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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.

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.

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.

Species of Mycobacteriaceae cause disease in animals and humans, including tuberculosis and leprosy. Individuals infected with organisms in the Mycobacterium tuberculosis complex (MTBC) or non-tuberculous mycobacteria (NTM) may present identical symptoms, however the treatment for each can be different. Although the NTM infection is considered less vital due to the chronicity of the disease and the infrequency of occurrence in healthy populations, diagnosis and differentiation among Mycobacterium species currently require culture isolation, which can take several weeks. The use of volatile organic compounds (VOCs) is a promising approach for species identification and in recent years has shown promise for use in the rapid analysis of both in vitro cultures as well as ex vivo diagnosis using breath or sputum. The aim of this contribution is to analyze VOCs in the culture headspace of seven different species of mycobacteria and to define the volatilome profiles that are discriminant for each species. For the pre-concentration of VOCs, solid-phase micro-extraction (SPME) was employed and samples were subsequently analyzed using gas chromatography–quadrupole mass spectrometry (GC-qMS). A machine learning approach was applied for the selection of the 13 discriminatory features, which might represent clinically translatable bacterial biomarkers.

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.

The present research reports on the development of a methodology to unravel the complex phytochemistry of cannabis. Specifically, cannabis inflorescences were considered and stir bar sorptive extraction (SBSE) was used for the preconcentration of the metabolites. Analytes were thermally desorbed into a comprehensive two-dimensional (2D) gas chromatography (GC × GC) system coupled with low- and high-resolution mass spectrometry (MS). Particular attention was devoted to the optimization of the extraction conditions, to extend the analytes' coverage, and the chromatographic separation, to obtain a robust data set for further untargeted analysis. Monoterpenes, sesquiterpenes, hydrocarbons, cannabinoids, other terpenoids, and fatty acids were considered to optimize the extraction conditions. The response of selected ions for each chemical class, delimited in specific 2D chromatographic regions, enabled an accurate and fast evaluation of the extraction variables (i.e., time, temperature, solvent, salt addition), which were then selected to have a wide analyte selection and good reproducibility. Under optimized SBSE conditions, eight different cannabis inflorescences and a quality control sample were analyzed and processed following an untargeted and unsupervised approach. Principal component analysis on all detected metabolites revealed chemical differences among the sample types which could be associated with the plant subspecies. With the same SBSE-GC × GC-MS methodology, a quantitative targeted analysis was performed on three common cannabinoids, namely, Δ9-tetrahydrocannabinol, cannabidiol, and cannabinol. The method was validated, giving correlation factors over 0.98 and

Gas chromatography (GC) coupled with electron ionization (EI) mass spectrometry (MS) is a well-established technique for the analysis of volatile and semi-volatile compounds. The main advantage is the highly repeatable fragmentation of the compounds into the ion source, generating intense and diagnostic fragmentation when the ionization is performed at 70 eV; this is considered the standard ionization condition and has been used for creating many established databases, which are of great support in the analyte identification process. However, such an intense fragmentation often causes the loss of the molecular ion or more diagnostic ions, which can be detrimental for the identification of homologous series or isomers, as for instance fatty acids. To obtain this information chemical or soft ionization can be used, but dedicated ion sources and conditions are required. In this work, we explored different ionization voltages in GC-EI-MS to preserve the intensity of the molecular ion using a conventional quadrupole MS. Twenty, 30, 50, and 70 eV were tested using a mixture of fatty acid methyl esters standards. Intensity and repeatability of the most informative ions were compared. Twenty and 70 eV were then used to analyze the fatty acid composition of six different strains of mycobacteria. Two approaches were used for elaborating the data: (1) a single average spectrum of the entire chromatogram was derived, which can be considered (in terms of concept) as a direct EI-MS analysis; (2) the actual chromatographic separation of the compounds was considered after automatic alignment. The results obtained are discussed herein. Graphical abstract ᅟ.

