Unravelling the origin of isoprene in the human body - a forty year odyssey.

In the breath research community's search for volatile organic compounds that can act as non-invasive biomarkers for various diseases, hundreds of endogenous volatiles have been discovered. Whilst these systemic chemicals result from normal and abnormal metabolic activities or pathological disorders, to date very few are of any use for the development of clinical breath tests that could be used for disease diagnosis or to monitor therapeutic treatments. The reasons for this lack of application are manifold and complex, and these complications either limit or ultimately inhibit the analytical application of endogenous volatiles for use in the medical sciences. One such complication is a lack of knowledge on the biological origins of the endogenous volatiles. A major exception to this is isoprene. Since 1984, i.e., for forty years, it has been generally accepted that the pathway to the production of human isoprene, and hence the origin of isoprene in exhaled breath, is through cholesterol biosynthesis via the mevalonate (MVA) pathway within the liver. However, various studies between 2001 and 2012 provide compelling evidence that human isoprene is produced in skeletal muscle tissue. A recent multi-omic investigation of genes and metabolites has revealed that this proposal is correct by showing that human isoprene predominantly results from muscular lipolytic cholesterol metabolism. Despite the overwhelming proof for a muscular pathway to isoprene production in the human body, breath research papers still reference the hepatic MVA pathway. The major aim of this perspective is review the evidence that leads to a correct interpretation for the origins of human isoprene, so that the major pathway to human isoprene production is understood and appropriately disseminated. This is important, because an accurate attribution to the endogenous origins of isoprene is needed if exhaled isoprene levels are to be correctly interpreted and for assessing isoprene as a clinical biomarker.

Volatilomic signatures of different strains of Helicobacter pylori.

BACKGROUND: Helicobacter pylori (H. pylori) infection is the most extensively studied risk factor for gastric cancer. As with any bacteria, H. pylori will release distinctive odors that result from an emission of volatile metabolic byproducts in unique combinations and proportions. Effectively capturing and identifying these volatiles can pave the way for the development of innovative and non-invasive diagnostic methods for determining infection. Here we characterize the H. pylori volatilomic signature, pinpoint potential biomarkers of its presence, and evaluate the variability of volatilomic signatures between different H. pylori isolates. MATERIALS AND METHODS: Using needle trap extraction, volatiles in the headspace above H. pylori cultures were collected and, following thermal desorption at 290°C in a splitless mode, were analyzed using gas chromatography-mass spectrometry. The resulting volatilomic signatures of H. pylori cultures were compared to those obtained from an analysis of the volatiles in the headspace above the cultivating medium only. RESULTS: Amongst the volatiles detected, 21 showed consistent differences between the bacteria cultures and the cultivation medium, with 11 compounds being elevated and 10 showing decreased levels in the culture's headspace. The 11 elevated volatiles are four ketones (2-pentanone, 5-methyl-3-heptanone, 2-heptanone, and 2-nonanone), three alcohols (2-methyl-1-propanol, 3-methyl-1-butanol, and 1 butanol), one aromatic (styrene), one aldehyde (2-ethyl-hexanal), one hydrocarbon (n-octane), and one sulfur compound (dimethyl disulfide). The 10 volatiles with lower levels in the headspace of the cultures are four aldehydes (2-methylpropanal, benzaldehyde, 3-methylbutanal, and butanal), two heterocyclic compounds (2-ethylfuran and 2-pentylfuran), one ketone (2-butanone), one aromatic (benzene), one alcohol (2-butanol) and bromodichloromethane. Of the volatile species showing increased levels, the highest emissions are found to be for 3-methyl-1-butanol, 1-butanol and dimethyl disulfide. Qualitative variations in their emissions from the different isolates was observed. CONCLUSIONS: The volatiles emitted by H. pylori provide a characteristic volatilome signature that has the potential of being developed as a tool for monitoring infections caused by this pathogen. Furthermore, using the volatilome signature, we are able to differentiate different isolates of H. pylori. However, the volatiles also represent potential confounders for the recognition of gastric cancer volatile markers.

Volatilomic profiles of gastric juice in gastric cancer patients.

