Fluorescent Approach to Quantitation of Reactive Oxygen Species in

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Anal. Chem. 2006, 78, 3097-3103

Fluorescent Approach to Quantitation of Reactive Oxygen Species in Mainstream Cigarette Smoke Boxin Ou† and Dejian Huang*,‡

Brunswick Laboratories, LLC, 6 Thatcher Ln, Wareham, Massachusetts 02751, and Food Science and Technology Program, Department of Chemistry, National University of Singapore, 3 Science Drive 3, Singapore 117543, Republic of Singapore

A novel approach to monitoring of mainstream smoke reactive oxygen species (ROS) has been developed and applied to the quantitation of smoke oxidants. Redoxactive fluorescent probe dihydrorhodamine 6G (DHR-6G) was selected as the molecular probe because it is sensitive to typical smoke ROS. The experimental system couples an automatic cigarette smoke machine fiber-optic fluorometer for real-time monitoring of the reaction progress between cigarette smoke and DHR-6G. Quantitation was achieved based on the amount of rhodamine 6G, which is the sole product from DHR-6G oxidation. With the optimization of the trapping efficiency, we detected 391 nmol of ROS/cigarette in the mainstream CS for a standard cigarette 2R4F under standard Federal Trade Commission smoking protocol. Applying this method, we quantified the ROS of selected cigarettes and found that the cigarettes made of burley tobacco have much (∼10 times) higher ROS content in the smoke than that in the tobacco made of bright tobacco. The smokeless cigarette, Eclipse, has comparable ROS with cigarettes made of bright tobacco. The primary hazard of cigarette smoking (either voluntarily or involuntarily) is oxidative stress, and it is suggested that reactive oxygen species (ROS) in the smoke is the culprit.1, 2 ROS in smoke includes peroxyl radicals, carbon-centered radicals, and nitric dioxides.3 Evidence has shown that ROS can damage DNA, initiate lipid peroxidation, and cause inflammation. As the oxidative stress accumulates over time for addicted smokers, it may trigger smoke-related diseases such lung cancer, pulmonary emphysema, cardiovascular diseases, and stroke. Surveys show that these diseases have a higher incidence rate among smokers.4 To better understand the impact of ROS to oxidative stress on smokers, it is important to accurately quantitate the ROS in smoke. Accurate quantitation of ROS in smoke will determine if the so-called reduced harm cigarettes, in the consumed, really have lower ROS * To whom correspondence should be addressed, E-mail: [email protected]. † Brunswick Laboratories. ‡ National University of Singapore. (1) Rahman, I. Lung Biol. Health Dis. 2004, 187, 211-255. (2) Karar, A.; Spira, A. Smoking and the molecular mechanisms of atherogenesis. In Molecular Mechanisms of Atherosclerosis; Loscalzo, J., Ed.; Taylor & Francis Ltd.: London, 2005; pp 209-219, (3) Church, D. F.; Pryor, W. A. Environ. Health Perspect. 1985, 64, 111-126. (4) CDC, Annual Smoking-Attributable Mortality, Years of Potential Life Lost, and Economic Costs-United States, 1995s1999. MMWR Weekly 2002, 51, 300-303. 10.1021/ac051993s CCC: $33.50 Published on Web 03/31/2006

