ESI–IT–MSn and DFT calculation for electron affinities of bimetallic oxovanadium complexes

Abstract  

The MS ⁿ spectra of three bimetallic oxovanadium complexes were obtained using an ion trap. The fragmentation pathways were elucidated. Common features and major differences between ESI–QTOF–MS/MS and ESI–IT–MS ⁿ spectra were compared. Electron affinities of several radical molecular anions were calculated by DFT and these could be used as an indicator of the ions’ stability.     

Spectral characterization of novel <Emphasis Type="Italic">bis</Emphasis> heterocycles comprising both piperidine and thiohydantoin nuclei

Abstract  

A series of bis heterocycles comprising both piperidine and thiohydantoin nuclei namely 3-(3-alkyl-2,6-diarylpiperin-4-ylidene)-2-thioxoimidazolidin-4-ones is synthesized and characterized by melting point, elemental analysis, MS, FT–IR, one-dimensional NMR (¹H, D₂O exchanged ¹H and ¹³C), two-dimensional HOMOCOSY, and NOESY spectroscopic data.     

Electron ionization mass spectral fragmentation study of sulfation derivatives of polychlorinated biphenyls

Background  

Polychlorinated biphenyls are persistent organic pollutants that can be metabolized via hydroxylated PCBs to PCB sulfate metabolites. The sensitive and selective analysis of PCB sulfate monoesters by gas chromatography-mass spectrometry (GC-MS) requires their derivatization, for example, as PCB 2,2,2-trichloroethyl (TCE) sulfate monoesters. To aid in the identification of unknown PCB sulfate metabolites isolated from biological samples, the electron impact MS fragmentation pathways of selected PCB TCE sulfate diesters were analyzed and compared to the fragmentation pathways of the corresponding methoxylated PCBs.     

Liquid chromatography/mass spectrometry based detection and semi‐quantitative analysis of INSL5 in human and murine tissues

Rationale

Insulin‐like peptide 5 (INSL5) is a hormone produced by enteroendocrine L‐cells in the colon that has recently been implicated in the control of metabolic homeostasis. However, research into its physiology has been hindered by the reported unreliability of commercially available immunoassays and additional detection assays would benefit this emerging field.

Methods

Peptides from purified murine L‐cells and homogenates from both human and mouse colonic tissues were extracted by precipitating larger proteins with acetonitrile. Untargeted liquid chromatography/tandem mass spectrometry (LC/MS/MS) analyses, followed by database searching, were used to detect and identify various INSL5 gene derived peptides and characterise their precise sequence. A similar approach was developed to quantify INSL5 levels in primary intestinal culture supernatants after purification and concentration by solid‐phase extraction.

Results

Mass spectral analysis of purified enteroendocrine cells and tissue homogenates identified the exact sequence of A and B chains of INSL5 endogenously expressed in L‐cells. Differences in the endogenously processed peptide and the Swissprot database entry were observed for murine INSL5, whereas the human sequence matched previous predictions from heterologous expression experiments. INSL5 was detected in the supernatant of human and mouse primary colonic cultures and concentrations increased after treatment with a known L‐cell stimulus.

Conclusions

The first LC/MS/MS‐based method capable of the detection and semi‐quantitative analysis of endogenous INSL5 using MS‐based techniques has been demonstrated. The methodology will enable the identification of stimulants for INSL5 secretion from murine and human primary colonic epithelial cultures.     

MALDI‐TOF MS Coupled with Collision‐Induced Dissociation (CID) Measurements of Poly(methyl methacrylate)

Matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) was chosen for an in‐detail analysis of poly(methyl methacrylate) (PMMA) in order to determine the possible fragmentation mechanism with the help of collision‐induced dissociation (CID). All experiments were performed on a well‐defined PMMA standard and were optimized for sample preparation and measurement conditions of both MS and MS/MS. In order to investigate the fragmentation pathways, two parent peaks—both charged with sodium (m/z = 1 625.9 and 2 226.2 Da, respectively)—were selected, thus permitting the examination of possible cleavages, and reaction pathways. For both chosen peaks, the MALDI‐TOF MS/MS spectra revealed four fragmentation series that could be explained by single or multiple main chain scissions and secondary reactions of the PMMA side groups. According to the molar mass of the fragments, a loss or migration of the side group to the end of the free radical, followed by a β‐scission, was favored. These insights are the first steps toward the construction of a library with fragments and fragmentation pathways, complementary to proteomics libraries, in order to obtain fast and automated identification of substances.

