A novel multiwalled carbon nanotube-cyclodextrin nanocomposite for solid-phase microextraction-gas chromatography-mass spectrometry determination of polycyclic aromatic hydrocarbons in snow samples.

Novel solid-phase microextraction coatings based on the use of multiwalled carbon nanotube-cyclodextrin (MWCNT-CD) nanocomposites were developed for the determination of 16-priority polycyclic aromatic hydrocarbons at ultratrace levels in snow samples. The performance of both ß- and γ-CD was tested to increase the detection capabilities towards the heaviest and most lipophilic compounds, i.e., five- and six-ring PAHs. To facilitate the interactions of MWCNTs with CDs, an oxidation procedure using both HNO3 and H2O2 was applied, obtaining superior results using MWCNTs-H2O2-γ-CD fiber. Detection and quantitation limits below 0.7 and 2.3 ng/L, RSD lower than 21%, and recoveries of 88(± 2)-119.8(± 0.4)% proved the reliability of the developed method for the determination of PAHs at ultratrace levels. The complexation capability of the γ-CD was also demonstrated in solution by NMR and fluorescence spectroscopy studies and at solid state by XRD analysis. Finally, snow samples collected in the ski area of Dolomiti di Brenta were analyzed, showing a different distribution of the 16 priority PAHs, being naphthalene, phenanthrene, fluoranthene, and pyrene the only compounds detected in all the analyzed samples.

Ultra-sensitive solid-phase Microextraction-Gas Chromatography-Mass spectrometry determination of polycyclic aromatic hydrocarbons in snow samples using a deep cavity BenzoQxCavitand.

A very sensitive and selective solid-phase microextraction-gas chromatography-mass spectrometry method based on the use of a deep cavity BenzoQxCavitand as innovative coating was developed and validated for the simultaneous determination of the 16 US-EPA priority pollutants polycyclic aromatic hydrocarbons (PAHs) in snow samples at ultra-trace levels. The presence of a 8.3 Å deep hydrophobic cavity allowed the engulfment of all the 16 PAHs, providing enhanced selectivity also in presence of interfering aromatic pollutants at high concentration levels. Validation proved the reliability of the method for the determination of the investigated compounds achieving detection limits in the 0.03-0.30 ng/L range, good precision, with relative standard deviations <18% and recovery rates in the 90.8(±2.1)%-109.6(±1.0)%. The detection of low-molecular weight PAHs in snow samples from Antarctica and Alps confirms the widespread occurrence of these compounds, thus assessing the impact of anthropogenic activities onto the environment.

A simple and efficient Solid-Phase Microextraction - Gas Chromatography - Mass Spectrometry method for the determination of fragrance materials at ultra-trace levels in water samples using multi-walled carbon nanotubes as innovative coating.

The occurrence of emerging contaminants is becoming of increasing importance to assess the impact of anthropogenic activities onto the environment. The present study reports for the first time the development and validation of an efficient method for the simultaneous determination of fragrance materials in water samples based on the use of a novel multiwalled carbon nanotubes (MWCNTs)-based solid-phase microextraction coating. Helical MWCNTs were selected as adsorbent material due to their outstanding extraction performance. The multicriteria method of desirability functions allowed the optimization of the experimental conditions in terms of extraction time and extraction temperature. Validation proved the reliability of the method for the determination of the analytes at ultra-trace levels, obtaining detection limits in the 0.2-13 ng/L range, good precision, with relative standard deviations lower than 20% and recovery rates in the 80 ± 12%-111 ± 11%. Superior enrichment factors compared to commercial fibers were also calculated. Finally, applicability to real sample analysis was demonstrated.

Superhydrophobic lab-on-chip measures secretome protonation state and provides a personalized risk assessment of sporadic tumour.

Secretome of primary cultures is an accessible source of biological markers compared to more complex and less decipherable mixtures such as serum or plasma. The protonation state (PS) of secretome reflects the metabolism of cells and can be used for cancer early detection. Here, we demonstrate a superhydrophobic organic electrochemical device that measures PS in a drop of secretome derived from liquid biopsies. Using data from the sensor and principal component analysis (PCA), we developed algorithms able to efficiently discriminate tumour patients from non-tumour patients. We then validated the results using mass spectrometry and biochemical analysis of samples. For the 36 patients across three independent cohorts, the method identified tumour patients with high sensitivity and identification as high as 100% (no false positives) with declared subjects at-risk, for sporadic cancer onset, by intermediate values of PS. This assay could impact on cancer risk management, individual's diagnosis and/or help clarify risk in healthy populations.

