Environ. Sci. Technol. 2005, 39, 471-478
Determination of 14 Polycyclic Aromatic Hydrocarbons in Mainstream Smoke from Domestic Cigarettes YAN S. DING, JENNA S. TROMMEL, XIZHENG J. YAN, DAVID ASHLEY, AND CLIFFORD H. WATSON* Emergency Response and Air Toxicants Branch, Division of Laboratory Science, National Center for Environmental Health, Centers for Disease Control and Prevention, 4470 Buford Highway NE, Mailstop F-47, Atlanta, Georgia 30341
Polycyclic aromatic hydrocarbons (PAHs) are a class of environmental pollutants created primarily from incomplete combustion of various organic materials including tobacco. Cigarette smoke is a complex mixture of various classes of compounds, including numerous PAHs, in both the mainstream and the sidestream smoke fractions. We measured the levels of 14 PAHs in mainstream smoke from unfiltered custom cigarettes made from individual tobacco types and 30 brands of domestic blended cigarettes using standardized smoking conditions, extraction from the Cambridge filter pads, and gas chromatography/mass spectrometry. Differences in smoke PAHs from cigarettes with selected tobacco blends were identified and illustrate how blend composition contributes to the overall mainstream smoke PAH profile. The PAH levels varied among the different commercial cigarette brands, with the amount of total mainstream smoke PAHs ranging from 1 to 1.6 µg per cigarette. Under machine smoking conditions, the mainstream smoke from domestic cigarettes had individual PAHs ranging from benzo[k]fluoranthene at levels below 10 ng/cigarette to naphthalene at levels of around 500 ng/cigarette. Low delivery cigarettes smoked with blocked filter vent holes dramatically increased the mainstream smoke PAH deliveries with respect to their unblocked counterparts. Inhalation of PAHs and other harmful chemicals from cigarette smoke are unique as they represent a routine voluntary exposure to common environmental pollutants.
Introduction Polycyclic aromatic hydrocarbons (PAHs) are environmental pollutants derived primarily from incomplete combustion of organic material. The U.S. Environmental Protection Agency (U.S. EPA) has identified 16 priority environmental PAH pollutants on the basis of evidence that they cause or may cause cancer. Among the PAHs, benzo[a]pyrene has been widely studied, and its ability to induce lung tumors is well-documented (1). The health consequences of other PAHs remain less clear. One significant source of PAH exposure is from tobacco products. To fully evaluate the contribution of total PAHs from tobacco smoke to the local * Corresponding author phone: (770)488-7638; fax: (770)488-0181; e-mail:
[email protected]. 10.1021/es048690k Not subject to U.S. Copyright. Publ. 2005 Am. Chem. Soc. Published on Web 12/09/2004
environment and to smokers, all sources of environmental tobacco smoke (ETS) should be investigated. ETS consists of mainstream smoke, the portion that leaves the mouth end of a cigarette; sidestream smoke, the portion released in the static burning period between puffs; and exhaled mainstream smoke. A commission by the California air recourses board (2) found that the levels of PAHs associated with cigarette smoking were 1.5-4 times higher than other indoor combustion sources. The average 24 h concentrations of benzo[a]pyrene associated with indoor combustion sources were gas heat, 0.4 ng/m3; fireplace use, 1.0 ng/m3; woodstove use, 1.2 ng/m3; and tobacco smoking, 2.2 ng/m3. Although procedures to measure mainstream smoke using standardized machine smoking protocols are well-established (3, 4), no universal method has been established to measure sidestream smoke (5). Several reports have examined the sidestream (SS) to mainstream (MS) smoke ratio of smoke components including selected PAHs (6). Depending on the type of cigarette and how the sidestream smoke was collected, the SS/MS for PAHs ranged from approximately 2 to 20. This is not too surprising because during the static burn period (in between puffs), the amount of oxygen available to the cigarette’s fire coal is diminished, and less complete combustion occurs (7). Measuring the levels of PAHs in ETS is a complex task and depends on several factors including the size, ventilation, and associated air-flow characteristics. For example, a person smoking outdoors or in a well-ventilated room would have relatively little exposure to sidestream smoke as compared to a more enclosed environment. In this paper, we concentrated our efforts on estimating the relative amounts of PAHs in mainstream smoke that a smoker voluntarily consumes during smoking. Cigarette smoke is a complex aerosol with multiple classes of chemical compounds, including numerous PAHs (8). All of the EPA’s 16 priority PAHs are present in tobacco smoke, and 14 of these have been cited for their carcinogenicity in tobacco smoke in the International Agency for Research on Cancer (IARC) monographs on the basis of either sufficient or limited evidence (9). Many studies that measured PAH levels in cigarette smoke reported only benzo[a]pyrene levels (1013), although one study also measured levels for the benzo[b/k]fluroanthenes (8). In analytical studies, benzo[a]pyrene often has been used