The main objective of the herein described research was focused on performing satisfactory flow modulation (FM), in comprehensive two-dimensional gas chromatography-mass spectrometry (GC×GC-MS), using an MS-compatible second-dimension gas flow of approx. 4 mL min(-1). The FM model used was based on that initially proposed by Seeley et al. [3]. The use of limited gas flows was enabled through fine tuning of the FM parameters, in particular the duration of the re-injection (or flushing) process. Specifically, the application of a long re-injection period (i.e., 700 ms) enabled efficient accumulation-loop flushing with gas flows of about 4 mL min(-1). It was possible to apply such extended re-injection periods by using different restrictor lengths in the connections linking the modulator to the auxiliary pressure source. FM GC×GC-MS applications were performed on a mixture containing C9-10 alkanes, and on a sample of essential oil. GC×GC-MS sensitivity was compared with that attained by using conventional GC-MS analysis, in essential oil applications. It was observed that signal intensities were, in general, considerably higher in the FM GC×GC-MS experiments.

We present a QR code paper microfluidic colorimetric assay that can exploit the hardware and software on mobile devices, and circumvent sample preparation by directly targeting volatile biomarkers. Our platform is a printable microarray of well-defined reaction regions, which outputs an instant diagnosis by directing the user to a URL containing their test result, while simultaneously storing epidemiological data for remote access and bioinformatics. To assist in the rapid identification of Escherichia coli in bloodstream infections, we employed an existing colorimetric reagent (p-dimethylaminocinnamaldehyde) and adapted its use to detect volatile indole, a biomarker produced by E. coli. Our assay was able to quantitatively detect indole in the headspace of E. coli culture after 12 h of growth (27.0 ± 3.1 ppm), assisting in species-level identification hours earlier than existing methods. Results were confirmed with headspace solid-phase microextraction (HS-SPME) two-dimensional gas chromatography time-of-flight mass spectrometry (GC×GC-ToFMS), which estimated indole concentration in E. coli culture to average 32.3 ± 5.2 ppm after 12 h of growth. This QR paper microfluidic platform represents a novel development in both telemedicine and diagnostics using volatile biomarkers. We envision that our QR code platform can be extended to other colorimetric assays for real-time diagnostics in low-resource environments.

The analysis of bacterial volatile organic compounds has gained attraction as a non-invasive way to identify disease-causing organisms, given that bacteria have unique metabolisms and volatile metabolic byproducts. In the present research, different adsorbent materials (Carbopack Y, X, B, Carboxen 1000 and Tenax TA), packed singularly or in combination, were compared in terms of sampling performance (sensitivity, repeatability and selectivity) for the extraction of standards and bacterial volatile metabolites in vitro (from Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli). After extraction, bacterial volatile organic compounds were desorbed and analyzed in a comprehensive two-dimensional gas chromatography system coupled to a time-of-flight mass spectrometer (GC × GC-ToF MS). The results show that Tenax has the greater ability to extract the standard mix as well as volatile organic compounds with better repeatability (4–26 RSD%), higher sensitivity (on average ∼24 fold) compared to Carbopack Y, X and Carboxen 1000 tube, which followed in terms of performance. In addition, Tenax confirmed the best sensitivity and discriminatory power with no misclassification in the untargeted and unsupervised analysis for the differentiation of the bacterial species.

The present research is focused on the use and evaluation of a novel helium ionization detector, defined as barrier discharge ionization detector (BID), within the context of (low-)flow modulation comprehensive two-dimensional gas chromatography (FM GC × GC). The performance of the BID device was compared to that of a flame ionization detector (FID), under similar FM GC × GC conditions. Following development and optimization of the FM GC × GC method, the BID was subjected to fine tuning in relation to acquisition frequency and discharge flow. Moreover, the BID performance was measured and compared to that of the FID, in terms of extra-column band broadening, sensitivity and dynamic range. The comparative study was carried out by using standard compounds belonging to different chemical classes, along with a sample of diesel fuel. Advantages and disadvantages of the BID system, also within the context of FM GC × GC, are critically discussed. In general, the BID system was characterized by a more limited dynamic range and increased sensitivity, compared to the FID. Additionally, BID and FID contribution to band broadening was found to be similar under the operational conditions applied. Particular attention was devoted to the behaviour of the FM GC × GC-BID system toward saturated and aromatic hydrocarbons, for a possible future use in the field of mineral-oil food contamination research.

