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Metabolomics represents an emerging topic that can be valuable in the knowledge of organism responses to different stimuli. Metabolomic studies of bivalves may reveal the constraints they are subjected to, and may help clarifying the functions most affected and the tolerance mechanisms triggered. In response to this approach, two-dimensional gas chromatography-time of flight mass spectrometry (GC×GC-ToFMS) combined with headspace solid phase microextraction (HS-SPME) was applied, for the first time to our knowledge, to the untargeted and comprehensive study of the volatile composition of clam species. Firstly, experimental parameters that influence the SPME extraction efficiency were evaluated: sample preparation mode, sample volume and SPME fiber coating. Taking into account the results from the optimization step, the metabolomic profiles were performed using 1 ml of clam soft tissues homogenized with N2 and diluted in deionised water (1:2, w/v), using the PDMS/DVB fiber coating. From a total of more than 200 compounds detected per sample, 63 were tentatively identified and distributed over the chemical families of hydrocarbons, ketones, aldehydes, alcohols, and terpenoids, which seems to arise from clams own metabolism, and/or from their activity as filter-feeders. The potential of this methodology to discriminate close related species was explored by comparing the volatile profiles of Venerupis philippinarum and Venerupis decussata from Ria de Aveiro, two clams belonging to the same genus. Both species experience different environment conditions, and physiological and biochemical characteristics, which may explain the observed differentiation between their metabolic profiles. To our knowledge, this is the most detailed information available so far about clam volatile composition, which represents a valuable data for future advanced studies in the ecology, toxicology and physiology of bivalves based on clams fingerprinting., (Copyright © 2013 Elsevier B.V. All rights reserved.)

