Cross-Validation of Metabolic Phenotypes in SARS-CoV-2 Infected Subpopulations Using Targeted Liquid Chromatography-Mass Spectrometry (LC-MS).

To ensure biological validity in metabolic phenotyping, findings must be replicated in independent sample sets. Targeted workflows have long been heralded as ideal platforms for such validation due to their robust quantitative capability. We evaluated the capability of liquid chromatography-mass spectrometry (LC-MS) assays targeting organic acids and bile acids to validate metabolic phenotypes of SARS-CoV-2 infection. Two independent sample sets were collected: (1) Australia: plasma, SARS-CoV-2 positive (n = 20), noninfected healthy controls (n = 22) and COVID-19 disease-like symptoms but negative for SARS-CoV-2 infection (n = 22). (2) Spain: serum, SARS-CoV-2 positive (n = 33) and noninfected healthy controls (n = 39). Multivariate modeling using orthogonal projections to latent structures discriminant analyses (OPLS-DA) classified healthy controls from SARS-CoV-2 positive (Australia; R2 = 0.17, ROC-AUC = 1; Spain R2 = 0.20, ROC-AUC = 1). Univariate analyses revealed 23 significantly different (p < 0.05) metabolites between healthy controls and SARS-CoV-2 positive individuals across both cohorts. Significant metabolites revealed consistent perturbations in cellular energy metabolism (pyruvic acid, and 2-oxoglutaric acid), oxidative stress (lactic acid, 2-hydroxybutyric acid), hypoxia (2-hydroxyglutaric acid, 5-aminolevulinic acid), liver activity (primary bile acids), and host-gut microbial cometabolism (hippuric acid, phenylpropionic acid, indole-3-propionic acid). These data support targeted LC-MS metabolic phenotyping workflows for biological validation in independent sample sets.

Maternal oxidative stress during pregnancy associated with emotional and behavioural problems in early childhood: implications for foetal programming.

Childhood mental disorders, including emotional and behavioural problems (EBP) are increasingly prevalent. Higher maternal oxidative stress (OS) during pregnancy (matOSpreg) is linked to offspring mental disorders. Environmental factors contribute to matOSpreg. However, the role of matOSpreg in childhood EBP is unclear. We investigated the associations between (i) matOSpreg and offspring EBP; (ii) social and prenatal environmental factors and matOSpreg; and (iii) social and prenatal factors and childhood EBP and evaluated whether matOSpreg mediated these associations. Maternal urinary OS biomarkers, 8-hydroxyguanosine (8-OHGua; an oxidative RNA damage marker) and 8-hydroxy-2'-deoxyguanosine (8-OHdG; an oxidative DNA damage marker), at 36 weeks of pregnancy were quantified by liquid chromatography-mass spectrometry in a population-derived birth cohort, Barwon Infant Study (n = 1074 mother-infant pairs). Social and prenatal environmental factors were collected by mother-reported questionnaires. Offspring total EBP was measured by Child Behavior Checklist Total Problems T-scores at age two (n = 675) and Strengths and Difficulties Questionnaire Total Difficulties score at age four (n = 791). Prospective associations were examined by multivariable regression analyses adjusted for covariates. Mediation effects were evaluated using counterfactual-based mediation analysis. Higher maternal urinary 8-OHGua at 36 weeks (mat8-OHGua36w) was associated with greater offspring total EBP at age four (ß = 0.38, 95% CI (0.07, 0.69), P = 0.02) and age two (ß = 0.62, 95% CI (-0.06, 1.30), P = 0.07). Weaker evidence of association was detected for 8-OHdG. Five early-life factors were associated with both mat8-OHGua36w and childhood EBP (P-range < 0.001-0.05), including lower maternal education, socioeconomic disadvantage and prenatal tobacco smoking. These risk factor-childhood EBP associations were partly mediated by higher mat8-OHGua36w (P-range = 0.01-0.05). Higher matOSpreg, particularly oxidant RNA damage, is associated with later offspring EBP. Effects of some social and prenatal lifestyle factors on childhood EBP were partly mediated by matOSpreg. Future studies are warranted to further elucidate the role of early-life oxidant damage in childhood EBP.

