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Here, we present a protocol for using Early Data Visualization Script, a user-friendly software tool to visualize complex volatile metabolomics data in clinical setups. We describe steps for tabulating data and adjusting visual output to visualize complex time-resolved volatile omics data using simple charts and graphs. We then demonstrate possible modifications by detailing procedures for the adaptation of four basic functions. For complete details on the use and execution of this protocol, please refer to Sukul et al. (2022) 1 and Remy et al. (2022). 2 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Due to their immediate exhalation after generation at the cellular/microbiome levels, exhaled volatile organic compounds (VOCs) may provide real-time information on pathophysiological mechanisms and the host response to infection. In recent years, the metabolic profiling of the most frequent respiratory infections has gained interest as it holds potential for the early, non-invasive detection of pathogens and the monitoring of disease progression and the response to therapy. Using previously unpublished data, randomly selected individuals from a COVID-19 test center were included in the study. Based on multiplex PCR results (non-SARS-CoV-2 respiratory pathogens), the breath profiles of 479 subjects with the presence or absence of flu-like symptoms were obtained using proton-transfer-reaction time-of-flight mass spectrometry. Among 223 individuals, one respiratory pathogen was detected in 171 cases, and more than one pathogen in 52 cases. A total of 256 subjects had negative PCR test results and had no symptoms. The exhaled VOC profiles were affected by the presence of Haemophilus influenzae , Streptococcus pneumoniae, and Rhinovirus. The endogenous ketone, short-chain fatty acid, organosulfur, aldehyde, and terpene concentrations changed, but only a few compounds exhibited concentration changes above inter-individual physiological variations. Based on the VOC origins, the observed concentration changes may be attributed to oxidative stress and antioxidative defense, energy metabolism, systemic microbial immune homeostasis, and inflammation. In contrast to previous studies with pre-selected patient groups, the results of this study demonstrate the broad inter-individual variations in VOC profiles in real-life screening conditions. As no unique infection markers exist, only concentration changes clearly above the mentioned variations can be regarded as indicative of infection or colonization.

Plants, animals and humans metabolically produce volatile isoprene (C 5 H 8 ). Humans continuously exhale isoprene and exhaled concentrations differ under various physio-metabolic and pathophysiological conditions. Yet unknown metabolic origin hinders isoprene to reach clinical practice as a biomarker. Screening 2000 individuals from consecutive mass-spectrometric studies, we herein identify five healthy German adults without exhaled isoprene. Whole exome sequencing in these adults reveals only one shared homozygous (European prevalence: <1%) IDI2 stop-gain mutation, which causes losses of enzyme active site and Mg 2+ -cofactor binding sites. Consequently, the conversion of isopentenyl diphosphate to dimethylallyl diphosphate (DMAPP) as part of the cholesterol metabolism is prevented in these adults. Targeted sequencing depicts that the IDI2 rs1044261 variant (p.Trp144Stop) is heterozygous in isoprene deficient blood-relatives and absent in unrelated isoprene normal adults. Wild-type IDI1 and cholesterol metabolism related serological parameters are normal in all adults. IDI2 determines isoprene production as only DMAPP sources isoprene and unlike plants, humans lack isoprene synthase and its enzyme homologue. Human IDI2 is expressed only in skeletal-myocellular peroxisomes and instant spikes in isoprene exhalation during muscle activity underpins its origin from muscular lipolytic cholesterol metabolism. Our findings translate isoprene as a clinically interpretable breath biomarker towards potential applications in human medicine., (© 2023. Springer Nature Limited.)

Being the proximal matrix, breath offers immediate metabolic outlook of respiratory infections. However, high viral load in exhalations imposes higher transmission risk that needs improved methods for safe and repeatable analysis. Here, we have advanced the state-of-the-art methods for real-time and offline mass-spectrometry based analysis of exhaled volatile organic compounds (VOCs) under SARS-CoV-2 and/or similar respiratory conditions. To reduce infection risk, the general experimental setups for direct and offline breath sampling are modified. Certain mainstream and side-stream viral filters are examined for direct and lab-based applications. Confounders/contributions from filters and optimum operational conditions are assessed. We observed immediate effects of infection safety mandates on breath biomarker profiles. Main-stream filters induced physiological and analytical effects. Side-stream filters caused only systematic analytical effects. Observed substance specific effects partly depended on compound's origin and properties, sampling flow and respiratory rate. For offline samples, storage time, -conditions and -temperature were crucial. Our methods provided repeatable conditions for point-of-care and lab-based breath analysis with low risk of disease transmission. Besides breath VOCs profiling in spontaneously breathing subjects at the screening scenario of COVID-19/similar test centres, our methods and protocols are applicable for moderately/severely ill (even mechanically-ventilated) and highly contagious patients at the intensive care., (© 2022. The Author(s).)

Breath volatile organics (VOCs) may provide immediate information on infection mechanisms and host response. We conducted real-time mass spectrometry-based breath profiling in 708 non-preselected consecutive subjects in the screening scenario of a COVID-19 test center. Recruited subjects were grouped based on PCR-confirmed infection status and presence or absence of flu-like symptoms. Exhaled VOC profiles of SARS-CoV-2-positive cases (n = 36) differed from healthy (n = 256) and those with other respiratory infections (n = 416). Concentrations of most VOCs were suppressed in COVID-19. VOC concentrations also differed between symptomatic and asymptomatic cases. Breath markers mirror effects of infections onto host's cellular metabolism and microbiome. Downregulation of specific VOCs was attributed to suppressive effects of SARS-CoV-2 onto gut or pulmonary microbial metabolism. Breath analysis holds potential for monitoring SARS-CoV-2 infections rather than for primary diagnosis. Breath profiling offers unconventional insight into host-virus cross-talk and infection microbiology and enables non-invasive assessment of disease manifestation., Competing Interests: The authors declare no competing interests., (© 2022 The Author(s).)

Background: While assumed to protect against coronavirus transmission, face masks may have effects on respiratory-haemodynamic parameters. Within this pilot study, we investigated immediate and progressive effects of FFP2 and surgical masks on exhaled breath constituents and physiological attributes in 30 adults at rest., Methods: We continuously monitored exhaled breath profiles within mask space in older (age 60-80 years) and young to middle-aged (age 20-59 years) adults over the period of 15 and 30 min by high-resolution real-time mass-spectrometry. Peripheral oxygen saturation ( S pO 2 ) and respiratory and haemodynamic parameters were measured (noninvasively) simultaneously., Results: Profound, consistent and significant (p≤0.001) changes in S pO 2 (≥60&#95;FFP2-15 min: 5.8±1.3%↓, ≥60&#95;surgical-15 min: 3.6±0.9%↓, <60&#95;FFP2-30 min: 1.9±1.0%↓, <60&#95;surgical-30 min: 0.9±0.6%↓) and end-tidal carbon dioxide tension ( P ETCO 2 ) (≥60&#95;FFP2-15 min: 19.1±8.0%↑, ≥60&#95;surgical-15 min: 11.6±7.6%↑, <60&#95;FFP2- 30 min: 12.1±4.5%↑, <60&#95;surgical- 30 min: 9.3±4.1%↑) indicate ascending deoxygenation and hypercarbia. Secondary changes (p≤0.005) to haemodynamic parameters ( e.g. mean arterial pressure (MAP) ≥60&#95;FFP2-15 min: 9.8±10.4%↑) were found. Exhalation of bloodborne volatile metabolites, e.g. aldehydes, hemiterpene, organosulfur, short-chain fatty acids, alcohols, ketone, aromatics, nitrile and monoterpene mirrored behaviour of cardiac output, MAP, S pO 2 , respiratory rate and P ETCO 2 . Exhaled humidity ( e.g. ≥60&#95;FFP2-15 min: 7.1±5.8%↑) and exhaled oxygen ( e.g. ≥60&#95;FFP2-15 min: 6.1±10.0%↓) changed significantly (p≤0.005) over time., Conclusions: Breathomics allows unique physiometabolic insights into immediate and transient effects of face mask wearing. Physiological parameters and breath profiles of endogenous and/or exogenous volatile metabolites indicated putative cross-talk between transient hypoxaemia, oxidative stress, hypercarbia, vasoconstriction, altered systemic microbial activity, energy homeostasis, compartmental storage and washout. FFP2 masks had a more pronounced effect than surgical masks. Older adults were more vulnerable to FFP2 mask-induced hypercarbia, arterial oxygen decline, blood pressure fluctuations and concomitant physiological and metabolic effects., Competing Interests: Conflict of interest: The authors have nothing to disclose., (Copyright ©The authors 2022.)

