1 Sample 1 10 19.3 96.5 0.0097 of qualitative and quantitative sco­ Sample 2 10 19.1 95.5 0.0096 Sm poletin studies to other commercial smok 3C tobacco products. This paper reports Sample 1 10 5.8 29.0 0.0029 cigar the identification of scopoletin in all arett, Sample 2 10 5.5 27.5 0.0028 the cigars, snuff, chewing tobaccos, 4C and I pipe tobaccos, pipe mixtures, and on a 1 Sample 1 10 7.3 36.5 0.0037 roll-your-own· cigarette tobaccos an­ Sample 2 10 7.3 36.5 0.0037 Comi: alyzed. Quantitative results are re­ to ac, Hand-rolled Cigarette Tobacco ported on selected samples of each To ti type of product as well as on the flasks lHC smoke from selected cigars. Sample 1 15 26.1 87.0 0.0087 flask, 0.0091 with Sample 2 15 27.3 91.0 Materials and Apparatus 2HC was , Sample 1 15 28.5 95.0 0.0095 Tobacm samples - Each tobacco and t Sample 2 15 28.0 93.3 0.0093 product tested was purchased locally the be on the open, retail market. Cigars stopp1 Pipe Mixtures studied were El Producto (Puri­ the si 1PM tano), Wm. Penn (Perfecto), Robt. stoppt Sample 1 15 23.7 79.0 0.0079 Burns (de Luxe), Robt. Burns (Cig­ added Sample 2 15 23.5 78.3 0.0078 arillo), White Owl (Perfecto), Roi­ down 2PM Tan (Perfecto), Roi-Tan (Golfer), :iide t Sample 1 15 26.0 86.7 0.0087 El Verso (Bouquet), El Verso (Mel­ tubes Sample 2 15 28.8 96.0 0.0096 low), King Edward, Melba, Melba third of 10( 3PM (Midget), Webster (Babies), Corina Sample 1 15 25.3 84.3 0.0084 (Magnolia), Corina (Larks), Corina Sample 2 15 24.4 81.3 0.0081 (Cigarillo), Coronitas (Perfecto), Experi1 Dutch Masters (Corona de Luxe), Pipe Tobaccos Pre: Dutch Masters (Crown), Dutch Mas­ sampl1 IP ters (Belvedere), Dutch Masters grourn (President), Muriel, Royalist (New Sample 1 15 17.8 59.3 0.0059 inill, ' Sample 2 15 18.7 62.3 0.0062 Duke), Cuesta-Ray, Antonio y Cleo­ were 2P patra, El Trelles, Red Dot (Per­ for th Sample 1 15 13.8 46.0 0.0046 fecto), Red Dot (Cigarillo), Missis­ 4 CH, Sample 2 15 14.4 48.0 0.0048 sippi River, Hunter (Imperial), and dried 3P grindi Sample 1 15 16.7 55.7 0.0056 of tht Sample 2 15 14.0 46.7 0.0047 1 This res..arch was supported, in part, by the were 1 Tobacco Industry Research Committee and the Atomic Eneruy Commission. indivic

(Tobacco Science 111) Little Fendrich (Buds). traction. were placed in the first, second, and Pipe tobaccos and mixtures and Each 4 g. extraction was carried third flasks, respectively. The Kjel­ hand-rolled cigarette tobaccos includ­ out in a separate Soxhlet extractor, dahl flasks used as traps were then ed Kite, Bugler, Revelation, Walnut, using 250 ml. of 85 percent isopropyl lowered into large evacuated flasks London Dock, Bond Street, Half and alcohol for aproximately 3 hrs. on a (modified Dewar) containing dry Half, George Washington, Edge­ steam bath. A second extraction was ice-acetone (approximately -77°) for worth, Dill's Best, Model, Sir Walter made on each sample, again using at least 30 minutes before smoking Raleigh, Prince Albert, Velvet, 250 ml. of 85 percent isopropyl alco­ and were kept in the cold baths Briggs, and Sutliff's Mixture No. 79. hol for 3 hrs. The two extracts of throughout the smoking. Snuffs tested were called Honest the 4 g. tobacco sample were com­ The puff duration, puff interval, Scotch Snuff, Scotch Snuff, Sweet bined, reduced to approximately 150 and volume of smoke per puff were Mird Snuff, and Copenhagen Snuff. ml. in vacuo, and the volume was 3.5 seconds, 30 seconds, and 53-57 The chewing tobaccos studied were then adjusted with 85 percent isopro­ ml, respectively. Exact volume of Bull of the Woods, Day's Work, W. pyl alcohol to 200 ml. in a volumetric smoke per puff, number of puffs, butt N. Tinsley, Tinsley's Natural Leaf, flask. Aliquots of this solution were length, humidity and room tempera­ Skoal WintergrePn