as a surrogate for other PAHs, and the abundant literature on it may tend to distract attention from other important PAHs (1, 9). We found only a few studies in the literature that measured 10 or more PAHs in mainstream cigarette smoke from Kentucky 1R4F reference cigarettes (University of Kentucky, Lexington, KY) or from generic tobacco samples (14-16). The most thorough study quantitated 17 PAHs by gas chromatography/mass spectrometry (GC/MS) in the mainstream smoke from 1R4F cigarettes (17). However, little information is available about PAH levels for specific commercial cigarette brands. Because many PAHs are potential carcinogens, reduction of their levels in cigarette smoke should be considered an important factor for reducing harm, especially in the development of new types of cigarettes including the potentially reduced emission products. Given the lack of information about chemical constituents in cigarettes available to the public, consumers have little or no resources addressing potential safety concerns associated with these compounds. Therefore, developing a modern and highthroughput quantitation method to accurately access levels of PAHs in contemporary cigarettes helps provide useful and much-needed information. VOL. 39, NO. 2, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
471
Previous researchers have used a variety of techniques to trap mainstream smoke and obtain quantitative information about PAHs. One technique used an acetone trap to collect smoke condensate, followed by liquid-liquid extractions and subsequent GC/MS (15, 16) or GC with flame ionization detection (14). Forehand et al. (18) analyzed PAHs in mainstream smoke total particulate matter (TPM) collected on glass fiber filters commonly known as Cambridge filter pads (CFP). After subsequent solid-phase extraction cleanup, PAHs were quantified either by high-performance liquid chromatography (HPLC) with fluorescence detection or GC/ MS. Most prior methods required collecting mainstream smoke from at least five cigarettes to obtain sufficient amounts of PAHs for quantitation (8, 10-12), although smoking 20 cigarettes was widely used in internal tobacco company research (17, 18). We developed an analytical technique for measuring PAHs in tobacco smoke particulate with extraction and quantitation methodologies primarily derived from the work of Gmeiner et al. (17) but with sufficient improvements to increase sensitivity where only three cigarettes are smoked per sample. We report here levels of 14 PAHs in smoke particulate from top-selling commercial cigarette brands from the four major tobacco companies in the United States. To ascertain the relative contribution from various tobacco types, we also examined nonfiltered cigarettes made exclusively from burley (air-cured), bright (flue-cured), oriental (Turkish or suncured), and reconstituted tobacco, as well as selected custom tobacco blends. Because filter ventilation is a key parameter influencing smoke deliveries, we also examined how the PAH delivery changed with blocked filter vent holes.
Experimental Procedures Safety. Personnel involved in weighing, diluting, or otherwise manipulating the compounds used were instructed in the safe handling of chemicals. These instructions included the wearing of personal protection items and proper laboratory practices. All compounds were handled in a fume hood, and personnel used appropriate protective safety glasses, gloves, and lab coats. Materials. PAHs used for calibrating standard solutions were obtained from Aldrich Chemical Co. (Milwaukee, WI). The U.S. EPA 16 PAH cocktail (13C, 99%, 5 µg/mL) used for internal standards was purchased from Cambridge Isotope Laboratories, Inc. (Andover, MA, catalog no. ES-4087). Solvents used included methanol (Tedia, Fairfield, OH), HPLC-grade water (J. T. Baker, Phillipsburg, NJ), and cyclohexane (Aldrich, 99.9+%, Milwaukee, WI). CFPs were obtained from Whatman (Maidstone, UK). Unfiltered custom blended cigarettes were purchased from Murty Pharmaceuticals, Inc. (Lexington, KY). Domestic commercial cigarettes were purchased from various retail sources in Atlanta, GA or were generously provided by the Massachusetts Department of Public Health. Unopened packs were individually sealed in plastic bags and stored at -70 °C until needed. Reference cigarettes used as quality control (QC) samples (2R4F) and in the accuracy studies (1R4F) were from the University of Kentucky (Lexington, KY). PAH Standard Solution. A standard stock solution containing all 14 PAHs (naphthalene, acenaphthylene, acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene, pyrene, benz[a]anthracene, chrysene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[e]pyrene, and benzo[a]pyrene) was prepared in methanol. For the calibration curve, seven blank CFPs (44 mm glass fiber filter pad) were spiked with varying amounts of PAH standard solution and 25 µL of the 13C PAH internal standard solution. Each CFP was then subjected to the same preparation procedure used for smoke samples. 472
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 39, NO. 2, 2005
