Volatile Organic Compounds Analysis in Breath Air in Healthy Volunteers and Patients Suffering Epidermoid Laryngeal Carcinomas
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- García, R.A., Morales, V., Martín, S. et al. Chromatographia (2014) 77: 501. doi:10.1007/s10337-013-2611-7
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Exhaled breath contains thousands of gaseous volatile organic compounds (VOCs) that may be used as non-invasive markers of head and neck epidermoid cancer. We hypothesized that solid phase micro-extraction coupled to gas chromatography–mass spectrometry can discriminate patients with epidermoid head and neck cancer from healthy controls by analyzing the gaseous volatile organic compounds, VOC-profile, in exhaled breath, thus identifying some non-invasive biomarkers to be used in early detection. Twenty healthy subjects participated in a cross-sectional study plus 11 patients with epidermoid supraglottic laryngeal cancer. VOCs from T3 supraglottic cancer were clustered distinctly from those of T1 and healthy subjects. Up to seven VOCs were detected differently from healthy volunteers, mainly 2-butanone and ethanol. Thus VOC-patterns of exhaled breath may discriminate patients with epidermoid head and neck cancer from healthy controls.
Neoplasms of the head and neck are particularly important because surgical treatment may cause extensive aesthetic deformities and impair important functions such as ingestion and speaking. Overall, head and neck carcinomas represent 5–10 % of malignant tumors diagnosed annually in Spain, and cause about 5 % of cancer deaths . About 500,000 new cases are diagnosed each year, representing the sixth most common cancer worldwide . Over 90 % of tumors in this region are epidermoid laryngeal carcinomas. The most common head and neck cancer is in the larynx, even though other points are the oral cavity, oropharynx, hypopharynx, and nasopharynx [3, 4]. The incidence of this group of tumors has been increasing over the past 30 years, with the most notable increase in tumors of the oral cavity and pharynx. Some 90 % of epidermoid carcinomas of head and neck are related to lifestyle, especially with the consumption of cigarettes and alcohol. The incidence of larynx cancer estimated in Spain in 2000 was 0.33 cases per 100,000 inhabitants for women, and 19.91 cases per 100,000 inhabitants for men, representing, in the case of males, the highest among European Union countries .
There are currently no effective programs for early detection of head and neck cancer. Close monitoring is recommended for people with known risk factors, such as heavy smokers and drinkers . On the other hand, no tumor marker has yet been able to identify epidermoid carcinomas of the head and neck to help early diagnosis. There are some early molecular events in carcinogenesis of head and neck, but their utility as a tumor marker has yet to be confirmed. Furthermore, the detection of some oncogenes, tumor suppressor genes, and DNA repair genes help us to provide prognostic information for some head and neck tumors, but do not allow early detection programs . Since the prognosis of these patients depends on the detection of cancer in early stages, it seems that it might of interest to develop a method for early diagnosis of disease in those patients with risk factors.
There exists strong evidence suggesting that some types of cancers may be detected through the analysis of exhaled air [7–10]. In 1971, Pauling et al.  identified over 200 volatile organic compounds (VOCs) present in exhaled air. This work has provided a starting point for research, allowing the association of these compounds with certain diseases [8–10]. Since 1985, further studies have been conducted in order to clarify the spectra of exhaled air, since a spectra of over 200 compounds may be very complicated to interpret. From statistical studies between healthy and cancer-diseased lungs, Gordon et al. and O’Neill et al. [10, 12] identified some potentially significant compounds for diagnosis. The search for organic compounds is not just limited to the information collected through exhaled air. This information is complementary to that obtainable from other samples: sweat, urine, and blood, which have already provided positive results in other applications such as drug analysis [13, 14].
The technique of solid phase micro-extraction (SPME) may be useful for the analysis of exhaled air. It is one of the methods used to concentrate volatile organic compounds emitted by different sources [15, 16]. The equipment is not expensive,is easy to use, and its small size makes it an easily transportable device [17, 18]. It was developed in the late 1980s by Arthur and Pawliszyn , and the method has been applied successfully in a wide variety of fields: environment, toxicology, pharmacology, and food and drug analysis. The coupling of gas chromatography with mass spectrometry allows the separation of the compounds to be carried into the chromatograph, while the identification is performed through the mass spectrometer [20–24].