Flavored, and then taken for qualitative and for ture for the quantitative studies Beech-Nut. quantitative analyses of scopoletin. have been recorded in table 2. Chrom.atography paper and cham­ Smoking procedure - Prior to After the cigar samples had been brrs-The chromatography paper smoking, 25 ml., 10 ml., and 10 ml. smoked, the trap system, still con­ Tobacco Science, 1958, 2-24, p. 111-114, ISSN.0082-4523.pdf used throughout these analyses has of an anhydrous acetone-absolute nected in series, was removed from been Schleicher and Schuell No. 589, ethyl alcohol (1 :1 v./v.) mixture the Dewar flasks, and was left until Red Ribbon. Purchased in 58x58 cm. sheets, the papers, unless otherwise noted, have been cut to a size approx­ imately 19x58 cm. each for use. Des­ Table 1-continued cending chromatography in conven­ tial 12"x24" Pyrex chromatography (I) (2) (3) (4) (5) jars and in the standard, stainless Pipe and Cigarette Tobaccos steel interior chromatography cab­ inets (approximately 27½"x26"x- lPC 19%" inside dimensions) has been Sample 1 15 11.6 38.7 0.0039 employed. Sample 2 15 10.2 34.0 0.0034 Smoking machine-To obtain cigar 2PC smoke for scopoletin analysis the Sample 1 15 10.0 3:L3 0.0033 cigar was smoked on a standard cig­ Sample 2 15 10.4 34.7 0.0035 arette smoking apparatus (Phipps 3PC and Bird, Inc., Richmond, Va.) based Sample 1 15 10.5 35.0 0.0035 on a design of the American Tobacco Sample 2 15 9.4 31.3 0.0031 Company. The holder was modified ~hewing Tobaccos to accommodate various cigar sizes. To trap the smoke, three Kjeldahl lCH flasks were used in series. The first Sample 1 20 5.!I 14.8 0.0015 flask, 300 ml. capacity, was equipped Sample 2 20 5.5 13.8 0.0014 with a spiral tube, one end of which 2CH was connected to the cigar holder Sample 1 20 6.0 15.0 0.0015 and the other end of which reached Sample 2 20 6.:I 15.8 0.0016 the bottom of the flask. A small glass 3CH stopper was attached to the end of Sample 1 20 8.7 21.8 0.0022 the side arm. During smoking, this Sample 2 20 9. L 22.8 0.0023 stopper could be removed and solvent 4CH** added from the side arm to wash Sample 1 20 10.0 25.0 0.0025 down the smoke which condensed in­ Sample 2 20 9.9 24.8 0.0025 Ride the spiral tube. Straight glass 5CH** tubes were used for the second and Sample 1 20 9.0 22.5 0.0023 third flasks, each of which flasks was Sample 2 20 8.8 22.0 0.0022 of 100 ml. capacity. Snuff Experimental IS Sample 1 20 10.4 26.0 0.0026 Prepamtion ond e.ctraction of Sample 2 20 11.7 29.3 0.0029 sample-Each tobacco product was 2S ground with a Wiley intermediate Sample 1 20 12.9 32.3 0.0032 mill, 40 mesh screen. The samples Sample 2 20 11.4 28.5 0.0029 were not dried beforehand, except 3S for three brands of chewing tobacco, Sample 1 20 10.0 25.0 0.0025 4 CH, 5 CH, and 6 CH. These were Sample 2 20 10.2 25.5 0.0026 dried at 50° for 48 l)rs. prior to grinding. Approximately 4 g. of each * In evety case reported, 4.00 g. of tobi1cco prodact was extract~d; total volume of each extract of these ground tobacco powders was 200 ml. •• The 4.00 g. are weights after drying at so•c for 48 hours. Brand 4CH lost 0.88% of its weight, were weighed and tra1jsferred into and SCH lost 8.23% of its wright prior to grinding. individual Soxhlet thimbles for ex-