Volatilomics is a powerful tool capable of providing novel biomarkers for the diagnosis of gastric cancer. The main objective of this study was to characterize the volatilomic signatures of gastric juice in order to identify potential alterations induced by gastric cancer. Gas chromatography with mass spectrometric detection, coupled with headspace solid phase microextraction as the pre-concentration technique, was used to identify volatile organic compounds (VOCs) released by gastric juice samples collected from 78 gastric cancer patients and two cohorts of controls (80 and 96 subjects) from four different locations (Latvia, Ukraine, Brazil, and Colombia). 1440 distinct compounds were identified in samples obtained from patients and 1422 in samples provided by controls. However, only 6% of the VOCs exhibited an incidence higher than 20%. Amongst the volatiles emitted, 18 showed differences in their headspace concentrations above gastric juice of cancer patients and controls. Ten of these (1-propanol, 2,3-butanedione, 2-pentanone, benzeneacetaldehyde, 3-methylbutanal, butylated hydroxytoluene, 2-pentyl-furan, 2-ethylhexanal, 2-methylpropanal and phenol) appeared at significantly higher levels in the headspace of the gastric juice samples obtained from patients; whereas, eight species showed lower abundance in patients than found in controls. Given that the difference in the volatilomic signatures can be explained by cancer-related changes in the activity of certain enzymes or pathways, the former set can be considered potential biomarkers for gastric cancer, which may assist in developing non-invasive breath tests for the diagnosis of this disease. Further studies are required to elucidate further the mechanisms that underlie the changes in the volatilomic profile as a result of gastric cancer.

Revisiting the rationale of mandatory masking.

In this perspective, we review the evidence for the efficacy of face masks to reduce the transmission of respiratory viruses, specifically severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and consider the value of mandating universal mask wearing against the widespread negative impacts that have been associated with such measures. Before the SARS-CoV-2 pandemic, it was considered that there was little to no benefit in healthy people wearing masks as prophylaxis against becoming infected or as unwitting vectors of viral transmission. This accepted policy was hastily reversed early on in the pandemic, when districts and countries throughout the world imposed stringent masking mandates. Now, more than three years since the start of the pandemic, the amassed studies that have investigated the use of masks to reduce transmission of SARS-CoV-2 (or other pathogens) have led to conclusions that are largely inconsistent and contradictory. There is no statistically significant or unambiguous scientific evidence to justify mandatory masking for general, healthy populations with the intention of lessening the viral spread. Even if mask wearing could potentially reduce the transmission of SARS-CoV-2 in individual cases, this needs to be balanced against the physical, psychological and social harms associated with forced mask wearing, not to mention the negative impact of innumerable disposed masks entering our fragile environment. Given the lack of unequivocal scientific proof that masks have any effect on reducing transmission, together with the evident harms to people and the environment through the use of masks, it is our opinion that the mandatory use of face masks in the general population is unjustifiable and must be abandoned in future pandemic countermeasures policies.

Establishing a cell-based screening workflow for determining the efficiency of CYP2C9 metabolism: moving towards the use of breath volatiles in personalised medicine.

The use of volatile biomarkers in exhaled breath as predictors to individual drug response would advance the field of personalised medicine by providing direct information on enzyme activity. This would result in enormous benefits, both for patients and for the healthcare sector. Non-invasive breath tests would also gain a high acceptance by patients. Towards this goal, differences in metabolism resulting from extensive polymorphisms in a major group of drug-metabolizing enzymes, the cytochrome P450 (CYP) family, need to be determined and quantified. CYP2C9 is responsible for metabolising many crucial drugs (e.g., diclofenac) and food ingredients (e.g., limonene). In this paper, we provide a proof-of-concept study that illustrates thein vitrobioconversion of diclofenac in recombinant HEK293T cells overexpressing CYP2C9 to 4'-hydroxydiclofenac. Thisin vitroapproach is a necessary and important first step in the development of breath tests to determine and monitor metabolic processes in the human body. By focusing on the metabolic conversion of diclofenac, we have been able to establish a workflow using a cell-based system for CYP2C9 activity. Furthermore, we illustrate how the bioconversion of diclofenac is limited in the presence of limonene, which is another CYP2C9 metabolising substrate. We show that increasing limonene levels continuously reduce the production of 4'-hydroxydiclofenac. Michaelis-Menten kinetics were performed for the diclofenac 4'-hydroxylation with and without limonene, giving a kinetic constant of the reaction,KM, of 103µM and 94.1µM, respectively, and a maximum reaction rate,Vmax, of 46.8 pmol min-1106cells-1and 56.0 pmol min-1106cells-1with and without the inhibitor, respectively, suggesting a non-competitive or mixed inhibition type. The half-maximal inhibitory concentration value (IC50) for the inhibition of the formation of 4'-hydroxydiclofenace by limonene is determined to be 1413µM.

