Effect of Cigarette Menthol Content on Mainstream ... - ACS Publications

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Effect of Cigarette Menthol Content on Mainstream Smoke Emissions S. M. Gordon,*,† M. C. Brinkman,† R. Q. Meng,‡ G. M. Anderson,‡ J. C. Chuang,†,|| R. R. Kroeger,† I. L. Reyes,† and P. I. Clark§ †

Tobacco Exposure Research Laboratory, Battelle Memorial Institute, 505 King Avenue, Columbus, Ohio 43201, United States Battelle Toxicology, Richland, Washington, United States § University of Maryland College Park, Maryland, United States ‡

ABSTRACT: The 2009 Family Smoking Prevention and Tobacco Control Act empowered the U.S. Food and Drug Administration to study “the impact of the use of menthol in cigarettes on the public health, including such use among children, African Americans, Hispanics and other racial and ethnic minorities,” and develop recommendations. Current scientific evidence comparing human exposures between menthol and nonmenthol smokers shows mixed results. This is largely because of the many differences between commercial menthol and nonmenthol cigarettes other than their menthol content. We conducted an innovative study using two types of test cigarettes: a commercial nonmenthol brand that we mentholated at four different levels, and Camel Crush, a commercial cigarette containing a small capsule in the filter that releases menthol solution into the filter when crushed. Cigarettes were machine-smoked at each of the menthol levels investigated, and the total particulate matter (TPM) was collected on a quartz fiber filter pad and analyzed by gas chromatography/mass spectrometry for menthol, nicotine, tobacco-specific nitrosamines (TSNAs), polycyclic aromatic hydrocarbons (PAHs), cotinine, and quinoline. The mainstream smoke was also monitored continuously in real time on a puff-by-puff basis for seven gas-phase constituents (acetaldehyde, acetonitrile, acrylonitrile, benzene, 1,3-butadiene, isoprene, and 2,5-dimethylfuran), using a proton transfer reactionmass spectrometer. Average yields (in micrograms/cigarette) for the analytes were determined. Menthol in the TPM samples increased linearly with applied menthol concentration, but the amounts of nicotine along with the target TSNAs, PAHs, cotinine, and quinoline in the cigarettes remained essentially unchanged. Similarly, yields of the targeted volatile organic compounds (VOCs) in whole smoke from the mentholated nonmenthol cigarettes that were measured in real-time were largely unaffected by their menthol levels. In the Camel Crush cigarettes, however, the VOC yields appeared to increase in the presence of menthol, especially in the gas phase. Although we succeeded in characterizing key mainstream smoke constituents in cigarettes that differ only in menthol content, further study is needed to definitively answer whether menthol affects exposure to selected cigarette constituents and thereby influences harm.

’ INTRODUCTION Tobacco manufacturers use approximately 600 substances as cigarette ingredients,1 purportedly to enhance tobacco smoke character.2 Menthol is the only additive that is actively marketed by manufacturers3 and about which consumers make conscious buying choices. It is an ingredient in almost all cigarettes, but those cigarettes with characterizing levels of menthol are particularly favored by youth and ethnic/racial minorities.4 There is growing evidence that menthol cigarettes are starter products for youth,5
8 impede cessation,5
7 increase relapse following cessation,7,8 and undermine social justice by the incessant targeted marketing of these products to communities of color.4,9 Menthol is the only cigarette flavoring still permitted under the recently enacted Family Smoking Prevention & Tobacco Control Act (FSPTCA; H.R. 1256, 2009), which gives the U.S. Food and Drug Administration (FDA) jurisdiction over the tobacco industry, but the law calls for research on the public health effects of its continued use in cigarettes. Tying menthol cigarette use to increased risk of tobacco-related diseases has r 2011 American Chemical Society

been difficult. Generally, epidemiologic studies are used to make that connection,7,10
17 but these tools may simply be too blunt to detect a difference in harm due to menthol in the cigarette in the presence of the overwhelming harm associated with smoking any tobacco product. Laboratory-based studies, however, have had mixed results. For instance, when comparing biomarkers of exposure (e.g., cotinine and carbon monoxide) between menthol and nonmenthol cigarette smokers, some studies showed decreased levels, some increased, and some no difference.18
25 These studies have also been hampered by smokers’ brand loyalties and the reluctance of established menthol or nonmenthol smokers to use the opposite cigarette style for the extended periods necessary to compare classic measures of toxicity.4 The answer may lie with the commercial cigarettes that were tested. There are likely many differences between menthol and nonmenthol cigarettes other than menthol levels. Our previous Received: July 13, 2011 Published: September 02, 2011 1744

