Tomasz Ligor

Determination of volatile organic compounds in exhaled breath of patients with lung cancer using solid phase microextraction and gas chromatography mass spectrometry

Abstract Background: Analysis of exhaled breath is a promising diagnostic method. Sampling of exhaled breath is non-invasive and can be performed as often as considered desirable. There are indications that the concentration and presence of certain of volatile compounds in exhaled breath of lung cancer patients is different from concentrations in healthy volunteers. This might lead to a future diagnostic test for lung cancer. Methods: Exhaled breath samples from 65 patients with different stages of lung cancer and undergoing different treatment regimes were analysed. Mixed expiratory and indoor air samples were collected. Solid phase microextraction (SPME) with carboxen/polydimethylsiloxane (CAR/PDMS) sorbent was applied. Compounds were analysed by means of gas chromatography (GC) and mass spectrometry (MS). Results: The method we used allowed identification with the spectral library of 103 compounds showing at least 15% higher concentration in exhaled breath than in inhaled air. Among those 103 compounds, 84 were confirmed by determination of the retention time using standards based on the respective pure compound. Approximately, one third of the compounds detected were hydrocarbons. We found aromatic hydrocarbons, alcohols, aldehydes, ketones, esters, ethers, sulfur compounds, nitrogen-containing compounds and halogenated compounds. Acetonitrile and benzene were two of 10 compounds which correlated with smoking behaviour. A comparison of results from cancer patients with those of 31 healthy volunteers revealed differences in the concentration and presence of certain compounds. The sensitivity for detection of lung cancer patients based on eight different compounds not seen in exhaled breath of healthy volunteers was 51% and the specificity was 100%. These eight potential markers for detection of lung cancer are 1-propanol, 2-butanone, 3-butyn-2-ol, benzaldehyde, 2-methyl-pentane, 3-methyl-pentane, n-pentane and n-hexane. Conclusions: SPME is a relatively insensitive method and compounds not observed in exhaled breath may be present at a concentration lower than LOD. The main achievement of the present work is the validated identification of compounds observed in exhaled breath of lung cancer patients. This identification is indispensible for future work on the biochemical sources of these compounds and their metabolic pathways. Clin Chem Lab Med 2009;47:550–60.

Analysis of exhaled breath from smokers, passive smokers and non-smokers by solid-phase microextraction gas chromatography/mass spectrometry.

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.

The analysis of healthy volunteers' exhaled breath by the use of solid-phase microextraction and GC-MS

We analysed breath and inhaled room air samples from 39 healthy volunteers (28 non-smokers, 8 smokers and 3 ex-smokers) by SPME-GC-MS. Mixed expiratory and indoor air samples were collected in freshly cleaned Tedlar bags. Eighteen millilitres of each sample were transferred into sealed, evacuated glass vials, preconcentrated by solid-phase microextraction (SPME, carboxen/polydimethylsiloxane) and investigated by gas chromatography with mass spectrometric detection (GC-MS). For the unequivocal identification of potential marker compounds, pure calibration mixtures of reference compounds (depending on commercial availability) were prepared to determine the retention time and mass spectra with respect to our analytical setting. Applying the adapted SPME-GC/MS method with limit of detection in the high ppb range (0.05-15.00 ppb), we succeeded in identifying altogether 38 compounds with concentrations in exhaled breath being at least 50% higher than concentration in inhaled air. From these 38 compounds, 31 were identified not only by the spectral library match but also by retention time of standards. A comparison of retention times and spectrum obtained for standards and determined compounds was performed. We found hydrocarbons (isoprene, 2-pentene, 2-methyl-1-pentene, benzene, toluene, p-cymene, limonene, 2,4-dimethylheptane, n-butane), ketones (acetone, hydroxypropanone, methylvinyl ketone), ethers (dimethyl ether, 1,3-dioxolane), esters (ethyl acetate), aldehydes (propanal, hexanal, heptanal, acrolein) and alcohols (ethanol, 2-metoxyethanol, isopropyl alcohol, 2,2,3,3- tetramethylcyclopropanemethanol, 3,4-dimethylcyclohexanol). Proper identification of compounds in different cohorts of patients and volunteers is the base for further investigation of origin, biochemical background and distribution of potential breath biomarkers.

Determination of volatile organic compounds as biomarkers of lung cancer by SPME–GC–TOF/MS and chemometrics

A method for qualitative and quantitative the determination of concentrations volatile organic compounds (VOCs) in human breath samples using solid phase microextraction (SPME) and gas chromatography-time of flight-mass spectrometry (GC-TOF/MS) has been carried out. They are employed for the preconcentration, separation and analysis of biological samples. The technique to rapid determination compounds present in human air, at the level of parts per billion (ppb) is applied. This method was optimized and evaluated. It showed linear correlations ranging from 0.83 to 234.05 ppb, limit of detection in the range of 0.31 to 0.75 ppb and precision, expressed as the RSD, was less then 10.00%. The unique combination of statistical methods allowed reduce the number of compounds to significant ones only and indicate the potential way to find the biomarkers of the lung cancer. Presented an analytical and statistical methods for detection composition of exhaled air could be applied as a potential non-intrusive tool for screening of lung cancer.