© 2006 American Chemical Society

in addition to lower levels of other toxins as claimed. A reliable quantitation method for ROS in smoke will be an indispensable tool for cigarette harm reduction and quality control. As an important type of ROS, free radicals in cigarette smoke (CS) were suggested and detected about a half-century ago. In 1958, Lyons and co-workers reported electron spin resonance (ESR) signals from CS condensate, and shortly afterward, free radicals were detected in whole cigarette smoke.5-6 Later, Pryor and co-workers further reported detection and estimation of free radical concentrations by applying spin trap phenyl tert-butyl nitrone (PBN), 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), and R-(3,5-di-tert-butyl-4-hydroxyphenyl)-N-tert-butyl nitrone (OHPBN). This trap is not reactive to peroxyl radicals,7 which are considered the dominant radicals according to kinetic analysis by Green,8 who suggested that carbon-centered radicals contribute only 1% of the total radicals, whereas peroxyl radicals should be dominant due to the fast reaction of R• and oxygen in the air. In addition to specificity, the spin trap method is rather tedious and labor intensive. It is desirable to have a convenient assay measuring free radical oxidants in smoke. Reported herein is such as approach using dihydrorhodamine 6G (DHR-6G) to quantify ROS in cigarette smoke. DHR-6G has been used as a marker of oxidative stress in biology. It is reactive with typical ROS (e.g., R•, RO•, ROO•) except H2O2.9 Therefore, it is logical to extend its application to measuring ROS in CS. The assay coupled the automatic smoking machine and in situ monitoring fluorescent spectrometer and can efficiently monitor ROS in the smoke and gas streams in real time with good reproducibility. EXPERIMENTAL SECTION Materials and Instrumentation. With the exception of the Research Cigarette (2R4F), which was obtained from the University of Kentucky, cigarettes were purchased at local stores in Massachusetts. DHR-6G, Redox Sensor, and dihydrorhodamine 123 (DHR-123) were obtained from Molecular Probes (Eugene, OR). Rhodamine 6G and Tween 20 were purchased from Aldrich (Milwaukee, WI). The washing bottles were obtained from Chemglass (Vineland, NJ) and modified by G. Finkenbeiner, Inc. (Waltham, MA) according to our specification. Solvents were (5) Lyons, M. J.; Gibson, J. F.; Ingram, D. J. E. Nature 1958, 181, 1003-1004. (6) Blum, A. L.; Weinstein, J; Sousa, J. A. Nature 1971, 229, 500-501. (7) Pryor, W. A.; Tamura, M.; Church, D. F. J. Am. Chem. Soc. 1984, 106, 5073-5079. (8) Flicker, T. M.; Green, S. A. Environ. Health Perspect. 2001, 109, 765-771. (9) Invitrogen Inc. The HandbooksA Guide to Fluorescent Probes and Labeling Technologies; http://probes.invitrogen.com/handbook/print/0101.html.

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Figure 1. Illustration of the experimental system used for real-time measurements of reactive oxygen species in cigarette smoke. The smoke was generated via an automatic smoking machine. The smoke passed through the gas-washing bottle adapted with a fluorescent reflective probe. The gas-washing bottle contained DHR-6G dissolved in DMA, water, and Tween-20 mixture. The reaction progress was monitored by a fiber-optic fluorescent spectrometer. Both the smoking machine and the spectrometer were controlled by computer software.

HPLC grade obtained from Sigma Co. (St. Louis, MO). Cigarette smoke was generated by an automated five-port smoker (KC Automation, Richmond, VA) that was operated according to the standard the Federal Trade Commission (FTC) protocol. The protocol specifies that the cigarette should be puffed in such a way that one puff would last 2 s with puff volume of 35 mL and frequency of 1 puff/min. The puff volume was calibrated using a bubble calibrator purchased from Bubble-O-Meter, LLC (Dublin, OH). Fluorescence intensity was recorded using an AvaSpec-2048 fiber-optic spectrometer equipped with a reflective probe (Avantes Inc, Boulder, CO). Chromatographic analyses were performed on an HP 1100 series (Hewlett-Packard, Palo Alto, CA) HPLC equipped with an autosampler/injector, binary pump, column heater, diode array detector, and HP ChemStation for data collection and manipulation. Reversed-phase separation was performed on a Zorbax (Hewlett-Packard) C18 column (2.1 × 150 mm, 3 µm) at 37 °C. Detection was recorded at 528 nm. The binary mobile phase consisted of the following: (A) water/acetonitrile/ acetic acid (89:9:2); (B) acetonitrile/water (80:20). The separation was performed using a linear gradient from 40 to 100% B in 7 min and then 100% B for 8 min. Selection of Fluorescent Probes. To evaluate the reactivity of these probes toward cigarette smoke, 0.3 mg/mL of each fluorescent probe in DMSO (20 mL) was bubbled with cigarette smoke (unfiltered) at a continuous flow rate of 110 ( 10 mL/min until 10 cigarettes were smoked. A 100-µL aliquot of the solution was sampled for measurement of fluorescent intensity using a microplate fluorescent reader with corresponding emission and excitation filters. The fluorescent intensity changes over time were plotted with the slopes of the curves being the indication of the sensitivity of the fluorescent probe toward oxidation by cigarette smoke. 3098

