Structural Characterization of Lipopeptides from Brevibacillus brevis HOB1

Brevibacillus brevis HOB1 was isolated from the formation water of an oil field and found to produce lipopeptides. The separation of lipopeptides was successfully achieved by reversed-phase high-performance liquid chromatography (HPLC) leading to nine separated peaks. The chemical structures of these lipopeptides were studied by means of electrospray ionization mass spectrometry (ESI-MS), gas chromatography-mass spectrometry (GC/MS), HPLC and electrospray ionization tandem mass spectrometry (ESI-MS/MS). As the results, all the lipopeptides had peptide parts with the same amino acid composition of Asp, Glu, Val, and Leu in the molar ratio 1:1:1:4, while the lipid part was composed of C₁₃–C₁₅ β-hydroxy fatty acids. As the sequence of fraction 1 was determined to be N-Glu-Leu-Leu-Val-Asp-Leu-Leu-C, the same as surfactin, they were proposed to be surfactin isoforms. Fraction 4 (C₁₅ surfactin) exhibited a good surface activity of 26.8 mN/m with CMC of 9?×?10^?6 M. Surfactin is a powerful biosurfactant possessing biological activities. As far as we know, Br. brevis is a new surfactin-producing species.     

Characterization of oligomeric compounds in secondary organic aerosol using liquid chromatography coupled to electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry

The components of secondary organic aerosols (SOAs) generated from the gas‐phase ozonolysis of two C₁₀H₁₆‐terpenes (α‐pinene; sabinene) and a cyclic C₆H₁₀ alkene (cyclohexene) were characterized by the use of a Fourier transform ion cyclotron mass spectrometer equipped with an electrospray ionization source operated in the negative ion mode. Reversed‐phase high‐performance liquid chromatography was used to achieve chromatographic separation of highly oxidized organic compounds. In addition to the well‐known group of low molecular weight oxidation products (monomers; e.g. dicarboxylic acids), higher molecular weight compounds (dimers) were also detected and their exact elemental compositions were determined. In order to provide additional information for the structural elucidation of these compounds, collision‐induced dissociation was applied. Based on the MS/MS spectra, two higher molecular weight products are proposed to be an ester and a peroxide. Molecular formulae calculated from the exact masses show that the SOA‐compounds are heavily oxidized and this information creates the background to a discussion of potential reaction pathways for the formation of higher molecular weight compounds. Copyright © 2009 John Wiley & Sons, Ltd.     

Production of Lipid and Fatty Acids during Growth of <Emphasis Type="Italic">Aspergillus terreus</Emphasis> on Hydrocarbon Substrates

An Aspergillus terreus, isolated from oil contaminated soil, could degrade a wide range of petroleum hydrocarbons including the immediate oxidation products of hydrocarbons, like alkanols and alkanals. Among all the linear chain carbon substrates, highest growth of 39.1 ± 3.8 g l⁻¹ (wet weight) was observed when n-hexadecane was used as the sole source of carbon. The growth of the fungus on this highly hydrophobic substrate was associated with the morphological change of the hyphae and increase production of lipid in the cells. The lipid production in the hydrocarbon (n-hexadecane) grown cells was sevenfold higher than the corresponding glucose grown cells. The fatty acid profile of the lipid content formed in the hydrocarbon grown cells was significantly different from the glucose grown cells and was composed of fatty acids with chain length C₁₄ to C₃₃ as revealed from the liquid chromatography electrospray ionization mass spectrometry analyses. Among the ranges, the fatty acids with chain lengths C₁₄ to C₁₈ were predominant in the profile. Considering the fatty acid profile and the high level of lipid production, this A. terreus mediated production of lipid is envisaged to have potential application in the oleochemical industries including the production of biodiesel.     

