Investigation of Tropane Alkaloids in Genetically Transformed <Emphasis Type="Italic">Atropa belladonna</Emphasis> L. Cultures

The purpose of the present study was to determine the tropane alkaloid content of genetically transformed hairy root cultures of Atropa belladonna L. Determination of alkaloids was performed by HPLC method. Samples were extracted with chloroform – methanol - cc. ammonia 15:5:1 (v/v/v). Crude extracts were purified on Extrelut columns. HPLC separation was performed on Luna C8 reversed phase column. An isocratic mixture of acetonitrile – 30 mM phosphate buffer - methanol 12.2:79.7:8.1(v/v/v) was used as eluent. Peaks were identified by addition of standards and diode-array detection. Hyoscyamine, scopolamine and apoatropine were determined by external standard method at 210 nm. We measured the alkaloid content of genetically transformed in vitro cultures (hairy roots and reorganised plants) cultivated on Gamborg B5 basic media. The highest hyoscyamine and scopolamine content was found in hairy root clone #K₅ (0,223 m/m%) and in hairy root clone #K₄ (0,018 m/m%) respectively. Alkaloid contents were higher in the hairy roots than in the reorganised plants.     

Production and Accumulation of 4-<Emphasis Type="Italic">O</Emphasis>-Methyl-α-<Emphasis Type="SmallCaps">d</Emphasis>-Glucuronosyl-Xylotriose by Growing Culture of Thermophilic <Emphasis Type="Italic">Anoxybacillus</Emphasis> sp. Strain JT-12

A thermophilic Anoxybacillus sp. strain JT-12, isolated from soil, produced acidic xylotriose, 4-O-methyl-α-d-glucuronosyl-xylotriose (MeGlcAX₃), as a main product from birchwood xylan and accumulated them in the culture under optimum conditions at pH 7.0 and 55 °C using 0.75% (w/v) birchwood xylan as a carbon source for 42–72 h. The acidic xylotriose was purified by ethanol precipitation and high-performance liquid chromatography using NH₂ Lichosher® 100 column. The results of electrospray ionization mass spectrometry, mass to charge ratio (m/z) 603.23, confirmed that the purified sample was acidic xylotriose that had benefits and applications in many fields.     

Chiral P,N-bidentate N-pyrrolylphophines as ligands with remarkable electronic properties

Previously unknown chiral P,N-bidentate N-pyrrolylphosphines and their chelate complexes [Rh(η²-P,N)(CO)Cl] and [Pd(Allyl)(η²-P,N)]BF₄ were synthesized by phosphorylation of (E,1R,2R,3R,5S)-2-[(2,6,6-trimethylbicyclo[3.1.1]heptyl-3-)iminomethyl]-1H-pyrrole. The composition and structures of the novel compounds were determined by the ¹H, ¹³C, and ³¹P NMR, IR, mass spectrometry (electrospray), and elemental analysis methods. N-pyrrolylphosphines were found to have unusual electronic properties, being simultaneously more strong π-acids and σ-bases as compared to phosphites.     

Composition of essential oils of four <Emphasis Type="Italic">Hedychium</Emphasis>species from Vietnam

Background

Vietnam is a country blessed with many medicinal plants widely used as food and for medicinal purposes, and they contain a host of active substances that contribute to health. However, the analysis of chemical constituents of these plant species has not been subject of literature discussion.

Results

In this study, the chemical compositions of essential oils of four Hedychium species, obtained by hydrodistillation, were determined by means of gas chromatography-flame ionization detector (GC-FID) and gas chromatography–mass spectrometry (GC-MS) techniques. Individually, α-pinene (52.5%) and β-pinene (31.8%) were present in the leaf oil of Hedychium stenopetalum Lodd., while linalool (45.2%), (E)-nerolidol (8.7%) and α-pinene (5.0%) were identified in the root. The leaf of Hedychium coronarium J. König was characterized by α-pinene (20.0%), linalool (15.8%), 1,8-cineole (10.7%), α-pinene (10.1%) and α-terpineol (8.6%); while α-pinene (23.6%), α-humulene (17.1%) and β-caryophyllene (13.0%) were identified in the root. Hedychium flavum Roxb., gave oil whose major compounds were α-pinene (22.5%), α-humulene (15.7%) and β-caryophyllene (10.4%) in the leaf; α-humulene (18.9%), β-caryophyllene (11.8%) and α-pinene (11.2%) in the stem, as well as α-pinene (21.8%), linalool (17.5%) and 1,8-cineole (13.5%) in the root. The main constituents of Hedychium ellipticum Buch.-Ham. ex Smith were (E)-nerolidol (15.9%), α-pinene (11.8%) and bornyl acetate (9.2%) in the leaf with 1,8-cineole (40.8%), α-pinene (18.3%) and α-pinene (11.0%) occurring in the root.

