Quantitative Studies of Scopoletin in Cigarette Smoke and Tobacco CHAO-HWA YANG, YASUSHI NAKAGAWA, and SIMON WENDER Chemistry Department, Universify of Oklahoma, Norman, Okla.
b Quantitative methods involving paper chromatography ond spectrophotometry have been devised for the determination of scopoletin in cigarette tobacco and smoke. Experimental results are reported on the tobacco of 24 brands of cigarettes and on the mainstream smoke arising from these cigarettes.
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recent discovery by Yang, Nakagawa, and Wender (4) that the mainstream smoke from cigarettes commonly used in the Cnited States contained scopoletin (6-niethoxy-7-hydroxycoumarin) has necessitated the development of procedures to determine quantitatively the amount of scopoletin present in the smoke from cigarettes and in the cigarette tobacco before smoking. This paper details the methods devised, and the experimental results obtained using these methods on the tobacco and on the mainstream smoke obtained from 24 brands of cigarettes. Many interfering substances were present in the samples, with only small amounts of scopoletin. Hence, extensive paper chromatography was employed for the separation and purification of scopoletin from the tobacco and smoke. Following repeated chromatography, the scopoletin was eluted with 50% ethyl alcohol-water and measured spectrophotometrically a t 344 mp. The scopoletin present was calculated from an experimentally determined standard curve prepared from known amounts of pure, synthetic scopoletin carried through the same procedure. Strict adherence to the details of the procedures described is essential for good reproducibility. The method reported by Tryon (2) to measure the scopoletin present in differentiating and nondzerentiating cultured tobacco tissue did not include the extensive chromatography found necessary to produce pure scopoletin and to give quantitative results on scopoletin present in cigarette tobacco and smoke. HE
coside of 6,7-dihydroxycoumarin), according to the method of Aghoramurthy and Seshadri (1). The absorption spectrum of a 3.90 X 10-5M solution of synthetic scopoletin in 50% aqueous alcohol (7.5 mg. per liter of 50y0 ethyl alcohol) showed a prominent absorption maximum a t 344 mp (Figure 1). The chromatography paper used was Schleicher & Schull No. 589 Red Ribbon, cut to a size of approximately 19 X 58 em., unless otherwise noted. Twenty-four different brands of cigarettes commonly used in the U. S. were analyzed individually or in composite. The brands of regular cigarettes analyzed were Camel, Lucky Strike, Philip Morris, Chesterfield, and Old Gold. The denicotinized cigarette studied was Sano, regular length with no filter tip. Menthol cigarettes included were Salem, Kool (regular and filter), Oasis, and Spud. The king-size nonfilter group consisted of Pall Mall, Raleigh, Philip Morris, Chesterfield, Herbert Tareyton, and Cavalier. Analyzed individually or in composite in the filter group were Viceroy, Winston, Marlboro, L &. M, Old Gold, Hit Parade, and Kent. Each cigarette was smoked on a standard smoking apparatus (Phipps and Bird, Inc., Richmond, Va.), based on a design of the American Tobacco Co. Capacity was four cigarettes per run. To trap the smoke, three Kjeldahl flasks were used in series. The first flask of 300-ml. capacity, was equipped
n-ith a spiral tube, one of which was connected to the cigarette holder while the other reached the bottom of the flask. A small glass stopper was attached to the end of the side arm. During smoking, this stopper could be moved and solvent added to wash d o m the smoke which condensed inside the spiral tube. Straight glass tubes were used for the second and third flasks of 100-ml. capacity. PROCEDURES Determination of Scopoletin in Cigarette Smoke. Two packs or boxes
of each brand of cigarettes were purchased from different local stores shortly before analysis. Just prior to actual smoking, the two packs of the brand to be analyzed were opened, and five cigarettes were taken from each package, and weighed. These ten cigarettes comprised one sample of that brand. A composite sample consisted of two cigarettes each from packages of five different brands of the same cigarette type. Prior to smoking, 25, 10, and 10 ml. of an anhydrous acetone-absolute ethyl alcohol (1 to 1 v./v.) mixture were placed in the first, second, and third Kjeldahl flasks, respectively, of the smoking apparatus, and these were then lowered into large evacuated flasks (modified Dewar) containing dry iceacetone (approximately -77" C.) for a t least 30 minutes before smoking, and were kept in the cold baths throughout the smoking.
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Figure 1. Absorption spectrum of scopoletin
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