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Real-Time Monitoring. To ensure convenient and accurate measurement, we monitored the reaction progress of smoke radicals with DHR-6G in real time by integrating a gas-liquid reactor with a fiber-optic fluorescent spectrometer equipped with a reflective probe. The principle of the fluorescent spectrometer and related technical information can be obtained from the manufacturer’s website at avantes.com. The fluorescent excitation is from a tungsten light source. The excitation light beam was passed through an inline filter with wavelength of 510 ( 10 nm before the light reached the fluorescent reflection probe emerged in the solution. The reflection probe also directs the emission light from the solution through a separate fiber-optic line to the spectrometer. The data are processed by Avasoft software. The captured fluorescent intensity data are automatically exported to an Excel spreadsheet for further calculations. The environmental light (from sunlight and laboratory lighting) gives a small background peak, and blank subtractions were made in all the measurements. Fluorescent data can be captured as fast as every second, but it is not necessary in this application as it takes 8-10 min to smoke one cigarette. Therefore, a fluorescent reading was taken every minute (equivalent to one reading per puff) or after every cigarette was smoked, depending on the experimental needs. Figure 1 shows the experimental system used in this study. Typical Smoke ROS Quantitation Procedure. In this paper, all the measurements were done without using the Cambridge filter; therefore, the smoke is considered whole smoke containing both the particulate phase and the gas phase. Each cigarette was smoked according to the FTC protocol until the char line was 3 mm from the tipping paper (approximately 8-10 puffs/cigarette). The smoke was passed through a gas dispersion tube connected to a frit disk to the trapping solvent containing DHR-6G (25 mg in 200 mL of 60% DMA in a water mixture containing 0.5% Tween-

Figure 2. Proposed reaction mechanism of DHR-6G with free radicals.

Figure 3. Fluorescent intensity changes over time during the reaction of DHR-123 with cigarette smoke (Marlboro regular). Experimental conditions are described in the text.

20). The change of DHR-6G fluorescence intensity was recorded at a certain time interval using an AvaSpec-2048 fiber-optic spectrometer equipped with a reflective probe and excitation (504 ( 10 nm) and emission filters (543 ( 10 nm) purchased from Omega Optical (Brattleboro, VT). All experiments were performed at room temperature (21-23 °C). After each measurement, the gas dispersion tube was thoroughly cleaned by flushing with water, then soaked in hot concentrated HNO3 for 20 min, and last rinsed thoroughly with plenty of water. RESULTS Probe Selection. There is a range of fluorescent probes commercially available for the detection of ROS in biological and chemical systems. The common feature of these probes is that they do not fluoresce until they are oxidized. One molecule of the probe can react with two radicals to form a highly fluorescent compound. For example, DHR-6G can be oxidized by peroxyl radical and lose two hydrogen atoms to form rhodamine 6G. The possible mechanism is shown in Figure 2. Redox Sensor, upon reaction with CS, shows small increase of fluorescent intensity until the fourth cigarette and then decreases (data not shown); this may indicate complex reactions forming nonfluorescent compounds. Therefore, it is not a good choice as a probe. The fluorescence intensity of hydroethidine or DHR-123 increases linearly with the number of cigarettes smoked. The DHR-123 is much more sensitive than that of hydroethidine (Figure 3). For DHR-6G (Figure 4), the fluorescence intensity increases and then levels off after five cigarettes. This fluorescent signal is so strong it saturates the fiber-optic probe. Indeed, the solution color is very intense compared with that of the DHR123. It may be possible that DHR-6G in the solution has been totally oxidized by the smoke. The structural difference of the

