Short CommunicationReceived: 17 June 2008, Revised: 14 August 2008, Accepted: 15 August 2008 Published online 27 November 2008 in Wiley Interscience (www.interscience.wiley.com) DOI 10.1002/bmc.1141 Analysis of exhaled breath from smokers, passive smokers and non-smokers by solidphase microextraction gas chromatography/ mass spectrometry John Wiley & Sons, Ltd. BogusÝaw Buszewski,a* Agnieszka Ulanowska,a,b Tomasz Ligor,a,b Natalia Denderza and Anton Amannb Analysis of exhaled breath by SPME GC/MS ABSTRACT: In this study, 38 samples of expired air were collected and analyzed from 20 non-smoking volunteers, four passive smokers and 14 smokers (21 women and 17 men). Measurements were carried out using solid-phase microextraction (SPME) as an isolation and preconcentration technique. The determination and identification were accomplished by gas chromatography coupled with mass spectrometry (GC/MS). Our data showed that ca 32% of all identified compounds in the breath of healthy non-smokers were saturated hydrocarbons. In the breath of smoking and passive smoking volunteers hydrocarbons were predominant, but also present were more exogenous analytes such as furan, acetonitrile and benzene than in the breath of non-smokers. Acetonitrile, furan, 3-methylfuran, 2,5-dimethylfuran, 2-butanone, octane and decane were identified in breath of smoking and passive smoking persons. Copyright © 2008 John Wiley & Sons, Ltd. Keywords: breath analysis; lung diseases; cigarette smoking; biomarkers Introduction Biomed. Chromatogr. 2009; 23: 551– 556 * Correspondence to: B. Buszewski, Chair of Environmental Chemistry and Bioanalytics, Faculty of Chemistry, Nicolaus Copernicus University, 7 Gagarin Str., PL-87-100 Toruñ, Poland. E-mail:
[email protected] a Chair of Environmental Chemistry and Bioanalytics, Faculty of Chemistry, Nicolaus Copernicus University, 7 Gagarin Str., PL-87-100 Toruñ, Poland b Department of Anesthesiology and Critical Medicine Care, Innsbruck Medical University, A-6020 Innsbruck, Austria Abbreviations used: COPD, chronic obstructive pulmonary disease; IARC, International Agency for Research on Cancer; VOCs, volatile organic compounds. Contract/grant sponsor: European Commission; Contract/grant number: BAMOD 19031. Contract/grant sponsor: Polish Ministry of Sciences and High Education; Contract/grant number: N204 165 31/3730. Contract/grant sponsor: CEEPUS-II scholarship; Contract/grant number: CIIPL-0004-01-0506-M-4479 and CII-PL-0004-02-0607-M-12188. Copyright © 2008 John Wiley & Sons, Ltd. 551 The role of tobacco smoking and involuntary smoking in cancer is very well documented in the scientific literature and was recently re-evaluated by the International Agency for Research on Cancer (IARC). The recent report concludes that tobacco smoking and tobacco smoke are carcinogenic to humans (IARC Group 1 classification) and that involuntary smoking (exposure to second-hand or ‘environmental’ tobacco smoke) is carcinogenic to humans (Group 1; Lewtas, 2007). Many nitrogen-containing substances have been found in the mutagenic fraction of cigarette smoke condensate. It is also known that smoking cigarettes increases the amount of free radicals in human body (Gelvan et al., 1995). The excess of free oxygen species causes the disturbance in intracellular balance which is commonly called oxidative stress. In pathological states DNA, other proteins and lipids are damaged. These changes in cells and genetic material accelerate the cells’ senescence and have the direct influence in forming many different diseases and also cancers. During metabolic processes occurring in live organisms many substances are generated. These compounds penetrate the bloodstream and circulate through the