Carbon-Centered Radicals in Cigarette Smoke - ACS Publications

Anal. Chem. 2009, 81, 631–641

Carbon-Centered Radicals in Cigarette Smoke: Acyl and Alkylaminocarbonyl Radicals Judit Bartalis,†,| Yi-Lei Zhao,‡,⊥ Jason W. Flora,§ John B. Paine III,§ and Jan B. Wooten*,§,# Philip Morris USA Postgraduate Research Program, Philip Morris Interdisciplinary Network of Emerging Science and Technology (INEST), and Philip Morris USA Research and Technology Center, 601 East Jackson Street, Richmond, Virginia 23219 The widely accepted mechanism of formation for carboncentered radicals in the gas-phase cigarette smoke involves reactions of NO2 and alkadienes. However, specific examples of such radicals have never been isolated from fresh cigarette smoke or their structure determined. We have identified two previously unrecognized classes of carbon-centered radicals, alkylaminocarbonyl and acyl radicals, that are unrelated to radicals that form by NOx chemistry. The combined abundance of these mainstream smoke radicals is significantly higher than the alkyl radicals previously quantified by electron paramagnetic resonance (EPR) solution spin-trapping methods. The new radicals were trapped directly from smoke with either 3-amino-proxyl (3AP) or 3-cyano-proxyl radical on a solid support and identified by combination of chemical synthesis, deuterium labeling, high-resolution mass spectrometry, nuclear magnetic resonance (NMR) spectroscopy, and ab initio quantum mechanical calculations. 3AP-R adducts were quantified both by high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) and by high-performance liquid chromatography with fluorescence detection (HPLC/ FLD). Seven acyl and 11 alkylaminocarbonyl radicals were identified in the whole smoke of cigarettes made from single tobacco varieties and blended tobacco research cigarettes. The overall yield of these radicals was measured to be 168-245 nmol/cigarette from machinesmoked cigarettes under Federal Trade Commission (FTC) conditions. The yield was significantly reduced when the gas-phase smoke was separated from whole smoke by filtration through a 0.1 µm Cambridge filter pad or upon aging whole smoke in an inert tube. Cigarette smoke, and smoke from biomass and fossil fuel combustion in general,1 is well-known to contain free radicals and * To whom correspondence should be addressed. Phone: 919-843-1496. E-mail: [email protected]. † Philip Morris USA Postgraduate Research Program. ‡ Philip Morris Interdisciplinary Network of Emerging Science and Technology. § Philip Morris USA Research and Technology Center. | Current address: Synthetic Genomics, Inc. 11149 North Torrey Pines Road, Suite 100, La Jolla, CA 92037. ⊥ Current address: NIST Center for Theoretical and Computational Nanosciences, NIST, 100 Bureau Drive MS 8380, Gaithersburg, MD 20899. # Current address: Department of Biomedical Engineering, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7178. 10.1021/ac801969f CCC: $40.75  2009 American Chemical Society Published on Web 12/18/2008

reactive oxygen and nitrogen species.2-9 Free radicals in particular have been noted as a source of both oxidative damage and oxidative stress.10,11 Pryor and co-workers made extensive measurements of radicals in cigarette smoke, focusing on the separate analysis of radicals in the particulate and gas-phase of smoke.4,6 For this purpose, the mainstream whole smoke exiting the filter end of the cigarette was separated into particulate-phase smoke, also called the total particulate matter (TPM), and gas-phase smoke by means of a Cambridge filter pad.12 The gas-phase cigarette smoke radicals were trapped by bubbling the gas-phase smoke into a solution containing the nitrone spin trap N-tert-butylR-phenylnitrone (PBN), and the radical adducts were quantified by electron paramagnetic resonance (EPR) spectroscopy. The predominant gas-phase radicals were shown to be alkoxyl (RO · ), and alkylperoxy (ROO · ) radicals, but Cueto and Pryor13 reported that carbon-centered radicals contribute ca. 30% to the total estimated yield. Using solid-phase radical trapping, an approach first applied by Church and Pryor6 and later by Flicker and Green,14,15 we recently showed that a significant amount of carbon-centered radicals is present in fresh mainstream cigarette smoke.16 Previously, EPR and FT-IR studies showed that the reaction between NO2 and dienes (e.g., isoprene, butadiene) plays a primary role in the formation of gas-phase cigarette smoke radicals resulting (1) Dellinger, B.; Pryor, W. A.; Cueto, R.; Squadrito, G. L.; Hegde, V.; Deutsch, W. A. Chem. Res. Toxicol. 2001, 14, 1371–1377. (2) Halliwell, B.; Gutteridge J. M. C. Free Radicals in Biology and Medicine, 3rd ed.; Oxford University Press: New York, 1999; pp 584-590. (3) Kodama, M.; Kaneko, M.; Aida, M.; Inoue, F.; Nakayama, T.; Akimoto, H. Anticancer Res. 1997, 17, 433–438. (4) Pryor, W. A.; Prier, D. G.; Church, D. F. Environ. Health Perspect. 298, 47, 345–355. (5) Pryor, W. A.; Tamura, M.; Church, D. F. J. Am. Chem. Soc. 1984, 106, 5073–5079. (6) Church, D. F.; Pryor, W. A. Environ. Health Perspect. 1985, 64, 111–126. (7) Zang, L. Y.; Stone, K.; Pryor, W. A. Free Radical Biol. Med. 1995, 19, 161– 167. (8) Maskos, Z.; Khachatryan, L.; Cueto, R.; Pryor, W. A.; Dellinger, B. Energy Fuels 2005, 19, 791–799. (9) Yan, F.; Williams, S.; Griffin, G. D.; Jagannathan, R.; Plunkett, S. E.; Shafer, K. H. J. Environ. Monit. 2005, 7, 681–687. (10) MacNee, W. Proc. Am. Thorac. Soc. 2005, 2, 50–60. (11) Wooten, J. B.; Chouchane, S.; McGrath, T. E.Tobacco Smoke Constituents Affecting Oxidative Stress. In Cigarette Smoke and Oxidative Stress; Halliwell, B. B., Poulsen, H. E., Eds.; Springer: Heidelberg, 2006; Chapter 2, pp 5-46. (12) Dube, M. F.; Green, C. R. Recent Adv. Tob. Sci. 1982, 8, 42–102. (13) Cueto, R.; Pryor, W. A. Vib. Spectros. 1994, 7, 97–111. (14) Flicker, T. M.; Green, S. A. Anal. Chem. 1998, 70, 2008–2012. (15) Flicker, T. M.; Green, S. A. Environ. Health Perspect. 2001, 109, 765–771. (16) Bartalis, J.; Chan, W. G.; Wooten, J. B. Anal. Chem. 2007, 79, 5103–5106.