Figure 4. Fluorescent intensity changes over time during the reaction of DHR-6G with cigarette smoke (Marlboro regular). Experimental conditions are described in the text.

two probes is such that DHR-6G has two electron-donating methyl groups attached to the arene carbons and is hence more electron rich than DHR-123. As such, DHR-6G may be more prone to oxidation. Therefore, we chose DHR-6G as the probe. It should be pointed out that hydroethidine and DHR-123 should work in principle. Choice of Reaction Media. Several factors were taken into account when we choose a solvent mixture for trapping the ROS. First, the solvent needed to have a high boiling point. During the reaction, smoke was passed through the solution at a rather large flow rate (17.5 mL/s on average during the puff). High boiling point solvents have low vapor pressure; therefore, the solvent carried away by the smoke is negligible. Second, the solvents selected should have good solubility for the lipophilic DHR-6G and good dispersion for the smoke, which is an aerosol, composed of mostly air, carbon dioxide, and water. Water compatibility of the solvent will enhance the dispersion of the aerosol. Third, the contact time between the smoke and the solvent should be as long as possible to ensure enough time for the ROS in the smoke phase to dissolve into the liquid phase. Surfactant Tween-20 was added to disperse the smoke into tiny bubbles; thus, the contact area between the smoke and liquid is much larger, and in addition, the small bubbles remain in the solution much longer than the larger bubbles. Overall, the surfactant greatly facilitates the interaction of smoke and the liquid phase. The solution was magnetically stirred to further maximize the contact time of the smoke in the trapping solution. Finally, the selected solvent should give optimal reaction rates between DHR-6G and the smoke. Taking these factors into consideration, we tested a range of solvent mixtures by following the fluorescent changes over time and found that the optimal mixture is 6:4 DMA in water containing 0.5% Tween-20 as the reaction rate (based on the slope of the fluorescent intensity plot vs reaction time) is the maximum. Adding Tween-20 significantly improved the fluorescent change rate. Therefore, we selected DMA (60% in water with 0.5% Tween20) for use in the quantitative measurements. Stability of DHR-6G to Air. DHR-6G is labeled as air-sensitivel therefore, before we applied it for ROS measurement, we tested its stability toward air bubbled according to the FTC protocol through the reactor for extended period (>1 h, Figure 5). Insignificant change of fluorescence intensity is observed. On the contrary, if cigarette smoke is passed through the solution, the fluorescence intensity changes were apparent and linearly increased with the amount of smoke passed. Therefore, we can conclude that, under the experimental conditions, air has little Analytical Chemistry, Vol. 78, No. 9, May 1, 2006

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Figure 7. Amount of rhodamine 6G formed vs the number of cigarettes smoked (Marlboro, regular). Figure 5. Typical reaction kinetics of cigarette smoke with DHR6G. Air was introduced by the smoking machine according to the FTC protocol. The fluorescence signal was monitored for an extended period. Apparently air does not cause significant oxidation of the probe. After introduction of smoke, the oxidation increased linearly yielding an increase of FL signal. The data were captured one point per minute.