Smoke Collection. Before being smoked, the cigarettes and CFPs were conditioned at 22 °C and 60% relative humidity for at least 24 h. Mainstream smoke TPM generated under Federal Trade commission (FTC) conditions (60 s puff interval, 2 s puff duration, and 35 mL puff volume) was collected on individual CFPs using a Cerulean (Milton Keynes, UK) ASM500 16-port smoking machine. The cigarettes were smoked to a butt length of 23 mm or the length of the filter overwrap plus 3 mm, whichever was longer, using the industry-standard Cambridge filter pad holder. Cigarettes (three each, randomly selected) from five different packs of each brand were smoked to obtain the average smoke particulate level for each PAH. During each smoking run, 2R4F cigarettes were smoked as QC samples. After a group of three cigarettes was smoked, each CFP was spiked with 25 µL of the 13C PAH internal standard solution and processed through the sample preparation scheme. Sample Preparation. The sample preparation scheme was based on a previously published method (17) with modifications listed next. After being smoked, each CFP was placed in 10 mL of methanol and shaken at 160 rpm for 1 h in a sealed 13 mL amber vial. The methanol extracts then were decanted, and the solvent volume was reduced to approximately 2 mL using a Zymark LV Evaporator (Hopkinton, MA). Any remaining particulate matter was removed with a 0.45 µm nylon syringe filter. Approximately 3.5 mL of HPLCgrade water was added to bring each sample to approximately 65:35 (v/v) water/methanol. Using a vacuum manifold (Visiprep 24-port from Supelco (Bellefonte, PA)), an ENVI18 SPE (Supelco) cartridge was conditioned with 8 mL of methanol followed by 8 mL of 65:35 (v/v) water/methanol. Smoke extract samples (∼65:35 (v/v) water/methanol) then were loaded and washed with 8 mL of water followed by 8 mL of 65:35 (v/v) water/methanol. These steps were all performed under atmospheric pressure. After being washed, a low vacuum was added to eliminate as much water as possible from the cartridges, and 4 mL of cyclohexane was added to the column for elution under very low vacuum. The cyclohexane extracts were evaporated to approximately 0.5 mL, and a 1 µL aliquot was used for GC/MS for analysis. GC/MS Analysis. GC separation was performed using a 30 m J & W Scientific (Folsom, CA) DB-5MS column (0.25 mm i.d.) having a 0.25 µm film thickness attached to an Agilent fused silica (deactivated) guard column (0.25 mm i.d., ∼3 m). The splitless injector was set to 270 °C. A constant flow of 1.7 mL/min of helium carrier gas was maintained through the column. The following temperature program was used: 15 °C/min ramp from 65 to 270 °C (hold for 1.5 min), 5 °C/ min ramp to 275 °C (hold for 1.5 min), 5 °C/min ramp to 285 °C (hold for 2.5 min), and a 25 °C/min ramp to 315 °C (hold for 2 min). The total run time was 25.37 min. An Agilent model 5973 mass spectrometer was used for data acquisition in selected ion monitoring mode (SIM). Accuracy Study. To determine the recovery for each PAH, two CFPs containing smoke TPM from three 1R4F cigarettes were spiked at two different concentrations of PAHs standard solutions, respectively. One CFP with TPM from three 1R4F without a spike was used as the blank. These experiments were performed in five replicate sets. All CFPs were then subjected to the standard sample preparation procedure and subsequent GC/MS analysis. Blocking Study. For the blocking experiment, filter ventilation holes of selected cigarette brands were sealed by a short strip of transparent tape (3M, St. Paul, MN). Modified cigarettes were then subjected to the same smoking regimen, sample preparation, and GC/MS analysis as regular cigarettes.
Results PAH SIM Group and Detection Limit. The combined total ion chromatogram of all 14 PAH analytes and their chemical
FIGURE 1. Combined total ion chromatogram of 14 polycyclic aromatic hydrocarbons and their chemical structures. Concentrations from A to N are 22.5, 12.3, 2.6, 17.6, 9.0, 4.7, 4.6, 3.4, 1.8, 1.8, 1.0, 0.4, 0.3, and 0.8 µg/mL, respectively.
TABLE 1. Analytical Parameters and SIMa Group of Target Polycyclic Aromatic Hydrocarbons PAH (SIM group) naphthalene (1)
13C
internal standard
ion mass ( m / z)
confirmation ion (m/z)
128.2
127.2
anthracene-13C6
ion mass ( m / z)
confirmation ion (m/ z)
retention time (min)
detection limit (ng)
184.2
182.2
5.44
155
7.89 8.17
50 13
acenaphthylene (2) acenaphthene (2)
152.2 154.2
151.2 152.2
acenaphthylene-13C fluoranthene-13C6
158.2 208.2
157.2 206.2
fluorene (3)
166.2
165.2
fluoranthene-13C6
208.2
206.2
9.00
84
phenanthrene (4) anthracene (4)
178.2 178.2
176.2 176.2
anthracene-13C6 anthracene-13C6
184.2 184.2
182.2 182.2
10.54 10.64
57 7
fluoranthene (5) pyrene (5)
202.3 202.3
200.3 200.3
fluoranthene-13C6 pyrene-13C3
208.2 205.2
206.2 203.2
12.48 12.75
6 15
benz[a]anthracene (6) chrysene (6)
228.3 228.3
114.3 226.3
benz[a]anthracene-13C6 chrysene-13C6
234.3 234.3
232.3 232.3
14.97 15.04
6 16
benzo[b]fluoranthene (7) benzo[k]fluoranthene (7)
252.3 252.3
250.3 250.3
benzo[b]fluoranthene-13C6 benzo[k]fluoranthene-13C6
258.3 258.3
256.3 256.3
17.71 17.80
16 5
benzo[e]pyrene (8) benzo[a]pyrene (8)
252.3 252.3
250.3 250.3
benzo[a]pyrene-13C4 benzo[a]pyrene-13C4
256.3 256.3
254.3 254.3
18.55 18.70
3 19
a
6
Selected ion monitoring for GC/MS.
