The main bacterial pathogens of cultivated mushroom as well as mushroom-associated bacteria, which were isolated 9 from Agaricus bisporus, Pleurotus ostreatus, and Pleurotus eryngii mushroom niches, were evaluated for the production of N-acyl-L-10 homoserine lactones (AHLs) by using four bioreporters. Furthermore, identification of AHLs by LC-ESI-FTICR MS was performed on culture filtrates of selected pathogens and mushroom-associated bacteria strains, which resulted in inducing at least one of the four bioreporters. Strains of Burkolderia gladioli pv agariciola, Pseudomonas agarici, and Pseudomonas gingeri, but not those of Pseudomonas tolaasii and Pseudomonas reactans, produced an array of AHLs depending on the strain. This is the first report of AHL production by mushroom bacterial pathogens. Forty-four of 236 bacterial isolates obtained from different niches of cultivated mushrooms, in part identified by the Biolog identification system, were demonstrated to produce AHLs. Among them, seven mushroom-associated bacterial species were for the first time demonstrated to produce the above signal molecules. In the culture filtrates of a certain number of isolates/strains the AHL-hydrolyzed forms were also present. The minimal signal inducing concentration (MSIC) of selected pure AHLs was also determined for the four bioreporters used in this study.

An approach is presented that can be of general applicability for structural elucidation of naturally occurring glucosinolates (GLSs) in crude plant extracts based on the fragmentation of isotopic A and A + 2 peaks. The most important fragmentation pathways were studied by tandem mass spectrometry (MS(n), n = 2, 3) using a linear quadrupole ion trap (LTQ) upon GLSs separation by optimized reversed-phase liquid chromatography (RPLC) and electrospray ionization (ESI) in negative ion mode. As the LTQ MS analyzer ensures high sensitivity and linearity, the fragmentation behavior under collision induced dissociation (CID) of the isotopic peaks A and A + 2 as precursor ions was carefully examined. All GLSs (R-C(7)H(11)O(9)NS(2)(-)) share a common structure with at least two sulfur atoms and significant isotopic abundance of (34)S. Thus, dissociation of the +2 Da isotopomeric ions results in several fragment ion doublets containing a combination of (32)S and (34)S. Accordingly, their relative abundances allow one to speed up the structural recognition of GLSs with great confidence, as it produces more structurally informative ions than conventional tandem MS performed on A ions. This approach has been validated on known GLSs bearing two, three, four, and six sulfur atoms by comparing expected and measured isotopic peak abundance ratios (I(A)/I(A+2)). Both group- and compound-specific fragments were observed; the predominant pathway of fragmentation of GLSs gives rise to species having the following m/z values, [M - SO(3) - H](-), [M - 196 - H](-), [M - 178 H](-), and [M - 162 - H](-) after H rearrangement from the R side chain. The present strategy was successfully applied to extracts of rocket salad leaves (Eruca sativa L.), which was sufficient for the chemical identification of a not already known 6-methylsulfonyl-3-oxohexyl-GLS, a long-chain-length aliphatic glucosinolate, which contains three sulfurs and exhibits a deprotonated molecular ion at m/z 494.1.

Many metabolomic applications use gas chromatography/mass spectrometry (GC/MS) under standard 70 eV electron ionization (El) parameters. However, the abundance of molecular ions is often extremely low, impeding the calculation of elemental compositions for the identification of unknown compounds. On changing the beam-steering voltage of the ion source, the relative abundances of molecular ions at 70 eV El were increased up to ten-fold for alkanes, fatty acid methyl esters and trimethylsilylated metabolites, concomitant with 2-fold absolute increases in ion intensities. We have compared the abundance, mass accuracy and isotope ratio accuracy of molecular species in El with those in chemical ionization (CI) with methane as reagent gas under high-mass tuning. Thirty-three peaks of a diverse set of trimethylsilylated metabolites were analyzed in triplicate, resulting in 342 ion species ([M+H](+), [M-CH(3)](+) for CI and [M](+), [M-CH(3)](+center dot) for EI). On average, CI yielded 8-fold more intense molecular species than El. Using internal recalibration, average mass errors of 1.8 +/- 1.6 mm/z units and isotope ratio errors of 2.3 +/- 2.0% (A+1/A ratio) and 1.7 +/- 1.8% (A+2/A ratio) were obtained. When constraining lists of calculated elemental compositions by chemical and heuristic rules using the Seven Golden Rules algorithm and PubChem queries, the correct formula was retrieved as top hit in 60% of the cases and within the top-3 hits in 80% of the cases.