Determination of meloxicam in human plasma by ultra-high-performance liquid chromatography–tandem mass spectrometry and its application in a pharmacokinetic study

A rapid, selective and sensitive ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) method was developed to detect meloxicam in human plasma. A triple quadrupole tandem mass spectrometer equipped with an electrospray ionization source was used in positive ion mode. Protein precipitation with acetonitrile was used for sample preparation. Meloxicam and ¹³C₆-meloxicam internal standard were analyzed on an Acquity CSH C₁₈ column with a mobile phase of acetonitrile and water in 0.1% formic acid using a gradient program for separation. The retention time of meloxicam was 1.1 min and the total run time was only 2.0 min. Detection was performed in multiple reaction monitoring mode using an electrospray ionization source with optimized mass spectrometry parameters. The calibration curves were linear in the range 10.0–3.00 × 10³ ng/ml (r ≥ 0.99). The within-run and between-run RSDs were ≤14.8%. The within-run and between-run REs ranged from −4.6 to 10.7%. There was no significant matrix effect, and the recovery rate was high. This method was fully validated, including reinjection reproducibility in human plasma. The method was applied to the pharmacokinetic study. All of the incurred sample reanalysis methods met the criteria.     

Observation of gas-phase molecular dications formed from neutral organics in solution via qemical electron-transfer reactions by using electrospray Ionization Mass Spectrometry

The first observation of organic dications formed by multiple electron loss in electrospray mass spectra is reported. The dications of β-carotene, canthaxanthine, cobalt(II) octaethylporphyrin, and nicke(II) octaethylporphyrin were created in solution via chemical electrontransfer reactions and detected in the gas phase by electrospray ionization mass spectrometry (ES-MS) using a flow-injection experiment. The analytes were injected into a flowing solvent-oxidant stream (10 μL/min) composed of dried methylene chloride containing ≈ 0.1% by volume trifluoroacetic acid and 0.1% by volume antimony pentafluoride (SbF₅). The dications created in this oxidizing solvent system were preserved for detection by rapidly transferring them from the reactive solvent-oxidant system to the gas phase, where, in the absence of the solvent system, they were “long-lived” and amenable to mass analysis. This work demonstrates means to produce ions novel to ES-MS and means to detect and study by ES-MS species that are short-lived in solution. In addition, this work shows that electrospray ionization can potentially be used to generate gas-phase dications for mass spectrometric study that are difficult to produce directly from gas-phase neutrals by other ionization techniques (e.g., M²⁺ from β-carotene).     

Ion chemistry of deprotonated phenylthiocarbamyl-phenylalanine

The fragmentation of an [M-H]^? anion of phenylthiocarbamyl (PTC)-phenylalanine (Phe) and of a deuterium labeled analog using electrospray ionization (ESI) in a negative ion mode was studied. Product ion experiments show that deprotonated PTC-Phe fragments mainly through three different pathways. Further tandem mass spectrometry experiments show that these three pathways actually correspond to the three different deprotonation sites in the molecule. These three ionization sites lead to the losses of, respectively, H₂S and (H₂S+CO₂), C₆H₅N=C=S, and C₆H₅NH₂ from [M-H]^? in the first steps. They yield anions stabilized by resonance. The fragment produced by the loss of C₆H₅NH₂ is relatively weak and originates from a less acidic site. By selecting these fragment anions successively as precursors from the ion source, detailed information on the fragmentation mechanism is obtained. The benzyl-type anion plays a stabilizing role through conjugation. The fragmentation process also involves some uncommon neutral losses. For example, losses of HCN involve reactions that may occur through fouror five-membered cyclic transition states and cyanide ion-molecule complexes. Similar losses of HCN may occur through both the carboxylate anion and one of its tautomeric forms. Deuterium labeling studies support all the mechanistic proposals. The fragments obtained following the loss of the derivatization reagent (C₆H₅N=C=S) show that it is the anion of the deprotonated free amino acid. Preliminary results have shown that this also occurs for other PTC amino acids.     

Comparative studies of the behavior of neutral and ionic ferrocene derivatives under different conditions of electrospray ionization