A Complete Pipeline for Untargeted Urinary Volatolomic Profiling with Sorptive Extraction and Dual Polar and Nonpolar Column Methodologies Coupled with Gas Chromatography Time-of-Flight Mass Spectrometry.

Volatolomics offers an opportunity for noninvasive detection and monitoring of human disease. While gas chromatography-mass spectrometry (GC-MS) remains the technique of choice for analyzing volatile organic compounds (VOCs), barriers to wider adoption in clinical practice still exist, including: sample preparation and introduction techniques, VOC extraction, throughput, volatolome coverage, biological interpretation, and quality control (QC). Therefore, we developed a complete pipeline for untargeted urinary volatolomic profiling. We optimized a novel extraction technique using HiSorb sorptive extraction, which exhibited high analytical performance and throughput. We achieved a broader VOC coverage by using HiSorb coupled with a set of complementary chromatographic methods and time-of-flight mass spectrometry. Furthermore, we developed a data preprocessing strategy by evaluating internal standard normalization, batch correction, and we adopted strict QC measures including removal of nonlinearly responding, irreproducible, or contaminated metabolic features, ensuring the acquisition of high-quality data. The applicability of this pipeline was evaluated in a clinical cohort consisting of pancreatic ductal adenocarcinoma (PDAC) patients (n = 28) and controls (n = 33), identifying four urinary candidate biomarkers (2-pentanone, hexanal, 3-hexanone, and p-cymene), which can successfully discriminate the cancer and noncancer subjects. This study presents an optimized, high-throughput, and quality-controlled pipeline for untargeted urinary volatolomic profiling. Use of the pipeline to discriminate PDAC from control subjects provides proof of principal of its clinical utility and potential for application in future biomarker discovery studies.

Systemic Perturbations in Amine and Kynurenine Metabolism Associated with Acute SARS-CoV-2 Infection and Inflammatory Cytokine Responses.

We performed quantitative metabolic phenotyping of blood plasma in parallel with cytokine/chemokine analysis from participants who were either SARS-CoV-2 (+) (n = 10) or SARS-CoV-2 (-) (n = 49). SARS-CoV-2 positivity was associated with a unique metabolic phenotype and demonstrated a complex systemic response to infection, including severe perturbations in amino acid and kynurenine metabolic pathways. Nine metabolites were elevated in plasma and strongly associated with infection (quinolinic acid, glutamic acid, nicotinic acid, aspartic acid, neopterin, kynurenine, phenylalanine, 3-hydroxykynurenine, and taurine; p < 0.05), while four metabolites were lower in infection (tryptophan, histidine, indole-3-acetic acid, and citrulline; p < 0.05). This signature supports a systemic metabolic phenoconversion following infection, indicating possible neurotoxicity and neurological disruption (elevations of 3-hydroxykynurenine and quinolinic acid) and liver dysfunction (reduction in Fischer's ratio and elevation of taurine). Finally, we report correlations between the key metabolite changes observed in the disease with concentrations of proinflammatory cytokines and chemokines showing strong immunometabolic disorder in response to SARS-CoV-2 infection.

Incomplete Systemic Recovery and Metabolic Phenoreversion in Post-Acute-Phase Nonhospitalized COVID-19 Patients: Implications for Assessment of Post-Acute COVID-19 Syndrome.

