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.     

Needle Trap Device-GC-MS for Characterization of Lung Diseases Based on Breath VOC Profiles

Volatile organic compounds (VOCs) have been assessed in breath samples as possible indicators of diseases. The present study aimed to quantify 29 VOCs (previously reported as potential biomarkers of lung diseases) in breath samples collected from controls and individuals with lung cancer, chronic obstructive pulmonary disease and asthma. Besides that, global VOC profiles were investigated. A needle trap device (NTD) was used as pre-concentration technique, associated to gas chromatography-mass spectrometry (GC-MS) analysis. Univariate and multivariate approaches were applied to assess VOC distributions according to the studied diseases. Limits of quantitation ranged from 0.003 to 6.21 ppbv and calculated relative standard deviations did not exceed 10%. At least 15 of the quantified targets presented themselves as discriminating features. A random forest (RF) method was performed in order to classify enrolled conditions according to VOCs’ latent patterns, considering VOCs responses in global profiles. The developed model was based on 12 discriminating features and provided overall balanced accuracy of 85.7%. Ultimately, multinomial logistic regression (MLR) analysis was conducted using the concentration of the nine most discriminative targets (2-propanol, 3-methylpentane, (E)-ocimene, limonene, m-cymene, benzonitrile, undecane, terpineol, phenol) as input and provided an average overall accuracy of 95.5% for multiclass prediction.     

Double-layer Tedlar bags: a means to limit humidity evolution of air samples and to dry humid air samples

Tedlar bags, which are widely used to collect air samples, especially VOCs and odorous atmospheres, can allow humidity to diffuse when relative humidity levels differ between the inside and outside. Starting with dry air inside the bag and humid air outside, we monitored equilibrium times under several conditions showing the evolution and influence of collected volumes and exposed surfaces. A double-film Tedlar bag was made, to limit the impact of external humidity on a sample at low humidity level. With the addition of a drying agent between both films, the evolution of humidity of a sample can be stopped for several hours. When a VOC mixture was monitored in a humid atmosphere, humidity was decreased but no significant evolution of VOC concentrations was observed.     

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.     

Detection of Paratuberculosis in Dairy Herds by Analyzing the Scent of Feces,Alveolar Gas,and Stable Air

Paratuberculosis is an important disease of ruminants caused by Mycobacterium avium ssp. paratuberculosis (MAP). Early detection is crucial for successful infection control, but available diagnostic tests are still dissatisfying. Methods allowing a rapid, economic, and reliable identification of animals or herds affected by MAP are urgently required. This explorative study evaluated the potential of volatile organic compounds (VOCs) to discriminate between cattle with and without MAP infections. Headspaces above fecal samples and alveolar fractions of exhaled breath of 77 cows from eight farms with defined MAP status were analyzed in addition to stable air samples. VOCs were identified by GC–MS and quantified against reference substances. To discriminate MAP-positive from MAP-negative samples, VOC feature selection and random forest classification were performed. Classification models, generated for each biological specimen, were evaluated using repeated cross-validation. The robustness of the results was tested by predicting samples of two different sampling days. For MAP classification, the different biological matrices emitted diagnostically relevant VOCs of a unique but partly overlapping pattern (fecal headspace: 19, alveolar gas: 11, stable air: 4–5). Chemically, relevant compounds belonged to hydrocarbons, ketones, alcohols, furans, and aldehydes. Comparing the different biological specimens, VOC analysis in fecal headspace proved to be most reproducible, discriminatory, and highly predictive.     

Pilot Study on Exhaled Breath Analysis for a Healthy Adult Population in Hawaii

Fast diagnostic results using breath analysis are an anticipated possibility for disease diagnosis or general health screenings. Tests that do not require sending specimens to medical laboratories possess capabilities to speed patient diagnosis and protect both patient and healthcare staff from unnecessary prolonged exposure. The objective of this work was to develop testing procedures on an initial healthy subject cohort in Hawaii to act as a range-finding pilot study for characterizing the baseline of exhaled breath prior to further research. Using comprehensive two-dimensional gas chromatography (GC×GC), this study analyzed exhaled breath from a healthy adult population in Hawaii to profile the range of different volatile organic compounds (VOCs) and survey Hawaii-specific differences. The most consistently reported compounds in the breath profile of individuals were acetic acid, dimethoxymethane, benzoic acid methyl ester, and n-hexane. In comparison to other breathprinting studies, the list of compounds discovered was representative of control cohorts. This must be considered when implementing proposed breath diagnostics in new locations with increased interpersonal variation due to diversity. Further studies on larger numbers of subjects over longer periods of time will provide additional foundational data on baseline breath VOC profiles of control populations for comparison to disease-positive cohorts.     