Breath analysis was coupled with ergo-spirometry for non-invasive profiling of physio-metabolic status under exhaustive exercise. Real-time mass-spectrometry based continuous analysis of exhaled metabolites along with breath-resolved spirometry and heart rate monitoring were executed while 14 healthy adults performed ergometric ramp exercise protocol until exhaustion. Arterial blood lactate level was analyzed at defined time points. Respiratory-cardiac parameters and exhalation of several blood-borne volatiles changed continuously with the course of exercise and increasing workloads. Exhaled volatiles mirrored ventilatory and/or hemodynamic effects and depended on the origin and/or physicochemical properties of the substances. At the maximum workload, endogenous isoprene, methanethiol, dimethylsulfide, acetaldehyde, butanal, butyric acid and acetone concentrations decreased significantly by 74, 25, 35, 46, 21, 2 and 2%, respectively. Observed trends in exogenous cyclohexadiene and acetonitrile mimicked isoprene profile due to their similar solubility and volatility. Assignment of anaerobic threshold was possible via breath acetone. Breathomics enabled instant profiling of physio-metabolic effects and anaerobic thresholds during exercise. Profiles of exhaled volatiles indicated effects from muscular vasoconstriction, compartmental distribution of perfusion, extra-alveolar gas-exchange and energy homeostasis. Sulfur containing compounds and butyric acid turned out to be interesting for investigations of combined diet and exercise programs. Reproducible metabolic breath patterns have enhanced scopes of breathomics in sports science/medicine., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Pugliese, Trefz, Weippert, Pollex, Bruhn, Schubert, Miekisch and Sukul.)

Regional anaesthesia is well established as a standard method in clinical practice. Currently, the local anaesthetics of amino-amide types such as prilocaine are frequently used. Despite routine use, complications due to overdose or accidental intravenous injection can arise. A non-invasive method that can indicate such complications early would be desirable. Breath gas analysis offers great potential for the non-invasive monitoring of drugs and their volatile metabolites. The physicochemical properties of o-toluidine, the main metabolite of prilocaine, allow its detection in breath gas. Within this study, we investigated whether o-toluidine can be monitored in exhaled breath during regional anaesthesia in an animal model, if correlations between o-toluidine and prilocaine blood levels exist and if accidental intravenous injections are detectable by o-toluidine breath monitoring. Continuous o-toluidine monitoring was possible during regional anaesthesia of the cervical plexus and during simulated accidental intravenous injection of prilocaine. The time course of exhaled o-toluidine concentrations considerably differed depending on the injection site. Intravenous injection led to an immediate increase in exhaled o-toluidine concentrations within 2 min, earlier peak and higher maximum concentrations, followed by a faster decay compared to regional anaesthesia. The strength of correlation of blood and breath parameters depended on the injection site. In conclusion, real time monitoring of o-toluidine in breath gas is possible by means of PTR-ToF-MS. Since simulated accidental intravenous injection led to an immediate increase in exhaled o-toluidine concentrations within 2 min and higher maximum concentrations, monitoring exhaled o-toluidine may potentially be applied for the non-invasive real-time detection of accidental intravenous injection of prilocaine.

The chemical composition of exhaled breath was examined for volatile organic compound (VOC) indicators of sexual arousal in human beings. Participants (12-male, 12-female) were shown a randomized series of three emotion-inducing 10-min film clips interspersed with 3-min neutral film clips. The films caused different arousals: sports film (positive-nonsexual); horror film (negative-nonsexual); and erotic (sexual) that were monitored with physiological measurements including genital response and temperature. Simultaneously the breath was monitored for VOC and CO 2 . While some breath compounds (methanol and acetone) changed uniformly irrespective of the film order, several compounds did show significant arousal associated changes. For both genders CO 2 and isoprene decreased in the sex clip. Some male individuals showed particularly strong increases of indole, phenol and cresol coincident with sexual arousal that decreased rapidly afterwards. These VOCs are degradation products of tyrosine and tryptophan, precursors for dopamine, noradrenalin, and serotonin, and therefore represent potential breath markers of sexual arousal., (© 2022. The Author(s).)

Healthy aging driven physio-metabolic events in females hold the key to complex in vivo mechanistic links and systemic cross talks. Effects from basic changes at genome, proteome, metabolome, and lipidome levels are often reflected at the upstream phenome (e.g., breath volatome) cascades. Here, we have analyzed exhaled volatile metabolites (measured via real time mass spectrometry based breathomics) data from 204 healthy females, aged between 07 and 80 years. Age related substance-specific differences were observed in breath biomarkers. Exhalation of blood-borne endogenous organosulfur, short-chain fatty acids, alcohols, aldehydes, alkene, ketones and exogenous nitriles, terpenes, and aromatics have denominated interplay between endocrine differences, energy homeostasis, systemic microbial diversity, oxidative stress, and lifestyle. Overall marker expressions were suppressed under daily oral contraception. Young homosexual/lesbian adults turned out as breathomic outliers. Previously proposed disease-specific breath biomarkers should be reevaluated upon aging effects. Breathomics offers a noninvasive window toward system-wide understanding and personalized monitoring of aging i.e., translatable to gerontology., Competing Interests: The authors declare no competing interests., (© 2022 The Author(s).)

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.

Analysis of volatile organic compounds (VOCs) is a novel approach to accelerate bacterial culture diagnostics of Mycobacterium avium subsp. paratuberculosis (MAP). In the present study, cultures of fecal and tissue samples from MAP-infected and non-suspect dairy cattle and goats were explored to elucidate the effects of sample matrix and of animal species on VOC emissions during bacterial cultivation and to identify early markers for bacterial growth. The samples were processed following standard laboratory procedures, culture tubes were incubated for different time periods. Headspace volume of the tubes was sampled by needle trap-micro-extraction, and analyzed by gas chromatography-mass spectrometry. Analysis of MAP-specific VOC emissions considered potential characteristic VOC patterns. To address variation of the patterns, a flexible and robust machine learning workflow was set up, based on random forest classifiers, and comprising three steps: variable selection, parameter optimization, and classification. Only a few substances originated either from a certain matrix or could be assigned to one animal species. These additional emissions were not considered informative by the variable selection procedure. Classification accuracy of MAP-positive and negative cultures of bovine feces was 0.98 and of caprine feces 0.88, respectively. Six compounds indicating MAP presence were selected in all four settings (cattle vs. goat, feces vs. tissue): 2-Methyl-1-propanol, 2-methyl-1-butanol, 3-methyl-1-butanol, heptanal, isoprene, and 2-heptanone. Classification accuracies for MAP growth-scores ranged from 0.82 for goat tissue to 0.89 for cattle feces. Misclassification occurred predominantly between related scores. Seventeen compounds indicating MAP growth were selected in all four settings, including the 6 compounds indicating MAP presence. The concentration levels of 2,3,5-trimethylfuran, 2-pentylfuran, 1-propanol, and 1-hexanol were indicative for MAP cultures before visible growth was apparent. Thus, very accurate classification of the VOC samples was achieved and the potential of VOC analysis to detect bacterial growth before colonies become visible was confirmed. These results indicate that diagnosis of paratuberculosis can be optimized by monitoring VOC emissions of bacterial cultures. Further validation studies are needed to increase the robustness of indicative VOC patterns for early MAP growth as a pre-requisite for the development of VOC-based diagnostic analysis systems., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Vitense, Kasbohm, Klassen, Gierschner, Trefz, Weber, Miekisch, Schubert, Möbius, Reinhold, Liebscher and Köhler.)