whole body. The barrier between blood and alveolar air is very thin, which is why metabolites can be identified in breath samples. Therefore, breath air is a complex mixture of volatile organic compounds (VOCs) (i.e. alcohols, aldehydes, hydrocabons, etc.), non-volatile organic compounds (i.e. leukotrienes and prostaglandins) and inorganic compound such as water vapor, carbon dioxide and nitrogen (Aghdassi and Allard, 2004; Bucchioni et al., 2004; Buszewski et al., 2007). Some of them are recognized as biomarkers. Biomarkers present in breath can be exogenous or endogenous. Benzene, ethylbenzene, xylene isomers, toluene and trichloromethane are supposed to be exogenous and they are probably inhaled with ambient air or ingested with food (Plebani et al., 1999). Endogenous biomarkers are produced in the body during lipid oxidation (e.g. ethane, pentane) or can be a by-product of synthesis for more complicated biomolecules such as cholesterol (Miekisch et al., 2004; Buszewski et al., 2007). Their presence and concentration provide valuable information about human health and can be an indicator of pathological states. For example, pentane, acetaldehyde, 2-propanol and heptanal were recognized as biomarkers of breast cancer (Phillips et al., 2006). Other endogenous compounds such as isoprene and acetone and also exogenous benzene and ethylbenzene were more often identified than in the air from non-smokers. Varian Inc. Its concentration is higher in the breath of passive smokers than in non-smokers. isoprene and hexane are endogenous and the remaining compounds are exogenous (Ligor et al. Our study shows that proportional content of particular compounds from different chemical class changed depending on the origin of the breath sample. Results and Discussion Fifty-six VOCs were found in all breath samples. has been identified in exhaled breath of smoking. 2. Middelburg. 2005). Afterwards... The origin of some substances was considered and substances characteristic of smokers and involuntary smokers are detailed. such as acetonitrile. 2-methyltiofene. e.e. However. 2007). acetone.e. 2005. hexane and toluene were found in all breath samples.interscience. 2009. a 10 mL of breath sample was transferred from the bag to the glass vial.5dimethylfuran. passive smoking and smoking healthy volunteers belonging to different chemical classes are presented in Table 2. Chen et al. carbon disulfide. A manual SPME holder and carboxen/polydimethylsiloxane (CAR/PDMS) (75 μm) coated fiber (Supelco. ethanol. 2002).wiley. passive smoking and smoking volunteers are presented. the chemical pathways of generation and the physiological meaning many of these compounds have not yet been explained. In breath samples of healthy non-smoking persons acetaldehyde. acetonitrile. Aldehydes (acetaldehyde). The oven temperature program was as follows: initial 40°C held for 2 min. i.and 3-methylpentane. The amount of 2-methylpentane in the breath of smokers was four-fold higher than in expired air from non-smokers. respectively. carbon disulfide. 2-butanone). acetone. carbon disulfide.. such as 2. benzene. Experimental Apparatus The GC/MS analysis was performed on Agilent 5975 Inert XL MSD coupled with 6890 N gas chromatograph (Agilent Technologies. are biomarkers of lung cancer (Poli et al. the same dependence also was observed (Poli et al.5-dimethylfuran. xylene isomers.and 3-methylpentane. ketones (acetone. Compounds present in cigarette smoke. Compounds such as ethanol. Waldbronn. acetonitrile and aromatic hydrocarbons like styrene. 