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from the oxidation of NO in smoke to NO2.5,13 The amount of NO in cigarette smoke is typically in the range of 100-600 µg/ cigarette.17 Nevertheless, using our highly efficient solid-phase trapping method, we did not find radicals of the nitroalkyl variety;18 rather, we identified only acyl and aminoalkylcarbonyl radicals that cannot form via the same mechanism. Several other groups have also reported EPR spectra for gasphase spin-trapped radical adducts.11,19,20 EPR spectroscopy, however, only allows differentiation between R · , RO · , and ROO · radicals based on their g-values and hyperfine coupling constants; it cannot provide precise structural identification. Structures of the individual radical species detected by the EPR spin-trapping approach have not previously been independently confirmed by isolation and spectrometric identification. Moreover, EPR analysis of gas-phase smoke radicals relies on the separation of gas-phase smoke from the TPM using Cambridge filter pads. Such filtration is known to induce changes in the chemistry of cigarette smoke.21,22 The present work presents a detailed investigation on the identification and quantification of carbon-centered radicals in the mainstream smoke from various single-component and blended tobacco cigarettes employing the solid-phase trapping method. We optimized the method of Flicker and Green14,15 for trapping efficiency, sample preparation, and high-performance liquid chromatography with fluorescence detection (HPLC/FLD) analysis, identified the carbon-centered radicals by utilizing high-resolution mass spectrometry, deuterium exchange, organic synthesis, and solution nuclear magnetic resonance (NMR) spectroscopy, and employed ab initio quantum mechanical calculations to confirm the structures. In addition, fast screening and semiquantification of some smoke samples was accomplished directly by highperformance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) without derivatization of the radical adducts with a fluorophore. We investigated the effects of various analytical parameters on the experimentally measured yield including oxidative atmosphere, temperature, filtration by the Cambridge pad, aging, and solution-phase trapping. Model cigarette smoke gas mixtures based on NOx chemistry were scrutinized to identify possible carbon-centered radicals matching the cigarette smoke radicals. MATERIALS AND METHODS Materials. HPLC-grade organic solvents (methanol, methylene chloride, acetonitrile) and water, NaCN, PBN, 0.2 µm Puradisc PVDF syringe filters (Whatman), toluene, 6 N certified hydrochloric acid, and solid borosilicate glass beads of 2 and 3 mm diameter were purchased from Fisher Scientific (Pittsburgh, PA), and Na2B4O7, 3-amino-proxyl radical (3AP), cyclopentylamine (17) Counts, M. E.; Morton, M. J.; Laffoon, S. W.; Cox, R. H.; Lipowicz, P. J. Regul. Toxicol. Pharmacol. 2005, 41, 185–227. (18) Atkinson, R.; Aschmann, S. M.; Winer, A. M.; Pitts, J. N., Jr Int. J. Chem. Kinet. 1984, 16, 697–706. (19) Baum, S. L.; Anderson, I. G. M.; Baker, R. R.; Murphy, D. M.; Rowlands, C. C. Anal. Chim. Acta 2003, 481, 1–13. (20) Culcasi, M.; Muller, A.; Mercier, A.; Cle´ment, J. L.; Payet, O.; Rockenbauer, A.; Marchand, V.; Pietri, S. Chem.-Biol. Interact. 2006, 164, 215–231. (21) Baker, R. R. Smoke Chemistry. In Tobacco. Production, Chemistry and Technology; Davies, E. L., Nielsen, M. T., Eds.; Blackwell Science: Oxford, U.K., 1999; pp 398-439. (22) Shorter, J. H.; Nelson, D. D.; Zahniser, M. S.; Parrish, M. E.; Crawford, D. R.; Gee, D. L. Spectrochim. Acta, Part A 2006, 63, 994–1001.

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(CPA), pure anhydrous Na2CO3, and 2,5-dimethoxyaniline (DMOAn) were from Acros Organics (Morris Place, NJ). The naphthalene-2,3-dicarboxaldehyde (NDA), 3-cyano-proxyl radical (3CP), ACS-grade chemicals (acetic anhydride, glacial acetic acid, ethyl acetate, and zinc, and methyl-isocyanate (MIC) (Supelco)) were purchased from Sigma-Aldrich (St. Louis, MO). All chemicals were purchased at their highest quality. The glass columns for trapping radicals and quartz column for pyrolysis were ordered from Research Glass (Richmond, VA). The smoke filter assembly consisted of a Cambridge Pad inserted into a disposable holder (Gelman Sciences, Ann Arbor, MI). Inert Kynar tubing of 0.25 in. i.d. (Cole Parmer Instrument Co., Vernon Hills, IL) was purchased through Fisher Scientific (Pittsburgh, PA). Cigarettes. Single tobacco component cigarettes with a similar construction to 2R4F research cigarettes were machine-made from bright (Br), burley (Bu), oriental (Or), reconstituted leaf (RL) tobaccos, and a blend (BL) of each of these four types of tobacco (30% Br, 30% Bu, 15% Or, and 25% RL). RL is manufactured from tobacco dust (80-90%) and binder, humectants, preservatives, and flavors (Gellatly et al.23). Base-web (BW) cigarettes are prepared from the warm water-insoluble part of the tobacco leaf stems. Kentucky 2R4F research-grade cigarettes were procured from the University of Kentucky (Davies and Vaught24). The design of 2R4F cigarettes is representative of domestic filtered cigarettes manufactured by major U.S. cigarette companies, and its smoke chemistry has been thoroughly investigated. The 2R4F blend composition has previously been described: 32.5% Br, 19.9% Bu, 11.0% Or, 27.1% RL, 1.2% Maryland, 2.8% glycerin, and 5.3% inverted sugar.17,25 Filter cigarettes containing cellulose as the only combustible leaf constituent were machine-made and contained ca. 40% cellulose and 60% Ca2CO3. All cigarettes were conditioned at 24 °C and 60% relative humidity in a controlled laboratory for at least 24 h prior to smoking. The 2R4F cigarettes are ventilated cigarettes with 29% ventilation achieved at the cigarette filter. Cigarette Smoking and Pyrolysis. Glass beads were coated with 3AP as follows: 330 g of beads placed into a round-bottom flask was covered slightly with acetone, and 18 mg of 3AP was added to it after dissolving in acetone. The content of the flask was mixed well manually, and the solvent was evaporated by rotary evaporator with mild rotation and 30 °C. The beads were transferred into a beaker and dried for 1 h in a desiccator at room temperature. The 110 g of beads were transferred into a trap that was plugged at the narrow end with glass wool without restricting the airflow. Three traps were prepared in a similar way for triplicate analysis, and a fourth trap was loaded with 110 g of uncoated beads as a control. Cigarette smoke samples were collected under standard Federal Trade Commission (FTC) conditions.26 The radical trapping device to collect whole smoke or gas-phase smoke is illustrated in Figure 1. The surface area of glass beads loaded into the trap of 35 mL void volume was 860 cm2 (3 mm diameter beads) for standard measurements. Additionally, 2 mm diam(23) Gellatly, G.; Keritsis, G.; Wrenn, S. E. U.S. Patent 5,724,998, 1988. (24) Davies, M. H.; Vaught, A. The Reference Cigarette. Kentucky Tobacco Research and Development Center; University of Kentucky: Lexington, KY, 1990. (25) Chen, P. X.; Moldoveanu, S. C. Beitr. Tabakforsch. 2003, 20, 448–458.