We report on the content of polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) in 15 breast milk samples of nursing women living in the city of Taranto (Southern, Italy) or nearby. Breast milk samples were collected over the 2008–2009 period and analyzed by gas chromatography coupled with high-resolution mass spectrometry (GC-HRMS) upon accelerated solvent extraction (ASE) using acetone/n-hexane mixture 1:1 (v/v). The method was validated demonstrating good performing features. Profiles of PCDD/PCDF congeners in breast milk samples exhibited a prevalence of PCDFs compared to PCDDs. Toxic equivalents (TEQs in picogram per gram fat) of four breast milk were far above the legal limit for human consumption of 3.0 pg/g; their estimated daily and weekly dietary intake were almost 5–20 and 10–40 times higher, respectively, than the tolerable intake values established by the World Health Organization.

GGlucosinolates (GLSs) are sulfur-rich plant secondary metabolites which occur in a variety of cruciferous vegetables and among various classes of them, genus Brassica exhibits a rich family of these phytochemicals at high, medium and low abundances. Liquid chromatography (LC) with electrospray ionization in negative ion mode (ESI-) coupled to a hybrid quadrupole linear ion trap (LTQ) and Fourier transform ion cyclotron resonance mass spectrometer (FTICRMS) was employed for the selective and sensitive determination of intact GLSs in crude sample extracts of broccoli (Brassica oleracea L. Var. italica), cauliflower (B. oleracea L. Var. Botrytis) and rocket salad (Eruca sativa L.) with a wide range of contents. When LTQ and FTICR mass analyzers are compared, the magnitude of the limit of detection was ca. 5/6-fold lower with the FTICR MS. In addition, the separation and detection by LC–ESI-FTICR MS provides a highly selective assay platform for unambiguous identification of GLSs, which can be extended to lower abundance (minor) GLSs without significant interferences of other compounds in the sample extracts. The analysis of Brassicaceae species emphasized the presence of eight minor GLSs, viz. 1-methylpropylGLS, 2-methylpropyl-GLS, 2-methylbutyl-GLS, 3-methylbutyl-GLS, n-pentyl-GLS, 3-methylpentyl-GLS, 4-methylpentyl-GLS and n-hexyl-GLS. The occurrence of these GLSs belonging to the saturated aliphatic side chain families C 4 ,C 5 and C , presumably formed by chain elongation of leucine, homoleucine and dihomoleucine as primary amino acid precursors, is described. Based on their retention behavior and tandem MS spectra, all these minor compounds occurring in plant extracts of B. oleracea L. Var. italica, B. oleracea L. Var. Botrytis and E. sativa L. were tentatively identified.

An unprecedented characterization of free fatty acids (FFA) in the lipid extracts of fresh or thermally treated mussels of sp. Mytilus galloprovincialis, including up to 128 saturated, mono- or poly-unsaturated and 63 oxidized (i.e., modified by hydroxylic, carbonylic and/or epoxylic groups) compounds, was achieved using reverse phase chromatography coupled to electrospray ionization-Fourier transform single and tandem mass spectrometry (RPC-ESI-FTMS,MS/MS). Subsequent Principal Components Analysis (PCA) evidenced several effects of thermal treatments on the mussel FFA profiles. In particular, death-inducing low temperature treatments (freezing at -16 °C or refrigeration at 4 °C for several days) induced a peculiar increase in the incidence of FFA, whereas the effect was absent in mussels undergoing death upon prolonged storage at room temperature (25 °C, 6 h) or fast cooking (100 °C, 5 min). Alive mussels, either fresh or resulting from short term (up to 48 h) refrigeration were actually indistinguishable by PCA, although subtle seasonal effects were observed.

