Investigating the Ionization of Dissolved Organic Matter by Electrospray.

Electrospray ionization (ESI) operating in the negative mode coupled to high-resolution mass spectrometry is the most popular technique for the characterization of dissolved organic matter (DOM). The vast molecular heterogeneity and the functional group diversity of this complex mixture prevents the efficient ionization of the organic material by a single ionization source, so the presence of uncharacterized material is unavoidable. The extent of this poorly ionizable pool of carbon is unknown, is presumably variable between samples, and can only be assessed by the combination of analysis with a uniform detection method. Charged aerosol detection (CAD), whose response is proportional to the amount of nonvolatile material and is independent from the physicochemical properties of the analytes, is a suitable candidate. In this study, a fulvic acid mixture was fractionated and analyzed by high-pressure liquid chromatography-mass spectrometry in order to investigate the polarity and size distributions of highly and poorly ionizable material in the sample. Additionally, DOM samples of terrestrial and marine origins were analyzed to evaluate the variability of these pools across the land-sea aquatic continuum. The relative response factor values indicated that highly ionizable components of aquatic DOM mixtures are more hydrophilic and have lower molecular weight than poorly ionizable components. Additionally, a discrepancy between the samples of terrestrial and marine origins was found, indicating that marine samples are better represented by ESI than terrestrial samples, which have an abundant portion of hydrophobic poorly ionizable material.

Online HPLC-ESI-HRMS Method for the Analysis and Comparison of Different Dissolved Organic Matter Samples.

Natural dissolved organic matter (DOM) is an ultracomplex mixture that is essential to global carbon cycling but is poorly understood because of its complexity. The most powerful tool for the DOM characterization is high-resolution mass spectrometry (HRMS) generally combined to direct infusion (DI) as sample introduction. Liquid chromatography (LC) represents a compelling alternative to DI; however, state-of-the-art techniques involve only offline LC-HRMS approaches, which have important logistical drawbacks that make DOM analysis more challenging. This study introduces a new method based on online coupling of liquid chromatography to high resolution mass spectrometry, able to overcome the disadvantages of usual approaches. It is characterized by high reproducibility (% Bray-Curtis dissimilarity among replicates ≈ 2.5%), and it reduces transient complexity and contaminant interferences, thus increasing the signal-to-noise ratio (S/N), leading to the identification of an overall larger number of formulas in the mixture. Moreover, the application of an in silico fractionation prior to the statistical analysis allows an easy, flexible, fast, and detailed comparison of DOM samples from a variety of sources with a single chromatographic run.

A General Route to ß-Substituted Pyrroles by Transition-Metal Catalysis.

An atom-efficient route to pyrroles substituted in the ß-position has been achieved in four high yielding steps by a combination of Pd, Ru, and Fe catalysis with only water and ethene as side-products. The reaction is general and gives pyrroles substituted in the ß-position with linear and branched alkyl, benzyl, or aryl groups in overall good yields. The synthetic route includes a Pd-catalyzed monoallylation step of amines with substituted allylic alcohols that proceeds to yield the monoallylated products in moderate to excellent yields. In a second step, unsymmetrical diallylated aromatic amines are generated from the reaction of a second allylic alcohol with high selectivity in moderate to good yields by control of the reaction temperature. Ru-catalyzed ring-closing metathesis performed on the diallylated aromatic amines yields the pyrrolines substituted in the ß-position in excellent yields. By addition of ferric chloride to the reaction mixture, a selective aromatization to yield the corresponding pyrroles substituted in the ß-position was achieved. A reaction mechanism involving a palladium hydride, generated from insertion of palladium to O-H of an allyl alcohol, that is responsible for the C-O bond cleavage to generate the π-allyl intermediate is proposed.

Brønsted acid-catalyzed intramolecular nucleophilic substitution of the hydroxyl group in stereogenic alcohols with chirality transfer.