Figure 1. Radical trapping device to collect whole smoke or gasphase smoke (dimensions 32.5 cm long including the 3 cm long narrower parts × 2.2 cm o.d.).

eter beads were employed in some experiments to evaluate the trapping efficiency of a larger surface area (1290 cm2). Three cigarettes were smoked in series on a smoking machine in a conditioned room. For the collection of aged smoke, variable lengths of Kynar tubing were inserted between the cigarette and the trap to age the smoke for 0.5, 1, 2, or 3 min. All smoking experiments involved three replicates and one control. The procedure for the pyrolysis experiments employed a tube furnace as described elsewhere.27 The radical trap was connected to the end of the quartz pyrolyzing tube via a short Swagelok stainless steel fitting. The cigarette filter and 3 mm of the tobacco rod was cut off the 2R4F research cigarette, and the remainder of the tobacco rod was placed inside the pyrolysis chamber in a ceramic boat. The tobacco sample was conditioned in the appropriate gas for 5 min, then heated for 10 min. Three cigarette rods were pyrolyzed in series, and the boat was cooled each time below 40 °C before the next heating cycle. Experiments were conducted at both 300 and 600 °C in He or 5% O2 in He as carrier gas at a flow rate of 50 mL/min. After smoke or pyrolysate collection, the beads were transferred into an Erlenmeyer flask containing 25 mL of 5% aqueous acetonitrile. The flask was closed and shaken gently for 30 min. Part of the sample was filtered through a 0.2 µm PVDF syringe filter and analyzed immediately or stored at +4 °C in a glass vial for later analysis. Solution Trapping of Smoke Radicals. The protocol of Pryor et al.4 was followed with modifications. The whole mainstream smoke was bubbled into an impinger containing 4 mL of toluene and 6 mg of 3AP. The trapping solvent was either held at room temperature or cooled with ice water. One cigarette was smoked per sample under FTC conditions. The 3AP-R adducts were extracted from toluene with 2 mL, 1 mL, and 1 mL of 0.01 N HCl, sequentially, and the extracts were combined. For chromatographic evaluation, the volume of the sample was normalized to smoke samples collected on glass beads by diluting 2 mL of sample with 2.15 mL of 5% aqueous acetonitrile. The HPLC analysis was conducted similar to other smoke samples. Derivatization and HPLC/FLD Analysis. An amount of 150 µL of samples collected on glass beads or by solution trapping was mixed with 50 µL of 10 mM NaCN in borate buffer (25 mM, (26) Pillsbury, H. C., Jr. Review of the Federal Trade Commission method for determining cigarette tar and nicotine yield. In The FTC cigarette test method for determining tar, nicotine, and carbon monoxide yields of U.S. cigarettes. Report of the NCI Expert Committee. National Cancer Institute Smoking and Tobacco Control Monograph. NIH Publication No. 96-4028; NIH: Bethesda, Maryland, 1996; pp 9-14. (27) McGrath, T. E.; Chan, W. G.; Hajaligol, M. R. J. Anal. Appl. Pyrolysis 2003, 66, 51–70.

pH 9.2) and 50 µL of 10 mM NDA in acetonitrile, and the volume was adjusted to 1 mL with borate buffer. An amount of 18 µL of 1 mg/mL 3AP in borate buffer was added to the control samples (smoke or pyrolysate). This solution was briefly vortexed and gently shaken for an additional 30 min, protected from light exposure by wrapping in aluminum foil. The reaction product was then diluted 10-fold with 50% aqueous methanol and analyzed by HPLC/FLD. The parameters on the HP-1100 HPLC system (Agilent Technologies) were as follows: autosampler at ambient temperature, injection volume 10 µL, column temperature 30 °C, and flow rate 0.5 mL/min. The LC column was Symmetry C18 (150 mm × 3.9 mm, 5 µm, Waters). The LC conditions were as follows: solvent A, water; solvent B, methanol; column equilibration time with 75% B 10 min before injection; elution gradient, 0-3 min 75% B; 3-20 min 75-90% B; 20-30 min 90% B. The FLD was operated at 420/480 nm excitation/emission wavelengths. The NDA adduct of CPA was used as the external standard for quantification. A linear regression curve based on peak areas was built out of six points for the CPA-NDA concentration range of 8.6-841.5 nM (r2 > 0.9999), with a limit of detection (LOD) of 0.2 nM (57.5 pg/mL). All compounds were quantified by considering a response factor 1.0 relative to the external standard. Collection of Radicals from Model Gas Mixtures. The model gas mixtures NO/isoprene/air, NO/isoprene/methanol/ air, and NO2/isoprene/N2 were prepared in a 2 L glass chamber. Commercially available gases (NO, NO2, isoprene, nitrogen) were supplied separately to the mixing chamber by a computerized multicomponent gas mixer (Environics Inc., Tolland, CT). Control samples were prepared containing no isoprene or methanol. The initial concentration of NO and NO2 in the 500 ppm NO/air mixture (at 0 s) was 426 and 9.4 ppm, respectively, as measured by lead salt mid-infrared tunable diode laser absorption spectrometry (MIR-TDL) (Aerodyne Research, Inc.). To obtain the desired gas mixtures, nitric oxide at 500 ppm was mixed with 0, 500, or 3000 ppm isoprene and 0 or 150 ppm methanol in air; NO2 at 500 ppm was mixed with either 500 or 1000 ppm isoprene in nitrogen. Several samples were collected for radical measurements before and after the expected half-lifetime of NO (t1/2). The concentration of NO and NO2 were recorded by MIR-TDL at reaction times ranging from 34.0 to 282.5 s in the NO/air mixtures in the presence or absence of either isoprene or methanol. The gas mixture was continuously admitted into a 20 m long Kynar tubing at a predetermined flow rate (30-1000 mL/min). The reaction time was determined by the time required for the gases to pass through the mixing chamber and tubing into the radical trap. The gas exiting the trap was continuously monitored for both NO and NO2 levels by MIR-TDL, which provides real-time detection of these gases with high sensitivity and selectivity (Harward et al.28). Synthesis of Model Compounds. 3CP-C(O)CH3. A mixture of 3CP (10 mg), acetic anhydride (100 µL), and glacial acetic acid (400 µL) in presence of excess zinc dust was stirred at ambient temperature. After 2 h, the mixture was diluted with 50% aqueous methanol (2 mL) and filtered. The filtrate was diluted with 5% (28) Harward, C. N.; Thweatt, W. D.; Baren, R. E.; Parrish, M. E. Spectrochim. Acta, Part A 2006, 63, 970–980.