2004). Among the identified VOCs were alcohols. The temperatures of the ion source and quadrupole were 190 and 150°C. isoprene. Some of these substances are toxic and carcinogenic. The scan rate was 3. non-smoking and passive smoking persons (Gelmont et al. acetone. The concentration of this compound also increases in patients with lung cancer and chronic obstructive pulmonary disease (COPD). then ramped at 5°C/min to 270°C and held for 3 min. The Netherlands) was used. 2005. Isoprene. octane and decane. Figure 1 shows a typical GC/MS chromatogram of breath from a healthy non-smoking volunteer. benzene. substances such as furan. Biomed. isoprene. 23: 551– 556 .. benzene. Copyright © 2008 John Wiley & Sons. hexane and toluene are usually identified. 2. Breath Collection 552 The exhaled air was collected and analyzed from 20 nonsmoking volunteers. Buszewski et al. Other compounds very often identified in breath samples are alcohols such as methanol. tetrahydrofuran. For many years it has been known that smoking cigarettes causes different diseases and also increases the risk of lung cancer. The exhaled breath was collected in 1 L Tedlar bags which were cleaned by flushing with nitrogen gas. In the exhaled breath of smoking and passive smoking persons many more exogenous substances were identified than in breath samples from non-smoking volunteers. the major hydrocarbon in human breath.. acetonitrile and xylene isomers.g. Poland). ethanol and 2-propanol. crimped glass vials were evacuated using a glass syringe. Before transferring. USA) was used.. 2. Helium was used as a carrier gas with linear velocity 40 cm/s. In the breath of non-smokers the most frequently observed chemicals were saturated hydrocarbons.. All patients were asked to fill in a questionnaire describing their current smoking status. Compounds identified in breath samples of non-smoking.25 mm × 3 μm capillary column (CP-Porabond-Q. toluene. hydrocarbons and sulfur compounds. In this paper compounds identified in exhaled breath of non-smoking. These analytes are supposed to be characteristic for smokers and some of them have been recognized as lung cancer biomarkers (Poli et al. 3methylfuran. The acquisition of chromatographic data was performed by means of Chemstation Software (Agilent). four passive smokers and 14 smokers (21 www. Ethanol. Germany). An SPME fiber was inserted in the vial and exposed to VOCs for 15 min. 2.5-dimethylfuran. The indoor air samples were also collected for background (Schlopp et al. These analytes have endogenous or exogenous origin.. Electron impact ionization was applied at 70 eV. can also be identified in smokers’ and passive smokers’ breath. toluene and styrene. dimethyl sulfide and 3-methylthio-1-propene) are also found in exhaled air samples. Ltd. 2007). 2-methyl1-propene. respectively. Germany) with a split–splitless injector.46 scans/s. passive smokers and the time elapsed since their last smoke. non-smokers. with scan range 15–220 amu. They are listed in Table 1. The classification to the appropriate group was done on the basis self-declaration of the volunteers. However. ketones. 1981). Solid-phase microextraction and gas chromatography coupled with mass spectrometry (SPME GC/MS) was developed for the analysis of volatiles in breath. 2007). aldehydes. active smokers. but is the highest in exhaled air from active smokers. then ramped at 10°C/min to 140°C. furan. ones. methylcyclopentane.B. sulfur-containing compounds (i. The temperature of the split–splitless injector was 200°C. They were not found in exhaled breath from non-smoking persons. A 25 m × 0. which were identified only in the breath of smoking and passive smoking persons.com/journal/bmc women and 17 men). The MS analyses were carried out in full-scan mode. Argon and nitrogen (purity 99.. pentane. In the case of pentane and 2-methylpentane. Bellefonte. the splitless time was 1 min and tje split ratio figure was 1:35. toluene. The saturated hydrocarbons were predominated. Chemicals and Materials Standard compounds were purchased from Sigma-Aldrich (Steinheim.. Chen et al.999%) were purchased from BOC Gazy (Bydgoszcz. The capnography method was used for the control of alveolar air sampling (Schubert et al. Chromatogr. Figures 2 and 3 show the GC/MS chromatograms of breath from a healthy smoker and a passive smoker. 