Dynamic Breath Limonene Sensing at High Selectivity.

Liver diseases (e.g., cirrhosis, cancer) cause more than two million deaths per year worldwide. This is partly attributed to late diagnosis and insufficient screening techniques. A promising biomarker for noninvasive and inexpensive liver disease screening is breath limonene that can indicate a deficiency of the cytochrome P450 liver enzymes. Here, we introduce a compact and low-cost detector for dynamic and selective breath limonene sensing. It comprises a chemoresistive sensor based on Si/WO3 nanoparticles pre-screened by a packed bed Tenax separation column at room temperature. We demonstrate selective limonene detection down to 20 parts per billion over up to three orders of magnitude higher concentrated acetone, ethanol, hydrogen, methanol, and 2-propanol in gas mixtures, as well as robustness to 10-90% relative humidity. Most importantly, this detector recognizes the individual breath limonene dynamics of four healthy volunteers following the ingestion (swallowing or chewing) of a limonene capsule. Limonene release and subsequent metabolization are monitored from breath measurements in real time and in excellent agreement (R2 = 0.98) with high-resolution proton transfer reaction mass spectrometry. This study demonstrates the potential of the detector as a simple-to-use and noninvasive device for the routine monitoring of limonene levels in exhaled breath to facilitate early diagnosis of liver dysfunction.

Detecting Hexafluoroisopropanol Using Soft Chemical Ionization Mass Spectrometry and Analytical Applications to Exhaled Breath.

Here we explore the potential use of proton transfer reaction/selective reagent ion-time-of-flight-mass spectrometry (PTR/SRI-ToF-MS) to monitor hexafluoroisopropanol (HFIP) in breath. Investigations of the reagent ions H3O+, NO+, and O2+• are reported using dry (relative humidity (rH) ≈ 0%) and humid (rH ≈ 100%)) nitrogen gas containing traces of HFIP, i.e., divorced from the complex chemical environment of exhaled breath. HFIP shows no observable reaction with H3O+ and NO+, but it does react efficiently with O2+• via dissociative charge transfer resulting in CHF2+, CF3+, C2HF2O+, and C2H2F3O+. A minor competing hydride abstraction channel results in C3HF6O+ + HO2• and, following an elimination of HF, C3F5O+. There are two issues associated with the use of the three dominant product ions of HFIP, CHF2+, CF3+, and C2H2F3O+, to monitor it in breath. One is that CHF2+ and CF3+ also result from the reaction of O2+• with the more abundant sevoflurane. The second is the facile reaction of these product ions with water, which reduces analytical sensitivity to detect HFIP in humid breath. To overcome the first issue, C2H2F3O+ is the ion marker for HFIP. The second issue is surmounted by using a Nafion tube to reduce the breath sample's humidity prior to its introduction into drift tube. The success of this approach is illustrated by comparing the product ion signals either in dry or humid nitrogen gas flows and with or without the use of the Nafion tube, and practically from the analysis of a postoperative exhaled breath sample from a patient volunteer.

Identification of Key Volatile Organic Compounds Released by Gastric Tissues as Potential Non-Invasive Biomarkers for Gastric Cancer.