The hydroxyl group of enantioenriched benzyl, propargyl, allyl, and alkyl alcohols has been intramolecularly displaced by uncharged O-, N-, and S-centered nucleophiles to yield enantioenriched tetrahydrofuran, pyrrolidine, and tetrahydrothiophene derivatives with phosphinic acid catalysis. The five-membered heterocyclic products are generated in good to excellent yields, with high degree of chirality transfer, and water as the only side-product. Racemization experiments show that phosphinic acid does not promote SN1 reactivity. Density functional theory calculations corroborate a reaction pathway where the phosphinic acid operates as a bifunctional catalyst in the intramolecular substitution reaction. In this mechanism, the acidic proton of the phosphinic acid protonates the hydroxyl group, enhancing the leaving group ability. Simultaneously, the oxo group of phosphinic acid operates as a base abstracting the nucleophilic proton and thus enhancing the nucleophilicity. This reaction will open up new atom efficient techniques that enable alcohols to be used as nucleofuges in substitution reactions in the future.

Speciation of inositol phosphates in lake sediments by ion-exchange chromatography coupled with mass spectrometry, inductively coupled plasma atomic emission spectroscopy, and 31P NMR spectroscopy.

A method for the detection and speciation of inositol phosphates (InsP(n)) in sediment samples was tested, utilizing oxalate-oxalic acid extraction followed by determination by high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) using electrospray ionization (ESI) in negative mode. The chromatographic separation was carried out using water and ammonium bicarbonate as mobile phase in gradient mode. Data acquisition under MS/MS was attained by multiple reaction monitoring. The technique provided a sensitive and selective detection of InsP(n) in sediment samples. Several forms of InsP(n) in the oxalate-oxalic acid extracted sediment were identified. InsP6 was the dominating form constituting 0.250 mg P/g DW (dry weight); InsP5 and InsP4 constituted 0.045 and 0.014 mg P/g DW, respectively. The detection limit of the LC-ESI-MS/MS method was 0.03 µM InsP(n), which is superior to the currently used method for the identification of InsP(n), (31)P nuclear magnetic resonance spectroscopy ((31)P NMR). Additionally sample handling time was significantly reduced.

Mechanistic insights into the Pd-catalyzed direct amination of allyl alcohols: evidence for an outer-sphere mechanism involving a palladium hydride intermediate.

The mechanism of direct amination of allyl alcohol by a palladium triphenylphosphite complex has been explored. Labelling studies show that the reaction proceeds through a π-allylpalladium intermediate. A second-order dependence of reaction rate on allyl alcohol concentration was observed. Kinetic isotope effect studies and ESI-MS studies are in agreement with a reaction proceeding through a palladium hydride intermediate in which both O-H bond and C-O bond cleavages are involved in rate-determining steps. A stereochemical study supports an outer-sphere nucleophilic attack of the π-allylpalladium intermediate giving complete chiral transfer from starting material to product.

Palladium(II)-catalyzed desulfitative synthesis of aryl ketones from sodium arylsulfinates and nitriles: scope, limitations, and mechanistic studies.

A fast and efficient protocol for the palladium(II)-catalyzed production of aryl ketones from sodium arylsulfinates and various organic nitriles under controlled microwave irradiation has been developed. The wide scope of the reaction has been demonstrated by combining 14 sodium arylsulfinates and 21 nitriles to give 55 examples of aryl ketones. One additional example illustrated that, through the choice of the nitrile reactant, benzofurans are also accessible. The reaction mechanism was investigated by electrospray ionization mass spectrometry and DFT calculations. The desulfitative synthesis of aryl ketones from nitriles was also compared to the corresponding transformation starting from benzoic acids. Comparison of the energy profiles indicates that the free energy requirement for decarboxylation of 2,6-dimethoxybenzoic acid and especially benzoic acid is higher than the corresponding desulfitative process for generating the key aryl palladium intermediate. The palladium(II) intermediates detected by ESI-MS and the DFT calculations provide a detailed understanding of the catalytic cycle.