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Chemical Research in Toxicology work has resulted in some intriguing contrasts in menthol and nonmenthol cigarettes. In the first study, which involved exposure of nonsmokers to sidestream smoke, we measured a greater number of small particles (0.3
0.5 μm) in the sidestream smoke from the menthol vs the nonmenthol test cigarettes.26,27 The second study, in which particles from mainstream smoke were monitored using a more refined particle counting technique, revealed that the test menthol cigarette produced significantly more ultrafine particles than the nonmenthol cigarette.28 The larger size particles that occur in the particulate/aerosol phase typically deposit in the upper part of the respiratory tract due to impaction, interception, gravitational sedimentation, and turbulent dispersion.29 Ultrafine particles, however, have high diffusivities and thus have a high probability of depositing in deeper regions of the respiratory tract and alveoli, so their clearance time may be much longer than for those particles in the upper respiratory tract.30
32 Therefore, the ultrafine particles are important from a health standpoint since many of the carcinogenic constituents attached to them in cigarette smoke are absorbed across the alveolar capillary membrane, and hence, their potential to contribute to health effects is greater. Although the cigarettes selected for our two previous studies had similar machinesmoked tar and nicotine yields, they were not identical; therefore, it is not known if the differences observed were the result of the effects of mentholation alone or the many other possible differences in the properties of commercial cigarettes. To adequately study the effects of mentholation of cigarettes on smoking behavior and smoke exposure biomarkers, we need to measure key mainstream smoke constituents in test cigarettes that differ only in menthol content. Of equal importance is the need to characterize cigarette smoke from its initial formation and as it ages since its composition and toxicity may change markedly with time because of the highly dynamic and reactive nature of the smoke.33 Consequently, smoke should be analyzed within a few seconds after formation instead of after it has aged for several minutes.34
36 Most previous studies of tobacco smoke constituents have relied on off-line, batch analysis techniques and have typically focused on measuring total cigarette smoke yields.37
42 As a result, shortterm changes in concentration that may occur during the smoking process go undetected. Several systems have been reported recently that allow real-time puff-resolved characterization of selected volatile organic compounds (VOCs) in filtered and unfiltered mainstream cigarette smoke.34
36,43 Our approach, which complements the systems used in previous studies, is based on the use of commercially available equipment and a flow-through system that minimizes sample carry-over and puffto-puff contamination. Cigarette smoke is a complex mixture containing numerous volatile organic compounds (VOCs) in the vapor phase and several specific classes of semivolatile organic compounds (SVOCs) attached to solid/liquid droplets that constitute the particulate/aerosol phase.39,44 Many compounds are partitioned between the two phases.33,45 Although the vapor phase accounts for 90
96% by weight of the mainstream smoke of a cigarette,39,46 the cancer slope factors47 of several smoke-related VOCs are generally much lower than those of the particle-bound, carcinogenic tobacco-specific nitrosamines (TSNAs) and polycyclic aromatic hydrocarbons (PAHs).48,49 Chronic exposure to these compounds contributes significantly to the health risks inherent in smoking.50 However, because of the much higher concentrations of the VOCs in mainstream smoke, compounds such as

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acetaldehyde, 1,3-butadiene, and acrylonitrile are associated with the highest calculated cancer risks of conventional cigarettes,49 and it has been suggested that this fraction is the most hazardous in mainstream smoke.48 Consequently, an assessment of exposure to toxins in cigarette smoke must address both vapor phase as well as particulate/aerosol phase constituents. The research reported in this article is, to the best of our knowledge, the first to describe the preparation of cigarettes that are equivalent except for variation in levels of menthol and the application of real-time techniques to characterize their emissions from machine-generated mainstream smoke. We selected two sets of cigarettes for this purpose: the first was a commercial nonmenthol cigarette, selected for its chemical and physical similarity to popular menthol brands, that we mentholated at low, medium, and high levels, thus providing four levels of menthol in the same cigarette. The second was a commercial cigarette that contains a small menthol capsule within the filter (Camel Crush). By squeezing the filter before or while smoking the cigarette, the smoker crushes the capsule and releases a menthol solution into the filter, essentially transforming a nonmenthol cigarette into a menthol cigarette. Camel Crush thus provides a readily available means of changing the menthol content of a cigarette while holding all other conditions constant. It also makes it easier for other research teams to replicate our work if they want to examine the effects of menthol in a cigarette in which the only difference is the menthol content. Camel Crush differs from conventional menthol cigarettes in that the menthol is confined to a small portion of the filter, instead of being distributed between the tobacco, filter, and paper. It therefore allows us to contrast the effects on mainstream smoke emissions of two different ways of releasing and delivering menthol to the user. It is not known whether this difference influences human smoking behavior and exposure. This article focuses on the innovative measurement of selected particle-bound chemicals in the total particulate matter (TPM) samples of machine-generated mainstream smoke from the cigarettes and on the yields of VOCs in the (unfiltered) whole smoke and in the (filtered) gas phase. Our real-time measurement technique is currently limited to the VOCs in the gas phase and the smoke; it does not extend to the particle-bound constituents.