We present a multivariate metabotyping approach to assess the functional recovery of nonhospitalized COVID-19 patients and the possible biochemical sequelae of "Post-Acute COVID-19 Syndrome", colloquially known as long-COVID. Blood samples were taken from patients ca. 3 months after acute COVID-19 infection with further assessment of symptoms at 6 months. Some 57% of the patients had one or more persistent symptoms including respiratory-related symptoms like cough, dyspnea, and rhinorrhea or other nonrespiratory symptoms including chronic fatigue, anosmia, myalgia, or joint pain. Plasma samples were quantitatively analyzed for lipoproteins, glycoproteins, amino acids, biogenic amines, and tryptophan pathway intermediates using Nuclear Magnetic Resonance (NMR) spectroscopy and mass spectrometry. Metabolic data for the follow-up patients (n = 27) were compared with controls (n = 41) and hospitalized severe acute respiratory syndrome SARS-CoV-2 positive patients (n = 18, with multiple time-points). Univariate and multivariate statistics revealed variable patterns of functional recovery with many patients exhibiting residual COVID-19 biomarker signatures. Several parameters were persistently perturbed, e.g., elevated taurine (p = 3.6 × 10-3 versus controls) and reduced glutamine/glutamate ratio (p = 6.95 × 10-8 versus controls), indicative of possible liver and muscle damage and a high energy demand linked to more generalized tissue repair or immune function. Some parameters showed near-complete normalization, e.g., the plasma apolipoprotein B100/A1 ratio was similar to that of healthy controls but significantly lower (p = 4.2 × 10-3) than post-acute COVID-19 patients, reflecting partial reversion of the metabolic phenotype (phenoreversion) toward the healthy metabolic state. Plasma neopterin was normalized in all follow-up patients, indicative of a reduction in the adaptive immune activity that has been previously detected in active SARS-CoV-2 infection. Other systemic inflammatory biomarkers such as GlycA and the kynurenine/tryptophan ratio remained elevated in some, but not all, patients. Correlation analysis, principal component analysis (PCA), and orthogonal-partial least-squares discriminant analysis (O-PLS-DA) showed that the follow-up patients were, as a group, metabolically distinct from controls and partially comapped with the acute-phase patients. Significant systematic metabolic differences between asymptomatic and symptomatic follow-up patients were also observed for multiple metabolites. The overall metabolic variance of the symptomatic patients was significantly greater than that of nonsymptomatic patients for multiple parameters (χ2p = 0.014). Thus, asymptomatic follow-up patients including those with post-acute COVID-19 Syndrome displayed a spectrum of multiple persistent biochemical pathophysiology, suggesting that the metabolic phenotyping approach may be deployed for multisystem functional assessment of individual post-acute COVID-19 patients.

Breath Volatile Organic Compound Profiling of Colorectal Cancer Using Selected Ion Flow-tube Mass Spectrometry.

OBJECTIVE: BACKGROUND:: Breath VOCs have the potential to noninvasively diagnose cancer. METHODS: Exhaled breath samples were collected using 2-L double-layered Nalophan bags, and were analyzed using selected-ion-flow-tube mass-spectrometry. Gold-standard test for comparison was endoscopy for luminal inspection and computed tomography (CT) to confirm cancer recurrence. Three studies were conducted: RESULTS:: CONCLUSION:: This study suggests the association of a single breath biomarker with the primary presence and recurrence of CRCa. Further multicenter validation studies are required to validate these findings.

Mass-Spectrometry Analysis of Mixed-Breath, Isolated-Bronchial-Breath, and Gastric-Endoluminal-Air Volatile Fatty Acids in Esophagogastric Cancer.

A noninvasive breath test has the potential to improve survival from esophagogastric cancer by facilitating earlier detection. This study aimed to investigate the production of target volatile fatty acids (VFAs) in esophagogastric cancer through analysis of the ex vivo headspace above underivatized tissues and in vivo analysis within defined anatomical compartments, including analysis of mixed breath, isolated bronchial breath, and gastric-endoluminal air. VFAs were measured by PTR-ToF-MS and GC-MS. Levels of VFAs (acetic, butyric, pentanoic, and hexanoic acids) and acetone were elevated in ex vivo experiments in the headspace above esophagogastric cancer compared with the levels in samples from control subjects with morphologically normal and benign conditions of the upper gastrointestinal tract. In 25 patients with esophagogastric cancer and 20 control subjects, receiver-operating-characteristic analysis for the cancer-specific VFAs butyric acid ( P < 0.001) and pentatonic acid ( P = 0.005) within in vivo gastric-endoluminal air gave an area under the curve of 0.80 (95% confidence interval of 0.65 to 0.93, P = 0.01). Compared with mixed- and bronchial-breath samples, all examined VFAs were found in highest concentrations within esophagogastric-endoluminal air. In addition, VFAs were higher in all samples derived from cancer patients compared with in the controls. Equivalence of VFA levels within the mixed and bronchial breath of cancer patients suggests that their origin within breath is principally derived from the lungs and, by inference, from the systemic circulation as opposed to direct passage from the upper gastrointestinal tract. These findings highlight the potential to utilize VFAs for endoluminal-gas biopsies and noninvasive mixed-exhaled-breath testing for esophagogastric-cancer detection.

