Analysis of Volatile Organic Compounds in Mainstream Cigarette

Mainstream cigarette smoke is a complex aerosol containing more than 4400 chemicals. The proliferation of new brands has necessitated development of f...
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Environ. Sci. Technol. 2007, 41, 1297-1302

Analysis of Volatile Organic Compounds in Mainstream Cigarette Smoke GREGORY M. POLZIN,* RACHEL E. KOSA-MAINES, DAVID L. ASHLEY, AND CLIFFORD H. WATSON Emergency Response and Air Toxicants Branch, Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention

Mainstream cigarette smoke is a complex aerosol containing more than 4400 chemicals. The proliferation of new brands has necessitated development of faster and more reliable methods capable of analyzing a wide range of compounds in cigarette smoke. Although the International Agency for Research on Cancer has classified whole cigarette smoke as a human carcinogen, many of the individual chemicals are themselves highly biologically active as carcinogens, teratogens, or have implications for cardiovascular disease. Among these chemicals are many volatile organic compounds (VOCs), e.g., benzene, ethylbenzene, and styrene. To analyze VOCs in mainstream cigarette smoke, we developed a novel headspace collection technique using polyvinylfluoride bags for sample collection followed by cannula transfer to evacuated standard 20-mL auto sampler vials. Coupling collection of the vapor-phase cigarette smoke with automated analysis by solid-phase microextraction and gas chromatography/ mass spectrometry enabled us to routinely quantify selected VOCs in mainstream cigarette smoke. This technique has similar reproducibility to previous cold trap and impinger collection methods with significantly higher sample throughput and virtually no solvent waste. In this report we demonstrate the method’s analytical capabilities by quantitatively analyzing 13 selected VOCs in mainstream cigarette smoke from top-selling domestic brands.

Introduction Cigarette smoking remains the leading preventable cause of premature death in the United States (1). From 1995 to 1999, an estimated 440 000 deaths were attributed each year to cigarette smoking (1). Although nicotine is the main chemical component of tobacco smoke that keeps people using the product (2), many of the other compounds in mainstream cigarette smoke are associated with cancer, birth defects, or heart disease (3). The combustion of cigarette tobacco filler during cigarette smoking creates an aerosol containing numerous chemical compounds (4, 5). Among the thousands of reported chemicals generated during smoking are several specific classes of hazardous compounds that merit concern (3, 4, 6-10). The International Agency for Research on Cancer (IARC) groups individual chemicals and chemical mixtures according to their carcinogenicity toward humans. Cigarette * Corresponding author phone: (770) 488-7292; fax: (770) 4880181; e-mail: [email protected]. 10.1021/es060609l Not subject to U.S. Copyright. Publ. 2007 Am. Chem. Soc. Published on Web 01/11/2007

smoke contains numerous compounds classified by IARC as known, probable, and possibly carcinogenic. Although whole cigarette smoke as a mixture has been classified as a human carcinogen (11), many of the individual volatile organic compounds (VOCs) present in whole smoke, such as benzene (12), ethylbenzene (13), and styrene (14), are known or potential human carcinogens. Although VOCs comprise only a small fraction (by weight) of mainstream cigarette smoke (3), smoking is a primary exposure source for many toxic volatile compounds, and this fraction has been proposed as the most hazardous fraction of mainstream smoke (15). For example, cigarette smoking accounts for nearly half of all Americans’ exposure to benzene, a known human carcinogen (16). Several cigarette design factors influence the delivery and composition of mainstream cigarette smoke. Because cigarette yields are determined by machine smoking, design differences that reduce machine yields may not reduce a smoker’s exposure. Filter ventilation reduces machine generated mainstream smoke yields but smokers often compensate by blocking the vent holes, taking larger puffs, or puffing more frequently (17). Similarly, changes to static burn rates, tobacco filler composition and weight, and different filter materials can result in a reduction in machine yields. Smokers who compensate in an effort to obtain the desired level of nicotine defeat these design features and obtain a much different exposure than estimated by standardized machine smoking measurements. An accurate assessment of cigarette design changes and their impact on mainstream smoke yields are required to help accurately estimate the health impact these changes may have. Earlier studies of VOCs in mainstream cigarette smoke were accomplished by using a variety of techniques for both collection and analysis. Previous researchers employed collection techniques such as solvent-filled impinger trains (18, 19), adsorbent materials (20-22), cold traps (23, 24), and direct injection of the gas sample (25, 26) to isolate the wide range of chemicals in cigarette smoke. Although these methods exhibited good reproducibility, the possibility of sample breakthrough, tedious cleanup steps, generation of solvent waste, and long sample-preparation times often limited their utility. Factors such as impinger solvent volumes, evaporation, reactivity, breakthrough, and perturbation of the machine puff also potentially contribute to increased variability in any analytical technique. Minimizing the influence of such variables improves accuracy and precision. We present a method for analyzing VOCs in mainstream cigarette smoke that allows for high-throughput analysis at a relatively low cost using commercially available materials and equipment. Key features of this method include the appropriate selection of internal standards, inert polyvinylfluoride (PVF) bags for vapor-phase collection, cannula transfer of smoke to an evacuated headspace vial, and subsequent automated solid-phase microextraction (SPME) and gas chromatography/mass spectrometry (GC/MS). Other advantages include minimal use of solvents, virtually no sample preparation or cleanup required, and no sample carryover. Method validation is discussed along with the analysis of ketones and arenes in 41 cigarette varieties that provide a representative sampling of domestic cigarettes spanning the current range of nicotine deliveries. We also examine the delivery of VOCs as a function of selected cigarette design features and their delivery relative to nicotine delivery. VOL. 41, NO. 4, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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mL puff of 2 s duration every 60 s) on an automated linear 16-port ASM 500 smoking machine (Cerulean, Milton Keynes, UK) to a butt length of either filter + 8 mm or filter overwrap + 3 mm, whichever was greater.