Conclusions

Ubiquitous monoterpenes and sesquiterpenes were identified as characteristic markers for Hedychium species. This work is of great importance for the evaluation of Hedychium essential oils grown in Vietnam.

     

Peculiarities of the Mass Spectrometric Detection of Anthocyanins in High-Performance Liquid Chromatography

The dependence of the degree of fragmentation of anthocyanin “molecular” ions on the cone voltage of a mass spectrometric electrospray ionization detector was studied. It was found that the voltage required for the fragmentation of 50% of original “molecular” ions, E _f (0.5), increased with the number of glycoside residues. The fragmentation of glycosides proceeds with the removal of the entire residue regardless of their structures. In the case of 3,5-diglycosides, two types of fragment ions formed due to the loss of glycosidic residues from different positions; the ratio of their peak intensities is reciprocal to the ratio of the masses of residues eliminated. The values of E _f (0.5) for monoglycosides (190 V), diglycosides (229 V), triglycosides (267 V), and for some acylated cyanidin-3,5-diglycosides are determined. These results were given for the gradient separation of anthocyanins by reversed-phase HPLC in aqueous acetonitrile containing 10 vol % of formic acid using quadrupole mass spectrometric detection.     

Subambient pressure electrospray ionization ion mobility spectrometry

The pressure dependence of sheath gas assisted electrospray ionization (ESI) was investigated based on two complementary experimental setups, namely an ESI-ion mobility (IM) spectrometer and an ESI capillary - Faraday plate setup housed in an optically accessible vacuum chamber. The ESI-IM spectrometer is capable of working in the pressure range between 300 and 1000 mbar. Another aim was the assessment of the analytical capabilities of a subambient pressure ESI-IM spectrometer. The pressure dependence of ESI was characterized by imaging the electrospray and recording current-voltage (I-U) curves. Qualitatively different behavior was observed in both setups. While the current rises continuously with the voltage in the capillary-plate setup, a sharp increase of the current was measured in the IM spectrometer above a pressure-dependent threshold voltage. The different character can be attributed to the detection of different species in both experiments. In the capillary-plate experiment, a multitude of charged species are detected while only desolvated ions attribute to the IM spectrometer signal. This finding demonstrates the utility of IM spectrometry for the characterization of ESI, since in contrast to the capillary-plate setup, the release of ions from the electrospray droplets can be observed. The I-U curves change significantly with pressure. An important result is the reduction of the maximum current with decreasing pressure. The connected loss of ionization efficiency can be compensated by a more efficient transfer of ions in the IM spectrometer at increased E/N. Thus, similar limits of detection could be obtained at 500 mbar and 1 bar.     

Acquiring Structural Information on Virus Particles with Charge Detection Mass Spectrometry

Charge detection mass spectrometry (CDMS) is a single-molecule technique particularly well-suited to measuring the mass and charge distributions of heterogeneous, MDa-sized ions. In this work, CDMS has been used to analyze the assembly products of two coat protein variants of bacteriophage P22. The assembly products show broad mass distributions extending from 5 to 15 MDa for A285Y and 5 to 25 MDa for A285T coat protein variants. Because the charge of large ions generated by electrospray ionization depends on their size, the charge can be used to distinguish hollow shells from more compact structures. A285T was found to form T = 4 and T = 7 procapsids, and A285Y makes a small number of T = 3 and T = 4 procapsids. Owing to the decreased stability of the A285Y and A285T particles, chemical cross-linking was required to stabilize them for electrospray CDMS.

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Quantification of Protein-Ligand Interactions by Laser Electrospray Mass Spectrometry

Laser electrospray mass spectrometry (LEMS) measurement of the dissociation constant (K_d) for hen egg white lysozyme (HEWL) and N,N′,N″-triacetylchitotriose (NAG₃) revealed an apparent K_d value of 313.2?±?25.9 μM for the ligand titration method. Similar measurements for N,N′,N″,N″’-tetraacetylchitotetraose (NAG₄) revealed an apparent K_d of 249.3?±?13.6 μM. An electrospray ionization mass spectrometry (ESI-MS) experiment determined a K_d value of 9.8?±?0.6 μM. In a second LEMS approach, a calibrated measurement was used to determine a K_d value of 6.8?±?1.5 μM for NAG₃. The capture efficiency of LEMS was measured to be 3.6?±?1.8% and is defined as the fraction of LEMS sample detected after merging with the ESI plume. When the dilution is factored into the ligand titration measurement, the adjusted K_d value was 11.3 μM for NAG₃ and 9.0 μM for NAG₄. The calibration method for measuring K_d developed in this study can be applied to solutions containing unknown analyte concentrations.