Enantiomeric separation in comprehensive two-dimensional gas chromatography with accurate mass analysis.

Chiral comprehensive two-dimensional gas chromatography (eGC×GC) coupled to quadrupole-accurate mass time-of-flight mass spectrometry (QTOFMS) was evaluated for its capability to report the chiral composition of several monoterpenes, namely, α-pinene, ß-pinene, and limonene in cardamom oil. Enantiomers in a standard mixture were fully resolved by direct enantiomeric-GC analysis with a 2,3-di-O-methyl-6-t-butylsilyl derivatized ß-cyclodextrin phase; however, the (+)-(R)-limonene enantiomer in cardamom oil was overlapped with other background components including cymene and cineole. Verification of (+)-(R)-limonene components based on characteristic ions at m/z 136, 121, and 107 acquired by chiral single-dimension GC-QTOFMS in the alternate MS/MSMS mode of operation was unsuccessful due to similar parent/daughter ions generated by interfering or co-eluting cymene and cineole. Column phases SUPELCOWAX, SLB-IL111, HP-88, and SLB-IL59, were incorporated as the second dimension column ((2)D) in chiral GC×GC analysis; the SLB-IL59 offered the best resolution for the tested monoterpene enantiomers from the matrix background. Enantiomeric ratios for α-pinene, ß-pinene, and limonene were determined to be 1.325, 2.703, and 1.040, respectively, in the cardamom oil sample based on relative peak area data.

Unravelling inulin molecules in food sources using a matrix-assisted laser desorption/ionization magnetic resonance mass spectrometry (MALDI-MRMS) pipeline.

Inulin, a polysaccharide characterized by a ß-2,1 fructosyl-fructose structure terminating in a glucosyl moiety, is naturally present in plant roots and tubers. Current methods provide average degrees of polymerization (DP) but lack information on the distribution and absolute concentration of each DP. To address this limitation, a reproducible (CV < 10 %) high throughput (<2 min/sample) MALDI-MRMS approach capable of characterizing and quantifying inulin molecules in plants using matched-matrix consisting of α-cyano-4-hydroxycinnamic acid butylamine salt (CHCA-BA), chicory inulin-12C and inulin-13C was developed. The method identified variation in chain lengths and concentration of inulin across various plant species. Globe artichoke hearts, yacón and elephant garlic yielded similar concentrations at 15.6 g/100 g dry weight (DW), 16.8 g/100 g DW and 17.7 g/100 g DW, respectively, for DP range between 9 and 22. In contrast, Jerusalem artichoke demonstrated the highest concentration (53.4 g/100 g DW) within the same DP ranges. Jerusalem artichoke (DPs 9-32) and globe artichoke (DPs 9-36) showed similar DP distributions, while yacón and elephant garlic displayed the narrowest and broadest DP ranges (DPs 9-19 and DPs 9-45, respectively). Additionally, qualitative measurement for all inulin across all plant samples was feasible using the peak intensities normalized to Inulin-13C, and showed that the ratio of yacón, elephant garlic and Jerusalem was approximately one, two and three times that of globe artichoke. This MALDI-MRMS approach provides comprehensive insights into the structure of inulin molecules, opening avenues for in-depth investigations into how DP and concentration of inulin influence gut health and the modulation of noncommunicable diseases, as well as shedding light on refining cultivation practices to elevate the beneficial health properties associated with specific DPs.

Integrated multidimensional and comprehensive 2D GC analysis of fatty acid methyl esters.