A mixture of native and oxidized phospholipids (PLs), generated by the soybean lipoxygenase type V-catalyzed partial oxidation of a lipid extract obtained from human platelets, was analyzed by HydrophilicInteraction Liquid Chromatography-ElectroSpray Ionization-Tandem Mass Spectrometry (HILIC–ESI-MS/MS). The complexity of the resulting mixture was remarkable, considering that the starting lipid extract, containing (as demonstrated in a previous study) about 130 native PLs, was enriched with enzy-matically generated hydroperoxylated derivatives and chemically generated hydroxylated forms of PLsbearing polyunsaturated side chains. Nonetheless, the described analytical approach proved to be verypowerful; indeed, focusing on phosphatidylcolines (PCs), the most abundant PL class in human platelets,about fifty different native/oxidized species could be identified in a single HILIC–ESI-MS/MS run. Low-energy collision induced dissociation tandem MS (CID-MS/MS) experiments on chromatographicallyseparated species showed single neutral losses of H2O2 and H2O to be typical fragmentation pathways of hydroperoxylated PCs, whereas a single H2O loss was observed for hydroxylated ones. Moreover, diag-nostic losses of n-hexanal or n-pentanol were exploited to recognize PCs hydroperoxylated on the last butfive carbon atom of a -6 polyunsaturated side chain. Despite the low resolution of the 3D ion trap mass analyzer used, the described HILIC–ESI-MS/MS approach appears very promising for the identification ofoxidized lipids in oxidatively stressed complex biological systems.

The identification of two unsaturated N-acylhomoserine lactones (AHLs) produced by Rhodobacter sphaeroides bacteria, based on liquid chromatography (LC) coupled to a hybrid quadrupole linear ion trap (LTQ)-Fourier transform ion cyclotron resonance (FTICR) mass spectrometer upon electrospray ionization (ESI), is presented. Along the confirmation of the signaling molecule already described in the literature, i.e. (Z)-N-tetradec-7-enoyl-homoserine lactone (C14:1-HSL), we have discovered the occurrence, at low yet significant levels, of another monounsaturated compound, C12:1-HSL, which may extend the number of small diffusible chemical signals known for R. sphaeroides. Both unsaturated AHLs were identified by high resolution FTICR mass spectrometry in extracts of bacterial culture media and the occurrence of a C=C bond was assessed upon their conversion to bromohydrins. Collision induced dissociation (CID) spectra were then collected on the LTQ mass analyser. A careful comparison of tandem MS spectra of monounsaturated (i.e., C12:1-HSL and C14:1-HSL) and saturated AHLs (i.e. C12-HSL and C14-HSL) led to emphasise two series of product ions, exhibiting 14 Da-spaced m/z ratios. Both series were referred to progressive fragmentations at the aliphatic end of the AHL acyl chains, followed by neutral losses of terminal alkenes (i.e., CH2=CH(CH2)nH). In particular, the series located at the higher end of the explored m/z range (> 200 Da), observed only for monounsaturated species, enabled the location of the C=C bond between carbons 7 and 8 of the acyl chain.

The purpose of this work is to explore the mass fragment characterization of commonly used drugs through a novel approach which involves isotope-selective tandem mass spectrometry (MS/MS). Collision induced dissociation (CID) was performed with a low-resolution linear-ion-trap mass spectrometer in positive electrospray ionization (ESI). Three pharmacologically active ingredients, i.e., omeprazole, meloxicam and brinzolamide, selected as model compounds in their own formulation, were investigated as a sodiated adduct [C17H19N3O3S+Na]+ (omeprazole) and as protonated adducts, [C14H13N3O4S2+H]+ and [C12H21N3O5S3+H]+, meloxicam and brinzolamide, respectively. Selecting a narrow window of 0.5 m/z units, precursor ion fragmentation by CID-MS/MS of isotopologues A+0, A+1 and A+2 was found very useful to confirm the chemical formula of product ions, thus aiding the establishment of characteristic fragmentation pathways of all three examined compounds. The correctness of putative molecular formula of product ions was easily demonstrated by exploiting the isotope peak abundance ratios (i.e., IF+0/IF+1 and IF+0/IF+2) as simple constraints in low resolution MS instrumentations.

Gram-negative bacteria use N-acylhomoserine lactones (AHLs) as their command language to coordinate population behavior during invasion and colonization of higher organisms. Although many different bacterial bioreporters are available for AHLs monitoring, in which a phenotypic response, e.g. bioluminescence, violacin production, and βgalactosidase activity, is exploited, mass spectrometry (MS) is the most versatile detector for rapid analysis of AHLs in complex microbial samples, with or without prior separation steps. In this paper we critically review recent advances in the application of high-resolution MS to analysis of the quorum sensing (QS) signaling molecules used by Gram-negative bacteria, with much emphasis on AHLs. A critical review of the use of bioreporters in the study of AHLs is followed by a short methodological survey of the capabilities of highresolution mass spectrometry (HRMS), including Fouriertransform ion cyclotron resonance (FTICR) MS and quadrupole time-of-flight (qTOF) MS. Use of infusion electrospray ultrahigh-resolution FTICR MS (12 Tesla) enables accurate mass measurements for determination of the elemental formulasofAHLsinAcidovorax sp. N35 and Burkholderia ubonensis AB030584. Results obtained by coupling liquid chromatography with a hybrid quadrupole linear ion trapFTICR mass spectrometer (LC–LTQ-FTICRMS, 7-T) for characterization of acylated homoserine lactones in the human pathogen Pseudomonas aeruginosa are presented. UPLC– ESI-qTOF MS has also proved to be suitable for identification of 3O-C HSL in Pseudomonas putida IsoF cell culture supernatant. Aspects of sample preparation and the avoidance of analytical pitfalls are also emphasized.

