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The reactivity of the sulfur-containing nucleoside 4-thio-(2 '-deoxy)-thymidine usually abbreviated as 4-thio-thymidine, (S-4-TdR) under Fenton conditions, ie, in the presence of H2O2 and catalytic amounts of Fe(II), was investigated by UV-vis spectroscopy and electrospray ionization single and tandem mass spectrometry (ESI-MS and MS/MS). S-4-TdR hydroxylated on the S atom was found to be a key reaction intermediate, ultimately leading to (2 '-deoxy)-thymidine usually abbreviated as thymidine, (TdR) as the main reaction product. This finding was in accordance with the outcome of the reaction between S-4-TdR and H2O2, previously investigated in our laboratory. On the other hand, the additional presence of center dot OH radicals, induced by the Fe(II)/H2O2 combination, led to the increased generation of another interesting S-4-TdR product, already observed after its reaction with H2O2 alone, ie, the covalent dimer including a S-S bridge between two S-4-TdR molecules. More importantly, multihydroxylated derivatives of S-4-TdR and TdR were detected as peculiar products obtained under Fenton conditions. Among them, a product bearing an OH group both on the methyl group linked to the thymine ring and on the C5 atom of the ring was found to prevail. The results obtained during this study, integrated by those found previously in our laboratory, indicate 4-thiothymidine as a promising molecular probe for the recognition, through a careful characterization of its reaction products, of the prevailing species among reactive oxygen species (ROS) corresponding to singlet-state oxygen, hydrogen peroxide, and hydroxylic radical.

Hydrophilic interaction liquid chromatography coupled to electrospray ionization-Fourier transform mass spectrometry was employed to study ceramide lipids occurring in mussels of sp. Mytilus galloprovincialis. Lipid extracts from alive mussels and mussels deliberately subjected to specific thermal treatments were analyzed. In particular, single and tandem MS measurements were performed on a hybrid quadrupole-Orbitrap mass spectrometer and then complemented by MS(n) analyses (n = 2, 3) achieved by a linear ion trap mass spectrometer. This approach enabled the characterization of 66 ceramide lipids, encompassing ceramide phosphoethanolamines (CPE), ceramide aminoethylphosphonates (CAEP) and N-monomethylated CAEP. The sphingoid and acyl chains of each ceramide lipid could be distinctly recognized in terms of numbers of carbon atoms and C=C bonds, and indications on the possible location of the latter on the sphingoid chain could be often inferred from fragmentation patterns. The occurrence of several species hydroxylated on the α carbon of the acyl chain was also discovered. On the other hand, the sphingoid chain of ceramide lipids was never found to be involved in oxidation processes, unless forced exposure of the mussel lipid extracts to atmospheric oxygen was performed. CPE(d19:3/16:0) and its hydroxylated form, CPE(d19:3/2-OH-16:0), were found to be the prevailing species among CPE, whereas CAEP(d18:2/16:0), CAEP(d19:3/16:0) and CAEP(d19:3/2-OH-16:0) were the most abundant CAEP. Finally, ceramide lipids showed a remarkably higher stability, compared with glycerophospholipids, in mussels subjected to different thermal treatments. This finding opens interesting perspectives on the role of ceramide-based lipids in the adaptation of aquatic organisms to thermal stresses. Copyright © 2016 John Wiley & Sons, Ltd.