TABLE 1. Linear Calibration Range, Calculated Limits of Detection (LOD), and Correlation Coefficients (R2) for the Vapor-Phase Portion of Mainstream Cigarette Smoke analyte

linear range (µg)

LOD (µg/cigarette)

R2

benzene p-xylene acetone o-xylene 2,3-butanedione 2-pentanone toluene styrene ethylbenzene 3-ethyltoluene 3-euten-2-one 3-pentanone 2-butanone

0.86-344 0.086- 34.4 2.78-1110 0.091-36.4 0.98-394 0.080-32.2 0.84-338 0.091-36.2 0.089-35.4 0.045-18.2 0.85-340 0.081-32.2 0.79-316

0.09 0.01 2.16 0.02 0.76 0.22 0.21 0.28 0.10 0.09 0.15 0.24 0.81

0.9999 0.9991 0.9986 0.9998 0.9999 0.9994 1.0000 0.9995 0.9996 1.0000 0.9999 0.9989 0.9994

Materials and Methods Materials. All target compounds were purchased from Aldrich Chemical Co. (Milwaukee, WI). Methanol was purchased from Tedia Company Inc. (Fairfield, OH). The ultrahigh purity gas helium was obtained from AirGas, Inc. (Atlanta, GA). Glass fiber Cambridge filter pads (CFP), 44 mm, were purchased from Whatman (Maidstone, England). Tedlar brand PVF bags, carboxen-polydimethylsiloxane (carboxen-PDMS) SPME fibers, and Mininert septum caps were purchased from Supelco (Bellefonte, PA). All chemicals and solvents were used without further purification. Internal Standards. An internal standard (IS) stock solution was prepared by adding 1 mL each of the perdeutero compounds acetone-d6, benzene-d6, styrene-d8, tetrahydrofuran-d8, toluene-d8, and p-xylene-d10 and diluting to 10 mL with methanol. Working solutions of 1:100 and 1:1000 dilutions, in methanol, were prepared monthly as ISs for the vapor and particulate phases respectively. These ISs were stored in airtight amber vials with Mininert septum caps and removed with a Hamilton (gas-tight) syringe as needed. A 10-µL aliquot of IS was added to each sample before analysis. Preparation of Standard Curves. We made a calibration stock solution by the addition of neat standards to methanol. The amount of each analyte added to the stock calibration mixture was recorded to the nearest 0.01 mg and diluted with methanol to a final volume of 10 mL. Serial dilutions of the calibration stock solution generated appropriate concentration ranges for each analyte (Table 1). These solutions were analyzed to prepare calibration curves for all 13 analytes in both the vapor and particulate phases. For the vapor phase, for which PVF bags were used to collect the smoke sample, calibration curves were generated by injection of a standard solution and IS into an empty bag and filled with 11 puffs of 35 mL from the smoking machine. The bag was then removed from the smoking machine and treated as a sample. Similarly, calibration curves for the particulate phase collected on the CFP were prepared by the addition of the standard solution and IS to a blank 20-mL headspace vial and treated as samples. All calibration curves were linear with R2 values exceeding 0.99. Calibration curves were prepared weekly and used for all analytical runs using the associated IS. Smoking Conditions. Prior to smoking, cigarette filter ventilation levels were measured with a QTM-5 (Cerulean, Milton Keynes, UK). For 24 h before smoking, cigarettes were conditioned in an environmental chamber (Parameter Generation Control, Inc, Black Mountain, NC) that was maintained, as specified in ISO 3402:1999, at a temperature of 22 °C and 60% relative humidity. Cigarettes were smoked according to the conditions detailed in ISO 3308:2000 (35 1298