Identification of volatile organic compounds secreted from cancer tissues and bacterial cultures

The early cancer diagnosis increases the possibility of total recovery. The infection of Helicobacter pylori is associated with gastric cancer, the second most common cancer in the world. The determination of volatile organic compounds (VOCs) excreted by stomach tissue and bacteria culture has been investigated. Solid-phase microextraction (SPME) was used for preconcentration and the determination was accomplished by gas chromatography coupled with mass spectrometry (GC/MS). The samples of tissue were taken from five patients (ten samples) with stomach cancer and normal (non-cancerous) segments from other parts of the stomach were used as a control. Eighteen compounds were identified in stomach tissue and seven of them were present both in healthy and cancer tissue. These compounds assumed to be endogenous and acetone ratio (AR) was calculated for ethanol, butane, carbon disulfide, 1-propanol, 2-butanone and 2-pentanone. The data shows that amount of 1-propanol and carbon disulfide in the gaseous composition is higher in cancer tissue than in normal tissue. Eight compounds were identified both in bacteria and tissue. These data suggest that bacteria present in the stomach might cause the increase in the concentration of 1-propanol and carbon disulfide in emission from cancer tissue.

Analysis of Exhaled Breath for Disease Detection

Breath analysis is a young field of research with great clinical potential. As a result of this interest, researchers have developed new analytical techniques that permit real-time analysis of exhaled breath with breath-to-breath resolution in addition to the conventional central laboratory methods using gas chromatography-mass spectrometry. Breath tests are based on endogenously produced volatiles, metabolites of ingested precursors, metabolites produced by bacteria in the gut or the airways, or volatiles appearing after environmental exposure. The composition of exhaled breath may contain valuable information for patients presenting with asthma, renal and liver diseases, lung cancer, chronic obstructive pulmonary disease, inflammatory lung disease, or metabolic disorders. In addition, oxidative stress status may be monitored via volatile products of lipid peroxidation. Measurement of enzyme activity provides phenotypic information important in personalized medicine, whereas breath measurements provide insight into perturbations of the human exposome and can be interpreted as preclinical signals of adverse outcome pathways.

Kinetic and equilibrium studies of phenol adsorption by natural and modified forms of the clinoptilolite

The contribution presents results of investigation of phenol adsorption by the raw, HDTMA- and NaOH-modified clinoptilolite. The experimental data on the sorption kinetic process are modelled using the particle and the film diffusion models with calculation of a rate constant of intraparticle mass transfer. Two main stages may be divided in the kinetic sorption process: (1) initial rapid sorption (amount of phenol sorbed 85-90%) extending over the first 60 min and (2) stage of slow approach to equilibrium covering about 8h. Equilibrium isotherms for phenol adsorption are modelled using the Langmuir, Freundlich and Dubinin-Radushkevich equations. The Freundlich isotherm describes in a greater degree phenol sorption from aqueous solutions of low phenol concentrations, while the Langmuir isotherm fits better to sorption at high initial concentrations.

Preliminary study of volatile organic compounds from breath and stomach tissue by means of solid phase microextraction and gas chromatography-mass spectrometry.

The determination of volatile organic compounds (VOCs) in exhaled air and stomach tissue emission for the detection of cancer has been investigated. Solid phase microextraction (SPME) was used for sample preconcentration. The method presented in this paper showed satisfactory precision (RSD below 11%), linearity in the range of 2.8-136 ppb and limit of detection ranging from 0.6 to 2.1 ppb. The breath and emission from cancer tissue were collected from three patients with stomach cancer. Acetone, carbon disulfide, 2-propanol, ethyl alcohol and ethyl acetate were identified in breath and tissue samples. These compounds have been assumed as endogenous. Acetone ratio (AR) was calculated for carbon disulfide, 2-propanol and n-butane. The AR for carbon disulfide was found to be higher for normal tissue (20.64-44.95) than for emission from cancer tissue (2.01-18.20). A limitation of this study is that only a few clinical samples were investigated. These results should be evaluated as preliminary because of the small number of patients examined.

Detection of volatile organic compounds as biomarkers in breath analysis by different analytical techniques

Breath is a rich mixture containing numerous volatile organic compounds at trace amounts (ppbv-pptv level) such as: hydrocarbons, alcohols, ketones, aldehydes, esters or heterocycles. The presence of some of them depends on health status. Therefore, breath analysis might be useful for clinical diagnostics, therapy monitoring and control of metabolic or biochemical cell cycle products. This Review presents an update on the latest developments in breath analysis applied to diagnosing different diseases with the help of high-quality equipment. Efforts were made to fully and accurately describe traditional and modern techniques used to determine the components of breath. The techniques were compared in terms of design, function and also detection limit of different volatile organic compounds. GC with different detectors, MS, optical sensor and laser spectroscopic detection techniques are also discussed.

Fibers with polypyrrole and polythiophene phases for isolation and determination of adrenolytic drugs from human plasma by SPME-HPLC

In this study, polypyrrole (PPy) and polythiophene (PTh) SPME coatings and their ability to extract selected adrenolytic drugs with different physico-chemical properties from standard solutions and human plasma samples were evaluated. In measurements metoprolol, oxprenolol, mexiletine, propranolol, and propaphenon were investigated. The main parameters such as extraction time, desorption conditions and pH influence were examined. Inter-day precisions were in range 0.1-2.0%, 1.1-2.9%, 1.3-2.6%, 0.1-2.6% and 0.3-2.1% for metoprolol, oxprenolol, mexiletine, propranolol and propaphenon, respectively. Accuracies were less than 15%, which was evaluated by analyzing preparation samples of five replicates. The method was successfully applied to human plasma samples spiked with selected adrenolytic drugs. The method was linear in the concentration range from 1 to 10microg/ml for all of studied adrenolytic drugs using human plasma samples. The PTh-SPME coating displayed higher extraction efficiency towards the target analytes in comparison to PPy-SPME. The reproducibility of the extraction using polypyrrole and polythiophene fibers was confirmed by variation coefficients lower than 8% and 3%, respectively.