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1H 4a Accession number 1521 Lorillard Research Center Greensboro A STUDY OF' THEI PUFF-EY-PUFF' D'ELIVERY OF MENTHOL, ANDNIOOTINE'~ ][N CIGARETTE SMOKE'AND~THE EFFECTS OF AIR D'ILUTIOlri' ON DELIVERY Submitted by: Rose A. Shoffner Report number: pate: la/23/80 Summary or Aimstract:. A simple extraction method for the simultaneous analysis : of menthol and nicotine has been developed. The method has been used to study the puff-by-puff delivery of menthol and nicotine .from several of our menthol brands along with dther low tar competitive brands. Experimental cigarettes were developed'in .order to study the effect of air dilution on the yield reduction, "of tar, menthol and nicotine. It has been found that by in- ~ creasing air dilution, the general effect is to enrich the ° nicotine content of tar more than the menthol content. Thee procedure also has applications for the analysis of tobacco filler for menthol and cigarettes for both menthol and1triacetin. /le Xc: Ms. M. S. Ireland Dr. H. J. Minnemeyer Dr. F. J. Schultz Mr. O. L. Tucker Library

INTRODUCTION A review of the literature shows comparatively few methods reported for the determination of menthol in cigarette smoke condensate.©ur currentmetho~d of menthol analysis1 isusedito determine menthol levels on the tobacco filler. Mitchell, Barbee and Irby reported a method for the determination of'rnenthol in smoke 2 which involved vaporiaation~from a Cambridge filter into a cold trap. Another vapor chromatographic method'3' was reported by Lyerly. A simple direct extraction method 4 has been reported by Ctarran for determining menthol in filters and on tobacco. A method similar to the one described by Curran wgs used by Parks f d i i - eterm n or ng menthol in smoke puff by-puff. , ~ The method described in this report is based on the simul- taneous extraction of menthol and nicotine using a binary organic solvent system. This report also g,ives information on the puff- by-puffana~lysis of several of our menthol brands, competiitive .brands, and experimental cigarettes. The~ experimental cigarettes were developed~in order to study the effects of different air filtration systems on the delivery of menthol and nicotine. EXPERIMENTAL Eaiuipment: 1. Filtrona 12-port smoking machine . Hewlett-Packard 583'0-A Gas Chiromatograph equipped, with flame ionization detector and'CC terminal, or .equivalent. A22aratus: . GC'column - 61 x 1/8" s:tainless steel, 10% ' Carbowax 20M on Anakrom ABS 80/90 mesh. . Cambridge filter pads and holders . Sample vials with rubber stoppers . 15 ml calibrated dispenser 5. 10, ul syringe

-2'- Extraction solution - 4:1 ratio of Trichlorotrifluoro- ethane (Freon 113) to isopropanol Internal standard solution - anethole (,= 2 mg/ml)' in . Freon~ 113 Menthol - 0'.3'60' mg/ml in~ Freon 113 Nicotine - 1.154 mg/ml in Freon 113 . GC'Calibration Solution:. 3 ml menthol standard 2' mi nicotine standard 1 ml internal standard 6 ml Freon 113 3' mil Isopropanol 1. Sample Preparation - Menthol cigarettes were conditioned' in packs overnight at 750 + 2oF and 60% + 2%relative humidity. They were sorted according,too pressure drop, percent air dilution and'weight then smoked im- mediately. Smoking - Cambridge filter pads and holders were weighed , before and after smoking in order to determine wet tar. Cigarettes were smoked on a puff-by-puff basis using 40.1cigarettes. The first cigarette was placed inithe ,first port and lighted as the machine took a puff. The cigarette was then moved to the second port and another cigarette placed'inithe first port. On the second cycle, the cigarette in the first port was lighted and then moved to the second port after the cigarette there was moved'to the third port. This process was repeated until all forty' cigarettes were smoked. Cigarettes were smoked toia standard butt length of tipping paper plus.3 mm. Puff counts were takeniand'averaged, 0QU'78379I4 3. Extraction - Cambridge filter pads were removed from the holders and placed in the sample vials containing 1 ml of the internal standard toawhichiwasadd'ed 15 mil of'the extraction solution,. The bottles were stoppered and shaken horizontally on a mechanical shaker for 30 minutes. The solutions were then injectedlinto the GC'.

