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The Peppermint Initiative seeks to inform the standardisation of breath analysis methods. Five Peppermint Experiments with gas chromatography-ion mobility spectrometry (GC-IMS), operating in the positive mode with a tritium 3 H 5.68 keV, 370 MBq ionisation source, were undertaken to provide benchmark Peppermint Washout data for this technique, to support its use in breath-testing, analysis, and research. Headspace analysis of a peppermint-oil capsule by GC-IMS with on-column injection (0.5 cm 3 ) identified 12 IMS responsive compounds, of which the four most abundant were: eucalyptol; β -pinene; α -pinene; and limonene. Elevated concentrations of these four compounds were identified in exhaled-breath following ingestion of a peppermint-oil capsule. An unidentified compound attributed as a volatile catabolite of peppermint-oil was also observed. The most intense exhaled peppermint-oil component was eucalyptol, which was selected as a peppermint marker for benchmarking GC-IMS. Twenty-five washout experiments monitored levels of exhaled eucalyptol, by GC-IMS with on-column injection (0.5 cm 3 ), at t = 0 min, and then at t + 60, t + 90, t + 165, t + 285 and t + 360 min from ingestion of a peppermint capsule resulting in 148 peppermint breath analyses. Additionally, the Peppermint Washout data was used to evaluate clinical deployments with a further five washout tests run in clinical settings generating an additional 35 breath samples. Regression analysis yielded an average extrapolated time taken for exhaled eucalyptol levels to return to baseline values to be 429 ± 62 min (±95% confidence-interval). The benchmark value was assigned to the lower 95% confidence-interval, 367 min. Further evaluation of the data indicated that the maximum number of volatile organic compounds discernible from a 0.5 cm 3 breath sample was 69, while the use of an in-line biofilter appeared to reduce this to 34., (Creative Commons Attribution license.)

Sampling of volatile organic compounds (VOCs) has shown promise for detection of a range of diseases but results have proved hard to replicate due to a lack of standardization. In this work we introduce the 'Peppermint Initiative'. The initiative seeks to disseminate a standardized experiment that allows comparison of breath sampling and data analysis methods. Further, it seeks to share a set of benchmark values for the measurement of VOCs in breath. Pilot data are presented to illustrate the standardized approach to the interpretation of results obtained from the Peppermint experiment. This pilot study was conducted to determine the washout profile of peppermint compounds in breath, identify appropriate sampling time points, and formalise the data analysis. Five and ten participants were recruited to undertake a standardized intervention by ingesting a peppermint oil capsule that engenders a predictable and controlled change in the VOC profile in exhaled breath. After collecting a pre-ingestion breath sample, five further samples are taken at 2, 4, 6, 8, and 10 h after ingestion. Samples were analysed using ion mobility spectrometry coupled to multi-capillary column and thermal desorption gas chromatography mass spectrometry. A regression analysis of the washout data was used to determine sampling times for the final peppermint protocol, and the time for the compound measurement to return to baseline levels was selected as a benchmark value. A measure of the quality of the data generated from a given technique is proposed by comparing data fidelity. This study protocol has been used for all subsequent measurements by the Peppermint Consortium (16 partners from seven countries). So far 1200 breath samples from 200 participants using a range of sampling and analytical techniques have been collected. The data from the consortium will be disseminated in subsequent technical notes focussing on results from individual platforms.

The production of volatile organic compounds (VOCs) during programmed cell death (PCD) is still insufficiently studied and their implication in the process is not well understood. The present study demonstrates that the release of VOSCs with presumed antioxidant capacity (methanethiol, dimethylsulfide and dimethyldisulfide) accompanies the cell death in chemical-stressed tobacco BY-2 suspension cultured cells. The cells were exposed to cell death inducers of biotic nature mastoparan (MP, wasp venom) and camptothecin (CPT, alkaloid), and to the abiotic stress agent CdSO 4 . The VOCs emission was monitored by proton-transfer reaction mass spectrometry (PTR-MS). The three chemicals induced PCD expressing apoptotic-like phenotype. The identified VOSCs were emitted in response to MP and CPT but not in presence of Cd. The VOSCs production occurred within few hours after the administration of the elicitors, peaked up when 20-50 % of the cells were dead and further levelled off with cell death advancement. This suggests that VOSCs with antioxidant activity may contribute to alleviation of cell death-associated oxidative stress at medium severity of cell death in response to the stress factors of biotic origin. The findings provide novel information about cell death defence mechanisms in chemical-challenged BY-2 cells and show that PCD related VOSCs synthesis depends on the type of inducer., Competing Interests: Declaration of Competing Interest There are no commercial links or other connections for each of the authors that could pose a conflict of interest in any way., (Copyright © 2020. Published by Elsevier GmbH.)