Fatty acid methyl ester (FAME) profiling in complex fish oil and milk fat samples was studied using integrated comprehensive 2D GC (GC × GC) and multidimensional GC (MDGC). Using GC × GC, FAME compounds--cis- and trans-isomers, and essential fatty acid isomers--ranging from C18 to C22 in fish oil and C18 in milk fat were clearly displayed in contour plot format according to structural properties and patterns, further identified based on authentic standards. Incompletely resolved regions were subjected to MDGC, with Cn (n = 18, 20) zones transferred to a (2)D column. Elution behavior of C18 FAME on various (2)D column phases (ionic liquids IL111, IL100, IL76, and modified PEG) was evaluated. Individual isolated Cn zones demonstrated about four-fold increased peak capacities. The IL100 provided superior separation, good peak shape, and utilization of elution space. For milk fat-derived FAME, the (2)D chromatogram revealed at least three peaks corresponding to C18:1, more than six peaks for cis/trans-C18:2 isomers, and two peaks for C18:3. More than 17 peaks were obtained for the C20 region of fish oil-derived FAMEs using MDGC, compared with ten peaks using GC × GC. The MDGC strategy is useful for improved FAME isomer separation and confirmation.

Targeted lipidomics coupled with machine learning for authenticating the provenance of chicken eggs.

Free-range eggs are ethically desirable but as with all high-value commercial products, the establishment of provenance can be problematic. Here, we compared a simple one-step isopropanol method to a two-step methyl-tert-butyl ether method for extracting lipid species in chicken egg yolks before liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The isopropanol method extracted 937 lipid species from 20 major lipid subclasses with high reproducibility (CV < 30 %). Machine learning techniques could differentiate conventional cage, barn, and free-range eggs using an external test dataset with an accuracy of 0.94, 0.82, and 0.82, respectively. Lipid species that differentiated cage eggs were predominantly phosphocholines and phosphoethanolamines whilst the free-range egg lipidomes were dominated by acylglycerides with up to three fatty acids. The lipid profiles were found to be characteristic of the cage, barns, and free-range eggs. The lipidomic analysis together with the statistical modeling approach thus provides an efficient tool for verifying the provenance of conventional chicken eggs.

System design for integrated comprehensive and multidimensional gas chromatography with mass spectrometry and olfactometry.

An integrated system having the combined capability to perform gas chromatography (GC), comprehensive two-dimensional GC (GC × GC), and target heart-cut multidimensional GC (MDGC) using olfactometry (O), flame ionization (FID), and/or mass spectrometry (MS) detection is described. This combines a number of contemporary GC methods into a single instrument to provide very high resolution profiling of a sample. This provides initial assessment of volatile compound composition through GC × GC analysis with FID, which can be correlated with GC analysis using parallel O and FID detection. Subsequent microfluidic (Deans) switching selects regions (heart-cuts) of the chromatographic elution from the first dimension ((1)D) column for further resolution on a long second dimension ((2)D(L)) column for parallel detection of O and MS. Various (2)D(L) operational conditions, as well as the effect of different heart-cut (H/C) duration, were compared. The favored mode involves cryotrapping of heart-cuts, cooling the oven, and reducing carrier flow to offer greater efficiency. An analytical strategy that incorporates GC-FID/O, GC × GC-FID, and MDGC-MS/O analyses with cumulative solid phase microextraction (SPME) sampling for volatile sample enrichment is presented in this work. Excellent qualitative and quantitative performance was demonstrated with a Shiraz wine sample and an allergens mixture, with tentative identification of acetic acid, octen-3-ol, and ethyl octanoate as aroma contributors in Shiraz wine and determination of ß-damascenone (floral odor) well separated from hexanoic acid (sweaty odor). A novel approach to obtain (2)D retention indices is reported, allowing matching of mass spectral, (1)I (retention index in (1)D) and (2)I (retention index in (2)D) data. The method employs the same olfactory detector at the end of the (1)D and (2)D(L) columns.

Universal method for online enrichment of target compounds in capillary gas chromatography using in-oven cryotrapping.

