Intestinal Absorption Study of a Granular Form of Ferric Pyrophosphate.

Iron deficiency is one of the most prevalent nutritional disorders worldwide. The standard treatment involves iron supplementation, but this task is challenging because of poor solubility and organoleptic issues. Moreover, the need to increase iron bioavailability represents a challenge for treating iron-related disorders. In this study, gastroresistance and iron intestinal absorption of an innovative granular formulation composed of ferric pyrophosphate, modified starch and phospholipids branded as Ferro Fosfosoma® was investigated. Gastroresistant properties were studied using standard protocols, and a bioaccessible fraction was obtained by exposing a food supplement to in vitro digestion. This fraction was used for investigating iron absorption in Caco-2 and human follicle-associated intestinal epithelium (FAE) models. Ferro Fosfosoma® showed an improved resistance to gastric digestion and higher intestinal absorption than ferric pyrophosphate salt used as a control in both models. In the FAE model, Ferro Fosfosoma® induces larger iron absorption than in the Caco-2 monolayer, most likely due to the transcytosis ability of M cells. The larger iron absorption in the Ferro Fosfosoma®-treated FAE model corresponds to higher ferritin level, proving physiological iron handling that was once delivered by granular formulation. Finally, the formulation did not induce any alterations in viability and barrier integrity. To conclude, Ferro Fosfosoma® favors iron absorption and ferritin expression, while preserving any adverse effects.

New insights in the slow ligand exchange reaction between Cr(III)-EDTA and Fe(III), and direct analysis of free and complexed EDTA in tannery wastewaters by liquid chromatography - Tandem mass spectrometry.

EDTA and soluble Cr(III) are usually both present in wastewaters coming from treatment plants handling tannery effluents. A well-established method to determine EDTA is based on the conversion of free and complexed EDTA into its Fe(III) complex. This procedure gives inconsistent data when Cr(III)-EDTA is present. This fact was here demonstrated by studying the kinetics of the exchange reaction between Fe(III) and Cr(III)-EDTA at 90 °C and various pH values, from acidic to neutral. The reaction is very slow (several weeks); the slow kinetics of conversion of Cr(III)-EDTA to Fe(III)-EDTA is even more accentuated at room temperature and the low concentrations of reactants in wastewaters. The presence of EDTA complexes of Fe(III) and Cr(III) was demonstrated in industrial effluents and wastewaters by developing a selective method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS), which was able to detect free and complexed EDTA at concentration levels <1 µM. A systematic underestimation of the EDTA expressed as Fe(III) complex was demonstrated in samples containing Cr(III)-EDTA. Cr(III)-EDTA was identified for the first time as a component of wastewater samples at a concentration level of about 2 µM and turned out to be an inert species that significantly contributes to the final soluble Cr amount. This study gives new insights into the inertness of Cr(III) toward metal exchange equilibria of EDTA complexes, resolves a bias in the analysis of total EDTA in samples containing Cr(III)-EDTA, allowing the direct determination of free and complexed EDTA by LC-MS.

Biocontrol traits of Bacillus licheniformis GL174, a culturable endophyte of Vitis vinifera cv. Glera.

BACKGROUND: Bacillus licheniformis GL174 is a culturable endophytic strain isolated from Vitis vinifera cultivar Glera, the grapevine mainly cultivated for the Prosecco wine production. This strain was previously demonstrated to possess some specific plant growth promoting traits but its endophytic attitude and its role in biocontrol was only partially explored. In this study, the potential biocontrol action of the strain was investigated in vitro and in vivo and, by genome sequence analyses, putative functions involved in biocontrol and plant-bacteria interaction were assessed. RESULTS: Firstly, to confirm the endophytic behavior of the strain, its ability to colonize grapevine tissues was demonstrated and its biocontrol properties were analyzed. Antagonism test results showed that the strain could reduce and inhibit the mycelium growth of diverse plant pathogens in vitro and in vivo. The strain was demonstrated to produce different molecules of the lipopeptide class; moreover, its genome was sequenced, and analysis of the sequences revealed the presence of many protein-coding genes involved in the biocontrol process, such as transporters, plant-cell lytic enzymes, siderophores and other secondary metabolites. CONCLUSIONS: This step-by-step analysis shows that Bacillus licheniformis GL174 may be a good biocontrol agent candidate, and describes some distinguished traits and possible key elements involved in this process. The use of this strain could potentially help grapevine plants to cope with pathogen attacks and reduce the amount of chemicals used in the vineyard.