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aqueous Na2CO3, and the product was extracted with CH2Cl2. The dried sample was redissolved in 2 mL of 35% aqueous methanol for HPLC-MS/MS analysis or in 0.5 mL deuterated chloroform for NMR analysis.16 3CP-C(O)NHCH3. A mixture of 3CP (10 mg), glacial acetic acid (50 µL), MIC (50 µL), and ethyl acetate (400 µL) in presence of excess zinc dust was stirred at ambient temperature. After 2 h, additional ethyl acetate (2 mL) was added and the solution filtered through a 0.2 µm PVDF syringe filter. The product was extracted into the aqueous phase by adding 2 mL of water and 2 mL of CH2Cl2 to the reaction mixture. The aqueous phase was neutralized with Na2CO3 and analyzed by HPLC-MS/MS. Hydrolysis of 3CP-Trapped Smoke Radicals. Smoke radicals were trapped with 3CP similar to 3AP on glass beads. The 3CP-R products were washed off the beads with 25 mL of 5% aqueous acetonitrile and filtered. One part of sample was mixed with two parts of Na2CO3 of 1 mg/mL and left to stand at room temperature for 1-2 days. Electrospray Ionization Tandem Mass Spectrometry (ESI(+)-MS/MS) Screening of Smoke Samples. Mass spectrometric analyses were conducted with a Micromass Quattro Ultima triple quadrupole MS (Waters Corp.) controlled by MassLynx 4.0 software (Waters Corp.). Smoke samples were infused in normal or deuterated 5% aqueous methanol (5% CH3OD in D2O) and 0.1% formic acid at 10 µL/min. The deuterated solvent was employed to determine number of active hydrogens for each MH+ and product ion by isotopic exchange. Ionization conditions were capillary 3 kV, cone 35 V, sheath gas at maximum, auxiliary gas at 120 L/min, source block 110 °C, desolvation 300 °C, ND collision potential 15 V. Precursor ion monitoring (PIM) was employed for both 3AP-R and 3CP-R samples, considering major fragments m/z 98 and 151, respectively. HPLC-MS/MS Separation and Quantification. For the separation of 3AP-R or 3CP-R samples the C18 Symmetry (100 mm × 2.1 mm, 3.5 µm, Waters) HPLC column was employed at a flow rate of 0.3 mL/min. Both eluent water (A) and methanol (B) contained 0.1% formic acid. Equilibration time was 10 min with the initial eluent mixture. Gradient elution for 3AP-R analysis is as follows: 0-5 min 5% B; 5-20 min 5-45% B; 20.1 min 75% B; 20.1-25 min 75% B. The gradient elution for 3CP-R analysis is as follows: 0-4 min 35% B; 4-20 min 35-65% B; 20-25 min 65-75% B. Precursor ion monitoring by focusing on the base peak (m/z 98 for 3AP-R, and m/z 151 for 3CP-R) permitted the detection of less abundant radical adducts. Multiple reaction monitoring (MRM) focused on the base peak permitted the radical adducts in various samples to be monitored. A capillary voltage of 1 kV and a cone voltage of 40 V were chosen for optimal signal intensity. The remaining parameters presented in the Hydrolysis of 3CP-Trapped Smoke Radicals section were unchanged. Direct mass spectrometric infusion and application of the PIM afforded highly selective spectra for 3AP-R (m/z 98) and 3CP-R (m/z 151) samples, respectively. Utilizing direct infusion, we were able to quickly screen smoke samples, compare various smoke samples by pattern recognition, confirm the presence of radicals, and determine the number of active hydrogens by isotopic labeling. For semiquantitative analysis, samples were diluted 1:10 with 4% aqueous methanol. The internal standard DMOAn at 50 ng/ 634


mL in 5% aqueous acetonitrile was added to the analyte to monitor signal stability. The HPLC run was divided into four different time segments (0-7.5 min, m/z 216 f 98, 230 f 98, and 154 f 139; 7.5-11 min, 201 f 98, 244 f 98, and 154 f 139; 11-15 min, 215 f 98, 258 > 610 > 98, and 154 f 139; 15-19 min, 229 f 98, 272 f 98, and 154 f 139). The response factor of alkylaminocarbonyl radicals relative to DMOAn either from whole smoke or gas-phase smoke samples was based on the concentration of methylaminocarbonyl radical determined by HPLC/FLD in the appropriate whole-smoke samples and fluctuated between 1-1.3 among various samples and runs. Similarly, the response factor of the acyl radicals was deduced from the concentration of acetyl radicals measured in whole smoke by LC/FLD and fluctuated between 1.5-2.5. Exact Mass Measurement by ESI(+)-LTQ-FTICR-MS. Pure HPLC fractions of 3AP-R and 3CP-R smoke samples were evaluated by direct infusion into a 7-T Thermo Finnigan (San Jose, CA) LTQ-FT hybrid linear ion trap Fourier transform ion cyclotron resonance mass spectrometer (LTQ-FTICR-MS) with XCalibur 2.0 software and a Thermo Finnigan ESI source. All samples contained 0.1% formic acid, were infused at a flow rate of 4 µL/min and ESI voltage of 3 kV. The instrument was set to 100 000 resolution (at 400 m/z), profile mode, positive ion mode, a microscan count of 1, and an m/z range of 50-400. External calibration was conducted using the automatic calibration feature in XCalibur tune mode on ions produced by a Thermo Finnigan recommended calibration mixture of caffeine, MRFA, and Ultramark. Mass accuracy confirmation was conducted by direct infusion of a caffeine standard (theoretical m/z 195.087 652) resulting in a mass accuracy of 0.420 ppm. Elemental compositions, mass accuracies, and peak resolutions were calculated by the XCalibur software. Quantum Mechanical Calculations. The newly discovered nitrogen-containing radicals were investigated by ab initio quantum mechanics, allowing us to predict and identify the correct isomeric forms. Specifically, the bond dissociation energy of all possible structures with a molecular formula of C2H4NO was computed. A robust complete basis set method (CBS-QB3)29 implemented in the “Gaussian-03” software package30 was employed for explorations of the nitrogen-containing radical structures. The computational method combines the advantages of speed and accuracy of density functional theory and wave function-based techniques and has been validated29 to a chemical accuracy of 4 kJ/mol for the G2 test set of organic compounds. RESULTS HPLC/FLD Evaluation of Smoke Samples. Cigarette smoke radicals were scavenged from whole smoke with the trapping agent 3AP coated on glass beads in a solvent-free atmosphere with the device shown in Figure 1. The 3AP-radical adducts derivatized with the NDA fluorophore and the NDA-3AP-R adducts were then separated and quantified by HPLC/FLD. Representative chromatograms for the 2R4F research cigarette mainstream smoke and appropriate control samples are shown in Figure 2. Ten radical adducts 1-10 were identified in the whole smoke by mass spectrometry as described below. Only (29) Montgomery, J. A.; Frisch, M. J.; Ochterski, J. W.; Petersson, G. A. J. Chem. Phys. 1999, 110, 2822–2827.

Figure 2. Representative HPLC/FLD chromatograms of fresh 2R4F whole smoke, gas-phase smoke, and whole-smoke control. The NDA-3AP-R adducts are labeled 1-10. Excess of NDA forms a large peak (/) that coelutes with compounds 5-7. NDA-3APH and the hydroxylamine NDA-3AP elute at 10.1 and 10.4 min, respectively. Column: Symmetry C18 (150 mm × 3.9 mm, 5 µm). Gradient elution at 0.5 mL/min with aqueous methanol: 0-3 min 75%, 3-20 min 75-90%, 20-30 min 90%. Fluorescence detection at 420 nm (Ex)/480 nm (Em).

