Profiling allergic asthma volatile metabolic patterns using a headspace-solid phase microextraction/gas chromatography based methodology

Allergicasthmarepresentsanimportantpublichealthissuewithsignificantgrowthovertheyears,especially in the paediatric population. Exhaled breath is a non-invasive, easily performed and rapid method forobtainingsamplesfromthelowerrespiratorytract.Inthepresentmanuscript,themetabolicvolatile 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 ofstudiesinbreathofallergicasthmaticchildrenbyHS-SPMEledtothedevelopmentofanexperimental 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 60min 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 allergicasthma(35),fromwhich13hadalsoallergicrhinitis,andhealthycontrolchildren(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 LeastSquares–DiscriminantAnalysis(PLS–DA)usingasetof28selectedmetabolitesanddiscrimination 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.

Allergic asthma exhaled breath metabolome: a challenge for comprehensive two-dimensional gas chromatography

Allergic asthma represents an important public health issue, most common in the paediatric population, characterized by airway inflammation that may lead to changes in volatiles secreted via the lungs. Thus, exhaled breath has potential to be a matrix with relevant metabolomic information to characterize this disease. Progress in biochemistry, health sciences and related areas depends on instrumental advances, and a high throughput and sensitive equipment such as comprehensive two-dimensional gas chromatography–time of flight mass spectrometry (GC × GC–ToFMS) was considered. GC × GC–ToFMS application in the analysis of the exhaled breath of 32 children with allergic asthma, from which 10 had also allergic rhinitis, and 27 control children allowed the identification of several hundreds of compounds belonging to different chemical families. Multivariate analysis, using Partial Least Squares-Discriminant Analysis in tandem with Monte Carlo Cross Validation was performed to assess the predictive power and to help the interpretation of recovered compounds possibly linked to oxidative stress, inflammation processes or other cellular processes that may characterize asthma. The results suggest that the model is robust, considering the high classification rate, sensitivity, and specificity. A pattern of six compounds belonging to the alkanes characterized the asthmatic population: nonane, 2,2,4,6,6-pentamethylheptane, decane, 3,6-dimethyldecane, dodecane, and tetradecane. To explore future clinical applications, and considering the future role of molecular-based methodologies, a compound set was established to rapid access of information from exhaled breath, reducing the time of data processing, and thus, becoming more expedite method for the clinical purposes.

Mass spectrometric characterization of two novel inflammatory peptides from the venom of the social wasp Polybia paulista

The social wasp P. paulista is relatively common in southeast Brazil causing many medically important stinging incidents. The seriousness of these incidents is dependent on the amount of venom inoculated by the wasps into the victims, and the characteristic envenomation symptoms are strongly dependent on the types of peptides present in the venom. In order to identify some of these naturally occurring peptides available in very low amounts, an analytical protocol was developed that uses a combination of reversed-phase and normal-phase high-performance liquid chromatography (HPLC) of wasp venom for peptide purification, with matrix-assisted laser desorption/ionization time-of-flight post-source decay mass spectrometry (MALDI-Tof-PSD-MS) and low-energy collision-induced dissociation (CID) in a quadrupole time-of-flight tandem mass spectrometry (QTof-MS/MS) instrument for peptide sequencing at the sub-picomole level. The distinction between Leu and Ile was achieved both by observing d-type fragment ions obtained under CID conditions and by comparison of retention times of the natural peptides and their synthetic counterparts (with different combinations of I and/or L at N- and C-terminal positions). To distinguish the isobaric residues K and Q, acetylation of peptides was followed by Q-Tof-MS analysis. The primary sequences obtained were INWLKLGKMVIDAL-NH2 (MW 1611.98Da) and IDWLKLGKMVMDVL-NH2 (MW 1658.98Da). Micro-scale bioassay protocols characterized both peptides as presenting potent hemolytic action, mast cell degranulation, and chemotaxis of poly-morphonucleated leukocyte (PMNL) cells. The primary sequences and the bioassay results suggest that these toxins constitute members of a new sub-class of mastoparan toxins, directly involved in the occurrence of inflammatory processes after wasp stinging. Copyright (C) 2004 John Wiley Sons, Ltd.

