Analysis of human breath with micro extraction techniques and continuous monitoring of carbon dioxide concentration

The detection of volatile organic compounds (VOCs) in human breath can be useful for the clinical routine diagnosis of several diseases in a non-invasive manner. Traditional methods of breath analysis have some major technical problems and limitations. Membrane extraction with a sorbent interface (MESI), however, has many advantages over current methods, including good selectivity and sensitivity, and is well suited for breath analysis. The aim of this project was to develop a simple and reproducible sampling device and method based on the MESI system for breath analysis. The feasibility and validity of the MESI system was tested with real human breath samples. Internal standard calibration methods were used for the quantitative analysis of various breath samples. Calibration curves for some main components (target analytes such as acetone and pentane) were determined in the research. The optimized stripping-side and feeding-side gas velocities were determined. The use of breath CO₂ as an internal standard for the analysis of breath VOCs is an effective method to solve the difficulties associated with variations in the target analyte concentrations in a sample, which are attributed to mass losses and different breathing patterns of different subjects. In this study, the concentration of breath acetone was successfully expressed normalized to CO₂ as in the alveolar air. Breath acetone of healthy males and females profiled at different times of the day was plotted using the MESI system, and results were consistent with the literature. This technique can be used for monitoring breath acetone concentrations of diabetic patients and for applications with other biomarker monitoring.     

Volatile Organic Compounds Analysis in Breath Air in Healthy Volunteers and Patients Suffering Epidermoid Laryngeal Carcinomas

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.     

Human exhaled air analytics: biomarkers of diseases

Over the last few years, breath analysis for the routine monitoring of metabolic disorders has attracted a considerable amount of scientific interest, especially since breath sampling is a non-invasive technique, totally painless and agreeable to patients. The investigation of human breath samples with various analytical methods has shown a correlation between the concentration patterns of volatile organic compounds (VOCs) and the occurrence of certain diseases. It has been demonstrated that modern analytical instruments allow the determination of many compounds found in human breath both in normal and anomalous concentrations. The composition of exhaled breath in patients with, for example, lung cancer, inflammatory lung disease, hepatic or renal dysfunction and diabetes contains valuable information. Furthermore, the detection and quantification of oxidative stress, and its monitoring during surgery based on composition of exhaled breath, have made considerable progress. This paper gives an overview of the analytical techniques used for sample collection, preconcentration and analysis of human breath composition. The diagnostic potential of different disease-marking substances in human breath for a selection of diseases and the clinical applications of breath analysis are discussed.     

Sampling and analysis of volatile organic compounds in bovine breath by solid-phase microextraction and gas chromatography-mass spectrometry

A relatively noninvasive method consisting of a face mask sampling device, solid-phase microextraction (SPME) fibers, and a gas chromatography-mass spectrometry (GC-MS) for the identification of volatile organic compounds (VOCs) in bovine breath was developed. Breath of three morbid steers with respiratory tract infections and three healthy steers were sampled seven times in 19 days for 15 min at each sampling. The breath VOCs adsorbed on the divinylbenzene (DVB)-Carboxen-polydimethyl siloxane (PDMS) 50/30 microm SPME fibers were transported to a laboratory GC-MS system for separation and identification with an in-house spectral library of standard chemicals. A total of 21 VOCs were detected, many of them for the first time in cattle breath. Statistical analyses using Chi-square test on the frequency of detection of each VOC in each group was performed. The presence of acetaldehyde (P < or = 0.05) and decanal (P < or = 0.10) were associated more with clinically morbid steers while methyl acetate, heptane, octanal, 2,3-butadione, hexanoic acid, and phenol were associated with healthy steers at P < or = 0.10. The results suggest that noninvasive heath screening using breath analyses could become a useful diagnostic tool for animals and humans.     

Ultrafast gas chromatography coupled to electronic nose to identify volatile biomarkers in exhaled breath from chronic obstructive pulmonary disease patients: A pilot study

An analytical method to identify volatile organic compounds (VOCs) in the exhaled breath from patients with a diagnosis of chronic obstructive pulmonary disease (COPD) using a ultrafast gas chromatography system equipped with an electronic nose detector (FGC eNose) has been developed. A prospective study was performed in 23 COPD patients and 33 healthy volunteers; exhalation breathing tests were performed with Tedlar bags. Each sample was analyzed by FCG eNose and the identification of VOCs was based on the Kovats index. Raw data were reduced by principal component analysis (PCA) and canonical discriminant analysis [canonical analysis of principal coordinates (CAP)]. The FCG eNose technology was able to identify 17 VOCs that distinguish COPD patients from healthy volunteers. At all stages of PCA and CAP the discrimination between groups was obvious. Chemical prints were correctly classified up to 82.2%, and were matched with 78.9% of the VOCs detected in the exhaled breath samples. Receiver operating characteristic curve analysis indicated the sensitivity and specificity to be 96% and 91%, respectively. This pilot study demonstrates that FGC eNose is a useful tool to identify VOCs as biomarkers in exhaled breath from COPD patients. Further studies should be performed to enhance the clinical relevance of this quick and ease methodology for COPD diagnosis.     