(Tobacco Science 112) -.:1 Table 2.-Determination of scopoletin in cigar smoke 10 Cigar type Weight (g.) Volume No. of puffs Butt Scoroletin Scopoletin and brand of cigar ± I ml. per cigar Length (mm.) (f,lg per (/,lg) per I g. 20 ml. aliquot cigar lC, cigarillo ·*Sample 1 11.90 54 35 30 8.0 3.4 *Sample 2 12.95 54 35 30 8.2 3.2 2C, perfecto *·*Sample 1 16.62 56 50 50 8.0 2.4 **Sample 2 16.69 56 50 50 4.5 1.4 For each sample: room temperature was 32°C; humidity, 24%; total volume of extract was 100 ml.; and 20 ml. aliquots were streaked on paper for analysis. * Each sample consisted of 4 cigarillos. ** Each sample consisted of 2 cigars. Tobacco Science, 1958, 2-24, p. 111-114, ISSN.0082-4523.pdf the temperature of the system reach­ tended paper chromatography for trace amount of scopoletin, two ed that of the room. The solvent al­ the quantitative separation and pur­ brands. Two cigars each of six ready in the three traps plus all the ification of the scopoletin, elution brands in group "a" which were acetone-absolute alcohol washings with 50 percent ethyl alcohol-water, considered likely to be highest in were combined and transferred quan­ and spectrophotometric measurement scopoletin content as estimated by titatively into a 100 ml. volumetric at 344 mµ.. The quantity of scopoletin the above method, were selected for Figure flask and made to volume. Twenty present was calculated from an ex­ quantitative analysis by the proce­ milliliter aliquots of this solution of perimentally determined standard dure of Yang et al. (in press). The tured smoke condensate were used for the curve prepared from known amounts quantitative results for four of these the ri scopoletin analyses. of pure, synthetic scopoletin carried samples are recorded in table 1. The 27.8 rette. Quali:tative analyses-For identifi­ through the same procedure as the other two brands had values of less cation purposes, the scopoletin was tobacco or smoke scopoletin. than 5 ftg per aliquot. Fn purified by extended paper chrom­ Analyses of the scopoletin content it is atography, using the basic procedure Results and Discussion -of five brands of regular cigarettes hand­ previously described (Yang, loc. cit.) (Camel, Lucky Strike, Philip Mor­ conta Data for the amount of scopo­ (colu The resulting scopoletin was checked letin found to be present in each to­ ris, Chesterfield, and Old Gold) for with an authentic sample synthesized the percentage of scopoletin in ciga­ 0.0061 bacco product containing at least 5 regul: by the procedure of Aghoramurthy f,lg of scopoletin per aliquot are re­ rette tobacco gave values in the range and Seshadri (1952). The infrared of 0.0066-0.012% (Yang, in press). This ported in table 1. Below this con­ brand spectrum of scopolet.in is shown in centration, the reproducibility and Only 1 C and 2C of the cigars tested figure 1. The absorption spectra fell in this range. These two were beled accuracy of the procedure become these, showed a prominent absorption max­ lower, and hence, for such samples thus selected for quantitative deter­ imum at 344 m1, (Yang loc. cit.) for mination of cigar smoke under the per Ii only the statement is made that Jar ci the synthetic and purified samples they contain less than 5 f,lg per ali­ smoking conditions described. Cigars Thi containing sufficient scopoletin. Each quot. Each value of column 3, 1 C and 2C actually carry the same scopoletin fraction from a tobacco brand name, but lC is the cigarillo brand table 1, is given in micrograms per pipe product was also subjected to one­ aliquot and represents the average and 2C is the perfecto of that brand. and two-dimensional mixed paper The smoke from 42 other cigars, slight of values obtained on three aliquots. comp~ chromatography with authentic In column 4 are recorded the micro­ including 19 other brands, were samples. Solvent systems used for grams of scopoletin per gram of to­ studied qualitatively for their scopo­ paper chromatography were 15 per­ bacco product analyzed. These values letin content. In cigar smoke there cent acetic acid-water; 60 percent have been expressed as percentages are impurities which seem to inter­ acetic acid-water; n-butyl alcohol­ in column 5. fere even more than usual with the acetic acid water (6:1:2 v/v); n­ All 31 brands of cigars analyzed determination of