Conformationally blocked quinoxaline cavitand as solid-phase microextraction coating for the selective detection of BTEX in air.

A tetraquinoxaline cavitand functionalized with methylenoxy bridges at the upper rim is proposed as selective solid-phase microextraction (SPME) coating for the determination of BTEX at trace levels in air. The SPME fibers were characterized in terms of film thickness, morphology, thermal stability and extraction capabilities. An average coating thickness of 35 (±4) µm, a thermal stability up to 350 °C and a good fiber-to-fiber and batch-to-batch repeatability with RSD lower than 15% were obtained. Excellent enrichment factors ranging from 360-700 × 10(3) were obtained for the investigated compounds. Finally, method validation proved the capabilities of the developed coating for the selective sampling of BTEX, achieving LOD values in the 0.4-1.2 ng m(-3) range.

Rapid desorption electrospray ionization-high resolution mass spectrometry method for the analysis of melamine migration from melamine tableware.

Migration of melamine into foods from melamine tableware has been object of recent Rapid Alert System for Food and Feed (RASFF) notifications. In this context, a rapid and sensitive desorption electrospray ionization-high resolution mass spectrometry (DESI-HRMS) method was developed and validated for the determination of melamine migration from plastic materials. The migration test was performed using acetic acid 3% (w/v) as food simulant. Evaluation of DESI parameters in terms of choice of support, motion profile, geometrical configuration and operating conditions coupled to the use of an orbitrap mass analyzer allowed to achieve significant improvements in terms of selectivity and accuracy obtaining detection and quantitation limits at low microgram per kilogram level. A LC-ESI-MS method was also developed for confirmatory purposes. Both methods were applied to 44 melamine tableware samples available on Italian market in order to assess their compliance with the law. Different concentration levels ranging from 0.00773±0.0006 to 3.0±0.1 mg/kg were found after the third exposure to the simulant in new and used tableware with two samples out of 44 being characterized by a melamine release higher than the legal limit, i.e. 2.5 mg/kg. A two tailed t-test allowed to assess the good agreement between the quantitative results obtained applying the DESI-MS method with those provided by LC-ESI-MS, thus proving reliability of DESI-HRMS as rapid technique for the study of melamine release from plastic materials.

Solid-phase microextraction of 2,4,6-trinitrotoluene using a molecularly imprinted-based fiber.

A molecularly imprinted polymer with trinitrotoluene as the template molecule was synthesized and used as the novel coating for solid-phase microextraction of the nitroaromatic explosive 2,4,6-trinitrotoluene for its selective determination. The fiber was characterized in terms of coating thickness, morphology, intra- and inter-batch repeatability and extraction efficiency. An average thickness of 50 ± 4 µm with a uniform distribution of the coating was obtained. Good performances of the developed procedure in term of both intra-batch and inter-batch repeatability with relative standard deviations <8% were obtained. Finally, detection and quantitation limits in the low nanogram per kilogram levels were achieved proving the superior extraction capability of the developed coating, obtaining gas chromatography-mass spectrometry responses about two times higher than those achieved using commercial devices.

Magnetic solid-phase extraction based on diphenyl functionalization of Fe3O4 magnetic nanoparticles for the determination of polycyclic aromatic hydrocarbons in urine samples.

Superparamagnetic Fe(3)O(4) diphenyl nanoparticles were prepared according to a solvothernal procedure and characterized by X-ray diffraction, infrared spectroscopy, surface area measurements, scanning electron microscopy, X-ray photoelectron spectroscopy and transmission electron microscopy. The magnetic phases present in the nanoparticle samples were analyzed by thermomagnetic analysis and the samples' magnetic properties were studied by vibrating sample magnetometry. The resulting nanoparticles having an average diameter of 200 nm were then used as solid-phase extraction sorbent for the determination of polycyclic aromatic hydrocarbons in urine samples. Method validation proved the feasibility of the developed beads for the quantitation of the investigated analytes at trace levels obtaining lower limit of quantitation values in the ng/l range. A good precision with coefficients of variations always lower than 15% was obtained. Finally, the superior extraction performance of the synthesized nanoparticles with respect to commercially available beads was proved.