’ EXPERIMENTAL PROCEDURES Cigarettes. For the commercial nonmenthol cigarette that we mentholated, we selected Camel “full flavor” King Filters (Camel King, 85 mm) because they closely matched the physical characteristics as well as the Federal Trade Commission (FTC) tar (17 mg/cigarette) and nicotine (1.3 mg/cigarette) levels of the most popular commercial menthol brands. To guard against regional variability, the cigarettes were acquired in same-lot, single-date purchases. All of the Camel King cigarettes were purchased from local retail dealers in 2010 in Richland, WA; the Camel Crush cigarettes were purchased in 2010 in Columbus, OH. For this study, four menthol-level cigarettes (prepared in-house) and two levels of Camel Crush cigarettes were analyzed for constituents in mainstream smoke generated via a smoking machine. Details of the preparation procedures and analysis techniques used are provided below. Mentholation of Nonmenthol Cigarettes. Celebucki et al.51 found that the menthol content of 48 popular brands of mentholated cigarettes commercially available in the US has a mean value of 3.89 ( 1.14 (SD) mg menthol/g tobacco. Previously, Giovino et al.52 reported that the menthol content of mentholated cigarettes ranged 1745

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Chemical Research in Toxicology from 0.1%
1.0% (expressed as percentage of dry tobacco weight). For this investigation, our goal was, therefore, to mentholate the selected nonmenthol Camel cigarettes to cover a range from 0% (i.e., untreated) to approximately 1.5%. Various methods are used to prepare menthol cigarettes.53
55 Common methods are the injection of a solution of menthol into the filter or tobacco paper or by spraying an alcohol-based menthol solution directly onto the shredded tobacco before the cigarettes are rolled and packed. Menthol cigarettes can also be prepared by direct deposition of vaporized flavorant directly onto the tobacco or by adding menthol to the paper side of the inner foil on the pack. Regardless of the mentholation procedure used, the result of all such applications is similar: during storage, the menthol steadily migrates through the entire cigarette from the point of application until equilibrium is reached between the menthol in the filter and the menthol in the tobacco rod.53,56,57 In our mentholation approach, the cigarettes were conditioned at 60 ( 3% relative humidity (RH) for 48
72 h, following which mentholation was accomplished by direct vapor deposition. L-Menthol crystals (Sigma, g99%) were ground to a powder in ∼100-g aliquots to increase the surface area and vapor output, then placed in mentholation chambers. Each mentholation chamber consisted of a 10 cm-high stainless steel insert pan in a Ziploc polyethylene bag that was evenly loaded with 500 g ( 10 g of the pulverized L-menthol. A single layer of 100 of the conditioned cigarettes (2 side-by-side rows of 50 cigarettes each) was placed in a wire rack in a mentholation chamber, which was maintained at 24 ( 1 °C and 45 ( 3% RH. The start of mentholation was temporally staggered to allow each chamber to reach the desired target concentration at about the same time. Cigarettes were mentholated at three levels. Additional cigarettes were maintained under the same conditions in a mentholation chamber to which no menthol was added, and these cigarettes served as reference cigarettes (0% mentholation). Each batch of mentholated cigarettes was stored in Ziploc bags at room temperature. Soon after preparation, three cigarettes were randomly drawn from the cigarettes at each mentholation level, to determine the extent of mentholation. After first performing a solvent extraction into isopropanol, the extracts were analyzed by gas chromatography with flame ionization detection (GC-FID), using anethole (Fluka, g99.5%) as the internal standard. The analytical system was calibrated with five serially diluted menthol standards that covered the range from 4 to 2,400 μg/mL. The calibration solutions were prepared in isopropanol and checked with independently prepared test standards at concentrations of 24 and 1,200 μg/mL. The calibration curve was linear (r2 > 0.9999), method specificity was high, with no coeluting peaks observed for either menthol or the internal standard, and the detection limit for the analysis was 0.5 μg/mL. Menthol in the tobacco and filter of the mentholated nonmenthol (MEN) cigarettes was extracted and measured separately. Smoking Machine and PTR-MS. A linear five-port smoking machine (Hawktech FP2000; Tri-City Machine Works, USA) was used to generate mainstream smoke samples from the test cigarettes. The instrument is equipped with five 140-mL glass syringe pumps, which mimic the human diaphragm during inhalation, thus avoiding the pulsation that is commonly associated with smoking machines that use peristaltic or diaphragm pumps. Smoke from each test cigarette was generated with the smoking machine using the ISO/FTC protocol (35 mL puff volume, 2 s puff duration, and one puff every 60 s). For the real-time, puff-by-puff analysis of VOCs in mainstream smoke, we used a compact proton transfer reaction-mass spectrometer (PTR-MS; Ionicon, Innsbruck, Austria). A detailed description of its operating principle and performance can be found elsewhere.58
60 Pure water vapor within the ion source of the instrument, a hollow cathode discharge, results in the production of high concentrations of H3O+ primary reactant ions. These primary ions pass into the drift tube, where they undergo mostly nondissociative proton transfer to the VOCs.