4-Chloro-α-cyanocinnamic acid (ClCCA) is a very useful matrix able to give the protonated adduct [M+H](+) of intact cyanocobalamin (CNCbl) as the base peak (m/z 1355.58) in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS). The only fragment observed is [M-CN + H](+•) formed through the facile (•) CN neutral loss reflecting the fairly low Co-C bond energy. All other investigated proton transfer matrices, including α-cyano-4-hydroxycinnamic acid, para-nitroaniline and 2,5-dihydroxybenzoic acid, give rise to a complete decyanation of CNCbl with concomitant formation of [M-CN + H](+•) , [M-CN + Na](+•) and [M-CN + K](+•) adducts at m/z 1329.57, 1351.55 and 1367.51, respectively. Depending on the matrix used, a variable degree of fragmentation involving the α-side axial ligand was observed. A plausible explanation of the specific behaviour of 4-chloro-α-cyanocinnamic acid as a soft matrix is discussed. Tandem mass spectra of both [M + H](+) and [M-CN + H](+•) ions were obtained and product ions successfully assigned. The possibility of detecting the protonated adduct of intact CNCbl was exploited in foodstuff samples such as cow milk and hen egg yolk by MALDI tandem MS upon sample extraction. We believe that our data provide strong basis for the application of MALDI tandem MS in the qualitative analysis of natural CNCbl, including fish, liver and meat samples. Copyright © 2016 John Wiley & Sons, Ltd.

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. 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. Copyright © 2014 John Wiley & Sons, Ltd.

A full characterization of sulfoquinovosyldiacylglycerols (SQDGs) in the lipid extract of spinach leaves has been achieved using liquid chromatography/electrospray ionization-linear quadrupole ion trap mass spectrometry (MS). Low-energy collision-induced dissociation tandem MS (MS/MS) of the deprotonated species [M − H]− was exploited for a detailed study of sulfolipid fragmentation. Losses of neutral fatty acids from the acyl side chains (i.e. [M − H − RCOOH]−) were found to prevail over ketene losses ([M − H − R'CHCO]−) or carboxylates of long-chain fatty acids ([RCOO]−), as expected for gas-phase acidity of SQDG ions. A new concerted mechanism for RCOOH elimination, based on a charge-remote fragmentation, is proposed. The preferential loss of a fatty acids molecule from the sn-1 position (i.e. [M − H − R1COOH]−) of the glycerol backbone, most likely due to kinetic control of the gas-phase fragmentation process, was exploited for the regiochemical assignment of the investigated sulfolipids. As a result, 24 SQDGs were detected and identified in the lipid extract of spinach leaves, their number and variety being unprecedented in the field of plant sulfolipids. Moreover, the prevailing presence of a palmitic acyl chain (16:0) on the glycerol sn-2 position of spinach SQDGs suggests a prokaryotic or chloroplastic path as the main route for their biosynthesis. Copyright © 2013 John Wiley & Sons, Ltd.

An liquid chromatography–mass spectrometry method using electrospray ionization in negative ion mode coupled with a hybrid quadrupole linear ion trap and Fourier transform ion cyclotron resonance (FTICR) mass spectrometer was applied to characterize of intact glucosinolates (GLSs) in crude sample extracts of wild bud flowers of Capparis spinosa (Capparis species, family Capparaceae). Structural information of GLSs was obtained upon precursor ions' isolation within the FTICR trapping cell and subsequent fragmentation induced by infrared multiphoton dissociation (IRMPD). Such a fragmentation was found very useful in terms of chemical identification of all precursor ions [M–H]− including sulfur-rich GLSs reported here for the first time. Along with most common GLSs already found in capers such as glucocapparin, isopropyl/n-propyl-GLS, mercapto-glucocapparin, and two indolic GLS, i.e., 4-hydroxyglucobrassicin and glucobrassicin, the occurrence of the uncommon glycinyl-glucocapparin as well as two sulfur-rich GLSs is reported. IRMPD showed an increased selectivity towards disulfide bond cleavages with thiol migration, suggesting the side chain structure of non-targeted compounds, i.e., disulfanyl-glucocapparin and trisulfanyl-glucocapparin. Glucocapparin [2.05 ± 0.25 mg/g, dry weight (dw)] was the most abundant GLS, followed by glucobrassicin (232 ± 18 µg/g, dw) and 4-hydroxyglucobrassicin (89 ± 12 µg/g, dw). All other compounds were present at very low content ranging from 0.5 to 1.5 µg/g dw. Copyright © 2012 John Wiley & Sons, Ltd.