Exhaled Breath and Oxygenator Sweep Gas Propionaldehyde in Acute Respiratory Distress Syndrome

Background: Oxidative stress-induced lipid peroxidation (LPO) due to neutrophil-derived reactive oxygen species plays a key role in the early stage of the acute respiratory distress syndrome (ARDS). Monitoring of oxidative stress in this patient population is of great interest, and, ideally, this can be done noninvasively. Recently, propionaldehyde, a volatile chemical compound (VOC) released during LPO, was identified in the breath of lung transplant recipients as a marker of oxidative stress. The aim of the present study was to identify if markers of oxidative stress appear in the oxygenator outflow gas of patients with severe ARDS treated with veno-venous extracorporeal membrane oxygenation (ECMO). Methods: The present study included patients with severe ARDS treated with veno-venous ECMO. Concentrations of acetone, isoprene, and propionaldehyde were measured in inspiratory air, exhaled breath, and oxygenator inflow and outflow gas at corresponding time points. Ion-molecule reaction mass spectrometry was used to measure VOCs in a sequential order within the first 24 h and on day three after ECMO initiation. Results: Nine patients (5 female, 4 male; age = 42.1 ± 12.2 year) with ARDS and already established ECMO therapy (pre-ECMO PaO₂/FiO₂ = 44.0 ± 11.5 mmHg) were included into analysis. VOCs appeared in comparable amounts in breath and oxygenator outflow gas (acetone: 838 (422–7632) vs. 1114 (501–4916) ppbv; isoprene: 53.7 (19.5–244) vs. 48.7 (37.9–108) ppbv; propionaldehyde: 53.7 (32.1–82.2) vs. 42.9 (24.8–122) ppbv). Concentrations of acetone, isoprene, and propionaldehyde in breath and oxygenator outflow gas showed a parallel course with time. Conclusions: Acetone, isoprene, and propionaldehyde appear in breath and oxygenator outflow gas in comparable amounts. This allows for the measurement of these VOCs in a critically ill patient population via the ECMO oxygenator outflow gas without the need of ventilator circuit manipulation.     

纳米材料在用于癌症诊断的呼出气挥发性有机物检测中的应用

细胞新陈代谢的变化会导致挥发性有机化合物（VOCs）类型及含量发生变化,因此可通过分析某些标志性VOCs简立起多种疾病早期诊断的模型. 人体呼出物中特征VOCs的检测作为一种非侵入性、无损的检测手段,近些年在疾病检测领域已成为世界范围内的研究热点. 其中,纳米材料可用于增强传感器性能,并使传感器便携式小型化,推进检测传感器进入临床. 在这篇综述中,作者将种类繁多的传感器中用到的纳米材料归纳总结为金属、金属氧化物、碳基、复合物和MOFs基纳米材料等几类,并讨论了不同类纳米材料在VOCs检测中的优劣势. 本文所建立起的分析方法及讨论有助于进一步了解检测技术的优越性与局限性. 最后,作者对利用VOCs的检测实现癌症早期筛选的研究及发展提出了个人观点.     

Use of poly(ethylene terephtalate) film bag to sample and remove humidity from atmosphere containing volatile organic compounds

Nalophan bags made from poly(ethylene terephtalate) film are often used to collect odorous gases. In this paper, the sample water removal method, based on humidity diffusion through the sample bag film, was applied using Nalophan bags and Tedlar bags to sample volatile organic compounds (VOCs) at low concentration (10 microg/m(3)). The removal of water with Nalophan bags enabled a reduction in relative humidity (RH) in a 10-L air sample from 80% to 20% in 2h at 20 degrees C. The use of Nalophan bags for the removal of water did not involve significant VOC loss among the 11 compounds studied.     

Modular Breath Analyzer (MBA): Introduction of a Breath Analyzer Platform Based on an Innovative and Unique,Modular eNose Concept for Breath Diagnostics and Utilization of Calibration Transfer Methods in Breath Analysis Studies

Exhaled breath analysis for early disease detection may provide a convenient method for painless and non-invasive diagnosis. In this work, a novel, compact and easy-to-use breath analyzer platform with a modular sensing chamber and direct breath sampling unit is presented. The developed analyzer system comprises a compact, low volume, temperature-controlled sensing chamber in three modules that can host any type of resistive gas sensor arrays. Furthermore, in this study three modular breath analyzers are explicitly tested for reproducibility in a real-life breath analysis experiment with several calibration transfer (CT) techniques using transfer samples from the experiment. The experiment consists of classifying breath samples from 15 subjects before and after eating a specific meal using three instruments. We investigate the possibility to transfer calibration models across instruments using transfer samples from the experiment under study, since representative samples of human breath at some conditions are difficult to simulate in a laboratory. For example, exhaled breath from subjects suffering from a disease for which the biomarkers are mostly unknown. Results show that many transfer samples of all the classes under study (in our case meal/no meal) are needed, although some CT methods present reasonably good results with only one class.     

