Generally, Soxhlet extraction is still the most used technique in the official methods of analysis for solid matrices. This traditional approach is expensive, time-consuming, and environmentally unfriendly. For some years, a great effort has been made to develop alternative and high-throughput analytical methods for the pollutant extraction from solids in compliance with the basic requirements of Green Analytical Chemistry (GAC). One of the most interesting approaches is the microwaveassisted extraction (MAE) because it allows for the rapid extraction of target molecules from solid matrices with low solvent consumption; besides, the working parameters can be controlled and properly optimized by univariate or multivariate approaches

paragrafo 4.2.8 Hybrid mass spectrometers. Breve descrizione delle diverse tipologie di spettrometri di massa ibridi

Polyamines are aliphatic amines with low molecular weight that are widely recognized as one of the most important cancer biomarkers for early diagnosis and treatment. The goal of the work herein presented is the development of a rapid and simple method for the quantification of free polyamines (i.e., putrescine, cadaverine, spermidine, spermine) and N-monoacetylated polyamines (i.e., N-Acetylspermidine, N-Acetylspermidine, and N-Acetylspermine) in human urine. A preliminary derivatization with propyl chloroformate combined with the use of solid phase microextraction (SPME) allowed for an easy and automatable protocol involving minimal sample handling and no consumption of organic solvents. The affinity of the analytes toward five commercial SPME coatings was evaluated in univariate mode, and the best result in terms of analyte extraction was achieved using the divinylbenzene/carboxen/polydimethylsiloxane fiber. The variables affecting the performance of SPME analysis were optimized by the multivariate approach of experimental design and, in particular, using a central composite design (CCD). The optimal working conditions in terms of response values are the following: extraction temperature 40 °C, extraction time of 15 min and no addition of NaCl. Analyses were carried out by gas chromatography-triple quadrupole mass spectrometry (GC-QqQ-MS) in selected reaction monitoring (SRM) acquisition mode. The developed method was validated according to the guidelines issued by the Food and Drug Administration (FDA). The satisfactory performances reached in terms of linearity, sensitivity (LOQs between 0.01 and 0.1 ?g/mL), matrix effect (68-121%), accuracy, and precision (inter-day values between -24% and +16% and in the range 3.3-28.4%, respectively) make the proposed protocol suitable to be adopted for quantification of these important biomarkers in urine samples.

In this work, organophosphate ester flame retardant (OPFRs) assay in environmental waters was addressed by using microextraction by packed sorbent (MEPS) and gas chromatography-tandem mass spectrometry (GC-MS/MS). Ten OPFRs with different physicochemical properties were taken into account as target compounds for a comprehensive method evaluation. Five MEPS cartridges (i.e., C2, C8, C18, Silica, and DVB) and seven solvents (i.e., methanol, ethyl acetate, methyl tert-butyl ether, hexane, acetonitrile, dichloromethane, and trichloromethane) were surveyed. The analysis was performed by using a gas chromatograph equipped with a programmed temperature vaporization injector (PTV). Univariate and multivariate approaches were exploited in order to optimize the parameters affecting the MEPS extraction and the PTV injection of the analytes into the gas chromatographic system. The optimal working conditions were achieved using DVB as sorbent material and acetonitrile as elution solvent. Internal standard calibration was carried out using TBP-d27 and TCEP-d12. Satisfactory values of accuracy and precision were generally obtained as well as limit of detection (2.7-99 pg/mL for tap water; 2.9-97 pg/mL for river water; 3-107 pg/mL for wastewater) and limit of quantification (0.01-0.2 ng/mL). The proposed protocol was evaluated on real case scenarios by analyzing tap water, river water and simulated wastewater samples. The developed method is not only eco-friendly due to the low use of organic solvents but also simple and automatable since the MEPS extraction procedure can be implemented in the autosampler routine.

