Detection of tuberculosis-associated compounds from human skin by GCxGC-TOFMS.

Tuberculosis (TB) remains a global health concern. This study aimed to investigate the potential of human skin volatile organic compounds (VOCs) as prospective biomarkers for TB diagnosis. It employed a non-invasive approach using a wearable silicone rubber band for VOC sampling, comprehensive gas chromatography - time of flight mass spectrometry (GCxGC-TOFMS), and chemometric techniques. Both targeted and untargeted biochemical screening was utilized to explore biochemical differences between healthy individuals and those with TB infection. Results confirmed a correlation between compounds found in this study, and those reported for TB from other biofluids. In a comparison to known TB-associated compounds from other biofluids our analysis established the presence of 27 of these compounds emanating from human skin. Additionally, 16 previously unreported compounds were found as potential biomarkers. The diagnostic ability of the VOCs selected by statistical methods was investigated using predictive modelling techniques. Artificial neural network multi-layered perceptron (ANN) yielded two compounds, 1H-indene, 2,3 dihydro-1,1,3-trimethyl-3-phenyl; and heptane-3-ethyl-2-methyl, as the most discriminatory, and could differentiate between TB-positive (n = 15) and TB-negative (n = 23) individuals with an area under the receiver operating characteristic curve (AUROC) of 92 %, a sensitivity of 100 % and a specificity of 94 % for six targeted features. For untargeted analysis, ANN assigned 3-methylhexane as the most discriminatory between TB-positive and TB- negative individuals. An AUROC of 98.5 %, a sensitivity of 83 %, and a specificity of 88 % were obtained for 16 untargeted features as chosen by high performance variable selection. The obtained values compare highly favourable to alternative diagnostic methods such as breath analysis and GeneXpert. Consequently, human skin VOCs hold considerable potential as a TB diagnostic screening test.

The metabolic consequences of HIV/TB co-infection.

BACKGROUND: The synergy between the human immunodeficiency virus (HIV) and Mycobacterium tuberculosis during co-infection of a host is well known. While this synergy is known to be driven by immunological deterioration, the metabolic mechanisms that contribute to the associated disease burden experienced during HIV/tuberculosis (TB) co-infection remain poorly understood. Furthermore, while anti-HIV treatments suppress viral replication, these therapeutics give rise to host metabolic disruption and adaptations beyond that induced by only infection or disease. METHODS: In this study, the serum metabolic profiles of healthy controls, untreated HIV-negative TB-positive patients, untreated HIV/TB co-infected patients, and HIV/TB co-infected patients on antiretroviral therapy (ART), were measured using two-dimensional gas chromatography time-of-flight mass spectrometry. Since no global metabolic profile for HIV/TB co-infection and the effect of ART has been published to date, this pilot study aimed to elucidate the general areas of metabolism affected during such conditions. RESULTS: HIV/TB co-infection induced significant changes to the host's lipid and protein metabolism, with additional microbial product translocation from the gut to the blood. The results suggest that HIV augments TB synergistically, at least in part, contributing to increased inflammation, oxidative stress, ART-induced mitochondrial damage, and its detrimental effects on gut health, which in turn, affects energy availability. ART reverses these trends to some extent in HIV/TB co-infected patients but not to that of healthy controls. CONCLUSION: This study generated several new hypotheses that could direct future metabolic studies, which could be combined with other research techniques or methodologies to further elucidate the underlying mechanisms of these changes.

Tuberculosis is associated with sputum metabolome variations, irrespective of patient sex or HIV status: an untargeted GCxGC-TOFMS study.

