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One convenient strategy to reduce environmental impact and pollution involves the reuse and revalorization of waste produced by modern society. Nowadays, global plastic production has reached 367 million tons per year and because of their durable nature, their recycling is fundamental for the achievement of the circular economy objective. In closing the loop of plastics, advanced recycling, i.e., the breakdown of plastics into their building blocks and their transformation into valuable secondary raw materials, is a promising management option for post-consumer plastic waste. The most valuable product from advanced recycling is a fluid hydrocarbon stream (or pyrolysis oil) which represents the feedstock for further refinement and processing into new plastics. In this context, gas chromatography is currently playing an important role since it is being used to study the pyrolysis oils, as well as any organic contaminants, and it can be considered a high-resolution separation technique, able to provide the molecular composition of such complex samples. This information significantly helps to tailor the pyrolysis process to produce high-quality feedstocks. In addition, the detection of contaminants (i.e., heteroatom-containing compounds) is crucial to avoid catalytic deterioration and to implement and design further purification processes. The current review highlights the importance of molecular characterization of waste stream products, and particularly the pyrolysis oils obtained from waste plastics. An overview of relevant applications published recently will be provided, and the potential of comprehensive two-dimensional gas chromatography, which represents the natural evolution of gas chromatography into a higher-resolution technique, will be underlined.

Systemic sclerosis is a rare autoimmune disease associated with rapidly evolving interstitial lung disease, responsible for the disease severity and mortality. Specific biomarkers enabling the early diagnosis and prognosis associated with the disease progression are highly needed. Volatile organic compounds in exhaled breath are widely available and non-invasive and have the potential to reflect metabolic processes occurring within the body. Comprehensive two-dimensional gas chromatography coupled to high-resolution mass spectrometry was used to investigate the potential of exhaled breath to diagnose systemic sclerosis. The exhaled breath of 32 patients and 30 healthy subjects was analyzed. The high resolving power of this approach enabled the detection of 356 compounds in the breath of systemic sclerosis patients, which was characterized by an increase of mainly terpenoids and hydrocarbons. In addition, the use of 4 complementary statistical approaches (two-tailed equal variance t-test, fold change, partial least squares discriminant analysis, and random forest) resulted in the identification of 16 compounds that can be used to discriminate systemic sclerosis patients from healthy subjects. Receiver operating curves were generated that provided an accuracy of 90%, a sensitivity of 92%, and a specificity of 89%. The chemical identification of eight compounds predictive of systemic sclerosis was validated using commercially available standards. The analytical variations together with the volatile composition of room air were carefully monitored during the timeframe of the study to ensure the robustness of the technique. This study represents the first reported evaluation of exhaled breath analysis for systemic sclerosis diagnosis and provides surrogate markers for such disease.

The high potential of exhaled breath for disease diagnosis has been highlighted in numerous studies. However, exhaled breath analysis is suffering from a lack of standardized sampling and analysis procedures, impacting the robustness of inter-laboratory results, and thus hampering proper external validation. The aim of this work was to verify compliance and validate the performance of two different comprehensive two-dimensional gas chromatography coupled to mass spectrometry platforms in different laboratories by monitoring probe metabolites in exhaled breath following the Peppermint Initiative guidelines. An initial assessment of the exhaled breath sampling conditions was performed, selecting the most suitable sampling bag material and volume. Then, a single sampling was performed using Tedlar bags, followed by the trapping of the volatile organic compounds into thermal desorption tubes for the subsequent analysis using two different analytical platforms. The thermal desorption tubes were first analyzed by a (cryogenically modulated) comprehensive two-dimensional gas chromatography system coupled to high-resolution time-of-flight mass spectrometry. The desorption was performed in split mode and the split part was recollected in the same tube and further analyzed by a different (flow modulated) comprehensive two-dimensional gas chromatography system with a parallel detection, specifically using a quadrupole mass spectrometer and a vacuum ultraviolet detector. Both the comprehensive two-dimensional gas chromatography platforms enabled the longitudinal tracking of the peppermint oil metabolites in exhaled breath. The increased sensitivity of comprehensive two-dimensional gas chromatography enabled to successfully monitor over a 6.5 h period a total of 10 target compounds, namely α-pinene, camphene, β-pinene, limonene, cymene, eucalyptol, menthofuran, menthone, isomenthone, and neomenthol.