radicals 1-4 were possible to quantify by LC/FLD as the remainder coeluted with interfering peaks as shown for the example of the control sample in Figure 2. The excess of NDA necessary for the derivatization31,32 created a large peak that coeluted with radicals 5-7; it also created additional peaks by reaction with excess 3AP, 3APH, and primary amines from smoke and primary amine impurities from the original 3AP stock solution. Although a few small HPLC peaks were sample-specific, they did not correspond to radical adducts and they may represent adducts of 3AP with compounds other than carbon-centered radicals, e.g., Br2-, CO3-, I2-, and · OH.33 The stability of the 3AP-R and NDA-3AP-R adducts was also investigated. 3AP-R samples dissolved in 5% acetonitrile stored at +4 °C demonstrated absolute stability for 1 week, which is consistent with an earlier report for adducts of another nitroxide with carbon-centered radicals.34,35 The NDA-3AP-R complex prepared for HPLC analysis did not degrade for at least 18 h. HPLC-MS Evaluation of Smoke Samples. The unambiguous detection of the three most abundant NDA-3AP-R adducts 1-3 by HPLC/FLD-MS afforded the mass units of these individual radicals. With the knowledge of these specific masses, (30) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Montgomery, J. A., Jr.; Vreven, T.; Kudin, K. N.; Burant, J. C.; Millam, J. M.; Iyengar, S. S.; Tomasi, J. J.; Barone, V.; Mennucci, B.; Cossi, M.; Scalmani, G.; Rega, N.; Petersson, G. A.; Nakatsuji, H.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Klene, M.; Li, X.; Knox, J. E.; Hratchian, H. P.; Cross, J. B.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Ayala, P. Y.; Morokuma, K.; Voth, A.; Salvador, P.; Dannenberg, J. J.; Zakrzewski, V. G.; Dapprich, S.; Daniels, A. D.; Strain, M. C.; Farkas, O.; Malick, D. K.; Rabuck, A. D.; Raghavachari, K.; Foresman, J. B.; Ortiz, J. V.; Cui, Q.; Baboul, A. G.; Clifford, S.; Cioslowski, J.; Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi, I.; Martin, R. L.; Fox, D. J.; Keith, T.; Al-Laham, M. A.; Peng, C. Y.; Nanayakkara, A.; Challacombe, M.; Gill, P. M. W.; Johnson, B.; Chen, W.; Wong, M. W.; Gonzalez, C.; Pople, J. A. Gaussian 03, revision C.02; Gaussian, Inc.: Wallingford, CT, 2004. (31) DeMontigny, P.; Stobaugh, J. F.; Givens, R. S.; Carlson, R. G.; Srinivasachar, K.; Sternson, L. A.; Higuchi, T. Anal. Chem. 1987, 59, 1096–1101. (32) Lai, F.; Sheehan, T. BioTechniques 1993, 14, 642–649. (33) Blough, N. V. Environ. Sci. Technol. 1988, 22, 77–82.

Figure 3. Representative LC-MS/MS results for 2R4F whole-smoke radicals trapped with 3AP and internal standard DMOAn. Four segments of multiple reaction monitoring (MRM) scans (3AP-R, m/z [M + 1] f 98; DMOAn, m/z 154 f 139) were run during which three sets of MRM scans were performed. Column: Symmetry C18 (100 mm × 2.1 mm, 3.5 µm). Gradient elution at 0.3 mL/min: 0-5 min 5% methanol with 0.1% formic acid (B) and water with 0.1% formic acid, 5-20 min 5-45% B, 20.1-25 min 75% B.

subsequent analysis by HPLC-MS of the underivatized 3AP-R adducts in smoke condensate recovered from the glass beads permitted the identification of the same radical species directly by mass spectrometry (Figure 3). The primary amine group on 3AP provides high sensitivity for MS detection using the positive ionization source. Collision-induced dissociation (CID) of 3AP-1-3AP-3 in the tandem mass spectrometer revealed a major ion with m/z 98(0) corresponding to a major fragment of the radical scavenger’s structure with molecular formula C6H12N+. The number in parentheses indicates the number of active hydrogens determined by deuterium exchange. By focusing on this fragment, all precursor ions could be monitored, and as a result, 20 peaks were tentatively identified as radicals. For all the molecular ions, peaks specific to 3AP ( m/z 72(0), 84(0), 124(0), and 141(2) were detected with the aid of product ion scanning. (34) Kieber, D. J.; Blough, N. V. Free Radical Res. Commun. 1990, 10, 109– 117.


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Table 1. Precise Mass, Chemical Structure, and Number of Exchangeable Hydrogens for Carbon-Centered Radicals in Smoke from 2R4F Cigarettes

a Number of active hydrogens determined by mass spectrometry by deuterium labeling. b Relative deviation in ppm between the experimentally determined and theoretical molecular masses of radical adducts. c Mass of radical calculated from the theoretical mass of radical adducts determined by FTICR.

To further eliminate false assignments, a second nitroxide with no amine group, 3-cyano-proxyl (3CP), was employed for the collection of additional smoke samples. Although the trapping efficiency of 3CP is an order of magnitude lower than that of 3AP33 and lacks a highly ionizing group, it nevertheless generates intense signals by HPLC-MS/MS. The CID of 3CP-R adducts resulted in a major fragment ion with m/z 151 (neutral loss of hydroxylated radical) common to all adducts. Ten radicals 1-10 in both 3AP-R and 3CP-R smoke samples were thus detected, and false assignments were removed from the previous 3AP-R set that can arise from the reaction between the amine group of the 3AP trapping agent and carboxyl or carbonyl compounds present in smoke. Additional new radicals at very low intensity (radicals 11-18) were detected in the 3CP-R samples but not confirmed in the 3AP-R samples due to interferences from possible products of 3AP with carbonyl or carboxyl compounds. Altogether, 18 radicals with masses varying from m/z 43 to 126 were positively identified. All the N-containing radicals exhibited the presence of a single active hydrogen. The results are summarized in Table 1. Identification of Chemical Structures. The FTICR-MS measurements of the masses of individual 3AP-R and 3CP-R adducts afforded highly accurate values for the quasi-molecular ions [M + H]+ with a resolution exceeding a 100 000 (Figure 4). Table 1 gives the mass units, chemical formulas, and mass accuracies of radicals in the order of elution for 3CP-R adducts. On the basis of the chemical formulas and elution pattern, two classes of radicals were distinguished in the smoke samples with (35) Kieber, D. J.; Blough, N. V. Anal. Chem. 1990, 62, 2275–2283.

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Figure 4. (A) FTICR-MS of fraction 14 with m/z range of 50-400 acquired in profile mode. Resolution of the 216.170 57 peak is approximately 220 000. The 215-219 m/z range is expanded to show the isotopic fine structure of the precursor ion. (B) High-resolution MS/MS where the isolation of 216.170 57 and CID occurred in the FTICR-MS.

the general chemical formulas CnH2nNO and CnH2n-1O. The most abundant radical of type CnH2n-1O, C2H3O (radical 3) was further scrutinized for structure identification. Its precise mass indicated two possible structures, · CH2COH or · C(O)-CH3. Subsequent comparison of the smoke-derived adducts and synthetic 3CP-C(O)CH3 by HPLC-MS/MS and NMR analysis confirmed this structural assignment.16 The presence of an ester bond between the trapping agent and the radical was

Table 2. Calculated Bond Dissociation Energies (BDE) and Relative Enthalpies (RE), with the CBS-QB3 Level of Theory, at 298 K and 1 atm for all Species Considered in This Studya

a The relative enthalpies of the radical species are reported with respect to · OH + CH3-CtN in the left column and those of parent molecules to H2O + CH3-CtN in the right column.