A method is described that permits automated online enrichment of injected compounds in multidimensional gas chromatography by using a microfluidic heart-cut (H-C) device to direct target compounds into a cryogenically cooled internal trap (cryotrap, CT). By performing multiple injections of a sample, selected compounds or regions of a primary column separation can be collected in the CT. Remobilizing the trapped species allows elution and further resolution on the second column. Using a well-balanced H-C device, compounds can be fully excluded from the collection step or quantitatively transferred to the CT. Peak areas of the remobilized compound correlate well with the number of sample injections. Trapping on various column phases shows the method is suited to quantitative trapping of alkanes of mass greater than about dodecane and fatty acid methyl esters greater than the C8 homologue. Caffeine and menthol standards of concentration 100 µg mL(-1) gave peak area correlation coefficients for 1-10 and 1-50 replicate split injections of 1 µL volume of 0.999 and 0.996, respectively. Peak height correlations were less favorable as a result of peak broadening on the second column, presumably due to overloading at greater collected mass. The method was applied to 0.2% solutions of peppermint oil (menthol; a major component; 44%) and 1.0% lavender oil (α-terpineol and neryl acetate; minor components of 1.05 and 0.42% abundance). The minor components gave good area and height correlations, and good recovery around 90% was observed for menthol compounds recovered from 15 accumulations. Response amplification was further demonstrated for menthol from mint oil headspace sampling using solid phase microextraction. This approach should be a valuable adjunct for improved detection specificity, for detectors of low sensitivity, and when prior sample concentration provides insufficient response of selected target analytes.

Techniques and application in comprehensive multidimensional gas chromatography - mass spectrometry.

In contrast to the well-known comprehensive two-dimensional gas chromatography (GC×GC) method, it is possible to define comprehensive multidimensional gas chromatography. 'Comprehensiveness' relates to analysis of the whole sample. Two-dimensional and multidimensional here refer to the use of at least two separation stages for analysis, however comprehensive 2DGC now appears to be reserved for the GC×GC method. This may be differentiated from comprehensive MDGC (CMDGC) simply by the analysis time assigned to the second (2D) column, although there does not appear to be a specific definition that relates to this analysis time parameter. A number of different implementation protocols for comprehensive MDGC are described here, that may involve either a single, or multiple, injection(s). In all cases, independent retention must be achieved on each dimension to ensure the probability of enhanced separation. An original application of a crude oil sample is presented to illustrate development of the MDGC approach that incorporates two Deans switches (DS) and a cryogenic trapping approach, performed using a sequential heart-cut (H/C) event method incremented by 0.5 min for each injection; a total of 40 injections is used to analyse the total sample. The higher peak capacity and consequently greater resolution on the long 2D column is illustrated, compared with that expected for conventional GC×GC, with tentative identification in order to classify chemical classes. Incorporating an approach to acquiring retention indices may be implemented, although its utility for petroleum hydrocarbons is limited. Structured groupings of different chemical classes, as exemplified by mono and diaromatics for the crude oil sample, were noted.

Cross-platform mass spectrometry annotation in breathomics of oesophageal-gastric cancer.

Disease breathomics is gaining importance nowadays due to its usefulness as non-invasive early cancer detection. Mass spectrometry (MS) technique is often used for analysis of volatile organic compounds (VOCs) associated with cancer in the exhaled breath but a long-standing challenge is the uncertainty in mass peak annotation for potential volatile biomarkers. This work describes a cross-platform MS strategy employing selected-ion flow tube mass spectrometry (SIFT-MS), high resolution gas chromatography-mass spectrometry (GC-MS) retrofitted with electron ionisation (EI) and GC-MS retrofitted with positive chemical ionisation (PCI) as orthogonal analytical approaches in order to provide facile identification of the oxygenated VOCs from breath of cancer patients. In addition, water infusion was applied as novel efficient PCI reagent in breathomics analysis, depicting unique diagnostic ions M+ or [M-17]+ for VOC identification. Identity confirmation of breath VOCs was deduced using the proposed multi-platform workflow, which reveals variation in breath oxygenated VOC composition of oesophageal-gastric (OG) cancer patients with dominantly ketones, followed by aldehydes, alcohols, acids and phenols in decreasing order of relative abundance. Accurate VOC identification provided by cross-platform approach would be valuable for the refinement of diagnostic VOC models and the understanding of molecular drivers of VOC production.