With the growing interest in the use of breath volatiles in the health sciences, the lack of standardization for the sampling and analysis of exhaled breath is becoming a major issue leading to an absence of conformity, reproducibility and reliability in spectrometric measurements. Through the creation of a worldwide 'peppermint consortium', the International Association of Breath Research has set up a task force to deal with this problem. Pharmacokinetic studies are proposed, and a real-time analytical technique that is being used is proton transfer reaction-time-of-flight-mass spectrometry (PTR-ToF-MS). This paper presents details on how the volatile compounds contained in a peppermint oil capsule, and hence on breath, appear in a PTR-ToF-MS. To aid that study, the key volatiles in the headspace of peppermint oil were first identified using gas chromatography-mass spectrometry, notably: menthol, menthone, 1,8-cineole, menthofuran, limonene, α-pinene and β-pinene. A PTR-ToF-MS analysis of these compounds has been undertaken, divorced from the complexity of the peppermint oil matrix using 'normal' and 'saturated' humidity drift-tube conditions, with the latter used to mimic breath samples, and over a range of reduced electric fields. There are no characteristic product ions that can distinguish monoterpenes and 1,8-cineole, and hence, without pre-separation, a combined washout for these volatiles can only be provided. By operating the drift tube above about 130 Td, there are characteristic product ions for menthone, menthofuran and menthol, namely m/z 155.14 (protonated menthone), m/z 151.11 (protonated menthofuran), m/z 139.15 (loss of H 2 O from protonated menthol) and m/z 83.09 (a fragment ion, C 6 H 11 + , from menthol). These have been used to monitor, with a high specificity, the temporal profile of these three compounds in breath following the ingestion of a peppermint oil capsule. To aid in the analyses, the proton affinities and gas-phase basicities for the key volatiles investigated have been determined using density functional theory.

Herbivore-induced plant volatiles (HIPVs) serve as specific cues to higher trophic levels. Novel, exotic herbivores entering native foodwebs may disrupt the infochemical network as a result of changes in HIPV profiles. Here, we analysed HIPV blends of native Brassica rapa plants infested with one of 10 herbivore species with different coexistence histories, diet breadths and feeding modes. Partial least squares (PLS) models were fitted to assess whether HIPV blends emitted by Dutch B. rapa differ between native and exotic herbivores, between specialists and generalists, and between piercing-sucking and chewing herbivores. These models were used to predict the status of two additional herbivores. We found that HIPV blends predicted the evolutionary history, diet breadth and feeding mode of the herbivore with an accuracy of 80% or higher. Based on the HIPVs, the PLS models reliably predicted that Trichoplusia ni and Spodoptera exigua are perceived as exotic, leaf-chewing generalists by Dutch B. rapa plants. These results indicate that there are consistent and predictable differences in HIPV blends depending on global herbivore characteristics, including coexistence history. Consequently, native organisms may be able to rapidly adapt to potentially disruptive effects of exotic herbivores on the infochemical network., (© 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.)

Volatile organic compounds emitted during the infection of apple (Malus pumila var. domestica) plants by Erwinia amylovora or Pseudomonas syringae pv. syringae were studied by gas chromatography-mass spectrometry and proton transfer reaction-mass spectrometry, and used to treat uninfected plants. Infected plants showed a disease-specific emission of volatile organic compounds, including several bio-active compounds, such as hexenal isomers and 2,3-butanediol. Leaf growth promotion and a higher resistance to the pathogen, expressed as a lower bacterial growth and migration in plant tissues, were detected in plants exposed to volatile compounds from E. amylovora-infected plants. Transcriptional analysis revealed the activation of salicylic acid synthesis and signal transduction in healthy plants exposed to volatiles produced by E. amylovora-infected neighbour plants. In contrast, in the same plants, salicylic acid-dependent responses were repressed after infection, whereas oxylipin metabolism was activated. These results clarify some metabolic and ecological aspects of the pathogenic adaptation of E. amylovora to its host., (© 2016 BSPP AND JOHN WILEY & SONS LTD.)