Fully automated solid-phase microextraction-fast gas chromatography-mass spectrometry method using a new ionic liquid column for high-throughput analysis of sarcosine and N-ethylglycine in human urine and urinary sediments.

A fully automated, non invasive, rapid and high-throughput method for the direct determination of sarcosine and N-ethylglycine in urine and urinary sediments using hexyl chloroformate derivatization followed by direct immersion solid-phase micro extraction and fast gas chromatography-mass spectrometric analysis was developed and validated. The use of a new ionic liquid narrow bore column, as well as the automation and miniaturization of the preparation procedure by a customized configuration of the utilized XYZ robotic system, allowed a friendly use of the GC apparatus achieving a quantitation limit of 0.06 µg L(-1) for sarcosine, good repeatability with CV always lower than 7% and reduced analysis times useful for point-of-care testing. The method was then applied for the analysis of 56 samples of urine and urinary sediments in healthy subjects, in those with benign prostatic hypertrophy and in patients with clinically localized prostate cancer. The results obtained showed that the medians of sarcosine/creatinine in urine were 103, 137 and 267 µg g(-1) respectively, thus assessing the potential use of sarcosine as urinary biomarker for prostate cancer detection. The highest values of sensitivity (79%) and specificity (87%) were obtained in correspondence of a cut-off value of 179 µg sarcosine(g creatinine)(-1), thus by using this cut-off threshold, sarcosine was significantly associated with the presence of cancer (p<0.0001). Finally, ROC analyses proved that the discrimination between clinically localized prostate cancer and patients without evidence of tumor is significantly correlated with sarcosine.

Development of a combined SEM and ICP-MS approach for the qualitative and quantitative analyses of metal nano and microparticles in food products [corrected].

An integrated approach based on the use of inductively coupled plasma mass spectrometry (ICP-MS) and scanning electron microscopy (SEM) for the qualitative and quantitative analyses of metal particles in foods was devised and validated. Different raw materials and food products, like wheat, durum wheat, wheat flour, semolina, cookies, and pasta were considered. Attention was paid to the development of sample treatment protocols for each type of sample to avoid potential artifacts such as aggregation or agglomeration. The analytical protocols developed followed by ICP-MS and SEM investigations allowed us the quantitative determination and the morphological and dimensional characterization of metal nano- and microparticles isolated from the raw materials and finished food products considered. The ICP-MS method was validated in terms of linearity (0.8-80 µg/g and 0.09-9 µg/g for Fe and Ti, respectively), quantification limits (0.73 µg/g for Fe and 0.09 µg/g for Ti), repeatability (relative standard deviation (RSD) % equal to 10% for Fe and 20% in a wheat matrix as an example), and extraction recoveries (93 ± 2-101 ± 2%). Validation of the scanning electron microscopy-energy dispersive X-ray spectroscopy (SEM-EDS) measurements was performed working in a dimensional range from 1 to 100 µm with an estimated error in the size determination equal to 0.5 µm. ICP-MS data as well as SEM measurements showed a decrease in the concentration of metal particles from wheat to flour and from durum wheat to semolina samples, thus indicating an external contamination of grains by metal particles. These findings were confirmed by environmental SEM analysis, which allowed investigation of particles of lower dimensions. Generally, the largest number of particles was found in the case of iron and titanium, whereas particles of copper and zinc were only occasionally found without any possibility of quantifying their number.

Planar solid-phase microextraction-ion mobility spectrometry: a diethoxydiphenylsilane-based coating for the detection of explosives and explosive taggants.