BACKGROUND: Volatilomics is a powerful tool capable of providing novel biomarkers for medical diagnosis and therapy monitoring. The objective of this study is to identify potential volatile biomarkers of gastric cancer. METHODS: The volatilomic signatures of gastric tissues obtained from two distinct populations were investigated using gas chromatography with mass spectrometric detection. RESULTS: Amongst the volatiles emitted, nineteen showed differences in their headspace concentrations above the normal and cancer tissues in at least one population of patients. Headspace levels of seven compounds (hexanal, nonanal, cyclohexanone, 2-nonanone, pyrrole, pyridine, and phenol) were significantly higher above the cancer tissue, whereas eleven volatiles (ethyl acetate, acetoin, 2,3-butanedione, 3-methyl-1-butanol, 2-pentanone, γ-butyrolactone, DL-limonene, benzaldehyde, 2-methyl-1-propanol, benzonitrile, and 3-methyl-butanal) were higher above the non-cancerous tissue. One compound, isoprene, exhibited contradictory alterations in both cohorts. Five compounds, pyridine, ethyl acetate, acetoin, 2,3-butanedione, and 3-methyl-1-butanol, showed consistent cancer-related changes in both populations. CONCLUSIONS: Pyridine is found to be the most promising biomarker candidate for detecting gastric cancer. The difference in the volatilomic signatures can be explained by cancer-related changes in the activity of certain enzymes, or pathways. The results of this study confirm that the chemical fingerprint formed by volatiles in gastric tissue is altered by gastric cancer.

Responses of Adult Hypera rumicis L. to Synthetic Plant Volatile Blends.

The behavioral responses of Hypera rumicis L. adults to varying blends of synthetic plant volatiles (SPVs) at various concentrations in lieu of single compounds are reported for the first time. For this study, Rumex confertus plants were treated with two blends of SPVs at different quantities that act as either attractants or repellents to insects. Blend 1 (B1) consisted of five green leaf volatiles (GLVs), namely (Z)-3-hexenal, (E)-2-hexenal, (Z)-3-hexenol, (E)-2-hexenol, and (Z)-3-hexen-1-yl acetate. Blend 2 (B2) contained six plant volatiles, namely (Z)-ocimene, linalool, benzyl acetate, methyl salicylate, ß-caryophyllene, and (E)-ß-farnesene. Each blend was made available in four different amounts of volatiles, corresponding to each compound being added to 50 µL of hexane in amounts of 1, 5, 25 and 125 ng. The effects of the two blends at the different concentrations on the insects were evaluated using a Y-tube olfactometer. Both sexes of the insects were found to be significantly repelled by the highest volatile levels of B1 and by two levels of B2 (25 and 125 ng). Females were also observed to be repelled using B2 with 5 ng of each volatile. Attraction was observed for both sexes only for B1 at the three lower volatile levels (1, 5 and 25 ng). In additional experiments, using only attractants, unmated females were found to be attracted to males, whereas mated females were only attracted to B1. Both unmated and mated males (previously observed in copula) were attracted only to females.

Modelling of Breath and Various Blood Volatilomic Profiles-Implications for Breath Volatile Analysis.

Researchers looking for biomarkers from different sources, such as breath, urine, or blood, frequently search for specific patterns of volatile organic compounds (VOCs), often using pattern recognition or machine learning techniques. However, they are not generally aware that these patterns change depending on the source they use. Therefore, we have created a simple model to demonstrate that the distribution patterns of VOCs in fat, mixed venous blood, alveolar air, and end-tidal breath are different. Our approach follows well-established models for the description of dynamic real-time breath concentration profiles. We start with a uniform distribution of end-tidal concentrations of selected VOCs and calculate the corresponding target concentrations. For this, we only need partition coefficients, mass balance, and the assumption of an equilibrium state, which avoids the need to know the volatiles' metabolic rates and production rates within the different compartments.

Induction of volatile organic compounds in Triticum aestivum (wheat) plants following infection by different Rhizoctonia pathogens is species specific.