Selective UV-filter detection with sensors based on stainless steel electrodes modified with polyaniline doped with metal tetrasulfonated phthalocyanine films

This work describes the construction and application of two amperometric sensors for sensitive UV-filter determination. The sensors were prepared using stainless steel electrodes in which polyaniline (PANI) was electrochemically polymerized in the presence of nickel (NiPcTS) or iron (FePcTS) tetrasulfonated phthalocyanines. The sensor surface characterizations were carried out using atomic force microscopy (AFM). The PANI/NiPcTS sensor was selective for the chemical UV-filter p-aminobenzoic acid (PABA) and the PANI/FePcTS sensor was selective for octyldimethyl-PABA (ODP), both in a mixture of tetrahydrofuran (THF) and 0.1 mol L(-1) H(2)SO(4) at a volume ratio of 30 : 70, and with an applied potential of 0.0 mV vs. Ag vertical bar AgCl. A detailed investigation of the selectivity was carried out for both sensors, in order to determine their responses for ten different UV filters. Finally, each sensor was successfully applied to PABA or ODP quantification in sunscreen formulations and water from swimming pools.

Gas Chromatographic Determination of Cyprodinil, Fludioxonil, Pyrimethanil, and Tebuconazole in Grapes, Must, and Wine

A rapid and simple gas chromatographic method for determinating cyprodinil, fludioxonil, pyrimethanil, and tebuconazole in grapes, must, and wine is described. An on-line microextraction method was used with a one-step extraction-partition procedure. Nitrogen-phosphorus and mass spectrometric detectors were used, because of their low sensitivity and high selectivity. Because of high selectivity of detector, no cleanup was necessary and the extract was concentrated 5 times. Recoveries from fortified grapes, must, and wine ranged from 93 to 110%. Limits of determination were 0.05 mg/kg for cyprodinil and pyrimethanil and 0.10 mg/kg for fludioxonil and tebuconazole.

Nebular gas drag and planetary accretion with eccentric high-mass planets

Aims.We investigate the dynamics of pebbles immersed in a gas disk interacting with a planet on an eccentric orbit. The model has a prescribed gap in the disk around the location of the planetary orbit, as is expected for a giant planet with a mass in the range of 0.1-1 Jupiter masses. The pebbles with sizes in the range of 1 cm to 3 m are placed in a ring outside of the giant planet orbit at distances between 10 and 30 planetary Hill radii. The process of the accumulation of pebbles closer to the gap edge, its possible implication for the planetary accretion, and the importance of the mass and the eccentricity of the planet in this process are the motivations behind the present contribution. Methods. We used the Bulirsch-Stoer numerical algorithm, which is computationally consistent for close approaches, to integrate the Newtonian equations of the planar (2D), elliptical restricted three-body problem. The angular velocity of the gas disk was determined by the appropriate balance between the gravity, centrifugal, and pressure forces, such that it is sub-Keplerian in regions with a negative radial pressure gradient and super-Keplerian where the radial pressure gradient is positive. Results. The results show that there are no trappings in the 1:1 resonance around the L 4 and L5 Lagrangian points for very low planetary eccentricities (e2 < 0.07). The trappings in exterior resonances, in the majority of cases, are because the angular velocity of the disk is super-Keplerian in the gap disk outside of the planetary orbit and because the inward drift is stopped. Furthermore, the semi-major axis location of such trappings depends on the gas pressure profile of the gap (depth) and is a = 1.2 for a planet of 1 MJ. A planet on an eccentric orbit interacts with the pebble layer formed by these resonances. Collisions occur and become important for planetary eccentricity near the present value of Jupiter (e 2 = 0.05). The maximum rate of the collisions onto a planet of 0.1 MJ occurs when the pebble size is 37.5 cm ≤ s < 75 cm; for a planet with the mass of Jupiter, it is15 cm ≤ s < 30 cm. The accretion stops when the pebble size is less than 2 cm and the gas drag dominates the motion. © 2013 ESO.