INTRODUCTION: Various studies have identified TB-induced metabolome variations. However, in most of these studies, a large degree of variation exists between individual patients. OBJECTIVES: To identify differential metabolites for TB, independent of patients' sex or HIV status. METHODS: Untargeted GCxGC/TOF-MS analyses were applied to the sputum of 31 TB + and 197 TB- individuals. Univariate statistics were used to identify metabolites which are significantly different between TB + and TB- individuals (a) irrespective of HIV status, and (b) with a HIV + status. Comparisons a and b were repeated for (i) all participants, (ii) males only and (iii) females only. RESULTS: Twenty-one compounds were significantly different between the TB + and TB- individuals within the female subgroup (11% lipids; 10% carbohydrates; 1% amino acids, 5% other and 73% unannotated), and 6 within the male subgroup (20% lipids; 40% carbohydrates; 6% amino acids, 7% other and 27% unannotated). For the HIV + patients (TB + vs. TB-), a total of 125 compounds were significant within the female subgroup (16% lipids; 8% carbohydrates; 12% amino acids, 6% organic acids, 8% other and 50% unannotated), and 44 within the male subgroup (17% lipids; 2% carbohydrates; 14% amino acids related, 8% organic acids, 9% other and 50% unannotated). Only one annotated compound, 1-oleoyl lysophosphaditic acid, was consistently identified as a differential metabolite for TB, irrespective of sex or HIV status. The potential clinical application of this compound should be evaluated further. CONCLUSIONS: Our findings highlight the importance of considering confounders in metabolomics studies in order to identify unambiguous disease biomarkers.

Lime and odour: A preliminary investigation into the effect of hydrated lime on the volatiles emitted from human remains.

The location of human remains is performed primarily through the aid of cadaver detection dogs, which rely on the malodour produced through decomposition of decaying bodies. Malefactors will attempt to conceal these putrefactive odours through chemical additions such as lime, which is also wrongly believed to accelerate decomposition and prevent the identification of the victim. Despite the frequency of lime in forensic applications, to date no research has been performed to determine its effect on the volatile organic compounds (VOCs) released during human decomposition. This research was therefore conducted to ascertain the effects of hydrated lime on the VOC profile of human remains. Two human donors were used in a field trial at the Australian Facility for Taphonomic Experimental Research (AFTER): one donor was covered with hydrated lime, and the other had no chemical additions acting as a control. VOC samples were collected over a period of 100 days and analysed using comprehensive two-dimensional gas chromatography coupled with time-of-flight mass spectrometry (GC×GC-TOFMS). The volatile samples were accompanied by visual observations of how decomposition progressed. The results showed that lime application decreased the rate of decomposition and decreased total carrion insect activity. Lime increased the abundance of VOCs during the fresh and bloat stages of decay, however the abundance of compounds plateaued during active and advanced decomposition and were much lower than those detected from the control donor. Despite this suppression of VOCs, the study found that dimethyl disulfide and dimethyl trisulfide, key sulfur-containing compounds, were still produced in high quantities, and can thus still be used to locate chemically altered human remains. Knowledge of the effects of lime on human decomposition can inform the training of cadaver detection dogs, and ensure a greater chance at locating victims of crimes or mass disasters.

Polychlorinated dibenzo-p-dioxin/dibenzofurans (PCDD/Fs) contamination in sediments and fish from Msimbazi River in Dar es Salam, Tanzania: patterns, sources, and their exposure to humans.

Results of a two-dimensional gas chromatograph time of flight mass spectroscopy (GCXGC-TOFMS) determination of polychlorinated dibenzo-p-dioxin (PCDDs) and polychlorinated dibenzofurans (PCDF) in sediments and catfish samples collected from the Msimbazi River are presented here. Samples were extracted using USEPA Method 1613. PCDD/Fs congeners in sediments ranged from 2.0 to 393.0 and 0.7 to 654.8 pg/g in the dry and wet seasons, respectively. 1,2,3,4,7,8,9-HepCDF was detected at the highest concentration, but all were lower than the USA action level of 1000 pg/g. Toxicity for each of the sampling points ranged from 19.7 to 36.5, with a mean concentration of 27.0 pg WHO 2005-TEQ g-1 in the dry season and 2.0 to 38.7 with a mean concentration of 20.7 pg WHO 2005-TEQ g-1 in the wet season. Analysis of variance (ANOVA) showed that there was no significant difference between PCDD/Fs TEQ during the dry and wet seasons (p = 0.08; α = 0.05). The highest TEQ value was estimated at Jangwani in the wet season. Toxicity of PCDD/Fs in catfish collected from the Msimbazi River ranged from 9.3 to 145.2, with a mean of 61.2 pg WHO2005-TEQg-1. Tetrachlorodibenzo dioxin (2, 3, 7, and 8-TCDD) was detected in all fish samples and ranged from 3.5 to 12.7 with a mean of 8.1 pg/g. The concentration of TCDD in fish exceeded the Agency for Toxic Substance and Diseases Registry MRL, thus posing a probable high risk to people whose dietary requirements depend on fish from the Msimbazi River.