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Figure 1. Schematic of the PTR-MS interfaced with the smoking machine. Organic trace compounds such as carbonyls, alcohols, aldehydes, nonpolar aromatics, and others can be monitored continuously in real time with a detection limit of about 1 part-per-billion by volume (ppbv). The sample stream in the PTR-MS does not require any preconcentration procedures, so no special sample preparation prior to the measurement is needed. Samples are introduced directly into the reaction chamber (drift tube), and analysis occurs rapidly. A schematic of the PTR-MS interfaced with the FP2000 smoking machine is shown in Figure 1. A constant stream of heated pure air (40 °C, AADCO Pure Air Generator, Cleves, OH), controlled by means of a mass flow controller at 5 L/min, is oriented orthogonally to the direction of the outlet flow of mainstream smoke from the smoking machine. An intermittent flow of mainstream smoke, i.e., 35 mL once every minute for 7 min, combines with the heated dilution air in the stainless steel tubing (3/400 od). This flowing mixture is sampled at 5 mL/min using a stainless steel probe (1/1600 od). To reduce the total concentration of the sample to a level that is compatible with the requirements of the PTR-MS, the probe outlet is connected to a T-piece, to which pure air is introduced (at 70 mL/min) that dilutes the sample by a factor of fifteen. The diluted mainstream smoke passes through a heated transfer line (150 °C) to prevent condensation and minimize surface losses of trace-level organics. The sample flows into the reaction chamber of the PTR-MS at a rate of ∼70 mL/min. Smoking Protocols. TPM Masses and Particle-Bound Chemicals in Mainstream Smoke. Using the smoking machine, we sequentially smoked four cigarettes (one cigarette per filter pad extracted) of each cigarette type at each menthol level according to the ISO/FTC regimen, seven puffs/cigarette. The TPM from each smoking sequence was collected on a standard 44-mm quartz fiber filter pad. After seven puffs, the cigarette was removed, and the filter was recovered for gravimetric and chemical analysis. To estimate particle mass, each filter was weighed in duplicate both before and after sampling using a microbalance (Model AT20, MettlerToledo, Columbus, OH; reading precision, 2 μg; reproducibility, 4 μg). Filters were weighed immediately after recovery in a temperature- and humidity-controlled room. After sampling and weighing, each filter was immediately transferred to a glass screw-cap jar and stored at
20 °C to reduce potential losses due to volatilization prior to chemical analysis. The difference between the final average mass of the sample filter and the initial average mass of the blank filter was used as the TPM mass. 1746