The most popular means of plant protection is the chemical method, but this control is often connected with the need for repeating chemical treatments. Thus, eco-friendly strategies should be developed where, under the European Green Deal, aromatic plants and their repellent properties seem to constitute a good alternative. In earlier studies, we have shown that insect injury, bacteria infestation and pathogen infection induce plant volatile organic compounds (VOCs) emission, which can provide defensive functions to plants. In this study, Triticum aestivum L. (Poaceae) cv. 'Jenga' wheat plants were intentionally infected with one of four Rhizoctonia species (R. cerealis, R. solani, R. zeae, and R. oryzae). The soil was inoculated by the pathogens during sowing, whereas shoots were inoculated at stage BBCH 33. In greenhouse experiments, we measured VOCs from wheat 3, 7 and 11 days following stem infestation, or 42 days following soil inoculation of Rhizoctonia spp. VOC emissions were found to be largest on days 7 or 11 post-stem inoculation (>3 days post-stem inoculation >42 days post-soil inoculation). T. aestivum infected by pathogens induced five common green leaf volatiles (GLVs), namely (Z)-3-hexenal = (Z)-3-HAL, (E)-2-hexenal = (E)-2-HAL, (Z)-3-hexen-1-ol = (Z)-3- HOL, (E)-2-hexenol = (E)-2-HOL, (Z)-3-hexen-1-yl acetate = (Z)-3-HAC], six common terpenes (ß-pinene = ß-PIN, ß-myrcene = ß-MYR, Z-ocimene = Z-OCI, linalool = LIN, benzyl acetate = BAC, ß-caryophyllene = ß-CAR), and indole = IND. We found that R. cerealis infested T. aestivum emitted the largest amounts of (Z)-3-HAL and (Z)-3-HAC, while T. aestivum infested by R. solani released the largest amount of LIN (7 or 11 days following stem infestation). VOCs released by the T. aestivum after R. cerealis (AGD I) and R. solani (AG 5) infestations were significantly larger in comparison to R. zeae (WAG-Z) and R. oryzae (WAG-O) for the volatiles (Z)-3-HAL, (E)-2-HAL, (Z)-3-HOL, (E)-2-HOL, (Z)-3-HAC, ß-PIN, ß-MYR, and LIN. With the exception of (E)-2-HOL, ß-MYR, LIN, BAC, ß-CAR, the other VOCs were emitted in similar amounts by infected T. aestivum 3 days following stem and soil inoculation. The quantities of induced VOCs were higher at days 7 and 11 than at 3 days post-infection, and greater when T. aestivum was infected with Rhizoctonia on the stem base than through the soil.

The peppermint breath test benchmark for PTR-MS and SIFT-MS.

A major challenge for breath research is the lack of standardization in sampling and analysis. To address this, a test that utilizes a standardized intervention and a defined study protocol has been proposed to explore disparities in breath research across different analytical platforms and to provide benchmark values for comparison. Specifically, thePeppermint Experimentinvolves the targeted analysis in exhaled breath of volatile constituents of peppermint oil after ingestion of the encapsulated oil. Data from thePeppermint Experimentperformed by proton transfer reaction mass spectrometry (PTR-MS) and selected ion flow tube mass spectrometry (SIFT-MS) are presented and discussed herein, including the product ions associated with the key peppermint volatiles, namely limonene,α- andß-pinene, 1,8-cineole, menthol, menthone and menthofuran. The breath washout profiles of these compounds from 65 individuals were collected, comprising datasets from five PTR-MS and two SIFT-MS instruments. The washout profiles of these volatiles were evaluated by comparing the log-fold change over time of the product ion intensities associated with each volatile. Benchmark values were calculated from the lower 95% confidence interval of the linear time-to-washout regression analysis for all datasets combined. Benchmark washout values from PTR-MS analysis were 353 min for the sum of monoterpenes and 1,8-cineole (identical product ions), 173 min for menthol, 330 min for menthofuran, and 218 min for menthone; from SIFT-MS analysis values were 228 min for the sum of monoterpenes, 281 min for the sum of monoterpenes and 1,8-cineole, and 370 min for menthone plus 1,8-cineole. Large inter- and intra-dataset variations were observed, whereby the latter suggests that biological variability plays a key role in how the compounds are absorbed, metabolized and excreted from the body via breath. This variability seems large compared to the influence of sampling and analytical procedures, but further investigations are recommended to clarify the effects of these factors.

Volatile Organic Compounds in Exhaled Breath as Fingerprints of Lung Cancer, Asthma and COPD.