Hedychium spicatum rhizome essential oil induces apoptosis in human prostate adenocarcinoma PC-3 cells via mitochondrial stress and caspase activation.

Hedychium spicatum is an essential oil bearing plant extensively used in the traditional system of medicine in several countries. Previous research has revealed H. spicatum essential oil (HSEO) to exhibit anti-tumor activity, although the mechanism of action is still unknown. Therefore, the current study was designed to carry out a comprehensive characterization of HSEO and evaluate the chemotherapeutic potential of HSEO against cancerous cells. The volatile constituents of HSEO was determined by one-dimensional gas chromatography with time-of-flight mass spectrometry (GC-TOFMS) and two-dimensional gas chromatography with time-of-flight mass spectrometry (GCxGC-TOFMS). In total, 193 phytocompounds could be detected, out of which 140 were identified for the first time. The major phytoconstituents detected by GCxGC-TOFMS were ß-pinene (10.94%), eucalyptol (6.45%), sabinene (5.48%) and trans-isolimonene (5.00%). GCxGC-TOFMS analysis showed two and half fold increase in the constituents over GC-TOFMS due to better chromatographic separation of constituents in the 2nd dimensional column. HSEO was tested for in vitro cytotoxic activity against cancerous (PC-3, HCT-116 and A-549) and normal (3T3-L1) cell, with HSEO being most selective for prostate cancer cell (PC-3) over non-tumorigenic fibroblast (3T3-L1) cell. HSEO treatment inhibited the colony formation ability of PC-3 cells. HSEO treatment caused apoptotic cell death and cell cycle arrest at G2/M and S phase in PC-3 cells. HSEO induced apoptosis via intracellular ROS accumulation, mitochondria depolarization and increased caspase-3, 8, and 9 levels in PC-3 cells. Additionally, HSEO treatment led to a decrease of Bcl-2 and Bcl-xL and increase of Bax and Bak protein levels. Overall, results from this study highlighted the anticancer potential of H. spicatum essential oil, which could be considered as a new agent for treating prostate cancer.

Analysis of sulfur compounds for crude oil fingerprinting using gas chromatography with sulfur chemiluminescence detector.

Oil spills are a severe problem worldwide due to the resulting damage to marine and coastal ecosystems and to local economies. Identification of the source of spilled oils can be challenging, especially if the oils have undergone severe weathering. Due to their high durability, biomarker compounds (e.g. hopanes, steranes) are widely used for oil fingerprinting. Some sulfur-containing heterocyclic compounds e.g. alkylated dibenzothiophenes are also considered to be highly resistant. In this study, the use of Gas Chromatography with Sulfur Chemiluminescence Detection was investigated as a means of oil fingerprinting using the distribution the sulfur compounds in five different fresh and weathered crude oils. Chemometric analysis was also performed. The results indicate that the sulfur compounds distribution is unique for each crude oil. The distributions of the heavy sulfur compounds (i.e., C2DBTs and C3DBTs) are unchanged after weathering. Therefore, the GC-SCD technique can be considered to support the oil spill identification.

Mutations in Rht-B1 Locus May Negatively Affect Frost Tolerance in Bread Wheat.

The wheat semi-dwarfing genes Rht (Reduced height) are widely distributed among the contemporary wheat varieties. These genes also exert pleiotropic effects on plant tolerance towards various abiotic stressors. In this work, frost tolerance was studied in three near-isogenic lines of the facultative variety 'April Bearded' (AB), carrying the wild type allele Rht-B1a (tall phenotype), and the mutant alleles Rht-B1b (semi-dwarf) and Rht-B1c (dwarf), and was further compared with the tolerance of a typical winter type variety, 'Mv Beres'. The level of freezing tolerance was decreasing in the order 'Mv Beres' > AB Rht-B1a > AB Rht-B1b > AB Rht-B1c. To explain the observed differences, cold acclimation-related processes were studied: the expression of six cold-related genes, the phenylpropanoid pathway, carbohydrates, amino acids, polyamines and compounds in the tricarboxylic acid cycle. To achieve this, a comprehensive approach was applied, involving targeted analyses and untargeted metabolomics screening with the help of gas chromatography/liquid chromatography­mass spectrometry setups. Several cold-related processes exhibited similar changes in these genotypes; indeed, the accumulation of eight putrescine and agmatine derivatives, 17 flavones and numerous oligosaccharides (max. degree of polymerization 18) was associated with the level of freezing tolerance in the 'April Bearded' lines. In summary, the mutant Rht alleles may further decrease the generally low frost tolerance of the Rht-B1a, and, based on the metabolomics study, the mechanisms of frost tolerance may differ for a typical winter variety and a facultative variety. Present results point to the complex nature of frost resistance.