The acid-insoluble salivary proteome obtained by addition of TFA to whole human saliva from adults, preterm and at-term newborns has been analysed by 2-DE in order to evidence differences among the three groups, and integrate data previously obtained on the acid-soluble fraction. 2-DE spots differentially expressed among the three groups were submitted to in-gel tryptic digestion and the peptide mixtures analysed by high resolution HPLC-ESI-MS/MS. By this strategy, we identified 3 over-expressed proteins in at-term newborns with respect to preterm newborns and adults (BPI fold-containing family A member 1, annexin A1, and keratin type 1 cytoskeletal 13), and several over-expressed proteins in adults (fatty acid-binding protein, S100 A6, S100 A7, S100 A9, prolactin-inducible protein, Ig kappa chain, cystatin SN, cystatin S/SA and ?-amylase 1). Four spots, already detected but not characterized by other authors in human saliva 2-DE, were attributed to different protein species of S100 A9 (long-type and long-type monophosphorylated, short-type and short-type monophosphorylated) by MS/MS analysis of tryptic peptides and sequential staining of 2-DE gels with Pro-Q Diamond, for specific detection of phosphoproteins, and total protein SYPRO Ruby stain

Homovanillic acid (HVA), vanylmandelic acid (VMA), and 5-hydroxyindoleacetic acid (5-HIAA) are the metabolites of some catecholamines such as epinephrine, nor-epinephrine, dopamine and serotonin and their quantification is used in the diagnosis and management of patients with neurocrine tumors. A novel approach in the assay of these biomarkers in human urine samples by solid phase microextraction (SPME) combined with gas chromatography-triple quadrupole mass spectrometry (GC-QqQ-MS) is presented. A preliminary derivatization with ethyl chloroformate/ethanol was used and the corresponding derivatives were then extracted by SPME in immersion mode. The performance of five SPME fibers and three chloroformates were evaluated in univariate mode and the best results were obtained using the polyacrylate fiber and ethyl chloroformate. The variables affecting the efficiency of SPME analysis were optimized by the multivariate approach of "Experimental design" and, in particular, a central composite design (CCD) was applied. The optimum working conditions in terms of response values were achieved by performing analysis at room temperature with addition of NaCl (9.5%) and with an extraction time of 25.8 min. Identification and quantification of analytes were carried out by using a gas chromatography-triple quadrupole mass spectrometry (GC-QqQ MS) system in multiple reaction monitoring (MRM) acquisition. An evaluation of all analytical parameters shows that the proposed method provides satisfactory results. Very good linearities were, in fact, achieved in the tested calibration ranges with correlation coefficient values >0.99 for all the analytes and accuracies and RSDs calculated for between-run and tested at concentrations of 1, 10, and 80 mg L-1 were ranging from 91.3% to 106.6%, and from 0.5 to 8.9%, respectively. Moreover, the LOD values obtained can be considered very satisfactory (1.3, 0.046 and 24.3 mu g L-1 for HVA, VMA and 5-HIAA, respectively). The developed protocol represents, therefore, a simple, rapid and selective tool for assaying these acidic biomarkers in urine samples for neuroendocrine cancer diagnosis. (C) 2012 Elsevier B.V. All rights reserved.

A new analytical approach, using paper spray tandem mass spectrometry, has been developed for assay of carnitine and acylcarnitines in urine. Paper spray (PS) is a very promising technique, especially in clinical investigations, because of its simplicity, low cost, and rapid sample preparation. A home-made paper spray device was used for assay of urinary acylcarnitines (C2-C18). The performance of solvents with different elution efficiency and paper substrates with different porosity grade and structure were tested by use of spiked synthetic urine. Tandem mass spectrometry in multiple reaction monitoring (MRM) mode was optimized to obtain better specificity and sensitivity. Analyte signals were evaluated for stability and reproducibility. Calibration with [H-2(3)]propionylcarnitine (C3-d3), [H-2(3)]octanoylcarnitine (C8-d3), and [H-2(3)] palmitoylcarnitine (C16-d3) as internal standards was used for quantification. Very good linearity was obtained, with correlation coefficients > 0.99 for C0-C12 and C16 acylcarnitines and > 0.96 for C14 and C18 acylcarnitines. Accuracy and precision (RSD, %) of the proposed procedure were tested at concentrations of 0.8, 8, and 20 mg L-1 with very satisfactory results: overall mean accuracy was 98.9 % and overall mean relative standard deviation 1 %. Limits of detection (LOD) between 6 and 208 mu g L-1 for propionylcarnitine and tetradecanoylcarnitine, respectively, can be regarded as very satisfactory. Application of the method to real urine proved that paper spray tandem mass spectrometry is a simple, rapid, and direct tool (no derivatization is required) for assay of carnitine and C2-C12 acylcarnitines in urine.