     

High-performance liquid chromatography-tandem mass spectrometry method for the analysis of N-oleoyl glycine and N-oleoyl alanine in brain and plasma

Interest has increased in the role of N-acyl amino acids in a variety of disease states and as potential pharmacotherapies. Recently, N-oleoyl glycine and N-oleoyl alanine have shown promise in reducing the rewarding effects of drugs of abuse and alleviating withdrawal signs in rodent models. Previously published methods for the quantitation of these analytes by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) in tissue were part of extensive lipidomic panels which may result in limited sensitivity and selectivity and also reported low recovery. Presented is a method for the extraction and HPLC-MS/MS analysis of N-oleoyl glycine and N-oleoyl alanine. The bias and precision of the assay were determined to be within ± 20%. The method was shown to be reliable and robust, with over 90% recovery for the low-level analytes. Increasing concentrations of N-oleoyl glycine and N-oleoyl alanine were quantitated in mouse brain and plasma following exogenous administration. This method was developed to serve to support studies investigating the pharmacokinetics and involvement of N-oleoyl glycine and N-oleoyl alanine in drug dependence and other diseases.     

Secondary α-deuterium kinetic isotope effects in [2+4] cycloaddition of (E)-2-phenylnitroethene to cyclopentadiene

Abstract  

Secondary α-deuterium kinetic isotope effects confirm that [2+4] cycloaddition between (E)-2-phenylnitroethene and cyclopentadiene occurs in concerted manner, on both the pathway leading to 6-endo-phenyl-5-exo-nitronorbornene and the pathway leading to the corresponding 6-exo-phenyl-5-endo-nitro isomer. According to Hammond terminology the transition states on competitive pathways should be considered in terms of symmetrical early states.     

Flavonoids activate pregnane × receptor-mediated <Emphasis Type="Italic">CYP3A4</Emphasis> gene expression by inhibiting cyclin-dependent kinases in HepG2 liver carcinoma cells

Background  

The expression of the drug-metabolizing enzyme cytochrome P450 3A4 (CYP3A4) is regulated by the pregnane × receptor (PXR), which is modulated by numerous signaling pathways, including the cyclin-dependent kinase (Cdk) pathway. Flavonoids, commonly consumed by humans as dietary constituents, have been shown to modulate various signaling pathways (e.g., inhibiting Cdks). Flavonoids have also been shown to induce CYPs expression, but the underlying mechanism of action is unknown. Here, we report the mechanism responsible for flavonoid-mediated PXR activation and CYP expression.     

Kinetic characterisation of arylamine <Emphasis Type="Italic">N</Emphasis>-acetyltransferase from <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

Background  

Arylamine N-acetyltransferases (NATs) are important drug- and carcinogen-metabolising enzymes that catalyse the transfer of an acetyl group from a donor, such as acetyl coenzyme A, to an aromatic or heterocyclic amine, hydrazine, hydrazide or N-hydroxylamine acceptor substrate. NATs are found in eukaryotes and prokaryotes, and they may also have an endogenous function in addition to drug metabolism. For example, NAT from Mycobacterium tuberculosis has been proposed to have a role in cell wall lipid biosynthesis, and is therefore of interest as a potential drug target. To date there have been no studies investigating the kinetic mechanism of a bacterial NAT enzyme.     

Analysis of ketone‐based neurosteroids by reactive low‐temperature plasma mass spectrometry

Rationale

Neurosteroids are important signalling molecules that modulate neuronal activity. Their low concentrations and low volatility make neurosteroid detection and quantification by ambient mass spectrometry challenging. Here we develop a reactive low‐temperature plasma mass spectrometry (LTP‐MS) method and demonstrate its potential for fast screening and quantification of neurosteroids in mouse brain.

Methods

Ketone‐based neurosteroids were analysed with the LTP‐MS method. The plasma of the LTP was heated in order to improve the desorption efficiency of low‐volatility neurosteroids. Methylamine with a concentration of 500 ppbv was employed as the reactive reagent. Neurosteroids in mouse brain tissue extracts were detected in 70 s with mass errors less than ±3 ppm due to coupling of the ion source with a high‐performance mass spectrometer.