The phospholipidome of blood microparticles (MPs) obtained from platelet-rich plasma of healthy individuals was characterized by hydrophilic interaction liquid chromatography (HILIC) coupled to electrospray ionization tandem mass spectrometry (ESI-MS/MS). The HILIC separation, performed on a silica stationary phase using an acetonitrile/methanol gradient, enabled the separation of several phospholipids (PL) classes, viz., phosphatidyl-cholines (PCs), -ethanolamines (PEs), -serines (PSs), -inositoles (PIs), sphyngomielins (SMs), and lyso forms of PCs and PEs. Structural characterization of species belonging to each class was performed by MS/MS measurements, in either positive or negative ion mode. The set of 131 phospholipids (including regioisomers) here identified represents the most comprehensive phospholipidomic characterization reported for human MPs. Although the phospholipidome composition of MPs and platelets, collected from the same donors, was found to be qualitatively the same, quantitative differences were evidenced for lyso-PCs, which appear to be significantly more abundant in MPs.

Samples of Mytilus galloprovincialis were examined to investigate the levels of polybrominated diphenyl ethers (PBDEs) in the Apulia's marine environment, a region in the South of Italy. The levels of nine PBDE congeners were measured in 40 mussel samples taken from the aquaculture farms in the South of Adriatic Sea and North Ionian Sea along the Apulia coast. While accelerated solvent extraction (ASE) was the extraction technique adopted using acetone/n-hexane (1:1, v/v), the content of PBDEs was evaluated by using gas chromatography coupled with high-resolution mass spectrometry (GC-HRMS) via electron ionization (EI) in the multiple ion monitoring (MIM) mode (two ions for each compound). The (13)C mass-labeled compounds were used to establish the levels of PBDEs in M. galloprovincialis samples. The total concentration of PBDEs (Sigma PBDEs) ranged from 0.2 to 6.9 ng/g dry mass, with the highest concentrations found around coastal areas of Salento. With regard to the composition of PBDE congeners, BDE-47, BDE-99 and BDE-100 were the predominant congeners in most of the samples. The present study is the first to report levels of PBDEs occurring in aquatic organisms living in aquaculture farms of Apulia region.

Oleuropein (Ole) has been claimed to mitigate cisplatin (CP) induced acute injury in kidney and liver of mice. In-vitro reactivity of hydrated CP species with Ole, and Ole metabolite hydroxytyrosol (HT) is of great interest as the preliminary step for gathering in-vivo information on the possible physiological role of the Ole/HT-cis-diammineplatinum (II) (Ole/HT-cis-DAP) conjugate.

Low temperature treatments commonly applied to seafood products have been shown to influence their phospholipid (PL) profile through enzymatic hydrolysis. In the present study, the generation of lysophospholipids (LPL) resulting from this process was systematically investigated for selected, commercially relevant seafood products, namely oysters, clams, octopuses, and shrimps. These products were subjected to thermal treatments like refrigeration or freezing after being purchased as fresh, defrozen, or frozen products depending on the case. The coupling between hydrophilic interaction liquid chromatography (HILIC) and electrospray ionization with high resolution/accuracy Fourier transform mass spectrometry (ESI-FTMS) was exploited to evaluate the PL profile of the cited products, especially the incidence of LPL related to the two main PL classes of seafood products-phosphatidylcholines (PC) and phosphatidylethanolamines (PE)-in the lipid extracts. The lyso forms of PE (LPE) were found to be generally more sensitive than those of PC (LPC) to thermal treatments, usually exhibiting a significant increase upon prolonged refrigeration at 4 °C in all types of investigated products except European flat oysters. Moreover, the distinction between fresh and frozen or defrozen products could be achieved in the case of octopuses and shrimps, respectively.