Domoic acid (DA) is a neurotoxin produced by different algae, including pennate diatoms, principally from the genus Pseudo-nitzschia, and it is the main cause of amnesic shellfish poisoning. Determination of this toxin in seawater samples is fundamental to define the real contamination risks for aquatic species. We have developed two very sensitive instrumental methods using hydrophilic interaction liquid chromatography coupled using tandem mass spectrometry in positive and negative polarity modes. Instrumental detection limits were 9 pg mL-1 for positive and 19 pg mL-1 for negative ionisation. A procedural method based on solid-phase extraction for the determination of dissolved DA present in seawater has been developed, and an extraction procedure was employed for the determination of the toxin in the particulate fraction. DA quantification was performed using the internal standard method to account for signals fluctuations and random errors during sample treatment. To our knowledge, this is the first study to use this quantification method for DA determination. Trueness, extraction yield, matrix effects, repeatability and procedural detection limits were evaluated during method validation. Procedural detection limits of 0.3 pg mL-1 (positive mode) and 0.6 pg mL-1 (negative mode) were found for the dissolved fraction, and absolute limits of 0.4 pg (positive mode) and 6.0 pg (negative mode) for particulate samples were obtained. The most sensitive method in positive mode was applied to define DA occurrence in the Venice Lagoon. Trace concentrations of domoic acid ranging from 1.5 to 16.2 pg mL-1 were found for the first time in the Venetian environment. [Figure not available: see fulltext.]

The work aims at developing a rapid and sensitive method for the quantification of perfluorocarboxylic acids in aqueous matrices. The proposed analytical approach is based on the use of solid phase microextraction in headspace mode after a fast derivatization of the carboxylate function by propylchloroformate/propanol mixture. Several fibers were evaluated and the optimization of the parameters affecting the SPME process was carried out using a central composite design. The optimum working conditions in terms of response values were achieved by performing analysis with CAR/PDMS fiber at room temperature, without addition of NaCl, with a sample volume of 6 ml and an extraction time of 10 min. Assay of PFCAs was performed by using a gas chromatography-triple quadrupole mass spectrometry (GC-QqQ MS) system in negative chemical ionization mode with ammonia as reagent gas. An overall evaluation of all analytical parameters shows that the proposed method provides satisfactory results. In particular, the observed accuracies, ranging from 84.4% to 116.8%, and the RSD values in the range 0.4% and 14.5% confirm the effectiveness of the developed protocol in the assay of PFCAs content in aqueous matrices. Moreover. LOD and LOQ values ranging from 0.08 to 6.6 ng I-1 and from 0.17 to 14.3 ng I-1, respectively, can be considered very satisfactory. None of the compounds were detected in six samples of river collected in Calabria. (C) 2012 Elsevier B.V. All rights reserved.

A simple and sensitive method was developed for the quantification of five carbamate pesticides in water samples using solid phase microextraction (SPME) combined with gas chromatography-triple quadrupole mass spectrometry (GC-QqQ-MS). The performance of five SPME fibers was tested in univariate mode whereas the other variables affecting the efficiency of SPME analysis were optimized by the multivariate approach of design of experiment (DoE) and, in particular, a central composite design (CCD) was applied. The optimum working conditions in terms of response values were achieved by performing analysis with polydimethylsiloxane/divinylbenzene (PDMS/DVB) fiber in immersion mode for 45 min at room temperature with addition of NaCl (10%). The multivariate chemometric approach was also used to explore the chromatographic behavior of the carbamates and to evaluate the importance of each variable investigated. An overall appraisement of results shows that the factor which gave a statistically significant effect on the response was only the injection temperature. Identification and quantification of carbamates was performed by using a gas chromatography-triple quadrupole mass spectrometry (GC-QqQ-MS) system in multiple reaction monitoring (MRM) acquisition. Since the choice of internal standard represented a crucial step in the development of method to achieve good reproducibility and robustness for the entire analytical protocol, three compounds (2,3,5-trimethacarb, 4-bromo-3,5-dimethylphenyl-n-methylcarbarnate (BDMC) and carbaryl-d7) were evaluated as internal standards. Both precision and accuracy of the proposed protocol tested at concentration of 0.08, 5 and 3 mu g l(-1) offered values ranging from 70.8% and 115.7% (except for carbaryl at 3 mu g l(-1)) and from 1.0% and 9.0% for accuracy and precision, respectively. Moreover, LOD and LOQ values ranging from 0.04 to 1.7 ng l(-1) and from 0.64 to 2.9 ng l(-1). respectively, can be considered very satisfactory. (C) 2012 Elsevier B.V. All rights reserved.