structures exhibited good sensitivity and chromatographic resolution (Figure 1). The total ion chromatographs from mainstream smoke extracts was much more complicated because of additional background chemical noise; however, the method yielded selected ion chromatograms that demonstrated good separation for our 14 analytes (Figure 2). Appropriate analytical parameters including retention times, and ion mass-to-charge ratios for the 14 PAHs measured were selected (Table 1). We determined relative response factors for target PAHs using 13C-labeled analogues as internal standards except naphthalene, acenaphthene, fluorene, phenanthrene, and benzo[e]pyrene that used surrogates. During the mass spectral data acquisition, the 14 PAHs were divided into eight SIM groups (Table 1) to improve overall sensitivity. Calibration curves were constructed for each PAH with detection levels ranging from low nanograms to micrograms. The detection limit (LOD) for each PAH was estimated from calibration curves as three times the standard deviations (from three lowest concentrations) extrapolated to zero concentration (19). The calculated LODs of PAHs ranged from 3 ng of benzo[e]pyrene to 155 ng of naphthalene (Table 1).
Mean Accuracy. The accuracy for each PAH was calculated by subtracting the PAH levels measured on a 1R4F sample from a 1R4F sample spiked with known amounts of analytes. For each PAH, we performed multiple spikes at low and high concentration levels (Table 2). We evaluated accuracy by comparing the measured increase in concentration above the unspiked sample with the theoretical amounts spiked (Table 2). For most PAHs, the mean accuracies ranged from 78 to 113% at both the low and the high spike levels, except for chrysene, which had a higher accuracy (127%) at the low spiked level, and acenaphthene, which had a large difference in accuracies at the low (53%) and high (112%) spike levels (Table 2). PAH Levels in Reference Cigarettes. We measured 14 PAHs levels in smoke particulate from 1R4F and 2R4F Kentucky reference cigarettes using our method (Table 3). A comparison of the 1R4F and 2R4F Kentucky reference cigarettes showed that most of PAH levels in mainstream cigarette are remarkably similar. The only obvious anomaly is that the level of some of the lower molecular weight PAHs, including naphthalene, in the 2R4F appears to be lower than in the 1R4F cigarettes. Because Kentucky reference cigarettes VOL. 39, NO. 2, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
473
FIGURE 2. Total ion chromatogram and selected reconstructed ion chromatograms of 14 PAHs in mainstream smoke TPM from a Kentucky reference 2R4F cigarette. such as the 1R4F and 2R4F are examples of American blended cigarettes with filler consisting of bright, burley, oriental, and reconstituted tobaccos, we also included the bright tobacco filler-based CM2 Virginia blended cigarette for reference. The PAH levels in CM2 monitor mainstream smoke were much higher than those from 1R4F and 2R4F, except for phenanthrene and anthracene, which were similar (Table 3). PAH Levels in Custom-Blended Cigarettes. To gain insight on PAH formation as a function of tobacco blend, we measured the levels of PAHs in a series of custom-blended unfiltered cigarettes. These cigarettes were divided into six groups on the basis of blend composition of different tobaccos (Table 4). When we compared the mainstream smoke deliveries of 100% burley (air-cured) to 100% bright (fluecured) cigarettes (Table 4), the 14 PAH levels fell into three clusters. The levels of naphthalene, acenaphthylene, and acenaphthene were higher in burley than in bright cigarettes; in the second cluster (fluorene, phenanthrene, anthracene, and fluoranthene), the two tobaccos had similar levels; in the last cluster (the remaining seven PAHs), PAH levels from the bright cigarettes were much higher than in burley 474
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 39, NO. 2, 2005
cigarettes. PAH levels in cigarettes made from 100% oriental were similar to those from 100% bright cigarettes except for fluorene and pyrene. Levels of these two PAHs were much higher in 100% oriental cigarettes than any other blended cigarettes. PAH levels in 100% reconstituted cigarettes were the lowest among all six blends. PAH levels in cigarettes made from various known combinations of four blended tobaccos also were determined (Table 4). PAH Levels in Domestic Cigarettes. Levels of PAHs in smoke particulate were measured in 30 cigarette brands. We also determined the percentage filter ventilation (%) and total PAHs, the sum of all 14 PAHs of each brand (Table 5). Graphical analysis of these results revealed several interesting observations (Figure 3A). The complete individual PAH results of these measurements are available as Supporting Information. In general, levels of PAHs were relatively consistent among cigarettes having similar FTC tar and nicotine deliveries from different tobacco companies (20). There was a decreasing trend in the levels of PAHs in smoke determined using the FTC method from full flavor to light to ultralight in the same brand families that correlates with the amount of filter
TABLE 2. Method Validation Parameters for Measuring Polycyclic Aromatic Hydrocarbons in Tobacco Smoke Particulatea compound naphthalene acenaphthylene acenaphthene fluorene phenanthrene anthracene fluoranthene pyrene benz[a]anthracene chrysene benzo[b]fluoranthene benzo[k]fluoranthene benzo[e]pyrene benzo[a]pyrene a
spiking level (ng)
mean accuracy (%)
precision (%)
786.0 3368 430 1842 90.7 388.5 615.2 2637 316.3 1356 163.8 702.0 161.2 690.9 118.0 505.8 63.4 271.5 64.4 276.0 33.6 144.0 14.2 60.9 11.7 50.1 29.1 124.8
91.8 97.3 88.7 88.3 53.1 112.0 79.2 113.3 109.5 105.2 105.0 105.7 99.9 113.4 100.3 102.4 102.0 103.1 127.6 111.4 111.5 106.0 104.6 101.1 78.3 78.4 109.1 104.0
2.9 5.8 12.6 8.8 3.8 2.1 5.4 2.9 6.0 4.8 7.6 4.0 4.5 3.1 13.9 8.7 2.6 1.6 5.7 2.6 3.6 2.9 8.4 2.2 10.7 4.5 4.4 2.9
For accuracy and precision measurements, n ) 5.