The present research is focused on the evaluation of a recently developed high-resolution time-of-flight mass spectrometer (HR TOF MS), under the challenging conditions of a flow-modulation comprehensive two-dimensional gas chromatography (FM GC × GC) experiment. The HR TOF MS instrument was operated at a spectral generation frequency of 30 Hz and a mass resolution of ≥25,000 (fwhm). The effluent exiting the second-dimension column was in the range 6-8 mL/min, with part directed to waste to avoid exceeding the maximum pumping capacity of the MS system. An FM GC × GC-HR TOF MS method was developed for the untargeted and targeted analysis of a sample of high complexity, namely, heavy gas oil. With regard to the untargeted results, these were satisfactory in relation to MS database searching and mass accuracies. Considering the targeted data, the high selectivity of the MS system was highlighted by the use of accurate mass extracted-ion-chromatograms with narrow mass windows (±5 and ±1 ppm), for specific classes of polyaromatic sulfur heterocycles (PASHs), namely, benzothiophenes and dibenzothiophenes. Finally, the instrumental performance was also evaluated through the injection of standard solutions of four classes of PASHs.

In the present research, the potential of breath analysis by comprehensive two-dimensional gas chromatography coupled to mass spectrometry (GC×GC-MS) was investigated for the discrimination between healthy and infected mice. A pilot study employing a total of 16 animals was used to develop a method for breath analysis in a murine model for studying Mycobacterium tuberculosis complex (MTBC) using the M. bovis bacillus Calmette-Guérin. Breath was collected in Tedlar bags and concentrated onto thermal desorption tubes for subsequent analysis by GC×GC-MS. Immunological test and bacterial cell count in bronchoalveolar lavage fluid and mice lung homogenate confirmed the presence of bacteria in the infected group. From the GC×GC-MS analysis, 23 molecules were found to mainly drive the separation between control and infected mice and their tentative identification is provided.This study shows that the overall used methodology is able to differentiate breath between healthy and infected animals, and the information herein can be used to further develop the mouse breath model to study MTBC pathogenesis, evaluate pre-clinical drug regimen efficacy, and to further develop the concept of breath-based diagnostics.

Untargeted metabolomics study of volatile organic compounds produced by different cell cultures is a field that has gained increasing attention over the years. Solid-phase microextraction has been the sampling technique of choice for most of the applications mainly due to its simplicity to implement. However, a careful optimization of the analytical conditions is necessary to obtain the best performances, which are highly matrix-dependent. In this work, five different solid-phase microextraction fibers were compared for the analysis of the volatiles produced by cell culture infected with the human respiratory syncytial virus. A central composite design was applied to determine the best time-temperature combination to maximize the extraction efficiency and the salting-out effect was evaluated as well. The linearity of the optimized method, along with limits of detection and quantification and repeatability was assessed. Finally, the effect of i) different normalization techniques (i.e. z-score and probabilistic quotient normalization), ii) data transformation (i.e. in logarithmic scale), and iii) different feature selection algorithms (i.e. Fisher ratio and random forest) on the capability of discriminating between infected and not-infected cell culture was evaluated.