Liquid chromatography-high resolution mass spectrometric methods for the surveillance monitoring of cyanotoxins in freshwaters.

A comprehensive risk management on human exposure to cyanotoxins, whose production is actually unpredictable, is limited by reliable analytical tools for monitoring as many toxic algal metabolites as possible. Two analytical approaches based on a LC-QTOF system for target analysis and suspect screening of cyanotoxins in freshwater were presented. A database with 369 compounds belonging to cyanobacterial metabolites was developed and used for a retrospective data analysis based on high resolution mass spectrometry (HRMS). HRMS fragmentation of the suspect cyanotoxin precursor ions was subsequently performed for correctly identifying the specific variants. Alternatively, an automatic tandem HRMS analysis tailored for cyanotoxins was performed in a single chromatographic run, using the developed database as a preferred precursor ions list. Twenty-five extracts of surface and drinking waters contaminated by cyanobacteria were processed. The identification of seven uncommon microcystins (M(O)R, MC-FR, MSer7-YR, D-Asp3MSer7-LR, MSer7-LR, dmAdda-LR and dmAdda-YR) and 6 anabaenopeptins (A, B, F, MM850, MM864, oscyllamide Y) was reported. Several isobaric variants, fully separated by chromatography, were pointed out. The developed methods are proposed to be used by environmental and health agencies for strengthening the surveillance monitoring of cyanotoxins in water.

Characterization of lipopeptides produced by Bacillus licheniformis using liquid chromatography with accurate tandem mass spectrometry.

RATIONALE: The plant endophyte Bacillus licheniformis, isolated from leaves of Vitis vinifera, was studied to individuate and characterize the presence of bioactive lipopeptides having amino acidic structures. METHODS: Crude extracts of liquid cultures were analyzed by ultra-high-performance liquid chromatography (UHPLC) coupled to a quadrupole time-of-flight (QTOF) mass analyzer. Chromatographic conditions were optimized in order to obtain an efficient separation of the different isobaric lipopeptides, avoiding merged fragmentations of co-eluted isomeric compounds and reducing possible cross-talk phenomena. Composition of the amino acids was outlined through the interpretation of the fragmentation behavior in tandem high-resolution mass spectrometry (HRMS/MS) mode, which showed both common-class and peculiar fragment ions. Both [M + H](+) and [M + Na](+) precursor ions were fragmented in order to differentiate some isobaric amino acids, i.e. Leu/Ile. Neutral losses characteristic of the iso acyl chain were also evidenced. RESULTS: More than 90 compounds belonging to the classes of surfactins and lichenysins, known as biosurfactant molecules, were detected. Sequential LC/HRMS/MS analysis was used to identify linear and cyclic lipopeptides, and to single out the presence of a large number of isomers not previously reported. Some critical issues related to the simultaneous selection of different compounds by the quadrupole filter were highlighted and partially solved, leading to tentative assignments of several structures. Linear lichenysins are described here for the first time. CONCLUSIONS: The approach was proved to be useful for the characterization of non-target lipopeptides, and proposes a rationale MS experimental scheme aimed to investigate the difference in amino acid sequence and/or in the acyl chain of the various congeners, when standards are not available. Results expanded the knowledge about production of linear and cyclic bioactive compounds from Bacillus licheniformis, clarifying the structures of isomeric forms, and enabling the use of selected endophytes to produce fungicides for eco-friendly biocontrol. Copyright © 2016 John Wiley & Sons, Ltd.

Characterization and quantification of N-(3-aminopropyl)-N-dodecyl-1,3-propanediamine biocide by NMR, HPLC/MS and titration techniques.

