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Kenneth BlowCHM 441004/13/2012

Quantitative Analysis of Selected Toxic Compounds in Cigarette Smoke

Cigarette smoke has been the main topic of discussion in health sciences for decades.

With over 150 ACS publications, there is still much to learn about cigarette smoke. Estimations

of the number of unidentified species are as high as 100,000.1 There are two types of cigarette

smoke: “whole smoke” and “gas-phase.” Whole smoke is a dynamic aerosol; a combination of

substances that partition between suspended particulate material and gaseous phases.2 When

compounds within the cigarette undergo combustion, gases travel along the cigarette rod towards

the cigarette filter. During travel, gases will cool off and condense together with water to form

particulate material3. The majority of the suspended particulate material is trapped by the

cigarette filter because of its size. However, compounds remaining strictly in the gas phase will

travel through the cigarette filter, reaching inhalation of the smoker. The gas phase consists of

approximately 400 to 500 individual compounds4.

In respect to the health effects of cigarette smoke, most research has been performed on

the particulate phase4. The particulate phase and gas phase of cigarette smoke contain different

characteristics and health effects between one another; therefore, knowledge about the phase

affiliation of smoke components is important2. Because of the transitions between gaseous and

particulate phases, semi-volatile compounds may be classified in both phases2.

The research performed follows an ideal method of analysis: study as many relevant

compounds as possible simultaneously (in both phases) on a real time basis (puff-by-puff).2

Because cigarette smoke is an extremely complex matrix, chemical reactions take place during

and as soon as the smoke is drawn. Therefore, instrumentation must be designed in such a

manner that the time spent during analyses is equivalent to the averaged time that a person would

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spend smoking a cigarette. The instrument must also be capable of accurately filtering out the

particulate-phase material so that gas and particulate phases may be individually analyzed

without any interference2. There are multiple methods to which the particulate phase can be

separated from the gas phase (Cambridge filter pad, electrostatic precipitation, jet impaction,

solid absorbents, cold traps); however, none of these methods are completely effective, due to

the highly dynamic matrix of the smoke.5

Zimmermann, R.2 states that the objective of the work was to develop a single

instrumental setup which best meets these requirements to analyze, at trace levels, several

volatile and semi-volatile compounds, which are believed to cause health effects for human

smokers. The main goal of the method described is a fast and comprehensive analysis which

interferes as little as possible with the complex smoke matrix.2

A quantitative analysis was performed on toxic compounds present in whole smoke and

in the gas phase. In order to make the results comparable to those found in other tobacco science

literature, similar conditions were met, including the use of a Cambridge filter pad at room

temperature.2

The compounds analyzed in this experiment were chosen based on their accessibility by

the instrument used, relativity towards smokers, and expected yields in tobacco smoke

publications6-12 used as a guideline. The compounds analyzed are nitric oxide, acetaldehyde, 1,3-

butadiene, acetone, isoprene, benzene, toluene, ethyl benzene and xylene.2

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Zimmermann, R.2

The method for the Quantitative Puff-by-Puff Characterization of Selected Toxic

Compounds in Cigarette Mainstream Smoke uses a laser-based single-photon-ionization (SPI) –

time of flight mass spectrometer (TOFMS) coupled to a smoking machine.2 This setup is based

off of a description by Mitschke, S.,13 who demonstrated with Zimmerman, R.2 that laser-based

photoionization methods can be applied in order to monitor volatile substances in cigarette

smoke.

Single Photon Ionization allows the detection of various aliphatic and aromatic

compounds in smoke up to ~120 m/z. The SPI technique uses photoionization, which causes a

soft ionization of target molecules within the sample by emitting a controlled wavelength of

radiation.13

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Zimmermann, R.2

In this experiment, vacuum ultraviolet (VUV) photons are generated by a Nd:YAG laser,

and a wavelength of 118 nanometers (equivalent to 10.49 electron volts) is projected onto the

sample.2

Any organic compounds with an ionization potential lower than 10.49 eV will be ionized.