ventilation (Table 5). This trend existed both in individual PAH and total PAHs. When normalized against mainstream smoke nicotine level of each brand (20), the decreasing trend from full flavor to light to ultralight was greatly diminished (Figure 3B). Blocking Study. To assess the relation between the percentage filter ventilation and levels of PAHs in different delivery types (full flavor, light, ultralight) within the same brand family, we selected one cigarette brand from each of the four major cigarette manufacturers to determine quantitatively the influence of filter ventilation on PAH mainstream smoke deliveries. To eliminate the effect of tobacco filler weight differences, we presented results as total PAHs per mg of tobacco smoked. For cigarettes machine smoked with the filter vent hole unobstructed, the levels of total PAHs
were generally higher for the full-flavored brands than for the light and ultralight variants (Figure 4A). One notable exception was Marlboro light for which the total PAH level exceeded the Marlboro full-flavored brand. When the filter vent holes were blocked with tape, the total PAH content in the mainstream smoke of the lights and ultralights increased significantly and more closely matched the levels in the fullflavored brands (Figure 4B).
Discussion The current method is suitable for analyzing 14 PAHs in a variety of modern cigarettes using commercially available equipment. An advantage of our method over previously published methods is sensitivity sufficient to quantify PAH levels in mainstream smoke by smoking only three cigarettes while maintaining good reproducibility and accuracy. In theory, improving sensitivity should be possible by collecting smoke from more cigarettes to increase the amount of PAHs trapped of the CFP. However, the amount of particulate matter that can be trapped before breakthrough is limited. Assuming breakthrough is not a problem, as the number of smoked cigarettes increases, the amounts of all the other approximately 4000 chemicals in smoke also increase proportionally. Because the concentrations of all constituents in the particulate matter remain constant, smoking more cigarettes may not improve sensitivity. In our experience, PAH sensitivity for these and higher molecular weight PAHs is best improved using a more thorough and time-consuming sample cleanup procedure or by more advanced mass spectral detection approaches, such as tandem MS or highresolution MS. The GC/MS total ion chromatograms obtained with relatively low-cost equipment showed good separation for these PAHs (Figures 1 and 2). The LODs are adequate to measure the lower PAHs from ultralight cigarettes smoked using standardized machine smoking protocols with a puff volume of 35 mL and a puff interval of 60 s. Method validation indicated good accuracy except for acenaphthene at the low spike level. Why the low acenaphthene recovery spike was only 53% is not known. Presumably, it results from the low level spike being near the background level. At the higher spike level, recovery of acenaphthene was similar to the other analytes. In general, the accuracy and precision was respectable considering that cigarettes are, for the most part, an agricultural product. The average low and high spiking experiments for all 14 analytes yielded average accuracies of 97.2 and 103.0%, respectively. The current method also demonstrated reasonably good precision with average relative standard deviations of 6.6% at the lower concentrations and 4.1% at the higher concentrations.