A new analytical approach is exploited in the assay of selenium speciation in selenized and not selenium enriched potatoes based on the widely available solid-phase microextraction (SPME) coupled to-GC-triple quadrupole mass spectrometry (SPME-GC-QqQ MS) method. The assay of selenomethionine (SeMet) and selenomethylselenocysteine (SeMeSeCys) in potatoes here reported provides clues to the effectiveness of SPME technique combined with gas chromatography-tandem mass spectrometry, which could be of general use. For the exploitation of the GC method, the selected analytes were converted into their N(O,S)-alkoxycarbonyl alkyl esters derivatives by direct treatment with alkyl chloroformate in aqueous extracts. The performance of five SPME fibers and three chloroformates were tested in univariate mode and the best results were obtained using the divinylbenzene/carboxen/polydimethylsiloxane fiber and propylchloroformate. The variables affecting the efficiency of SPME analysis were optimized by the multivariate approach of design of experiment (DoE) and, in particular, a central composite design (CCD) was applied. Tandem mass spectrometry in selected reaction monitoring (SRM) has allowed the elimination of matrix interferences, providing reconstructed chromatograms with well-resolved peaks and the achievement of very satisfactory detection and quantification limits. Both precision and recovery of the proposed protocol tested at concentration of 8 and 40 g kg-1 (dry matter), offered values ranging from 82.3 to 116.3% and from 8.5 to 13.1% for recovery and precision, respectively. The application of the method to commercial samples of selenized and not selenium enriched potatoes proved that the Se fertilization increases significantly the concentration of these bioavailable selenoamino acids.

A method has been developed for the determination of acrylamide at the picogram per cubic metre level in particulate-phase outdoor aerosol using highperformance liquid chromatography with triple quadrupole tandem mass spectrometric detection. Acrylamide was identified by positive ion electrospray mass spectrometry using m/z 72.00/54.90 as monitoring ion transition. The limit of detection, defined as three times the standard deviation of the procedural blanks, was 0.4 pg m-3 (173 pg absolute amount injected); the repeatability was 8% (evaluated as the relative standard deviation of five consecutive measurements on cleaned quartz fibre filters of acrylamide standard spikes) and the recovery was 52

Sarcosine is an amino acid derivative of N-methylglycine and is involved in the amino acid metabolism and methylation processes that are enriched during prostate cancer progression. It could also serve as a new target to be measured during therapeutic interventions and help in the identification of aggressive tumors for radical treatment. In this study, we present a new urine test that can help early diagnosis of prostate cancer. The method for the quantification of sarcosine in urine consists of a solid-phase microextraction (SPME) step followed by gas chromatography-triple quadrupole mass spectrometry analysis. We used a preliminary derivatization step with ethyl chloroformate/ethanol and the corresponding ester was then extracted by SPME in immersion mode. Several fibers were evaluated and the optimization of the parameters affecting the SPME process was carried out using an experimental design. The optimal values were 20 min extraction time, 10% NaCl, and 270°C using a divinylbenzene/Carboxen/ polydimethylsiloxane fiber. The triple quadrupole analyzer acquired data in selected reaction monitoring mode, allowing us to obtain reconstructed chromatograms with well-defined chromatographic peaks. The accuracy and precision of this method were evaluated at concentrations of 70, 250, and 800 ng/ml and were found to be acceptable. Very satisfactory values (0.10 and 0.16 ng/ml, respectively) were also achieved for the limit of detection and the limit of quantification. The proposed protocol represents a rapid, simple, selective, and sensitive tool to quantify sarcosine in urine samples for prostate cancer diagnosis and for a screening test. © 2011 Springer-Verlag.