Pressurised hot water extraction in continuous flow mode for thermolabile compounds: extraction of polyphenols in red onions.

Extraction and analysis of labile compounds in complex sample matrices, such as plants, is often a big analytical challenge. In this work, the use of a "green and clean" pressurised hot water extraction (PHWE) approach performed in continuous flow mode is explored. Experimental data for extraction and degradation kinetics of selected compounds were utilised to develop a continuous flow extraction (CFE) method targeting thermolabile polyphenols in red onions, with detection by high-performance liquid chromatography (HPLC)-diode array detection (DAD)-mass spectrometry (MS). Water containing ethanol and formic acid was used as extraction solvent. Method performance was focused on extraction yield with minimal analyte degradation. By adjusting the flow rate of the extraction solvent, degradation effects were minimised, and complete extraction could be achieved within 60 min. The CFE extraction yields of the polyphenols investigated were 80-90 % of the theoretically calculated quantitative yields and were significantly higher than the yields obtained by conventional methanol extraction and static batch extraction (70-79 and 58-67 % of the theoretical yields, respectively). The precision of the developed method was lower than 8 % expressed as relative standard deviation.

Decarboxylative palladium(II)-catalyzed synthesis of aryl amidines from aryl carboxylic acids: development and mechanistic investigation.

A fast and convenient synthesis of aryl amidines starting from carboxylic acids and cyanamides is reported. The reaction was achieved by palladium(II)-catalysis in a one-step microwave protocol using [Pd(O2 CCF3 )2 ], 6-methyl-2,2'-bipyridyl and trifluoroacetic acid (TFA) in N-methylpyrrolidinone (NMP), providing the corresponding aryl amidines in moderate to excellent yields. The protocol is very robust with regards to the cyanamide coupling partner but requires electron-rich ortho-substituted aryl carboxylic acids. Mechanistic insight was provided by a DFT investigation and direct ESI-MS studies of the reaction. The results of the DFT study correlated well with the experimental findings and, together with the ESI-MS study, support the suggested mechanism. Furthermore, a scale-out (scale-up) was performed with a non-resonant microwave continuous-flow system, achieving a maximum throughput of 11 mmol h(-1) by using a glass reactor with an inner diameter of 3 mm at a flow rate of 1 mL min(-1) .

Extraction of antioxidants from spruce (Picea abies) bark using eco-friendly solvents.

INTRODUCTION: Antioxidants are known to avert oxidation processes and they are found in trees and other plant materials. Tree bark is a major waste product from paper pulp industries; hence it is worthwhile to develop an extraction technique to extract the antioxidants. OBJECTIVE: To develop a fast and environmentally sustainable extraction technique for the extraction of antioxidants from bark of spruce (Picea abies) and also to identify the extracted antioxidants that are abundant in spruce bark. METHODOLOGY: A screening experiment that involved three different techniques was conducted to determine the best technique to extract antioxidants. The antioxidant capacity of the extracts was determined with DPPH (2,2-diphenyl-1-picrylhydrazyl) assay. Pressurised fluid extraction (PFE) turned out to be the best technique and a response surface design was therefore utilised to optimise PFE. Furthermore, NMR and HPLC-DAD-MS/MS were applied to identify the extracted antioxidants. RESULTS: PFE using water and ethanol as solvent at 160 and 180°C, respectively, gave extracts of the highest antioxidant capacity. Stilbene glucosides such as isorhapontin, piceid and astringin were identified in the extracts. CONCLUSION: The study has shown that PFE is a fast and environmentally sustainable technique, using water and ethanol as solvent for the extraction of antioxidants from spruce bark.

Mineral Phosphorus Supply in Piglets Impacts the Microbial Composition and Phytate Utilization in the Large Intestine.