An automated 96-thin-film solid phase microextraction system was optimized for high throughput analysis of phenolic compounds in wine, berry, and grape samples. Liquid chromatography and tandem mass spectrometry methods were optimized and applied for separation and detection of compounds. Evaluation of five different stationary phases showed that polystyrene-divinylbenzene-polyacrylonitrile (PS-DVB-PAN) is the optimum extraction phase for the extraction of phenolic compounds under study. The thin-film PS-DVB-PAN SPME coating provided almost exhaustive extraction recovery for all phenolics compounds under study, except for naringenin with 80% recovery. Extraction efficiency, inter- and intra-day reproducibility, sensitivity, linearity, limit of detection and quantitation, and matrix effect were evaluated. Intra-day and inter-day reproducibility were in the respective range of 4-8 and 7-13% relative standard deviation (RSD) for all eight phenolic compounds. Limits of detection and quantitation of the proposed SPME-LC-MS/MS system for the analysis of analytes under study were found in the range of 0.2-3 and 0.5-10 ng/mL, respectively. Standard addition calibration was applied for the quantitative determination of unknown phenolic compounds from wine, berry, and grape samples. The assessment of matrix effect showed significant reduction of ion suppression/enhancement using SPME method in comparison with that of solvent extraction technique., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The volatiles (VOCs) and semi-volatile organic compounds (SVOCs) responsible for aroma are mainly present in skin of grape varieties. Thus, the present investigation is directed towards the optimisation of a solvent free methodology based on headspace-solid-phase microextraction (HS-SPME) combined with gas chromatography-quadrupole mass spectrometry (GC-qMS) in order to establish the global volatile composition in pulp and skin of Bual and Bastardo Vitis vinifera L. varieties. A deep study on the extraction-influencing parameters was performed, and the best results, expressed as GC peak area, number of identified compounds and reproducibility, were obtained using 4 g of sample homogenised in 5 mL of ultra-pure Milli-Q water in a 20 mL glass vial with addition of 2g of sodium chloride (NaCl). A divinylbenzene/carboxen/polydimethylsiloxane fibre was selected for extraction at 60°C for 45 min under continuous stirring at 800 rpm. More than 100 VOCs and SVOCs, including 27 monoterpenoids, 27 sesquiterpenoids, 21 carbonyl compounds, 17 alcohols (from which 2 aromatics), 10 C(13) norisoprenoids and 5 acids were identified. The results showed that, for both grape varieties, the levels and number of volatiles in skin were considerably higher than those observed in pulp. According to the data obtained by principal component analysis (PCA), the establishment of the global volatile signature of grape and the relationship between different part of grapes-pulp and skin, may be an useful tool to winemaker decision to define the vinification procedures that improves the organoleptic characteristics of the corresponding wines and consequently contributed to an economic valorization and consumer acceptance., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Allergic asthma represents an important public health issue with significant growth over the years, especially in the paediatric population. Exhaled breath is a non-invasive, easily performed and rapid method for obtaining samples from the lower respiratory tract. In the present manuscript, the metabolic volatile profiles of allergic asthma and control children were evaluated by headspace solid-phase microextraction combined with gas chromatography-quadrupole mass spectrometry (HS-SPME/GC-qMS). The lack of studies in breath of allergic asthmatic children by HS-SPME led to the development of an experimental design to optimize SPME parameters. To fulfil this objective, three important HS-SPME experimental parameters that influence the extraction efficiency, namely fibre coating, temperature and time extractions were considered. The selected conditions that promoted higher extraction efficiency corresponding to the higher GC peak areas and number of compounds were: DVB/CAR/PDMS coating fibre, 22 °C and 60 min as the extraction temperature and time, respectively. The suitability of two containers, 1L Tedlar® bags and BIOVOC®, for breath collection and intra-individual variability were also investigated. The developed methodology was then applied to the analysis of children exhaled breath with allergic asthma (35), from which 13 had also allergic rhinitis, and healthy control children (15), allowing to identify 44 volatiles distributed over the chemical families of alkanes (linear and ramified) ketones, aromatic hydrocarbons, aldehydes, acids, among others. Multivariate studies were performed by Partial Least Squares-Discriminant Analysis (PLS-DA) using a set of 28 selected metabolites and discrimination between allergic asthma and control children was attained with a classification rate of 88%. The allergic asthma paediatric population was characterized mainly by the compounds linked to oxidative stress, such as alkanes and aldehydes. Furthermore, more detailed information was achieved combining the volatile metabolic data, suggested by PLS-DA model, and clinical data., (Copyright © 2011 Elsevier B.V. All rights reserved.)

In this work, a methodology to characterise the volatile and semi-volatile compounds from marine salt by headspace solid-phase microextraction (HS-SPME) and comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry (GCxGC/TOFMS) was developed. Samples from two saltpans of Aveiro, in Portugal, with diverse locations, obtained over three years (2004, 2005, and 2007) were analysed. A 50/30microm divinylbenzene/carboxen/polydimethylsiloxane SPME fibre was used. The volatiles present in the headspace of the solid salt samples (crystals) were equilibrated overnight at 60 degrees C and extracted for 60min prior to injection in the GCxGC/TOFMS. 157 compounds, distributed over the chemical groups of hydrocarbons, aldehydes, esters, furans, haloalkanes, ketones, ethers, alcohols, terpenoids, C(13) norisoprenoids, and lactones were detected across the samples. Furans, haloalkanes and ethers were identified for the first time in marine salt. The large number of co-elutions on the first column that were resolved by the GCxGC system revealed the complexity of marine salt volatile composition. The existence of a structured 2D chromatographic behaviour according to volatility, in the first dimension ((1)D), and primarily polarity, in the second dimension ((2)D), was demonstrated, allowing more reliable identifications. The resolution and sensitivity of GCxGC/TOFMS enabled the separation and identification of a higher number of volatile compounds compared to GC-qMS, allowing a deeper characterisation of this natural product., (Copyright 2010. Published by Elsevier B.V.)