Lung cancer, chronic obstructive pulmonary disease (COPD) and asthma are inflammatory diseases that have risen worldwide, posing a major public health issue, encompassing not only physical and psychological morbidity and mortality, but also incurring significant societal costs. The leading cause of death worldwide by cancer is that of the lung, which, in large part, is a result of the disease often not being detected until a late stage. Although COPD and asthma are conditions with considerably lower mortality, they are extremely distressful to people and involve high healthcare overheads. Moreover, for these diseases, diagnostic methods are not only costly but are also invasive, thereby adding to people's stress. It has been appreciated for many decades that the analysis of trace volatile organic compounds (VOCs) in exhaled breath could potentially provide cheaper, rapid, and non-invasive screening procedures to diagnose and monitor the above diseases of the lung. However, after decades of research associated with breath biomarker discovery, no breath VOC tests are clinically available. Reasons for this include the little consensus as to which breath volatiles (or pattern of volatiles) can be used to discriminate people with lung diseases, and our limited understanding of the biological origin of the identified VOCs. Lung disease diagnosis using breath VOCs is challenging. Nevertheless, the numerous studies of breath volatiles and lung disease provide guidance as to what volatiles need further investigation for use in differential diagnosis, highlight the urgent need for non-invasive clinical breath tests, illustrate the way forward for future studies, and provide significant guidance to achieve the goal of developing non-invasive diagnostic tests for lung disease. This review provides an overview of these issues from evaluating key studies that have been undertaken in the years 2010-2019, in order to present objective and comprehensive updated information that presents the progress that has been made in this field. The potential of this approach is highlighted, while strengths, weaknesses, opportunities, and threats are discussed. This review will be of interest to chemists, biologists, medical doctors and researchers involved in the development of analytical instruments for breath diagnosis.

Breath Biopsy Assessment of Liver Disease Using an Exogenous Volatile Organic Compound-Toward Improved Detection of Liver Impairment.

INTRODUCTION: Liver cirrhosis and its complication - hepatocellular carcinoma (HCC) - have been associated with increased exhaled limonene. It is currently unclear whether this increase is more strongly associated with the presence of HCC or with the severity of liver dysfunction. METHODS: We compared the exhaled breath of 40 controls, 32 cirrhotic patients, and 12 cirrhotic patients with HCC using the Breath Biopsy platform. Breath samples were analyzed by thermal desorption-gas chromatography-mass spectrometry. Limonene levels were compared between the groups and correlated to bilirubin, albumin, prothrombin time international normalized ratio, and alanine aminotransferase. RESULTS: Breath limonene concentration was significantly elevated in subjects with cirrhosis-induced HCC (M: 82.1 ng/L, interquartile range [IQR]: 16.33-199.32 ng/L) and cirrhosis (M: 32.6 ng/L, IQR: 6.55-123.07 ng/L) compared with controls (M: 6.2 ng/L, IQR: 2.62-9.57 ng/L) (P value = 0.0005 and 0.0001, respectively) with no significant difference between 2 diseased groups (P value = 0.37). Levels of exhaled limonene correlated with serum bilirubin (R = 0.25, P value = 0.0016, r = 0.51), albumin (R = 0.58, P value = 5.3e-8, r = -0.76), and international normalized ratio (R = 0.29, P value = 0.0003, r = 0.51), but not with alanine aminotransferase (R = 0.01, P value = 0.36, r = 0.19). DISCUSSION: Exhaled limonene levels are primarily affected by the presence of cirrhosis through reduced liver functional capacity, as indicated by limonene correlation with blood metrics of impaired hepatic clearance and protein synthesis capacity, without further alterations observed in subjects with HCC. This suggests that exhaled limonene is a potential non-invasive marker of liver metabolic capacity (see Visual abstract, Supplementary Digital Content 1, http://links.lww.com/CTG/A388).

Effect of inhaled acetone concentrations on exhaled breath acetone concentrations at rest and during exercise.

Real-time measurements of the differences in inhaled and exhaled, unlabeled and fully deuterated acetone concentration levels, at rest and during exercise, have been conducted using proton transfer reaction mass spectrometry. A novel approach to continuously differentiate between the inhaled and exhaled breath acetone concentration signals is used. This leads to unprecedented fine grained data of inhaled and exhaled concentrations. The experimental results obtained are compared with those predicted using a simple three compartment model that theoretically describes the influence of inhaled concentrations on exhaled breath concentrations for volatile organic compounds with high blood:air partition coefficients, and hence is appropriate for acetone. An agreement between the predicted and observed concentrations is obtained. Our results highlight that the influence of the upper airways cannot be neglected for volatiles with high blood:air partition coefficients, i.e. highly water soluble volatiles.

