Beitrage zur Tabakforschung International · Volume 11 · No. 4 · August 1982

Cigarette Smoke Formation Studies VII. The Fate of (S-14C)·Methoprene In Cigarette Smoke* by Andrew F. Frisch, Bobby W. Francis, Marie K. Chavis and Robert W. jenkins, Jr. Philip Morris Research Center, Richmond, Virginia, U.S.A.

INTRODUCTION Figure 1. Structure of (S-14C)~methoprene.

(5-14C)•-methoprene The economic consequences of damage to tobacco prod­ ucts by the cigarette beetle ( Lasioderma serricorne) and tobacco moth (Ephestia elutella) have long been of con­ cern to the tobacco industry. Considerable effort has 0~ been expended to effect control of these pests at all 14 stages of processing and storage. Nevertheless, with all (5· 0)-lsopropyl (2E ,4E)-11-methoxy-3,7,11-tri methyl-2,4-dodecad lenoate the controls currently in use, the total dollar loss per year from beetle infestation remains large. In late 1973, Strong and Diekman (1) reported the ap­ parent effective control of the cigarette beetle and sev­ eral pests of stored products by insect growth regulators EXPERIMENTAL (IGR). Additional testing (2) of a material called metho­ prene+ demonstrated excellent control of the tobacco Radiochemicals beetle. 14 Methoprene (Figure 1) or isopropyl (2£,4£)-11-me­ The (5- C)-methoprene (5) was provided by Zoecon thoxy-3,7,11-trimethyl-2,4-dodecadienoate is classified Corporation of Palo Alto, California. The chemical as a juvenile hormone mimic which prevents the larvae purity as received was 99.2 Ofo (2£,4£)-methroprene, of a series of insects from developing into normal adults 0.4 41/o (2Z,4E)-methoprene. The 500 11Ci (58 mCi/ (3). This material· is unique as an insecticide since it mmol) sample was received in a solution of hexane/ exhibits a morphogenetic rather than a terminal toxic benzene. The solvents were removed at water aspirator effect upon the target insect. Long et al. (4) have re­ vacuum with a warm water bath. The residual (5-uq. ported that methoprene, when applied to laboratory methoprene was then transferred to 50 ml absolute 14 stored tobacco, is completely effective in controlling the ethanol and refrigerated. The C-activity of the ethano­ cigarette beetle for up to thirty months following the lic solution was 10 11Ci/ml. initial application. Most of the IGR is retained on the tobacco. It follows that some of this residual metho­ Cigarette Preparation prene would find its way into cigarettes. This work describes the total distribution of (5-14C)-methoprene in University of Kentucky (1R1) cigarettes were employed cigarette smoke and its combustion and pyrolysis prod­ in this study. The filler was removed from the paper ucts in mainstream smoke. tubes by hand and sprayed by means of a technique previously described (6). The level of application for • Presented, in part, at the 31st Tobacco Chemists' Research Conference at Greensboro, North Carolina, U.S.A., in October 1977. + Methoprene is the active ingredient in KABAT• and in Altosid•; both Received: 27th November 1981 - accepted: 23rd June 1982. are trademarks of Zoecon Corporation, Palo Alto, California.