An analytical methodology based on headspace solid phase microextraction (HS-SPME) combined with comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry (GC x GC-ToFMS) was developed for the identification and quantification of the toxic contaminant ethyl carbamate (EC) directly in fortified wines. The method performance was assessed for dry/medium dry and sweet/medium sweet model wines, and for quantification purposes, calibration plots were performed for both matrices using the ion extraction chromatography (IEC) mode (m/z 62). Good linearity was obtained with a regression coefficient (r(2)) higher than 0.981. A good precision was attained (R.S.D. <20%) and low detection limits (LOD) were achieved for dry (4.31 microg/L) and sweet (2.75 microg/L) model wines. The quantification limits (LOQ) and recovery for dry wines were 14.38 microg/L and 88.6%, whereas for sweet wines were 9.16 microg/L and 99.4%, respectively. The higher performance was attained with sweet model wine, as increasing of glucose content improves the volatile compound in headspace, and a better linearity, recovery and precision were achieved. The analytical methodology was applied to analyse 20 fortified Madeira wines including different types of wine (dry, medium dry, sweet, and medium sweet) obtained from several harvests in Madeira Island (Portugal). The EC levels ranged from 54.1 microg/L (medium dry) to 162.5 microg/L (medium sweet)., (2010 Elsevier B.V. All rights reserved.)

The establishment of geographic origin chemical biomarkers for the marine salt might represent an important improvement to its valorisation. Volatile compounds of marine salt, although never studied, are potential candidates. Thus, the purpose of this work was the development of a headspace solid phase microextraction (SPME) combined with gas chromatography-quadrupole mass spectrometry (HS-SPME/GC-qMS) methodology to study the volatile composition of marine salt. A 65mum carbowax/divinylbenzene SPME coating fibre was used. Three SPME parameters were optimised: extraction temperature, sample quantity, and presentation mode. An extraction temperature of 60 degrees C and 16g of marine salt in a 120mL glass vial were selected. The study of the effect of sample presentation mode showed that the analysis of an aqueous solution saturated with marine salt allowed higher extraction efficiency than the direct analysis of salt crystals. The dissolution of the salt in water and the consequent effect of salting-out promote the release of the volatile compounds to the headspace, enhancing the sensitivity of SPME for the marine salt volatiles. The optimised methodology was applied to real matrices of marine salt from different geographical origins (Portugal, France, and Cape Verde). The marine salt samples contain ca. 40 volatile compounds, distributed by the chemical groups of hydrocarbons, alcohols, phenols, aldehydes, ketones, esters, terpenoids, and norisoprenoids. These compounds seem to arise from three main sources: algae, surrounding bacterial community, and environment pollution. Since these volatile compounds can provide information about the geographic origin and saltpans environment, this study shows that they can be used as chemical biomarkers of marine salt.

A headspace-solid phase microextraction followed by gas chromatographic analysis (HS-SPME-GC) was developed to be applied in the study of the interactions between the wine polymeric fraction and the ethyl esters: ethyl hexanoate, ethyl octanoate, and ethyl decanoate. Wine models (WM) were prepared with 10% (v/v) aqueous ethanol at pH 3.5 with distinct wine polymeric concentrations prepared from white wine of Vitis vinifera L. var. Fernão-Pires: 1.0 g L(-1) (PWM1), with a polymeric concentration approaching the real one in wine; 10.0 g L(-1) (PWM10); and 30.0 g L(-1) (PWM30), saturated with polymeric fraction. A reference wine model (RWM) was prepared without polymeric fraction. Each volatile compound (4.0 mg L(-1)) was added separately to the RWM and to the WM with the three levels of polymeric material (PWM). From the retention index (RI) calculated for each compound using the formula: [RI = 1 - (C(RWM) - C(PWM))/C(RWM)], where C(RWM) is the concentration of the compound in the RWM and C(PWM) is the concentration of the compound in the given PWM, the retention capacity of each wine polymeric fraction towards the three esters was established. The higher retention indexes were observed for ethyl decanoate, the more hydrophobic compound, and for the PWM with higher concentration. Furthermore, this study also suggested that the retained compounds are dosed to the headspace, which may promote the perception of their aroma for a longer period of time.