TABLE 3. Comparison of Polycyclic Aromatic Hydrocarbon Levels (ng/cigarettea) Measured in Reference Cigarettes
a
PAH
1R4Fa (RSD,b %)
2R4Fc (RSD,b %)
CM2 monitor no.2d (RSD,b %)
1R4Fe (RSD,b %)
1R4Ff (RSD,e %)
naphthalene acenaphthylene acenaphthene fluorene phenanthrene anthracene fluoranthene pyrene benz[a]anthracene chrysene benzo[b]fluoranthene benzo[k]fluoranthene benzo[e]pyrene benzo[a]pyrene
350.3 (6) 116.9 (7) 84.8 (3) 217.5 (4) 134.8 (4) 74.9 (7) 74.4 (5) 48.6 (6) 13.4 (5) 15.7 (2) 9.4 (3) 1.5 (9) 2.9 (5) 10.3 (4)
192.0 (23) 88.3 (11) 51.3 (14) 156.3 (12) 145.2 (9) 69.8 (12) 63.0 (17) 49.5 (14) 16.5 (9) 19.7 (12) 10.6 (12) 1.9 (15) 3.2 (8) 11.0 (7)
407 (46) 153 (20) 88 (19) 257 (10) 144 (10) 74 (12) 101 (12) 77.0 (30) 22.6 (11) 31.4 (9) 18.3 (13) 3.9 (18) 6.2 (6) 14.7 (8)
236.0 (8) 50.4 (8) 25.3 (5) 119.0 (4) 110.0 (3) 38.1 (6) 46.2 (4) 33.2 (5) 13.2 (4) 21.8 (4) 8.6 (3) 1.5 (5) 4.0 (3) 7.9 (3)
361.7 (3) 239.0 (2) 147.7 (3) 35.8 (3) 51.6 (5) 32.1 (3) 14.0 (3) 11.2 (3) 11.2g (3) 6.4 (6) 7.6 (3)
n ) 5. b Relative standard deviation. c n ) 50. d n ) 5. e Ref 17. f Ref 18. g Benzo[b]fluoranthene + benzo[k]fluoranthene. VOL. 39, NO. 2, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
475
TABLE 4. Polycyclic Aromatic Hydrocarbon Levels (ng/cigarette) in Mainstream Smoke among Custom-Blended Unfiltered Cigarettes PAH
100% burley
100% bright
100% oriental
100% recona
40% bri, 30% bur, 15% ori, reb
25% bri, bur, ori, reb
naphthalene acenaphthylene acenaphthene fluorene phenanthrene anthracene fluoranthene pyrene benz[a]anthracene chrysene benzo[b]fluoranthene benzo[k]fluoranthene benzo[e]pyrene benzo[a]pyrene
1055 194 134 416 221 148 109 48.9 25.2 37.6 27.5 3.8 6.7 21.9
725 175 99 466 243 136 118 71.2 43.1 75.9 34.9 6.7 10.5 36.3
725 208 201 625 300 150 142 138.4 48.0 78.0 37.5 9.1 9.4 28.7
230 54 70 229 89 62 79 56.0 15.6 20.2 14.5 3.4 4.8 12.3
795 168 130 440 214 136 115 83.6 30.1 53.2 31.1 3.2 8.3 28.4
633 138 94 349 212 125 100 78.8 34.3 48.7 28.0 5.6 8.8 27.4
total (ng/cigarette)
2448
a
Reconstituted tobacco.
b
2241
2700
brand
% filter total ventilationa (ng/cigarette)
Philip Morris
Marlboro
full flavor light ultralight Basic full flavor light ultralight Virginia Slims Benson & Hedges
14.0 22.3 46.7 2.2 15.0 32.0 19.9 15.1
1149 1070 594 1114 859 477 1185 1598
RJ Reynolds
Camel
23.7 26.6 52.3 18.3 31.6 49.7 2.1 12.6
1292 1012 597 1124 942 745 1432 1248
1.6 16.4 21.0 43.5 2.0 20.8 82.0
1232 1011 751 561 1274 1165 87
6.0 18.6 21.5 22.5 68.4
1146 921 660 832 401
full flavor light ultralight Winston full flavor light ultralight Salem Doral (full flavor)
Brown & GPC Williamson
full flavor medium light ultralight Kool Misty (full flavor) Carlton
Lorillard
Newport
other
Omni King (full flavor) Omni 100s Light
full flavor medium light Kent True
0.2b 0.2b
904 824
a Filter ventilation of an individual cigarette was determined on QTM 5 ventilation measurement apparatus purchased from Filtrona (Richmond, VA). b Charcoal filter.
Comparison of our results of the 1R4F reference cigarettes with levels from previously published reports (17, 18) shows that the levels we determined for anthracene, fluoranthene, and pyrene are somewhat higher than expected. The others generally fell within the range of values established for 1R4F cigarettes. Some previous results were obtained by smoking 20 cigarettes on a 92 mm CFP; with this high number of cigarettes and larger volume of smoke drawn through the CFP, some compounds may have saturated the CFP, and their levels may have been lower because of breakthrough. 476
9
2236
1881
bri ) bright, bur ) burley, ori ) oriental, re ) reconstituted tobacco.