The present paper reports the determination of the tri-amine N-(3-aminopropyl)-N-dodecyl-1,3-propanediamine (TA) present in a raw material called LONZABAC used to formulate various, widely used commercial biocides. The active principle, TA, is present in LONZABAC together with other molecules at lower concentration levels. Three independent analytical approaches, namely solution NMR spectroscopy, liquid chromatography coupled to high resolution mass spectrometry (LC/HRMS) and acid-base titration in mixed solvent, were used to overcome the problem of the non-availability of the active principle as high purity standard. NMR analysis of raw material, using a suitable internal standard, evidenced in all analyzed lots the presence of the active principle, the N-dodecyl-1,3-propanediamine (DA) and the n-dodecylamine (MA) and the absence of non-organic, NMR-inactive species. NMR peak integration led to a rough composition of the MA:DA:TA as 1:9:90. The LC/HRMS analysis allowed the accurate determination of DA and MA and confirmed in all samples the presence of the TA, which was estimated by difference: MA=1.4±0.3%, DA=11.1±0.7%, TA=87.5±1.3%. The obtained results were used to setup an easy, rapid and cheap acid-base titration method able to furnish a sufficiently accurate evaluation of the active principle both in the raw material and in diluted commercial products. For the raw material the results were: TA+MA=91.1±0.8% and DA-MA=8.9±0.8%, statistically coherent with LC/MS ones. The LC/MS approach demonstrated also its great potentialities to recognize trace of the biocide components both in environmental samples and in the formulated commercial products.

Degradation products from naturally aged paper leaves of a 16th-century-printed book: a spectrochemical study.

In this work, we present a wide-range spectrochemical analysis of the degradation products from naturally aged paper. The samples obtained from wash waters used during the de-acidification treatment of leaves from a 16th-century-printed book were analysed through NMR, IR, Raman UV/Vis, EPR and X-ray fluorescence (XRF) spectroscopy and HPLC-MS and inductively coupled plasma (ICP) analysis. By these methods we also studied some of the previous samples treated by acidification (sample AP) and catalytic hydrogenation (sample HP). Crossing all the data, we obtained precise indications about the main functional groups occurring on the degraded, water-soluble cellulose oligomers. These results point out that the chromophores responsible for browning are conjugated carbonyl and carboxyl compounds. As a whole, we show that the analysis of wash waters, used in the usual conservation treatments of paper de-acidification, gives much valuable information about both the conservation state of the book and the degradation reactions occurring on the leaves, due to the huge amount of cellulose by-products contained in the samples. We propose therefore this procedure as a new very convenient general method to obtain precious and normally unavailable information on the cellulose degradation by-products from naturally aged paper.

Application of LC-MS and LC-MS-MS to the analysis of photo-decomposed crystal violet in the investigation of cultural heritage materials aging.

In this work, the accurate liquid chromatography-ultraviolet-visible (LC-UV-Vis), LC-mass spectrometry (MS) and LC-MS-MS analysis of the photo-degradation products of crystal violet (CV) is reported. CV is a light fugitive early synthetic dye which had a widespread diffusion into the market starting from the end of the XIX century and was used among others by V. Van Gogh and P. Gauguin in their writings, drawings or paintings. On-line photodiode array detector enabled simultaneous UV-Vis spectra acquisition. Many degradation compounds were identified through their exact mass (2 ppm accuracy) and MS-MS technique. In particular, all CV demethylated products, demethylated Michler's ketone and particularly some compounds that most likely contain oxygen, such as N-oxides, were found. Fragmentation products are all justified by the proposed fragmentation scheme, in term of precursor exact mass and isotopic profile, characteristic losses in fragmentation and rebuilt structure formula. In particular, we hypothesized the presence of N-imido oxides and hydroxylamine derivates, never reported before, together with the demethylated derivatives of the studied dyes. All these compounds, although at trace level in our samples, contribute to the discoloration and fading of works of arts made with CV. In particular, demethylation of CV by UV light leads to formation of compounds absorbing at shorter wavelengths than CV (blue shift) or no-absorbing in visible range (yellow-colourless) with an overall effect that may appear reddish-brown. This phenomenon justifies drawings appearing grey or brown on aged yellowed paper, when CV-based inks or paints were used. The final aim was to better characterize the photo-degradation of early synthetic dyes (in particular of CV) and to gain a better insight into the discoloration and fading of purple ink strokes made of CV.