Results

Reaction between neurosteroids and methylamine, seeded into the LTP gas stream, resulted in the formation of protonated methylamine–neurosteroid adducts with 5‐ to 100‐fold abundances, compared to [M + H]⁺ ions detected in non‐reactive LTP‐MS. The lowest detectable concentrations of neurosteroid standards were in the range of ng/mL. Concentrations of neurosteroids in male and female mouse brain extracts as determined with reactive LTP‐MS were on the level of ng/g, comparable to results obtained with high‐performance liquid chromatography–tandem mass spectrometry.

Conclusions

The developed reactive LTP‐MS is capable of providing sensitive identification and quantification of ketone‐based neurosteroids in mouse brain extracts with minimal sample treatment, and showcases the potential of reactive LTP‐MS as a tool for fast screening of neurosteroid levels in brain.

     

Heterocycles [h]-fused to 4-oxoquinoline-3-carboxylic acid. Part VII: synthesis of some 6-oxoimidazo[4,5-h]quinoline-7-carboxylic acids and esters

Abstract  

A series of ethyl 2-(substituted)-9-cyclopropyl-4-fluoro-6-oxo-1H-imidazo[4,5-h]quinoline-7-carboxylates has been prepared from ethyl 7,8-diamino-1,4-dihydroquinoline-3-carboxylate via thermally induced reactions with model alkanoic acids or via microwave-assisted cyclocondensation with some arene carboxaldehydes. Acid-catalysed hydrolysis of the resulting ester derivatives furnished the corresponding imidazoquinoline-7-carboxylic acids. The structures of these new acid and ester derivatives are based on microanalytical and spectral (IR, MS, and NMR) data.     

Modes of complexation of rutin with the flavonoid reagent diphenylborinic acid 2-aminoethyl ester

Abstract  

The interaction of the model flavonoid glycoside rutin with the flavonoid reagent diphenylborinic acid 2-aminoethyl ester (DPBA) was investigated using a combination of HPLC–DAD–ESI–MS analysis, UV–visible spectroscopy, and semiempirical calculations. Mass spectra and spectroscopic data made it possible to describe the complexation pathway as addition of diphenylboron groups to the two available 3′,4′-o-diphenolic and the 5-hydroxy-4-keto coordinating sites of rutin. Semiempirical calculations were carried out to obtain the conformation of the most stable DPBA/rutin adducts. The results showed that a number of complexation dynamics can occur as a function of the characteristics of the medium (type and pH of the solvent) and of the amount of DPBA. This work suggests the possibility of substantially improving existing procedures for recognition of flavonoid compounds by choice of suitable experimental conditions.     

Limited proteolysis of human histone deacetylase 1

Background  

Histone deacetylase (HDAC) proteins are associated with cell proliferation, differentiation, apoptosis, and cancer. Specifically, HDAC1 is linked with cell growth, a hallmark of cancer formation. HDAC1 is a phosphoprotein and phosphorylation at S421 and S423 promotes HDAC1 enzymatic activity and protein association. While single and double point mutants of HDAC1 at S421 and S423 appear functionally similar, the evidence suggests that HDAC1 is phosphorylated simultaneously at both S421 and S423 in vivo. Additional experiments are necessary to probe the role of double phosphorylation of HDAC1 at S421 and S423.     

A novel bifunctional N-acetylglutamate synthase-kinase from <Emphasis Type="Italic">Xanthomonas campestris</Emphasis> that is closely related to mammalian N-acetylglutamate synthase

Background  

In microorganisms and plants, the first two reactions of arginine biosynthesis are catalyzed by N-acetylglutamate synthase (NAGS) and N-acetylglutamate kinase (NAGK). In mammals, NAGS produces an essential activator of carbamylphosphate synthetase I, the first enzyme of the urea cycle, and no functional NAGK homolog has been found. Unlike the other urea cycle enzymes, whose bacterial counterparts could be readily identified by their sequence conservation with arginine biosynthetic enzymes, mammalian NAGS gene was very divergent, making it the last urea cycle gene to be discovered. Limited sequence similarity between E. coli NAGS and fungal NAGK suggests that bacterial and eukaryotic NAGS, and fungal NAGK arose from the fusion of genes encoding an ancestral NAGK (argB) and an acetyltransferase. However, mammalian NAGS no longer retains any NAGK catalytic activity.     