The Impact of a Graded Maximal Exercise Protocol on Exhaled Volatile Organic Compounds: A Pilot Study

Exhaled volatile organic compounds (VOCs) are of interest due to their minimally invasive sampling procedure. Previous studies have investigated the impact of exercise, with evidence suggesting that breath VOCs reflect exercise-induced metabolic activity. However, these studies have yet to investigate the impact of maximal exercise to exhaustion on breath VOCs, which was the main aim of this study. Two-litre breath samples were collected onto thermal desorption tubes using a portable breath collection unit. Samples were collected pre-exercise, and at 10 and 60 min following a maximal exercise test (VO_2MAX). Breath VOCs were analysed by thermal desorption-gas chromatography-mass spectrometry using a non-targeted approach. Data showed a tendency for reduced isoprene in samples at 10 min post-exercise, with a return to baseline by 60 min. However, inter-individual variation meant differences between baseline and 10 min could not be confirmed, although the 10 and 60 min timepoints were different (p = 0.041). In addition, baseline samples showed a tendency for both acetone and isoprene to be reduced in those with higher absolute VO_2MAX scores (mL(O₂)/min), although with restricted statistical power. Baseline samples could not differentiate between relative VO_2MAX scores (mL(O₂)/kg/min). In conclusion, these data support that isoprene levels are dynamic in response to exercise.     

Comparison of Targeted and Untargeted Approaches in Breath Analysis for the Discrimination of Lung Cancer from Benign Pulmonary Diseases and Healthy Persons

The aim of the present study was to compare the efficiency of targeted and untargeted breath analysis in the discrimination of lung cancer (Ca+) patients from healthy people (HC) and patients with benign pulmonary diseases (Ca−). Exhaled breath samples from 49 Ca+ patients, 36 Ca− patients and 52 healthy controls (HC) were analyzed by an SPME–GC–MS method. Untargeted treatment of the acquired data was performed with the use of the web-based platform XCMS Online combined with manual reprocessing of raw chromatographic data. Machine learning methods were applied to estimate the efficiency of breath analysis in the classification of the participants. Results: Untargeted analysis revealed 29 informative VOCs, from which 17 were identified by mass spectra and retention time/retention index evaluation. The untargeted analysis yielded slightly better results in discriminating Ca+ patients from HC (accuracy: 91.0%, AUC: 0.96 and accuracy 89.1%, AUC: 0.97 for untargeted and targeted analysis, respectively) but significantly improved the efficiency of discrimination between Ca+ and Ca− patients, increasing the accuracy of the classification from 52.9 to 75.3% and the AUC from 0.55 to 0.82. Conclusions: The untargeted breath analysis through the inclusion and utilization of newly identified compounds that were not considered in targeted analysis allowed the discrimination of the Ca+ from Ca− patients, which was not achieved by the targeted approach.     

Organic metabolites in exhaled human breath—A multivariate approach for identification of biomarkers in lung disorders

The gas chromatographic profiles of exhaled air from lung cancer patients have been investigated. The breath from healthy volunteers, smokers and non-smokers, and lung cancer patients without treatment and under radio and/or chemotherapy, was collected using Tedlar bags. Different profiles for healthy people and cancer patients could be recognized by multivariate analysis and significant diagnostic compounds could be established. Target compounds showed to be linear and branched hydrocarbons between C₁₄ and C₂₄. Solid phase microextraction (SPME) coupled to gas chromatography mass spectrometry GC–(TOF)-MS was used. The method showed good precision (RSD below 26%) and limit of detection ranged from 0.04 to 8.0 ppb. These findings show a high potential for establishment of laboratorial screening methods. Validation studies in a larger number of patients are being done.     

呼气中痕量挥发性有机物的质子转移反应质谱在线检测研究

通过对自主研制的大气成分在线检测质子转移反应质谱的进样管路系统进行改造,建立了可在线检测呼气中痕量挥发性有机物的质子转移反应质谱装置。通过对呼气进样系统的旁路流量控制,实现对进样速度的调控,既可提高进样速度,以满足实时监测呼气中指定成分浓度变化;也可适时关闭旁路,以降低进样速度,从而对呼气成分进行全谱分析,避免采样袋采样和浓缩的复杂程序和潜在干扰。以作者呼出气体作为研究对象,对装置性能进行测试,结果表明:装置最快响应时间可达1s,对呼气中丙酮的探测灵敏度高达每10-9(V/V)浓度的信号强度为14.6counts/s,多次呼气测量重复性好,有望广泛应用于呼气疾病诊断研究。     