-3- 4. Gas Chromatog;raphic Conditions - GC oven conditions and'iprogram conditions are shownlin Figure 1. RESULTS ANDDISCUSSIOiyd Recovery of Menthol - Recovery of menthol from Cambridge filter pads was checkediby smoking five nonmentholated cigarettes on each pad and then adding 1 ml of the menthol •stock solution to the pads. The pads were then placed in, sample vials,, stopperediand allowed to sit 30 minutes before extracting., Results shown in Table 1 indicate a recovery of 100%. Recovery of Nicotine - Recovery of nicotine from Cambridge fi]lter pads was checked by smoking,five low tar/nicotine cigarettes on each pad and thenadiding 1 m,l of the nicotine stock solutions to the pads. The pads were then placed in sample vials, stoppered and allowed'to:sit 30 minutes before extracting. Results are shown in Table 2. Precision of the Method - Precision of the method for nicotine was.checked by smoking 24 samples (5 cigarettes/pad). one- half of these samples was analyzed for nicotine by the method described above and the other half by our smoking; lab's GC standard procedure for nicotine. TabSe.3' shows the statistical comparisons of the data collected. No statisticall difference was found in precision of the two methods at a 95% confidence level. Gas Chromatography Figure 2 shows a typical chromatographic separation of' a sample. Menthol is eluted at 2.2'minutes, followediby the!internal standard, anethole, at 4.6 minutes and nicotine at 5.5 minutes. The small peak that elutes after nicotine was identified using,GC/FT-IR and GC/MS as neophiytadiene. In order tolkeep this peak from interfering with nicotine, it was necessary to run the program isothermally at 170u. Puff-By-Puff Analysis a0'783*795 1. Menthol - preliminary results of menthol analysis appeared to be low when compared with results found in the literature. 3' Several experiments were run to determine why these results were low. After running!analysis on!freshly opened packs of Triumph Menthol and comparing those results with, previous ones, it was hypothesized that menthol levels could be dropping due:to time spent in the conditioning room

-4- in opened trays. This hypothesis was checked'by analyzing Triumph Menthol-cigarettes that had been placed in the con- ditioning, chamber and allowed to~ sit open for six days., Results shown in Table 4 indicated that there was a loss of menthol during the conditioning period. In order to over- come this problem, the procedure was changed so that samples were conditioned in unopened packs and opened immediately before smoking. Figures 31 and 4 show the puff-by-puff delivery of menthol for our brands and competitive brands, respectively. The total amount of menthol delivexed/cigarette for our brands ranged from 301 pg for Newport Lig,ht -to 152 _ pg for Triumph. -The competitive brands gave slightly lower deliveries than our average.• Table 5I shows the calculated values for percent delivery of menthol per puff basedionn weight. 2. Nicotine - puff-by-puff analysis of nicotine for our brands and'competitive brands is shown in Figiures 5 and 6, respectively. Salem Light 85's delivered the highest total amount of nicotine at 938'Ug followed by Newport. Light 85's at 760 jng,. The nicotine results foundiby using this procedure were within the range reported by our smoking lab. The percent delivery of nicotine per puff is shown in Table 6., Air Dilution Versus Delivery Experimental cigarettes were developed in order to study e t+he effocts off' air dilution on the delivery of menthol andlnicotine. The cigarettes were made using the same blend and filter construction with the only difference being the number of lines of'perforations in the tipping paper. Cigarettes were selected'so that the air d'ilution. .values ranged from 0-50%. Figure 7 shows the comparative rates at which~ menthol and nicotine are reduced in~ the smoke condensate as a function of increasing air dilution., When looking at the total amount delivered, we found that the amount of nicotine present in smoke drops faster with in- creasing air dilution than that of menthol. Figures 8 and 9 show the puff-by-puffdeliveryof menthol and nicotine,, respectively. Table 7 shows the comparative differences on the experimental cigarettes and the percent yield reductions for tar, menthol, andlnicotine. These values are shown graphically iniFigure 10'as percent yield reduction vs. per- cent air d'ilution,. As expected, the yield reduction for 0 , tar approaches the 1:1 ratio line when comparedito air O' ~ G0 W. Q' ~

toiair dilution. On aipercent reduction basis, menthol is reduced more than nicotine. Therefore, increasing air dilution enriches the nicotine content of tar more than the menthol content. FURTHER' STUDIES Experiments are being conducted'in order to:determine the applications of this method to include the analyses of tobacco filler for menthol and cigarettes for both menthol and triacetin. Production samples are being analyzed froin the same groups oif samples takenifor analysis by our Quality Control lab. Preliminary results indicate that this method may be acceptable but no definite conclusions can be drawn until data have been analyzed for comparison of the two methods. REFERENCES 1 Wagner„ J. R. and Thaggard, N. A.,, "An Automated Method for the Gas Chromatographic Determination of Nicotine in Smoke°', TobaccoiScience, 18', 63-66 (1974) . Mitchell, B. C'..,, Barbee„ E. P., and Irby, R. M., "The Chromatographic Determination of Menthol in Cigarettes and Cigarette Smoke" , Tobacco Science, 7, 6'4'-70 (1963), . Lyerly, L: A., "Direct Vapor Chromatographic Determination of Menthol, Propylene Glycol, Nicotine and Triacetin in Cigarette Smoke'"', Tobacco. Science, 11, 49-.51 (1967). C'urrani,, J. G., "Delivery of Menthol from Cigarettes Containing Either a Mentholated Filter or Mentholated Tobacco"', Tobacco Science, 16,, 40-42' (1972). . Parks, R. Mi., "'Menthol in Smoke II"',, Lorillard Research Report, Accessi:oniNbi. 445 (;19.73).