Electrochemical behavior of catechol in the presence of 2-methyl-1,3-cyclopentanedione: application to electrosynthesis

Abstract  

Electro-oxidation of catechol in the presence of 2-methyl-1,3-cyclopentanedione as a nucleophile was investigated in water–acetonitrile (90:10 v/v) solution. The results indicate that the o-benzoquinone electrogenerated participates in a Michael addition reaction with this nucleophile. The electrosynthesis of 2-(3,4-dihydroxyphenyl)-2-methylcyclopentane-1,3-dione was carried out. The product was characterized by NMR, MS, FT-IR, and elemental analysis. An EC mechanism was deduced from voltammetric and spectroscopic data. Also, the Michael addition reaction rate constant (k _m) was estimated using digital simulation of voltammograms.     

A DFT study of the hydrolysis of hydantoin

Density functional theory (DFT) calculations were made on the hydrolysis of hydantoin (2,4-imidazolidinedione). In the neutral hydrolysis, reacting systems composed of hydantoin and (H₂O)_n with n = 1+3, 2+3, 3+3, and 4+3 were adopted. Three water molecules (“+3”) participate in the in-plane hydrogen-bond circuit, and the n–3 = 1, 2, 3 or 4 water cluster works for the out-of-plane nucleophilic attack onto the carbonyl carbon of hydantoin. Transition states (TSs) involving bond interchanges prompted by proton transfers were determined. The reaction path with n = 3+3 containing N-carbamoyl glycine, N-carboxy glycine and three tetrahedral intermediates was found to be most likely. In the acid-catalyzed hydrolysis, a reacting system composed of hydantoin and H₃O⁺(H₂O)₇ was employed. Ten TSs and nine intermediates were obtained. N-carbamoyl glycine and N-carboxy glycine were confirmed to be detectable stable species. The product consists of glycine, carbonic acid (not CO₂), NH₄⁺, and (H₂O)₅. It has the exothermic energy, whereas the product in the neutral hydrolysis is of the endothermic one for all n values. For both neutral (n = 3+3) and acid-catalyzed hydrolyses, the rate-determining steps were calculated to be for formation of the tetrahedral intermediate, HOOC-CH₂-NH-C(OH)₂NH₂. The pattern of proton transfers along hydrogen bonds was carefully investigated.     

Fragmentation of deprotonated d-ribose and d-fructose in MALDI—Comparison with dissociative electron attachment

We present a detailed, collaborative study on the fragmentation of deprotonated native d-ribose and d-fructose and the isotopically labelled 1-¹³C-d-ribose, 5-¹³C-d-ribose and C-1-d-d-ribose. The fragmentation is studied in a matrix assisted laser desorption/ionization time of flight mass spectrometer (MALDI ToF MS), both in in-source decay (ISD) and post-source decay (PSD) mode and compared with fragmentation through dissociative electron attachment (DEA). Fragmentation of deprotonated monosaccharides formed in the MALDI process, as well as their transient molecular anions formed upon electron attachment are characterized by loss of different numbers of H₂O and CH₂O units. Two different fragmentation pathways leading to cross-ring cleavage are identified. Metastable decay of deprotonated d-ribose proceeds either via an X-type cleavage yielding fragment anions at m/z = 119, 100 and 89, or via an A-type cleavage resulting in m/z = 89, 77 and 71. A fast and early metastable cross-ring cleavage of deprotonated d-ribose observed in in-source decay is dominated by X-type cleavage leading mainly to m/z = 100 and 71. For dissociative electron attachment to d-ribose a sequential dissociation was identified that includes metastable decay of the dehydrogenated molecular anion leading to m/z = 89. All other fragmentation reactions in DEA to d-ribose are likely to proceed directly and on a faster timescale (below 400 ns).     

Direct analysis of volatile organic compounds in foods by headspace extraction atmospheric pressure chemical ionisation mass spectrometry

Rationale

The rapid screening of volatile organic compounds (VOCs) by direct analysis has potential applications in the areas of food and flavour science. Currently, the technique of choice for VOC analysis is gas chromatography/mass spectrometry (GC/MS). However, the long chromatographic run times and elaborate sample preparation associated with this technique have led a movement towards direct analysis techniques, such as selected ion flow tube mass spectrometry (SIFT‐MS), proton transfer reaction mass spectrometry (PTR‐MS) and electronic noses. The work presented here describes the design and construction of a Venturi jet‐pump‐based modification for a compact mass spectrometer which enables the direct introduction of volatiles for qualitative and quantitative analysis.

Methods

Volatile organic compounds were extracted from the headspace of heated vials into the atmospheric pressure chemical ionization source of a quadrupole mass spectrometer using a Venturi pump. Samples were analysed directly with no prior sample preparation. Principal component analysis (PCA) was used to differentiate between different classes of samples.