Polychlorodibenzo-p-dioxins (PCDDs), polychloro-dibenzofurans (PCDFs), dioxin-like (DL), and non dioxin-like (NDL) polychlorinated biphenyls (PCBs) are currently regulated in food and feed within the European territory (EU 2017/644-771). The confirmatory methods of analysis for checking compliance with maximum levels (MLs) for these involve either the historically-established GC-magnetic sector high-resolution mass spectrometry (GC-HRMS) and, more recently, GC-triple quadrupole mass spectrometry operating in tandem mode (GC-QQQMS/MS). In this study, the performance of a novel triple quadrupole GC-QQQMS/MS system equipped with a programable temperature vaporization (PTV) injector was evaluated for the analysis of regulated PCDD/Fs and PCBs in food and feed. The MS analyzer was equipped with a titanium ionization chamber and a new short collision cell capable to accumulate and eject ions by means of very narrow pulses that allow to minimize the noise and to adapt accumulation times for sensitive multiple reaction monitoring (MRM). The analytical capability of the system was confronted by the strict requirements (selectivity, reproducibility, linearity, quant/qual MRM transitions, accuracy, robustness) set by the EU Regulation for a range of standards, quality control (QC) and food/feed samples. In this respect, the approach showed high precision (1.9&ndash, 15% relative standard deviation (RSD) at low pg/&micro, L) and accuracy (&gt, 80%, except for one hexa-CDD). The quantitative results were also compared to the most used GC-HRMS. In this case, comparable results in terms of single congener concentration basis and total toxic equivalent (TEQ) basis for PCDD/Fs and DL-PCBs were obtained for the QC samples analyzed.

Microorganisms catabolize carbon-containing compounds in their environment during growth, releasing a subset of metabolic byproducts as volatile compounds. However, the relationship between growth media and the production of volatile compounds has been largely unexplored to-date. To assess the core and media-specific components of the Klebsiella pneumoniae volatile metabolome via growth in four in vitro culture media. Headspace volatiles produced by cultures of K. pneumoniae after growth to stationary phase in four rich media (brain heart infusion broth, lysogeny broth, Mueller-Hinton broth, and tryptic soy broth) were analyzed using comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry (GC×GC-TOFMS). Differences in the composition of headspace volatiles as a function of growth media were assessed using hierarchical clustering analysis (HCA) and principal component analysis (PCA). A total of 365 volatile compounds were associated with the growth of K. pneumoniae across all media, of which 36 (10%) were common to all growth media, and 148 (41%) were specific to a single medium. In addition, utilizing all K. pneumoniae-associated volatile compounds, strains clustered as a function of growth media, demonstrating the importance of media in determining the metabolic profile of this organism. K. pneumoniae produces a core suite of volatile compounds across all growth media studied, although the volatile metabolic signature of this organism is fundamentally media-dependent.

The present research is based on the development of an effective, environmentally-friendly and low-cost method for investigation of minor components in vegetable oils, exploiting the advantages of a miniaturized solid-phase extraction (SPE) and the potential of flow modulation (FM) comprehensive two-dimensional gas chromatography (GC×GC), coupled to a mass spectrometer (MS) and a flame ionization detector (FID). The initial sample preparation step was carried out using a miniaturized approach characterized by a SPE process in a Pasteur pipette. Then, the isolated fraction was injected into an FM GC×GC system. Tentative identification was carried out by means of MS spectral information, while quantification was carried out by using the FID data. Four different vegetable oil samples were analyzed using the proposed method, namely an extra virgin olive oil (EVO), a hazelnut oil (HO), a borage oil, and an EVO/HO mixture.

The first description of a comprehensive two-dimensional gas chromatography (GC×GC) elucidation of an essential oil appeared in 2000 (J. High Resol. Chromatogr. 2000;23:208-214); since then, a variety of investigations have been published also involving complex samples related to essential oils, such as perfumes. In many instances, there was no mismatch between the use of GC×GC, and the analytical scope; on the other hand, in some specific cases the use of GC×GC appeared to be less adequate. The present contribution is focused on the application of GC×GC, within the context of essential oil analysis. Perfumes are also tightly related to essential oils, and so are herein considered. Rather than to describe and critically-review all the research related to such investigations, attention has been devoted to the general analytical usefulness of GC×GC in the aforementioned application fields, over more established approaches, such as conventional GC and heart-cutting multidimensional GC.