TABLE 5. Polycyclic Aromatic Hydrocarbon Levels (ng/ cigarette) in Domestic Cigarettes company
941
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 39, NO. 2, 2005
Data from custom-blended cigarettes suggest that the tobacco filler blend significantly influences the PAH profiles. Reconstituted tobacco is made from tobacco dust, fines, and particles and from ground ribs and stems; various additives such as cellulose fiber are added during the reconstituted sheet manufacturing process. Such additives can substantially reduce the cigarette smoke yields of tar, nicotine, phenols, and PAHs (21). Our result showed that 100% reconstituted tobacco has the lowest levels of PAHs. The predominately used three tobacco types, burley, bright, and oriental, are all cured differently. Burley is air-cured; bright (also called Virginia or blond) is flue-cured by artificial heat; and oriental (Turkish) is sun-cured (21). The pyrosynthesis of benzo[a]pyrene reportedly is higher in bright or oriental than that in burley tobaccos (21), and we found that the levels of four- and five-membered ring PAHs including benzo[a]pyrene produced in burning bright or oriental tobaccos were much higher than those in burley tobaccos (Table 4). The phenomenon may result from the generally lower nitrate contents in bright or oriental tobaccos than in burley tobaccos (21). The nitrogen oxides formed during the burning of tobacco serve as free-radical scavengers that help prevent formation of precursors for the pyrosynthesis of high-end PAHs. With increasing nitrate content of the cigarette tobacco, the pyrosynthesis of high-end PAHs (i.e., benzo[a]pyrene) is correspondingly inhibited. Interestingly, the average amounts of two- or three-membered ring PAHs in bright or oriental tobaccos were lower than in the burley tobacco suggesting that tobacco type or processing (flue-cured, sun-cured, or air-cured) may play a role in the final distribution of PAHs in mainstream smoke. We analyzed CM2 monitor cigarettes because of their different tobacco blend composition as compared to the 1R4F and 2R4F Kentucky reference cigarettes. The PAH profiles between CM2 monitor and 2R4F/1R4F are similar to those between 100% bright cigarettes and blended cigarettes, respectively (Tables 3 and 4). We also calculated the expected PAH levels for the two custom-blended cigarettes on the basis of the levels in the custom single blends. Calculations of PAH levels, based on known blend composition and the PAH contribution from each blend component, to that measured in the mainstream smoke from the customblended cigarettes were in excellent agreement. The plot of PAH-calculated levels versus the measured values yielded a correlation coefficient of 0.999 (data not shown). We believe such good agreement further establishes the robustness of our method. Such findings may provide a way to estimate
FIGURE 3. Total polycyclic aromatic hydrocarbons in domestic cigarettes from four major tobacco manufacturers. F ) full flavor; L ) light; UL ) ultralight; RJR ) RJ Reynolds; BW ) Brown and Williamson; PM ) Philip Morris; and LR ) Lorillard. (A) Total PAH (ng/cigarette) and (B) total PAH levels normalized by mainstream nicotine delivery.
FIGURE 4. Total polycyclic aromatic hydrocarbons in selected cigarettes (different variants) from four major tobacco manufacturers. F ) full flavor; L ) light; UL ) ultralight; RJR ) RJ Reynolds; BW ) Brown and Williamson; PM ) Philip Morris; and LR ) Lorillard. (A) Regular smoking condition and (B) filter vent holes taped closed before smoking. tobacco filler mixture compositions from measured PAH levels. We observed substantial variation in the mainstream smoke levels of PAHs in 30 domestic brands. However, the main influence for modern domestic, American-blended cigarettes appears to arise from differences in the amount of filter ventilation. When machine smoked with a 35 mL puff volume, 60 s puff interval, and a 2 s puff duration, cigarettes with decreased PAH levels are most likely those with higher amounts of filter ventilation typically associated with light and ultralight brand variants. This is not surprising because, under machine smoking conditions, light and ultralight brands have reduced smoke deliveries of nicotine and tar. As the amount of filter ventilation increases, the mainstream smoke is more diluted with air drawn through the filter vent holes, resulting in lower deliveries of all smoke constituents. Blocking the filter vent holes resulted in substantial increases in PAHs. However, the levels of PAHs in the lower delivery cigarettes do not always equal those in the full-flavor brands. Other sources of PAH reduction including increased paper porosity and less tobacco filler, on a weight basis, typically used in lower delivery brands, may reduce PAH deliveries. The differences resulting from the filler masses can be minimized by normalizing the PAH levels with the grams of tobacco consumed during smoking. Also, differences in PAH levels could reflect possible differences in filler efficiencies or longer filters associated with many of the lower delivery brands. Although physical design characteristics, such as more filter ventilation holes in lower delivery cigarettes, reduces the levels of PAHs in mainstream smoke under FTC conditions (no vent blocking), it does not necessarily reduce deliveries to a smoker who obstructs the vent holes by fingers or lips. Kozlowski et al. have shown that smokers can easily
block ventilation holes during smoking (22, 23). When total PAH was normalized against nicotine delivery for each brand, differences among full-flavor, light, and ultralight significantly decreased (Figure 3B). The filter vent hole blocking experiments also revealed similar trends by increasing the delivery of mainstream smoke constituents. In addition, modern cigarettes typically are smoked more aggressively than the machine smoking protocol used here. In fact, several researchers have shown that lighter delivery cigarettes typically are smoked with a 40-45 mL puff volume and a 30 s puff interval (21-24). Therefore, the amount of PAHs inhaled by smokers is likely to be substantially higher than that reported here. Our findings suggest that if smokers adjust their smoking behavior to achieve a consistent nicotine intake, they will be exposed to an equivalent amount of PAHs regardless of whether they smoke full-flavor, light, or ultralight varieties of a particular brand. To make machine smoke measurements more meaningful, additional toxicity and information about cigarette consumption are required. Sources of toxicity data such as the IARC monograph series are valuable for selected compounds but in general provide only limited information. Detailed studies of individual smoking behaviors for smokers with a range of different ages and races and of both sexes are needed to meaningfully describe modern smoking practices. Accurate smoking topography data are required to better estimate deliveries of tar, nicotine, PAHs, and other smoke constituents. Data on the 14 PAH profiles from the different types of tobacco suggest that simply switching from one tobacco type or blends may not reduce the PAH carcinogenic burden. Also, using benzo[a]pyrene as a surrogate for total PAHs may not be entirely appropriate, especially when comparing cigarettes of different blends, because good correlations do VOL. 39, NO. 2, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
477
not exist with the levels of all of the other 13 PAHs (Table 4) analyzed in this study. Additional higher molecular weight PAHs in cigarette smoke may also depend on the tobacco variety, curing, or blend composition. Many of these larger PAHs are more difficult to quantify with the current methodology but could be potential carcinogens and should be evaluated. To improve our specificity and sensitivity on the analysis of higher molecular weight PAHs, we are evaluating liquid chromatography combined with tandem MS. Although such equipment significantly increases the cost of analysis, the isolation, purification, and quantification of additional PAHs is needed to provide additional information about exposure assessments and harm potential.