verified by hydrolysis of 3CP-3 in a weak basic solution; 16.9% of the 3CP-acetyl adduct hydrolyzed during a 1 day period (RSD ) 2.8%, n ) 3) and 73.0% in two days (RSD ) 0.8%, n ) 3). The most abundant N-radical, · C2H4NO (radical 1), exhibited higher polarity than the acetyl radical of similar carbon count based on the elution order on the C18 HPLC columns in all three forms, 3AP-R, NDA-3AP-R, and 3CP-R. The higher polarity was also characteristic of the rest of the N-radicals. Isotopic labeling experiments indicated the presence of an active hydrogen in each case, suggesting the presence of a secondary amine group or hydroxyl group that corroborates the higher polarity. Such observations allowed us to delimit the number of possible structures for the N-radicals. The 3CP adducts with methylaminocarbonyl, 3CP-C(O)NHCH3, were synthesized and found to match the LC-MS/MS characteristics of 3CP-1. This synthesis yielded as a side product some acetyl adduct due to the presence of acetic acid in the reaction mixture. The remaining two potential oxime structures were excluded by 1H NMR analysis as previously described.16 The 3CP-1 demonstrated complete stability in a weakly basic solution monitored over 24 h. All possible structures for C2H4NO were evaluated by ab initio quantum mechanical calculations (Table 2). Among them were two energetically low-lying peptide-bond-related radicals with formula C2H4NO: an N-centered acetamino radical (CH3-C(dO)-NH( · )) and a carbon-centered alkylaminocarbonyl (CH3NHC( · )dO) radical. The carbon-centered radical was lower in energy by 29 kJ/mol than the N-centered radical. Moreover, the O-centered acetaldoxime radical (CH3CHdN-O · ), the next low-lying species, was at much higher energy, 127 kJ/mol above CH3NHC( · )dO. Therefore,

these nitrogen-containing radicals resonate between two types of peptide bonds with different alkyl substituents (C2-C6). The exceptional stability of these radicals stems from delocalization in the N-CdO structure. Factors Affecting Radical Yield Measurements. The 10 most intense radicals in the mainstream smoke sample of 2R4F cigarettes trapped with 3AP were quantified by HPLC-MS/MS; radicals 1-3 were the most abundant, representing 87% of the total (Figure 5). The collective abundance of radicals 1-10 approximates the total yield, since the remaining radicals were only observed at trace levels. For the collection of gas-phase smoke samples, a Cambridge filter pad was placed in the conventional way between the cigarette and the trap to filter out TPM. Interestingly, the acyl radicals were almost entirely removed by the Cambridge pad, with only ca. 5% remaining in smoke residue, while the abundance of nitrogen-containing radicals was reduced by half relative to their yield in whole smoke. In comparison with 2R4F whole smoke, the fresh smoke from cigarettes made of cellulose alone consisted of mainly acyl radicals. When the 2R4F whole smoke was bubbled into toluene containing 3AP at room temperature, only ca. 30% of the radicals relative to solvent-free trapping were detected (Figure 6). Cooling the trapping solvent with ice water decreased the yield of acyl radicals to 20%, keeping the abundance of N-radicals at ca. 30%. In separate experiments, whole and gas-phase smoke samples were collected in inert Kynar tubing and aged for 1 min before collection. Intriguingly, the yield of radicals was dramatically reduced in these samples as observed in fresh gas-phase smoke (Figure 5). Additional aging experiments (0.5, 2, and 3 min) were conducted only on whole smoke. Since the puffing interval deviated from the 1 min standard time, the number of puffs varied Analytical Chemistry, Vol. 81, No. 2, January 15, 2009

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Figure 5. Abundance of radicals 1-10 in the whole smoke and gas-phase smoke from 2R4F cigarettes and in the whole smoke from cellulose cigarettes. Yields are compared between standard trapping and trapping on a larger surface and between fresh smoke and smoke aged for 1 min (the error bars represent the standard deviations between two experiments, each with three independently prepared replicas).

Figure 6. Yield of 2R4F fresh whole-smoke radicals collected in solvent-free environment on glass beads compared to the yield of radicals trapped by bubbling whole smoke into toluene at room temperature or cooled in an ice bath (the error bars represent the standard deviations between five independently prepared replicas).

from that of the standard experiments. Longer intervals involved longer smoldering time that reduced the number of puffs. A gradual, second-order polynomial decline in the abundance of N-radicals was recorded between 0-3 min aging periods, providing an average half-lifetime t1/2 ) 1 ± 0.5 min. The experiment implies a relatively large error since a carryover of a small fraction of fresh smoke from the tubing to the trap was observed during puffing, which was considered for calculating t1/2. The abundance of acyl radicals dropped sharply at 0.5 min to ca. 5%, and the t1/2 could not be estimated. The three major radicals were also detected in tobacco pyrolysate, in both inert and oxidative environment (Figure 7). For samples heated at 300 °C, unlike at 600 °C, a significant increase in the yield was detected when 5% O2 was supplied to the heating atmosphere. It is notable that during pyrolysis in inert atmosphere at 600 °C, a significant surge in radical abundance was observed relative to pyrolysis at the lower heating temperature. We investigated the relative and absolute efficiency of solventfree trapping using 2R4F cigarettes. High trapping efficiency requires complete monolayer surface coverage (2 × 1014 molecules/ cm2)36 of the beads by the trapping agent. The amount of 3AP used (6 mg) represents a significant excess (28 times) over the amount required to fully cover the glass beads. As expected, (36) Hines, A. L.; Maddox, R. N. Mass Transfer Fundamentals and Applications; Prentice-Hall Inc.: Englewood Cliffs, NJ, 1985; pp 462-463.

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Figure 7. Profile of the three most intense radicals in the 2R4F cigarette rod pyrolysate as a function of temperature and carrier gas (He and He with 5% O2), recorded by HPLC/FLD (the error bars represent the standard deviations between three independently prepared replicas).

doubling the amount of 3AP did not influence the yield, although it enhanced the background noise during HPLC/FLD analysis. When the 3AP-exposed surface area of the trap was increased by a factor of 1.50 by switching from 3 to 2 mm glass beads, the yield increased only by 1.16 (Figure 5). Doubling the amount of 3AP for the larger surface of the 2 mm beads did not affect the result; consequently, the 3 mm beads were used for all subsequent experiments. To measure the trapping efficiency of the tube, two traps were attached in tandem and the chromatographic peak areas recorded for both traps separately. Only ca. 4% of the yield measured in the first trap was found in the second trap. Consequently, the first tube’s trapping efficiency is 96% and a single trap appears to be able to trap radicals almost in their entirety. Model Gas Mixtures. Cueto and Pryor13 applied FT-IR spectroscopy to monitor the evolution of NO and NO2 in various mixtures of NO, isoprene, and methanol in air. A mixture of NO, isoprene, and methanol in air was found to be the best model system resembling gas-phase cigarette smoke in terms of the persistence of NO, the evolution NO2, and the rate of formation of nitroalkoxy radicals measured by EPR spintrapping with PBN.5,6 Accordingly, we used TDL absorption spectroscopy to measure the concentration of NO and NO2 in