Despite the same basic ingredients used in brewing, there is a significant variation in beer styles. With the rapid increase in craft brewing, beer styles have become even more numerous and complex in the recent past. A GC-MS/VUV (post-column split for dual detection) instrument with headspace high capacity SPME was used to investigate 21 different beers which represent three beer styles - India pale ales, blondes, and hefeweizens. Since results from untargeted studies can be affected by the sorbent material used, the extraction performances of three high capacity SPME fibers, i.e., polydimethylsiloxane, polydimethylsiloxane/carbon wide range, and polydimethylsiloxane/carbon wide range/divinylbenzene, were evaluated. Good reproducibility (10% RSD) was obtained for each high capacity fiber using both detectors. The tandem MS/VUV detection coupled with GC separation proved to be particularly valuable for compound identification, especially for isomers and compounds with similar structures. The evaluation of VUV detection for untargeted analysis led to similar performances as MS detection. Both the VUV and the MS were able to effectively differentiate between beer styles using principal component analysis. In addition, the use of 3 different statistical approaches, one-way ANOVA (p-value 0.05), partial least square discriminant analysis, and random forest, universally identified 12 of the components most influential in distinguishing the three beer styles (e.g., β-myrcene, linalool, isopentyl acetate, 2,4-di-tert-butylphenol). This is the first reported evaluation of VUV detection and the first comparison of simultaneous VUV and MS detection for untargeted classification of complex mixtures using GC.

In this review, we consider and discuss the affinity and complementarity between a generic sample preparation technique and the comprehensive two-dimensional gas chromatography process. From the initial technical development focus (e.g., on the GC×GC and solid-phase microextraction techniques), the trend is inevitably shifting toward more applied challenges, and therefore, the preparation of the sample should be carefully considered in any GC×GC separation for an overreaching research. We highlight recent biomedical, food, and plant applications (2016-July 2020), and specifically those in which the combination of tailored sample preparation methods and GC×GC-MS has proven to be beneficial in the challenging aspects of non-targeted analysis. Specifically on the sample preparation, we report on gas-phase, solid-phase, and liquid-phase extractions, and derivatization procedures that have been used to extract and prepare volatile and semi-volatile metabolites for the successive GC×GC analysis. Moreover, we also present a milestone section reporting the early works that pioneered the combination of sample preparation techniques with GC×GC for non-targeted analysis.

Exhaled breath analysis has a high potential for early non-invasive diagnosis of lung inflammatory diseases, such as asthma. The characterization and understanding of the inflammatory metabolic pathways involved into volatile organic compounds (VOCs) production could bring exhaled breath analysis into clinical practice and thus open new therapeutic routes for inflammatory diseases. In this study, lung inflammation was simulated in vitro using A549 epithelial cells. We compared the VOC production from A549 epithelial cells after a chemically induced oxidative stress in vitro, exposing the cells to H2O2, and a biological stress, exposing the cells to an inflammatory pool of sputum supernatants. Special attention was devoted to define proper negative and positive controls (8 different types) for our in vitro models, including healthy sputum co-culture. Sputum from 25 asthmatic and 8 healthy patients were collected to create each pool of supernatants. Each sample type was analyzed in 4 replicates using solid-phase microextraction (SPME) comprehensive two-dimensional gas chromatography hyphenated to time-of-flight mass spectrometry (GC×GC-TOFMS). This approach offers high resolving power for complex VOC mixtures. According to the type of inflammation induced, significantly different VOCs were produced by the epithelial cells compared to all controls. For both chemical and biological challenges, an increase of carbonyl compounds (54%) and hydrocarbons (31%) was observed. Interestingly, only the biological inflammation model showed a significant cell proliferation together with an increased VOC production linked to asthma airway inflammation. This study presents a complete GC×GC-TOFMS workflow for in vitro VOC analysis, and its potential to characterize complex lung inflammatory mechanisms.

Headspace gas chromatography is frequently used for aroma profiling thanks to its ability to naturally exploit the volatility of aroma compounds, and also to provide chemical information on sample composition. Its main advantages rely on simplicity, no use of solvent, amenability to automation, and the cleanliness of the extract. In the present contribution, the most effective sampling (dynamic extraction), separation (multidimensional gas chromatography), and detection (mass spectrometry) techniques for untargeted analysis are exploited in combination, showing their potential in unraveling aroma profiles in fruit beers. To complete the overall analytical process, a neat workflow for data analysis is discussed and used for the successful characterization and identification of five different beer flavors (berries, cherry, banana, apple, and peach). From the technical viewpoint, the coupling of purge-and-trap, comprehensive two-dimensional gas chromatography, and mass spectrometry makes the global methodology unique, and it is for the first time discussed. A (low-)flow modulation approach allowed for the full transfer into the second dimension with mass-spectrometry compatible flow (< 7 mL/min), avoiding the need of splitting before detection and making the overall method sensitive (1.2-5.2-fold higher signal to noise ratio compared to unmodulated gas chromatography conditions) and selective.