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VOC Collection-Vapor Phase. The vapor-phase portion of mainstream cigarette smoke was collected in individual 1-L PVF bags attached directly to the exhaust ports of the ASM 500 puffing engines. To reduce background and sample carryover, the smoking machine was programmed to complete 30 blank puffs per port before attachment of the PVF bags. Background levels of all analytes were measured on a per port basis and corrections, if needed, were applied to all results for that day’s analytical runs. Prior to smoking, 10 µL of the 1:100 IS solution was added to each 1-L PVF bag. After smoking, the PVF bags were closed and immediately removed from the smoking machine. A portion of the smoke sample, collected in the PVF bag, was subsequently transferred to an evacuated 20-mL headspace vial (Microliter Corporation, Atlanta, GA) by a 27 gauge cannula. Vials, containing the analytical samples were subsequently loaded onto a LEAP Combi-Pal auto sampler (LEAP Technologies, Carrboro, NC) equipped with SPME sampling for analysis by GC/MS. VOC Collection-Particulate Phase. The particulate phase of the mainstream cigarette smoke was collected on a standard 44-mm CFP. After smoking, the CFP was removed from the holder and placed in a 20-mL headspace vial and spiked with 10 µL of the 1:1000 IS solution. The sample was then loaded on a LEAP Combi-Pal autosampler and analyzed by automated SPME/GC/MS. VOC Analysis. VOCs were quantitatively analyzed using an Agilent 6890 gas chromatograph coupled to an Agilent 5973 mass spectrometer. The sample was incubated at 30 °C for 2 min before the vial was sampled for 30 s with a 75-µm carboxen-PDMS SPME fiber. Analytes were then desorbed from the fiber at 260 °C in a heated inlet and focused onto an Agilent DB-624 capillary column (30.0 m × 320 µm × 1.80 µm). Helium flow was maintained in constant flow mode at an average linear velocity of 46 cm/sec. For the analysis of vapor-phase samples, the GC oven, equipped with liquid nitrogen cryo cooling, was programmed to start at -20 °C, hold for 2 min, and ramp to 200 °C at 8 °C/min, for a total run time of 29.50 min. The GC analysis of the particulate phase samples differed in that the oven started at 40 °C, was held constant for 2 min, and then ramped to 230 °C at 6 °C/min, for a total run time of 35 min. Both methods used a transfer line temperature of 255 °C and source and quadrupole temperatures of 230 °C and 150 °C, respectively. Data were acquired in full-scan mode over 30-200 amu. Cigarettes for Testing. We selected premium and value cigarette brands, ranging from full-flavor to ultralight, including both mentholated and non-mentholated varieties, from the top four domestic cigarette producers for analysis. The cigarettes were purchased at commercial retail outlets in the Atlanta metropolitan area or were provided by the Massachusetts Department of Public Health. The 1R4F and 2R4F research cigarettes were purchased from the University of Kentucky Tobacco and Health Research Institute (Lexington, KY). All cigarettes were stored in their original packaging at -70 °C until being conditioned prior to analysis. Quality Control Materials. The 2R4F research cigarette was used as a quality control (QC) material. QC cigarettes were smoked daily and analyzed with all runs performed that day. All 13 analytes measured in the smoke of the 2R4F cigarettes were characterized to determine the mean and the 95th and 99th confidence intervals for each analyte studied. Acceptance criteria for QC and blank samples followed the criteria prescribed by Taylor (27).

TABLE 2. Analyte Yields (in Micrograms Per Cigarette), Standard Deviation (σ), and Percent Relative Standard Deviation (%RSD) for the 2R4F Kentucky Research Cigarette over a 6 Month Period (N ) 100)

FIGURE 1. Vapor-phase sample stability as a function of time.

Results and Discussion Calibration, detection, and quantification of the 13 analytes were straightforward. The linear quantification range, LOD, and linear regression coefficients were more than adequate to provide quantitative data for the analytes studied (Table 1). The LODs for these analytes, calculated as three times the standard deviation extrapolated to zero concentration (27), ranged from 0.01 µg to 2.12 µg per cigarette. Although not needed for this study, switching to single ion monitoring rather than a full spectral scan should improve LODs. The results for each analyte in this study are presented on a percigarette basis which simplifies data analysis and provides for direct cigarette-to-cigarette comparisons. Accurate quantification of the chemicals in mainstream cigarette smoke dictates the analysis of both the particulate and vapor phases. We accomplished this by parallel analysis of the particulate matter located on the CFP and the vaporphase sample. Because of the highly volatile nature of the analytes selected for this study, we found the fraction in the particulate phase to be extremely low. Quantification of the 13 analytes in the particulate- and vapor-phase samples for the 2R4F research cigarettes confirmed that these analytes resided mainly in the vapor phase. In fact, the levels of 12 of the 13 analytes on the CFP had values below the method limit of detection (LOD). The only measurable analyte in the particulate phase, 2,3-butanedione, represented less than 2% of the measured vapor sample. Because of the nearly complete gas-phase partitioning, we only report levels measured in the vapor phase for the remainder of the analyses.