This proves SPI to be quite useful in this particular case. Gases, for example N2, O2, CO2, and

H2O, have a higher concentration than the target compounds and, therefore, would normally

overload the detector. Because these higher concentrated gases have a higher ionization potential

than 10.49 eV, the detector cannot record them. Any neutral molecules that reach the detector

will not be recorded. Therefore, the spectral data will not be overloaded with signals.2

In order to transfer the cigarette smoke into the ionization chamber, a smoking machine is

necessary. Borgwaldt single port smoking machines have been designed for instrumental

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applications involving cigarette smoke. This instrument was chosen for use, and necessary

modifications were made to decrease contaminations and memory effects of the commercially

available machine. The smoking machine consists of a filter holder, to which the cigarette is

attached. Connected to the filter holder, a smoking machine valve is attached, which allows air

intake. Between the smoking valve and pump, a heated transfer line consisting of a deactivated

silica capillary is attached. This line connects the smoking machine to the ionization chamber.

The purpose of the heated line is to evaporate the organic volatile and semi-volatile particles that

are deposited on any particles within the smoke. This allows the accessibility of these volatile

compounds by the SPI-TOFMS. To analyze the gas phase components, a Cambridge filter pad

was attached to the filter holder2. The Cambridge filter pad causes separation between particulate

and gas smoke phases. The quartz fiber filter pad is stabilized by an organic binder, which

collects the particulate phase of cigarette smoke.5

The procedure was carried out using 2R4F research cigarettes, available from the

University of Kentucky, Kentucky Tobacco Research and Development Center (KTRDC).

35mm puff measurements with 2s puff durations were delivered to the SPI-TOFMS once every

60s until 8mm of the cigarette remained (not including the cigarette filter). The cigarette was lit

with a Borgwaldt electric lighter.2 The cigarettes were stored for several days under controlled

conditions of 60% relative air humidity and 22 oC.14 During the experiment, three mass spectra

were obtained every second, and the signal intensities of each compound were summed for each

puff. Three gas phases and whole smoke replicates were obtained for every cigarette measured,

and the corresponding mean and standard deviation were calculated. Because a separation

between ethyl benzene and xylene is not possible (same molar mass), their yields were summed

together.2

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A three-dimensional illustration of the time-resolved SPI-TOFMS analysis of puffs 4, 5,

and 6 is shown below:

Zimmermann, R.2

The 180 (3 spectra per second for 60s) mass spectra, recorded during an individual puff,

were summed into a single spectrum. These three spectra show differences in the quantity of

each of the 9 chemicals. For example, the largest peak (acetaldehyde) increases with each

consecutive puff. This illustration helps represent the variation between the mass spectra on a

puff – by – puff real – time basis.

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A single mass spectrum is shown below, featuring all cigarette smoke species accessible

by SPI-TOFMS:

Zimmermann, R.2

No explanations were given as to when this spectrum was obtained. The purpose of this

illustration merely was to show what a mass spectrum of cigarette smoke looks like when SPI –

TOFMS were used. Note that the signals of xylene and ethyl benzene (~106 m/z) overlap each

other. That is why their values were combined as one during this study.

Due to varying lighting behaviors, the 2R4F research cigarette required eight to nine

puffs to complete. Three cigarettes were recorded for the whole smoke phase, and three for the

gas phase. The mean value of the number of puffs per cigarette is 8.7 for whole smoke, and 8.3

for the gas phase. Therefore, there was no standard deviation calculated for the ninth puff for the

gas phase.2 The contamination and memory problems caused by the smoking machine are even

greater when analyzing the gas phase. This is because of the Cambridge filter pad.13

When the filter pad is used during gas phase analyses, volatile organic material trapped in

the filter may evaporate and contaminate the smoking machine. A more accurate approach to this

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method would require a new filter pad for every new puff.2

Total smoke yields for each 2R4F research cigarette were recorded and compared to

those in other literature. The yields were in good agreement, so single puff quantifications were

carried out.2 The following graph shows a puff-by-puff comparison between the gas phase and

whole smoke.

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Zimmermann, R.2

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The gas-phase and whole smoke values of nitric oxide, acetaldehyde, acetone, benzene,

toluene, and the C2 benzenes gradually increase with each consecutive puff. This trend is caused

by the reduction in cigarette length. As the cigarette gets shorter, the condensation of the gases

decreases.2 Because whole smoke is a combination of both the gas phase and the particulate

phase, whole smoke values are expected to be larger than the gas-phase values.

However, there should be one exception: Nitric oxide. Nitric oxide has a boiling point of

-151.8 oC and, as a result, should remain completely in the gas-phase at room temperature. This

means the gas phase values of NO should be as large as the whole smoke values. These results

must reflect a reaction that removes nitric oxide. The Cambridge filter pad was only used to

analyze the gas phase, so one possibility could be that the Cambridge filter pad is directly

reacting with the NO.2

Nitric oxide oxidizes to NO2 in smoke over a short period of time.2 Reports15-16 show that

both NO and NO2 are capable of reacting with other compounds in cigarette smoke.