203 the ethanolic solution of (5-14C)-methoprene was 1 ml/ g Mainstream 14C-Smoke Analyses of filler. After evaporation of the solvent and equi­ a) Gas Phase: Evaluation of the mainstream gas phase libration at 600fo relative humidity and 24 °C, the was conducted using a smoking machine similar to that labeled filler was reinserted in the 1R1 paper tubes described by Newman et al. (8) and a trapping arrange­ using a Laredo~ (Brown & Williamson Tobacco Corp.) ment as shown in Figure 3. This trapping system was cigarette maker. Two sets of (5-14C)-methoprene ciga­ similar in design and operation to that reported by Lau­ rettes were prepared in this fashion. One set had a rene et al. (9). The trap consisted of 0.635 cm X 1.22 m 30 mm butt section which contained unlabeled 1R1 filler coiled copper tube immersed in liquid nitrogen. The col­ and a forward 55 mm section prepared from (5-14C)­ lection of gas phase was accomplished by smoking one methoprene sprayed filler; the second set contained the cigarette to a 23 mm butt length through a 4.5 cm di­ 14C-labeled filler uniformly distributed throughout the ameter Cambridge pad. After the cigarette butt was entire length of the 1R1 tobacco rod. All the cigarettes removed from the Cambridge pad holder. one clearing containing sprayed filler were refrigerated until required puff was taken through the pad. The shunt valve (Fig­ for smoking. The amount of ( 5-14C)-methoprene present ure 3} was closed and both valves 1 and 2 were simul­ in the equilibrated cigarettes was determined by ex­ taneously rotated 180° to direct the helium flow through tracting the filler in 25 ml of 95 Ofo ethanol and count·· the cold trap. The trap was purged with helium for 10 ing an aliquot of the extract. minutes in order to remove air. At this time the liquid nitrogen was removed and the trap heated for 5 minutes with a heat gun. At the end of the heating period the Total Smoke Distribution Studies gas chromatographic program was initiated. All ga~ phase analyses were obtained using a Fisher Victoreen The distribution of the (5-14C)-methoprene and its prod­ 4800 gas chromatograph with a 0.32 cm outside di­ ucts in cigarette smoke were determined using a total ameter X 7.2 m stainless steel Porapak~ Q 80/100 mesh recovery smoking machine (7) as illustrated in Figure 2. column. Helium flow was 30 cm8 / min, and the program The total particulate matter (TPM) was determined by rate was 20°C/min from -150°C to 0°C and 5°C/ weighing the Cambridge filter pad before and after min from 0 °C to 230 °C. The column effluent was split smoking. After the final weighing, the pad was imme­ 9:1, with nine parts to the flow proportional counter for diately extracted in 25 ml of 95 Ofo ethanol. The radio­ 14C detection, and one part to the flame ionization de­ activity in the 23 mm unconsumed tobacco butt was tector. The balance of the analytical procedure and also determined by extraction into 25 ml of 95 Ofo description of the equipment have been previously re­ ethanol for 24 hours. All extracts were subsequently ported (10). radioassayed by liquid scintillation counting. The sidestream and mainstream gas phases were con­ b) Semi-Volatiles: The analysis of the semi-volatile portion (11} of mainstream smoke was accomplished verted to carbon dioxide (C02} and absorbed directly into Hyamine 10-X~ (Rohm and Haas) previously using an injection port machined from stainless steel and cooled to 0°C. Aliquots of the Hyamine 10-X~ absorbent capable of accepting a 12 mm outside diameter X 5 cm solution and of the extracts from the Cambridge pads quartz sleeve (Figure 4). For any given analysis the in­ were taken for liquid scintillation counting. jection port was pre-heated to 200 °C, and the gas radio­ chromatograph was always pre-cooled to -50 °C. A portion of the Cambridge pad containing particulate

Figure 2. Total recovery smoking machine. Figure 3. Gas phase collection system.

To GLC Solenoid} column 1

CuO (750° C) To balloon -4---6;~

Hyamlne Hyamine 10-X• 10-X•

204 Figure 4. Injection port assembly for seml-volatlles Liquid Scintillation Counting analysis. A Packard Tricarb liquid scintillation spectrometer (Model 3390) with optimized settings was used. Aliquots of all ethanol extracts were counted in a scintillator solution containing 4 g PPO [2,5-diphenyloxazole] and 50 mg POPOP [p-bis-2-(5-phenyloxazoyl)-benzene] per liter of toluene. All Hyamine 10-X181 aliquots were placed in lnsta-GeliBi (Packard) scintillator cocktail prior to counting. Counting times were vari.ed to establish good counting statistics.