analyte

average

σ

%RSD

benzene toluene styrene o-xylene m/p-xylene 3-ethylbenzene 3-ethyltoluene acetone 3-buten-2-one 2,3-butanedione 2-butanone 2-pentanone 3-pentanone

44.1 57.4 2.2 1.7 9.9 4.4 1.6 366.9 45.7 89.3 86.3 12.8 6.1

5.1 5.8 0.4 0.3 1.3 0.6 0.6 64.2 7.5 13.1 11.8 2.4 1.3

11.7 10.2 17.0 14.8 13.6 13.3 37.1 17.5 16.3 14.7 13.7 18.6 20.8

TABLE 3. Vapor-phase Mainstream Cigarette Smoke Yields of Previously Reported Volatile Organic Compounds from a Kentucky 1R4F Research Cigarette. Literature Reported Values Included for Comparison. Values Are Shown ( Standard Deviation (n ) 5) analyte

delivery (µg/cig)

benzene toluene styrene xylenes acetone

41 ( 4 60 ( 7 2.9 ( 0.6 12.9 ( 1.9 340 ( 41

a

From ref 23.

b

previously Reported Results (µg/cig) 45,a 51,b 59,d 41e 68,a 73,b 61e 2.1,a 1.8e 10.6e 284,c 380d

From ref 28. c From ref 8.

d

From ref 26. e From ref

24.

Because of the potential for sample loss and the reactivity of selected analytes in this study, the choice of an appropriate IS was important. Isotopically labeled ISs for each analyte was not practical because of the large number and wide concentration ranges of the chemicals in cigarette smoke. Limiting the number of ISs simplifies sample preparation times and increases SPME sensitivity. Also, the use of a limited number of ISs can greatly reduce the cost per analysis. Therefore, we selected six perdeutero ISs for quantification of VOCs in mainstream cigarette smoke. Carbonyl compounds were quantified using either acetone-d6 or THF-d8 and aromatic compounds using benzene-d6, toluene-d8,

FIGURE 2. A typical vapor-phase full scan chromatogram for the 2R4F research cigarette. Compounds quantified in this study are labeled. For clarity, signal before 7 min and after 21 min are not shown. VOL. 41, NO. 4, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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TABLE 4. Delivery of Aromatic Volatile Organic Compounds in Mainstream Cigarette Smoke of 41 U.S. Brandsa % vent

benzene

toluene

styrene

o-xylene

m/p-xylene

Et-benzene

3-Et-toluene

Basic Camel (M) Newport (M) Salem (M) Camel Jade (M) Kool (M) GPC Marlboro Benson & Hedges Camel Marlboro (M) Kent Doral Winston

0 0 0 0 1 1 5 12 13 15 16 20 22 23

49.8 ( 5.9 52.9 ( 3.6 50.7 ( 2.5 56.8 ( 6.9 57.1 ( 3.5 50.5 ( 4.5 56.4 ( 0.7 46.8 ( 0.5 50.7 ( 2.4 56.6 ( 0.9 42.8 ( 3.3 38.3 ( 2.1 50.0 ( 7.4 51.5 ( 1.5

69.0 ( 9.6 74.6 ( 5.8 72.3 ( 3.7 81.2 ( 12.7 82.4 ( 3.7 73.1 ( 3.1 75.0 ( 2.8 67.0 ( 3.6 73.8 ( 6.4 76.1 ( 2.0 62.4 ( 6.2 57.2 ( 5.8 66.0 ( 12.0 68.9 ( 1.7

4.3 ( 0.4 5.0 ( 0.6 4.3 ( 0.0 5.3 ( 1.1 5.4 ( 0.4 4.2 ( 0.2 4.7 ( 0.2 3.9 ( 0.3 3.8 ( 0.9 4.8 ( 0.1 3.2 ( 0.2 3.0 ( 0.4 4.6 ( 0.7 4.1 ( 0.3

2.4 ( 0.1 2.7 ( 0.3 2.4 ( 0.0 2.9 ( 0.5 3.0 ( 0.3 2.3 ( 0.2 2.6 ( 0.1 2.2 ( 0.1 2.4 ( 0.4 2.7 ( 0.1 1.9 ( 0.2 1.7 ( 0.1 2.7 ( 0.4 2.4 ( 0.1

13.1 ( 1.2 15.0 ( 1.3 13.2 ( 0.4 16.0 ( 2.8 16.4 ( 1.4 13.4 ( 0.9 14.4 ( 0.8 12.8 ( 0.4 14.1 ( 2.2 14.7 ( 0.6 11.1 ( 1.0 9.8 ( 0.3 14.6 ( 1.5 13.7 ( 0.1

6.1 ( 0.7 6.9 ( 0.6 6.3 ( 0.3 7.5 ( 1.2 7.8 ( 0.7 6.2 ( 0.3 6.9 ( 0.3 5.6 ( 0.2 6.1 ( 0.9 6.7 ( 0.2 5.1 ( 0.4 4.6 ( 0.7 6.3 ( 0.6 6.1 ( 0.2