A sufficient supply of phosphorus (P) to pigs in livestock farming is based on the optimal use of plant-based phytate and mineral P supplements to ensure proper growth processes and bone stability. However, a high P supplementation might bear the risk of higher environmental burden due to the occurrence of excess P and phytate degradation products in manure. In this context, the intestinal microbiota is of central importance to increase P solubility, to employ non-mineral P by the enzymatic degradation of phytate, and to metabolize residual P. A feeding experiment was conducted in which piglets were fed diets with different P levels, resulting in three groups with low, medium (covering requirements), and high concentrations of available P. Samples from caecum and colon digesta were analysed for microbial composition and phytate breakdown to estimate the microbial contribution to metabolize P sources. In terms of identified operational taxonomic units (OTU), caecum and colon digesta under the three feeding schemes mainly overlap in their core microbiome. Nevertheless, different microbial families correlate with increased dietary P supply. Specifically, microbes of Desulfovibrionaceae, Pasteurellaceae, Anaerovoracaceae, and Methanobacteriaceae were found significantly differentially abundant in the large intestine across the dietary treatments. Moreover, members of the families Veillonellaceae, Selenomonadaceae, and Succinivibrionaceae might contribute to the observed phytate degradation in animals fed a low P diet. In this sense, the targeted manipulation of the intestinal microbiota by feeding measures offers possibilities for the optimization of intestinal phytate and P utilization.

Efforts to improve detection sensitivity for capillary electrophoresis coupled to atmospheric pressure photoionization mass spectrometry.

Electrospray ionization performs best with volatile buffers. However, generally the best separation performance for capillary electrophoresis (CE) is achieved with non-volatile buffers. Hyphenation of CE with mass spectrometry (MS) utilizing atmospheric pressure photoionization (APPI) enables use of a wider range of separation buffers without compromising detection sensitivity. As APPI is considered to be mass flow sensitive, the use of a larger inner diameter separation capillary (75 microm) allows larger volumes to be injected, without decreased separation performance, thus providing improved sensitivity (approx. a factor of 10), compared to the use of a 25 microm capillary. However, nebulizing gas flow and position of capillary tip in the sprayer have to be carefully optimized to prevent excessive band broadening. Further improvement in sensitivity (approx. a factor of 2) was obtained by decreasing the distance between the sprayer and ionization region, indicating that a specially designed CE/APPI-MS interface for low flow rates will be favourable.

Oxidation of 4-chloroaniline studied by on-line electrochemistry electrospray ionization mass spectrometry.

The oxidation of 4-chloroaniline (4-CA) has been studied by electrochemistry (EC) coupled on-line with electrospray ionization mass spectrometry (ESI-MS) using two electrochemical flow cells of different design. The experimental results, which generally verify previously suggested oxidation pathways for 4-CA, also indicate the presence of an up to now unrecognized comproportionation reaction. The oxidation of 4-CA (m/z 128.2) was found to give rise to the formation of both an oxidized dimer, 4-[(4-chlorophenyl)imino]-2,5-cyclohexadien-1-imine (m/z 217.2), and a reduced dimer, 4-amino-4'-chlorodiphenylamine (m/z 219.2), in addition to a dimer intermediate (m/z 253.2). The unexpected formation of the reduced dimer is shown to stem from a comproportionation reaction involving 4-CA and the oxidized dimer. The presence of the latter reaction was clearly seen by comparing results obtained with two thin-layer flow cells, both with conversion efficiencies of 50% under mass transport controlled conditions but of different design with respect to the influence of the counter electrode reaction on the reaction at the working electrode. The experimental results demonstrate that the formation of the reduced dimer is favored by a decrease in the local pH in the flow cell, and the influence of the pH on the oxidation of 4-CA was also investigated in the pH range between 2.0 and 6.0 using off-line voltammetry. It is concluded that EC/ESI-MS is a powerful tool for the study of the present type of reactions and that studies of the reaction pathways of these systems are best carried out under noncoulometric experimental conditions as the latter facilitates the detection of reaction intermediates. Comproportionation reactions, similar to the reaction present in the 4-CA system, can also be expected to be present during the formation of conducting polymers such as polyaniline and polypyrrole.