RESULTS AND DISCUSSION To GLC column The cigarettes labeled with (5-14C)-methoprene were first extracted in ethanol to determine the activity level matter from one cigarette was placed into a quartz sleeve, per cigarette. The uniform distribution of the labeled the injection port opened, the sleeve with pad in place methoprene throughout the cigarette was also checked inserted and the port resealed immediately. The injection by the extraction techniques described earlier and found port was swept with helium for 20 minutes. At the end to fall within acceptable limits (6). . of this period the oven temperature of the chromato­ The cigarettes were smoked on a total smoke recovery graph was advanced to 0 °C. The oven equilibrated in machine, and the average distribution of activity in the about 2 minutes at which time programming was started various portions of smoke for six replicate runs is pre­ at 5 °C per min to a maximum temperature of 240 °C sented in Table 1. The percent distributions are calculated (OV-101 column). The distribution of activity from the from the amount of tobacco consumed during smoking. Cambridge pad was determined using two different The mainstream contributions from methoprene are columns: similar to those of added menthol which has been re­ ported by ]enkins et al. (12). The amount of 14C-activity 1) Carbowax181 20M-TPA, 50fo on Chromosorb G-HP for uniformly labeled (U) menthol in the mainstream 80/100 mesh, 3.2 mm outside diameter X 4 m with 30 cm3 I gas phase was 1.4 °/o compared to 1.30/o for methoprene, min helium flow (maximum temperature of column: while the activity in the mainstream particulate phase for 220°C). menthol was 39.00fo compared to 38.2°/o for metho­ 2) OV-101, 30fo on Chromosorb G-HP 80/100 mesh, prene. It should also be pointed out that the menthol 3.2 mm outside diameter X 3 m with 30 cm3 I min delivery to the particulate phase was due in part to a helium flow. 2.3 Ofo depletion of menthol in the butt. The converse has been found with methoprene, where 8.1 °/o of the activity Counting efficiency for the flow proportional counting consumed is filtered out by the unburned tobacco column system was determined using 14G-toluene (New England (23 mm butt). This value was attained by measuring the Nuclear) after each gas radiochromatographic run. Main­ total activity in the butt after smoking and subtracting stream gas phase and semi-volatile compound identifi­ the amount of radioactivity present before smoking. In cations w.ere obtained with a Finnigan GC/MS (Model order to confirm this 8.1 °/o value, cigarettes were pre­ 3300) in an electron impact mode using previously in­ pared which contained unlabeled or "cold" filler in the dicated gas chromatographic parameters. last 30 mm of the tobacco rod. Five additional smoking

Table 1. Distribution of radioactivity (ftCI) In smoke from clgareHes labeled with (5-UC)-metho- prene (based on tobacco consumed). Cigarette No. Percent Smoke phase Average distribution 1 I 2 I 3 I 4 I 5 I 6 Mainstream gas 0.04 0.04 0.04 0.04 0.04 0.04 0.04 1.3 Mainstream total particulate matter 1.11 1.26 1.12 1.17 1.24 1.18 1.18 38.2 Sidestream gas 0.51 0.57 0.50 0.51 0.65 0.55 0.55 17.8 Sidestream total partlculate matter 1.07 1.06 1.08 1.06 1.10 1.07 1.07 34.6 Butt* 0.45 0.26 0.18 0.25 0.10 0.25 0.25 8.1

Total recovery** 3.18 3.19 2.92 3.03 3.13 3.09 3.09 100

• Value obtained by subtracting calculated activity present on 23 mm butt length prior to smoking. •• Calculated activity recovery based on tobacco consumed during smoking = 3.09 ~tCI/ cigarette.