2.3 ( 0.3 2.8 ( 0.7 2.2 ( 0.6 2.9 ( 0.9 2.7 ( 1.1 1.8 ( 0.4 2.4 ( 0.0 2.3 ( 0.3 2.4 ( 0.8 2.6 ( 0.2 1.6 ( 0.3 1.3 ( 0.9 2.9 ( 0.7 2.2 ( 0.1

medium or mild brands GPC Marlboro (M) Newport (M) Marlboro

12 13 20 22

43.8 ( 3.6 43.9 ( 3.1 42.2 ( 4.2 41.9 ( 4.3

57.9 ( 8.5 62.0 ( 5.2 58.5 ( 6.1 57.6 ( 7.5

3.1 ( 0.2 3.0 ( 0.4 3.2 ( 0.5 3.0 ( 0.3

1.7 ( 0.1 1.9 ( 0.2 1.8 ( 0.2 1.9 ( 0.1

9.7 ( 0.9 10.8 ( 0.9 10.0 ( 1.3 11.0 ( 0.9

4.8 ( 0.3 5.2 ( 0.5 4.7 ( 0.6 4.9 ( 0.5

1.3 ( 0.2 1.5 ( 0.2 1.7 ( 0.3 1.6 ( 0.2

light brands Basic GPC Marlboro Newport (M) Camel Jade (M) Camel (M) Marlboro (M) Doral Camel Winston Misty Misty (M)

15 21 22 23 24 25 25 26 30 30 49 50

42.0 ( 0.7 38.2 ( 3.5 40.4 ( 3.0 31.6 ( 3.1 42.8 ( 3.8 39.6 ( 3.9 37.0 ( 5.9 38.2 ( 5.8 39.4 ( 3.5 44.5 ( 3.4 30.7 ( 2.6 25.4 ( 1.5

55.1 ( 1.4 50.3 ( 4.3 55.4 ( 6.5 43.2 ( 5.4 60.3 ( 4.7 57.1 ( 9.3 51.3 ( 9.4 53.4 ( 6.9 52.6 ( 6.6 57.2 ( 5.7 39.9 ( 3.3 32.3 ( 1.9

2.9 ( 0.2 2.5 ( 0.4 2.7 ( 0.1 1.8 ( 0.4 3.4 ( 0.3 2.8 ( 0.6 2.3 ( 0.3 2.9 ( 0.6 2.6 ( 0.3 2.9 ( 0.4 1.8 ( 0.3 1.2 ( 0.3

1.8 ( 0.1 1.5 ( 0.2 1.8 ( 0.2 1.2 ( 0.1 1.9 ( 0.2 1.6 ( 0.3 1.4 ( 0.3 1.8 ( 0.3 1.6 ( 0.2 1.9 ( 0.2 1.2 ( 0.1 0.8 ( 0.1

10.1 ( 0.3 8.5 ( 1.0 10.4 ( 1.2 6.8 ( 0.9 10.9 ( 1.1 9.7 ( 1.8 8.6 ( 1.7 10.0 ( 1.4 9.0 ( 1.1 10.4 ( 1.4 6.9 ( 0.8 4.8 ( 0.5

4.7 ( 0.1 4.1 ( 0.5 4.6 ( 0.5 3.3 ( 0.5 5.3 ( 0.5 4.8 ( 1.0 4.1 ( 0.6 4.4 ( 0.7 4.2 ( 0.5 4.8 ( 0.5 3.2 ( 0.3 2.2 ( 0.2

1.6 ( 0.4 1.4 ( 0.3 1.8 ( 0.3 0.9 ( 0.2 1.6 ( 0.4 1.1 ( 0.6 0.9 ( 0.5 1.8 ( 0.2 1.4 ( 0.3 1.5 ( 0.2 0.9 ( 0.1 0.5 ( 0.0

ultralight brands Basic Marlboro GPC Camel Marlboro (M) Winston Doral Misty (M) True Carlton Carlton (M)

32 47 49 52 53 55 61 67 68 77 78

25.6 ( 5.0 25.5 ( 2.8 26.7 ( 3.5 29.3 ( 1.1 27.3 ( 2.6 26.6 ( 0.2 19.7 ( 1.9 15.7 ( 1.3 15.0 ( 1.1 6.3 ( 0.6 3.7 ( 0.9

32.0 ( 6.7 33.6 ( 4.8 34.5 ( 4.0 39.7 ( 2.5 33.3 ( 3.3 31.7 ( 1.8 27.7 ( 2.9 21.0 ( 3.1 18.8 ( 2.1 7.3 ( 0.7 4.5 ( 1.3

1.4 ( 0.5 1.4 ( 0.3 1.9 ( 0.3 2.1 ( 0.3 1.2 ( 0.3 1.5 ( 0.3 1.2 ( 0.2 0.7 ( 0.2 0.9 ( 0.3 0.3 ( 0.1 0.3 ( 0.3