Identification and characterization of polyphenolic antioxidants using on-line liquid chromatography, electrochemistry, and electrospray ionization tandem mass spectrometry.

It is demonstrated that electrochemistry (EC) coupled to liquid chromatography (LC) and electrospray ionization tandem mass spectrometry (LC/EC/ESI-MS/MS) can be used to rapidly obtain information about the antioxidant activity (i.e., oxidation potential) and capacity (i.e., amount) of polyphenolic compounds, including catechin, kaempferol, resveratrol, quercetin, and quercetin glucosides. The described on-line LC/EC/ESI-MS/MS method facilitates the detection and characterization of individual antioxidants based on a combination of the obtained m/z values for the antioxidants and their oxidation products, the potential dependences for the ion intensities, and correlations between the retention times in the LC, EC, and MS chromatograms. As these results provide patterns that can be used in rapid screening for antioxidants in complex samples, the method should be a valuable complement to chemical assays commonly used to determine the total antioxidant capacity of samples. It is shown that the antioxidant capacity for a mixture of polyphenolic compounds depends on the redox potential employed in the evaluation, and this should consequently be taken into account when comparing results from different total antioxidant capacity assays. It is also demonstrated that the inherent antioxidant capacities of phenolic compounds increase with an increasing number of hydroxyl groups and that the potential needed to oxidize the remaining hydroxyl groups increases successively upon oxidation of the compound. Unlike chemical assays, which generally do not provide any information about the identities of the compounds on the molecular level, the present screening method can be used to identify individual antioxidants, rank compounds with respect to their ease of oxidation, and to study the antioxidant capacity at any redox potential of interest.

Synthesis of styrenes by palladium(II)-catalyzed vinylation of arylboronic acids and aryltrifluoroborates by using vinyl acetate.

One Heck of a reaction: Treatment of arylboronic acids or aryltrifluoroborates with vinyl acetate by using a palladium(II) catalyst gives the corresponding styrenes (see scheme). No palladium reoxidant is needed and the vinylation is performed under non-inert conditionsReactions of aromatic and heteroaromatic boronic acids or aryltrifluoroborate salts with vinyl acetate in the presence of a palladium(II) catalyst give the corresponding styrenes in good yields. This Heck reaction proceeds with microwave heating in less than 30 min at 140 degrees C in the absence of base and tolerates a variety of substituents. No palladium reoxidant is needed and the vinylation is performed under non-inert conditions. Mass spectrometry (electrospray ionization mass spectrometry (ESIMS) and tandem mass spectrometry (MS/MS)) was used to identify cationic palladium-containing complexes in ongoing reactions. The key intermediates that have been detected, together with experiments that used deuterated vinyl acetate, support the existence of catalytically active palladium hydride species, and that it is the arylation of ethylene, not vinyl acetate, which generates the styrene product. The mechanism of the reaction is discussed in terms of the palladium(II) intermediates mentioned above.

Microwave-promoted palladium(II)-catalyzed C-P bond formation by using arylboronic acids or aryltrifluoroborates.

The first Pd(II)-catalyzed P arylation has been performed by using palladium acetate, the rigid bidentate ligand dmphen (dmphen=2,9-dimethyl-1,10-phenanthroline), and without the addition of base or acid. Couplings of arylboronic acids or aryl trifluoroborates with H-phosphonate dialkyl esters were conducted in 30 min with controlled microwave (MW) heating under non-inert conditions. Aryl phosphites were also synthesized at room temperature with atmospheric air as the sole reoxidant. The arylated phosphonates were isolated in 44-90 % yields. The excellent chemoselectivity of the method was illustrated in the synthesis of a Mycobacterium tuberculosis glutamine synthetase (MTB-GS) inhibitor. Online ESIMS was used to detect cationic palladium species in ongoing reactions directly, and a catalytic cycle has been proposed based on these results.

Screening for organic phosphorus compounds in aquatic sediments by liquid chromatography coupled to ICP-AES and ESI-MS/MS.