Chemical Changes in the Broccoli Volatilome Depending on the Tissue Treatment.

The storage of plant samples as well as sample preparation for extraction have a significant impact on the profile of metabolites, however, these factors are often overlooked during experiments on vegetables or fruit. It was hypothesized that parameters such as sample storage (freezing) and sample pre-treatment methods, including the comminution technique or applied enzyme inhibition methods, could significantly influence the extracted volatile metabolome. Significant changes were observed in the volatile profile of broccoli florets frozen in liquid nitrogen at -20 °C. Those differences were mostly related to the concentration of nitriles and aldehydes. Confocal microscopy indicated some tissue deterioration in the case of slow freezing (-20 °C), whereas the structure of tissue, frozen in liquid nitrogen, was practically intact. Myrosinase activity assay proved that the enzyme remains active after freezing. No pH deviation was noted after sample storage - this parameter did not influence the activity of enzymes. Tissue fragmentation and enzyme-inhibition techniques applied prior to the extraction influenced both the qualitative and quantitative composition of the volatile metabolome of broccoli.

Elucidating the Antimycobacterial Mechanism of Action of Decoquinate Derivative RMB041 Using Metabolomics.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), still remains one of the leading causes of death from a single infectious agent worldwide. The high prevalence of this disease is mostly ascribed to the rapid development of drug resistance to the current anti-TB drugs, exacerbated by lack of patient adherence due to drug toxicity. The aforementioned highlights the urgent need for new anti-TB compounds with different antimycobacterial mechanisms of action to those currently being used. An N-alkyl quinolone; decoquinate derivative RMB041, has recently shown promising antimicrobial activity against Mtb, while also exhibiting low cytotoxicity and excellent pharmacokinetic characteristics. Its exact mechanism of action, however, is still unknown. Considering this, we used GCxGC-TOFMS and well described metabolomic approaches to analyze and compare the metabolic alterations of Mtb treated with decoquinate derivative RMB041 by comparison to non-treated Mtb controls. The most significantly altered pathways in Mtb treated with this drug include fatty acid metabolism, amino acid metabolism, glycerol metabolism, and the urea cycle. These changes support previous findings suggesting this drug acts primarily on the cell wall and secondarily on the DNA metabolism of Mtb. Additionally, we identified metabolic changes suggesting inhibition of protein synthesis and a state of dormancy.

Elucidating the Antimycobacterial Mechanism of Action of Ciprofloxacin Using Metabolomics.

In the interest of developing more effective and safer anti-tuberculosis drugs, we used a GCxGC-TOF-MS metabolomics research approach to investigate and compare the metabolic profiles of Mtb in the presence and absence of ciprofloxacin. The metabolites that best describe the differences between the compared groups were identified as markers characterizing the changes induced by ciprofloxacin. Malic acid was ranked as the most significantly altered metabolite marker induced by ciprofloxacin, indicative of an inhibition of the tricarboxylic acid (TCA) and glyoxylate cycle of Mtb. The altered fatty acid, myo-inositol, and triacylglycerol metabolism seen in this group supports previous observations of ciprofloxacin action on the Mtb cell wall. Furthermore, the altered pentose phosphate intermediates, glycerol metabolism markers, glucose accumulation, as well as the reduction in the glucogenic amino acids specifically, indicate a flux toward DNA (as well as cell wall) repair, also supporting previous findings of DNA damage caused by ciprofloxacin. This study further provides insights useful for designing network whole-system strategies for the identification of possible modes of action of various drugs and possibly adaptations by Mtb resulting in resistance.

Mycobacterium tuberculosis curli pili (MTP) is associated with significant host metabolic pathways in an A549 epithelial cell infection model and contributes to the pathogenicity of Mycobacterium tuberculosis.