We have studied the behavior of ferrocene CpFeCp (FcH), ferrocenium triiodide [FcH]⁺I₃⁻, dimethylaminomethylferrocene FcCH₂NMe₂ and its trimethylammonium salt [FcCH₂NMe₃]⁺I⁻ under the conventional conditions of electrospray ionization (ESI), when the substance solution is subjected to spraying, and in two versions of desorption electrospray ionization (DESI), when the sprayed solvent bombards the surface of solid or liquid samples. In addition to these techniques, the behavior of neutral compounds under conditions of electrospray ionization of vapors of the studied compounds in a gas phase (ESI_V) has been investigated. It has been shown using the examples of ferrocene and its dimethylaminomethyl derivative that the detection limits for these compounds occurring in a gas phase are comparable within an order of magnitude with their detection limits under the ESI and DESI conditions of solid and liquid samples. The high effectiveness of ionization of analyte vapors makes it possible to use the ESI method not only in combination with liquid (conventional ESI technology) and thin layer chromatography (DESI), but also with gas liquid chromatography (ESI_V). Thus, the electrospray ionization becomes a universal method allowing studies of a compound under the natural conditions in any state of aggregation, that is, solid, liquid, and gas. With the help of statistical methods for designing experiments (complete factorial experiment), quantitative evaluation of the influence of experimental parameters on the ion-formation processes under different ESI conditions has been carried out, which makes it possible to purposefully select the optimal conditions to record the ESI mass spectra with a minimum number of experiments. Moreover, analysis of the dependences of the mass spectra on the experimental parameters can serve as an instrument for studying the details of the ion-formation mechanisms depending upon different ways of ionization.     

Comparative ESI FT-MS and MALDI-TOF structural analyses of representative human <Emphasis Type="Italic">N</Emphasis>-linked glycans

Modern glycan analysis is primarily based on mass spectrometry, where instruments based on electrospray or matrix-assisted laser desorption ionization are currently the most frequently used. In the present study, electrospray ionization (ESI) coupled with a high-resolution Fourier transform mass spectrometer (LTQ Orbitrap) and matrix-assisted laser desorption/ionization (MALDI) coupled with a time-of-flight (TOF/TOF) detector were used to analyze two N-glycan standards with intact free reducing ends (disialo biantennary and asialo triantennary) and representative PA-labeled human serum N-glycan structures isolated by hydrophilic interaction anion-exchange chromatography (HIAX), confirmed by ¹H NMR analysis and consequently compared with the ProteinScape Glycome database. Different combinations of ion sources with fragmentation devices results in various fragmentation patterns and adducts. Also, the effect of sample derivatization on the acquired signals is discussed. Compared to the MALDI technique, free glycans did not lose labile sialic acids easily in the ESI source. On the other hand, fluorescent PA-labeling leads to improved core fragmentation and signal intensities; linkage-specific ethyl esterification leads to reduced adduct and fragment formation and enhanced stability of sialic acids in the MALDI ion source. Thereby, both methods have their advantages and disadvantages in terms of detection, fragmentation and robustness.     

Features of the First and Second Order Electrospray Ionization Mass Spectra for Salt-Like Products Derived from Monools and Diols Using Combined Reagents Based on ω-Bromoacyl Chlorides and Nitrogen Bases

Derivatization by composite reagents based on ω-bromoacyl chlorides [ClCO(C₂) _n Br (n = 1–4)] and pyridine was applied to study aliphatic and alicyclic alcohols and diols by ordinary and tandem electrospray ionization (ESI) mass spectrometry. The applied derivatization involves the simultaneous acylation of hydroxyl groups with an acyl chloride moiety and the quaternization of pyridine with a terminal bromoalkyl group. Under the ESI conditions, quaternary salts produce corresponding mono and diammonium cations, which are detected in the first-order mass spectra. Collision-induced dissociation (CID) of primary cations generated from monool derivatives gives rise to ammonium cations of the corresponding acids HOOC(CH₂) _n –N⁺(C₅H₅). The CID of primary dications affords the same cations which are also eliminated from dications to form mono-charged fragments.     