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Chemical Research in Toxicology Chemical analysis of the collected TPM samples was based on procedures developed previously in our laboratory for similar matrices and chemical classes.61,62 The samples were extracted with 50% dichloromethane in ethyl acetate, and the resulting sample extracts were divided into two aliquots. Aliquot 1 was solvent exchanged into 100 mM ammonium acetate and analyzed by liquid chromatography
tandem mass spectrometry (LC-MS/MS) for the TSNAs, N0 -nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Aliquot 2 was diluted or subjected to additional cleanup, as needed, using solid phase extraction, then analyzed by automated gas chromatography/mass spectrometry (GC/MS) for the remaining smokerelated analytes, nicotine, menthol, cotinine, quinoline, and the PAHs, pyrene and benzo[a]pyrene (BaP).61,63 VOCs in Mainstream Smoke. To measure gas phase VOCs generated with the smoking machine on a puff-by-puff basis from each test cigarette, a 44-mm Cambridge-type quartz fiber filter in its holder was placed directly behind the cigarette holder to separate the particulate phase of the smoke aerosol from the (filtered) gas phase.64 We investigated the effect of the particulate on the VOCs from the Camel Crush cigarettes by removing the filter and repeating the measurements on the (unfiltered) whole smoke. The PTR-MS was adjusted to simultaneously monitor the following target ion masses that we associate with the analytes shown in parentheses: m/z 45 (acetaldehyde), m/z 42 (acetonitrile), m/z 54 (acrylonitrile), m/z 79 (benzene), m/z 55 (1,3-butadiene), m/z 69 (isoprene), and m/z 97 (2,5-dimethylfuran). Using the instrument’s data acquisition software, data from the cigarettes were recorded continuously every 1
2 s. Five of the VOCs (acetaldehyde, acrylonitrile, benzene, 1,3-butadiene, and isoprene)50 are known carcinogens and respiratory, cardiovascular, and/or reproductive or developmental toxicants,65 while two (2,5-dimethylfuran and acetonitrile) are recognized markers of tobacco smoke exposure.64,66
68 Absolute concentrations were calculated directly without the need for independent calibration or the use of standards because most of the target VOCs have proton affinities much greater than that of H2O, and proton transfer thus occurs on every collision with well-established rate constants.60 Data Analysis. To determine menthol in the unburned tobacco and filter of the MEN cigarettes and the particle-bound target analytes in the TPM from the mainstream smoke of the smoked cigarettes, we used ChemStation software (Agilent Technologies) to quantify the GC/MS selected ion chromatograms. Peak areas were integrated, and the results were inspected and manually reintegrated if necessary. Menthol content of the MEN cigarettes was expressed as a percentage of dry tobacco weight; mean values and standard deviations for the compounds in the mainstream smoke were estimated at each of the menthol levels of interest. Identification of the target particle-bound SVOCs in the GC/MS data from the analysis of the TPM samples was based on their GC retention times relative to internal standards and the relative abundances of the monitored ion masses. Quantification of each target analyte was achieved by comparison of the integrated ion current response of the target ion to that of the internal standard using the average response factor of the target analyte generated from a standard calibration curve.61 For each cigarette type and at each mentholation level, we calculated the average yield and standard deviation for the analyte (in ng or μg per cigarette). All particle-bound SVOC yields were normalized to the mass of collected TPM in the mainstream smoke. To estimate the total yields of the VOCs per cigarette in the (filtered) gas phase and the (unfiltered) whole smoke, we first corrected the measured PTR-MS concentrations by replacing the default protontransfer reaction rate constant (k = 2.0  10
9 cm3 s
1) with the known measured or calculated value for the compound.60,69 We used the default value for acrylonitrile and 2,5-dimethylfuran, for which no rate constants

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Table 1. Menthol Levels in Unburned Mentholated Nonmenthol Cigarettes menthol in

menthol

total

mentholation

mentholation

tobacco

in filter

menthola

level

duration (h)

(μg/cig.)

(μg/cig.)

(%)

Cigarette Set 1 1b

0

0

0

0.00

2

22

1,170

440

0.16

3

61

1,950

920

0.32

4

240

8,600

2,600

1.55

Cigarette Set 2 b

1

0

0

0

0.00

2

21

460

289

0.10

3

62

854

675

0.20

4

240

7,285

2,984

1.40

Total menthol weight (in filter + tobacco) to tobacco weight. b Control cigarette (Camel “full flavor” King Filter), nonmenthol. a

were available. Then, we integrated and summed the areas under the peaks of the recorded puff-by-puff ion mass signals from the PTR-MS for each cigarette (seven puffs per cigarette) and multiplied the results by 15 to account for the dilution of the sample stream flowing into the PTR-MS (cf. Figure 1). For each cigarette type and at each mentholation level (i.e., four for the MEN cigarettes and two for the Camel Crush cigarettes), we averaged the mean summed peak areas and standard deviations from the cigarettes (usually four replicates at each mentholation level) for each analyte. Conversion of the summed PTR-MS peak areas (in ppbv 3 s) to total yield of each smoke constituent (in μg/cigarette) was based on the molecular weight of the chemical, an assumed temperature and pressure of 25 °C and 1 atm, respectively, and a sample flow rate through the stainless steel tubing of 5.039 L/min for the period during which mainstream smoke was being generated (374 s). Finally, we calculated the average yield and standard deviation for each analyte (μg/cigarette). We did not conduct any additional tests to determine whether any differences were statistically significant because of the relatively small number (e4) of cigarettes that were smoked and evaluated at each menthol level.