Molecules slow down as they pass through the Cambridge filter pad. As a result; more

time is required for the smoke to reach the ionization chamber. The lack of nitric oxide could

also be caused by any reactions taking place between NO other molecules in cigarette smoke.2

In conclusion, SPI-TOFMS coupled to a smoking machine has proven useful for a quick

and broad quantitative analysis of toxic compounds in cigarette smoke. Results obtained have

shown phase affiliation of compounds from one puff to another. In this study, 9 chemicals were

attained, but in principle, a greater number of molecules may be analyzed.2

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REFERENCES

(1) Zimmermann, R. (2006). Quantitative puff-by-puff-resolved characterization of selected toxic compounds in cigarette mainstream smoke. Chem. Res. Toxicol. 17, pp 511-520.

(2) Wakeham, H. (1971) Recent trends in tobacco and tobacco smoke research. In Proceedings of the Symposium on the Chemical Composition of Tobacco and Tobacco Smoke (Schmeltz, I., Ed.) pp 1-20, 162nd National Meeting of the American Chemical Society, Washington, DC.

(3) Baker, R. R. (1999) Smoke chemistry. In Tobacco: Production, Chemistry, and Technology (Davis, L. D., and Nielson, M. T., Eds.), pp 398-439, Blackwell Science, Oxford, U.K.

(4) Gaworski, C. L., Dozier, M. M., Eldridge, S. R., Morrissey, R., Rajendran, N., and Gerhart, J. M. (1998) Cigarette smoke vapor-phase effects on the rat upper respiratory tract. Inhalation Toxicol. 10, 857- 873.

(5) Dube, M. F., and Green, C. R. (1982) Methods of collection of smokefor analytical purposes. Recent Adv. Tob. Sci. 8, 42-102.

(6) Hoffmann, D., and Hecht, S. S. (1990) Advances in tobacco carcinogenesis.In Chemical Carcinogenesis and Mutagenesis I (Cooper, C. S., and Grover, P. L., Eds.) pp 307-364, Springer, London.

(7) Hoffmann, D., and Hoffmann, I. (1997) The changing cigarette: 1950-1995. J. Toxicol. EnViron. Health 50, 307-364.

(8) Hoffmann, D., and Hoffmann, I. (1998) Letters to the editor: tobaccosmoke components. Beitr. Tabakforsch. Int. 18 (1), 49-52.

(9) Vorhees, D. J., Heiger-Bernays, W., and McClean, M. D. (1997)Human health risk associated with cigarette smoke: the link betweensmoke constituents and additives. Menzie-Cura & Associates, Chelmsford,MA.

(10) Fowles, J., and Bates, M. (2000) The chemical constituents in cigarettesand cigarette smoke: priorities for harm reduction. A report to theNew Zealand Ministry of Health. Kenepuru Science Centre, Porirua,New Zealand.

(11) Fowles, J., and Dybing, E. (2003) Application of toxicological riskassessment principles to the chemical constituents of cigarette smoke.Tob. Control 12, 424-430.

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(12) Rodgman, A., and Green, C. R. (2003) Toxic chemicals in cigarettemainstream smoke hazards and hoopla. Beitr. Tabakforsch. Int. 20(8), 481-545.

(13) Mitschke, S., Adam, T., Streibel, T., Baker, R. R., and Zimmermann,R. (2005) Application of time-of-flight mass spectrometry with laser-basedphotoionization methods for time-resolved on-line analysis ofmainstream cigarette smoke. Anal. Chem. 77 (8), 2288-2296.

(14) Baker, R. R. (2002) The development and significance of standardsfor smoking-machine methodology. Beitr. Tabakforsch. Int. 20 (1), 23-41.

(15) Vilcins, G., and Lephardt, J. O. (1975) Aging processes of cigarettesmoke: formation of methyl nitrite. Chem. Ind. (London) 22, 974-975.

(16) Cueto, R., and Pryor, W. A. (1994) Cigarette smoke chemistry:Conversion of nitric oxide to nitrogen dioxide and reactions of nitrogenoxide with other smoke components as studied by Fourier TransformInfrared Spectroscopy. Vib. Spectrosc. 7, 97-111.