Passive sampling of airborne furan indoors by solid-phase microextraction

Furan may be formed in food under heat treatment and is highly suspected to appear in indoor air. The possible exposure to indoor furan raises concerns because it has been found to cause carcinogenicity and cytotoxicity in animals. To determine airborne furan, solid-phase microextraction (SPME) technique was utilised as a diffusive sampler. The Carboxen/Polydimethylsiloxane (CAR/PDMS, 75 μm) fibre was used, and the SPME fibre assembly was inserted into a polytetrafluoroethene tubing. Furan of known concentrations was generated in Tedlar gas bags for the evaluation of SPME diffusive samplers. After sampling, the sampler was inserted into the injection port of a gas chromatograph coupled with a mass spectrometer (GC/MS) for thermal desorption and analysis. Validation of the SPME device with active sampling by charcoal tube was performed side by side as well. The charcoal tube was desorbed by acetone before analysis with GC/MS. The experimental sampling constant of the sampler was found equal to (9.93 ± 1.28) × 10^?3 (cm³ min^?1) at 25°C. Furthermore, side-by-side validations between SPME device and charcoal tube showed linear relationship with r = 0.9927. The designed passive sampling device for furan has the advantages of both passive sampling and SPME technique and looks suitable for assessing indoor air quality.     

Human breath analysis: methods for sample collection and reduction of localized background effects

Solid-phase microextraction (SPME) was applied, in conjunction with gas chromatography–mass spectrometry, to the analysis of volatile organic compounds (VOCs) in human breath samples without requiring exhaled breath condensate collection. A new procedure, exhaled breath vapor (EBV) collection, involving the active sampling and preconcentration of a breath sample with a SPME fiber fitted inside a modified commercial breath-collection device, the RTube™, is described. Immediately after sample collection, compounds are desorbed from the SPME fiber at 250 °C in the GC-MS injector. Experiments were performed using EBV collected at −80 °C and at room temperature, and the results compared to the traditional method of collecting exhaled breath condensate at −80 °C followed by passive SPME sampling of the collected condensate. Methods are compared in terms of portability, ease-of-use, speed of analysis, and detection limits. The need for a clean air supply for the study subjects is demonstrated using several localized sources of VOC contaminants including nail polish, lemonade, and gasoline. Various simple methods to supply clean inhaled air to a subject are presented. Chemical exposures are used to demonstrate the importance of providing cleaned air (organic vapor respirator) or an external air source (tubing stretched to a separate room). These techniques allow for facile data interpretation by minimizing background contaminants. It is demonstrated herein that this active SPME breath-sampling device provides advantages in the forms of faster sample collection and data analysis, apparatus portability and avoidance of power or cooling requirements, and performance for sample collection in a contaminated environment.      

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.     

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.     

Breath Biomarkers in Diagnostic Applications

The detection of chemical compounds in exhaled human breath presents an opportunity to determine physiological state, diagnose disease or assess environmental exposure. Recent advancements in metabolomics research have led to improved capabilities to explore human metabolic profiles in breath. Despite some notable challenges in sampling and analysis, exhaled breath represents a desirable medium for metabolomics applications, foremost due to its non-invasive, convenient and practically limitless availability. Several breath-based tests that target either endogenous or exogenous gas-phase compounds are currently established and are in practical and/or clinical use. This review outlines the concept of breath analysis in the context of these unique tests and their applications. The respective breath biomarkers targeted in each test are discussed in relation to their physiological production in the human body and the development and implementation of the associated tests. The paper concludes with a brief insight into prospective tests and an outlook of the future direction of breath research.     

Investigation of the storage stability of selected volatile sulfur compounds in different sampling containers

The suitability of various sample containers (i.e. standard Tedlar sample bags, black/clear layered Tedlar sample bags and Silcosteel sample cylinders) was examined for a gaseous multicomponent standard containing methylmercaptan, ethylmercaptan, dimethyl sulfide, ethylmethyl sulfide, 2-propylmercaptan, 1-propylmercaptan, 2-butylmercaptan, diethyl sulfide and 1-butylmercaptan (1 mg/m3 each in nitrogen). In the black/clear layered Tedlar sample bags, significant losses (up to 10% for methylmercaptan as the most critical component) were observed after 2 days, whereas in the standard Tedlar sample bags the recovery of methylmercaptan was approximately 90% even after 1 week. The Silcosteel sample cylinders were suitable for sampling of volatile sulfur compounds with respect to the stability of the analytes, but the recoveries exceeded 100% especially for the higher boiling compounds, which was attributed to enrichment effects on parts of the sampling system.