New phenolic compounds from Olea europaea, identified by high performance liquid chromatography/electrospray ionization tandem mass spectrometry, are suitable markers for differentiation between different varieties of olive trees cultivated in the same geographical area (Rende, Italy). Five cultivars (Carolea, Cassanese, Coratina, Nocellara and Leccino) were considered for the discrimination. Samples of Carolea, cultivated in three different geographical zones (Rende, Mirto and Spoleto, Italy), were as well checked to evaluate possible differences. Three supervised pattern recognition procedures, linear discriminant analysis (LDA), soft independent modelling of class analogy (SIMCA) and K-nearest neighbours (KNN) were used to classify samples in five groups corresponding to the five cultivars and in three groups corresponding to the three areas of production. The results show that KNN provides a model unable to predict a proper assignment of the cultivar, at least for those olive trees considered in this work, whereas LDA and SIMCA allow the achievement of good percentage of prediction for the cultivars as well as cultivation zones. © 2009 Elsevier Ltd. All rights reserved.

A simple and sensitive liquid chromatography-tandem mass spectrometry (negative ion-electrospray ionization) methodology to determine sphingosine 1-phosphate (S1P) and sphinganine 1-phosphate (DH-S1P) in biological samples is described. The method has been validated over the linearity range of 2-100 ng/ml (r > 0.999) using synthetic C17-sphingosine 1-phosphate (C17-S1P) as an internal standard. In multiple reaction monitoring analysis (378.2 > 79.2), the lower limit of quantification for S1P was 5.0 ng/ml but the detection limit for the bioactive lipid was below 5 pg (12 fmol). Chromatographic separation was achieved on a UPLC BEH Hilic column with a binary mobile phase consisting of 30mM ammonium acetate (pH 4.0) and acetonitrile/MeOH/30mM ammonium acetate buffer (pH 4.0). The methodology detected 176.7±54.0 ng/ml of S1P and 81.2±23.3 ng/ml of DH-S1P in human plasma, as well as 201.0±72.0 ng/ml of S1P and 96.5±20.1 ng/ml of DH-S1P in mice plasma.

A proteomics-based approach was used for characterizing wheat gliadins from an Italian common wheat (Triticum aestivum) cultivar. A two-dimensional gel electrophoresis (2-DE) map of roughly 40 spots was obtained by submitting the 70% alcohol-soluble crude protein extract to isoelectric focusing on immobilized pH gradient strips across two pH gradient ranges, i.e., 3-10 or pH 6-11, and to sodium dodecyl sulfate-polyacrylamide electrophoresis in the second dimension. The chymotryptic digest of each spot was characterized by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and nano electrospray ionization-tandem mass spectrometry (MS/MS) analysis, providing a "peptide map" for each digest. The measured masses were subsequently sought in databases for sequences. For accurate identification of the parent protein, it was necessary to determine de novo sequences by MS/MS experiments on the peptides. By partial mass fingerprinting, we identified protein molecules such as alpha/beta-, gamma-, omega-gliadin, and high molecular weight-glutenin. The single spots along the 2-DE map were discriminated on the basis of their amino acid sequence traits. alpha-Gliadin, the most represented wheat protein in databases, was highly conserved as the relative N-terminal sequence of the components from the 2-DE map contained only a few silent amino acid substitutions. The other closely related gliadins were identified by sequencing internal peptide chains. The results gave insight into the complex nature of gliadin heterogeneity. This approach has provided us with sound reference data for differentiating gliadins amongst wheat varieties.

Ionspray (IS) and fast atom bombardment (FAB) positive ionization mass spectrometry (MS) of 1:1 ?-cyclodextrin (?-CD)-melatonin (MLT) host-guest complex allowed the detection of gaseous protonated 1:1 ?-CD-MLT. Tandem MS collision-induced dissociation (CID) of such protonated 1:1 ?-CD-MLT species showed the proton (charge) to be retained to a significant extent by the host and by its cage fragmentation products, in spite of the higher proton affinity of MLT with respect to that of ?-CD. This requires an endothermic guest-to-host proton transfer to occur within the gaseous association. Collisional activation could be accounted for by the promotion of such an endothermic process; however, the proton affinity decrease of the guest determined by the loss of the elements of acetamide, which is a dominant MS dissociation reaction of pure protonated MLT, could also provide a rationale for such an endothermic guest-to-host proton transfer. This proposal parallels the reaction scheme we had previously formulated for the analogous MS and tandem MS behaviour of 1:1 ?-CD-5-methoxytryptamine inclusion complex with the protonated 5-methoxytryptamine guest undergoing deamination. Copyright © 2001 John Wiley & Sons, Ltd.