Figure 6. Standard curve of rhodamine 6G in DMA, water, and Tween-20 (60:40, 0.5%). The fluorescence intensity was measured under the same conditions as the smoke experiment to eliminate possible variance.

contribution to oxidation of DHR-6G; instead, smoke ROS should be responsible for the formation of rhodamine 6G. The oxidation products of DHR-6G were determined by HPLC, and rhodamine 6G appears to be the sole product (data not shown). This finding greatly simplifies the quantitation of the ROS. From the reaction mechanism, one DHR-6G reacts with two radicals to form rhodamine 6G. The added DHR is fixed at concentration of 146 µM. (25 mg of DHR-6G/ampule dissolved in 200 mL of solvent) This concentration is over 100 times more than the possible ROS concentration in a typical cigarette mainstream smoke. Therefore, DHR-6G is not the limiting factor in the reaction. Quantitation of the ROS Concentrations. The concentration of rhodamine 6G formed in the reaction with smoke is calculated from a standard curve obtained by plotting the known rhodamine 6G (purchased separately) concentration versus the fluorescent intensity measured DMA/water (6:4) mixture with 0.5% Tween20 using the fiber-optic fluorometer with instrumental settings identical to that of the reaction. The linearity ranges from 0.65 to 5.2 µM as shown in Figure 6. Using this standard curve, the concentrations of rhodamine 6G formed during the reaction of smoke with DHR-6G can be calculated. The fluorescence intensity increases linearly with the number of cigarettes smoked and can be converted to the nanomoles of rhodamine 6G formed against the number of cigarettes smoked giving a linear curve as shown in Figure 7. The slope of the curve times two (stoichiometry of 3100 Analytical Chemistry, Vol. 78, No. 9, May 1, 2006

Table 1. ROS Contents of Different Cigarette Smoke cigarette brands

ROS (nmmol/cig)

2R4F Marlboro (regular) Camel (with filters) Parliament Chonghua Red Tower Mountain Double Happiness Eclipse

384 ( 50 414 ( 42 288 ( 20 416 ( 10 44 ( 2 36 ( 3 43 ( 5 32 ( 2

between DHR-6G and free radicals) gives the amount of ROS generated in the mainstream smoke of each cigarette. In this case, the 2R4F research cigarette has 384 ()2 × 187) nmol/cigarette radicals in the mainstream. Reproducibility. For each run, a standard curve was constructed daily to correct any system error. The ROS produced by 2R4F cigarettes were quantified on 12 consecutive days. The mean value was 391 nmol/cigarette, and the relative standard deviation was 7.96%. Hence, this assay is highly reproducible especially considering that each cigarette may be slightly different even though they are considered to be a standardized research cigarette. Radicals in Different Cigarette Smoke. Using the apparatus, we have measured the oxidants in smoke of 2R4F, a standard research cigarette, and a list of commercial cigarettes from United States tobacco producers and from Chinese tobacco companies. Each measurement was repeated three times, and the ROS concentrations are tabulated in Table 1. Overall, the ROS concentrations of the Chinese CS are consistently lower than that of U.S. counterpart by as much as 10 times. The smokeless cigarette, Eclipse, has ROS concentrations comparable with the Chinese cigarette. DISCUSSION ROS in Cigarette Smoke. The combustion of organic matter involves primarily radical chain reactions between the organic matter and oxygen. The end products are ideally water and carbon dioxide. However, this ideal combustion is never achieved in cigarette smoke as evidenced by the many organic compounds that have been discovered in CS. Reactive oxygen species, including free radicals, are among them. The ROS perhaps are the most damaging toxins in CS since they can oxidize protein, damage DNA, and induce lipid peroxidation. Quantitation of ROS in CS would yield critical information on accessing cigarette toxicity.10 (10) Smith, C. J.; Martin, P. Recent Adv. Tob. Sci. 2002, 28, 166-190.

Quantitation of ROS in CS. The most frequently applied methods for measuring free radicals use spin trap and electron spin resonance which was also used to detect free radicals in smoke condensates. Lyons and co-workers reported ESR signals from CS condensate, and shortly afterward, free radicals were detected in whole cigarette smoke.5.6 Pryor detected an ESR signal in aqueous extracts of cigarette “tar” (ACT) and assigned the chemical structure of the radical to be a long-lived semiquinone based on the similarity of the ESR signals of ACT and that of an aged catechol solution.11 The authors also applied the spin trap methodology for identifying and quantifying gas-phase radicals. In the study, PBN, DMPO, and OHPBN were used to trap the highly reactive radicals according to following reaction (eq 1):