Colorimetric Artificial Nose for Identification of Breath Volatile Organic Compounds of Patients with Lung Cancer

A novel and highly sensitive colorimetric sensor array was developed for the detection and identification of breath volatile organic compounds（VOCs） of patients with lung cancer.Employing dimeric metalloporphyrins,metallosalphen complexes,and chemically responsive dyes as the sensing elements,the developed sensor array of artificial nose shows a unique pattern of colorific changes upon its exposure to eight less-reactive VOCs and their mixture gas at a concentration of 735 nmol/L within 3 min.Potential of quantitative analysis of VOCs samples was proved.A good linear relationship of 490-3675 nmol/L was obtained for benzene vapor with a detection limit of 49 nmol/L（S/N=3）.Data analysis was carried out by Hierarchical cluster analysis（HCA） and principal component analysis（PCA）.Each category of breath VOCs clusters together in the PCA score plot.No errors in classification by HCA were observed in 45 trials.Additionaly,the colorimetric sensor array showed good reproducibility under the cyclic sensing experiments.These results demonstrate that the developed colorimetric artificial nose system is an excellent sensing platform for the identification and quantitative analysis of breath VOCs of patients with lung cancer.     

人体呼出气中挥发性有机化合物的检测方法

呼出气检测作为一种潜在的新型临床检测手段受到广泛关注。本文详细综述了人体呼出气中挥发性有机化合物(VOCs)的各类检测方法和技术,分别对色谱法、质谱法和光谱及传感器法的原理、特点和最新研究进展进行了介绍,对照总结了目前已确定的异戊二烯、丙酮等疾病生物标志物的各种分析方法和实测数据,并展望了未来的研究动向。     

Analysis of biogenic volatile organic compounds in human health and disease

The analysis of volatile biomarkers of disease in breath is attractive because breath analysis is non-invasive and quick, and allows for repeated sampling. Challenges faced in the development of breath analysis include developing techniques that can measure analytes at very low concentrations, gaining an understanding of the exhalation physiology of individual volatiles, and determining the relationship between the proposed biomarker and the underlying condition. A small number of breath tests are used in clinical practice, but there is great potential for the development and wider application of clinical breath analysis in infection, inflammation, cancer and metabolic disease.     

Breath Analysis: Comparison among Methodological Approaches for Breath Sampling

Despite promising results obtained in the early diagnosis of several pathologies, breath analysis still remains an unused technique in clinical practice due to the lack of breath sampling standardized procedures able to guarantee a good repeatability and comparability of results. The most diffuse on an international scale breath sampling method uses polymeric bags, but, recently, devices named Mistral and ReCIVA, able to directly concentrate volatile organic compounds (VOCs) onto sorbent tubes, have been developed and launched on the market. In order to explore performances of these new automatic devices with respect to sampling in the polymeric bag and to study the differences in VOCs profile when whole or alveolar breath is collected and when pulmonary wash out with clean air is done, a tailored experimental design was developed. Three different breath sampling approaches were compared: (a) whole breath sampling by means of Tedlar bags, (b) the end-tidal breath collection using the Mistral sampler, and (c) the simultaneous collection of the whole and alveolar breath by using the ReCIVA. The obtained results showed that alveolar fraction of breath was relatively less affected by ambient air (AA) contaminants (p-values equal to 0.04 for Mistral and 0.002 for ReCIVA Low) with respect to whole breath (p-values equal to 0.97 for ReCIVA Whole). Compared to Tedlar bags, coherent results were obtained by using Mistral while lower VOCs levels were detected for samples (both breath and AA) collected by ReCIVA, likely due to uncorrected and fluctuating flow rates applied by this device. Finally, the analysis of all data also including data obtained by explorative analysis of the unique lung cancer (LC) breath sample showed that a clean air supply might determine a further confounding factor in breath analysis considering that lung wash-out is species-dependent.     