Rationale: Analysis of exhaled breath for asthma phenotyping using endogenously generated volatile organic compounds (VOCs) offers the possibility of noninvasive diagnosis and therapeutic monitoring. Induced sputum is indeed not widely available and markers of neutrophilic asthma are still lacking.Objectives: To determine whether analysis of exhaled breath using endogenously generated VOCs can be a surrogate marker for recognition of sputum inflammatory phenotypes.Methods: We conducted a prospective study on 521 patients with asthma recruited from the University Asthma Clinic of Liege. Patients underwent VOC measurement, fraction of exhaled nitric oxide (FENO) spirometry, sputum induction, and gave a blood sample. Subjects with asthma were classified in three inflammatory phenotypes according to their sputum granulocytic cell count.Measurements and Main Results: In the discovery study, seven potential biomarkers were highlighted by gas chromatography-mass spectrometry in a training cohort of 276 patients with asthma. In the replication study (n = 245), we confirmed four VOCs of interest to discriminate among asthma inflammatory phenotypes using comprehensive two-dimensional gas chromatography coupled to high-resolution time-of-flight mass spectrometry. Hexane and 2-hexanone were identified as compounds with the highest classification performance in eosinophilic asthma with accuracy comparable to that of blood eosinophils and FENO. Moreover, the combination of FENO, blood eosinophils, and VOCs gave a very good prediction of eosinophilic asthma (area under the receiver operating characteristic curve, 0.9). For neutrophilic asthma, the combination of nonanal, 1-propanol, and hexane had a classification performance similar to FENO or blood eosinophils in eosinophilic asthma. Those compounds were found in higher levels in neutrophilic asthma.Conclusions: Our study is the first attempt to characterize VOCs according to sputum granulocytic profile in a large population of patients with asthma and provide surrogate markers for neutrophilic asthma.

In the host, pathogenic microorganisms have developed stress responses to cope with constantly changing environments. Stress responses are directly related to changes in several metabolomic pathways, which could hamper microorganisms' unequivocal identification. We evaluated the effect of various in vitro stress conditions (acidic, basic, oxidative, ethanolic, and saline conditions) on the metabolism of Staphylococcus aureus, Bacillus cereus, and Pseudomonas aeruginosa, which are common lung pathogens. The metabolite profiles of the bacteria were analyzed using liquid chromatography coupled to triple quadrupole and quadrupole time-of-flight mass spectrometry. The advantages of targeted and untargeted analysis combined with univariate and multivariate statistical analysis (principal component analysis, hierarchical cluster analysis, partial least square discriminant analysis, random forest) were combined to unequivocally identify bacterial species. In normal in vitro conditions, the targeted methodology, based on the analysis of primary metabolites, enabled the rapid and efficient discrimination of the three bacteria. In changing in vitro conditions and specifically in presence of the various stressors, the untargeted methodology proved to be more valuable for the global and accurate differentiation of the three bacteria, also considering the type of stress environment within each species. In addition, species-specific metabolites (i.e., fatty acids, polysaccharides, peptides, and nucleotide bases derivatives) were putatively identified. Good intra-day repeatability and inter-day repeatability (< 10% RSD and < 15% RSD, respectively) were obtained for the targeted and the untargeted methods. This untargeted approach highlights its importance in unusual (and less known) bacterial growth environments, being a powerful tool for infectious disease diagnosis, where the accurate classification of microorganisms is sought.