INTRODUCTION: A clear understanding of the metabolome of Mycobacterium tuberculosis and its target host cell during infection is fundamental for the development of novel diagnostic tools, effective drugs and vaccines required to combat tuberculosis. The surface-located Mycobacterium tuberculosis curli pili (MTP) adhesin forms initial contact with the host cell and is therefore important for the establishment of infection. OBJECTIVE: The aim of this investigation was to determine the role of MTP in modulating pathogen and host metabolic pathways in A549 epithelial cells infected with MTP proficient and deficient strains of M. tuberculosis. METHODS: Uninfected A549 epithelial cells, and those infected with M. tuberculosis V9124 wild-type strain, Δmtp and the mtp-complemented strains, were subjected to metabolite extraction, two-dimensional gas chromatography time-of-flight mass spectrometry (GCxGC-TOFMS) and bioinformatic analyses. Univariate and multivariate statistical tests were used to identify metabolites that were significantly differentially produced in the WT-infected and ∆mtp-infected A549 epithelial cell models, comparatively. RESULTS: A total of 46 metabolites occurred in significantly lower relative concentrations in the Δmtp-infected cells, indicating a reduction in nucleic acid synthesis, amino acid metabolism, glutathione metabolism, oxidative stress, lipid metabolism and peptidoglycan, compared to those cells infected with the WT strain. CONCLUSION: The absence of MTP was associated with significant changes to the host metabolome, suggesting that this adhesin is an important contributor to the pathogenicity of M. tuberculosis, and supports previous findings of its potential as a suitable drug, vaccine and diagnostic target.

A Metabolomic Approach for Predicting Diurnal Changes in Cortisol.

Introduction: The dysregulation of cortisol secretion has been associated with a number of mental health and mood disorders. However, diagnostics for mental health and mood disorders are behavioral and lack biological contexts. Objectives: The goal of this work is to identify volatile metabolites capable of predicting changes in total urinary cortisol across the diurnal cycle for long-term stress monitoring in psychological disorders. Methods: We applied comprehensive two-dimensional gas chromatography coupled with time-of-flight mass spectrometry to sample the urinary volatile metabolome using an untargeted approach across three time points in a single day for 60 subjects. Results: The finalized multiple regression model includes 14 volatile metabolites and 7 interaction terms. A review of the selected metabolites suggests pyrrole, 6-methyl-5-hepten-2-one and 1-iodo-2-methylundecane may originate from endogenous metabolic mechanisms influenced by glucocorticoid signaling mechanisms. Conclusion: This analysis demonstrated the feasibility of using specific volatile metabolites for the prediction of secreted cortisol across time.

Mapping polychlorinated dibenzo-p-dioxins/dibenzofurans in soils around Pugu municipal dump site in Dar es Salaam, Tanzania: Implications on dermal and soil ingestion exposure for people in the peripheral.

Experimental data on the contribution of a dump site in Tanzania as a point source of the 17 possible congeners of PCDD/Fs to the environment is presented. Dry and wet season samples were collected around Pugu municipal dump site followed by GCxGC-TOFMS analysis. The dominant congeners were OctaCDD, 1,2,3,4,6,7,8-HepCDF; 1,2,3,4,6,7,8-HeptaCDD and 1,2,4,7-PeCDD. The concentrations of the congeners expressed as TEQ WHO2005 ranged from 11.69 to 48.97 pg/g with a mean of 29.44 pg/g for the dry season and TEQ WHO2005 4.13-85.82 pg/g with a mean of 41.51 pg/g for the wet season. These levels were speculated high enough to accumulate in free-range chickens and cause harmful effects to humans that consumed them especially residents around Pugu dump site. Exposure of people to PCDD/Fs through dermal absorption and soil ingestion were estimated using the VLIER-HUMAAN Mathematical model. Exposure through dermal absorption was estimated to be 1.2 × 10-4 and 9.8 × 10-6 ng TEQ/kg day for children and adults respectively while through soil ingestion via consumption of contaminated foods and other sources was 0.0045 and 0.27 ng TEQ/kg day for children and adults respectively. These values however were well below the WHO tolerable daily intake. Generally, there was no significant variation for total PCDD/Fs in the dry and wet season (α = 0.08). Strong positive correlation (r = 0.94) between total PCDD/Fs and organic matter content was observed during the wet season.