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 (ΣPBDEs) 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-1 00 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.

N-acyl-homoserine-lactones (AHSLs) are widely conserved signal molecules present in quorum sensing systems of Gram-negative bacteria such as Pseudomonas aeruginosa. We present here the results obtained with a hybrid linear trap/Fourier transform ion cyclotron resonance (LTQ-FTICR) mass spectrometer used to investigate the occurrence of AHSLs and cognate N-acyl-homoserines (AHSs) in bacterial isolates of P. aeruginosa (strain PAO1). Two hydrolysed AHSs were found in significant amounts, most likely formed through the lactone opening of N-3-oxo-decanoyl-L-homoserine-lactone (3OC10-HSL) and N-3-oxo-dodecanoyl-L-homoserine-lactone (3OC12-HSL). Structure elucidation of these ring-opened molecules, i.e. N-3-oxo-decanoyl-L-homoserine (3OC10-HS), and N-3-oxo-dodecanoyl-L-homoserine (3OC12-HS), which are not detected by bacterial biosensors, was performed by high-resolution and accurate mass measurements upon liquid chromatography (LC) and confirmed by tandem MS in the LTQ analyser. Assignment of chemical formula, with mass spectra in the form of [M + H]+, was significantly expedited by extracted ion chromatograms (XICs) because the number of potentially plausible formulae for each protonated signalling molecule was considerably reduced a priori by the LC behaviour, the high mass measurement accuracy available in FTICR mass spectra and the isotopic patterns. At least two concentration levels were observed in spent culture supernatants of P. aeruginosa: compounds at a relatively high content (5–15 µM) that is C4-HSL, 3OC10-HS, and 3OC12-HS and those occurring at a lower content (

A method for the comprehensive profiling of the N-acyl-homoserine lactone (AHL) family of bacterial quorum-sensing molecules is presented using liquid chromatography (LC) coupled to a hybrid quadrupole linear ion trap (LTQ) and Fourier-transform ion-cyclotron-resonance mass spectrometer (FTICR). We demonstrate an increase in signal intensity in MS with electrospray ionization (ESI) of the protonated molecules, [M + H](+), by using acetonitrile (ACN) instead of methanol (MeOH) as the organic solvent under the conditions in which the samples were supplied to the probe by direct infusion at constant flow rates. The presence of ACN prevents the formation of methanol adducts such as [M + MeOH + H](+) and [M + MeOH + Na](+), while also lowering the signal intensity of sodiated [M + Na](+) ions. Sensitivity of these signaling molecules in terms of signal-to-noise ratio (S/N) using low-resolution LTQ-MS and high-resolution FTICR-MS were compared under reversed-phase (RP) LC separations with ESI interface. Special emphasis was paid to the choice of the separation column, its elution conditions and detection of the major AHL compounds produced by the Serratia liquefaciens strain ATCC 27592. The most promising results were obtained using a RP C16-amide column eluted with a linear mobile phase gradient ACN/H(2)O containing 0.1% formic acid. The whole set of AHL homologs in bacterial extracts was detected in the extracted-ion chromatographic (XIC) mode, and the calculations of molecular formulae were performed by including the isotopic pattern. This mode of displaying data, with a very narrow mass-to-charge ratio window (i.e. +/- 0.0010 as m/z unit) around each selected ion, has allowed the identification of all the eight known homoserine lactones, viz. C(4)-HSL, 3-oxo-C(6)-HSL, C(6)-HSL, 3-oxo-C(8)-HSL, C(8)-HSL, C(10)-HSL, C(12)-HSL and C(14)-HSL. In addition, at least four uncommon signaling mediators previously unreported, namely, 3-oxo-C(10:1)-HSL, 3-oxo-C(11:2)-HSL, 3-oxo-C(13:2)-HSL and 3-OH-C(16)-HSL, were identified and characterized; their roles in cell-to-cell communication has to be elucidated.