Background: Isoprene (C 5 H 8 ) is a clinically important breath metabolite. Although, hundreds of studies have reported differential expressions in isoprene exhalation as breath biomarker for diverse diseases, the substance couldn't enter to clinical practice as diagnostic marker. Moreover, many experimental/basic observations upon breath isoprene remained unrelated to the corresponding pathophysiological effects on its putative metabolic origin (i.e. mevalonate pathway). Here, we investigated the fundamental reason that hindered the rational interpretation and translation of this marker from basic to clinical science., Methods: Via high-resolution mass-spectrometry based breathomics in 1026 human subjects, we discovered adults with significant deficiency (order of magnitude lower than the normal) and complete absence of breath isoprene. We prospectively applied real-time breathomics, quantitative gene expression analysis of the mevalonate pathway enzymes, lipid-profiling and hemodynamic monitoring on those isoprene deficient subjects and controls. Additionally, the subject with absence of isoprene was followed up throughout different phases of her womanhood., Results: In contrast to convention, we witnessed that adults can live healthy without exhaling isoprene or with significant deficiency. This rare phenotype represents a recessive inheritance. Despite physio-metabolic changes during menstrual cycle (that is known to profoundly affect isoprene exhalation) and profoundly increased plasma cholesterol during pregnancy and after childbirth, isoprene remained absent. All genes of mevalonate pathway enzymes were normally expressed in all participants, without any down-regulation or compensatory up-regulation., Conclusions: Absence/deficiency of isoprene despite normal lipid profiles and no mevalonate pathway malfunction disqualifies the long-believed metabolic origin of isoprene from cholesterol biosynthesis. Thus, clinical translation of breath isoprene expressions should not be generally attributed to corresponding pathophysiological effects onto mevalonate/cholesterol pathway. Our finding has refined and optimized the clinical interpretation of isoprene as biomarker in volatile metabolomics and breathomics. Future studies will address the correct metabolic origin of isoprene to imply this important marker to routine practice., Competing Interests: The authors declare no conflict of interest., (© 2021 The Authors.)

Breath analysis holds promise for non-invasive in vivo monitoring of disease related processes. However, physiological parameters may considerably affect profiles of exhaled volatile organic substances (VOCs). Volatile substances can be released via alveoli, bronchial mucosa or from the upper airways. The aim of this study was the systematic investigation of the influence of different sampling sites in the respiratory tract on VOC concentration profiles by means of a novel experimental setup. After ethical approval, breath samples were collected from 25 patients undergoing bronchoscopy for endobronchial ultrasound or bronchoscopic lung volume reduction from different sites in the airways. All patients had total intravenous anaesthesia under pressure-controlled ventilation. If necessary, respiratory parameters were adjusted to keep P ET CO 2 = 35-45 mm Hg. 30 ml gas were withdrawn at six sampling sites by means of gastight glass syringes: S1 = Room air, S2 = Inspiration, S3 = Endotracheal tube, S4 = Trachea, S5 = Right B6 segment, S6 = Left B6 segment (S4-S6 through the bronchoscope channel). 10 ml were used for VOC analysis, 20 ml for PCO 2 determination. Samples were preconcentrated by solid-phase micro-extraction (SPME) and analysed by gas chromatography-mass spectrometry (GC-MS). PCO 2 was determined in a conventional blood gas analyser. Statistically significant differences in substance concentrations for acetone, isoprene, 2-methyl-pentane and n-hexane could be observed between different sampling sites. Increasing substance concentrations were determined for acetone (15.3%), 2-methyl-pentane (11.4%) and n-hexane (19.3%) when passing from distal to proximal sampling sites. In contrast, isoprene concentrations decreased by 9.9% from proximal to more distal sampling sites. Blank bronchoscope measurements did not show any contaminations. Increased substance concentrations in the proximal respiratory tract may be explained through substance excretion from bronchial mucosa while decreased concentrations could result from absorption or reaction processes. Spatial mapping of VOC profiles can provide novel insights into substance specific exhalation kinetics and mechanisms.

Proton transfer reaction time-of-flight mass spectrometry (PTR-ToF-MS) is a powerful tool for real-time monitoring of trace concentrations of volatile organic compounds (VOCs). The sensitivity of PTR-ToF-MS also depends on the ability to effectively focus and transmit ions from the relatively high-pressure drift tube (DT) to the low-pressure mass analyzer. In the present study, a modular ion-funnel (IF) is placed adjacent to the DT of a PTR-ToF-MS instrument to improve the ion-focusing. IF consists of a series of electrodes with gradually decreasing orifice diameters. Radio frequency (RF) voltage and direct current (DC) electric field are then applied to the electrodes to get the ions focused. We investigated the effect of the RF voltage and DC field on the sensitivity of a pattern of VOCs including hydrocarbons, alcohols, aldehydes, ketones, and aromatic compounds. In a proof-of-concept study, the instrument operating both as normal DT (DC-mode) and at optimal IF conditions (RF-mode) was applied for the breath analysis of 21 healthy human subjects. For the range of investigated VOCs, an improvement of one order of magnitude in sensitivity was observed in RF-mode compared with DC-mode. Limits of detection could be improved by a factor of 2-4 in RF-mode compared with DC-mode. Operating the instrument in RF-mode allowed the detection of more compounds in the exhaled air compared with DC-mode. Incorporation of the IF considerably improved the performance of PTR-ToF-MS allowing the real-time monitoring of a larger number of potential breath biomarkers. Graphical abstract.

Control of breathing is automatic and its regulation is keen to autonomic functions. Therefore, involuntary and voluntary nervous regulation of breathing affects ventilatory variations, which has profound potential to address expanding challenges in contemporary pulmonology. Nonetheless, the fundamental attributes of the aforementioned phenomena are rarely understood and/or investigated. Implementation of unconventional approach like breathomics may leads to a better comprehension of those complexities in respiratory medicine. We applied breath-resolved spirometry and capnometry, non-invasive hemodynamic monitoring along with continuous trace analysis of exhaled VOCs (volatile organic compounds) by means of real-time mass-spectrometry in 25 young and healthy adult humans to investigate any possible mirroring of instant ventilatory variations by exhaled breath composition, under varying respiratory rhythms. Hemodynamics remained unaffected. Immediate changes in measured breath compositions and corresponding variations occurred when respiratory rhythms were switched between spontaneous (involuntary/unsynchronised) and/or paced (voluntary/synchronised) breathing. Such changes in most abundant, endogenous and bloodborne VOCs were closely related to the minute ventilation and end-tidal CO 2 exhalation. Unprecedentedly, while preceded by a paced rhythm, spontaneous rhythms in both independent setups became reproducible with significantly (P-value ≤ 0.005) low intra- and inter-individual variation in measured parameters. We modelled breath-resolved ventilatory variations via alveolar isoprene exhalation, which were independently validated with unequivocal precision. Reproducibility i.e. attained via our method would be reliable for human breath sampling, concerning biomarker research. Thus, we may realize the actual metabolic and pathophysiological expressions beyond the everlasting in vivo physiological noise. Consequently, less pronounced changes are often misinterpreted as disease biomarker in cross-sectional studies. We have also provided novel information beyond conventional spirometry and capnometry. Upon clinical translations, our findings will have immense impact on pulmonology and breathomics as they have revealed a reproducible pattern of ventilatory variations and respiratory homeostasis in endogenous VOC exhalations.

Assessment and treatment of postoperative pain can be challenging as objective examination techniques to detect and quantify pain are lacking. We aimed to investigate changes of exhaled volatile organic compounds (VOCs) in patients with postoperative pain before and after treatment with opioid analgesics. In an observational study in 20 postoperative patients, we monitored for postoperative pain, hemodynamic parameters, and catecholamines before and during treatment. VOCs in the patients were determined by direct real-time proton transfer reaction time-of-flight mass spectrometry prior (0 min) and after piritramide application (15 min as well as 30 min). Cardiovascular variables changed and norepinephrine levels decreased during treatment. The VOCs acetonitrile (<0.001), acetaldehyde ( p = 0.002), benzopyran ( p = 0.004), benzene ( p < 0.001), hexenal ( p = < 0.001), 1-butanethiol ( p = 0.004), methanethiol ( p < 0.001), ethanol ( p = 0.003), and propanol ( p = < 0.001) changed significantly over time. Patients with Numeric Rating Scale (NRS) < 4 showed a significantly lower concentration of hexenal compared to patients with NRS > 4 at the time points 15 min (45.0 vs. 385.3 ncps, p = 0.047) and 30 min (38.3 vs. 334.6 ncps, p = 0.039). Breath analysis can provide additional information for noninvasive monitoring for analgesic treatment in postoperative patients.