2-butanone. carbon disulfide. There are some compounds. 3-methylfuran. ex.5-Dimethylfuran 3-(Methylthio)-1-propene 2.4-Dimethylheptane 4-Methylheptane Octane Decane The frequency of appearance in breath sample (%) Non-smoker Passive smoker Smoker Origin 30 (6) 45 (9) 100 (20) 20 (4) 30 (6) 10 (2) 0 30 (6) 0 100 (20) 20 (4) 95 (19) 30 (6) 15 (3) 25 (5) 10 (2) 10 (2) 15 (3) 100 (20) 60 (12) 5 (1) 5 (1) 15 (3) 15 (3) 55 (11) 0 10 (2) 0 50 (10) 25 (5) 95 (19) 100 (20) 65 (13) 90 (17) 100 (20) 70 (14) 5 (1) 0 5 (1) 45 (9) 5 (1) 20 (4) 15 (3) 75 (15) 75 (15) 5 (1) 5 (1) 10 (2) 35 (7) 20 (10) 5 (1) 40 (8) 5 (1) 5 (1) 0 0 0 25 (1) 100 (4) 0 25 (1) 25 (1) 100 (4) 50 (2) 25 (1) 100 (4) 50 (2) 100 (4) 0 25 (1) 50 (2) 25 (1) 25 (1) 0 100 (4) 50 (2) 0 0 25 (1) 25 (1) 50 (2) 0 25 (1) 0 75 (3) 0 100 (4) 100 (4) 50 (2) 100 (4) 100 (4) 100 (4) 0 0 0 50 (2) 0 25 (1) 25 (1) 50 (2) 25 (1) 0 0 0 50 (2) 50 (2) 25 (1) 25 (1) 0 0 25 (1) 25 (1) 21 (3) 14 (2) 100 (14) 14 (2) 14 (2) 14 (2) 100 (14) 50 (7) 7 (1) 100 (14) 29 (4) 100 (14) 100 (14) 29 (4) 29 (4) 0 7 (1) 7 (1) 79 (11) 79 (11) 7 (1) 0 7 (1) 7 (1) 43 (6) 7 (1) 14 (2) 7 (1) 14 (2) 38 (5) 100 (14) 100 (14) 79 (11) 86 (12) 100 (14) 38 (5) 0 7 (1) 0 29 (4) 0 21 (3) 14 (2) 79 (11) 79 (11) 14 (2) 0 7 (1) 50 (7) 64 (9) 14 (2) 43 (6) 7 (1) 7 (1) 14 (2) 7 (1) en en en ? ? ? ex en ex en ex en en/ex en ex ex ? ex en en ? ? ? ex ex ex ex en ? ? ? ? ex ? en ex ? ex ? ? ? ? ? ex ? ? ? ? ex ex ex ex ? ? en en a Biomed. en. 2009. Chromatogr.wiley.3-Pentadiene Isoprene Pentane 1-Pentene 2-Methyl-2-propenal Cyclopentane 2-Methylfuran Trichloromethane 3-Methylfuran Tetrahydofuran (THF) 2-Butanone Ethyl acetate 2. 23: 551– 556 Copyright © 2008 John Wiley & Sons. Ltd.interscience. www. endogenous.4.4-Dimethylpentane 2-Methylthiofene 2-Methylhexane 3-Methylhexane Toluenea 2. Compounds identified in exhaled breath of smoking.5-Dimethyl-2-hexene Ethylbenzene* p-Xylene Styrene o-Xylene 2.Analysis of exhaled breath by SPME GC/MS Table 1. The number in parentheses is the number of breath samples the compound was found in. the origin is not known.2-Dietoxyethane 2-Methylbutane 1. . The total number of examined non-smokers was 20.com/journal/bmc 553 Confirmed with standards. passive smokers 4 and active smokers 14 Compound a Methanol Acetaldehyde Ethanola Isobutane 2-Methyl-1-propene 2-Butene Acetonitrilea Butane Furan Acetonea Methylene chloride Carbon disulfide 2-Propanola Dimethyl sulfide (DMS) Diethyl ether 1.2-Dimethylbutane 2-Methylpentane 3-Methylpentane Benzenea Methylcyclopentane Hexanea Cyclohexane Isopropyloctane 2. non-smoking and passive smoking volunteers.4-Trimethyl-1-pentene 5-Methyl-3-heptene 3-Methyl-2-heptene 5. exogenous.?. Chromatogr. 13) hexane. 13) hexane. 6) isoprene. 16) ethylbenzene.. Another study showed that the concentration of benzene in exhaled air from smokers rapidly increases after smoking a cigarette. Benzene and toluene are commonly identified in breath samples independent on their origin. 7) pentane. benzene is regarded as toxic and carcinogenic and it is always identified in greater amounts in the breath of smokers. 3) butane. 2007). 4) acetone.com/journal/bmc micrograms per cigarette. Substances such as benzene.interscience. styrene. 5) carbon disulfide. Biomed. 4) acetone. 