Results from a 15-year study on hydrocarbon concentrations in water and sediment from Admiralty Bay, King George Island, Antarctica

Admiralty Bay on the King George Island hosts the Brazilian, Polish and Peruvian research stations as well as the American and Ecuadorian field stations. Human activities in this region require the use of fossil fuels as an energy source, thereby placing the region at risk of hydrocarbon contamination. Hydrocarbon monitoring was conducted on water and sediment samples from the bay over 15 years. Fluorescence spectroscopy was used for the analysis of total polycyclic aromatic hydrocarbons (PAHs) in seawater samples and gas chromatography with flame ionization and/or mass spectrometric detection was used to analyse individual n-alkanes and PAHs in sediment samples. The results revealed that most sites contaminated by these Compounds are around the Brazilian and Polish research stations due to the intense human activities, mainly during the summer. Moreover, the sediments revealed the presence of hydrocarbons from different sources, suggesting a mixture of the direct input of oil or derivatives and derived from hydrocarbon combustion. A decrease in PAH concentrations occurred following improvement of the sewage treatment facilities at the Brazilian research station, indicating that the contribution from human waste may be significant.

Automated analysis of lidocaine and its metabolite in plasma by in-tube solid-phase microextraction coupled with LC-UV for pharmacokinetic study

A sensitive, selective, and reproducible in-tube solid-phase microextraction and liquid chromatographic (in-tube SPME/LC-UV) method for determination of lidocaine and its metabolite monoethylglycinexylidide (MEGX) in human plasma has been developed, validated, and further applied to pharmacokinetic study in pregnant women with gestational diabetes mellitus (GDM) subjected to epidural anesthesia. Important factors in the optimization of in-tube SPME performance are discussed, including the draw/eject sample volume, draw/eject cycle number, draw/eject flow rate, sample pH, and influence of plasma proteins. The limits of quantification of the in-tube SPME/LC method were 50 ng/mL for both metabolite and lidocaine. The interday and intraday precision had coefficients of variation lower than 8%, and accuracy ranged from 95 to 117%. The response of the in-tube SPME/LC method for analytes was linear over a dynamic range from 50 to 5000 ng/mL, with correlation coefficients higher than 0.9976. The developed in-tube SPME/LC method was successfully used to analyze lidocaine and its metabolite in plasma samples from pregnant women with GDM subjected to epidural anesthesia for pharmacokinetic study.

Application of comprehensive gas chromatography (GCxGC) to measurements of volatile organic species in ambient air

The focus of this thesis was the in-situ application of the new analytical technique "GCxGC" in both the marine and continental boundary layer, as well as in the free troposphere. Biogenic and anthropogenic VOCs were analysed and used to characterise local chemistry at the individual measurement sites. The first part of the thesis work was the characterisation of a new set of columns that was to be used later in the field. To simplify the identification, a time-of-flight mass spectrometer (TOF-MS) detector was coupled to the GCxGC. In the field the TOF-MS was substituted by a more robust and tractable flame ionisation detector (FID), which is more suitable for quantitative measurements. During the process, a variety of volatile organic compounds could be assigned to different environmental sources, e.g. plankton sources, eucalyptus forest or urban centers. In-situ measurements of biogenic and anthropogenic VOCs were conducted at the Meteorological Observatory Hohenpeissenberg (MOHP), Germany, applying a thermodesorption-GCxGC-FID system. The measured VOCs were compared to GC-MS measurements routinely conducted at the MOHP as well as to PTR-MS measurements. Furthermore, a compressed ambient air standard was measured from three different gas chromatographic instruments and the results were compared. With few exceptions, the in-situ, as well as the standard measurements, revealed good agreement between the individual instruments. Diurnal cycles were observed, with differing patterns for the biogenic and the anthropogenic compounds. The variability-lifetime relationship of compounds with atmospheric lifetimes from a few hours to a few days in presence of O3 and OH was examined. It revealed a weak but significant influence of chemistry on these short-lived VOCs at the site. The relationship was also used to estimate the average OH radical concentration during the campaign, which was compared to in-situ OH measurements (1.7 x 10^6 molecules/cm^3, 0.071 ppt) for the first time. The OH concentration ranging from 3.5 to 6.5 x 10^5 molecules/cm^3 (0.015 to 0.027 ppt) obtained with this method represents an approximation of the average OH concentration influencing the discussed VOCs from emission to measurement. Based on these findings, the average concentration of the nighttime NO3 radicals was estimated using the same approach and found to range from 2.2 to 5.0 x 10^8 molecules/cm^3 (9.2 to 21.0 ppt). During the MINATROC field campaign, in-situ ambient air measurements with the GCxGC-FID were conducted at Tenerife, Spain. Although the station is mainly situated in the free troposphere, local influences of anthropogenic and biogenic VOCs were observed. Due to a strong dust event originating from Western Africa it was possible to compare the mixing ratios during normal and elevated dust loading in the atmosphere. The mixing ratios during the dust event were found to be lower. However, this could not be attributed to heterogeneous reactions as there was a change in the wind direction from northwesterly to southeasterly during the dust event.