This work is intended to investigate the viability for detecting some VOCs in the air breathed out by patients, with the potential for them to be used as biomarkers in the early detection of epidermoid laryngeal carcinomas. The SPME for analysis of exhaled air combined with gas chromatography–mass spectrometry (SPME/GC–MS) was used as our analytical technique.
Chemical and Materials
Analytical standards used were: indoor air standard 50 component, 1,000 μg mL−1 each component in methanol:water (97:3) (Aldrich); C1–C6 n-paraffins, 15 ppm each component in nitrogen (Fluka); and BTEX mix in nitrogen, 10 ppm each component in nitrogen (Fluka). All SPME fibers assemblies, fiber holders for manual and automatic sampling, 0.75 mm liners for SPME, and 5-L Tedlar Bags were from Supelco.
Description of the subject studied
Supraglottic extirpated 1 year ago
Once the patient air breath has been transferred to the Tedlar Bags, SPME sorption was performed using several fibers: CAR/PDMS, DVB/PDMS, PDMS, and CAR/PDMS/DVB. The extraction was performed at 25 °C within 15 and up to 120 min in order to optimize the adsorption time. The fiber was introduced into the GC injector for analysis immediately after sampling. Two bags of each sample were analyzed by this procedure to ensure reproducibility. The fibers were initially conditioned according to the instructions of the manufacturer in order to remove contaminants and to stabilize the solid phase. Conditioning was carried out in an extra split/splitless port with helium carrier gas prior to each adsorption.
To obtain the experimental data, a mass spectrometer Saturn 2000 ion trap GC/MS (Varian) was employed. The chromatographic column used was of type VF-5 ms capillary FOUR factor (Varian). The column length was 60 m, internal diameter 0.25 mm, and the thickness of the stationary phase was 0.25 μm. A splitless injection mode was selected with a valve off-time of 5 min; during this time, the SPME fiber was inserted into the gas chromatograph injector at 175 °C for 5 min, since the compounds adsorbed by the fiber are extracted with increasing temperature. The initial temperature of the column was held at 35° for 5 min, and then programmed to rise to 115° at 5 °C min−1 and to 250 °C at 20 °C min−1. Helium was used as the carrier gas at a flow rate of 1 mL min−1. The mass spectrometer was operated in electron impact ionization mode at 70 eV. The electron-impact (EI) mass spectra of the analytes were recorded in scan mode (scan range 35–280 m/z) to determine retention times and characteristic mass fragments.
Results and Discussion
To conduct this study, ten healthy non-smokers and ten healthy smokers were voluntarily chosen, in order to perform a preliminary study of the VOCs present in the exhaled air of healthy people, and, therefore, to confirm that the air exhaled by all subjects analyzed follows a certain pattern. The comparison was made by age, and exhaled air bags from people with an age range between 20 and 60 years were analyzed. After studying the characteristics, in terms of the composition of the exhaled air from a population of healthy smokers and non-smokers at different ages, the exhaled air from people with larynx cancer at different stages was analyzed: four patients with supraglottic epidermoid larynx cancer T3, six patients with supraglottic epidermoid larynx cancer T1, and one patient with supraglottic epidermoid larynx cancer T3 who had been operated on and the tumor removed.
The technique of SPME is based on the adsorption of compounds from the sample of exhaled air to the adsorbent fiber, placed on the SPME equipment, leaving a sufficient contact time for the analyte concentration to either reach or get close to adsorption equilibrium. Thus, to test real samples, it is necessary to optimize the method of compound adsorption/concentration with the aim of identifying the greatest number of VOCs present in the air. Thus, two parameters that exert significant influence on the SPME technique with gas samples were optimized: the nature of the adsorbent phase SPME fiber and the time of adsorption.