Influenza A is a serious pathogen itself, but often leads to dangerous co-infections in combination with bacterial species such as Streptococcus pyogenes. In comparison to classical biochemical methods, analysis of volatile organic compounds (VOCs) in headspace above cultures can enable destruction free monitoring of metabolic processes in vitro. Thus, volatile biomarkers emitted from biological cell cultures and pathogens could serve for monitoring of infection processes in vitro. In this study we analysed VOCs from headspace above (co)-infected human cells by using a customized sampling system. For investigating the influenza A mono-infection and the viral-bacterial co-infection in vitro, we analysed VOCs from Detroit cells inoculated with influenza A virus and S. pyogenes by means of needle-trap micro-extraction (NTME) and gas chromatography mass spectrometry (GC-MS). Besides the determination of microbiological data such as cell count, cytokines, virus load and bacterial load, emissions from cell medium, uninfected cells and bacteria mono-infected cells were analysed. Significant differences in emitted VOC concentrations were identified between non-infected and infected cells. After inoculation with S. pyogenes, bacterial infection was mirrored by increased emissions of acetaldehyde and propanal. N-propyl acetate was linked to viral infection. Non-destructive monitoring of infections by means of VOC analysis may open a new window for infection research and clinical applications. VOC analysis could enable early recognition of pathogen presence and in-depth understanding of their etiopathology.

Reactive exhaled volatile organic compounds (VOCs) such as nitrogen- and sulfur-containing substances may be related to diseases, metabolic processes and bacterial activity. As these compounds may interact with any surface of the analytical system, time-resolved monitoring and reliable quantification is difficult. We describe a proton transfer reaction time-of-flight mass spectrometry (PTR-ToF-MS) based analytical method for direct breath-resolved monitoring of reactive compounds. Aliphatic amines were used as test substances. Matrix adapted gas standards were generated by means of a liquid calibration unit. Calibration conditions were adapted in terms of materials, temperature and equilibration time. PTR-ToF-MS conditions were optimized in terms of inlet materials, transfer line and drift tube temperature and drift tube reduced electric field (E/N). Optimized PTR conditions in combination with inert materials and high temperatures considerably reduced the interactions of compounds with the surfaces of the analytical system. Good linearity (R2 > 0.99, RSDs < 5%) with LODs between 0.15 ppbV and 1.23 ppbV and LOQs between 0.24 ppbV and 1.94 ppbV could be achieved. The method was then applied to breath-resolved monitoring of reactive compounds in 17 healthy subjects after high and low oral protein challenge. Exhaled concentrations of trimethylamine, indole, methanethiol, dimethylsulfide, acetone, 2-propanol, 2-butanone and phenol showed significant changes after protein intake. Methanethiol concentrations increased 6-fold within minutes after the protein intake. Optimization of methods and instrument design enabled reliable breath-resolved PTR-MS based analysis of exhaled reactive VOCs in the sub-ppbV range. Continuous in vivo monitoring of exhaled amines and sulphur containing compounds may provide novel non-invasive insight into endogenous and gut bacteria driven protein metabolism.

Monitoring metabolic adaptation to type 1 diabetes mellitus in children is challenging. Analysis of volatile organic compounds (VOCs) in exhaled breath is non-invasive and appears as a promising tool. However, data on breath VOC profiles in pediatric patients are limited. We conducted a cross-sectional study and applied quantitative analysis of exhaled VOCs in children suffering from type 1 diabetes mellitus (T1DM) (n = 53) and healthy controls (n = 60). Both groups were matched for sex and age. For breath gas analysis, a very sensitive direct mass spectrometric technique (PTR-TOF) was applied. The duration of disease, the mode of insulin application (continuous subcutaneous insulin infusion vs. multiple daily insulin injection) and long-term metabolic control were considered as classifiers in patients. The concentration of exhaled VOCs differed between T1DM patients and healthy children. In particular, T1DM patients exhaled significantly higher amounts of ethanol, isopropanol, dimethylsulfid, isoprene and pentanal compared to healthy controls (171, 1223, 19.6, 112 and 13.5 ppbV vs. 82.4, 784, 11.3, 49.6, and 5.30 ppbV). The most remarkable differences in concentrations were found in patients with poor metabolic control, i.e. those with a mean HbA 1c above 8%. In conclusion, non-invasive breath testing may support the discovery of basic metabolic mechanisms and adaptation early in the progress of T1DM.

An analysis of exhaled volatile organic compounds (VOC) may deliver systemic information quicker than available invasive techniques. Metabolic aberrations in pediatric type 1 diabetes (T1DM) are of high clinical importance and could be addressed via breathomics. Real-time breath analysis was combined with continuous glucose monitoring (CGM) and blood tests in children suffering from T1DM and age-matched healthy controls in a highly standardized setting. CGM and breath-resolved VOC analysis were performed every 5 minutes for 9 hours and blood was sampled at pre-defined time points. Per participant ( n = 44) food intake and physical activity were identical and a total of 22 blood samples and 93 minutes of breath samples were investigated. The inter-individual variability of glucose, insulin, glucagon, leptin, and soluble leptin receptor relative to food intake differed distinctly between patients and controls. In T1DM patients, the exhaled amounts of acetone, 2-propanol, and pentanal correlated to glucose concentrations. Of note, the strength of these correlations strongly depended on the interval between food intake and breath sampling. Our data suggests that metabolic adaptation through postprandial hyperglycemia and related oxidative stress is immediately reflected in exhaled breath VOC concentrations. Clinical translations of our findings may enable point-of-care applicability of online breath analysis towards personalized medicine.

Analysis of volatile organic compounds (VOC) derived from bacterial metabolism during cultivation is considered an innovative approach to accelerate in vitro detection of slowly growing bacteria. This applies also to Mycobacterium avium subsp. paratuberculosis (MAP), the causative agent of paratuberculosis, a debilitating chronic enteritis of ruminants. Diagnostic application demands robust VOC profiles that are reproducible under variable culture conditions. In this study, the VOC patterns of pure bacterial cultures, derived from three independent in vitro studies performed previously, were comparatively analyzed. Different statistical analyses were linked to extract the VOC core profile of MAP and to prove its robustness, which is a prerequisite for further development towards diagnostic application. Despite methodical variability of bacterial cultivation and sample pre-extraction, a common profile of 28 VOCs indicating cultural growth of MAP was defined. The substances cover six chemical classes. Four of the substances decreased above MAP and 24 increased. Random forest classification was applied to rank the compounds relative to their importance and for classification of MAP versus control samples. Already the top-ranked compound alone achieved high discrimination (AUC 0.85), which was further increased utilizing all compounds of the VOC core profile of MAP (AUC 0.91). The discriminatory power of this tool for the characterization of natural diagnostic samples, in particular its diagnostic specificity for MAP, has to be confirmed in future studies., Competing Interests: The authors have declared that no competing interests exist.

Volatile organic compounds (VOCs) emitted from breath, faeces or skin may reflect physiological and pathological processes in vivo. Our setup employs real-time proton-transfer-reaction time-of-flight mass spectrometry (PTR-TOF-MS) to explore VOC emissions of dairy cows in stable air under field conditions. Within one herd of 596 cows, seven groups (8-117 cows per group) were assessed. Groups differed in milk yield and health status (two contained cows with paratuberculosis, a chronic intestinal infection). Each group arrived one after another in the area of air measurement in front of the milking parlour. A customised PTR-TOF-MS system with a 6 m long and heated transfer line, was used for measuring VOCs continuously for 7 h, 1.5 m above the cows. Three consecutive time periods were investigated. Twenty-seven VOCs increased while the animals were gathering in the waiting area, and decreased when the animals entered the milking parlour. Linear correlations between the number of animals present and VOC concentrations were found for (C 4 H 6 )H + and (C 3 H 6 O)H + . A relatively high concentration of acetone above the cows that had recently given birth to a calf might be related to increased fat turnover due to calving and different nutrition. Changes in VOC emissions were related to the presence of animals with paratuberculosis, to different average milk yields per group and to the time of the day (morning versus noon milking time). We found that VOC monitoring of stable air may provide additional immediate information on an animal's metabolic or health status and foster novel applications in the field of breath research.

