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.     

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.     

氧、氮、氢分析仪校准方法探讨

建立了氧、氮、氢分析仪的校准方法,介绍了氧、氮、氢分析仪的工作原理、校准要求、校准用标准物质.以北京纳克分析仪器有限公司生产的ON-3000氧氮分析仪和H-3000氢分析仪作为校准对象,对校准方法进行了验证.该方法已作为评价氧、氮、氢分析仪的依据并得到认可.     

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.     

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.     

Towards a modular,versatile and portable sensor system for measurements in gaseous environments based on microcantilevers

Microfabricated silicon cantilever sensor arrays represent a powerful platform for sensing applications in physics, chemistry, material science, biology and medicine. The sensor response is mechanical bending due to absorption of molecules. In gaseous environment, polymer-coated microcantilevers are used as electronic nose for characterization of vapors, resulting in cantilever bending due to polymer swelling upon exposure. Medical applications involve fast characterization of exhaled patient's breath samples for detection of diseases, based on the presence of certain chemicals in breath. We present a portable, compact, modular microcantilever setup, which uses a micropump for aspiration and a bluetooth interface for remote data acquisition.     

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.     

S671智能化钾钠分析仪的研究与应用

应用微电子技术和离子选择电极测量原理实现人体血液和尿中钾、钠离子分析的自动化和智能化。该仪器能自动完成电极的校正、清洗、测量及报警,并对测量值进行计算和输出分析结果。本文应用S671智能化钾钠分析仪对水样、质控血清及人体血清中的钾、钠进行了全自动的测量,具有很大的临床意义。     

A procedure for calibration transfer between near-infrared instruments--a worked example using a transmittance single tablet assay for piroxicam in intact tablets

A procedure was developed for different modes of calibration transfer in near-infrared (NIR) spectroscopy, which included a method for the selection of a subset of samples appropriate for transfer. As a worked example, these guidelines were applied to the transfer of a multivariate calibration model, representing a validated NIR single tablet assay for the active within an intact pharmaceutical product, between three equivalent dispersive NIR transmission instruments. Transfer was first evaluated between two instruments, representing the situation where both were available during calibration development. A spectral correction method alone, applied to the transfer instrument, was not sufficient to facilitate transfer, with further optimisation of the calibration model using a novel wavelength selection algorithm necessary to remove regions of the spectral range that resulted in skewed predictions on the second instrument. Through this approach, a single calibration model was found to be equally accurate and precise on the two instruments. A procedure, using the Kennard-Stone algorithm, is described for determining a reduced number of samples as a transfer set using only the spectral information from the original instrument. The purpose of the subset was to permit transfer to a new instrument where that instrument was not available until after calibration development or where it was undesirable to re-measure the full sample set (i.e. due to excessive reference chemistry). Utilising the transfer set, transfer to a third instrument was evaluated. The calibration model, optimised between the first two instruments, was not directly applicable for the third instrument, with further wavelength selection required to remove a small region of spectral data. On completion, using a full statistical evaluation, a single calibration model was found to be equally accurate and precise on all three instruments.     

Detection of Δ9-Tetrahydr0Cannabin0L in Human Breath Following Marijuana Smoking

Abstract

Two techniques were used for enriching human breath samples for δ⁹ -tetrahydrocannabinol (I). One method utilized a polyethylene foam wafer which was positioned in front of the expired air stream using a modified face mask. The second technique was a cryogenic trap containing ethanol. Analysis of the enriched breath samples following smoking of a marijuana cigarette was accomplished using a previously described hplc-ms procedure. Results demonstrated that (I) was detectable with either enrichment method following marijuana smoking.     

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.     

Breath acetone monitoring by portable Si:WO3 gas sensors

Breath analysis has the potential for early stage detection and monitoring of illnesses to drastically reduce the corresponding medical diagnostic costs and improve the quality of life of patients suffering from chronic illnesses. In particular, the detection of acetone in the human breath is promising for non-invasive diagnosis and painless monitoring of diabetes (no finger pricking). Here, a portable acetone sensor consisting of flame-deposited and in situ annealed, Si-doped epsilon-WO₃ nanostructured films was developed. The chamber volume was miniaturized while reaction-limited and transport-limited gas flow rates were identified and sensing temperatures were optimized resulting in a low detection limit of acetone (∼20 ppb) with short response (10–15 s) and recovery times (35–70 s). Furthermore, the sensor signal (response) was robust against variations of the exhaled breath flow rate facilitating application of these sensors at realistic relative humidities (80–90%) as in the human breath. The acetone content in the breath of test persons was monitored continuously and compared to that of state-of-the-art proton transfer reaction mass spectrometry (PTR-MS). Such portable devices can accurately track breath acetone concentration to become an alternative to more elaborate breath analysis techniques.     

Performance of Amperometric Platinized‐Nafion Based Gas Phase Sensor for Determining Nitric Oxide (NO) Levels in Exhaled Human Nasal Breath

Nitric oxide (NO) levels in exhaled breath are a non‐invasive marker that can be used to diagnose various respiratory diseases and monitor a patient's response to given therapies. A portable and inexpensive device that can enable selective NO concentration measurements in exhaled breath samples is needed. Herein, the performance of an amperometric Pt‐Nafion‐based gas phase sensor for detection of NO in exhaled human nasal breath is examined. Enhanced selectivity over carbon monoxide and ammonia is achieved via an in‐line zinc oxide‐based filter. Exhaled nasal NO levels measured in 21 human samples with the sensor are shown to correlate well with those obtained using a chemiluminescence reference method (R²=0.9836).     