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Application of high-performance liquid chromatography to the determination of the concentration of lichen secondary metabolites

Preparative isolation of aromatic metabolites from lichens of the Cladonia genus (C. stellaris, C. arbuscula, C. amaurocraea, and C. rangiferina) growing in Central Yakutia was carried out. Identification by IR, UV, and time-of-flight mass spectrometry has shown that the isolated compounds belong to the group of lichen substances. It was shown that the component composition of the studied lichens corresponds to the previously described one. At the same time, the concentration of perlatolic and barbatic acids in C. stellaris and C. amaurocrae lichens, respectively, growing in Central Yakutia found by HPLC is higher than in similar types of temperate climatic zones.     

Top-Down LESA Mass Spectrometry Protein Analysis of Gram-Positive and Gram-Negative Bacteria

We have previously shown that liquid extraction surface analysis (LESA) mass spectrometry (MS) is a technique suitable for the top-down analysis of proteins directly from intact colonies of the Gram-negative bacterium Escherichia coli K-12. Here we extend the application of LESA MS to Gram-negative Pseudomonas aeruginosa PS1054 and Gram-positive Staphylococcus aureus MSSA476, as well as two strains of E. coli (K-12 and BL21 mCherry) and an unknown species of Staphylococcus. Moreover, we demonstrate the discrimination between three species of Gram-positive Streptococcus (Streptococcus pneumoniae D39, and the viridans group Streptococcus oralis ATCC 35037 and Streptococcus gordonii ATCC35105), a recognized challenge for matrix-assisted laser desorption ionization time-of-flight MS. A range of the proteins detected were selected for top-down LESA MS/MS. Thirty-nine proteins were identified by top-down LESA MS/MS, including 16 proteins that have not previously been observed by any other technique. The potential of LESA MS for classification and characterization of novel species is illustrated by the de novo sequencing of a new protein from the unknown species of Staphylococcus.

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Functionalization of sterically hindered catechol and <Emphasis Type="Italic">o</Emphasis>-benzoquinone with 2,2,6,6-tetramethylpiperidine 1-oxyl

Sterically hindered 4,6-di-tert-butyl-3-formylcatechol and 3,6-di-tert-butyl-o-benzoquinone react with 4-amino-2,2,6,6-tetramethylpiperidine 1-oxyl to give new chelate ligands of the o-quinone type bearing a 2,2,6,6-tetramethylpiperidine 1-oxyl neutral radical moiety. Structures of the synthesized compounds were established by ESR spectroscopy, IR spectroscopy, mass spectrometry, and X-ray diffraction analysis.     

Straightforward and solvent-free synthesis of 2-amino-4<Emphasis Type="Italic">H</Emphasis>-chromenes in the presence of a choline-based magnetic ionic liquid as catalyst

Choline tetrachloroferrate with a magnetic response property is applied in multicomponent one-pot synthesis of 2-amino-4H-chromenes as effective catalyst. The catalyst is characterized by the UV–Vis and Raman spectroscopies and by the electrospray ionization mass spectrometry.     

Formation of heterometallic compounds in mixed solutions of nickel(II)-Schiff base complexes and lanthanide nitrates: Electrospray ionization mass spectrometry data

Atmospheric-pressure electrospray ionization mass spectrometric data are reported for methanolic Ni(SB) + Ln(NO₃)₃ · nH₂O solutions (H₂SB = N,N′-ethylene-bis(salicylaldiimine), N,N′-ethylene-bis(3-methoxysalicylaldiimine), N,N′-ethylene-bis(acetylacetonediimine); Ln = La, Eu, Lu). Dinuclear and trinuclear heterometallic complexes of composition [(Ni(SB)) _x Ln(NO₃)₃] (x = 1, 2) form in these solutions. The dinuclear-totrinuclear ion intensity ratio depends on the natures of the lanthanide and the Schiff base.     