Assessment of a Noninvasive Exhaled Breath Test for the Diagnosis of Oesophagogastric Cancer.

Importance: Early esophagogastric cancer (OGC) stage presents with nonspecific symptoms. Objective: The aim of this study was to determine the accuracy of a breath test for the diagnosis of OGC in a multicenter validation study. Design, Setting, and Participants: Patient recruitment for this diagnostic validation study was conducted at 3 London hospital sites, with breath samples returned to a central laboratory for selected ion flow tube mass spectrometry (SIFT-MS) analysis. Based on a 1:1 cancer:control ratio, and maintaining a sensitivity and specificity of 80%, the sample size required was 325 patients. All patients with cancer were on a curative treatment pathway, and patients were recruited consecutively. Among the 335 patients included; 172 were in the control group and 163 had OGC. Interventions: Breath samples were collected using secure 500-mL steel breath bags and analyzed by SIFT-MS. Quality assurance measures included sampling room air, training all researchers in breath sampling, regular instrument calibration, and unambiguous volatile organic compounds (VOCs) identification by gas chromatography mass spectrometry. Main Outcomes and Measures: The risk of cancer was identified based on a previously generated 5-VOCs model and compared with histopathology-proven diagnosis. Results: Patients in the OGC group were older (median [IQR] age 68 [60-75] vs 55 [41-69] years) and had a greater proportion of men (134 [82.2%]) vs women (81 [47.4%]) compared with the control group. Of the 163 patients with OGC, 123 (69%) had tumor stage T3/4, and 106 (65%) had nodal metastasis on clinical staging. The predictive probabilities generated by this 5-VOCs diagnostic model were used to generate a receiver operator characteristic curve, with good diagnostic accuracy, area under the curve of 0.85. This translated to a sensitivity of 80% and specificity of 81% for the diagnosis of OGC. Conclusions and Relevance: This study shows the potential of breath analysis in noninvasive diagnosis of OGC in the clinical setting. The next step is to establish the diagnostic accuracy of the test among the intended population in primary care where the test will be applied.

Relating pressure tuned coupled column ensembles with the solvation parameter model for tunable selectivity in gas chromatography.

The differential pressure drop of carrier gas by tuning the junction point pressure of a coupled column gas chromatographic system leads to a unique selectivity of the overall separation, which can be tested using a mixture of compounds with a wide range of polarity. This study demonstrates a pressure tuning (PT) GC system employing a microfluidic Deans switch located at the mid-point of the two capillary columns. This PT system allowed variations of inlet-outlet pressure differences of the two columns in a range of 52-17psi for the upstream column and 31-11psi for the downstream column. Peak shifting (differential migration) of compounds due to PT difference are related to a first order regression equation in a Plackett-Burman factorial study. Increased first (upstream) column pressure drop makes the second column characteristics more significant in the coupled column retention behavior, and conversely increased second (downstream) column pressure drop makes the first column characteristics more apparent; such variation can result in component swapping between polar and non-polar compounds. The coupled column system selectivity was evaluated in terms of linear solvation energy relationship (LSER) parameters, and their relation with different pressure drop effects has been constructed by applying multivariate principle component analysis (PCA). It has been found that the coupled column PT system descriptors provide a result that shows a clear clustering of different pressure settings, somewhat intermediate between those of the two commercial columns. This is equivalent to that obtained from a conventional single-column GC analysis where the interaction energy contributed from the stationary phases can be significantly adjusted by choice of midpoint PT. This result provides a foundation for pressure differentiation for selectivity enhancement.

Review of the role and methodology of high resolution approaches in aroma analysis.