The structures of organic phosphorous (P) compounds in aquatic sediments are to a large extent unknown although these compounds are considered to play an important role in regulating lake trophic status. To enhance identification of these compounds, a liquid chromatography (LC) method for their separation was developed. The stationary phase was porous graphitic carbon (PGC), and the mobile phases used in the gradient elution were compatible with both inductive coupled plasma atomic emission spectroscopy (ICP-AES) and electrospray ionization tandem mass spectrometry (ESI-MS/MS). With LC-ICP-AES, eight different P containing peaks could be observed in the P chromatogram indicating that at least eight different P compounds were separated. With the setup of an information dependent acquisition (IDA) with ESI-MS/MS, the mass over charge ( m/ z) of compounds containing a phosphate group (H 2PO 3 (-), m/ z 97) could be measured and further fragmentation experiments gave additional information on the structure of almost 40 separated P compounds, several were verified to be nucleotides. ICP-AES was very suitable in the development of the LC method and allowed screening and quantification of P compounds. The presented LC-ESI-MS/MS technique was able to identify several sediment organic P compounds.

Simultaneous analysis of five antidepressant drugs using direct injection of biofluids in a capillary restricted-access media-liquid chromatography-tandem mass spectrometry system.

Direct analysis, with minimal sample pretreatment, of antidepressant drugs, fluoxetine, imipramine, desipramine, amitriptyline, and nortriptyline in biofluids was developed with a total run time of 8 min. The setup consists of two HPLC pumps, injection valve, capillary RAM-ADS-C18 pre-column and a capillary analytical C18 column connected by means of a six-port valve in backflush mode. Detection was performed with ESI-MS/MS and only 1 microm of sample was injected. Validation was adequately carried out using FLU-d(5) as internal standard. Calibration curves were constructed under a linear range of 1-250 ng mL(-1) in plasma, being the limit of quantification (LOQ), determined as 1 ng mL(-1), for all the analytes. With the described approach it was possible to reach a quantified mass sensitivity of 0.3 pg for each analyte (equivalent to 1.1-1.3 fmol), translating to a lower sample consumption (in the order of 10(3) less sample than using conventional methods).

Characterisation of anthocyanins in red cabbage using high resolution liquid chromatography coupled with photodiode array detection and electrospray ionization-linear ion trap mass spectrometry.

The aim of this work was to analyse and tentatively identify anthocyanin species in red cabbage using HPLC/DAD-ESI/Qtrap MS. The extraction was realized by using a pressurized liquid technique and the separation of the pigments was achieved by a high resolution liquid chromatography system with a 1.8µm particles C-18 column. Photodiode array detection was employed to determine the UV/Vis spectral characteristic of the pigments. Electrospray ionization-linear ion trap mass spectrometry allowed the specific determination of the fragmentation patterns of the anthocyanins, by performing different ion scan modes. Twenty four anthocyanins were separated and identified, all having cyanidin as aglycon, represented as mono- and/or di-glycoside, and acylated, or not, with aromatic and aliphatic acids. Nine anthocyanins were identified for the first time in red cabbage.

Using standard additions to improve extraction and quantification of inositol hexakisphosphate in sediment samples by ion chromatography electrospray ionization mass spectrometry.

Several key aspects for the analysis of inositol hexakisphosphate (InsP6) have been investigated in order to establish a suitable method for the study of sediment samples from different aquatic systems. Apparent matrix effects for the ion chromatography electrospray ionization tandem mass spectrometric detection (IC-ESI-MS/MS) method were accounted for with a standard addition approach, which also compensated for variation in extraction efficiency. Several parameters of the extraction method were optimized to improve the extraction efficiency for different sediment types. We observed an improvement in the extraction efficiency between 18% and 720%. Finally, the method was used to gain first insights into the relevance of InsP6 in two aquatic systems located at the German Baltic coastal area. InsP6 was detected in several sediment samples with concentrations between 2.3 and 15.2 µg InsP6-P/g dry weight (DW).