LC-MS Analysis of Antioxidant Plant Phenoloids

Extracts of selected medicinal plants with promising integral antioxidative capacity were examined by high-performance liquid chromatography (HPLC) coupled with diode-array detection (DAD) and on-line mass spectrometry (ESI-MS or APCI-MS). These techniques allowed determination of the main components of each extract, which may serve us thereby providing a typical “finger-print” in the identification of the plants. More specifically various flavonol aglycones, flavon- and flavonol-glycosides, flavonol-diglycosides were detected in herbs of Solidago canadensis chemovarieties, in leaves of Filipendula ulmaria and in the herb of Viola tricolor species.     

Mass Spectrometric Detection of Charged Silver Nanoclusters with Hydrogen Inclusions Formed by the Reduction of AgNO<Subscript>3</Subscript> in Ethylene Glycol

In the problem of the production silver nanoparticles, mass spectrometry allows one to identify nanoclusters as nuclei or intermediates in the synthesis of nanoparticles and to understand the mechanisms of their formation. Using low-temperature secondary emission mass spectrometry, we determined the cluster composition of a system formed in the microwave treatment of a solution of AgNO₃ in ethylene glycol (M). Along with silver ion–ethylene glycol associates М _m ? Ag⁺ (m = 1–5) and small silver clusters AgM _n ⁺ (n = 1–9), unusual silver clusters with one hydrogen atom [Ag _n H]⁺ (n = 2, 4) were observed. Possible pathways for the formation of silver nanoparticles taking into account hydrogen-containing cluster intermediates are discussed.     

New Insights on Non-Enzymatic Oxidation of Ganglioside GM1 Using Mass Spectrometry

Gangliosides are acidic glycosphingolipids that are present in cell membranes and lipid raft domains, being particularly abundant in central nervous systems. They participate in modulating cell membrane properties, cell–cell recognition, cell regulation, and signaling. Disturbance in ganglioside metabolism has been correlated with the development of diseases, such as neurodegenerative diseases, and in inflammation. Both conditions are associated with an increased production of reactive oxidation species (ROS) that can induce changes in the structure of biomolecules, including lipids, leading to the loss or modification of their function. Oxidized phospholipids are usually involved in chronic diseases and inflammation. However, knowledge regarding oxidation of gangliosides is scarce. In order to evaluate the effect of ROS in gangliosides, an in vitro biomimetic model system was used to study the susceptibility of GM1 (Neu5Acα2-3(Galβ1-3GalNAcβ1-4)Galβ1-4Glcβ1Cer) to undergo oxidative modifications. Oxidation of GM1 under Fenton reaction conditions was monitored using high resolution electrospray ionization-mass spectrometry (ESI-MS) and tandem mass spectrometry (ESI-MS/MS). Upon oxidation, GM1 underwent oxidative cleavages in the carbohydrate chain, leading to the formation of other gangliosides GM2 (GalNAcβ1-4Gal(Neu5Acα2-3)1-4Glcβ1Cer), GM3 (Neu5Acα2-3Galβ1-4Glcβ1Cer), asialo-GM1 (Galβ1-3GalNAcβ1-4Galβ1-4Glcβ1Cer), asialo-GM2 (GalNAcβ1-4Galβ1-4Glcβ1Cer), of the small glycolipids lactosylceramide (LacCer), glucosylceramide (GlcCer), and of ceramide (Cer). In addition, oxygenated GM1 and GM2 (as keto and hydroxy derivatives), glycans, oxidized glycans, and oxidized ceramides were also identified. Nonenzymatic oxidation of GM1 under oxidative stress contributes to the generation of other gangliosides that may participate in the imbalance of gangliosides metabolism in vivo, through uncontrolled enzymatic pathways and, consequently, play some role in neurodegenerative processes.

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Processes of hydrogenation of trifluoromethylfullerenes in the mass spectrometer ion source

The processes of hydrogenation of a mixture of trifluoromethylfullerenes was studied in situ by means of positive-and negative-ion mass spectrometry. The effective addition of 1, 5, and 11 hydrogen atoms was revealed. The appearance energies of positive trifluoromethylfullerene ions C ₆₀ (CF₃) _n ⁺ (n = 1–8) and C₆₀(CF₃) _n H⁺ (n = 1, 3, 5, 7) were determined.     