Kinetic analysis of the gas-phase radical reactions led the authors to conclude that there were 5000 nmol of radicals in the smoke of one Marlboro cigarette under FTC protocol. Carbon-centered radicals are believed to contribute only 1% of the total radicals. Spin trapping coupled with ESR or HPLC did provide evidence that there are free radicals in CS. However, it is not a convenient quantitation method for radicals because spin trapping methods involve tedious experimental procedures. Spin trap is selectively reactive toward certain radicals and neglects the more abundant peroxyl radicals. If one were to use spin traps to measure different types of radicals, multiple spin traps would be needed. In addition, spin traps do react with nonradical reactive oxidants, which can be equally harmful to humans. Unlike spin traps, the fluorescent probe used in this method is sensitive to the common free radicals including peroxyl and alkoxyl and carbon-centered and hydroxyl radicals. These radicals possess strong oxidizing power whereas RHD-6G is a good reductant (eq 4).

2ROS + DHR-6G f rhodamine 6G + reduced ROS (4) Based on the ESR signal patterns and intensity, the authors further concluded that the R• groups included alkoxyl radicals, carbon-centered radicals, and nitric oxide. Based on the ESR data, the authors also estimated that there were 5 nmol of carboncentered radicals generated in the gas phase of each 1R1 research tobacco smoked.7 No peroxyl radical (ROO•) was detected by the authors. This is consistent with a separate finding by Saito and co-workers,12 who showed that ROO• was not reactive toward PBN; presumably, it is not as nucleophilic as a carbon-centered radical and RO•. ROO•, a reaction product from R• and oxygen in the air, is believed to be the predominant radical in CS. Green and coworkers detected carbon-centered radicals in CS using spin trap 3-amino-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (3AP) (eqs 2 and 3).

3AP is specific to carbon-centered radicals.11 In that experiment, CS was passed through a column packed with glass beads coated with 3AP. The trapped nonradical amine (3AP-R) was washed from the beads. The isolated 3AP-R was further converted to a fluorescent compound by reaction with naphthalenedicarboxaldehyde (NDA) to give the fluorescent adduct 3AP-R-NDA. Finally, the concentrations of 3AP-R-NDA were quantified by HPLC coupled with a fluorescent detector. Their results suggest that the concentration carbon-centered radicals is 10 times higher (54 nmol) in one Marlboro cigarette than in one 1R1 cigarette.8 (11) Pryor, W. A. Environ. Health Perspect. 1987, 105 (Suppl. 4) 875-882.

With real-time monitoring, the experimental procedure is greatly simplified in comparison with the spin trapping methods. In addition, we have demonstrated the validity of the method in terms of reproducibility and linearity. Using this method, quantitation of ROS in one cigarette smoked only takes 30 min. In contrast, it would take much longer time for spin trap method to measure one data point. Different trapping methods have been applied in the quantitation of smoke related toxins such as carbon monoxide, nitrosoamines, 13 and other small-molecule compounds.14 These compounds have rather long lifetimes, and real-time monitoring is not essential. Free radical oxidants, on the other hand, are often transient species and can only be captured with fast chemical reactions. The major difference between a free radical probe and DHR-6G is that the oxidation of DHR-6G may not be necessarily caused by free radicals. Nonradical oxidants can also oxidize DHR6G. It is reasonable to assume that free radical oxidants are the dominant ROS in CS as combustion is a free radical reaction process. The radical intermediates in the combustion can conveniently escape into the smoke as the smoking cigarette is puffed. Spin trap and ESR methods have been applied to quantify free radicals in CS. For example, quantitation of free radicals from heated cigarettes (Eclipse) has been carried out using a PBN/ benzene solution to form a stable radical species. By measuring the electron spin resonance signal intensities, the amount of free radicals was estimated. Analysis of cigarettes that included a new carbon filter and an experimental tobacco blend demonstrated a vapor-phase free radical reduction on the order of 80% when compared to conventional equivalent tar cigarettes. Free radical reductions for a new type of cigarette, which heats rather than (12) Saito, K.; Yoshioka, H.; Kazama, S.; Cutler, R. G. Biol. Pharm. Bull. 1998, 21, 401-404. (13) Wang, J.; Chan, W. G.; Haut, S. A.; Krauss, M. R.; Izac, R. R.; Hempfling, W. P. J. Agric. Food Chem. 2005, 53, 4686-4691. (14) Hoffman, D.; Hoffman, I.; El-Bayoumy, K. Chem. Res. Toxicol. 2001, 14, 767-790.