The 1 : 1 inclusion complex of ?-cyclodextrin (?CD) with 5-methoxytryptamine (5MTA) hydrochloride was studied by mass spectrometry (MS) and tandem-MS with fast atom bombardment (FAB), electrospray (ES) and ionspray (IS) ionisation and triple quadrupole or ion trap analysers. A protonated 1 : 1 ?CD/MTA gaseous association was always observed; the protonated species of deaminated 5MTA and of some typical ?CD fragments were obtained, in addition to the expected protonated 5MTA, as tandem-MS dissociation products. A reaction pattern starting from the deamination of protonated 5MTA, as a recently described high pressure chemical ionisation or ES process of primary amines, was suggested, which accounts for the formation of excited protonated ?CD and its fragmentation by tandem-MS, even under unimolecular conditions.

(-)-Menthol-?-(D)-glucopyranoside 1 and neohesperidin dihydrochalcone 2 are glycoconjugates of practical interest. Compound 1 releases (-)-menthol in foods by slow hydrolysis and 2 is a sweetener used as an alternative to sucrose for diet food and beverages. Their molecular encapsulation in ?-cyclodextrin (?CD) is currently under investigation with the purpose of obtaining long lasting properties in the final product. From a mass spectrometric point of view, the study of the protonated gaseous 1 : 1 host-guest complexes of 1 and 2 with ?CD is of particular interest, since the guest molecules possess an apolar aglycon, likely to interact with the lipophilic cavity of ?CD, and a saccharidic part, whose capability of interaction with the polar external surface of ?CD has not been investigated so far. We carried out a fast-atom bombardment (FAB) mass spectrometric and tandem mass spectrometric (MS/MS) study in thioglycerol on 1 and 2 and their gaseous protonated 1 : 1 association complexes with ?CD. The FAB and tandem mass spectrometry results are later presented and discussed. Collisionally-activated decomposition data from native 1 and 2 are compared with those of the complexes of 1 and 2 with ?CD. The experimental data indicate that the presence of ?CD may dramatically alter the fragmentation pattern of the two glycoconjugates. The observed fragmentation patterns are consistent with an attractive interaction between the polar external surface of ?CD and the sugar residues of the guest molecules 1 and 2. The mass spectrometric data not only provide information on the nature of non-covalent interaction in controlling binding phenomena, but also point out a role for carbohydrate-carbohydrate interactions, possibly mediated via hydrogen bonding.

[object Object]2-Acetyl-1-pyrroline (1), 2-propionyl-1-pyrroline (2) and 5-acetyl-2,3-dihydro-1,4-thiazine (3), roast smelling odorants in food, form stable inclusion compounds with ?-cyclodextrin. Fast atom bombardment (FAB) mass spectra of such complexes in thioglycerol showed abundant [G+Hs+Mx+H]+ ions, where G=guest (1, 2 or 3), Hs=host (?-cyclodextrin) and Mx=one molecule of matrix, consistent with protonated non-covalent three-component adducts, and nearly negligible 1:1 associations of the type [G+Hs+H]+. Collision-activated decomposition (CAD) experiments indicated that [G+Hs+Mx+H]+ are made of neutral ?-cyclodextrin and protonated 1:1 guest-matrix adducts. The nature of these latter adducts was investigated by FAB mass spectrometric experiments on 1, 2 and 3 in thioglycerol without ?-cyclodextrin. In all cases the most intense signals are due to [G+Mx+H]+ and [G+H]+, with a small contribution of [G+Mx-H2O+H]+ to the total ion current. CAD experiments on [G+Mx+H]+ afforded protonated guest molecules as the base peak, consistent with the decomposition of protonated non-covalent 1:1 guest-matrix associations, possibly mediated by an intermolecular hydrogen bond. According to these data, there is a significant contribution of non-covalent three-component associations to [G+Hs+Mx+H]+ complexes, although the possibility of the formation of covalent guest-matrix adducts is not ruled out definitely, as discussed in the text. © 1997 by John Wiley & Sons, Ltd.