Simultaneous determination and pharmacokinetics study of four quinones in rat plasma by ultra high performance liquid chromatography with electrospray ionization tandem mass spectrometry after the oral administration of Qianzhi capsules

A sensitive, specific, and accurate ultra high‐performance liquid chromatography with electrospray ionization tandem mass spectrometry method was developed and validated for the simultaneous quantification of purpurin, munjistin, mollugin, and alizarin from Qianzhi capsules in rat plasma. Chromatographic separation was performed on an Agilent Eclipse Plus C₁₈ RRHD column with a mobile phase consisting of methanol and 5 mM ammonium acetate/water with gradient elution. The analytes were quantified on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring mode and switching the electrospray ion source polarity with positive electrospray ionization in a single run. Samples were pretreated by liquid–liquid extraction with cyclohexane. The intra‐ and interday precision and accuracy of the assay were within acceptable ranges. Matrix effects for all of the analytes were between 90.16 and 100.21%. The average recovery ranged from 75.38 to 88.96%. This method was successfully applied to study the pharmacokinetic parameters of the four compounds in rat plasma after oral administration of Qianzhi capsules. Four quinones could be rapidly absorbed into blood (t_max, 0.80–1.93 h) and eliminated relatively slowly (t_1/2, 8.07–11.97 h). The results might be helpful for guiding the clinical application of Qianzhi capsules in the future.     

Conformational changes of ubiquitin during electrospray ionization as determined by in‐ESI source H/D exchange combined with high‐resolution MS and ECD fragmentation

In the paper, we have demonstrated the possibility of performing hydrogen/deuterium (H/D) exchange of proteins in the region of gas‐phase ion formation in an electrospray ion source by saturating the electrospray ionization source with vapors of a deuterating agent (D₂O or MeOD). In this region, charged droplets are shrinking and the protein ions transfer into the gas phase. As a model protein, we have used ubiquitin whose ion mobility spectrometry and gas‐phase H/D exchange in the vacuum part of a mass spectrometer demonstrated the presence of gas‐phase conformers with different cross sections and H/D exchange rates. In our experiments, we observed monomodal deuterium distributions for all solvents, charge states, desolvating capillary temperature and types of deuterating agent. Also, we found that the number of H/D exchanges increases with an increasing desolvating capillary temperature and decreasing charge state. We observed that solution composition (49 : 50 : 1 H₂O : MeOH : formic acid or 99 : 1 H₂O : formic acid) influences the charge‐state distribution but did not change the degree of H/D exchange for the same charge state. Electron‐capture dissociation fragmentation shows that higher charge states contain a segment that is protected from access by the deuterating agent. Copyright © 2014 John Wiley & Sons, Ltd.     

Gas‐phase fragmentation reactions of protonated benzofuran‐ and dihydrobenzofuran‐type neolignans investigated by accurate‐mass electrospray ionization tandem mass spectrometry

We have investigated gas‐phase fragmentation reactions of protonated benzofuran neolignans (BNs) and dihydrobenzofuran neolignans (DBNs) by accurate‐mass electrospray ionization tandem and multiple‐stage (MSⁿ) mass spectrometry combined with thermochemical data estimated by Computational Chemistry. Most of the protonated compounds fragment into product ions B ([M + H–MeOH]⁺), C ([ B –MeOH]⁺), D ([ C –CO]⁺), and E ([ D –CO]⁺) upon collision‐induced dissociation (CID). However, we identified a series of diagnostic ions and associated them with specific structural features. In the case of compounds displaying an acetoxy group at C‐4, product ion C produces diagnostic ions K ([ C –C₂H₂O]⁺), L ([ K –CO]⁺), and P ([ L –CO]⁺). Formation of product ions H ([ D –H₂O]⁺) and M ([ H –CO]⁺) is associated with the hydroxyl group at C‐3 and C‐3′, whereas product ions N ([ D –MeOH]⁺) and O ([ N –MeOH]⁺) indicate a methoxyl group at the same positions. Finally, product ions F ([ A –C₂H₂O]⁺), Q ([ A –C₃H₆O₂]⁺), I ([ A –C₆H₆O]⁺), and J ([ I –MeOH]⁺) for DBNs and product ion G ([ B –C₂H₂O]⁺) for BNs diagnose a saturated bond between C‐7′ and C‐8′. We used these structure‐fragmentation relationships in combination with deuterium exchange experiments, MSⁿ data, and Computational Chemistry to elucidate the gas‐phase fragmentation pathways of these compounds. These results could help to elucidate DBN and BN metabolites in in vivo and in vitro studies on the basis of electrospray ionization ESI‐CID‐MS/MS data only.     