The mystery of the discolored flints. New molecules turn prehistoric lithic artifacts blue.

Prehistoric artifacts turning blue in the store rooms of the Natural History Museum in Verona, Italy recently raised serious issues for heritage materials conservation. Our analytical investigation showed that the unusual discoloration process of the flint tools is caused by the surface presence of at least three previously unknown pigmenting molecules of the triphenylmetane dyes class: 6-(bis(2,2,4-trimethyl-1,2-dihydroquinolin-6-yl)methylene)-2,2,4-trimethyl-2,6-dihydroquinolinium and its hydrogenated derivatives 2,2,4-trimethyl-6-((2,2,4-trimethyl-1,2,3,4-tetrahydroquinolin-6-yl)(2,2,4-trimethyl-1,2-dihydroquinolin-6-yl)methylene)-2,6-dihydroquinolinium and 6-(bis(2,2,4-trimethyl-1,2,3,4-tetrahydroquinolin-6-yl)methylene)-2,2,4-trimethyl-2,6-dihydroquinolinium. The peculiar formation of the molecules is possibly catalyzed within the silica pore surface starting from a well-known rubber stabilizer 2,2,4-trimethyl-1,2-dihydroquinoline released by the plastic pads flooring the storing cabinets. The investigated reaction and its surprising blue product represent a case study of the application of modern materials science to conservation and a serious warning towards the unpredictable challenges faced in the preservation of our cultural heritage.

Helicobacter pylori acidic stress response factor HP1286 is a YceI homolog with new binding specificity.

HP1286 from Helicobacter pylori is among the proteins that play a relevant role in bacterial colonization and persistence in the stomach. Indeed, it was demonstrated to be overexpressed under acidic stress conditions, together with other essential virulence factors. Here we describe its crystal structure, determined at 2.1 A resolution. The molecular model, a dimer characterized by two-fold symmetry, shows that HP1286 structurally belongs to the YceI-like protein family, which in turn is characterized by the lipocalin fold. The latter characterizes proteins possessing an internal cavity with the function of binding and/or transport of amphiphilic molecules. Surprisingly, a molecule of erucamide was found bound in the internal cavity of each monomer of recombinant HP1286, cloned and expressed in an Escherichia coli heterologous system. The shape and length of the cavity indicate that, at variance with other members of the family, HP-YceI has a binding specificity for amphiphilic compounds with a linear chain of about 22 carbon atoms. These features, along with the fact that the protein is secreted by the bacterium and is involved in adaptation to an acidic environment, suggest that its function could be that of sequestering specific fatty acids or amides from the environment, either to supply the bacterium with the fatty acids necessary for its metabolism, or to protect and detoxify it from the detergent-like antimicrobial activity of fatty acids that are eventually present in the external milieu.

Quantitative determination of chlorophenols in leather by pressurized liquid extraction and liquid chromatography with diode-array detection.

Pressurized liquid extraction (PLE) with acetonitrile was used for the recovery of chlorophenols (4-chloro-3-methylphenol, 4-chloro-2-methylphenol, 2,4-dichlorophenol, 2-phenylphenol, 2,4,6-trichlorophenol, 2,3,4,6-tetrachlorophenol and pentachlorophenol) present as biocides in leather. After a single cycle PLE treatment, solutions underwent pre-concentration by evaporation of the solvent under vacuum and clean-up treatment with solid-phase extraction cartridges. Quantitative analysis of the target compounds was carried out by liquid chromatography with gradient elution and UV spectrophotometric detection at variable wavelength for the various analytes in the range 190-240 nm. Instrumental detection limits and operative detection limits in the real matrices were determined according to the Hubaux-Vos approach and to the US Environmental Protection Agency procedures. The detection limits for the seven analytes ranged from 10 to 70 microg kg(-1). Linearity was very good in the explored range (10(-7)-10(-5)M) giving R(2) values from 0.995 to 1.000 for pentachlorophenol and 2,4-dichlorophenol, respectively. Repeatability was satisfactory, 2-5% for a 1 x 10(-6)M level of concentration, on five repeated measurements on the sample. Recovery yield values with the proposed procedure were determined using spiked samples. Overall recovery ranged from 88 to 97%. The method was used for routine analysis of real leather samples.