Results

The interface is shown to be able to routinely detect problem analytes such as fatty acids and biogenic amines without the requirement of a derivatisation step, and is shown to be able to discriminate between four different varieties of cheese with good intra and inter‐day reproducibility using an unsupervised PCA model. Quantitative analysis is demonstrated using indole standards with limits of detection and quantification of 0.395 μg/mL and 1.316 μg/mL, respectively.

Conclusions

The described methodology can routinely detect highly reactive analytes such as volatile fatty acids and diamines without the need for a derivatisation step or lengthy chromatographic separations. The capability of the system was demonstrated by discriminating between different varieties of cheese and monitoring the spoilage of meats.     

Photodamage to Multidrug-resistant Gram-positive and Gram-negative Bacteria by 870 nm/930 nm Light Potentiates Erythromycin,Tetracycline and Ciprofloxacin

We have previously shown that 870 nm/930 nm wavelengths cause photodamage at physiologic temperatures in methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli via generation of endogenous radical oxygen species (ROS) and decreased plasma membrane potentials (ΔΨp). We tested MRSA (Strain HSJ216) in vitro with sublethal 870 nm/930 nm laser energy and subinhibitory concentrations of erythromycin, tetracycline, penicillin, rifampin and trimethoprim to surmise whether photodamage could potentiate these antimicrobials. We also tested patient isolates of fluoroquinolone-resistant MRSA and E. coli with subinhibitory concentrations of ciprofloxacin. In MRSA (Strain HSJ216) we observed 97% potentiation (a 1.5 log₁₀ CFU decrease) with erythromycin and tetracycline. In patient isolates of E. coli, we observed 100% potentiation (>3 log₁₀ CFU decrease) in all irradiated samples with ciprofloxacin. To assess whether staphyloxanthin pigment conferred protection against the generated ROS, we created an isogenic carotenoid-deficient mutant of S. aureus that was significantly less tolerant of 870 nm/930 nm exposure than the wild type strain (P < 0.0001). We suggest that antibiotic potentiation results from a photobiological attenuation of ATP-dependent macromolecular synthetic pathways, similar to that observed with daptomycin, via disruption of ΔΨp and endogenous generation of ROS. With erythromycin, tetracycline and ciprofloxacin, attenuation of energy-dependent efflux systems is also a possibility.     

Endogenous high‐performance liquid chromatography/tandem mass spectrometry interferences and the case of perfluorohexane sulfonate (PFHxS) in human serum; are we overestimating exposure?

Perfluorinated acids have received increasing scientific attention due to their widespread global distribution, environmental persistence and bioaccumulation in wildlife and humans. For perfluorohexane sulfonate (PFHxS, C₆F₁₃SO, m/z 399), all existing human data have been generated using high‐performance liquid chromatography (HPLC) and its most sensitive tandem mass spectrometric (MS/MS) transitions (m/z 399/80 [SO₃]^? or m/z 399/99 [SO₃F]^?), but this may be problematic because of co‐eluting endogenous steroid sulfates that share common fragmentation pathways. We examined the magnitude of over‐reporting for PFHxS in pregnant women (n = 29), and in pooled serum of males, non‐pregnant and pregnant females (n = 3, 100 samples per pool), by comparing m/z 399/80 and 399/99 data with an interference‐free transition, m/z 399/119. PFHxS concentrations in pregnant women determined using m/z 399/80 and 399/99 (p < 0.05), but not m/z 399/119, were positively correlated to the response of the steroid sulfates. This led to an average overestimation of PFHxS by 1.5‐ and 4.7‐fold, using m/z 399/80 and 399/99, respectively, and validated the use of m/z 399/119 for the first time. The interferences were a problem in all human serum samples, and analysis of pooled serum revealed statistically significant over‐reporting by m/z 399/80 and 399/99 for pregnant women > non‐pregnant women > men. The magnitude of over‐reporting here represents a worst‐case scenario, but the extent to which the published literature values are biased is unknown due to limited details of methods in existing reports. Instead of using the less sensitive m/z 399/119 transition, we showed that an alternative selection of column and mobile phase can allow for sufficient chromatographic separation of the interferences. In conclusion, it was shown that routine analytical methods are prone to systematically overestimating PFHxS concentrations in serum of men or women, but that this can be avoided by alternative chromatographic steps or MS/MS transitions. Copyright © 2009 John Wiley & Sons, Ltd.