Supporting Information Available Two tables. This material is available free of charge via the Internet at http://pubs.acs.org.
Literature Cited (1) Hecht, S. S. J. Natl. Cancer Inst. 1999, 91, 1194. (2) Sheldon, L.; Clayton, A.; Keever, J.; Perritt, R.; Whitaker, D. Indoor Concentrations of Polycyclic Aromatic Hydrocarbons in California Residences. Contract A033-132. Final Report; Air Resources Board: Sacramento, CA, 1993. (3) Pillsbury, H. C.; Bright, C. C.; O’Connor, J.; Irish, F. W. J. Assoc. Off. Anal. Chem. 1969, 52, 458. (4) ISO. Tobacco and Tobacco ProductssRoutine Analytical CigaretteSmoking MachinesDefinitions, Standard Conditions, and Auxiliary Equipment; ISO 3308, 1997. (5) Proctor, C. J.; Martin, C.; Beven, J. L.; Dymond, H. F. Analyst 1988, 113, 1509. (6) Baker, R. R. In Smoke chemistry; Davis, D. L., Nielsen, M. T., Eds.; World Agriculture Series; Tobacco Production, Chemistry, and Technology. Blackwell Science: London, 1999. (7) Browne, C. L. The Design of Cigarettes; Hoechst Celanese Corporation: Charlotte, NC, 1990. (8) Evans, W. H.; Thomas, N. C.; Boardman, M. C.; Nash, S. J. Sci. Total Environ. 1993, 136, 101.
478
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 39, NO. 2, 2005
(9) IARC. IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans. Tobacco Smoking; IARC: Lyon, France, 1986; Vol. 38. (10) Dumont, J.; Larocque-Lazure, F.; Iorio, C. J. Chromatogr. Sci. 1993, 31, 371. (11) Kayali, M. N.; Rubio-Barroso, S. J. Liq. Chromatogr. 1995, 18, 1617. (12) Risner, C. H. J. Chromatogr. Sci. 1988, 26, 113. (13) Tomkins, B. A.; Jenkins, R. A.; Griest, W. H.; Reagan, R. R.; Holladay, S. K. J. Assoc. Off. Anal. Chem. 1985, 68, 935. (14) Grimmer, G.; Naujack, K. W.; Dettbarn, G. Toxicol. Lett. 1987, 35, 117. (15) Lee, M. L.; Novotny, M.; Bartle, K. D. Anal. Chem. 1976, 48, 405. (16) Schmid, E. R.; Bachlechner, G.; Varmuza, K.; Klus, H. Fresenius Z. Anal. Chem. 1985, 322, 213. (17) Gmeiner, G.; Stehlik, G.; Tausch, H. J. Chromatogr. A 1997, 767, 163. (18) Forehand, J. B.; Dooly, G. L.; Moldoveanu, S. C. J. Chromatogr. A 2000, 898, 111. (19) Taylor, J. K. Quality Assurance of Chemical Measurement; Lewis Publishers: Chelsea, MI, 1987; pp 79-82. (20) Federal Trade Commission. Report of tar, nicotine, and carbon monoxide of the smoke of 1294 Varieties of Domestic Cigarettes For the Year 1998, issued 2000. http://www.ftc.gov/reports/ tobacco/1998tar&nicotinereport.pdf. (21) Hoffmann, D.; Hoffmann, I. J. Toxicol. Environ. Health 1997, 50, 307. (22) Kozlowski, L. T.; Rickert, W. S.; Pope, M. A.; Robinson, J. C.; Frecker, R. C. Br. J. Addict. 1982, 77, 159. (23) Kozlowski, L. T.; O’Connor, R. J. Tobacco Control 2002, 11 (Suppl 1), I40-I50. (24) Kozlowski, L. T.; Pillitteri, J. L. Tobacco Control 2001, 10 (Suppl 1), i12-i16.
Received for review August 20, 2004. Revised manuscript received October 22, 2004. Accepted October 26, 2004. ES048690K