A novel diethoxydiphenylsilane-based coating for planar solid-phase microextraction was developed using sol-gel technology and used for ion mobility spectrometric detection of the explosives 2,4,6-trinitrotoluene, 2,4-dinitrotoluene, and of the explosive taggant ethylene glycol dinitrate. The trap was characterized in terms of coating thickness, morphology, inter-batch repeatability, and extraction efficiency. An average thickness of 143 ± 13 µm with a uniform distribution of the coating was obtained. Good performances of the developed procedure in terms of both intra-batch and inter-batch repeatability with relative standard deviations <7% were obtained. Experimental design and desirability function were used to find the optimal conditions for simultaneous headspace extraction of the investigated compounds: the optimal values were found in correspondence of a time and a temperature of extraction of 45 min and 40 °C, respectively. Detection and quantitation limits in low nanogram levels were achieved proving the superior extraction capability of the developed coating, obtaining ion mobility spectrometric responses at least two times higher than those achieved using commercial teflon and paper traps.

Identification of in vivo nitrosylated phytochelatins in Arabidopsis thaliana cells by liquid chromatography-direct electrospray-linear ion trap-mass spectrometry.

Reversed-phase liquid chromatography (RPLC) and electrospray (ESI)-linear ion trap (LIT) mass spectrometry was applied to the direct characterization of in vivo S-nitrosylated (SNO) phytochelatins (PCs) expressed in cadmium-stressed Arabidopsis thaliana cells. Cys-nitrosylation is under discussion as in vivo redox-based post-translational modification of proteins and peptides in plants in which the -NO group is involved as signal molecule in different biological functions. The gas-phase ion chemistry of in vivo and in vitro generated SNO-PC(s) was compared with the aim of evaluating NO binding stability and improving MS knowledge about peptide nitrosation. Using RPLC separation and ESI-LIT-MS, mono-nitrosylated PCs were identified in in vivo cadmium treated A. thaliana cells without derivatization. The in vivo binding of the NO group to PC(2), PC(3) and PC(4) resulted to occur selectively on only one cystein residue. The fragmentation pathway energies of the in vitro GSNO-generated NO-PCs with respect to the in vivo NO-PCs were investigated, suggesting the presence of a different internal stability for these molecules. By carrying out MS(2) experiments on these quasi-symmetric peptides, the different stability degree of the NO group was demonstrated to be correlated with the PC chain length. In addition, the data obtained highlight a putative role of the adjacent Glu/Cys motif in the gas-phase stability of the NO-containing molecule.

A novel headspace solid-phase microextraction method using in situ derivatization and a diethoxydiphenylsilane fibre for the gas chromatography-mass spectrometry determination of urinary hydroxy polycyclic aromatic hydrocarbons.

An innovative and simple headspace solid-phase microextraction method using a novel diethoxydiphenylsilane fibre based on in situ derivatization with acetic anhydride was optimized and validated for the gas chromatography-mass spectrometry determination of some monohydroxy metabolites of polycyclic aromatic hydrocarbons, namely 1-hydroxynaphthalene, 2-hydroxynaphtalene, 9-hydroxyfluorene, 2-hydroxyfluorene and 1-hydroxypyrene at trace levels in human urine. Enzymatic hydrolysis was applied before derivatization, whereas extraction conditions, i.e. the effects of time and temperature of extraction and salt addition were investigated by experimental design. Regression models and desirability functions were applied to find the experimental conditions providing the highest global extraction response. These conditions were found in correspondence of an extraction temperature of 90 degrees C, an extraction time of 90 min and 25% NaCl added to urine samples. The capabilities of the developed method were proved obtaining limit of quantitations in the 0.1-2 microg/l range, thus allowing the bio-monitoring of these compounds in human urine. A good precision was observed both in terms of intra-batch and inter-batch repeatability with RSD always lower than 14%. Recoveries ranging from 98(+/-3) to 121(+/-1)% and extraction yields higher than 72% were also obtained. Finally, the analysis of urine specimens of coke-oven workers revealed analytes' concentrations in the 2.2-164 microg/l range, proving the exposure to PAHs of the involved workers.

Determination of food emulsifiers in commercial additives and food products by liquid chromatography/atmospheric-pressure chemical ionisation mass spectrometry.

A new, reliable liquid chromatography/atmospheric-pressure chemical ionisation mass spectrometry (LC-APCI-MS) method was developed for the quantitative determination of food emulsifiers composed of mono- and diacylglycerols of fatty acids (E471 series) in complex food matrices. These additives are extremely interesting for the food industry because of their useful properties. Indeed, they improve the manufacture of products by acting as foams and creams stabilisers, crumb-softeners, or antistaling agents. The proposed method also allows us to qualitatively characterise new food emulsifiers composed of other acid esters of mono- and diacylglycerols (E472 series). The validation of the method was performed on blank minicake spiked samples for detection limits (reaching ppm levels), linearity, recovery, precision, and accuracy. The method was then successfully applied to commercial additives containing mixtures of emulsifiers, as well as to food products such as margarines and minicakes.