205 Table 2. Identification of the radioactive components In Table 3. Mainstream smoke gas phase comparison. mainstream smoke gn phase from '(S-14C)·methoprene treated clgareHes. Labeled additive Component %of total (5-14C)-methoprene 14C-tobacco Peak Identification activity In mainstream Carbon monoxide 23.5% 23.0% gas phase* Carbon dioxide 62.3% 62.1% Other gases 14.2% 14.9% Carbon monoxide 23.5 ± 2.0 2 Methane 1.3 ± 0.4 Fraction of total activity 3 Carbon dioxide 62.3 ± 1.5 consumed during smoking 1.3% 13.7% 4 Ethylene 0.9 ± 0.3 5 Ethane 0.7 ± 0.2 6 Propene 0.2 ± 0.1 Table 4. Percent distribution of radioactive components 7 1,3-Butadiene (plus some C =C-C= C In the semi-volatile portion of smoke from (5·14C)-metho­ and C ::C-C :: C) 0.9 ± 0.3 prene treated clgareHea. 8 n-Butane (plus some 1· or 2-butene) 0.9 ± 0.3 %of 0/G. of 9 Isoprene (mainly) 2.0 ± 0.4 Compound mainstream sidestream 10 1,3-Pentadiene (possibly some C6H10 particulate particulate and C6H12) 0.8 ± 0.2 11 Benzene 3.9 ± 0.5 Unknown "A" 1.1 ± 0.4 14.2 ± 2.0 12 Toluene 2.6 ± 0.4 (2Z,4E) *-methoprene 1.8 ± 0.5 4.0 ± 1.0 (2E,4E)-methoprene 96.8 ± 2.0 81.5 ± 2.0 • These figures represent 1.3°/o of total activity consumed during Residual unknown "B" smoking. 0.3 ± 0.1 0.3 ± 0.1

• 0.4 Dfa of the added (5-ICC)·methoprene was Initially present as (2Z,4E)·methoprene. runs using these cigarettes produced excellent agreement with the data in Table 1. The butt filtration in this instance was 8.60/o. Even with the careful attention that The particulate contribution to mainstream and side­ was given to insure the proper intertwining of tlie stream smoke was also evaluated by gas radiochromato­ tobacco shreds between the labeled and unlabeled filler, graphy. These data are shown in Table 4. A 30/o OV-101 the possibility always exists that an abnormal interface column was used to generate the data. A comparative or junction resulted. Therefore, the value of 8.1 Ofo is run was conducted using a 5 °/o Carbowax® 20M-TPA thought to better reflect the true value for butt filtra­ column which gave a similar value (96.2°/o} for the un­ tion. This relatively high value of 8.f0fo is comparable changed methoprene. These data demonstrate that metho­ to the 8.3 Ofo reported for anthracene by Thornton (13), prene transfers essentially intact to the mainstream par­ and 8.5 Ofo reported by Jenkins (12} for 1-proline. Both ticulate phase. In the sidestream particulate phase, the of these materials were shown to undergo sublimation degree of pyrolytic breakdown is enhanced, but the and subsequent vapor phase condensation when exposed methoprene still to a large degree undergoes distillation. to the lower temperatures in the butt. This supports the Unknown "A", a compound eluting ahead of metho­ premise that methoprene exists to a large degree in the prene, was not identified. Unknown ''B" is the residual volatile portion of mainstream particulate smoke. activity remaining on the TPM pad after sweeping with The chemical composition of the radioactivity in the helium. No chemical analyses were performed on the mainstream smoke was studied by gas radiochromato­ residuals. graphy. The mainstream smoke gas phase components A small percentage of the (2£,4£)-methoprene under­ arising from the added labeled methoprene (1.3 Ofo of went isomerization to the (2Z,4E) form during the activity consumed during smoking) are detailed in distillation process. A small amount of the (2Z,4E) isomer Table 2. It is apparent that almost 86 Ofo of the labeled (0.40/o) was initially present in the (5-14C)-methoprene. gas phase is composed of CO and C02• The balance of gas phase constituents containing 14C-activity indicate no single component greater than 4 Ofo of the total gas phase. SUMMARY Listed in Table 3 is a comparison of the mainstream gas phases from (5-14C)-methoprene and 14C-bright tobacco An insect growth regulator, (5-14C)-methoprene [5-14C­ (U). Although much less total mass is contributed by the isopropyl (2£,4£)-11-methoxy-3,7,11-trimethyl-2,4-do­ methoprene (1.3 Ofo of the 12.4 tJ.g methoprene consumed decadienoate], has been appVed to tobacco filler from vs. 13.70/o of the 730 mg of tobacco consumed), the which cigarettes were made. The Kentucky 1R1 reference COJCO ratios are identical, indicating no selective cigarette was employed in this study to determine the 14 generation of either C02 or CO by the added metho­ distribution of the (5- C)-methoprene and its com­ prene. bustion and pyrolytic products in cigarette smoke.