Metabolic characterization of human adipose tissue-derived mesenchymal stromal/stem cells (ASCs) is of importance in stem cell research. The monitoring of the cell status often requires cell destruction. An analysis of volatile organic compounds (VOCs) in the headspace above cell cultures might be a noninvasive and nondestructive alternative to in vitro analysis. Furthermore, VOC analyses permit new insight into cellular metabolism due to their view on volatile compounds. Therefore, the aim of our study was to compare VOC profiles in the headspace above nondifferentiating and adipogenically differentiating ASCs. To this end, ASCs were cultivated under nondifferentiating and adipogenically differentiating conditions for up to 21 days. At different time points the headspace samples were preconcentrated by needle trap micro extraction and analyzed by gas chromatography/mass spectrometry. Adipogenic differentiation was assessed at equivalent time points. Altogether the emissions of 11 VOCs showed relevant changes and were analyzed in more detail. A few of these VOCs, among them acetaldehyde, were significantly different in the headspace of adipogenically differentiating ASCs and appeared to be linked to metabolic processes. Furthermore, our data indicate that VOC headspace analysis might be a suitable, noninvasive tool for the metabolic monitoring of (mesenchymal stem) cells in vitro., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Proton-transfer-reaction-time-of-flight-mass-spectrometry (PTR-ToF-MS) is a powerful tool for real-time monitoring of volatile organic compound (VOC) profiles in human breath. However, varying oxygen concentrations in the sample matrix may influence results from VOC analysis by PTR-ToF-MS. Elevated oxygen concentrations are likely to occur in clinical settings, but also when respiratory masks or breathing apparatus are used (e.g. in scuba diving, aviation, firefighting). Oxygen concentration may vary between subjects or within the course of a measurement or study and thus bias results. We systematically assessed the effect of high O 2 concentrations (up to 90%) in the sample matrix on the results of PTR-MS analysis for a pattern of VOCs including aromatics, aldehydes and ketones in dry and humid samples. In vivo experiments in healthy volunteers and mechanically ventilated animals were done to test the effect under real-life conditions. H 3 O + count significantly decreased by more than 40% when the amount of oxygen in the sample matrix was increased. Almost all investigated VOCs were significantly effected by varying oxygen concentrations and differences in signal intensities of more than 50% could be observed. The effect was generally more pronounced in dry samples but still significant under humid conditions. A linear dependency of sensitivity on the oxygen concentration in the sample matrix was observed for a number of VOCs (e.g. aldehydes) possibly enabling a factor based correction. VOC intensities were also influenced under in vivo conditions, e.g. ethanol decreased up to 71%. When PTR-MS analysis is carried out under oxygen supply, these issues need to be carefully considered.

Influenza is one of the most common causes of virus diseases worldwide. Virus detection requires determination of Influenza RNA in the upper respiratory tract. Efficient screening is not possible in this way. Analysis of volatile organic compounds (VOCs) in breath holds promise for non-invasive and fast monitoring of disease progression. Breath VOC profiles of 14 (3 controls and 11 infected animals) swine were repeatedly analyzed during a complete infection cycle of Influenza A under high safety conditions. Breath VOCs were pre-concentrated by means of needle trap micro-extraction and analysed by gas chromatography mass spectrometry before infection, during virus presence in the nasal cavity, and after recovery. Six VOCs could be related to disease progression: acetaldehyde, propanal, n-propyl acetate, methyl methacrylate, styrene and 1,1-dipropoxypropane. As early as on day four after inoculation, when animals were tested positive for Influenza A, differentiation between control and infected animals was possible. VOC based information on virus infection could enable early detection of Influenza A. As VOC analysis is completely non-invasive it has potential for large scale screening purposes. In a perspective, breath analysis may offer a novel tool for Influenza monitoring in human medicine, animal health control or border protection.

Volatile organic compounds (VOCs) emitted from in vitro cultures may reveal information on species and metabolism. Owing to low nmol L -1 concentration ranges, pre-concentration techniques are required for gas chromatography-mass spectrometry (GC-MS) based analyses. This study was intended to compare the efficiency of established micro-extraction techniques - solid-phase micro-extraction (SPME) and needle-trap micro-extraction (NTME) - for the analysis of complex VOC patterns. For SPME, a 75 μm Carboxen®/polydimethylsiloxane fiber was used. The NTME needle was packed with divinylbenzene, Carbopack X and Carboxen 1000. The headspace was sampled bi-directionally. Seventy-two VOCs were calibrated by reference standard mixtures in the range of 0.041-62.24 nmol L -1 by means of GC-MS. Both pre-concentration methods were applied to profile VOCs from cultures of Mycobacterium avium ssp. paratuberculosis. Limits of detection ranged from 0.004 to 3.93 nmol L -1 (median = 0.030 nmol L -1 ) for NTME and from 0.001 to 5.684 nmol L -1 (median = 0.043 nmol L -1 ) for SPME. NTME showed advantages in assessing polar compounds such as alcohols. SPME showed advantages in reproducibility but disadvantages in sensitivity for N-containing compounds. Micro-extraction techniques such as SPME and NTME are well suited for trace VOC profiling over cultures if the limitations of each technique is taken into account., (Copyright © 2018 John Wiley & Sons, Ltd.)

Natural menstrual cycle and/or oral contraception diversely affect women metabolites. Longitudinal metabolic profiling under constant experimental conditions is thereby realistic to understand such effects. Thus, we investigated volatile organic compounds (VOCs) exhalation throughout menstrual cycles in 24 young and healthy women with- and without oral contraception. Exhaled VOCs were identified and quantified in trace concentrations via high-resolution real-time mass-spectrometry, starting from a menstruation and then repeated follow-up with six intervals including the next bleeding. Repeated measurements within biologically comparable groups were employed under optimized measurement setup. We observed pronounced and substance specific changes in exhaled VOC concentrations throughout all cycles with low intra-individual variations. Certain blood-borne volatiles changed significantly during follicular and luteal phases. Most prominent changes in endogenous VOCs were observed at the ovulation phase with respect to initial menstruation. Here, the absolute median abundances of alveolar ammonia, acetone, isoprene and dimethyl sulphide changed significantly (P-value ≤ 0.005) by 18.22↓, 13.41↓, 18.02↑ and 9.40↓%, respectively. These VOCs behaved in contrast under the presence of combined oral contraception; e.g. isoprene decreased significantly by 30.25↓%. All changes returned to initial range once the second bleeding phase was repeated. Changes in exogenous benzene, isopropanol, limonene etc. and smoking related furan, acetonitrile and orally originated hydrogen sulphide were rather nonspecific and mainly exposure dependent. Our observations could apprehend a number of known/pre-investigated metabolic effects induced by monthly endocrine regulations. Potential in vivo origins (e.g. metabolic processes) of VOCs are crucial to realize such effects. Despite ubiquitous confounders, we demonstrated the true strength of volatolomics for metabolic monitoring of menstrual cycle and contraceptives. These outcomes may warrant further studies in this direction to enhance our fundamental and clinical understanding on menstrual metabolomics and endocrinology. Counter-effects of contraception can be deployed for future noninvasive assessment of birth control pills. Our findings could be translated toward metabolomics of pregnancy, menopause and post-menopausal complications via breath analysis.