Study of the Influence of Adsorbent Fiber Type
Identified compounds in terms of the SPME fiber type in use
Acetone, toluene, α-pinene, β-pinene, n-decane, limonene,n-undecane, n-tridecane, n-tetradecane, n-pentadecane, n-hexadecane
Acetone, 2-butanone, n-hexane, bromodicloromethane, isoctane, n-heptane, toluene, ethylbencene, m+p-xylene, o-xylene, α-pinene, β-pinene, 1,2,4-trimethylbencene, n-decane, limonene
Acetone, isopropanol, ethyl acetate, n-heptane, toluene, butyl acetate, n-octane, ethylbencene, m+p-xylene, estirene, o-xylene, n-nonane, α-pinene, 3-ethyltoluene+4-ehyltoluene, 1,3,5-trimethylbencene, 2-ethyltoluene, β-pinene, 1,2,4- trimethylbencene, n-decane, 1,4-dichlorobencene, limonene, n-undecane, n-DODECANE, n-tridecane, n-tetradecane
Acetone, isopropanol, 2-butanone, bromodicloromethane, isoctane, n-heptane, toluene, butyl acetate, n-octane, ethylbencene, m+p-xylene, o-xylene, n-nonane, α-pinene, 2-ethyltoluene, β-pinene, 1,2,4-trimethylbencene, n-decane, 1,4-diclorobencene, limonene, n-undecane, n-dodecane, n-tridecane, n-tetradecane
Adsorption Time Optimization
Once having established the type of fiber to be used, the adsorption time featuring each pair of analytes/fibers was the next parameter to be considered. To determine the optimal, four different times were chosen from 15 to 120 min, using the fiber DVB/PDMS chosen as optimal in the previous section.
In the analyzed chromatograms (not shown), the influence of adsorption time is much more pronounced in the compounds shown in the third part of the chromatogram, in which a significant increase of compounds, from 15 to 30 min, is noticed. After examining the results, in general, the chromatographic peak area of the compounds augments as the adsorption time increases. The longer the contact time between the fiber and the sample, the greater the concentration retained. However, this trend is fulfilled up to near the equilibrium time, when the analyte concentration remains constant. A maximum area was achieved by 60 min, and therefore this was established as optimal for the further studies.
Determination of the Limit of Detection (LOD) in the SPME/GC–MS Process
The Limit of Detection (LOD) is the lowest concentration of analyte in a sample that can be detected. The instrument LOD is an important factor to be determined because the lack of certain compounds may be due either to their absence in the exhaled air, or because their concentration is below the LOD. In both cases, the analytes would be undetectable by the analytical equipment used.
n-Hexane was used as reference compound, by diluting a standard mixture at different concentrations (500, 100, 50, and 10 ng mL−1), and thereafter concentrated with the SPME technique, and analyzed by GC–MS. In the proposed method, the LOD was 40 ng mL−1, which may be sufficient for the analysis of VOCs, taking into account that the concentration of the exhaled air is estimated in the literature to be in the range 10−9–10−12 M [7, 26].
Feasibility Study of Tedlar Bags Storage
As noted above, the exhaled air is stored in Tedlar bags, which are sealed and easily transportable. However, during the analysis of the samples, it was observed that the chemical composition of the bag interfered with the results obtained, due to adsorption of volatile compounds from the bag into the SPME fiber.
The chromatographic analysis showed that, during the first 20 min, no presence of compounds was detected, and therefore the identified peaks in real samples in this time interval are inherent to the composition of exhaled air, and no subtraction of the blank is needed. After 20 min, two main compounds were detected: dimethyl acetamide and phenol, with a relatively high intensity. In addition, during the study of the real samples, these two compounds have the highest intensities in the chromatographic analysis. As a consequence, the different compounds released by the sample bag were subtracted from the real samples, since these compounds came from the decomposition of plastic bags [27–29].
Analysis of Real Samples of Healthy Non-Smokers and Smokers
From the study of the chromatograms, it can be concluded that the air exhaled by healthy people, both smokers and non-smokers, is characterized by exhibiting similar chromatograms, making possible the comparison between samples, and allowing to the creation of a typical pattern for the analysis of air exhaled by healthy people. An average of 45 VOCs present in exhaled air was detected for each subject. These results are consistent with those obtained by Phillips  for the analysis of VOCs present in air exhaled by humans, these authors identifying up to 100 compounds using other systems for sample pre-concentration.
Studying the patient ages, small differences can be found, as shown in the chromatograms of Fig. 1 for non-smoker subjects. Subjects over 50 (1, 2, and 3) display slightly greater numbers of VOCs than subjects over 20–30, during the first 20 min of analysis. At least, two clear compounds are distinguished, ethyl acetate and acetic acid. Additionally, a 15 % higher intensity in the peaks areas from the older subjects can be clearly seen.
In the chromatograms obtained when analyzing smokers patients, no significant differences comparing the patient’s age are noticed, indicating that the age factor for this population is not significant. In this category, the exhaled air is conditioned by the consumption of cigarettes, obtaining higher intensities in the subjects over 50–60 years. However, performing a comparison between smoker and non-smoker subjects aged between 20 and 30, there are clear differences in the peaks of the chromatograms, not only regarding the different compounds but also in the intensity of some common analytes to both families of subjects.