Effect of eluent composition and pH and chemiluminescent reagent pH on ion chromatographic selectivity and luminol-based chemiluminescence detection of Co(2+), Mn(2+) and Fe(2+) at trace levels.

The alkaline-luminol/H(2)O(2)-based chemiluminescent (CL) detection of Fe(2+), Co(2+), and Mn(2+), separated with a Dionex CS5A ion chromatographic phase was studied by means of a multi-pump flow system allowing the variation of the post-column solution composition. A perchlorate gradient at pH 1.9 (with HCl) was used to separate cations partially complexed with 5.6mM oxalate present in the eluent and necessary for the chosen separation phase. A 0.91mM luminol, 3.3mM H(2)O(2) in 0.25M carbonate buffer at pH 10.5 composition was chosen as CL reagent solution. The chosen pH value warrants signal repeatability and wider linearity range although absolute signal is not maximum. The CL signal was related to the pH of the two post-column mixing solutions. Calibration plots of Co(2+) and Fe(2+) were linear in the chosen concentration range whilst a parabolic model was the best fit for Mn(2+). Detection limits were 0.24, 0.50 and 375nM for Co(2+), Fe(2+) and Mn(2+), respectively. The method was used to determine Co(2+) at trace level in commercial copper chelates used for animal feeding. A comparison with a chromatographic method with spectrophotometric detection was made giving results comparable both in absolute values and accuracy.

A pH-stat study of the reaction of some transition metal cations with disodium ethylenedinitrilotetraacetate (EDTA) and its analytical application.

The pH-stat titration technique is an autonomous and very powerful tool for performing and monitoring chelatometric titrations of metal cations with great accuracy, poorly known, however, and seldom exploited. Based on measurement of the amount of strong base required to keep the pH of the test solution at a selected value during stepwise known additions of ethylenedinitrilotetraacetate (EDTA), it requires a glass electrode as the only sensor and is easily implemented on potentiometric titrators. It was introduced a quarter of century ago on an empirical basis for a very peculiar purpose (determination of calcium in diary products), but only very recently it was generalised and its fundamentals were thoroughly examined. In this work, pH-static titrations of some transition metal cations of analytical relevance (Co(2+), Cu(2+), Mn(2+), Zn(2+)) were thoroughly investigated in the acid pH range between 2.3 and 5 or 7 (the highest pH depending on the metal hydroxide or carbonate solubility). The results at higher acidity showed unsuspected properties of such chelation reactions. At moderately acid pH (generally >/=4), indeed, pH-static titrations yield results of high precision and accuracy. On decreasing pH, however, the reaction stoichiomety deviates more and more from the 1:1 ratio between chelating agent and cation, seemingly because of formation of binuclear complexes, an occurrence very seldom mentioned in the current literature. The optimal titration conditions for each metal are defined, and directions for establishing a laboratory protocol for quantitative determinations are given.

Determination of biogenic amines in chocolate by ion chromatographic separation and pulsed integrated amperometric detection with implemented wave-form at Au disposable electrode.

A rapid and selective cation exchange chromatographic method coupled to integrated pulsed amperometric detection (PAD) has been developed to quantify biogenic amines in chocolate. The method is based on gradient elution of aqueous methanesulfonic acid with post column addition of strong base to obtain suitable conditions for amperometric detection. A potential waveform able to keep long time performance of the Au disposable electrode was set up. Total analysis time is less than 20min. Concentration levels of dopamine, serotonin, tyramine, histamine and 2-phenylethylamine were measured, after extraction with perchloric acid from 2g samples previously defatted twice with petroleum ether. The method was used to determine the analytes in chocolate real matrices and their quantification was made with standard addition method. Only dopamine, histamine and serotonin were found in the analysed real samples. Repeatabilities of their signals, computed on their amounts in the real samples, were 5% for all of them. Repeatabilities of tyramine and phenethylamine were relative to standard additions to real samples (close to 1mg/l in the extract) and were 7 and 3%, respectively. Detection limits were computed with the 3s of the baseline noise combined with the calibration plot regression parameters. They were satisfactorily low for all amines: 3mg/kg for dopamine, 2mg/kg for tyramine, 1mg/kg for histamine, 2mg/kg for serotonin, 3mg/kg for 2-phenylethylamine.