206 The distribution of smoke activity as determined using et que 8,1 °/o est retenue par filtrage clans le megot a total smoke recovery device showed 38.2 percent of (23 mm). the 14C-activity to be in mainstream smoke with 52.4 L'analyse de la phase particulaire et de la phase gazeuse percent in sidestream and 8.1 percent filtered by the du courant principal par radiochromatographie en phase tobacco butt (23 mm). gazeuse montre que la plus grande partie de l'activitC Analyses of mainstream particulate matter and gas phase trouvCe dans le courant principal est du (5-14C)-mthho­ by gas radiochromatography indicated the major portion prene indtange (96,8 OJo). Des analyses plus limitks de la of the activity found in mainstream smoke to be un­ phase particulaire du courant secondaire ont ete aussi c:hanged (5-UC)-methoprene (96.8 percent). Limited conduites. Une analyse de la phase gazeuse du courant sidestream particulate matter analyses were also con­ principal (1,3 GJo de l'activit~ totale), montre que CO et ducted. Analysis of the mainstream gas phase (1.3 per­ COr interviennent pour 86 OJo de l'activitC de cette phase. cent of total activity) for carbon-H disuibution showed Les 14°/o restants sont repartis sur dix autres constituants that carbon monoxide and carbon dioxide accounted for de la phase gazeuse. 86 percent of the gas phase activity. The remaining 14 percent was distributed among ten additional gas phase components. REFERENCES