Rapid Analysis of 27 Components of Isodon serra by LC–ESI-MS–MS

A new method based on high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry has been developed for simultaneous analysis of 27 components (eighteen diterpenoids, six phenolic acids, and three flavonoids) of Isodon serra, a famous traditional Chinese medicine. Separation on a C₁₈ column was achieved by gradient elution with water and methanol both containing 0.1% formic acid. Identification and quantification of the analytes were achieved by use of a hybrid quadrupole linear ion-trap mass spectrometer. Multiple-reaction monitoring (MRM) was used for quantification, with switching of electrospray ion source polarity between positive and negative modes in the same chromatographic run. An information-dependent acquisition (IDA) method was used to trigger product ion scans above the MRM signal threshold so that the 27 compounds could be identified by use of enhanced product-ion scans (EPI). The method was fully validated (for linearity, precision, accuracy, and limits of detection and quantification). The results indicated that this simple method was rapid, specific, and reliable. The method was successfully applied to analysis of 45 batches of Isodon serra samples from different sources, and quantification of the compounds in Isodon serra was achieved.     

Secondary Electrospray Ionization of Complex Vapor Mixtures. Theoretical and Experimental Approach

In secondary electrospray ionization (SESI) systems, gaseous analytes exposed to an electrospray plume become ionized after charge is transferred from the charging electrosprayed particles (the charging agent) to the vapor species. Currently available SESI models are valid for simplified systems having only one type of electrosprayed species, which ionizes only one single vapor species, and for the limit of low vapor concentration. More realistic models require considering other effects. Here we develop a theoretical model that accounts for the effects of high vapor concentration, saturation effects, interferences between different vapor species, and electrosprays producing different types of species from the liquid phase. In spite of the relatively high complexity of the problem, we find simple relations between the different ionic species concentrations that hold independently of the particular ion source configuration. Our model suggests that an ideal SESI system should use highly concentrated charging agents composed preferably of only one dominating species with low mobility. Experimental measurements with a MeOH-H₂O-NH₃ electrospray and a mixture of fatty acids and lactic acid served to test the theory, which gives good qualitative results. These results also suggest that the SESI ionization mechanism is primarily based on ions rather than on charged droplets.     

Internal energy effects in collision induced dissociation spectra

The effect of the precursor ion internal energy on the branching ratios obtained from collision induced dissociation fragmentation patterns was examined for [NH₃]⁺ and [C₂H₄N]⁺. The ion internal energy was changed by varying both the chemical ionization reagent gas and the ion source pressure. Effects observed in the collision induced dissociation fragmentation patterns as a function of the ion source pressure are explained by the reaction exothermicities and by collisional deactivation of internally excited ions (at high pressure).     

Olefin Metathesis of a Ruthenium Carbene Complex by Electrospray Ionization in the Gas Phase

Surprisingly similar reactions in the gas phase and in solution [Eq. (a)]: The ion 1 obtained by electrospray ionization behaves in the gas phase analogously to the corresponding complex [RuCl₂(=CHPh)(PCy₃)₂] in solution. Measured relative rates for ring-opening metathesis are interpreted under the assumption of an intramolecular π complex, which leads to an estimation of the equilibrium constant for π complexation.     

A new window on fullerene chemistry: An electrospray mass spectrometric study of the amination of C60

Polyamines derived from C₆₀ were characterized by electrospray ionization mass spectrometry in 1:1 methanoltoluene containing 2.5% trifluoroacetic acid. Peak assignments are consistent with the formula C₆₀O_n(RNH₂)_m for the protonated products. The preferential formation of the hexaaminated product C₆₀O₆(RNH₂)₆ was also observed.     