Studies on aboveground (AG) plant organs have shown that volatile organic compound (VOC) emissions differ between simultaneous attack by herbivores and single herbivore attack. There is growing evidence that interactive effects of simultaneous herbivory also occur across the root-shoot interface. In our study, Brassica rapa roots were infested with root fly larvae (Delia radicum) and the shoots infested with Pieris brassicae, either singly or simultaneously, to study these root-shoot interactions. As an analytical platform, we used Proton Transfer Reaction Mass Spectrometry (PTR-MS) to investigate VOCs over a 3 day time period. Our set-up allowed us to monitor root and shoot emissions concurrently on the same plant. Focus was placed on the sulfur-containing compounds; methanethiol, dimethylsulfide (DMS), and dimethyldisulfide (DMDS), because these compounds previously have been shown to be biologically active in the interactions of Brassica plants, herbivores, parasitoids, and predators, yet have received relatively little attention. The shoots of plants simultaneously infested with AG and belowground (BG) herbivores emitted higher levels of sulfur-containing compounds than plants with a single herbivore species present. In contrast, the emission of sulfur VOCs from the plant roots increased as a consequence of root herbivory, independent of the presence of an AG herbivore. The onset of root emissions was more rapid after damage than the onset of shoot emissions. The shoots of double infested plants also emitted higher levels of methanol. Thus, interactive effects of root and shoot herbivores exhibit more strongly in the VOC emissions from the shoots than from the roots, implying the involvement of specific signaling interactions.

Insect herbivores trigger various biochemical changes in plants, and as a consequence, affect other organisms that are associated with these plants. Such plant-mediated indirect effects often involve herbivore-induced plant volatiles (HIPVs) that can be used as cues for foraging herbivores and their natural enemies, and are also known to affect pollinator attraction. In tightly co-evolved systems, the different trophic levels are expected to display adaptive response to changes in HIPVs caused by native herbivores. But what if a new herbivore invades such a system? Current literature suggests that exotic herbivores have the potential to affect HIPV production, and that plant responses to novel herbivores are likely to depend on phylogenetic relatedness between the invader and the native species. Here we review the different ways exotic herbivores can disrupt chemically mediated interactions between plants and the key users of HIPVs: herbivores, pollinators, and members of the third (i.e. predators and parasitoids) and fourth (i.e. hyperparasitoids) trophic levels. Current theory on insect invasions needs to consider that disruptive effects of invaders on infochemical networks can have a short-term impact on the population dynamics of native insects and plants, as well as exerting potentially negative consequences for the functioning of native ecosystems., (© 2014 John Wiley & Sons Ltd.)

Ralstonia solanacearum (Rs) and Clavibacter michiganensis subsp. sepedonicus (Cms) are the bacterial causal agents of potato brown and ring rot, respectively, and are included in the A2 list of quarantine pathogens in Europe. Identification by GC-MS analysis of volatile organic compounds from Rs or Cms cultured on different nutrient media was performed. GC-MS and PTR-MS analysis were carried out also on unwounded potato tubers infected with the same pathogens. Infected tubers were produced by experimental inoculations of the plants. In in vitro experiments, Rs or Cms emitted volatile compounds, part of which were specific disease markers of potato (2-propanol and 3-methylbutanoic acid), mainly originating from bacterial metabolism (i.e., amino acid degradation, carbohydrate and fatty acid oxidation). In potato tubers, pathogen metabolism modified the volatile compound pattern emitted from healthy samples. Both bacteria seem to accelerate metabolic processes ongoing in potatoes and, in the case of Rs, disease markers (1-hepten-3-ol, 3,6-dimethyl-3-octanone, 3-ethyl-3-methylpentane, 1-chloroctane, and benzothiazole) were identified.

Plants emit various volatile organic compounds (VOCs) upon herbivore attack. These VOC emissions often show temporal dynamics which may influence the behavior of natural enemies using these volatiles as cues. This study analyzes on-line VOC emissions by roots of Brassica nigra plants under attack by cabbage root fly larvae, Delia radicum. Root emitted VOCs were detected using Proton-Transfer-Reaction Mass Spectrometry (PTR-MS) and Gas Chromatography-Mass Spectrometry (GC-MS). These analyses showed that several sulfur containing compounds, such as methanethiol, dimethyl sulfide (DMS), dimethyl disulfide (DMDS), dimethyl trisulfide (DMTS) and glucosinolate breakdown products, such as thiocyanates (TC) and isothiocyanates (ITC), were emitted by the roots in response to infestation. The emissions were subdivided into early responses, emerging within 1-6 h after infestation, and late responses, evolving only after 6-12 h. The marker for rapid responses was detected at m/z 60. The ion detected at m/z 60 was identified as thiocyanic acid, which is also a prominent fragment in some TC or ITC spectra. The emission of m/z 60 stopped when the larvae had pupated, which makes it an excellent indicator for actively feeding larvae. Methanethiol, DMS and DMDS levels increased much later in infested roots, indicating that activation of enzymes or genes involved in the production of these compounds may be required. Earlier studies have shown that both early and late responses can play a role in tritrophic interactions associated with Brassica species. Moreover, the identification of these root induced responses will help to design non-invasive analytical procedures to assess root infestations., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Root herbivores are notoriously difficult to study, as they feed hidden in the soil. However, root herbivores may be traced by analyzing specific volatile organic compounds (VOCs) that are produced by damaged roots. These VOCs not only support parasitoids in the localization of their host, but also may help scientists study belowground plant-herbivore interactions. Herbivore-induced VOCs are usually analyzed by gas-chromatography mass spectrometry (GC-MS), but with this off-line method, the gases of interest need to be preconcentrated, and destructive sampling is required to assess the level of damage to the roots. In contrast to this, proton-transfer-reaction mass spectrometry (PTR-MS) is a very sensitive on-line, non-invasive method. PTR-MS already has been successfully applied to analyze VOCs produced by aboveground (infested) plant parts. In this review, we provide a brief overview of PTR-MS and illustrate how this technology can be applied to detect specific root-herbivore induced VOCs from Brassica plants. We also specify the advantages and disadvantages of PTR-MS analyses and new technological developments to overcome their limitations.