A comprehensive look into the volatile exometabolome of enteroxic and non-enterotoxic Staphylococcus aureus strains.

Staphylococcal food poisoning is a disease that originates significant health and economic losses and is caused by Staphylococcus aureus strains able to produce enterotoxins. The aim of this work is to go further on the study of the volatile exometabolome of S. aureus using an advanced gas chromatographic technique. Enterotoxic and non-enterotoxic strains were assessed. The volatile exometabolome profile comprised 240 volatiles belonging to ten chemical families. This volatiles were mainly by-products of branched-chain amino acids and methionine degradation, pyruvate metabolism, diacetyl pathway, oxidative stress and carotenoid cleavage. Metabolites released by the first two pathways were produced in higher contents by the enterotoxic strains. This study add further insights to S. aureus volatile exometabolome, and also shows that by applying it, it is possible to distinguish strains of S. aureus by the number of produced enterotoxins, which is especially important from the food safety point of view.

Validation of a modified QuEChERS method for the analysis of organochlorine pesticides in fatty biological tissues using two-dimensional gas chromatography.

The monitoring of organic contaminants in the environment has gained tremendous traction in recent times, however, little attention has focused on the development of appropriate methods for the analysis of biological tissues from key sentinel species. In this study, a rapid, low-cost method is presented for the analysis of organochlorine pesticides (OCPs) in fatty biological tissues using a modified QuEChERS (Quick, Easy, Cheap, Efficient, Rugged and Safe) approach. The adapted extraction procedure is tested on biological samples of varying fat content, including fish muscle tissue and two previously untested matrices; coral and adipose tissue. Residue analyses were conducted by two-dimensional gas chromatography-time-of-flight mass spectrometry (GCxGC-TOF-MS). The method was fully validated through the evaluation of recoveries, limits of detection, linearity and precision. Mean recoveries (n = 3) for all 18 target compounds ranged between 69 and 102% across all matrices, with relative standard deviations (RSD) <10% in most cases. Limits of detection (LOD) ranged from 0.1 to 2.0 ng g-1. The method was successfully applied to the analysis of real samples collected from iSimangaliso Wetland Park World Heritage Site, South Africa.

Comparison of a disposable sorptive sampler with thermal desorption in a gas chromatographic inlet, or in a dedicated thermal desorber, to conventional stir bar sorptive extraction-thermal desorption for the determination of micropollutants in water.

The presence of micropollutants in the aquatic environment is a worldwide environmental concern. The diversity of micropollutants and the low concentration levels at which they may occur in the aquatic environment have greatly complicated the analysis and detection of these chemicals. Two sorptive extraction samplers and two thermal desorption methods for the detection of micropollutants in water were compared. A low-cost, disposable, in-house made sorptive extraction sampler was compared to SBSE using a commercial Twister sorptive sampler. Both samplers consisted of polydimethylsiloxane (PDMS) as a sorptive medium to concentrate micropollutants. Direct thermal desorption of the disposable samplers in the inlet of a GC was compared to conventional thermal desorption using a commercial thermal desorber system (TDS). Comprehensive gas chromatography coupled to time-of-flight mass spectrometry (GC × GC-TOFMS) was used for compound separation and identification. Ten micropollutants, representing a range of heterogeneous compounds, were selected to evaluate the performance of the methods. The in-house constructed sampler, with its associated benefits of low-cost and disposability, gave results comparable to commercial SBSE. Direct thermal desorption of the disposable sampler in the inlet of a GC eliminated the need for expensive consumable cryogenics and total analysis time was greatly reduced as a lengthy desorption temperature programme was not required. Limits of detection for the methods ranged from 0.0010 ng L-1 to 0.19 ng L-1. For most compounds, the mean (n = 3) recoveries ranged from 85% to 129% and the % relative standard deviation (% RSD) ranged from 1% to 58% with the majority of the analytes having a %RSD of less than 30%.

Qualitative analyses of less-volatile organic molecules from female skin scents by comprehensive two dimensional gas chromatography-time of flight mass spectrometry.