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burns tobacco, were even greater (88-97%).15 It was later shown that the Eclipse vapor phase has a radical concentration of 2.22 × 1014 spin/cigarette. This value is 95% lower than that of research cigarette, 1R4F (4.85 × 1015 spin/cigarette).16 In agreement with this result, our data show that ROS in Eclipse smoke is ∼92% less than 2R4F. The major difference lies in an absolute number. If we were to convert the nanomoles of ROS into number of spins, Eclipse has 1.9 × 1016 spins/cigarette (32 × 10-9 (mol) × 6.02 × 1023), which is ∼100 times higher than the value obtained by the spin trap method. The difference illustrates that the spin trapping method dramatically underestimates the amount of the radicals by ∼10 times, likely due to the specificity of the probe as it is not sensitive to the more dominant peroxyl radicals, ROO•. Our data also reveal that, in the mainstream CS, 2R4F has 48 (2.3 × 1017 spin/cigarette) times higher ROS concentration than that in 1R4F measured using spin trap. Pryor’s seminal report estimated there were 17 nmol of spin/ cigarette radicals for gas-phase CS of 1R1 research cigarette.7 Green’s result suggested there were 54 nmol of spin/cigarette in CS of Marlboro (regular).8 Our results place the concentration of ROS at ∼400 nmol of spin/cigarette for the whole cigarette mainstream smoke generated from 2R4F or from Marlboro. The difference is the trapping method and the probes used. DHR-6G is a general probe and can thus detect more radicals than the spin trap PNB or 3-AP. The cigarettes manufactured by Chinese tobacco companies have strikingly lower (∼10 times) ROS concentrations than that of Marlboro and similar brands manufactured in the United States. We suggest that the reason is likely caused by the tobacco leaves being treated differently. Chinese companies normally use bright tobacco leaves whereas the Marlboro and 2R4R used burley tobacco leaves. Study is underway to unveil the chemical composition difference and their effect on ROS concentration in the two types of tobaccos. The fact that the Chinese cigarettes have ROS concentration comparable to that of smokeless cigarette, Eclipse, implies there are other ways to obtain lower ROS contents than just reducing the temperature. Trapping Efficiency. Our original plan was to use three tandem traps for the smoke to pass consecutively to ensure complete trapping of the free radicals. However, smoke radical concentrations change over time due to secondary reactions of the smoke components.7 Under the standard FTC protocol of a 35-mL puff in 2 s and 1 puff/min, the smoke was trapped between the space of two traps and aged for 1 min before it is passed to the second trap. None-FTC protocol is still important because this method can in principle be extended to measurement of ROS in other gaseous streams like automobile exhaust and ROS in indoor air radical pollution. We thus tried using a continuous smoking devise generated through an air aspirator powered by compressed air. The continuous flow rate was set to 35 mL/min. This flow rate is very small compared to the conditions under the FTC protocol (35 mL/2 s, or 1050 mL/min during the 2-s puffing). The smoke was passed through the reactor in much slower motion, (15) Borgerding, M. F.; Blakley, R. L.; Winkler, L. S.; Henry, D. D.; Bowman, G. D.; Smith, D. H. The 49th annual meeting of the Tobacco Chemists' Research Conference, Lexington, KY, 1995. (16) R. J. Reynolds Co. Eclipse, A cigarette that primarily heats rather than burns the tobacco, Summary of scientific tests; 2000.