The Volatilomic Footprints of Human HGC-27 and CLS-145 Gastric Cancer Cell Lines.

The presence of certain volatile biomarkers in the breath of patients with gastric cancer has been reported by several studies; however, the origin of these compounds remains controversial. In vitro studies, involving gastric cancer cells may address this problem and aid in revealing the biochemical pathways underlying the production and metabolism of gastric cancer volatile indicators. Gas chromatography with mass spectrometric detection, coupled with headspace needle trap extraction as the pre-concentration technique, has been applied to map the volatilomic footprints of human HGC-27 and CLS-145 gastric cancer cell lines and normal Human Stomach Epithelial Cells (HSEC). In total, 27 volatile compounds are found to be associated with metabolism occurring in HGC-27, CLS-145, and HSEC. Amongst these, the headspace concentrations of 12 volatiles were found to be reduced compared to those above just the cultivating medium, namely there was an observed uptake of eight aldehydes (2-methylpropanal, 2-methyl-2-propenal, 2-methylbutanal, 3-methylbutanal, hexanal, heptanal, nonanal, and benzaldehyde), three heterocyclic compounds (2-methyl-furan, 2-ethyl-furan, and 2-pentyl-furan), and one sulfur-containing compound (dimethyl disulphide). For the other 15 volatiles, the headspace concentrations above the healthy and cancerous cells were found to be higher than those found above the cultivating medium, namely the cells were found to release three esters (ethyl acetate, ethyl propanoate, and ethyl 2-methylbutyrate), seven ketones (2-pentanone, 2-heptanone, 2-nonanone, 2-undecanone, 2-tridecanone, 2-pentadecanone, and 2-heptadecanone), three alcohols (2-methyl-1-butanol, 3-methyl-1-butanol, and 2-ethyl-1-hexanol), one aromatic compound (toluene), and one sulfur containing compound [2-methyl-5-(methylthio) furan]. In comparison to HSEC, HGC-27 cancer cell lines were found to have significantly altered metabolism, manifested by an increased production of methyl ketones containing an odd number of carbons. Amongst these species, three volatiles were found exclusively to be produced by this cell line, namely 2-undecanone, 2-tridecanone, and 2-heptadecanone. Another interesting feature of the HGC-27 footprint is the lowered level of alcohols and esters. The CLS-145 cells exhibited less pronounced changes in their volatilomic pattern compared to HSEC. Their footprint was characterized by the upregulated production of esters and 2-ethyl-hexanol and downregulated production of other alcohols. We have therefore demonstrated that it is possible to differentiate between cancerous and healthy gastric cells using biochemical volatile signatures.

Studies pertaining to the monitoring of volatile halogenated anaesthetics in breath by proton transfer reaction mass spectrometry.

Post-operative isoflurane has been observed to be present in the end-tidal breath of patients who have undergone major surgery, for several weeks after the surgical procedures. A major new non-controlled, non-randomized, and open-label approved study will recruit patients undergoing various surgeries under different inhalation anaesthetics, with two key objectives, namely (1) to record the washout characteristics following surgery, and (2) to investigate the influence of a patient's health and the duration and type of surgery on elimination. In preparation for this breath study using proton transfer reaction time-of-flight mass spectrometry (PTR-TOF-MS), it is important to identify first the analytical product ions that need to be monitored and under what operating conditions. In this first paper of this new research programme, we present extensive PTR-TOF-MS studies of three major anaesthetics used worldwide, desflurane (CF3CHFOCHF2), sevoflurane ((CF3)2CHOCH2F), and isoflurane (CF3CHClOCHF2) and a fourth one, which is used less extensively, enflurane (CHF2OCF2CHFCl), but is of interest because it is an isomer of isoflurane. Product ions are identified as a function of reduced electric field (E/N) over the range of approximately 80 Td to 210 Td, and the effects of operating the drift tube under 'normal' or 'humid' conditions on the intensities of the product ions are presented. To aid in the analyses, density functional theory (DFT) calculations of the proton affinities and the gas-phase basicities of the anaesthetics have been determined. Calculated energies for the ion-molecule reaction pathways leading to key product ions, identified as ideal for monitoring the inhalation anaesthetics in breath with a high sensitivity and selectivity, are also presented.