1.0 ( 0.3 1.0 ( 0.2 1.2 ( 0.2 1.4 ( 0.1 1.0 ( 0.1 1.1 ( 0.1 1.0 ( 0.0 0.7 ( 0.1 0.6 ( 0.1 0.3 ( 0.1 0.2 ( 0.1

5.6 ( 1.7 5.9 ( 1.1 6.5 ( 1.0 7.6 ( 0.6 5.8 ( 0.5 6.2 ( 0.4 5.7 ( 0.3 4.0 ( 0.8 3.7 ( 0.3 1.9 ( 0.3 1.5 ( 0.4

2.7 ( 0.7 2.7 ( 0.3 3.0 ( 0.4 3.4 ( 0.3 2.7 ( 0.3 2.8 ( 0.3 2.5 ( 0.2 1.7 ( 0.2 1.7 ( 0.2 1.0 ( 0.1 0.8 ( 0.2

0.7 ( 0.4 0.8 ( 0.4 1.1 ( 0.4 1.3 ( 0.4 0.7 ( 0.1 0.9 ( 0.1 0.8 ( 0.2 0.4 ( 0.3 0.5 ( 0.0 0.2 ( 0.1 0.0 ( 0.0

full flavored brands

a

All analytes are expressed as micrograms per cigarette along with the corresponding standard deviation, (M) denotes a mentholated brand.

p-xylene-d10, or styrene-d8. Appropriate pairing of IS to analyte was determined by a similarity in vapor pressure, structure, and as was the case for styrene, reactivity, to achieve optimal linear response. Sample stability was of great concern because of numerous reactive compounds associated with mainstream cigarette smoke. Previous reports have indicated problems with sample stability over time (24, 26). To examine sample stability, a 2R4F cigarette was smoked and aliquots of the vapor phase were transferred by cannula into 10 individual headspace vials. These vials were sequentially sampled and analyzed over the following 20 h. During this time, a steady decrease in the raw area counts was observed for all analytes. Although both analyte and IS ion counts decreased over time, change in the analyte relative response ratio was less than 15% for all analytes over a 20-hr period (Figure 1). Therefore, to minimize temporal variability, all samples were analyzed within 20 h of collection. Concentration of the sample on the SPME fiber, coupled with the relatively high abundances of the chosen analytes in mainstream cigarette smoke, provided sufficient sensitivity to allow full-scan mass spectral detection. Using full-scan data acquisition provided quantitative analysis of the analytes while retaining spectral information for qualitative analysis 1300

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of additional smoke constituents. The total ion chromatogram for a 2R4F research cigarette (Figure 2) demonstrates the good separation, high signal response, and adequate run time for all 13 analytes. Given the chemical complexity of smoke and the large number of chemicals readily identified in the vapor phase, this method could be readily expanded to analyze additional, volatile, cigarette smoke constituents. Method reproducibility was assessed by evaluating the results generated over a 6 month period for all 13 analytes when a 2R4F cigarette was machine smoked. During that time more than 100 analytical determinations were made for the mainstream smoke of 2R4F cigarettes. The average deliveries and standard deviations for the analytes studied were determined from these data (Table 2). The calculated relative standard deviation for all 13 compounds was, on average, 16.9% for the 2R4F cigarette. Day-to-day variability in this method can be attributed both to the variability in the analytical process and sample-to-sample variability among the 2R4F cigarettes. The recent replacement of the 1R4F reference cigarette with the 2R4F research cigarette made delivery comparisons with other researchers tenuous. To make meaningful comparisons, we also analyzed some of the older 1R4F cigarettes. However, of the 13 analytes reported here, only values for

TABLE 5. Delivery of Volatile Organic Ketones in Mainstream Cigarette Smoke of 41 U.S. Brandsa % vent

acetone

Basic Camel (M) Newport (M) Salem (M) Camel Jade (M) Kool (M) GPC Marlboro Benson & Hedges Camel Marlboro (M) Kent Doral Winston

full flavored brands

0 0 0 0 1 1 5 12 13 15 16 20 22 23

401.4 ( 43.5 511.7 ( 34.7 488.7 ( 6.0 534.2 ( 47.3 541.4 ( 39.6 494.0 ( 81.9 477.8 ( 25.8 402.7 ( 11.7 438.1 ( 27.0 470.7 ( 10.0 376.9 ( 31.4 376.0 ( 51.3 472.5 ( 97.0 433.3 ( 36.2

105.4 ( 3.5 128.1 ( 9.0 115.1 ( 8.7 128.0 ( 15.7 124.9 ( 12.5 107.2 ( 30.6 115.5 ( 3.9 101.3 ( 15.0 100.4 ( 10.6 122.2 ( 5.0 106.3 ( 9.5 95.0 ( 6.2 145.0 ( 71.0 119.6 ( 10.3