Instrumental human scent analysis is undoubtedly desirable for many forensic as well medical applications. Most of the previous human scent studies were focused on volatile organic compounds (VOCs) which were analysed by head space solid phase micro-extraction gas chromatography/mass spectrometry (HS-SPME-GC/MS). This method is, however, significantly less sensitive to "heavier" less volatile compounds emitted from the human skin. These less volatile organic scent molecules probably create the basis of the individual human scent signature, and therefore, our attention is focused mainly on these "heavier" compounds. The human scent was adsorbed onto purified glass beads and samples were prepared as hexane solutions obtained by extraction from the sampled glass beads. To resolve a lot of very similar molecules, the comprehensive two-dimensional gas chromatography with time-of-flight mass spectrometer (GCxGC-TOFMS) was used to analyse the hexane scent solutions. Using this technique, more than 137 less volatile molecules including organic fatty acids, ketones, aldehydes, simple esters, alcohols, and especially various fatty acid esters with different carbon chains were identified. A considerable number of these molecules were identified in the scent samples for the first time.

Thermal desorption comprehensive two-dimensional gas chromatography coupled to variable-energy electron ionization time-of-flight mass spectrometry for monitoring subtle changes in volatile organic compound profiles of human blood.

Blood is a matrix with high potential for forensic investigations and human rescue. Its volatile signature can be used in search exercises to locate injured or deceased individuals. Little is known, however, about the volatile organic compound (VOC) profile of blood, except that it is complex and varies while blood ages. In the present study, we used thermal desorption (TD) and comprehensive two-dimensional gas chromatography (GCxGC) coupled to variable-energy electron ionization time-of-flight mass spectrometry (TOFMS) to monitor VOC signatures of human blood. A highly complex reference standard (Century Mix) containing 108 compounds of various chemical functionalities and several homologue series of compounds was used for the purpose of transposing our previously developed cryogenically modulated GCxGC-TOFMS methods into the use of a reverse fill/flush (RFF) flow modulator. The average peak width at half height was 340ms and the average tailing factor was 1.16. Light VOCs (down to C4) were effectively flow modulated and exhibited minimal breakthrough over a large dynamic range spanning four orders of magnitude. Mass spectrometric detection was performed using electron impact ionization (EI) carried out at 70eV and lower energies (12, 14, and 16eV). The use of variable-energy (ve) EI allowed mass spectra to be produced with less fragmentation and an increased presence of structurally significant ions and the molecular ion. This provided additional confidence in peak assignments, especially for closely eluting isomers often observed in the profiling of the headspace of blood. Variable-energy EI TD-GCxGC-TOFMS blood data sets were statistically processed using principal component analyses (PCA) and hierarchical cluster analyses (HCA). These techniques demonstrated that the effect of aging was greater than the inter-individual variation on the blood VOC profile. The combination of retention indices, low and high EI MS spectra served as a strong basis to gain more confidence in analytical identification by excluding identities proposed by mass spectral databases (70eV) for compounds contributing to the separation of blood of different ages.

Anti-inflammatory and anti-oxidant effects of Cardamom (Elettaria repens (Sonn.) Baill) and its phytochemical analysis by 4D GCXGC TOF-MS.

Elleteria repens is a large cardamom used in the culinary preparations. In the present study, we have evaluated in vitro antioxidant and free radical scavenging activities E. repens hexane extract (ERH) exhibited DPPH and metal chelating activity with IC50 values of 464±28.3µg/ml, 199±7.2µg/ml whereas the reducing power and antioxidant activities are found to be 289±14.6 AAE/mg, 468±22.7 GAE/mg. The observed antioxidant activities could be correlated with metabolites such as polyphenol, flavonoid, and terpenoid group of compounds identified in hexane fraction of E. repens by 4D GCXGC TOF-MS. Further ERH was evaluated for its protective properties against macromolecules such as DNA, protein and lipid damage. The extract showed protection against H2O2 induced DNA damage and inhibited AAPH induced protein oxidation and lipid peroxidation. Moreover, ERH administration to rats at 50 and 100mg/kg inhibited carrageenan-induced paw edema, and down-regulated cytokines such as COX-2, IL-6, and TNF-α and inhibited i-NOS mediated NO generation. E. repens also exhibited antioxidant effects by restoring SOD, catalase, GSH levels and inhibited lipid peroxidation in carrageenan challenged rats. Overall, the results suggest that E. repens may be useful in combating inflammation and oxidative stress.