Volatile organic compound (VOC) profiles emitted in trace concentrations from bacteria or cells has gained increasing importance over the decades. Analysis of VOCs in the headspace does not interfere with in vitro systems and, therefore, offers new options for non-invasive monitoring of cultures. Currently there is not any available standardized in vitro sampling system which considers effects of dilution and contamination onto ppbV to pptV VOC concentrations during. In this study a new in vitro system for online and offline headspace measurement of biological cultures was designed. The system was built from inert materials, equipped with universal sampling ports and easily adjustable volume options. Standard VOC mixtures in the system were analyzed by means of proton-transfer-reaction time-of-flight mass spectrometry and needle-trap-microextraction coupled with gas chromatography/mass spectrometry with a variance of 5%-14% and 10%-15%, respectively. In a proof of concept setup volatile emissions over cell cultures and pure media were assessed. The newly developed system enabled reliable and reproducible headspace analyses of in vitro cultures. As parallel application of different analytical methods is possible and confounding factors could be minimized, this set-up represents an important step towards standardization of headspace analysis over biological cultures.

Background: The analysis of volatile organic compounds (VOCs) in breath allows non-invasive investigations of diseases. Animal studies are conducted as a model to perform research of VOCs and their relation to diseases. In large animal models ruminants were often used as experimental targets. The effect of their physiological eructation on VOC exhalation has not been examined yet and is the objective of this study., Methods: Continuous breath profiles of two young cattle, four adult goats and four adult sheep were measured through a mask, covering mouth and nose, in real-time (200 ms) by means of proton transfer reaction time of flight mass spectrometry. Each animal was analysed twelve times for 3 consecutive minutes., Results: Real-time monitoring yielded a distinction of different episodes in the breath profiles of ruminants. An algorithm to separate eructation episodes and alveolar breath was established. In the first exhalation after eructation at least 19 VOC concentrations increased (up to 36-fold) and went back to initial levels in subsequent exhalations in all investigated ruminants. Decay of concentrations was substance specific. In goats, less VOCs were affected by the eructation compared to cattle and sheep. Breath profiles without exclusion of eructation episodes showed higher variations and median values than profiles where eructation episodes were excluded., Conclusion: Real-time breath analysis of ruminants enables the discrimination and characterisation of alveolar breath and eructation episodes. This leads to a better understanding of variation in breath data and possible origins of VOCs: breath or digestion related. To avoid impairment of breath gas results and to gain further information on bacterial products from the rumen, eructation and alveolar breath data should be analysed separately.

Background: Paraplegia due to spinal cord ischemia (SCI) is a serious complication after repair of thoracoabdominal aortic aneurysms. For prevention and early treatment of spinal ischemia, intraoperative monitoring of spinal cord integrity is essential. This study was intended to improve recognition of SCI through a combination of transcranial motor-evoked potentials (tc-MEPs), serum markers, and innovative breath analysis., Methods: In 9 female German Landrace pigs, tc-MEPs were captured, markers of neuronal damage were determined in blood, and volatile organic compounds (VOCs) were analyzed in exhaled air. After thoraco-phrenico-laparotomy, SCI was initiated through sequential clamping (n = 4) or permanently ligating (n = 5) SAs of the abdominal and thoracic aorta in caudocranial orientation until a drop in the tc-MEPs to at least 25% of the baseline was recorded. VOCs in breath were determined by means of solid-phase microextraction coupled with gas chromatography-mass spectrometry. After waking up, clinical and neurological status was evaluated (Tarlov score). Spinal cord histology was obtained in postmortem., Results: Permanent vessel ligature induced a worse neurological outcome and a higher number of necrotic motor neurons compared to clamping. Changes of serum markers remained unspecific. After laparotomy, exhaled acetone and isopropanol showed highest concentrations, and pentane and hexane increased during ischemia-reperfusion injury., Conclusions: To mimic spinal ischemia occurring in humans during aortic aneurysm repair, animal models have to be meticulously evaluated concerning vascular anatomy and function. Volatiles from breath indicated metabolic stress during surgery and oxidative damage through ischemia reperfusion. Breath VOCs may provide complimentary information to conventional monitoring methods., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Proton transfer reaction time-of-flight mass spectrometry (PTR-ToF-MS) represents an attractive tool for the real-time analysis of VOC profiles in human breath. Quantification of breath VOCs by means of direct MS may be affected by the matrix, as human breath not only contains several hundred VOCs at the ppbV-pptV level, but is water saturated and contains percentage levels of CO 2 . Investigation of breath biomarkers in clinical studies requires quantitative and comparable results. We therefore systematically assessed the effect of humidity, CO 2 and O 2 on the results of PTR-MS analysis. We investigated more than 20 VOCs, including aldehydes, ketones, aromatic compounds and hydrocarbons with different sample humidity, CO 2 and O 2 content. The influence of data processing (e.g. normalization to the H 3 O + ion count) was also addressed. An increase of the H 3 O + count of about 20% was observed when the humidity in the sample was increased to breath levels. Large differences regarding the measured VOC intensities were found between the dry and humid samples. Data normalization to the H 3 O + or water-clusters could not fully compensate for the humidity-dependent effects. However, as the determination of most VOCs linearly depends on the humidity over the whole investigated range, factor-based correction seems possible. The effects of CO 2 were more pronounced in the dry samples than in the humid samples but only had a minor influence on the results. The same was true for the influence of O 2 . For the reliable quantification of VOCs in clinical studies and for the standardization of VOC research, well-adapted calibration standards are required for PTR-MS analysis.

Background: Contemporary resuscitation guidelines for basic life support recommend an immediate onset of cardiac compressions in case of cardiac arrest followed by rescue breaths. Effective ventilation is often omitted due to fear of doing harm and fear of infectious diseases. In order to improve ventilation a pre-stage of an automatic respirator was developed for use by laypersons., Methods: Fifty-two healthy volunteers were ventilated by means of a prototype respirator via a full-face mask in a pilot study. The pre-stage public access ventilator (PAV) consisted of a low-cost self-designed turbine, with sensors for differential pressure, flow, FO 2 , FCO 2 and 3-axis acceleration measurement. Sensor outputs were used to control the respirator and to recognize conditions relevant for efficiency of ventilation and patients' safety. Different respiratory manoeuvres were applied: a) pressure controlled ventilation (PCV), b) PCV with controlled leakage and c) PCV with simulated airway occlusion. Sensor signals were analysed to detect leakage and airway occlusion. Detection based upon sensor signals was compared with evaluation based on clinical observation and additional parameters such as exhaled CO 2 ., Results: Pressure controlled ventilation could be realized in all volunteers. Leakage was recognized with 93.5% sensitivity and 93.5% specificity. Simulated airway occlusion was detected with 91.8% sensitivity and 91.7% specificity., Conclusion: The pre-stage PAV was able to detect potential complications relevant for patients' safety such as leakage and airway occlusion in a proof of principle study. Prospectively, this device provides a respectable basis for the development of an automatic emergency respirator and may help to improve bystander resuscitation.

Modern statistical methods which were developed for pattern recognition are increasingly being used for data analysis in studies on emissions of volatile organic compounds (VOCs). With the detection of disease-related VOC profiles, novel non-invasive diagnostic tools could be developed for clinical applications. However, it is important to bear in mind that not all statistical methods are equally suitable for the investigation of VOC profiles. In particular, univariate methods are not able to discover VOC patterns as they consider each compound separately. The present study demonstrates this fact in practice. Using VOC samples from a controlled animal study on paratuberculosis, the random forest classification method was applied for pattern recognition and disease prediction. This strategy was compared with a prediction approach based on single compounds. Both methods were framed within a cross-validation procedure. A comparison of both strategies based on these VOC data reveals that random forests achieves higher sensitivities and specificities than predictions based on single compounds. Therefore, it will most likely be more fruitful to further investigate VOC patterns instead of single biomarkers for paratuberculosis. All methods used are thoroughly explained to aid the transfer to other data analyses.