By comparing both age families, a greater number of compounds are detected in the chromatograms of subjects aged between 50 and 60, regardless of their status as a smoker or not. Focusing on the subjects between 50 and 60, certain differences between smokers and non-smokers are found, similar to the subjects in their 20–30s. For some common compounds, there are differences in intensities. Concentrations of certain compounds, in the case of smokers, are indeed relevant; for example, n-nonane with an increase of the peak area around 50 %.
Additionally, four possible potential compounds that may be indicative of potential marker for smokers have been detected. These compounds were only identified in these samples, while undetected in the breathed out air from non-smokers [8, 25]. The compounds are benzene (diagnostic ions at m/z 78 and 51), furaldehyde (diagnostic ions at m/z 96, 67, and 39), 4-isobutyl-1-(1-hydroxyethyl)-benzene diagnostic ions at m/z 122, 91, and 65) and 2,3,5-trimethylhexane (diagnostic ions at m/z 85, 57, and 43).
Analysis of Exhaled Air by People with Larynx Cancer
Potentially markers compounds of laryngeal carcinoma
Retention time (min)
45, 31, 29
57, 43, 29
57, 45, 43, 29
109, 82, 55
83, 70, 55
93, 80, 39
101, 94, 91, 79
Meaningfully, no presence of significant concentrations of these compounds in the case of supraglottic epidermoid larynx cancer T1 was found, showing a chromatographic pattern very similar to healthy subjects. Table 3 shows the potential markers that have been found from the analysis of subjects with supraglottic epidermoid larynx cancer T3, after comparison with the compounds found in patients with supraglottic epidermoid larynx cancer T1, and healthy patients, both smokers and non-smokers.
The study of the exhaled air of patients with laryngeal cancer at different stages determines that the analysis by GC–MS is a useful technique for identifying compounds that are potential biomarkers of epidermoid carcinomas of head and neck. Up to seven different compounds have been identified, regarding patients with the same disease in a less advanced stage, and healthy subjects, the most significant being ethanol and 2-butanone. Analyzing the results, we conclude that VOCs appear only in locally advanced tumors. Further investigations improving the analytical technique and the concentrations to be detected will be necessary to extend the results to small tumors and to the histological characteristics of the tumor.
Indeed, although these results are preliminary, an interesting path is open to explore the development of devices (electronic noses) to detect volatile substances that are potential markers of respiratory diseases, in this case of epidermoid larynx carcinomas of the head and neck.
In the light of the results, we can conclude that the optimized method of coupling the techniques of solid phase micro-extraction, SPME, and GC–MS can identify volatile organic compounds in exhaled air, and also discriminate between subjects of different ages, and smokers and non-smokers. The method is feasible as a starting point for a study of patients with laryngeal cancer, since some compounds that may have potential utility as biomarkers in these diseases have been identified.
The sample should always be collected in the same room where the ambient air has been previously analyzed. The patient must be fasting 8 h before blowing, and no alcohol or cigarettes must be consumed. The technique of solid phase microextraction, SPME, concentrated the compounds present in exhaled air samples between 100 and 500 times. Additionally, the variables that most influence the effectiveness of the process were optimized, such as the type of adsorbent phase, concluding that the fiber DVB/PDMS provides the best results, and the extraction time, which is set at 60 min.
A total of 31 common VOCs in non-smokers have been determined, whereas smokers reach up to 45 VOCs, thus differentiating the two types of populations. The comparative analysis between healthy smokers and non-smokers reveals four marker compounds related to cigarette consumption, since they have only been detected in samples from smokers. These compounds are: benzene, furaldehyde, 4-isobutyl-1-(1-hydroxyethyl)-benzene, and 2,3,5-trimethylhexane.
The study of the exhaled air of patients with laryngeal cancer at different stages determines that the analysis by GC–MS is a useful technique for identifying compounds that are potential biomarkers of epidermoid carcinomas of the head and neck. Up to seven different compounds have been identified, regarding patients with the same disease in a less advanced stage, and healthy subjects, the most significant being ethanol and 2-butanone.
We would like to thank all the participants in our study, especially those who are suffering from cancer. The financial support of Fundación Mútua Madrileña and the Spanish government (CTQ2008-05909/PPQ and CTQ2011-22707 projects) is gratefully acknowledged.