Deamination of protonated amines to yield protonated imines

Primary and secondary amines, when examined in atmospheric pressure chemical ionization, electrospray ionization, or chemical ionization, display protonated imines in their mass spectra. These products arise formally by nucleophilic substitution at the α-carbon with loss of both ammonia and molecular hydrogen. Collision-induced dissociation (CID) is used to characterize the product ions by comparison with authentic protonated imines. Gas-phase ion/molecule reactions of protonated amines with neutral amines also yield products that correspond to protonated imines (deamination and dehydrogenation), as well as providing simple deamination products. The reaction mechanism was investigated further by reacting the deamination product, the alkyl cation, with a neutral amine. The observed dehydrogenation of the nascent protonated secondary amine indicates that the reaction sequence is loss of ammonia followed by dehydrogenation even though the isolated protonated secondary amines did not undergo dehydrogenation upon CID. Formation of the deamination products in the protonated amine/amine reaction is competitive with proton-bound dimer formation. The proton-bound dimers do not yield deamination products under CID conditions in the ion trap or in experiments performed using a pentaquadrupole instrument. This demonstrates that the geometry of the proton-bound dimer, in which the α-carbons of the alkylamines are well separated [C _a -N-H-N-C _a ], is an unsuitable entry point on the potential energy hypersurface for formation of the imine [C _a -N-C _a ]. Isolation of the proton-bound dimers in the quadrupole ion trap is achieved with low efficiency and this characteristic can be used to distinguish them from their covalently bound isomers.     

A targeted/non-targeted screening method for perfluoroalkyl carboxylic acids and sulfonates in whole fish using quadrupole time-of-flight mass spectrometry and MSe

A new method for measuring perfluoroalkyl contaminants (PFCs) in biological matrices has been developed. An ultra-high pressure liquid chromatograph equipped with a quadrupole time-of-flight mass spectrometer (UPLC-QToF) was optimized using a continuous precursor/product ion monitoring mode. Unlike traditional targeted studies that isolate precursor/product ion pairs, the current method alternates between two ionization energy channels to continuously capture standard electrospray ionization (low energy) and collision induced dissociation (high energy) spectra. The result is the indiscriminant acquisition of paired low and high energy spectra for all constituents eluting from the chromatographic system. This technique was evaluated for the routine analysis of perfluoroalkyl species. Using this technique, linear perfluoroalkyl carboxylic acids (C₄ to C₁₄) and perfluoroalkyl sulfonates (C₄, C₆, C₈ and C₁₀) exhibited a linear range spanning over three orders of magnitude and were detectable at levels less than 1 pg on column with a root mean squared signal to noise ratio of 5 to 20. Lake trout (Salvelinus namaycush) and National Institutes of Standards and Technology Standard Reference Material 1946 were used to evaluate matrix effects and the accuracy of this method when applied to a whole fish extract. The current method was also evaluated as a diagnostic tool to identify unknown PFCs using experimental fragmentation patterns, mass defect filtering and Kendrick plots.

Peroxide Bond Driven Dissociation of Hydroperoxy-Cholesterol Esters Following Collision Induced Dissociation

Oxidative modification of polyunsaturated fatty acids, which occurs through enzymatic and nonenzymatic processes, is typically initiated by the attachment of molecular oxygen to an unsaturated fatty acyl chain forming a lipid hydroperoxide (LOOH). Enzymatic pathways are critical for cellular homeostasis but aberrant lipid peroxidation has been implicated in important pathologies. Analysis of primary oxidation products such as hydroperoxides has proven to be challenging for a variety of reasons. While negative ion electrospray ionization has been used for the specific detection of some LOOH species, hydroperoxide dehydration in the ion source has been a significant drawback. Here we describe positive ion electrospray ionization of ammoniated 13-hydroperoxy-9Z, 11E-octadecadienoyl cholesterol and 9-hydroperoxy-10E, 12Z-octadecadienoyl cholesterol, [M + NH₄]⁺, following normal phase high-pressure liquid-chromatography. Dehydration in the ion source was not prevalent and the ammoniated molecular ion was the major species observed. Collisionally induced dissociation of the two positional isomers yielded unique product ion spectra resulting from carbon–carbon cleavages along their acyl chains. Further investigation of this behavior revealed that complex collision induced dissociations were initiated by scission of the hydroperoxide bond that drove subsequent acyl chain cleavages. Interestingly, some of the product ions retained the ammonium nitrogen through the formation of covalent carbon–nitrogen or oxygen–nitrogen bonds. These studies were carried out using hydroperoxy-octadecadienoate cholesteryl esters as model compounds, however the observed mechanisms of [LOOH + NH₄]⁺ ionization and dissociation are likely applicable to the analysis of other lipid hydroperoxides and may serve as the basis for selective LOOH detection as well as aid in the identification of unknown lipid hydroperoxides.