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but unfortunately, the water vapor generated in the combustion of the cigarette condensed on the cigarette filter and generated enough resistance so that the flow rate could not be maintained constant enough for meaningful data to be reported. The FTC protocol has taken into account mimicking of real smoking situations. Change to a different puffing protocol alters the cigarette combustion dynamics and the smoke compositions. It is mandatory that analysis of smoke toxins follows exactly the FTC protocol. Therefore, we used one trap but optimized the trapping efficiency by using the largest possible volume of solution and applying surfactants to ensure longer contact time of the gas and the liquid. The liquid in the trap creates a resistance for the smoking machine. If more liquid was placed, the resistance of the liquid will make it impossible for the smoking machine to generate a 35-mL puff in 2 s. Even with all these considerations, it is still possible that some of the radicals escaped into the gas stream and thus may lower the accuracy of the results. This does not compromise the precision of the data as demonstrated in our day-to-day variability study and the comparison of the data for different CS samples. Since all the measurements are done under the same conditions, it is reasonable to expect a constant ratio between the ROS that escapes and that trapped for different cigarettes. Limitation of This Method and Potential Improvements. It should be pointed out that DHR-6G does not directly react with diluted H2O2 or lipid peroxides, which at low concentration are not as reactive as the other radical species. The oxidative power of H2O2 normally is amplified when it is combined with a transition metal (Fe(II) or Cu(I)), which would induce a Fenton-type reaction (H2O2 + Fe(II) f HO• + Fe(III) + HO-). The generated hydroxyl radical would react with DHR-6G. It is therefore conceivable that one can add a small amount of Fe(II) salt to the trapping solution so that H2O2 and ROOH can also be measured. Work is underway to this end. Alternatively, H2O2 detection in cigarette smoke has been reported using an indirect colorimetric assay.17 The trapping efficiency of the assay needs further investigation. Along the same line, use of the expensive DHR-6G is not feasible for measurements of a large number of samples at the current prototype. It is envisioned that a gas splitter could in principle be used to divert the CS into two streams so that only a small fraction of the CS passes through a miniaturized trap. This would be a potential solution to reduce the amount of DHR-G usage in each measurement and the volume of the trapping solution for optimal trapping efficiency and ensure the accuracy of the measurement. CONCLUSIONS The method described herein provides a practical alternative to quantify reactive oxygen species in cigarette smoke. Our method adds a powerful tool for clinical researchers to investigate the relationship between oxidative stress and ROS in cigarette smoke and other gaseous pollutants with a more accurate ROS dosage that smokers are inhaling. It has been suggested that different types of cigarettes may have significantly different effects on lung cancer.18 The relationship between the ROS concentrations and the type of cigarette will be useful information for the epidemiological study of disease and smoke. In addition, our (17) Yan, F.; Williams, S.; Griffin, G. D.; Jagannathan, R.; Plunkett, S. E.; Shafer, K. H.; Vo-Dinh, T. J. Environ. Monit. 2005, 7 (7), 681-687. (18) Lee, P. N. Inhalation Toxicol. 2001, 13, 951-976.

approach is general and can be applied to measure reactive oxidants in other gaseous streams such as wood smoke, exhausts from automobile, and power plants that burn fossil fuels. This method also allows one to rapidly screening the activity of radical scavengers in mitigating the oxidants in CS and air pollutions.19 (19) Nishizawa, M.; Kohno, N.; Nishimura, N.; Kitagawa, A.; Niwano, Y. Chem. Pharm. Bull. (Tokyo) 2005, 53 (7), 796-799.

ACKNOWLEDGMENT The authors thank Dr. Geoffrey Chan for stimulating discussions and Philip Morris USA for partial financial support.

Received for review November 9, 2005. Accepted March 6, 2006. AC051993S

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