15.1 ( 0.4 21.4 ( 2.2 18.8 ( 2.6 21.2 ( 2.9 21.2 ( 3.2 16.8 ( 4.9 18.2 ( 0.5 15.1 ( 2.5 15.9 ( 2.3 18.6 ( 1.6 14.8 ( 1.4 12.0 ( 3.0 22.5 ( 8.5 16.5 ( 2.9

95.5 ( 6.4 126.2 ( 9.5 116.8 ( 7.5 127.9 ( 13.8 129.5 ( 13.5 107.7 ( 24.3 114.7 ( 4.4 94.8 ( 9.1 101.2 ( 8.8 115.4 ( 5.3 92.1 ( 7.0 83.3 ( 2.1 129.6 ( 46.3 104.3 ( 12.7

6.6 ( 0.4 9.7 ( 0.9 8.5 ( 1.0 9.8 ( 1.2 10.3 ( 1.8 8.3 ( 2.3 8.1 ( 0.4 6.7 ( 0.9 7.4 ( 1.5 8.5 ( 1.5 6.7 ( 0.7 5.3 ( 1.6 9.7 ( 3.0 6.9 ( 1.4

60.7 ( 2.7 78.2 ( 8.6 74.4 ( 3.5 81.5 ( 8.7 80.1 ( 8.7 79.1 ( 18.8 79.3 ( 6.0 64.0 ( 9.8 64.6 ( 5.8 73.5 ( 5.5 53.5 ( 5.8 49.2 ( 2.9 94.8 ( 47.5 68.7 ( 6.4

medium or mild brands GPC Marlboro (M) Newport (M) Marlboro

12 13 20 22

356.1 ( 21.4 358.6 ( 31.3 375.3 ( 31.0 332.2 ( 35.5

82.4 ( 9.9 88.6 ( 13.6 94.1 ( 12.9 79.8 ( 2.3

10.9 ( 0.8 12.8 ( 1.8 14.5 ( 3.2 12.2 ( 0.7

77.6 ( 1.6 83.9 ( 9.4 91.2 ( 13.8 78.5 ( 6.2

4.7 ( 0.3 5.6 ( 0.4 6.8 ( 1.3 5.3 ( 0.4

50.5 ( 6.4 47.7 ( 9.3 55.5 ( 7.7 53.2 ( 3.8

light brands Basic GPC Marlboro Newport (M) Camel Jade (M) Camel (M) Marlboro (M) Doral Camel Winston Misty Misty (M)

15 21 22 23 24 25 25 26 30 30 49 50

336.9 ( 3.2 304.7 ( 17.7 321.2 ( 20.9 301.6 ( 28.8 353.2 ( 27.8 353.4 ( 59.8 313.0 ( 20.3 294.5 ( 34.7 299.6 ( 28.6 350.1 ( 21.0 255.5 ( 17.9 232.6 ( 21.3

84.0 ( 5.7 75.8 ( 8.6 75.0 ( 6.7 73.5 ( 4.1 88.9 ( 5.1 78.7 ( 3.9 76.8 ( 2.8 73.7 ( 6.8 72.6 ( 2.5 88.3 ( 3.0 68.0 ( 1.2 64.7 ( 18.8

12.3 ( 0.9 10.7 ( 1.9 12.0 ( 1.4 10.2 ( 0.9 13.1 ( 0.4 10.7 ( 0.5 9.9 ( 1.8 10.2 ( 0.6 9.8 ( 1.0 12.1 ( 0.6 8.9 ( 0.4 7.5 ( 2.4

79.4 ( 2.2 70.5 ( 8.4 76.1 ( 5.7 68.0 ( 4.9 84.8 ( 4.1 76.9 ( 8.5 69.6 ( 7.0 67.5 ( 6.3 68.0 ( 3.1 81.7 ( 4.1 59.3 ( 3.0 52.1 ( 9.8

5.4 ( 0.4 4.7 ( 0.7 5.2 ( 0.7 4.6 ( 0.3 6.4 ( 0.3 4.6 ( 0.9 4.3 ( 0.8 4.5 ( 0.5 4.7 ( 0.8 5.2 ( 0.6 3.7 ( 0.3 3.6 ( 1.2

50.6 ( 1.5 44.8 ( 6.2 50.1 ( 2.3 41.2 ( 1.2 50.1 ( 3.2 44.9 ( 4.9 40.5 ( 2.7 42.4 ( 3.9 42.3 ( 3.4 52.2 ( 2.5 38.8 ( 1.4 38.3 ( 14.8

ultralight brands Basic Marlboro GPC Camel Marlboro (M) Winston Doral Misty (M) True Carlton Carlton (M)

32 47 49 52 53 55 61 67 68 77 78

194.0 ( 21.4 202.3 ( 25.1 213.6 ( 24.2 228.8 ( 12.7 197.4 ( 15.9 195.2 ( 5.8 156.1 ( 13.8 139.9 ( 6.2 122.2 ( 9.0 55.2 ( 4.7 45.6 ( 10.6