Mass spectrometric identification of Varicella-Zoster Virus (VZV) proteins recognized by human T cells

Primary varicella-zoster virus (VZV) infection during childhood leads to varicella commonly known as chickenpox. After primary infection has occurred VZV establishes latency in the host. During subsequent lifetime the virus can cause reactivated infection clinically known as herpes zoster or shingles. In immunodeficient patients’ dissemination of the virus can lead to life-threatening disease. Withdrawal of acyclovir drug prophylaxis puts allogeneic hematopoietic stem-cell transplantation (HSCT) patients at increased risk for herpes zoster as long as VZV-specific cellular immunity is impaired. Although an efficient live attenuated VZV vaccine for zoster prophylaxis exists, it is not approved in immunocompromised patients due to safety reasons. Knowledge of immunogenic VZV proteins would allow designing a noninfectious nonhazardous subunit vaccine suitable for patients with immunodeficiencies. The objective of this study was to identify T cell defined virus proteins of a VZV-infected Vero cell extract that we have recently described as a reliable antigen format for interferon-gamma (IFN-γ) enzyme-linked immunosorbent spot (ELISpot) assays (Distler et al. 2008). We first separated the VZV-infected/-uninfected Vero cell extracts by size filtration and reverse-phase high performance liquid chromatography (RP-HPLC). The collected fractions were screened for VZV reactivity with peripheral blood mononuclear cells (PBMCs) of VZV-seropositive healthy individuals in the sensitive IFN-γ ELISpot assay. Using this strategy, we successfully identified bioactive fractions that contained immunogenic VZV material. VZV immune reactivity was mediated by CD4+ memory T lymphocytes (T cells) of VZV-seropositive healthy individuals as demonstrated in experiments with HLA blockade antibodies and T cell subpopulations already published by Distler et al. We next analyzed the bioactive fractions with electrospray ionization mass spectrometry (ESI-MS) techniques and identified the sequences of three VZV-derived proteins: glycoprotein E (gE); glycoprotein B (gB), and immediate early protein 62 (IE62). Complementary DNA of these identified proteins was used to generate in vitro transcribed RNA for effective expression in PBMCs by electroporation. We thereby established a reliable and convenient IFN-γ ELISPOT approach to screen PBMCs of healthy donors and HSCT patients for T cell reactivity to single full-length VZV proteins. Application in 10 VZV seropositive healthy donors demonstrated much stronger recognition of glycoproteins gE and gB compared to IE62. In addition, monitoring experiments with ex vivo PBMCs of 3 allo-HSCT patients detected strongly increased CD4+ T cell responses to gE and gB for several weeks to months after zoster onset, while IE62 reactivity remained moderate. Overall our results show for the first time that VZV glycoproteins gE and gB are major targets of the post-transplant anti-zoster CD4+ T cell response. The screening approach introduced herein may help to select VZV proteins recognized by memory CD4+ T cells for inclusion in a subunit vaccine, which can be safely used for zoster prophylaxis in immunocompromised HSCT patients.