17) p-xylene. The GC/MS chromatogram of exhaled breath from a healthy smoking person: 1) ethanol. 8) 3-methylfuran. 14) toluene. 2) acetonitrile. but decline to values similar to those of non-smokers within an hour Copyright © 2008 John Wiley & Sons.wiley. Ltd. Figure 1.. toluene. 2. 3) 2-methyl-1-propene. 7) pentane. 5) carbon disulfide. 2007). 12) methylcyclopentane. However.4. 23: 551– 556 . 10) 2-methylpentane. The GC/MS chromatogram of exhaled breath from a healthy non-smoking person: 1) acetaldehyde. 15) 2. The chromatographic searching showed that in cigarette fume about 3500 can be identified and over 40 have carcinogen properties (Charles et al. 18) o-xylene. 11) benzene. 10) 3-methylpentane. Buszewski et al. 8) trichloromethane. Figure 2. 9) ethyl acetate.5-dimethylfuran and other compounds can be detected in amounts of about a few hundreds of www. 6) isoprene. 554 The increasing number of exogenous compounds in the air from smokers and passive smokers was caused by smoking cigarette (Charles et al. 2009. 2) ethanol. 9) 2-methylpentane.B.4-trimethyl-1pentene. 12) methylcyclopentane. 14) toluene. 11) 3-methylpentane. Acknowledgements This work is supported by the European Commission (project BAMOD no.2 Conclusions Chromatographic measurements showed that in the breath of smokers and passive smokers many exogenous compounds can be identified. 9) tetrahydrofuran.6 1. These substances originate from cigarette smoke.5-dimethylfuran) are toxic and carcinogenic and might increase the probability of appearance of lung cancer.1 10. once smoking stops.8 5. Human exhaled air analytics: biomarkers of diseases.0 10.5 8. 2.1 7. Breath alkanes as a marker of oxidative stress in different clinical conditions.6 2. Chromatogr. 6) dimethyl sulfide. Furan.g.7 0.7 6.2 3.wiley. Csoma Z. Thus they were identified more often in smokers’ and passive smokers’ breath than in exhaled air from non-smokers.3 13. In contrast.6 3.0 20.7 10. . Financial support from Foundation for Polish Sciences (FNP) Professor’s Subsidy. 5) carbon disulfide.2 19. Respiratory Medicine 2004.8 0. Barnes PJ and Kharitonov SA. References Biomed. 17) 2.8 2.Analysis of exhaled breath by SPME GC/MS Figure 3. Table 2. passive smoking and smoking healthy volunteers belonging to different chemical classes Percentage compounds in chemical class (%) Chemical class Hydrocarbon Hydrocarbon unsaturated Hydrocarbon cyclic Alcohol Aldehyde Ketone Nitril Furan-related Chlorinated compound Sulfur-containig compound Ether Ester Aromatic Nonsmoker Passive smoker Smoker 32. Some of these analytes (e.interscience. 16) toluene. 1995).9 7. Bucchioni E. Ligor T and Amann A. Ltd.3 3. Composition and emissions of VOCs Copyright © 2008 John Wiley & Sons. These carcinogen substances can be used to distinguish between smokers and non-smokers (Gordon et al.3 8. 10) ethyl acetate.3 4. Chung KF. The most common identified exogenous compounds were benzene.1 14. 2009. Charles SM.com/journal/bmc 555 (Plebani et al.7 4. 23: 551– 556 Aghdassi E and Allard JP.9 8. 13) methylcyclopentane. 1999). 3) butane. Free Radical Biology and Medicine 2000. 2002). Buszewski B. 2) acetonitrile.6 3. N204 165 31/3730) and CEEPUS-II scholarship CII-PL-0004-01-0506-M-4479 and CII-PL-0004-02-0607-M-12188. xylene isomers. 19) p-xylene. Batterman SA and Jia C. Number of compounds identified in breath samples of non-smoking. 11) 2-methylpentane. (Mistrz programme) is gratefully acknowledged.5-dimethylfuran were identified in the breath of smokers and passive smokers..2 14. Biomedical Chromatography 2007.6 6. 14) hexane. 2-methylfuran and 2.. 7) isoprene.2 1. 8) 2-methylfuran. ethylbenzene. 28(6): 880–886.3 4.4. benzene. 