Analysis of the odour complexity in food and beverage products demands high resolution approaches for distinguishing individual aroma-impact compound(s), and for assessing their contribution to the global aroma of a sample. This paper aims to review current applications incorporating different advanced separation methodologies, and their roles in achieving high resolution aroma analysis. This includes prior low resolution gas chromatography-olfactometry (GC-O) with fractionation procedures using chemical manipulation, adsorption chromatography and ion exchange separation. Innovative multidimensional gas chromatography (MDGC) arrangements that are appropriately designed with olfactometry are of specific focus here. The revelation of resolved components using these integrated approaches provides significantly improved knowledge of aroma composition in samples.

Application of integrated comprehensive/multidimensional gas chromatography with mass spectrometry and olfactometry for aroma analysis in wine and coffee.

Component coelution in chromatographic analysis complicates identification and attribution of individual odour-active volatile molecules in complex multi-component samples. An integrated system incorporating comprehensive two-dimensional gas chromatography (GC × GC) and multidimensional gas chromatography (MDGC), with flame ionisation, olfactometry and mass spectrometry detection was developed to circumvent data correlation across different systems. Identification of potent odorants in Shiraz wine and the headspace of ground coffee are demonstrated as selected applications. Multiple solid-phase microextraction (SPME) sampling with GC-O located odour-active regions; GC × GC established the complexity of odour-active regions; MDGC provided high-resolution separation for each region; simultaneous 'O' and MS detection completed the analysis for target resolved peaks. Seven odour regions in Shiraz were analysed with MDGC-O/MS detection, revealing 11 odour volatiles through matching of mass spectrometry and retention indices from both separating dimensions, including acetic acid; octen-3-ol; ethyl octanoate; methyl-2-oxo-nonanoate; butanoic acid, 2-methylbutanoic acid, and 3-methylbutanoic acid; 3-(methylthio)-1-propanol; hexanoic acid; ß-damascenone; and ethyl-3-phenylpropanoate. A capsicum odour in ground coffee was identified as 2-methoxy-3-isobutylpyrazine with a 5-fold increase in S/N of the odorant when acquired using a 6-time cumulative SPME sampling approach.

Evaluation of comprehensive two-dimensional gas chromatography with accurate mass time-of-flight mass spectrometry for the metabolic profiling of plant-fungus interaction in Aquilaria malaccensis.

To explore the possible obligate interactions between the phytopathogenic fungus and Aquilaria malaccensis which result in generation of a complex array of secondary metabolites, we describe a comprehensive two-dimensional gas chromatography (GC × GC) method, coupled to accurate mass time-of-flight mass spectrometry (TOFMS) for the untargeted and comprehensive metabolic profiling of essential oils from naturally infected A. malaccensis trees. A polar/non-polar column configuration was employed, offering an improved separation pattern of components when compared to other column sets. Four different grades of the oils displayed quite different metabolic patterns, suggesting the evolution of a signalling relationship between the host tree (emergence of various phytoalexins) and fungi (activation of biotransformation). In total, ca. 550 peaks/metabolites were detected, of which tentative identification of 155 of these compounds was reported, representing between 20.1% and 53.0% of the total ion count. These are distributed over the chemical families of monoterpenic and sesquiterpenic hydrocarbons, oxygenated monoterpenes and sesquiterpenes (comprised of ketone, aldehyde, oxide, alcohol, lactone, keto-alcohol and diol), norterpenoids, diterpenoids, short chain glycols, carboxylic acids and others. The large number of metabolites detected, combined with the ease with which they are located in the 2D separation space, emphasises the importance of a comprehensive analytical approach for the phytochemical analysis of plant metabolomes. Furthermore, the potential of this methodology in grading agarwood oils by comparing the obtained metabolic profiles (pattern recognition for unique metabolite chemical families) is discussed. The phytocomplexity of the agarwood oils signified the production of a multitude of plant-fungus mediated secondary metabolites as chemical signals for natural ecological communication. To the best of our knowledge, this is the most complete information available so far about essential oils of A. malaccensis, which represents a valuable extension to available data for advanced studies on microbial-mediated biotransformation of terpenes, and offers promise for potential discovery of unanticipated phytochemicals, and biotechnological exploitation.