Selective Reagent Ion Mass Spectrometric Investigations of the Nitroanilines.

This paper presents an investigation of proton and charge transfer reactions to 2-, 3- and 4-nitroanilines (C6H6N2O2) involving the reagent ions H3O+·(H2O)n (n = 0, 1 and 2) and O2+, respectively, as a function of reduced electric field (60-240 Td), using Selective Reagent Ion-Time-of-Flight-Mass Spectrometry (SRI-ToF-MS). To aid in the interpretation of the H3O+·(H2O)n experimental data, the proton affinities and gas-phase basicities for the three nitroaniline isomers have been determined using density functional theory. These calculations show that proton transfer from both the H3O+ and H3O+·H2O reagent ions to the nitroanilines will be exoergic and hence efficient, with the reactions proceeding at the collisional rate. For proton transfer from H3O+ to the NO2 sites, the exoergicities are 171 kJ mol-1 (1.8 eV), 147 kJ mol-1 (1.5 eV) and 194 kJ mol-1 (2.0 eV) for 2-, 3- and 4-nitroanilines, respectively. Electron transfer from all three of the nitroanilines is also significantly exothermic by approximately 4 eV. Although a substantial transfer of energy occurs during the ion/molecule reactions, the processes are found to predominantly proceed via non-dissociative pathways over a large reduced electric field range. Only at relatively high reduced electric fields (> 180 Td) is dissociative proton and charge transfer observed. Differences in fragment product ions and their intensities provide a means to distinguish the isomers, with proton transfer distinguishing 2-nitroaniline (2-NA) from 3- and 4-NA, and charge transfer distinguishing 4-NA from 2- and 3-NA, thereby providing a means to enhance selectivity using SRI-ToF-MS.

Compendium of the Reactions of H3O+ With Selected Ketones of Relevance to Breath Analysis Using Proton Transfer Reaction Mass Spectrometry.

Soft chemical ionization mass spectrometric techniques, such as proton transfer reaction mass spectrometry (PTR-MS), are often used in breath analysis, being particularly powerful for real-time measurements. To ascertain the type and concentration of volatiles in exhaled breath clearly assignable product ions resulting from these volatiles need to be determined. This is difficult for compounds where isomers are common, and one important class of breath volatiles where this occurs are ketones. Here we present a series of extensive measurements on the reactions of H3O+ with a selection of ketones using PTR-MS. Of particular interest is to determine if ketone isomers can be distinguished without the need for pre-separation by manipulating the ion chemistry through changes in the reduced electric field. An additional issue for breath analysis is that the product ion distributions for these breath volatiles are usually determined from direct PTR-MS measurements of the compounds under the normal operating conditions of the instruments. Generally, no account is made for the effects on the ion-molecule reactions by the introduction of humid air samples or increased CO2 concentrations into the drift tubes of these analytical devices resulting from breath. Therefore, another motivation of this study is to determine the effects, if any, on the product ion distributions under the humid conditions associated with breath sampling. However, the ultimate objective for this study is to provide a valuable database of use to other researchers in the field of breath analysis to aid in analysis and quantification of trace amounts of ketones in human breath. Here we present a comprehensive compendium of the product ion distributions as a function of the reduced electric field for the reactions of H3O+. (H2O)n (n = 0 and 1) with nineteen ketones under normal and humid (100% relative humidity for 37 °C) PTR-MS conditions. The ketones selected for inclusion in this compendium are (in order of increasing molecular weight): 2-butanone; 2-pentanone; 3-pentanone; 2-hexanone; 3-hexanone; 2-heptanone; 3-heptanone; 4-heptanone; 3-octanone; 2-nonanone; 3-nonanone; 2-decanone; 3-decanone; cyclohexanone; 3-methyl-2-butanone; 3-methyl-2-pentanone; 2-methyl-3-pentanone; 2-methyl-3-hexanone; and 2-methyl-3-heptanone.

Proton transfer reaction time-of-flight mass spectrometric measurements of volatile compounds contained in peppermint oil capsules of relevance to real-time pharmacokinetic breath studies.

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 ß-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 H2O from protonated menthol) and m/z 83.09 (a fragment ion, C6H11 +, 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.