15) cyclohexane.0 4.8 25.3 0 2.0 0. Allegra L. Thus the analysis of acetonitrile in breath sample is the most suitable indicator of whether or not a given subject is a smoker (Jordan et al.8 3. the Polish Ministry of Sciences and High Education (grant no. 4) acetone. styrene. The GC/MS chromatogram of exhaled breath from a healthy passive smoking person: 1) ethanol. the concentration of acetonitrile in exhaled air of smokers takes almost a week to decrease to that of non-smokers. 21: 553– 566.2 1. Kèsy M.5 18. The large number of aromatic compounds in exhaled air from passive smokers can be also a reason for increased risk of lung cancer. 98: 651– 655.4-trimethyl-1-pentene. Adenosine 50-monophosphate increases levels of leukotrienes in breath condensate in astma. 12) 3-methylpentane. 20) o-xylene. www. acetonitrile and furan derivatives. 18) ethylbenzene.2 4. 19031).. Greenberg J. Plebani C. Balbi B. Clopton DA. Chromatogr. Prediction of breast cancer using volatile biomarkers in the breath. Ligor T. Pan Y. 99: 1456–1460. Volatile organic compounds as breath biomarkers for active and passive smoking.interscience. Gunawardena R. Chen Q and Saltman P. Cancer 2007. Tranfo G. Biochemistry Biophysical Research Communications 1995. Stein RA and Mead JF. Salerno A. Schubert JK. Journal of Breath Research 2007.and side-stream smoke of research cigarettes. and cardiovascular effects. Tietje O and Wong C. Geiger K and Noldge-Schomburg GF. Kwon CS. Talanta 1999. Biochemistry Biophysical Research Communications 1981. Bianchi L. reproductive. Lewtas J. A study of the volatile organic compounds exhaled by lung cancer cells in vitro for breath diagnosis. Acampa O. 110(7): 689–698.com/journal/bmc Copyright © 2008 John Wiley & Sons. 40: 587–594. 347: 25–39. Carbognani P. Buszewski et al. Gordon SM. Szeliga J. Miekisch W. Jackowski M and Buszewski B. Mutation Research 2007. 636: 95–133. Biomed. Fisher P. Miekisch W and Geiger K. Acetonitrile and benzene in the breath of smokers and non-smokers investigated by proton transfer reaction mass spectrometry (PTR-MS). International Journal of Mass Spectrometry and Ion Processes 1995. Breath analysis in critically ill patients: potential and limitations. Scholpp J. Holzinger R and Lindinger W. 71(6): 1–10. Lu D. Schubert JK. Respiratory Research 2005. Air pollution combustion emissions: characterization of causative agents and mechanisms associated with cancer. Goldoni M. 23: 551– 556 . Poli D. Hansel A. Brinkman MC. 110: 835–844. Atmospheric Environment 2007. 556 www. Breath markers and soluble lipid peroxidation markers in critically ill patients. Exhaled volatile organic compounds in patients with non-small cell lung cancer: cross sectional and nested short-term follow-up study. Sites and mechanism of low-level oxidative stress in cultured cells. 148: 1–3. Rusca and Mutti A. Miekisch W. Wang P. Wallace LA. Diagnostic potential of breath analysis—focus on volatile organic compounds. Ying K. Isoprene—the main hydrocarbon in human breath. Environmental Health Perspectives 2002. Schubert JK and Noeldge-Schomburg GFE. 50: 409–412. Chen X.wiley. Expert Review of Molecular Diagnostics 2004. as a biomarker for occupational exposure. Breast Cancer Research and Treatment 2006. Preliminary study of volatile organic compounds from breath and stomach tissue by means of solid phase microextraction and gas chromatography–mass spectrometry. 1: 1–6. Corradi M. Wang Y. Xu F. An optimized sampling and GC–MS analysis method for benzene in exhaled breath. Gelvan D. Cataneo RN. 206(1): 421–428.B. 4: 619–629. Chen E and Zhang W. Moreno V. Panebianco A and Marcelloni AM. Clinical Chemistry Laboratory Medicine 2002. Jordan A. Callahan PJ and Kenny DV. Phillips M. 99: 19–21. Ditko BA. 41: 5371–5384. Ltd. 2009. Clinica Chimica Acta 2004. Gelmont D. in main.