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.

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.

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).

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.

Separation of inositol phosphate isomers in environmental samples by ion-exchange chromatography coupled with electrospray ionization tandem mass spectrometry.

A method for isomeric separation of inositol phosphates (InsPn) in environmental samples originating from different sources such as soil, manure/compost, and aquatic sediments has been developed. The method includes a single NaOH-EDTA extraction step, centrifugation and direct injection of a particle free solution into an ion chromatographic column. Isomeric liquid chromatographic separation was achieved with an ammonium carbonate gradient compatible with electrospray ionization tandem mass spectrometric detection (LC-ESI-MS/MS). The detection limits of the LC-ESI-MS/MS method were between 0.03-0.16µM for the different InsPn, corresponding to 37-99ng P/g sample DW. The method has shown to be able to analyze more than 200 samples from soil, manure and sediment without any severe matrix effects. This will allow future studies of the fate of inositol phosphates in the environment.

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.

One-pot synthesis of keto thioethers by palladium/gold-catalyzed click and pinacol reactions.

An atom-efficient synthesis of keto thioethers was devised via tandem gold/palladium catalysis. The reaction proceeds through a regioselective thiol attack at the ß-position of the alcohol, followed by an alkyl, aryl, or benzyl 1,2-shift. Both acyclic and cyclic systems were studied, in the latter case leading to the ring expansion of cyclic substrates.

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.

Strategies for differentiation of isobaric flavonoids using liquid chromatography coupled to electrospray ionization mass spectrometry.

Flavonoids are a class of secondary plant metabolites existing in great variety in nature. Due to this variety, identification can be difficult, especially as overlapping compounds in both chromatographic separations and mass spectrometric detection are common. Methods for distinguishing isobaric flavonoids using MS(2) and MS(3) have been developed. Chromatographic separation of various plant extracts was done with RP-HPLC and detected with positive ESI-MS operated in information-dependent acquisition (IDA) mode. Two methods for the determination of flavonoid identity and substitution pattern, both featuring IDA criteria, were used together with the HPLC equipment. A third method where the collision energy was ramped utilized direct infusion. With the developed strategies, it is possible to differentiate between many isobaric flavonoids. Various classes of flavonoids were found in all of the plant extracts, in the red onion extract 45 components were detected and for 29 of them the aglycone was characterized, while the substituents were tentatively identified for 31 of them. For the strawberry extract, those numbers were 66, 30 and 60, and for the cherry extract 99, 56 and 71. The great variety of flavonoids, several of them isobaric, found in each of the extracts highlights the need for reliable methods for flavonoid characterization. Methods capable of differentiating between most of the isobars analyzed have been developed.

Equilibrium Study of Pd(dba)2 and P(OPh)3 in the Pd-Catalyzed Allylation of Aniline by Allyl Alcohol.

Reaction of Pd(dba)2 and P(OPh)3 shows a unique equilibrium where the Pd[P(OPh)3]3 complex is favored over both Pd(dba)[P(OPh)3]2 and Pd[P(OPh)3]4 complexes at room temperature. At a lower temperature, Pd[P(OPh)3]4 becomes the most abundant complex in solution. X-ray studies of Pd[P(OPh)3]3 and Pd(dba)[P(OPh)3]2 complexes show that both complexes have a trigonal geometry with a Pd-P distance of 2.25 Å due to the π-acidity of the phosphite ligand. In solution, pure Pd(dba)[P(OPh)3]2 complex equilibrates to the favored Pd[P(OPh)3]3 complex, which is the most stable complex of those studied, and also forms the most active catalytic species. This catalyst precursor dissociates one ligand to give the reactive Pd[P(OPh)3]2, which performs an oxidative addition of nonmanipulated allyl alcohol to generate the π-allyl-Pd[P(OPh)3]2 intermediate according to ESI-MS studies.

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.

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.

Extraction and quantification of phosphorus derived from DNA and lipids in environmental samples.

Understanding the flux and turnover of phosphorus (P) in the environment is important due to the key role P plays in eutrophication and in the ambition to find cost-effective measures to mitigate it. Orthophosphate diesters, including DNA and phospholipids (PLs), represent a potentially degradable P pool that could support future primary production and eutrophication. In this study, extraction techniques were optimized and combined with colorimetric determination of extracted P to provide a selective quantification method for DNA-P and PL-P in agricultural soil, sediment and composted manure. The proposed method is rapid and reproducible with an RSD of <10%. Recovery, evaluated by spiking the sample matrices with DNA and PL standards, was over 95% for both DNA and PLs. The method can be used for the determination of the pool size of the two organic P fractions. Results show that DNA-P comprises 3.0% by weight of the total P (TP) content in the studied soil, 10.4% in the sediment and 8.4% in the compost samples. The values for PL-P are 0.5%, 6.0% and 1.7% for soil, sediment and compost, respectively.

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) .

Degradation effects in the extraction of antioxidants from birch bark using water at elevated temperature and pressure.

Experiments with birch bark samples have been carried to enable a distinction between extraction and degradation effects during pressurised hot water extraction. Two samples, E80 and E180, contained birch bark extracts obtained after extraction at 80 and 180°C for up to 45 min, respectively. Two other samples, P80 and P180, were only extracted for 5 min at the two temperatures and were thereafter filtered and hydrothermally treated at 80 and 180°C, respectively. During the latter treatment, samples were collected at different times to assess the stability of the extracted compounds. An offline DPPH (2,2-diphenyl-1-picrylhydrazyl) assay, as well as a high performance liquid chromatographic separation coupled to an electrochemical detector, were used to determine the antioxidant capacity of the processed samples. The results obtained with the different techniques were compared to assess the yield of the extraction and degradation processes. In addition, an online hyphenated system comprising high performance liquid chromatography coupled to diode-array; electrochemical; and tandem mass spectrometric detection (HPLC-DAD-ECD-MS/MS) was used to study the compositions of the extracts in more detail. The results for the samples processed at 80°C showed that the extraction reached a steady-state already after 5 min, and that the extracted compounds were stable throughout the entire extraction process. Processing at 180°C, on the other hand, gave rise to partly degraded extracts with a multitude of peaks in both the diode array and electrochemical detectors, and a higher antioxidant capacity compared to for the extracts obtained at 80°C. It is concluded that HPLC-DAD-ECD is a more appropriate technique for the determination of antioxidants than the DPPH assay. The mass spectrometric results indicate that one of the extracted antioxidants, catechin, was isomerised to its diastereoisomers; (+)-catechin, (-)-catechin, (+)-epicatechin, and (-)-epicatechin.

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.

Anthocyanin characterization utilizing liquid chromatography combined with advanced mass spectrometric detection.

Anthocyanins are naturally occurring polyphenolic plant pigments. To analyze the anthocyanin content of samples, rapid and reliable methods for separation and detection are needed. In this work an LC-DAD-ESI-MS/MS instrument was used to develop a new tandem MS data acquisition strategy for anthocyanin characterization which was subsequently compared to more conventional measurements. It has been shown that the newly developed strategy, multiple reaction monitoring-initiated anthocyanin characterization (MIAC), can successfully be used in anthocyanin analysis and has various advantages compared to some more traditional measurements, such as enhanced selectivity, better signal-to-noise ratio and simplified data evaluation. Furthermore, the number of relevant MS/MS data increased significantly with the MIAC method compared with a more common information dependent MS experiment strategy.