1. Strong, R. B., and J. Diekman: Comparative ef­ fectiveness of fifteen insect growth regulators against ZUSAMMENFASSUNG several pests of stored products; J. Economic En­ tomology 66 (1973) 1167-1173. Der Wirkstoff (5-uC)-Methopren (5-14C-Isopropyl 2. Manzelli, M. A.: Personal communication, Decem­ (2E,4E)-11-methoxy-3,7,11-trimethyl-2,4-dodecadienoat] ber 1975. zur Regulierung des Wac:hstums von Insekten wurde auf 3. Henrick, C. A., G. B. Staal and J. B. Siddall: Alkyl den Cigarettenfiilltabak der Versuc:hscigarette 1R1 der 3,7,11-trimethyl-2,4-dodecadienoates, A new class Universitiit von Kentucky gespriiht, in deren Rauc:h die of potent insect growth regulators with juvenile Verteilung von (5-14C)-Methopren und dessen Verbren.­ hormone activity; J. Agr. Food Chem. 21 (1973) nungs- und Pyrolyseprodukten untersuc:ht wurde. 354-359. Unter Benutzung einer Wiederauffindungsvorric:htung fiir 4. Long, J. S., R. M. Lehman, M. H. Tickle and M. A. den Gesamtraudt wurden 38,2 O/o der uc-Aktivit1it im Manzelli: The efficacy of KABAT* for control of Hauptstromraud:a, 52,4 °/o im Nebenstromraudt und the cigarette beetle under severe infestation condi­ 8,1 °/o im Tabakstummel (23 mm) gefunden. tions; Tobacco Science 24 (1980) 119-121. Die Untersudtung der Partikelphase und der Gasphase 5. Schooley, D. A., B. J. Bergot, L. L. Durham and des Hauptstromrauc:hes durch Gasradiochromatographie ]. B. Siddall: Environmental degradation of the ergab, daB der grOBte Teil der im Hauptstromrauch auf­ insect growth regulator methoprene (isopropyl 14 14 gefundenen C-Aktivitli.t unverli.ndertes (5- C)-Metho­ (2£,4£)-11-methoxy-3,7, 11-trimethyl- 2,4-dodecadi­ pren ist (96,8 °/o). In begrenztem Malle wurde auch die enoate), 11. Metabolism by aquatic micro-organisms; Partikelphase des Nebensuomraudtes untersucht. Die J. Agr. Food Chem. 23 (1975) 293-298. uc-Aktivitli.t der Gasphase des Hauptstromrauches 6. Jenkins, R. W., Jr., R. T. Bass, G. C. Newel!, Jr., (1,3 °/o der Gesamtaktivit1it) ri.ihrt zu 86 GJo von Kohlen­ T. Segura and R. H. Newman: Recommendations monoxid und Kohlendioxid her. Die restlichen 14 O/o ver­ for the standardized preparation of carbon-14label­ teilen sidt auf zehn andere Gasphasenbestandteile. ed cigarettes; Tobacco Science 19 (1975) 123-126. 7. Jenkins, R. W., Jr., M. K. Chavis, R. H. Newman and F. A. Morrell: The Quantitative recovery of smoke from radioactively labeled cigarettes; Int. J. R£SUM£ Appl. Radiat. lsot. 22 (1971) 691-697. 8. Newman, R. H., W. L. }ones and R. W. Jenkins, Un regulateur de croissance des insects, le (5- 14 C)-m~tho­ Jr.: Automatic device for the evaluation of total prene [ 5-14C-isopropyl (2E,4E)-11-mCthoxy-3,7,11-tri­ mainstream cigarette smoke; Anal. Chem. 41 (1969) methyl.:.2,4-dodkadienoate J a Cte applique au tabac a 543-545. partir duquel on a fabrique des cigarettes. La cigarette 9. Laurene, A. H., L. A. Lyerly and G. W. Young: de reference 1R1 (UniversitC de Kentucky) a etC utilisCe Direct vapor chromatographic determination of dans cette etude pour dCterminer la rCpartition du acetaldehyde, acrolein, and acetone in cigarette (5-14C)-methoprene et de ses produits de combustion et smoke; Tobacco Science 8 (1964) 150-153. de pyrolyse dans la fumee de cigarette. 10. Jenkins, R. W., Jr., R. H. Newman, R. D. Carpen­ La distribution de l'activit6 de la fumCe, dMinie au ter and T. S. Osdene: Cigarette smoke formation moyen d'un appareillage collectant la totalite de la fu­ studies, I. Distribution and mainstream products mCe, montre que 38,2 OJo de l'activit6 due :\ 14C est dans from added 14C-dotriacontane-16,17; Beitr. Tabak­ le courant principal, 52,4 °/t dans le courant secondaire forsch. 5 (1970) 295-298.

207 11. Levins, R. J., and R. M. Ikeda: The separation of Acknowledgements volatiles from solids in the injection port of a gas The application of (J-14C)-methoprene to tobacco fillet duomatograph; J. Gas Chromatogr. 6 (1968) 331- and cigarette fabrication by Mr. R. Bass and Mr. G. 335. Newell and GC/MS assistance of Dr. R. Kornfeld are 12. Jenkins, R. W., Jr., R. A. Comes and R. T. Ba~: The use of carbon-14 labelled compounds in smoke gratefully acknowledged. prcx:ursor studies - A review; Recent Advances in Tobacco Science 1 (1975) 1-30 (29th Tobacco Chemists' Researdt Conference, Beltsville - College Authors' address: Park, Maryland, U.S.A., 1975. 13. Thornton, R. E., and C. Valentine: Determination Philip Morris Research Center, of the balance of carbon-14 activity added to ciga­ P.O. Box 26583, rettes; Beitr. Tabakforsch. 4 (1968) 287-2'11. Richmond, Virginia, 23261, U.S.A.

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