Differentiation of the volatile profile of microbiologically contaminated canned tomatoes by dynamic headspace extraction followed by gas chromatography-mass spectrometry analysis.

The aromatic profile of microbiologically contaminated canned tomatoes was analyzed by the dynamic headspace extraction technique coupled with gas chromatography-mass spectrometry. Canned tomatoes contaminated with Escherichia coli, Saccharomyces cerevisiae and Aspergillus carbonarius were analyzed after 2 and 7 days. About 100 volatiles were detected, among which alcohols, aldehydes and ketones were the most abundant compounds. Gas chromatographic peak areas were used for statistical purposes. First, principal component analysis was carried out in order to visualize data trends and clusters. Then, linear discriminant analysis was performed in order to detect the set of volatile compounds ables to differentiate groups of analyzed samples. Five volatile compounds, i.e. ethanol, beta-myrcene, o-methyl styrene, 6-methyl-5-hepten-2-ol and 1-octanol, were found to be able to better discriminate between uncontaminated and contaminated samples. Prediction ability of the calculated model was estimated to be 100% by the "leave-one-out" cross-validation. An electronic nose device was then used to analyze the same contaminated and not contaminated canned tomato samples. Preliminary results were compared with those obtained by dynamic headspace gas chromatography-mass spectrometry, showing a good agreement.

Hydroxyestrogens inhibit angiogenesis in swine ovarian follicles.

The rapid, controlled, and cyclical nature of angiogenesis in the ovarian follicle suggests the potential for sex steroids to influence neovascularization. Angiogenesis is regulated by a local balance between the levels of endogenous stimulators and inhibitors. Multiple lines of evidence suggest that estrogens stimulate angiogenesis via effects on endothelial cells. However, it is of outstanding value to investigate the negative control of this process. Since the main estrogen metabolites, 2-hydroxyestradiol and 4-hydroxyestradiol (4-OHE2) have been demonstrated to function as anti-estrogen in several estrogen-dependent organs; the aim of this study was to investigate their potential involvement in the modulation of follicular angiogenesis. Hydroxyestrogens were quantified in swine follicular fluid and their effects were studied on granulosa cell vascular endothelial growth factor (VEGFA) production and tested in an angiogenesis bioassay. Our study documents that these molecules are physiologically present in swine follicular fluid and their concentrations significantly (P<0.001) increase during follicle development. Moreover, angiogenesis bioassay revealed that both hydroxyestrogens significantly (P<0.001) inhibited new vessel growth. We evidenced that the most potent negative effect is mediated by 4-OHE2. The anti-angiogenic potential of this molecule is also supported by its ability to inhibit (P<0.001) VEGFA production by granulosa cells. Increased knowledge in this area is of utmost importance for future therapeutic options to contrast infertility disorders associated with aberrant angiogenesis; this would be also very useful for the treatment of diseases characterized by disregulated angiogenesis and vascular regression.

Experimental design for the optimization of the extraction conditions of polycyclic aromatic hydrocarbons in milk with a novel diethoxydiphenylsilane solid-phase microextraction fiber.

An innovative solid-phase microextraction coating based on the use of diethoxydiphenylsilane synthesized by sol-gel technology was used for the determination of polycyclic aromatic hydrocarbons at trace levels in milk. The effects of time and temperature of extraction and acetone addition were investigated by experimental design. Regression models and desirability functions were applied to find the experimental conditions providing the highest global extraction response. The capabilities of the developed fiber were proved obtaining limit of quantitation values in the low microg/l range, enabling the direct analysis of complex matrices like milk and a complete desorption of high-boiling compounds without carryover effects.

Use of specific peptide biomarkers for quantitative confirmation of hidden allergenic peanut proteins Ara h 2 and Ara h 3/4 for food control by liquid chromatography-tandem mass spectrometry.