Respiratory parameters such as flow or rate have complex effects on the exhalation of volatile substances and can hamper clinical interpretation of breath biomarkers. We have investigated the effects of progressively applied upper-airway resistances on the exhalation of volatile organic compounds (VOCs) in healthy humans. We performed real-time mass-spectrometric determination of breath volatiles in 50 subjects with parallel, non-invasive hemodynamic monitoring, breath-resolved spirometry and capnometry during controlled tidal breathing (12 breaths/min). Airway resistance was increased by changing the mouthpiece diameters from 2.5 cm to 1.0 cm and to 0.5 cm. At the smallest diameter, oxygen uptake increased (35%↑). Cardiac output decreased (6%↓) but end-tidal PCO 2 (8%↑) and exhalation of blood-borne isoprene (19%↑) increased. Carbon dioxide production remained constant. Furan, hydrogen sulphide mirrored isoprene. Despite lowered minute ventilation (4%↓) acetone concentrations decreased (3%↓). Exogenous acetonitrile, propionic acid, isopropanol, limonene mimicked acetone. VOC concentration changes could be modelled through substance volatility. Airway resistance-induced changes in hemodynamics, and ventilation can affect VOC exhalation and thereby interfere with breath biomarker interpretation. The effects of collateral ventilation, intra-alveolar pressure gradients and respiratory mechanics had to be considered to explain the exhalation kinetics of CO 2 and VOCs. Conventional breath sampling via smaller mouthpiece diameters (≤1.0 cm, e.g. via straw in Tedlar bags or canisters, etc) will immediately affect VOC exhalation and thereby mislead the analysis of the obtained results. Endogenous isoprene may probe respiratory muscle workload under obstructive conditions. Breath-gas analysis might enhance our understanding of diagnosis and management of obstructive lung diseases in the future.

Monitoring metabolic adaptation to chronic kidney disease (CKD) early in the time course of the disease is challenging. As a non-invasive technique, analysis of exhaled breath profiles is especially attractive in children. Up to now, no reports on breath profiles in this patient cohort are available. 116 pediatric subjects suffering from mild-to-moderate CKD (n = 48) or having a functional renal transplant KTx (n = 8) and healthy controls (n = 60) matched for age and sex were investigated. Non-invasive quantitative analysis of exhaled breath profiles by means of a highly sensitive online mass spectrometric technique (PTR-ToF) was used. CKD stage, the underlying renal disease (HUS; glomerular diseases; abnormalities of kidney and urinary tract or polycystic kidney disease) and the presence of a functional renal transplant were considered as classifiers. Exhaled volatile organic compound (VOC) patterns differed between CKD/ KTx patients and healthy children. Amounts of ammonia, ethanol, isoprene, pentanal and heptanal were higher in patients compared to healthy controls (556, 146, 70.5, 9.3, and 5.4 ppbV vs. 284, 82.4, 49.6, 5.30, and 2.78 ppbV). Methylamine concentrations were lower in the patient group (6.5 vs 10.1 ppbV). These concentration differences were most pronounced in HUS and kidney transplanted patients. When patients were grouped with respect to degree of renal failure these differences could still be detected. Ammonia accumulated already in CKD stage 1, whereas alterations of isoprene (linked to cholesterol metabolism), pentanal and heptanal (linked to oxidative stress) concentrations were detectable in the breath of patients with CKD stage 2 to 4. Only weak associations between serum creatinine and exhaled VOCs were noted. Non-invasive breath testing may help to understand basic mechanisms and metabolic adaptation accompanying progression of CKD. Our results support the current notion that metabolic adaptation occurs early during the time course of CKD.

Breath analysis not only holds great potential for the development of new non-invasive diagnostic methods, but also for the identification and follow up of drug levels in breath. This is of interest for both, forensic and medical science. On the one hand, the detection of drugs of abuse in exhaled breath-similar to the well-known breath alcohol tests-would be highly desirable as an alternative to blood or urine analysis in situations such as police controls for drugged driving. The non-invasive detection of drugs and their metabolites is thus of great interest in forensic science, especially since marijuana is becoming legalized in certain parts of the US and the EU. The detection and monitoring of medical drugs in exhaled breath without the need of drawing blood samples on the other hand, is of high relevance in the clinical environment. This could facilitate a more precise medication and enable therapy control without any burden to the patient. Furthermore, it could be a step towards personalized medicine. This review gives an overview of the current state of drug detection in breath, including both volatile and non-volatile substances. The review is divided into two sections. The first section deals with qualitative detection of drugs (drugs of abuse), while the second is related to quantitative drug detection (medical drugs). Chances and limitations are discussed for both aspects. The detection of the intravenous anesthetic propofol is presented as a detailed example that demonstrates the potential, requirements, pitfalls and limitations of therapeutic drug monitoring by means of breath analysis.

Analysis of exhaled VOCs can provide information on physiology, metabolic processes, oxidative stress and lung diseases. In critically ill patients, VOC analysis may be used to gain complimentary information beyond global clinical parameters. This seems especially attractive in mechanically ventilated patients frequently suffering from impairment of gas exchange. This study was intended to assess (a) the effects of recruitment maneuvers onto VOC profiles, (b) the correlations between electrical impedance tomography (EIT) data and VOC profiles and (c) the effects of recruitment onto distribution of ventilation. Eleven mechanically ventilated patients were investigated during lung recruitment after cardiac surgery. Continuous breath gas analysis by means of PTR-ToF-MS, EIT and blood gas analyses were performed simultaneously. More than 300 mass traces could be detected and monitored continuously by means of PTR-ToF-MS in every patient. Exhaled VOC concentrations varied with recruitment induced changes in minute ventilation and cardiac output. Ammonia exhalation depended on blood pH. The improvement in dorsal lung ventilation during recruitment ranged from 9% to 110%. Correlations between exhaled concentrations of acetone, isoprene, benzene sevoflurane and improvement in regional ventilation during recruitment were observed. Extent and quality of these correlations depended on physico-chemical properties of the VOCs. Combination of continuous real-time breath analysis and EIT revealed correlations between exhaled VOC concentrations and distribution of ventilation. This setup enabled immediate recognition of physiological and therapeutic effects in ICU patients. In a perspective, VOC analysis could be used for non-invasive control and optimization of ventilation strategies.

Mycobacterium avium ssp. paratuberculosis (MAP) causes chronic granulomatous enteritis in ruminants. Bacterial growth is still the diagnostic 'gold standard', but is very time consuming. MAP-specific volatile organic compounds (VOCs) above media could accelerate cultural diagnosis. The aim of this project was to assess the kinetics of a VOC profile linked to the growth of MAP in vitro. The following sources of variability were taken into account: five different culture media, three different MAP strains, inoculation with different bacterial counts, and different periods of incubation. Needle-trap microextraction was employed for pre-concentration of VOCs, and gas chromatography-mass spectrometry for subsequent analysis. All volatiles were identified and calibrated by analysing pure references at different concentration levels. More than 100 VOCs were measured in headspaces above MAP-inoculated and control slants. Results confirmed different VOC profiles above different culture media. Emissions could be assigned to either egg-containing media or synthetic ingredients. 43 VOCs were identified as potential biomarkers of MAP growth on Herrold's Egg Yolk Medium without significant differences between the tree MAP strains. Substances belonged to the classes of alcohols, aldehydes, esters, ketones, aliphatic and aromatic hydrocarbons. With increasing bacterial density the VOC concentrations above MAP expressed different patterns: the majority of substances increased (although a few decreased after reaching a peak), but nine VOCs clearly decreased. Data support the hypotheses that (i) bacteria emit different metabolites on different culture media; (ii) different MAP strains show uniform VOC patterns; and (iii) cultural diagnosis can be accelerated by taking specific VOC profiles into account.

Breath volatile organic compound (VOC) analysis can open a non-invasive window onto pathological and metabolic processes in the body. Decades of clinical breath-gas analysis have revealed that changes in exhaled VOC concentrations are important rather than disease specific biomarkers. As physiological parameters, such as respiratory rate or cardiac output, have profound effects on exhaled VOCs, here we investigated VOC exhalation under respiratory manoeuvres. Breath VOCs were monitored by means of real-time mass-spectrometry during conventional FEV manoeuvres in 50 healthy humans. Simultaneously, we measured respiratory and hemodynamic parameters noninvasively. Tidal volume and minute ventilation increased by 292 and 171% during the manoeuvre. FEV manoeuvre induced substance specific changes in VOC concentrations. pET-CO2 and alveolar isoprene increased by 6 and 21% during maximum exhalation. Then they decreased by 18 and 37% at forced expiration mirroring cardiac output. Acetone concentrations rose by 4.5% despite increasing minute ventilation. Blood-borne furan and dimethyl-sulphide mimicked isoprene profile. Exogenous acetonitrile, sulphides, and most aliphatic and aromatic VOCs changed minimally. Reliable breath tests must avoid forced breathing. As isoprene exhalations mirrored FEV performances, endogenous VOCs might assure quality of lung function tests. Analysis of exhaled VOC concentrations can provide additional information on physiology of respiration and gas exchange.