Analysis of volatile organic compounds in wastewater during various stages of treatment for high-tech industries

Summary A protocol combining purge-and-trap (P&T) and solid-phase microextraction (SPME) was established for the analysis of volatile organic compounds (VOCs) in the wastewater plant of high-tech industries. Over 60 VOCs could be analyzed by P&T coupled to gas chromatography/mass spectrometry (P&T-GC-MS). Four polar VOCs commonly used in the high-tech industries were determined by SPME coupled to gas chromatography/flame ionization detection (SPME-GC-FID). The limits of detection for the analytes were less than 1.1 μg L⁻¹ with P&T-GC-MS, and between 1.5 and 12.5 μg L⁻¹ with SPME-GC-FID. Satisfactory recoveries (83% to 130%) were obtained. Real samples were analyzed from a wastewater treatment plant during various stages of treatment. The major pollutant in the wastewater influent was found to be acetone (>4 ppm).     

Significant different volatile biomarker during bronchoscopic ion mobility spectrometry investigation of patients suffering lung carcinoma

Exhaled breath of patients suffering non-small bronchial carcinoma contains volatile organic compounds (VOC) different from healthy people. VOCs could be detected using ion mobility spectrometry down to the pg/L range even in air directly. To date, the origin of the different VOCs found is insecure. Such VOCs could be a direct product of the metabolism of the tumor or relatable to mostly present co-factors like infections or necrosis or a reaction of the human organism to the tumor (e.g. oxidativ stress). In the present study the breath of 19 patients suffering from confirmed NSCLC (non-small-cell lung carcinoma) with different histological types was investigated. In all cases flexible video-chip bronchoscopy was realized. Before taking samples for histological investigations in the lung on both main bronchi, samples of air were taken using a polytetrafluoroethylene (PTFE or Teflon) tube as catheter directly from the working channel of a bronchoscope and connected directly to the inlet of the ion mobility spectrometer. The measurement was started immediately. In total, 72 common peaks could be identified. 5 Peaks were significantly varying between the tumor site and the collateral lung. Considering adenocarcinoma, one peak separates both sites clearly and was relatable to the dimer of n-Dodecane. Two peaks were found on squamous cell carcinoma and relatable to 2-Butanol or 2-Methylfuran and Nonanal. The sensitivity, specificity, positive and negative predictive values were, for adenocarcinoma 100%, 75%, 80% and 100%, respectively – for squamous cell carcinoma 78%/78%, 67%/78%, 70%/80% and 75%/88%, for 2-Butanol and Nonanal respectively. Therefore, VOCs obtained from bronchoscopic sampling of breath could be detected using ion mobility spectrometry. The present study suggests that lung carcinoma with different histology will be represented by different volatile analytes.     

Coupling of stir bar sorptive extraction with single photon ionization mass spectrometry for determination of volatile organic compounds in water

A home-made stir bar sorptive extraction (SBSE) apparatus was combined to a single photon ionization time-of-flight mass spectrometer (SPI-TOFMS) for rapid and sensitive determination of trace volatile organic compounds (VOCs) in water. The home-made SBSE bar, low-cost and disposable, was used for VOCs extraction. A thermal desorption (TD) device was designed to desorb the analytes from the SBSE bar, and a high throughput interface was developed to transfer the analytes into the ionization chamber of the SPI-TOFMS. The combination of large extraction volume of SBSE bar, and the direct measurement power of SPI-TOFMS enable a short analysis time for VOCs in water with high sensitivity, for example the limits of detection (LODs) were in the range of 7.4-11.1 ng L(-1) for benzene, toluene, and p-xylene (BTX) within 15 min. BTX aqueous solutions were chosen to demonstrate the quantitative capability, the linear range was from 0.05 to 100 μg L(-1) and the correlation coefficients were better than 0.996. The proposed method was successfully applied for the analysis of VOCs in urban river water.     