scopoletin. And. butyl alcohol-benzene-pyridine-water qualitatively were found to contain because of the apparently very low (5: 1 :3 :3 v/v); and nitromethane­ some scopoletin, though apparently scopoletin content of the smoke from benzene-water (2 :3 :5 v/v). Typical in different amounts. For prelimi­ these other cigars, quantitative stud­ R, values for scopoletin in these sol­ nary classification purposes as to ies on the smoke from cigars have vent systems, respectively, using the scopoletin content, the relative size been limited to lC and 2C. The re­ S & S No. 589 paper and a tempera­ of the purified scopoletin zone and sults of these analyses are shown in ture of 28° ± 3° were 0.47; 0.47; the intensity of its fluorescence were table 2. In the case of 2C, definite 0.82; 0.82 and 0.69. used as guides, in a manner similar variations were found in individual Quantitative analy,ies-The quan­ to that employed for the estimation cigars analyzed. titative determinations of scopoletin of rutin in tobacco Ramples by Penn The values in table 2 indicate that in the various tobacco products and and Weybrew (1958). The brands lC and 2C, by far the highest of all in cigar smoke were performed by were thus placed in one of the fol­ the cigars tested in their scopoletin the analytical procedures already de­ lowing groups: (a) relatively higher content, produce a relatively lower scribed by Yang et al. for scopoletin scopoletin content, nine brands; (b) scopoletin content in the smoke than in cigarette tobacco and smoke, resp. medium, eight brands; (c) low sco­ was found in smoke from a compara­ (Yang, in press). These involved ex- poletin content, 12 brands; and (d) ble amount of tobacco in manufac-

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Figure I. hfrared spectrum of scopoletin (from Perkin-Elmer Infra cord). tured cigarettes. In the latter case, from cigarettes. Literature Cited the range was found to be from 10.3- The amount of scopoletin in the Aghoramurthy, K. and Seshadri, 27.8 pg scopoletin per 1 g. of ciga­ snuff and chewing tobacco samples T. R., "A convenient synthesis of rette. analyzed was even lower than that scopoletin." J. Sci. Ind. Research From the values shown in table 1, of the pipe tobaccos studied. Two (India), llB: 411-13 ( 1952). it is evident that both brands of brands, however, (4S and 6CH) were hand-rolled cigarette tobacco tested considerably lower in scopoletin con­ Penn, P. T. and Weybrew, J. A., contained scopoletin in an amount tent than the other snuff and chew­ "Some factors affecting the con­ (column 5) that falls in the range of ing tobacco samples analyzed. Both tent of the principal polyphenols in 0.0066-0.012% which was found for had less than 5 µ.g of scopoletin per tobacco leaves." Tobacco Science regular. manufactured cigarettes. aliquot. 2: 20-24 (1958). This was also the case for the 3 Yang, Chao-Hwa, Nakagawa, Y. and brands ( 1PM, 2PM, and 3PM) la­ Summary Wender. S. R., "Identification of beled a,: pipe mixtures. None of Scopoletin has been found to be scopoletin in cigarette tobacco and these, however, approached the up­ present in all cigars, snuff, chewing per limits found for tobacco in regu­ smoke." J. Org. Chem. 23: 204-205 tobaccos, pipe tobaccos, pipe mix­ (1958) lar cigarettes. tures, and roll-your-own cigarette The scopoletin content of all the tobaccos analyzed. Yang, Chao-Hwa, Nakagawa, Y., and brands labeled as pipe tobacco or Quantitative results have been re­ Wender, S. H., "Quantitative stud­ pipe and cigarette tobacco was ported on selected samples of each ies of scopoletin in cigarette smoke slightly lower than that found in a type of tobacco product as well as on and tobacco." Anal. Chem. (In comparable weight of the tobacco the smoke from selected cigars. press).

{Tobacco Science 114)