In this pilot study, volatile molecules produced by cultures of Mycobacterium tuberculosis were evaluated to determine whether they could be used to discriminate between uninfected and M. tuberculosis-infected macaques. Thirty seven of the culture biomarkers were detectable in macaque breath and were shown to discriminate between uninfected and infected animals with an area under the curve (AUC) of 87%. An AUC of 98% was achieved when using the top 38 discriminatory molecules detectable in breath. We report two newly discovered volatile biomarkers, not previously associated with M. tuberculosis, that were selected in both our in vitro and in vivo discriminatory biomarker suites: 4-(1,1-dimethylpropyl) phenol and 4-ethyl-2,2,6,6-tetramethylheptane. Additionally, we report the detection of heptanal, a previously identified M. tuberculosis breath biomarker in humans, as an in vitro culture biomarker that was detected in every macaque breath sample analyzed, though not part of the in vivo discriminatory suite. This pilot study suggests that molecules from the headspace of M. tuberculosis culture show potential to translate as breath biomarkers for macaques infected with the same strain.

The present contribution is focused on the evolution and current trends of comprehensive two-dimensional gas chromatography-mass spectrometry (GC × GC-MS), with respect to a review that described this specific methodology published at the beginning of 2008 (Mondello et al., 2008). In fact, since then there has been considerable evolution in the MS field, certainly exceeding that observed in GC × GC. In particular, the present paper will cover the combination of novel MS machines [single quadrupole (Q) and triple quadrupole, isotope ratio, low- and high-resolution time-of-flight (ToF), hybrid (Q-ToF)] to GC × GC systems, and will position comprehensive two-dimensional gas chromatography within the wider context of separation science. © 2014 Wiley Periodicals, Inc. Mass Spec Rev 35:524-534, 2016.

The present research is based on the development and use of a flow-modulation (FM) comprehensive two-dimensional gas chromatography-quadrupole mass spectrometry (GC×GC-qMS) method for the determination of recently-highlighted (by the Scientific Committee on Consumer Safety) fragrance allergens (54) in cosmetics. FM GC×GC-qMS conditions were finely tuned to generate flow conditions (≈7 mL min(-1)) compatible with the qMS system used. Six-point calibration curves, over the range 1, 5, 10, 20, 50, 100 mg L(-1), were constructed for the 54 target allergens, with satisfactory linearity observed in all cases. Absolute quantification was performed by using extracted ions; target analyte identification was performed through measurement of ion ratios (qualifier/quantifier), full-scan MS database matching and the use of linear retention indices. Additional analytical figures of merit subjected to measurement were intra-day repeatability, accuracy at the 25 and 5 mg L(-1) levels, and limits of detection and quantification. The number of data points per peak, along with mass spectral skewing, was also subjected to evaluation. Finally, the FM GC×GC-qMS method was used not only for the quantification of target allergens in five commercial perfumes, but also for general qualitative profiling.

Phthalates are a group of synthetic compounds mainly used as plasticizers, which have been classified as endocrine-disrupting chemicals and potential human-cancer causing agents. They can be found in high amounts in foods, deriving mainly from plastic packaging. The analytical determination of these compounds is very challenging since they are ubiquitous. Therefore, minimization of sample manipulation is highly desirable. The present work exploited the application of a solid-phase microextraction method for the analysis of phthalates in vegetable oil. A preliminary comparison between a polydimethylsiloxane (PDMS) and a Carbopack Z/PDMS fiber was carried out both in the headspace and direct immersion extraction modes. Before immersing the fiber, a rapid liquid–liquid extraction was performed using acetonitrile to remove the bulk of triglycerides. PDMS in the direct immersion mode showed the best performance. The method was fully validated obtaining a good linearity with a coefficient of correlation of over 0.9960 for all compounds, repeatability and accuracy values generally better than 10%, and very good limit of quantification values.