’ RESULTS AND DISCUSSION Menthol Content of Mentholated Cigarettes. Two separate sets of MEN cigarettes were prepared for this project. Menthol in the tobacco and filter of these cigarettes was extracted and measured; the results obtained are summarized in Table 1. In each case, approximately 70% of the menthol was found in the tobacco and about 30% in the cigarette filter. This distribution illustrates the consistency of the mentholation preparation method used and is in good agreement with results reported previously.57 Yields of Particle-Bound SVOCs from Mainstream Smoke. The average normalized analyte yields and their standard deviations for the target SVOCs in the two sets of MEN cigarettes prepared at four mentholation levels are presented in Figure 2. The results show generally good agreement between the two data sets, providing support for the technique used to mentholate the cigarettes and the procedures used to measure the constituent concentrations and yields. The measured levels of menthol in the TPM samples increase approximately linearly with applied 1747

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Figure 2. Effect of menthol at four mentholation levels on measured yields of particle-bound constituents in mainstream smoke from two sets of mentholated nonmenthol cigarettes. Asterisks indicate concentrations below the method detection limit.

Figure 3. Effect of menthol on measured yields of particle-bound constituents in mainstream smoke in Camel Crush cigarettes measured in the uncrushed (nonmenthol) and crushed (menthol) modes. Asterisks indicate concentrations below the method detection limit. The amount of menthol measured in the crushed mode (∼65 mg/g TPM) is estimated to correspond to about 0.6% mentholation.

menthol concentration, except at the highest levels. The menthol yield measured in set 1 is lower than that in set 2, which disagrees with the results in Table 1 and is puzzling. We speculate that this may be because the menthol levels measured in the TPM of the cigarettes in set 1 were carried out roughly three weeks after the cigarettes had been mentholated and were received in our laboratory, whereas those from set 2 were analyzed soon after preparation and receipt. The delay in analyzing the set 1 samples may have resulted in some loss of menthol from the unburned, mentholated cigarettes during storage. The results in Figure 2 suggest that the amounts of nicotine as well as the amounts of the target TSNAs, PAHs, cotinine, and quinoline are largely unaffected by changes in the menthol levels of the cigarettes. Figure 3 shows the results obtained for the target analytes in the TPM samples from the Camel Crush cigarettes in the uncrushed (nonmenthol) and crushed (menthol) modes. A comparison with the data in Figure 2 suggests that the amount of menthol released into the filter in the crushed mode (∼65 mg/g TPM) corresponds to about 0.6% menthol. The yields of nicotine and the remaining target SVOCs appear to be about the same as those in the MEN cigarette. Also, as in the previous case, there is no

Figure 4. PTR-MS time
concentration profiles of masses 55 (1, 3-butadiene), 79 (benzene), 54 (acrylonitrile), and 97 (2,5-dimethylfuran) recorded while machine-smoking a single nonmenthol (0% mentholation) test cigarette (traces are offset vertically for clarity).

apparent difference in the yields of the analytes in the Camel Crush between the menthol and nonmenthol modes. Puff-by-Puff Characterization and Total Yields of Select VOCs in Mainstream Smoke Gas Phase and Whole Smoke. To illustrate the response time and resolution of the PTR-MS, Figure 4 shows the puff-by-puff profiles for four of the analytes of interest, namely, 1,3-butadiene, benzene, acrylonitrile, and 2,5dimethylfuran, obtained while smoking a single nonmenthol (0% mentholation) test cigarette. To facilitate comparisons between cigarettes within a cigarette set, each smoking sequence was limited to seven puffs. The plots show that the peaks from each puff rise and fall together before returning to their original baseline levels between puffs; the individual traces are offset vertically to more clearly show the responses. This demonstrates that the puffs are not affected by contamination and that there is no carryover of material between puffs. As a result, we did not need to include cleaning puffs between successive puffs, as described by Adam et al.34 The plots also illustrate the wide dynamic range of the PTR-MS and its fine time resolution. Under the conditions that applied to these measurements, the instrument sampled the smoke stream once every 1
2 s. 1748

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Figure 5. Effect of menthol on measured yields of whole smoke (unfiltered) VOCs in mainstream smoke from machine-smoked mentholated nonmenthol cigarettes (cf. Table 1, set 2).