The availability of rapid and accurate methods to assess fruit flavor is of utmost importance to support quality control especially in the breeding phase. Breeders need more information and analytical tools to facilitate selection for complex multigenic traits such as flavor quality. In this study, it is shown that proton-transfer reaction mass spectrometry (PTR-MS) is a suitable method to monitor at high sensitivity the emission of volatiles determining the tomato aromatic profile such as hexanal, hexenals, methanol, ethanol, and acetaldehyde. The volatiles emitted by 14 tomato varieties (at red stage) were analyzed by 2 solvent-free headspace methods: solid-phase microextraction/gas chromatography MS and PTR-MS. Multivariate statistics (principal component analysis and cluster analysis) of the PTR-MS results allow an unambiguous separation between varieties, especially with a clear fingerprinting separation between the different tomato types: round truss, cocktail, and cherry tomatoes. PTR-MS was also successfully used to monitor the changes in volatile profiles during postharvest ripening and storage., (© 2012 Institute of Food Technologists®)

Rationale: When performing trace gas analysis, it is not always possible to bring the source of volatiles and the gas analyzer together. In these cases, volatile storage containers, such as thermal desorption (TD) tubes, can be used for off-line measurement. TD is routinely combined with gas chromatography/mass spectrometry (GC/MS), but so far not with proton-transfer reaction mass spectrometry (PTRMS), which has a faster response., Methods: A PTR-quadrupole-MS instrument and a PTR-ion-trap-MS instrument were separately coupled to a TD unit for off-line analysis of trace volatiles in air. Carbograph 1TD/Carbopack X sorbent tubes were filled with different concentrations of a trace gas mixture containing low molecular weight volatiles (32 g/mol up to 136 g/mol) and measured with the above-mentioned combinations. The carrier gas in the TD unit was changed from helium to nitrogen to be able to combine this instrument with the mass spectrometer., Results: Good linearity and reproducibility with the amount of gas stored were obtained. The storage capacity over time (up to 14 days) showed larger variability (<11% for all compounds, except for acetone 27%). Several tubes were filled with breath of different persons, and the breath of a smoker showed increased levels of acetonitrile and benzene. The combination of the PTR ion-trap instrument with the TD unit was also investigated. Due to its higher sampling rate, the ion-trap system showed higher throughput capabilities than the quadrupole system., Conclusions: The combination of TD with PTRMS using both a quadrupole and an ion trap for off-line volatile analysis has been validated. TD tubes can be a robust and compact volatile storage method when the mass spectrometry and the sampling cannot be performed in the same place, for example in large screening studies. In addition, a higher measurement throughput than with GC/MS could be obtained., (Copyright © 2012 John Wiley & Sons, Ltd.)

Bacteria emit a wealth of volatiles. The combination of coupled gas chromatography/mass spectrometry (GC/MS) and proton-transfer-reaction mass spectrometry (PTR-MS) analyses provided a most comprehensive profile of volatiles of the rhizobacterium Serratia odorifera 4Rx13. An array of compounds, highly dominated by sodorifen (approximately 50%), a bicyclic oligomethyl octadiene, could be detected. Other volatiles included components of the biogeochemical sulfur cycle such as dimethyl disulfide (DMDS), dimethyl trisulfide and methanethiol, terpenoids, 2-phenylethanol, and other aromatic compounds. The composition of the bouquet of S. odorifera did not change significantly during the different growth intervals. At the beginning of the stationary phase, 60 μg of volatiles per 24 h and 60 easily detectable components were released. Ammonia was also released by S. odorifera, while ethylene, nitric oxide (NO) and hydrogen cyanide (HCN) could not be detected. Dual culture assays proved that 20 μmol DMDS and 2.5 μmol ammonia, individually applied, represent the IC(50) concentrations that cause negative effects on Arabidopsis thaliana.