热脱附-气相色谱-质谱法测定环境空气中67种挥发性有机物

采用热脱附-气相色谱-质谱法,建立了同时分析环境空气中67种挥发性有机物的分析方法。对比了5种不同填充材料不锈钢吸附管对78种挥发性有机物的吸附能力。填充材料为Tenax TA和Carbograph 1TD的混合填料吸附管对分析物的捕集效果最好,在30 mL/min高纯He气持续吹脱45 min的情况下,未发生穿透（即穿透率小于10%）的化合物达67种,分析物的种类包括芳香烃、脂肪烃、卤代烃和含氧挥发性有机物等。优化了使用该吸附管测定67种目标物时的热脱附条件。在5~100 ng范围内,目标化合物的色谱响应值与其量间具有良好的线性关系,其相关系数（r）均在1.0000~0.9977之间。方法检出限为0.3~2.4 ng,以采样体积1 L计算,检出限为0.3~2.4 μg/m³。加标量为20 ng时,7次重复实验目标化合物回收率均在81.6%~114.9%之间,目标化合物的相对标准偏差为1.2%~10.1%。采用该方法对某车厢内空气进行了检测,检出了包括酯类、卤代烷烃、卤代烯烃以及芳香族化合物在内的19种目标化合物,其范围为1.1~84.1 μg/m³。该方法准确、可靠、灵敏度高,实现了对环境空气中67种目标污染物的准确定量。     

Comprehensive Two-Dimensional Gas Chromatography–Mass Spectrometry Analysis of Exhaled Breath Compounds after Whole Grain Diets

Exhaled breath is a potential noninvasive matrix to give new information about metabolic effects of diets. In this pilot study, non-targeted analysis of exhaled breath volatile organic compounds (VOCs) was made by comprehensive two-dimensional gas chromatography–mass spectrometry (GCxGC-MS) to explore compounds relating to whole grain (WG) diets. Nine healthy subjects participated in the dietary intervention with parallel crossover design, consisting of two high-fiber diets containing whole grain rye bread (WGR) or whole grain wheat bread (WGW) and 1-week control diets with refined wheat bread (WW) before both diet periods. Large interindividual differences were detected in the VOC composition. About 260 VOCs were detected from exhaled breath samples, in which 40 of the compounds were present in more than half of the samples. Various derivatives of benzoic acid and phenolic compounds, as well as some furanones existed in exhaled breath samples only after the WG diets, making them interesting compounds to study further.     

Diagnosis of Helicobacter pylori infection.

A number of reliable methods are currently available for the diagnosis of Helicobacter pylori infection. These diagnostic tests can be classified into invasive methods that require endoscopy and gastric biopsy, and noninvasive methods. Invasive methods include gastric mucosal biopsies at endoscopy for bacteriologic culture, histology, and the rapid urease test. Noninvasive methods include the urea breath test and serologic tests. Each of these diagnostic tests has its advantages and disadvantages. Histologic examination remains the gold standard for diagnosis. It can also detect coccoidal forms of the bacteria and be used to assess the severity of gastritis. Culture of H pylori should be performed if antibiotic sensitivity of the organism is required. A rapid urease test is the quickest test for H pylori status. The urea breath test detects urease activity in the entire stomach, thus eliminating the possibility of a sampling error, which occurs in random gastric biopsies. Serologic tests using either ELISA or latex-agglutination methods are excellent for diagnosis of H pylori infection, but not useful for monitoring effects of therapy. Recently, the polymerase chain reaction has been applied to fixed-tissue biopsies, as well as body secretions in the diagnosis of H pylori infection.     

Statistical and bioinformatical methods to differentiate chronic obstructive pulmonary disease (COPD) including lung cancer from healthy control by breath analysis using ion mobility spectrometry

Human breath analysis is a powerful and especially a non-invasive technique for the monitoring and hopefully also for the diagnosis of respiratory diseases, including chronic obstructive pulmonary disease (COPD). The exhaled breath of 95 patients suffering COPD and of 35 healthy controls was investigated using an Ion Mobility Spectrometer (IMS) coupled to a Multi-Capillary Column (MCC) without any pre-separation or pre-enrichment. Starting with the results from a Mann–Whitney-Wilcoxon rank sum test to find analytes with the highest potential with respect to differentiation, box and whisker plots, metabolic maps and probability charts were introduced and compared. In addition, the sensitivity, specificity, positive and negative predictive values and the accuracy of the relation were also summarized. The findings were compared to the results of a principal component analysis. Finally, decision trees were introduced to visualize the interdependencies between the analytes and the classifications. The application of these biostatistical methods with simultaneous inclusion of several VOCs for disease classification by ion mobility spectrometry of human breath will provide much more information than using single peaks and single concentration dependencies for disease classification and discrimination of various groups. Towards the future application of potential biomarkers for clinical diagnostic procedures, complex analytical methods, such as ion mobility spectrometry, need statistical and bioinformatical tools which are simple in application, visualize the results and support decisions on the basis of the data obtained from measurements of analytes in exhaled human breath.     