Several previous studies have noted that the concentrations of some of the VOCs in mainstream smoke increase with the number of puffs.70
72 We observed a similar trend in this work. The effect has been ascribed both to a decrease in filtration for compounds in the particulate phase of the smoke by the unburned (tobacco) portion of the cigarette, which decreases as the cigarette is burned, along with a buildup in the filter and an increased release of volatile species as the paper is consumed. VOC Yields. The total yield per cigarette of each VOC in whole smoke from the second set of MEN cigarettes prepared for this study (cf. Table 1) is summarized in Figure 5. For the cigarettes with characterizing menthol levels, i.e., 0.10%, 0.20%, and 1.40%, there is no clear dependence of VOC yield on menthol content, except in the case of the peak at mass 55. Here, the m/z 55 ion yield, which we associated with 1,3-butadiene, does not appear to change at the intermediate mentholation levels but increases by a factor of approximately 3.5 from 0% mentholation to 1.40% mentholation. This effect is influenced by the menthol fragment ion at m/z 55 as discussed below. The total yield per cigarette of the target VOCs in the gas phase and whole smoke from the Camel Crush cigarettes in the uncrushed (i.e., nonmentholated) and crushed (i.e., mentholated) modes is presented in Figure 6. Contrary to the effect observed with the MEN cigarettes in Figure 5, all of the VOC yields from the Camel Crush cigarettes increased in the presence of menthol, especially in the gas phase. As noted earlier, the m/z 55 ion yield, associated with 1,3-butadiene, increased strongly with mentholation in the whole smoke samples. Again, this behavior is affected by the menthol fragment ion at m/z 55 as discussed below. A comparison of the gas phase and whole smoke total yields for the target compounds shows that, for all of the analytes monitored, the levels in whole smoke are quite similar to those in the gas phase. In an earlier study, Adam et al.34 reported that VOC yields were roughly twice as high in whole smoke than in the gas phase. In their study, they used the 2R4F Kentucky reference research cigarette and conducted quantitative puff-by-puff and yield measurements of selected toxicants in the mainstream smoke from this cigarette using a soft single photon ionization
time-of-flight mass spectrometric technique. They found that, in the whole-smoke mode,

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successive puffs were affected by contamination and memory effects so that they were forced to follow each puff with a cleaning puff in an effort to account for the contribution of the contamination to the total yield. Differences in the cigarette types examined and in the fact that our system did not require cleaning puffs may help to explain the differences noted between their results and ours. Monitoring m/z 55 for 1,3-Butadiene with the PTR-MS. A disadvantage of the PTR-MS technique is that it only provides information on product ion mass. In a matrix as complex as mainstream cigarette smoke, the product ions frequently are not unique to a single compound, so the identification of compounds measured with the PTR-MS is usually tentative and must be interpreted with caution.58,73,74 Cluster ion formation, product ion fragmentation, and the presence of different species of the same mass can all lead to significant overlap.73 The PTR-MS forms the reagent water cluster ion (H2O)2H3O+ at m/z 55.58,73,75 The abundance of this peak and the overall performance of the instrument (i.e., sensitivity and product ion distribution) is strongly dependent on the electric field energy (E/N) of the drift tube, which is the ratio of the applied electric field strength (V/cm) to the gas number density (molecules/cm3).73 Increasing the E/N ratio produces more energetic collisions, which reduces the formation of water cluster ions, but it may also cause additional product ion fragmentation, so the choice of a suitable E/N operating ratio is a compromise.58,73 Figure 7a shows the effect of varying the E/N ratio on the relative intensity of the m/z 55 peak in the laboratory air. At an E/N ratio of 133 Td (where 1 Td = 10
17 cm2 V
1), which corresponds to a drift tube operating pressure of 2.1 mbar and an applied potential of 600 V, the m/z 55 relative intensity is negligible. It is only after E/N is reduced below 111 Td that the m/z 55 intensity begins to increase, becoming quite significant at the lowest E/N ratio examined (E/N = 88.5 Td). Clearly, higher E/N ratios result in more energetic collisions, which reduce the proportion of water cluster ions such as (H2O)2H3O+ in the drift tube to negligible levels. To determine the effect of the electric field energy (E/N) on menthol, we measured the full-scan PTR-MS mass spectrum of a headspace sample of pure L-menthol powder as a function of the E/N ratio and under the same operating conditions that were used to generate the mainstream smoke VOC data shown earlier. The results are shown in Figure 7b. Although many proton transfer processes involving primary H3O+ ions form only one product ion species for each neutral fragment, corresponding to the protonated parent molecule, Spanel and Smith76,77 have shown that in many alcohols, H3O+ protonation is followed by the ejection of a water molecule. In the case of menthol, they found that a single product ion was observed at m/z 139.76 Under our instrument conditions, Figure 7b shows that menthol undergoes considerable fragmentation, despite the use of a soft ionization technique and that fragmentation occurs over the entire range of E/N ratios considered. Since all of our gas phase and whole smoke measurements discussed earlier were made using an E/N ratio of 133 Td, the increase observed in the apparent yield of 1,3-butadiene as the level of menthol was increased in the whole smoke samples (cf. Figure 6) was clearly due to the menthol fragment ion at m/z 55. There is no comparable effect observed in the gas phase samples because most of the menthol occurs in the particulate phase, and it is almost entirely trapped by the filter pad. For the whole smoke samples measured under our operating conditions, setting the 1749

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Figure 6. Effect of menthol on measured yields of gas-phase (filtered) and whole smoke (unfiltered) VOCs in mainstream smoke from Camel Crush cigarettes machine-smoked in the uncrushed (nonmenthol) and crushed (menthol) modes.