Background: Sepsis is associated with coagulation abnormalities, and a high content of intravascular tissue factor (TF) may contribute to the development of multisystem organ failure. Circulating microvesicles (MVs) are increased during sepsis and characterized by their phosphatidylserine content. It is unclear whether MVs-as a part of the host response to the infection-are beneficial or rather contribute to systemic complications in sepsis. In the present prospective clinical pilot study, we investigated whether plasma TF and MVs are associated with the risk of multiple organ failure and mortality., Methods: Thirty patients diagnosed with sepsis, severe sepsis, or septic shock were enrolled and classified as 19 survivors and 11 non-survivors. Blood samples were collected on the day of admission and then daily for up to 2 weeks. MVs and TF were quantified in plasma by ELISA., Results: Non-survivors had significantly higher TF concentrations on day 3 compared to survivors. Logistic regression analysis revealed that patients with high amounts of TF had significantly increased risk for severity of disease, according to high Simplified Acute Physiology Score II (SAPS II) scores (odds ratio 18.7). In contrast, a higher content of phosphatidylserine-rich MVs were apparently associated with a lower risk for mortality and multiple organ failure, although this was only a trend and the odds ratios were not significant., Conclusions: This study showed that a high amount of TF in septic patients is significantly associated with increased risk for disease severity, according to a high SAPS II score. Quantification of total MVs in plasma, independent from their cell origin, might be indicative for the outcome of patients in sepsis.

Concentrations of exhaled volatile organic compounds (VOCs) may depend not only on biochemical or pathologic processes but also on physiological parameters. As breath sampling may be done in different body positions, effects of the sampling position on exhaled VOC concentrations were investigated by means of real-time mass spectrometry. Breaths from 15 healthy volunteers were analyzed in real-time by PTR-ToF-MS-8000 during paced breathing (12/min) in a continuous side-stream mode. We applied two series of body positions (setup 1: sitting, standing, supine, and sitting; setup 2: supine, left lateral, right lateral, prone, and supine). Each position was held for 2 min. Breath VOCs were quantified in inspired and alveolar air by means of a custom-made algorithm. Parallel monitoring of hemodynamics and capnometry was performed noninvasively. In setup 1, when compared to the initial sitting position, normalized mean concentrations of isoprene, furan, and acetonitrile decreased by 24%, 26%, and 9%, respectively, during standing and increased by 63%, 36%, and 10% during lying mirroring time profiles of stroke volume and pET-CO2. In contrast, acetone and H2S concentrations remained almost constant. In setup 2, when compared to the initial supine position, mean alveolar concentrations of isoprene and furan increased significantly up to 29% and 16%, respectively, when position was changed from lying on the right side to the prone position. As cardiac output and stroke volume decreased at that time, the reasons for the observed concentrations changes have to be linked to the ventilation/perfusion ratio or compartmental distribution rather than to perfusion alone. During final postures, all VOC concentrations, hemodynamics, and pET-CO2 returned to baseline. Exhaled blood-borne VOC profiles changed due to body postures. Changes depended on cardiac stroke volume, origin, compartmental distribution and physico-chemical properties of the substances. Patients' positions and cardiac output have to be controlled when concentrations of breath VOCs are to be interpreted in terms of biomarkers.

Mycobacterium avium ssp. paratuberculosis (MAP) is the causative agent of a chronic enteric disease of ruminants. Available diagnostic tests are complex and slow. In vitro, volatile organic compound (VOC) patterns emitted from MAP cultures mirrored bacterial growth and enabled distinction of different strains. This study was intended to determine VOCs in vivo in the controlled setting of an animal model. VOCs were pre-concentrated from breath and feces of 42 goats (16 controls and 26 MAP-inoculated animals) by means of needle trap microextraction (breath) and solid phase microextraction (feces) and analyzed by gas chromatography/ mass spectrometry. Analyses were performed 18, 29, 33, 41 and 48 weeks after inoculation. MAP-specific antibodies and MAP-specific interferon-γ-response were determined from blood. Identities of all marker-VOCs were confirmed through analysis of pure reference substances. Based on detection limits in the high pptV and linear ranges of two orders of magnitude more than 100 VOCs could be detected in breath and in headspace over feces. Twenty eight substances differed between inoculated and non-inoculated animals. Although patterns of most prominent substances such as furans, oxygenated substances and hydrocarbons changed in the course of infection, differences between inoculated and non-inoculated animals remained detectable at any time for 16 substances in feces and 3 VOCs in breath. Differences of VOC concentrations over feces reflected presence of MAP bacteria. Differences in VOC profiles from breath were linked to the host response in terms of interferon-γ-response. In a perspective in vivo analysis of VOCs may help to overcome limitations of established tests.

Bulky and hyphenated laboratory-based analytical instrumentation such as gas chromatography/mass spectrometry is still required to trace breath biomarkers in the low ppbV level. Innovative sensor-based technologies could provide on-site and point-of-care (POC) detection of volatile biomarkers such as breath aldehydes related to oxidative stress and cancer. An electrochemical sensor system was developed for direct detection of the total abundance of aldehydes in exhaled breath in the ppbV level and for simultaneous determination of the airway inflammation markers carbon monoxide (CO) and nitric oxide (NO). The sensor system was tested in vitro with gaseous standard mixtures and in vivo in spontaneously breathing patients and under mechanical ventilation in an animal model. The sensor system provided in vitro and in vivo detection of trace levels of aldehydes, CO and NO. Inertness of the tubing system was important for reliable results. Sensitivity of the aldehyde sensor increased with humidity. Response time for analysis of breath samples was about 22 s and relative standard deviations of sensor amplitudes were <5%. Detection limits in the low ppbV range and a linear range of more than two orders of magnitude could be achieved for volatile aldehydes. Cross sensitivities were moderate for alcohols such as ethanol or isopropanol and negligible for other typical breath volatile organic compounds such as acetone, isoprene or propofol. In proof of concept analyses in patients suffering from lung cancer and diabetes, aldehyde and CO sensor signals differed between the groups. Elevated CO levels indicated previous smoking. In a mechanically ventilated pig, continuous monitoring of breath aldehyde concentrations in the low ppbV was realized. Cumulative aldehyde measurements may add interesting and complementary information to the conventional parameters used in clinical breath research. POC applicability, easy handling and low cost of sensors facilitate measurements in large patient cohorts.

A fast detection method to analyze gaseous organic compounds in complex gas mixtures was developed, using a needle trap device (NTD) in conjunction with thermal-desorption photoionization time-of-flight mass spectrometry (TD-PI-TOFMS). The mass spectrometer was coupled via a deactivated fused silica capillary to an injector of a gas chromatograph. In the hot injector, the analytes collected on the NTD were thermally desorbed and directly transferred to the PI-TOFMS ion source. The molecules are softly ionized either by single photon ionization (SPI, 118 nm) or by resonance enhanced multiphoton ionization (REMPI, 266 nm), and the molecular ion signals are detected in the TOF mass analyzer. Analyte desorption and the subsequent PI-TOFMS detection step only lasts ten seconds. The specific selectivity of REMPI (i.e., aromatic compounds) and universal ionization characteristics render PI-MS as a promising detection system. As a first demonstrative application, the alveolar phase breath gas of healthy, nonsmoking subjects was sampled on NTDs. While smaller organic compounds such as acetone, acetaldehyde, isoprene, or cysteamine can be detected in the breath gas with SPI, REMPI depicts the aromatic substances phenol and indole at 266 nm. In the breath gas of a healthy, smoking male subject, several xenobiotic substances such as benzene, toluene, styrene, and ethylbenzene can be found as well. Furthermore, the NTD-REMPI-TOFMS setup was tested for breath gas taken from a mechanically ventilated pig under continuous intravenous propofol (2,6-diisopropylphenol, narcotic drug) infusion.