One-year time series of investigations of analytes within human breath using ion mobility spectrometry

Ion mobility Spectrometry is used to detect volatile analytes within human breath directly. Many volatile organic compounds (VOC) show significant day-to-day variation in the signal height related to the concentration of the analyte, although the breath collection had been performed under the same conditions with respect to similar sampling procedure, similar dead volume, similar measurement time, and measurement conditions. Variations of 8 different analytes are investigated over a time period of 11 months in the exhaled breath of the same person in the same room environment. The individual variability is reported for Benzothiazole; D-Limonene; Eucalyptol; Decamethylcyclopentasiloxane; Decanal; 1-Hexanol, 2-ethyl-; Cyclohexanone, 5-methyl-2-(1-methylethyl) and Nonanal. The paper shows, that the individual variability must be taken into consideration to relate the findings to medical questions. Therefore, the room air concentration of VOCs must be taken into account, so that the difference between exhaled and inhaled air has to be used as indicator. Finally, starting with individual variabilities, the normal variation related to the specific analyte should be considered in addition.     

二次纳流电喷雾电离耦合超高分辨质谱检测人体呼出气中相对高分子量化合物

二次电喷雾电离源耦合超高分辨质谱(SESI-UHRMS)有望检出人体呼出气中分子量大于300的相对高分子量化合物,这些化合物的发现将有助于更准确地理解呼出气中挥发性有机化合物(VOCs)的来源、产生机制以及SESI源电离机理,更好地实现SESI-UHRMS的转化应用.本研究自组装nanoSESI源(尚无商业产品)耦合四极杆-静电场轨道阱质谱(最高质量分辨率1.2×10~5),考察了该装置对健康人体呼出气中分子量为300~500化合物的检出情况.结果表明,所搭建的nanoSESI-UHRMS装置检测人体呼出气的重现性好、灵敏度高,可检出数十种分子量为300~500的化合物.根据这些化合物在单次呼气过程中信号强度随时间变化的趋势,推断其来源分别为内源性和外源性;各化合物的元素组成主要包括C,H,N和O,环-双键当量(RDB)的均值为(4.5±3.1),表明检出的化合物可能为醛酮或不饱和脂肪酸,容易在SESI源中被电离.本研究初步验证了自组建nanoSESI-UHRMS检测人体呼出气中相对高分子量化合物的可行性,为后续进一步开展方法应用奠定了基础.     

Oral malodor,assessed by closed-loop,gas chromatography,and ion-trap technology

A method is described for the analysis of volatile organic compounds in saliva and tongue coating samples. The techniue is based on an off-line preconcentration step by means of a closed-loop trapping system followed by gas chromatography-ion trap detection. With the closed-loop technique, the volatile organic compounds(VOCs) are released from the matrix and trapped on an adsorbent without interference of water. The VOCs are released from the adsorbent into the gas chromatograph by thermdesorption. After separation, identification of the compounds is performed by ion trap technology. By this technique 82 compounds could be demonstrated in saliva and tongue coating samples. The technique is also used to demonstrate the formation of volatile bacterial fermentation compounds when a protein substrate is added to tongue coating samples. It is considered a very promising tool in further research on oral malodor.     

Multiple headspace solid-phase microextraction for the quantitative determination of volatile organic compounds in multilayer packagings

The theory of multiple headspace solid-phase microextraction (HS-SPME) and a method based on multiple HS-SPME for the quantitative determination of volatile organic compounds (VOCs) in packaging materials is presented. The method allows the direct analysis of solid samples without using organic solvents to extract analytes. Multiple headspace solid-phase microextraction is a stepwise method proposed to eliminate the influence of the sample matrix on the quantitative analysis of solid samples by HS-SPME. Different amounts of packaging and different volumes of standard solution were studied in order to remove a substantial quantity of analytes from the headspace at each extraction and obtain the theoretical exponential decay of the peak area of the four successive extractions and, thus, the total area was calculated from these four extractions. In addition, two fibres were compared: carboxen-polydimethylsiloxane (CAR-PDMS) and divinylbenzene-carboxen-polydimethylsiloxane (DVB-CAR-PDMS), as they showed differences in the linearity of the exponential decay with the number of extractions depending on the compound. The CAR-PDMS fibre was better for the VOCs with a low molecular mass, whereas the DVB-CAR-PDMS fibre was better for the VOCs with a high molecular mass. Finally, the method was characterised in terms of linearity, detection limit and reproducibility and applied to analyse four multilayer packaging samples with different VOCs contents.