An analytical approach for on-site analysis of breath samples for Δ9-tetrahydrocannabinol (THC)

Increased acceptance of cannabis containing the psychoactive component, Δ9-tetrahydrocannabinol (THC), raises concerns about the potential for impaired drivers and increased highway accidents. In contrast to the “breathalyzer” test, which is generally accepted for determining the alcohol level in a driver, there is no currently accepted roadside test for THC in a motorist. There is a need for an easily collectible biological sample from a potentially impaired driver coupled with an accurate on-site test to measure the presence and quantity of THC in a driver. A novel breath collection device is described, which includes three separate sample collectors for collecting identical A, B, and C breath samples from a subject. A simple one-step ethanol extraction of the “A” breath collector sample can be analyzed by UHPLC/selected ion monitoring (SIM) liquid chromatography/mass spectrometry (LC/MS) to provide qualitative and quantitative determination of THC in breath sample in less than 4 min for samples collected up to 6 h after smoking a cannabis cigarette. SIM LC/MS bioanalyses employed d3-THC as the stable isotope internal standard fortified in negative control breath samples for quantitation including replicates of six calibrator standards and three quality control (QC) samples. Subsequent confirmation of the same breath sample in the B collectors was then confirmed by a reference lab by LC/MS/MS analysis. Fit-for-purpose bioanalytical validation consistent with pharmaceutical regulated bioanalyses produced pharmacokinetic (PK) curves for the two volunteer cannabis smokers. These results produced PK curves, which showed a rapid increase of THC in the breath of the subjects in the first hour followed by reduced THC levels in the later time points. A simpler single-point calibration curve procedure with calibrators and QC prepared in ethanol provided similar results. Limitations to this approach include the higher cost and operator skill sets for the instrumentation employed and the inability to actually determine driver impairment.     

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.     

Breath analysis: trends in techniques and clinical applications

Over the last few years, breath analysis for the routine monitoring either of metabolic disorders caused by specific diseases or occupational exposure to toxic substances has become more and more attractive due to its noninvasiveness.Several approaches have been proposed for sample collection, treatment, and analysis. This paper gives a critical overview of the analytical devices and procedures used for breath analysis. Results on the identification of chemicals in human breath for a selection of diseases are discussed.     

多批次肝衰竭患者呼出气体的电喷雾萃取电离质谱检测及代谢组学数据分析

采用高分辨电喷雾萃取电离质谱(EESI-MS)技术对肝衰竭患者和健康志愿者呼出气体样本进行快速检测, 结合多块偏最小二乘分析(MB-PLS)方法, 对多批次获取的呼出气体代谢数据进行统计建模分析, 并与传统的PLS方法进行比较. 结果表明, MB-PLS方法能有效消除批次差异对统计建模的影响. 此外, 利用MB-PLS模型变量VIP值对变量进行筛选, 可降低数据的冗余, 消除无关变量对模型的影响, 从而有效提高了模型的性能.     

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.     

The Influence of Smoking Status on Exhaled Breath Profiles in Asthma and COPD Patients

Breath analysis using eNose technology can be used to discriminate between asthma and COPD patients, but it remains unclear whether results are influenced by smoking status. We aim to study whether eNose can discriminate between ever- vs. never-smokers and smoking <24 vs. >24 h before the exhaled breath, and if smoking can be considered a confounder that influences eNose results. We performed a cross-sectional analysis in adults with asthma or chronic obstructive pulmonary disease (COPD), and healthy controls. Ever-smokers were defined as patients with current or past smoking habits. eNose measurements were performed by using the SpiroNose. The principal component (PC) described the eNose signals, and linear discriminant analysis determined if PCs classified ever-smokers vs. never-smokers and smoking <24 vs. >24 h. The area under the receiver–operator characteristic curve (AUC) assessed the accuracy of the models. We selected 593 ever-smokers (167 smoked <24 h before measurement) and 303 never-smokers and measured the exhaled breath profiles of discriminated ever- and never-smokers (AUC: 0.74; 95% CI: 0.66–0.81), and no cigarette consumption <24h (AUC 0.54, 95% CI: 0.43–0.65). In healthy controls, the eNose did not discriminate between ever or never-smokers (AUC 0.54; 95% CI: 0.49–0.60) and recent cigarette consumption (AUC 0.60; 95% CI: 0.50–0.69). The eNose could distinguish between ever and never-smokers in asthma and COPD patients, but not recent smokers. Recent smoking is not a confounding factor of eNose breath profiles.     

Directional Photomanipulation of Breath Figure Arrays

Porous polymeric films are of paramount importance in many areas of modern science and technology. However, processing methods typically based on direct writing, imprint, and lithography techniques have low throughput and are often limited to specific fabricated shapes. Herein, we demonstrate the directional photomanipulation of breath figure arrays (BFAs) formed by an azobenzene‐containing block copolymer to address the aforementioned problems. Under the irradiation of linearly polarized light, the round pores in the BFAs were converted to rectangular, rhombic, and parallelogram‐shaped pores in 30 min, due to the anisotropic mass migration based on the photo‐reconfiguration of the azobenzene units. Through a secondary irradiation after rotating the sample by 90°, the transformed pores were apparently recovered. Therefore, this non‐contacted, directional photomanipulation technique in conjunction with breath figure processing opens a new route to nano/microporous films with finely tuned features.