Development and application of mass-spectrometric methods for the quantification and characterization of organic compounds in ice cores

Eisbohrkerne stellen wertvolle Klimaarchive dar, da sie atmosphärisches Aerosol konservieren. Die Analyse chemischer Verbindungen als Bestandteil atmosphärischer Aerosole in Eisbohrkernen liefert wichtige Informationen über Umweltbedingungen und Klima der Vergangenheit. Zur Untersuchung der α-Dicarbonyle Glyoxal und Methylglyoxal in Eis- und Schneeproben wurde eine neue, sensitive Methode entwickelt, die die Stir Bar Sorptive Extraction (SBSE) mit der Hochleistungsflüssigchromatographie-Massenspektrometrie (HPLC-MS) kombiniert. Zur Analyse von Dicarbonsäuren in Eisbohrkernen wurde eine weitere Methode entwickelt, bei der die Festphasenextraktion mit starkem Anionenaustauscher zum Einsatz kommt. Die Methode erlaubt die Quantifizierung aliphatischer Dicarbonsäuren (≥ C6), einschließlich Pinsäure, sowie aromatischer Carbonsäuren (wie Phthalsäure und Vanillinsäure), wodurch die Bestimmung wichtiger Markerverbindungen für biogene und anthropogene Quellen ermöglicht wurde. Mit Hilfe der entwickelten Methoden wurde ein Eisbohrkern aus den Schweizer Alpen analysiert. Die ermittelten Konzentrationsverläufe der Analyten umfassen die Zeitspanne von 1942 bis 1993. Mittels einer Korrelations- und Hauptkomponentenanalyse konnte gezeigt werden, dass die organischen Verbindungen im Eis hauptsächlich durch Waldbrände und durch vom Menschen verursachte Schadstoffemissionen beeinflusst werden. Im Gegensatz dazu sind die Konzentrationsverläufe einiger Analyten auf den Mineralstaubtransport auf den Gletscher zurückzuführen. Zusätzlich wurde ein Screening der Eisbohrkernproben mittels ultrahochauflösender Massenspektrometrie durchgeführt. Zum ersten Mal wurden in diesem Rahmen auch Organosulfate und Nitrooxyorganosulfate in einem Eisbohrkern identifiziert.

Enantioselective determination of ondansetron and 8-hydroxyondansetron in human plasma from recovered surgery patients by liquid chromatography-tandem mass spectrometry

A liquid chromatographic-mass spectrometric assay with atmospheric pressure chemical ionization for quantification of ondansetron and its main metabolite 8-hydroxyondansetron in human plasma was presented. The enantiomeric separation was achieved on a Chiralcel OD-R column containing cellulose tris-(3,5-dimethylphenylcarbamate). The validation data were within the required limits. The assay was successfully applied to authentic plasma samples. Quantitative results from postoperative patients receiving ondansetron demonstrated a great interindividual variability in postoperative plasma drug concentrations, the metabolites were not detected in their unconjugated form. A wide variation in the S-(+)-/R-(-)-ondansetron concentration ratio between 0.14 and 7.18 is indicative for a stereoselective disposition or metabolism. In further studies CYP2D6 and CYP3A4 genotype dependent metabolism of ondansetron enantiomers as well as of co-administered drugs and clinical efficacy of the medication should be tested.

On-line SPE LC-MS/MS for the quantification of Δ9-tetrahydrocannabinol (THC) and its two major metabolites in human peripheral blood by liquid chromatography tandem mass spectrometry

A universal and robust analytical method for the determination of Δ9-tetrahydrocannabinol (THC) and two of its metabolites Δ9-(11-OH)-tetrahydrocannabinol (11-OH-THC) and 11-nor-Δ9-carboxy-tetrahydrocannabinol (THC-COOH) in human whole blood was developed and validated for use in forensic toxicology. Protein precipitation, integrated solid phase extraction and on-line enrichment followed by high-performance liquid chromatography separation and detection with a triple quadrupole mass spectrometer were combined. The linear ranges used for the three cannabinoids were from 0.5 to 20 ng/mL for THC and 11-OH-THC and from 2.5 to 100 ng/mL for THC-COOH, therefore covering the requirements for forensic use. Correlation coefficients of 0.9980 or better were achieved for all three analytes. No relevant hydrolysis was observed for THC-COOH glucuronide with this procedure--in contrast to our previous GC-MS procedure, which obviously lead to an artificial increase of the THC-COOH concentration due to the hydrolysis of the glucuronide-conjugate occurring at high pH during the phase-transfer catalyzed methylation step.