Gas-Phase Chemistry of Benzyl Cations in Dissociation of <Emphasis Type="BoldItalic">N</Emphasis>-Benzylammonium and <Emphasis Type="BoldItalic">N</Emphasis>-Benzyliminium Ions Studied by Mass Spectrometry

In this study, the fragmentation reactions of various N-benzylammonium and N-benzyliminium ions were investigated by electrospray ionization mass spectrometry. In general, the dissociation of N-benzylated cations generates benzyl cations easily. Formation of ion/neutral complex intermediates consisting of the benzyl cations and the neutral fragments was observed. The intra-complex reactions included electrophilic aromatic substitution, hydride transfer, electron transfer, proton transfer, and nucleophilic aromatic substitution. These five types of reactions almost covered all the potential reactivities of benzyl cations in chemical reactions. Benzyl cations are well-known as Lewis acid and electrophile in reactions, but the present study showed that the gas-phase reactivities of some suitably ring-substituted benzyl cations were far richer. The 4-methylbenzyl cation was found to react as a Brønsted acid, benzyl cations bearing a strong electron-withdrawing group were found to react as electron acceptors, and para-halogen-substituted benzyl cations could react as substrates for nucleophilic attack at the phenyl ring. The reactions of benzyl cations were also related to the neutral counterparts. For example, in electron transfer reaction, the neutral counterpart should have low ionization energy and in nucleophilic aromatic substitution reaction, the neutral counterpart should be piperazine or analogues. This study provided a panoramic view of the reactions of benzyl cations with neutral N-containing species in the gas phase.     

Physicochemical studies on bioactive Cr(III) coordination compounds with esters of hydrazine carboxylic acid as hetero donor ligands

Reaction between hydrazine derivative ligands (HrzE) or (HrzB) and chromium salt in 1:2 (metal:ligand) molar ratio yielded monometallic trivalent coordination compounds with general formula [Cr(HrzE)₂X₂]X and [Cr(HrzB)₂X₂]X, where (HrzE) = hydrazine carboxylic acid ethyl ester, (HrzB) = hydrazine carboxylic acid tert-butyl ester, and X = Cl^?, Br^? or F^?. Elemental analysis, conductivity measurements, magnetic moment measurements, and various spectroscopic techniques, viz. infrared, ultraviolet–visible, and electrospray ionization mass spectrometry, were applied to illustrate the structure and composition of the coordination compounds. Analytic and conductivity results were consistent with 1:1 electrolytic behavior and the proposed formulas of the coordination compounds. Electronic absorption data and magnetic moment parameters indicated octahedral geometry surrounding the metal ion in the coordination compounds. The in vitro antimicrobial behavior of the ligands and coordination compounds was screened using four bacterial strains (Staphylococcus aureus, Bacillus subtilis, Escherichia coli, and Salmonella typhi) and two fungal strains (Candida parapsilosis and Saccharomyces cerevisiae). The results indicated improved activity of the coordination compounds compared with the free ligands against the studied bacteria and fungi.     

Dicarboxylic acids and hydroxy fatty acids in different species of fungi

Secondary metabolites of fungi can be responsible for allergies; therefore, the identification of compounds produced by these organisms is very important. Fungi produce large amounts of secondary metabolites, which belong to groups of chemicals such as: dicarboxylic acids, hydroxy acids, alcohols, hydrocarbons, esters, fatty acids, sterols, amino acids and mycotoxins. The presence of all these compounds in human proximity contributes to many diseases. Therefore, the aim of the study was a qualitative and quantitative analysis of hydroxy and dicarboxylic acids produced by fungi occurring in student hostel in Poland, in the province of Pomerania. The following species of fungi were subjected to extraction: Aspergillus niger, Aspergillus fumigatus, Aspergillus candidus, Rhizopus sp., Geotrichum candidum, and Penicillium chrysogenum. A mixture of ethyl acetate and methanol was used for the extraction. The obtained extracts were further analyzed by gas chromatography mass spectrometry (GC–MS). In all samples of fungi, the presence of a total of 22 acids, including 13 dicarboxylic and 9 hydroxy acids, was confirmed. Most acids (17 different acids) were identified in A. fumigatus. Only 10 acids were identified in the mycelium of G. candidum and A. niger. Acids which were identified in all samples of the mycelium were 22-hydroxydocosanoic acid, 24-hydroxytetracosanoic acid and adipic acid. The most abundant compounds were 22-hydroxydocosanoic acid in A. fumigatus, A. candidus, Rhizopus sp., G. candidum and P. chrysogenum, and succinic acid in A. niger. More experiments are needed to understand the physiological role of hydroxy and dicarboxylic acids. We hope that our results are an important contribution to further studies on the human health.