Figure 7. Effect of PTR-MS electric field energy (E/N) on (a) reagent water cluster ion (H2O)2H3O+ at m/z 55 in laboratory air; and (b) major product ions in a PTR-MS mass spectrum obtained from a headspace sample of pure L-menthol powder.

drift tube voltage at 500 V (i.e., E/N = 111 Td) may have offered the best compromise.

’ CONCLUSIONS Obtaining conclusive results from human exposure studies on menthol cigarettes is challenging. The differences in concentrations that have been observed in previous menthol studies may be attributed to real physical and chemical differences between the commercial menthol and nonmenthol cigarettes used in testing, as well as to the highly dynamic and reactive nature of cigarette smoke where the concentrations of key constituents and toxicity may change as the smoke ages. To overcome these challenges and isolate the effects of menthol on exposure, we conducted a study using two types of cigarettes that differ only in menthol content. Menthol in the TPM samples obtained from the cigarettes at each of the menthol levels investigated showed a linear increase with applied menthol concentration, but the amounts of nicotine, target TSNAs, PAHs, cotinine, and quinoline in the cigarettes remained essentially unchanged. Similarly,

yields of the targeted VOCs in mainstream smoke from the MEN cigarettes that were measured in real-time on a puff-by-puff basis in whole smoke were largely unaffected by the levels of menthol present in the cigarettes. This is in contrast to the VOC yields from the Camel Crush cigarettes, which appear to increase in the presence of menthol, especially in the gas phase. This may be due to the unique difference in the way in which menthol is generated and delivered in the Camel Crush cigarettes. More studies are needed to explore this difference. Although we have successfully demonstrated techniques to mentholate cigarettes at different concentrations and perform real-time measurements of key mainstream smoke constituents in the cigarettes, further study is needed to definitively answer whether menthol affects exposure to selected cigarette constituents and thereby influences harm. We recommend such menthol studies use test cigarettes that differ only in menthol content and apply the real-time measurement techniques described here to assess the effects of menthol on human exposure. A comprehensive suite of measurements should include particle size distribution, 1750

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Chemical Research in Toxicology mass, and composition; percent deposition of mainstream smoke in a smoker’s respiratory tract; and real-time analysis of volatiles in mainstream smoke and exhaled breath. Although no exhaled breath or other human exposure measurements were made in this study, the PTR-MS instrument is ideally suited to the high humidity of the exhaled breath matrix. These and related measurements will provide the evidence base needed to make timely and critical regulatory decisions on tobacco products for the protection of public health.

’ AUTHOR INFORMATION Corresponding Author

*Phone: (614)424-5278. Fax: (614)458-5278. E-mail: gordon@ battelle.org. Funding Sources

This work was supported by Battelle internal research and development funds. )

Notes

Retired.

’ ACKNOWLEDGMENT We gratefully acknowledge helpful advice and support from Herbert Bresler and Catherine Smith. ’ ABBREVIATIONS FSPTCA, Family Smoking Prevention & Tobacco Control Act; H.R., House Resolution; FDA, U.S. Food and Drug Administration; VOC, volatile organic compound; SVOC, semivolatile organic compound; TSNA, tobacco-specific nitrosamine; PAH, polycyclic aromatic hydrocarbon; TPM, total particulate matter; FTC, Federal Trade Commission; RH, relative humidity; GC-FID, gas chromatography
flame ionization detection; MEN, mentholated nonmenthol cigarette; ISO, International Organization for Standardization; PTR-MS, proton transfer reaction-mass spectrometer; LC-MS/MS, liquid chromatography
tandem mass spectrometry; NNN, N 0 -nitrosonornicotine; NNK, 4-(methylnitrosamino)1-(3-pyridyl)-1-butanone; BaP, benzo[a]pyrene; ppbv, parts per billion by volume; E/N, drift tube electric field energy; Td, unit of electric field energy (1 Td = 10
17 cm2 V
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