similar gas mixtures but employed the 3AP solid-phase trap to measure potential carbon-centered radicals in the residue that collects on the glass beads. We collected samples using the 3AP trap at various time points from 0 to 300 s after the initial mixing of the gases. In a 500 ppm NO, 500 ppm isoprene in air mixture after a 34 s of reaction time, we recorded 422.0 and 35.8 ppm, respectively, of NO and NO2 with the TDL spectrometer (NO t1/2 ) 98 s). In a second mixture, 500 ppm NO, 3000 ppm isoprene, 150 ppm methanol in air, we recorded 388.0 and 45.6 of NO and NO2 ppm, respectively, at the same reaction time (NO t1/2 ) 102 s). Therefore, conditions were favorable, in principle, for the formation of nitroalkyl radicals by NOx chemistry. Flicker and Green measured the total carbon-centered radical yield in a gas mixture containing 500 ppm NO and 590 ppm isoprene in air as a function of reaction time.15 Using the entire fluorescence chromatogram of the NDA-3AP-R adducts for quantification for a 20 s reaction time, they recorded a maximum of 0.018 nmol R · /mL gas for this mixture and 0.015 nmol R · /mL with the inclusion of 85 ppm methanol. Surprisingly, although we detected many chromatographic peaks by FLD or MS in similar gas mixtures, evaluation of our chromatograms did not reveal the presence of any carbon-centered radicals under the specified conditions. In our mixture of 500 ppm NO and 500 ppm isoprene, oxidation of NO is slow and its t1/2 was estimated to be ca. 4 min. Nitric oxide in the absence of isoprene oxidized at half of this rate (experimental t1/2 ca. 8 min), confirming isoprene has catalytic effect over the oxidation. The theoretical half-lifetime of NO in air is 11.3 min (k ) 7.25 × 103 M-2 s-1),40 which is comparable to the experimental value. To achieve a shorter oxidation time, we increased the concentration of isoprene significantly relative to NO. As a result, for a mixture of 500 ppm NO and 3000 ppm isoprene in air, we measured a t1/2 of 98 s (1.6 min). The rate of NO2 increase did not parallel the decrease of NO because the former was partially removed by the isoprene. In the presence of methanol (150 ppm), the oxidation rate of NO did not increase, but the maximum concentration of NO2 decreased from ca. 85 to ca. 70 ppm, which suggests that methanol partially removed NO2 by forming methyl nitrite.13 The HPLC/FLD analysis of our model gas samples produced numerous chromatographic peaks, but none of these peaks corresponded to smoke radicals. This result was also confirmed by HPLC-MS/MS. However, it is nevertheless puzzling that we did not detect any type of C-centered radicals. All peaks present in our samples were also recorded in an appropriate control sample, which refutes the presence of any C-centered radicals. Flicker and Green investigated the NO/isoprene mixture in hydrated air.15 Although they suggested the presence of radicals, some possibly similar to smoke radicals, the optimum reaction time of 20 s for the presented mixture was hardly sufficient to initiate any NO oxidation and consequently the formation of any radicals. The theoretical t1/2 of NO for this gas mixture would be significantly longer. Furthermore, the level of NO and NO2 in situ was not monitored, which makes the results presented by Flicker and Green difficult to reconcile with our own, and further investigations are warranted.

Figure 8. Radical yields for smoke from single-component and blended cigarette samples in nanomoles per cigarette. (A) Radical yields in whole smoke. (B) Radical yields in gas-phase smoke. The yellow bars represent the acetyl radical, the most abundant acyl radical (the error bars represent the standard deviations between three independently prepared replicas).

Variations between Cigarette Types. We evaluated the whole mainstream smoke and gas-phase smoke from various singlecomponent and blended tobacco research cigarettes for carboncentered radicals. The data for whole and gas-phase smoke radicals reported in nanomoles per cigarette are presented in Figure 8. Overall, the combined abundance of acyl radicals and the combined abundance of N-radicals showed large variations in radical yield due to tobacco chemistry. Interestingly, the concentration of N-radicals from the single-component cigarettes gave values very close (σ ) 10%) to that the blended tobacco cigarettes, but the concentration of acyl radicals dropped by 30% regardless of the normalization method. In our preliminary report, we showed that cellulose and polysaccharides are an abundant source of acyl radicals that appear to form primarily from their corresponding pyrolytic aldehydes.16 We estimated a 0.6% abundance of acetyl radicals relative to the abundance of acetaldehyde reported for smoke from 2R4F cigarettes. The so-called base-web cigarettes produced the highest level of acyl radicals among the different cigarette types due to its high polysaccharide content, and its radical profile is the closest of all the tobaccos tested to the radical profile of smoke from cellulose cigarettes (Figures 5 and 7). The oriental tobacco cigarettes produced the second highest yield of acyl radicals. Oriental tobacco leaves are much smaller than either bright or burley tobacco leaves, and they are processed without removing the stems. Since the stems are rich in cellulose,37 cigarettes made from 100% oriental tobacco produce relatively more acyl radicals than cigarettes made from either bright or burley tobaccos. For all of the cigarette types, the concentration of acyl radicals dropped 80-90% in the gas-phase smoke in comparison to whole smoke. (37) Bokelman, G. H.; Ryan, W. S., Jr. Beitr. Tabakforsch. 1985, 13, 29–36.


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By contrast, the concentration of the N-radicals dropped by only 50-60%, with the exception of oriental tobacco, for which the abundance drops by only 27%. DISCUSSION Using our solid-phase trapping method, we isolated and identified two homologous series of saturated carbon-centered radicals in cigarette smoke consisting of acyl, C(O)R (R, C1-C4), and alkylaminocarbonyl, C(O)NHR (R, C1-C5), radicals. Within both series, radicals containing greater than four carbon atoms were found in different structural isomeric forms. Additionally, two unsaturated alkylaminocarbonyl radicals, 12 and 15, were detected (Table 1). The discovery of the carbon-centered radicals reported here was the result of serendipity, since we expected that the radicals would be related to the well-established NOx gasphase chemistry. Nevertheless, none of the carbon-centered radicals found in the smoke or pyrolysate from any of the blended tobacco or single-component cigarettes from any of the samples (fresh, aged, whole, or gas-phase) contained a nitro group. Also, nitro-radicals were not detected in any of the model gas mixtures. In the cigarette smoking experiments, the number of radicals decreased upon aging, contrary to what is generally expected on the basis of NOx chemistry. Despite that NO2 has been confirmed experimentally to be present in gas-phase smoke (i.e., whole smoke passed through a 0.1 µm Cambridge filter),22 the concentration of radicals in our experiments on gas-phase smoke was significantly less compared to fresh whole smoke. Furthermore, in our experiments only acyl and aminocarbonyl type radicals were detected in every case. Currently, it is not clear whether the gas-phase radicals trapped by the EPR spin-trapping methods are related to the whole-smoke radicals trapped by the direct solid-phase trapping approach since the structures of individual PBN radical adducts trapped from cigarette smoke have never been determined. Additional studies are needed to determine whether the carbon radical adducts from the two different trapping methods may include some of the same radical species. Differences in reaction kinetics and experimental setups are likely to be major factors affecting the discrepancy between the HPLC and EPR yields in measurements of gas-phase cigarette smoke radicals. The reaction rate of alkyl radicals with PBN (k ) 1.3 × 105 M-1 s-1)38 is at least five orders of magnitude lower than the reaction rate with 3AP. Furthermore, PBN is less likely to trap alkyl radicals in the presence of a high concentration of alkoxyl radicals, which react 10 times faster with PBN.33 Certain smoke constituents can also deactivate the newly formed PBN-R radicals,19,39 thus further reducing the number of radicals measured by EPR and PBN spin trapping. On the other hand, the high reactivity of nitroxides toward carbon-centered radicals affords diffusion-limited trapping of carbon-centered radicals from smoke. The solvent-free medium and very large surface area of the 3AP-coated glass beads further enhances the trapping efficiency, leading to larger measured yields. In our experiments employing 3AP in toluene solution to trap the carboncentered radicals gas-phase smoke, we recorded a 70% drop in (38) Schmid, P.; Ingold, K. U. J. Am. Chem. Soc. 1978, 100, 2493–2500. (39) Janzen, E. G.; Krygsman, P. H.; Lindsay, D. A.; Haire, D. L. J. Am. Chem. Soc. 1990, 112, 8279–84.