48.8 ( 6.8 48.0 ( 5.3 56.7 ( 8.4 57.3 ( 2.0 44.3 ( 3.7 49.5 ( 2.1 36.5 ( 0.7 36.4 ( 3.4 32.6 ( 2.4 15.1 ( 1.9 12.7 ( 2.0

6.0 ( 1.4 6.8 ( 1.3 7.1 ( 1.4 7.6 ( 0.8 6.1 ( 0.4 5.7 ( 0.2 4.8 ( 0.4 3.9 ( 0.6 3.3 ( 0.2 1.4 ( 0.1 1.1 ( 0.2

43.1 ( 6.6 45.5 ( 6.6 50.0 ( 7.3 52.3 ( 1.7 43.7 ( 2.6 42.7 ( 1.4 34.0 ( 1.6 29.7 ( 2.4 26.3 ( 1.6 11.7 ( 1.0 10.0 ( 1.8

2.7 ( 0.5 3.0 ( 0.5 3.3 ( 0.7 3.6 ( 0.5 2.6 ( 0.3 2.5 ( 0.1 2.1 ( 0.2 1.7 ( 0.3 1.5 ( 0.1 0.6 ( 0.0 0.5 ( 0.0

25.4 ( 4.3 30.5 ( 5.3 29.2 ( 5.5 31.1 ( 2.4 27.9 ( 3.6 26.2 ( 0.2 20.1 ( 2.4 17.7 ( 1.0 15.2 ( 0.6 7.5 ( 1.0 5.5 ( 0.9

a

2,3-butanedione 2-pentanone

2-butanone

3-pentanone 3-buten-2-one

All analytes are expressed as micrograms per cigarette along with the corresponding standard deviation, (M) denotes a mentholated brand.

acetone (8, 26), benzene (22-24, 26, 28), styrene (23, 24), toluene (23, 24, 28), and total xylenes (24) were reported in previous studies for the 1R4F research cigarette. Quantified values for the deliveries of these five analytes in 1R4F cigarette smoke were comparable to previously reported values (Table 3). These previous methods used a variety of smoke collection techniques, including the use of solvent filled cold traps as well as directly sampling the cigarette vapor phase or smoke, which differed from the PVF bag technique used in the current study. The agreement with previous work helps to support the validity of our reported results. Additionally, the measured values of all 13 analytes for the 1R4F research cigarette are comparable to the newer 2R4F research cigarette (Tables 4 and 5) and should provide a useful future reference. Brand and manufacturer differences among top-selling domestic cigarettes were investigated for the analytes in this study. Delivery data, along with filter ventilation are reported in Tables 4 and 5. When separated into groups on the basis of their FTC mainstream smoke delivery designations (fullflavor, medium, light, or ultralight), no statistical differences (p < 0.05) existed between the individual VOC smoke concentrations in the 41 commercial brands examined from the four major domestic manufacturers for brands grouped together according to comparable delivery. Further statistical analysis of analyte delivery for all 41 cigarette brands showed

that filter ventilation was the main cigarette design parameter influencing delivery of the 13 analytes. For example, benzene delivery, when analyzed as a function of filter ventilation (Figure 3), showed a good linear relation for all 41 brands, as did the other 12 VOCs analyzed in this study. In comparison, physical characteristics such as filler mass and filter length were much poorer predictors of the overall mainstream smoke delivery of these analytes. One limitation of the current study is the smoking parameters were based on standardized machine smoking methods, which do not accurately estimate smoke deliveries for modern cigarettes (29). Kozlowski et al. (17) have shown that smokers of low delivery cigarettes may compensate to obtain sufficient nicotine by blocking the vent holes or by taking more frequent, larger, and deeper puffs. Such compensation behavior would result in much higher deliveries of all constituents in cigarette smoke. If the present results are normalized to the reported nicotine delivery of the cigarette, all cigarettes studied had, within standard deviation, nearly identical deliveries of all 13 analytes (Figure 4). Work is under way in our laboratories to define the relationship between the amount of nicotine delivered under a wide range of smoking conditions and the resultant potential exposure to these and other VOCs in mainstream cigarette smoke. VOL. 41, NO. 4, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 3. The relation between filter ventilation and the level of benzene in the vapor phase of mainstream cigarette smoke for top selling domestic cigarettes. Each data point is the result of at least three measurements and error bars representing the standard deviation are included. A linear regression of the points in provided for reference.

FIGURE 4. Average selected analyte deliveries normalized to Federal Trade Commission (FTC) nicotine values. Errors bars are at one standard deviation.

Disclaimers Use of trade names is for identification only and does not imply endorsement by the Centers for Disease Control and Prevention or by the U.S. Department of Health and Human Services. The findings and conclusions in this report are those of the author(s) and do not necessarily represent the views of the Centers for Disease Control and Prevention.

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Received for review March 15, 2006. Revised manuscript received October 18, 2006. Accepted October 18, 2006. ES060609L