TABLE 1 - RECOVERY OF' MENTHOL Sample % Recovery 1 9'9 . 2. 2 97.2 3 102.5' 4 _ 100.6 5 98.3 6 100'. 3 7 102.,2 & 100. 0'. 100.0' Average . TABLE 2' - RECOVERY OF NICOTINE Sample 1 3'. 5'. 100.4! Average

Statistical P'arameter Smoking Lab N 12 Mean 1.36~53 Variance 01.00369 Std., Dev. 0. 0'599 Std. Error of Mean 0.0173 Coeff. of Variatzon 4.3.889~ TABLE 3 - PRECISION OF' TH'EI METHOD TABLE'4 - LOSS OF MENTHOL DURING CONDITIONING Cigarette Triumph - 5 cig/pad - not conditioned - Triumph - 5 cig/pad 6 days opened in, cond', chamber Menthol ('mg/'cig.) .2'S50 .2'682' .0498' .0'3'96

Triumph True Green 85 True Green 100 Golden Light 10!.0~ Newport Light 85 Golden Light 85 Salem Light 85 Merit 85 Kool Super Light Carlton~ 85 Doral II TABLE' 5 - MENTHOL DELIVERY $ AIR D'TL ~. WT. OF' MENT . ON TOBACCO (mg/Cig.j %DEL 2' . IIVERY , 3' . ON' E 41 . ACEi' FU , 5'~ . FF 6 69 5.09 ' . 0 9' .,2 2 .26. . 28 .40 5 '. .541 52 3.3'6 .29' .82 1.11 1. 2' 1 1.4'2 1.,51 1.62 34 4.04 .30 .79~ 91 1.01 1.27 1.40' 1.78 56 1.96 .105 .18' . 3!9'. .53 .56 .68' .9'6 36 3'.19 ~ .61 1. 0'2' 1.2'1 1.30 1.55 1.7'0 2.05 42 3'.12 . 2'9, . 83 ' 1.06' 1.1~7 1.2'4. 1.,44 18' 2'.62. .,46 1.17 1.3'6 1.22' I 1. 5.4. 1.73 6 1.86 30 3'.83' .32 .8'3' 1.02 1.01 1.32 1.47 1.80' 49 3'.. 34 .13 .38I .49 .53' .71 .83 1.20 8'0. 8.9'0 .,02. . 0.6 .11 .13'. .210 .25 56 3'. 4 5 .22. .59. .76 . 7 3' 1.0'1 1.17 1.16 .61 1.15 1.90

TABLE 6 - NICOTINE'DELIVERY $, T., OF NIC. - IR ' ON TOBACCQ ' % DELIVERY/PUFF BRAND' IL. (mg/Cig. y 1 2' 3 4 5 6 7 8 , ~~- . ~ . ~ ~~ Trium h~ P 69 ~ 17.2 1 . ~0~6~ .12' ~ .15 ~ .17 ! .25~~ ~ .29! ._ ~ .32' .~3'6 True~ Green 85 ~152 ' ]12'.16~~ .17' .~4!2' .53 .58 ~ .69' I .~75~' ~ .78 ' - True Green 100 34 14.84 .2'4 .46 .54 .55 ' .66 , .T6 .912 I - Golden Light 100' 56 17.60, I .08 .14, .26 .30 .35 .41 .46 .54 Newport Light 85 '3I6 14.,85 ~ . 34! . 59. .,69 . 72 . 88 .92 1. 0'0 - ~ . . Golden Light 8~~5~ ~4,2~~ ~ ]14~.~9~~2~~ .22 ~~ .52' I .6'~7 .73~~, ~~ -7~& .89 - ~ - Salem Light 85 18' 13'.93' .34 I .63 .80 I.71 .,97' 1.04 1.0'9. 1.~16 Merit 85 30 10.91 ' .28 '' .,5'8 . 72'. . 68' .92 . 98 .94 - bCool ' Super Light 49 ' 13.96 .14 . 27 .36 . 39' .51 I. 57' .66 ;Carlton 85 8'.0 I 9.76 .04 .08' .12 .14 .2'.1 .24 - - .'.,,~ . .. ~ . r Y . ~ ~ I • i~ II ,=`IDoal 6 ~. 33 ~ 1 16 ~ .32 ! 44 . '~ .44 4~4 .~ 631 .74 6~6 .66 ~ - V•'~•) ~