Headspace solid phase micro extraction GC-ECD determination of volatile organic chlorinated hydrocarbons in soils.

Soil samples were suspended in a suitable aqueous solvent and a solid phase microextraction (SPME) fibre was used to sample the headspace (HS) for five volatile chlorinated compounds (VOX). Their determination was made by GC-ECD technique in the splitless mode. Preliminary studies on the effects of methanol and of the sand/clay ratio on the fibre extraction were made. Four experimental factors, namely, extraction time, extraction temperature, pH and NaCl%, able to affect distribution of the analytes among the four different phases, were varied in suitable ranges. A multivariate approach applied to the face centred cube (FCC) experimental design, was used to try to optimise the overall sample response. The suitable set of factors found for the determination of chloroform, 1,2-dichloroethane, trichloroethylene, 1,1 ,2-trichloroethane, 1,1,2,2-tetrachloroethane, was a compromise among the relevant optimal factor sets of the single analytes. Detection limits of 0.003 ng, 0.022 ng, 0.001 ng, 0.015 ng and 0.002 ng were found respectively for the five cited analytes. The method was successfully used to determine the analyte contents in two real soils sampled in an industrial area.

Non-linear and non-constant variance calibration curves in analysis of volatile organic compounds for testing of water by the purge-and-trap method coupled with gas chromatography/mass spectrometry.

Currently used operating conditions for analysing volatile organic compounds (VOCs) by purge-and-trap gas chromatography/mass spectrometry (GC/MS) produced non-linear calibration curves with non-uniform variance. Second-order polynomial models therefore had to be used in weighted regression analysis of measurements of replicates spiked at various concentrations. A transparent procedure based on a reported method of very low computational complexity allowed calculation of the parameters of second-order models, confidence bands of regression lines, prediction bands, and confidence intervals of discriminated analyte concentrations. Tolerance intervals were introduced for this last purpose. Critical, detection and quantification levels drawn from the calibration curves were compared with those calculated by the EPA method.

Evidence of Cr(VI) formation during analysis of leather Proposal of an alternative method of analysis through the ion-chromatographic approach and post-column reaction.

The formation of Cr(VI) in Cr(III) tanned leather, in neutral and alkaline solution, has been demonstrated by means of crossed experiments using different pH buffers, ethylenediaminetetraacetic acid as Cr(III) complexing agent and NaCl solutions. According to the found results the composition of the extracting solution suitable to extract Cr(VI) amount present in leather was pH 4.4 (which is also the tanned leather natural pH) and 5% NaCl (w/v). Interferences coming from coloured compounds have been eliminated with suitable SPE cartridges. A new protocol for the analysis of Cr(VI) based on ion chromatography and a diphenylcarbazide post-column reaction has been implemented. The use of a large volume injection loop (500mul) allowed to obtain a very low quantification limit (0.15mgkg(-1)) despite the low amount of leather extracted (0.2g with respect to 2.0g used by the IUC 18 official method). Evidence of the transient nature of Cr(VI) in leather requires using the external calibration procedure for the correct quantification of the species.

Kinetic considerations on the electrogenerated luminescence of luminol at platinum electrode in the presence of hydrogen peroxide and oxygen.

The ECL behavior of the luminol/H2O2 and luminol/O2 systems was evaluated at Pt electrode by using different electroanalytical techniques such as chronoamperometry, cyclic and rotating disk electrode (RDE) voltammetry. Diffusive and kinetic parameters such as the diffusion coefficient of luminol, D, the number of exchanged electrons, n, and the apparent heterogeneous rate constant, kap, were determined in the maximum light emission conditions achieved at pH 11, at an electrode potential of 750 mV vs. SCE. The experimental order of reaction were determined from the relation between the reactant concentrations and the emitted light intensity.