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the yield per cigarette. However, the use of this aprotic solvent is not compatible with the HPLC analysis; consequently, solventphase trapping required an additional extraction step with a protic solvent prior to the final analysis. With regard to the trapping efficiency by the solid-phase method, the abundance of trapped 2R4F mainstream cigarette smoke radicals increased only by a factor of 1.16 upon switching from 3 to 2 mm glass beads with a larger surface area. Consequently, it is highly unlikely that overall yield of trapped radicals would significantly change by an additional increase in the trapping surface area. In this regard, the yield of radicals that we presented for the various cigarettes (Figure 7), although not adjusted for the larger surface area of the 2 mm glass beads, is close to the maximum achievable yield. Evidently, the strong competition between O2 and 3AP for the smoke radicals limits the number of radicals trapped to those in close proximity to the trapping agent. On the basis of our kinetic estimates, the amount of radicals reacting with O2 is larger by a factor of ca. 23 than the radicals reacting with the trapping agent. Alkylaminocarbonyl and acyl radicals are most likely intermediate radical fragments or degradation products of metastable or excited molecules in smoke. It is apparent that these radicals are not primary radicals from thermal decomposition because any small nonaromatic carbon-centered radicals that form in the cigarette coal exhibit exceedingly short lifetimes. Consequently, the carbon-centered radicals that we detect by the solid-phase trapping method are likely to form directly and rapidly in the smoke during the time frame of the nucleation and the formation of the smoke aerosol. Acyl radicals in particular have lifetimes measured in nanoseconds, a lifetime comparable to the highly reactive hydroxyl radical.40 By comparison, the alkylaminocarbonyl radicals are more stable due to the resonance stabilization of the N-CdO bonds. Our ab initio quantum mechanical calculations show that these radicals have the highest stability among all possible radicals with the same chemical formula (Table 2). The differential removal of these two types of radicals by the Cambridge pad suggests that both types of radicals form, all or in part, in front of the filter pad, i.e., inside the cigarette. Our data, and the data collected in previous studies,21,22 provide cogent evidence that the Cambridge pad induces significant qualitative and quantitative changes to smoke. The fact that a major part of N-containing radicals and most of the acyl radicals disappear upon aging suggests that molecular oxygen in air is the major species that accounts for disappearance of radicals at the filter pad. Therefore, our solid-phase trapping utilizing nitroxide reagents in fresh whole smoke appears to be a more accurate method for quantifying carbon-centered radicals than other spintrapping methods utilizing gas-phase smoke. During the thermal degradation of tobacco, a significant surge in the level of various smoke species in presence of oxygen was reported by Burton and Childs.41 This surge was attributed to the formation of ROO · , a powerful branching agent that can significantly enhance various radical reactions in the presence of oxygen. The pyrolysis experiments we conducted suggest two parallel mechanisms of radical formation, one being oxygendependent and the other oxygen-independent. Although oxygen (40) U.S. NIST (National Institute of Standards and Technology) Kinetics Database. http://kinetics.nist.gov. (accessed June 1, 2007). (41) Burton, H. R.; Childs, G., Jr. Beitr. Tabakforsch 1977, 9, 45–52.

was removed from the pyrolyzing chamber, there could be some air trapped inside the cigarette rod. This level of air could contribute partially to the formation of radicals at the two temperature points. However, the boost in the number of radicals at 600 °C is severalfold higher compared to samples heated at 300 °C, suggesting an oxygen-independent mechanism (Figure 6). The alkylaminocarbonyl radicals most likely derive from multiple sources of nitrogen. On the basis of previous studies, inorganic nitrates (mainly at lower temperature) and amino acids, peptides, proteins, and alkaloids are involved in the formation of various nitrogenous smoke constituents.21,42 It is notable that fragments of R-NH-C(O) · arise from alkoxyl radicals formed at the R-carbon position of the polymeric peptide or protein chain via rapid rearrangement and subsequent fragmentation reactions during γ-irradiation in presence of oxygen.43 Such a reaction mechanism is plausible during cigarette smoke considering the significant concentration of protein in tobacco.37 In this regard, the level of alkylaminocarbonyl radicals recorded during pyrolysis at the lower temperature point is contributed mainly to inorganic nitrates.44 The presence of proteins and other N-containing organic tobacco constituents boost this level significantly at the higher temperature point. CONCLUSIONS The alkylaminocarbonyl and acyl radicals reported here are the first specific carbon-centered radical species to be positively (42) Chortyk, O. T.; Schlotzhauer, W. S. Beitr. Tabakforsch. 1973, 7, 165–178. (43) Davies, M. J. Arch. Biochem. Biophys. 1996, 336, 163–172. (44) Johnson, W. R.; Hale, R. W.; Clough, S. C.; Chen, P. H. Nature 1973, 243, 223–225.

identified in fresh mainstream cigarette smoke. These same radicals were also detected in the pyrolysate from the thermal degradation of tobacco in both inert and oxidative atmospheres. Separation of gas-phase smoke from whole smoke using a Cambridge filter pad was shown to reduce the yield of these radicals. The abundance of the newly discovered radicals as measured by HPLC is significantly higher on a per cigarette basis than the best estimates of the abundance of the nonspecific alkyl radicals previously measured by EPR spin-trapping methods. The current analytical approach, which employs solid-phase trapping with nitroxide radical scavengers and HPLC-MS, offers significant advantages over EPR spin-trapping analysis for these classes of radical species. These advantages include the capability to analyze whole smoke, the very high efficiency of trapping in a solvent-free system, the high stability of the radical adducts, and the capability to monitor individual radical species. ACKNOWLEDGMENT The authors benefited from discussions with Dr. Salem Chouchane, Dr. Justin Heynekamp, Dr. Jeffrey Seeman, Professor Sarah Green, Professor Barry Dellinger, and Professor W. A. Pryor. We also thank Dr. William D. Thweatt for his assistance in measuring NO and NO2 levels in gas model mixtures by TDL. Yi-Lei Zhao thanks PM USA for financial support and NIH for supercomputer time on the Beowulf Cluster. Received for review September 17, 2008. Accepted November 25, 2008. AC801969F


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Carbon-Centered Radicals in Cigarette Smoke - ACS Publications

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