The aim of this study was to investigate volatile organic compounds (VOCs) in exhaled breath as possible non-invasive markers to monitor the inflammatory response in inflammatory bowel disease (IBD) patients as a result of repeated and prolonged moderate-intensity exercise. We included 18 IBD patients and 19 non-IBD individuals who each completed a 30, 40, or 50 km walking exercise over three consecutive days. Breath and blood samples were taken before the start of the exercise event and every day post-exercise to assess changes in the VOC profiles and cytokine concentrations. Proton transfer reaction time-of-flight mass spectrometry (PTR-ToF-MS) was used to measure exhaled breath VOCs. Multivariate analysis, particularly ANOVA-simultaneous component analysis (ASCA), was employed to extract relevant ions related to exercise and IBD. Prolonged exercise induces a similar response in breath butanoic acid and plasma cytokines for participants with or without IBD. Butanoic acid showed a significant correlation with the cytokine IL-6, indicating that butanoic acid could be a potential non-invasive marker for exercise-induced inflammation. The findings are relevant in monitoring personalized IBD management. [ABSTRACT FROM AUTHOR]

Volatile organic compounds (VOCs) in exhaled breath provide insights into various metabolic processes and can be used to monitor physiological response to exercise and medication. We integrated and validated in situ a sampling and analysis protocol using proton transfer reaction time-of-flight mass spectrometry (PTR-ToF-MS) for exhaled breath research. The approach was demonstrated on a participant cohort comprising users of the cholesterol-lowering drug statins and non-statin users during a field campaign of three days of prolonged and repeated exercise, with no restrictions on food or drink consumption. The effect of prolonged exercise was reflected in the exhaled breath of participants, and relevant VOCs were identified. Most of the VOCs, such as acetone, showed an increase in concentration after the first day of walking and subsequent decrease towards baseline levels prior to walking on the second day. A cluster of short-chain fatty acids including acetic acid, butanoic acid, and propionic acid were identified in exhaled breath as potential indicators of gut microbiota activity relating to exercise and drug use. We have provided novel information regarding the use of breathomics for non-invasive monitoring of changes in human metabolism and especially for the gut microbiome activity in relation to exercise and the use of medication, such as statins. [ABSTRACT FROM AUTHOR]

Abstract: The aim of this study was to develop an analytical system to study the tomato aroma profile. An artificial chewing device coupled to a PTR-MS was developed to mimic, as close as possible, the release of volatiles during chewing in the human mouth and the retronasal olfaction perception. VOC profiles of 9 tomato lines, selected based on flavor characteristics by a sensory panel, were acquired by both a PTR-MS system following artificial chewing and by SPME–GC–MS and compared to the quantitative descriptive analysis (QDA) measured by the trained sensory panel. Based on multivariate statistical analysis, data obtained by the PTR-MS system showed a better correlation to the outcome of the QDA than SPME–GC–MS, especially for the descriptive parameters “tomato fragrance” and “tomato flavor”. The great advantage of such an analytical system was the possibility to study the release kinetics of volatiles during eating and the possibility to consider volatile concentration similar to in vivo condition resulting to an improved characterization of the aroma profile. [Copyright &y& Elsevier]

Abstract: Proton-transfer reaction mass spectrometry (PTR-MS) was used to study ultraviolet (UV) light-induced lipid peroxidation in human skin, in vivo. Emissions of volatile organic compounds (VOCs) in the mass range between 20 and 150amu in the headspace of the skin of 16 healthy volunteers were monitored before, during and after irradiation in an on-line and non-invasive fashion. From these experiments, five volatile substances were found to reflect the damage caused by UV-radiation. The two major compounds (monitored at mass 45 and 59amu) were identified as acetaldehyde and propanal using a combination of Tenax-based gas chromatographic pre-separation with PTR-MS. The other volatiles (with characteristic ions at, among others, masses 73 and 87amu) could not be identified. Simultaneous measurement of the established lipid peroxidation biomarker ethene using laser-based photoacoustic trace gas detection revealed a similar pattern and statistically significant correlations between VOC production measured with PTR-MS and ethene. Variations in UV-radiation intensity were reflected by the amount of acetaldehyde and propanal emitted from the skin. Our results show that acetaldehyde and propanal can be used as biomarkers for lipid peroxidation. [Copyright &y& Elsevier]