Sophie Graff,1 Delphine Zanella,2 Pierre-Hugues Stefanuto,2 Monique Henket,1 Virginie Paulus,1 Francoise Guissard,1 Catherine Moermans,1 Kristel Van Steen,3 Renaud Louis,1 Florence Schleich1 1Department of Respiratory Medicine, GIGA I3, CHU Sart-Tilman, Liege, Belgium; 2Organic and Biological Analytical Chemistry Group, University of Liege, Liege, Belgium; 3BIO3-Systems Genetics, GIGA-R, University of Liege, Liege, BelgiumCorrespondence: Sophie GraffDepartment of Respiratory Medicine, CHU Sart-Tilman, GIGA +4, CHU - B34, Avenue de l’Hôpital, 11, Liège 4000, BelgiumTel +32 4366 45 92Email sgraff@uliege.be

The increased attraction of biological volatile compounds has opened the route to a wide variety of sampling techniques, amongst which trap tubes packed with adsorbent materials are commonly used. Many types of adsorbent materials are available and the choice of the adsorbent can impact the obtained results in untargeted analysis. Therefore, a proper combination of the adsorbent material and the sample is necessary to increase the robustness and reproducibility of biological studies. In this study, the sampling performance of thermal desorption tubes with six common adsorbent material combinations, i.e., Tenax® TA, Tenax® TA/Carbopack™ B, Tenax® TA/Sulficarb, Tenax® TA/Carbograph™ 5TD, Tenax® TA/Carbograph™ 1TD/Carboxen® 1003, and Carboxen® 1016/Carbograph™ 5TD, was evaluated in two different setups: in vitro and in vivo sampling. The in vitro setup consisted of the headspace dynamic extraction of spiked serum, and a mixture of 19 standards was evaluated in terms of response and reproducibility. The in vivo setup consisted into two parts: the first one was based the evaluation of the standard mixture, which was flash-vaporised into Tedlar® bags containing exhaled breath; the second part was based on the longitudinal monitoring of breath metabolites originating from a beverage intake (i.e., brewed coffee), over a 90 min time period. The tubes were all desorbed and analysed in a comprehensive two-dimensional gas chromatography system coupled to a high-resolution time-of-flight mass spectrometer (GC × GC-HR ToF MS). In both sampling setups, the widest analytes coverage and the overall best extraction yield on the selected compounds were obtained using Tenax® TA, followed by Tenax® TA/Carbopack™ B. Tenax® TA provided the highest sampling reproducibility with 12 %RSD, 10 %RSD and5 %RSD of the response during the experiments using the in vitro setup, the in vivo setup, and during the longitudinal tracking, respectively.

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 Marie Sklodowska-Curie Actions Innovative Training Network: Ion-Molecule Processes for Analytical Chemistry Technologies (IMPACT) (www.impact-h2020itn.com), which has supported BH, MM and GP as Early Stage Researchers through the European Commission's HORIZON 2020 Programme under Grant Agreement Number 674911. The EMBER consortium for support in the dissemination of the Peppermint Initiative; researchers involved in the application of the protocol to specific techniques (to be published in due course) Abigail Joyce, Amisha Manek, Bellagambi Francesca Giuseppa, Bonsignori Alessio, Ghimenti Silvia and Biagini Denise, Liesl Carr, Luke Bryant and Rebecca Cordell. MW and SJF are supported by the NIHR Manchester Biomedical Research Centre. The group from Li`ege University is supported by the FNRS through the EOS and the FRIA initiatives. Ruszkiewicz, DM (, orresponding author), Loughborough Univ, Ctr Analyt Sci, Dept Chem, Loughborough, Leics, England ; Univ Birmingham, Sch Phys & Astron, Mol Phys Grp, Birmingham, W Midlands, England. D.M.Ruszkiewicz2@lboro.ac.uk

Exhaled breath analysis has become an important part of volatilomics, with high potential for health applications, particularly as a noninvasive diagnostic tool. Nevertheless, this approach faces numerous challenges, the first being the need to employ powerful analytical techniques to properly resolve the breath volatilome and to extract the relevant information, and the second consisting of establishing a high-quality and reliable data analysis workflow. This chapter highlights the capabilities of comprehensive two-dimensional gas chromatography (GC×GC) as the ideal analytical technique for the analysis of exhaled breath. The coupling of GC×GC with mass spectrometry (MS) and the enhanced identification power using high-resolution (HR) MS is also discussed. In addition, an emphasis is placed on the importance of the data processing workflow, as well as the need to produce high data quality in order to perform useful statistical analyses. Finally, some successful applications of GC×GC-(HR)MS in volatilomics are presented.

VOCs are surrogate markers for salbutamol and OCS use, which are part of the definition of poor asthma control. This suggests that VOCs are promising biomarkers that could possibly be used to detect poor adherence to inhaled therapy.http://bit.ly/2SgE44R