A liquid chromatography-electrospray-tandem mass spectrometry (LC-ESI-MS-MS) method based on the detection of biomarker peptides from allergenic proteins was devised for confirming and quantifying peanut allergens in foods. Peptides obtained from tryptic digestion of Ara h 2 and Ara h 3/4 proteins were identified and characterized by LC-MS and LC-MS-MS with a quadrupole-time of flight mass analyzer. Four peptides were chosen and investigated as biomarkers taking into account their selectivity, the absence of missed cleavages, the uniform distribution in the Ara h 2 and Ara h 3/4 protein isoforms together with their spectral features under ESI-MS-MS conditions, and good repeatability of LC retention time. Because of the different expression levels, the selection of two different allergenic proteins was proved to be useful in the identification and univocal confirmation of the presence of peanuts in foodstuffs. Using rice crisp and chocolate-based snacks as model food matrix, an LC-MS-MS method with triple quadrupole mass analyzer allowed good detection limits to be obtained for Ara h 2 (5 microg protein g(-1) matrix) and Ara h 3/4 (1 microg protein g(-1) matrix). Linearity of the method was established in the 10-200 microg g(-1) range of peanut proteins in the food matrix investigated. Method selectivity was demonstrated by analyzing tree nuts (almonds, pecan nuts, hazelnuts, walnuts) and food ingredients such as milk, soy beans, chocolate, cornflakes, and rice crisp.

Validation of an ion-pair liquid chromatography-electrospray-tandem mass spectrometry method for the determination of heterocyclic aromatic amines in meat-based infant foods.

A method based on ion-pair liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS) is reported for determining heterocyclic aromatic amines (HAAs) in meat-based infant foods. The HAAs encompassed quinoline (IQ, MeIQ), quinoxaline (MeIQx), pyridine (PhIP), and carboline derivatives (AalphaC, Harman, Norharman) with d(3)-IQ, (13)C(2)-MeIQx, and d(3)-PhIP used as labelled internal standards. The method used extraction into acetone followed by a clean-up on an SCX solid-phase extraction column. LC separation was performed on a TSKgel ODS-80TS column (250 x 2.0 mm, 5 microm), the mobile phase being an ammonium formate-formic acid buffer (3.03 mM ammonium formate, pH = 2.8) aqueous solution-acetonitrile gradient at a flow rate of 0.2 ml min(-1). For unequivocal identification of each analyte, three ions were detected and chosen for selected reaction monitoring (SRM). Validation was carried out on lyophilized meat samples. Mean recoveries ranged between 78 +/- 4% and 98 +/- 2% for different analytes. Limits of quantification generally lower than 8 ng g(-1) were demonstrated in meat samples for the analytes investigated. The method exhibited a good linearity and repeatability. Robustness testing identified those factors which were statistically significant in influencing chromatographic separation and response, and indicated which parameters have to be strictly controlled for a reliable analysis of HAAs. In particular, the mobile-phase flow rate was found to be statistically significant (alpha = 0.05) for the capacity factor (k') of all analytes except for AalphaC peak, whereas the mobile-phase pH resulted to be a critical parameter for the k' values of IQ, MeIQ, and Norharman. The method was proved to be robust vs. resolution between IQ and MeIQ peaks. Among mass-spectrometric parameters, collision energy was found to significantly affect quantitative response of all analytes except that of IQ. The applicability of the method to the analysis of meat-based infant food samples was demonstrated.

Porcine follicular fluids: comparison of solid-phase extraction and matrix solid-phase dispersion for the GC-MS determination of hormones during follicular growth.

The capabilities of solid-phase extraction (SPE) and matrix solid-phase dispersion (MSPD) for the determination of the hormones 17beta-estradiol, 2-hydroxyestradiol, 4-hydroxyestradiol and 2-methoxyestradiol by gas chromatography-mass spectrometry (GC-MS) in a very complex matrix like porcine follicular fluids were compared, thus proving the highest effectiveness of the SPE technique. Validation was carried out in terms of limit of quantitation (LOQ), precision, accuracy, recovery and stability. LOQ values in the low microg kg(-1) were achieved, with all the other parameters satisfying the acceptance criteria for the validation of bioanalytical methods. The applicability of the method to the determination of the hormones in porcine follicular fluids was demonstrated, thus allowing to observe an increase of the concentration of the hormones during the follicular growth.