Mass spectrometric characterization of two novel inflammatory peptides from the venom of the social wasp Polybia paulista

The social wasp P. paulista is relatively common in southeast Brazil causing many medically important stinging incidents. The seriousness of these incidents is dependent on the amount of venom inoculated by the wasps into the victims, and the characteristic envenomation symptoms are strongly dependent on the types of peptides present in the venom. In order to identify some of these naturally occurring peptides available in very low amounts, an analytical protocol was developed that uses a combination of reversed-phase and normal-phase high-performance liquid chromatography (HPLC) of wasp venom for peptide purification, with matrix-assisted laser desorption/ionization time-of-flight post-source decay mass spectrometry (MALDI-Tof-PSD-MS) and low-energy collision-induced dissociation (CID) in a quadrupole time-of-flight tandem mass spectrometry (QTof-MS/MS) instrument for peptide sequencing at the sub-picomole level. The distinction between Leu and Ile was achieved both by observing d-type fragment ions obtained under CID conditions and by comparison of retention times of the natural peptides and their synthetic counterparts (with different combinations of I and/or L at N- and C-terminal positions). To distinguish the isobaric residues K and Q, acetylation of peptides was followed by Q-Tof-MS analysis. The primary sequences obtained were INWLKLGKMVIDAL-NH(2) (MW 1611.98 Da) and IDWLKLGKMVMDVL-NH(2) (MW 1658.98 Da). Micro-scale bioassay protocols characterized both peptides as presenting potent hemolytic action, mast cell degranulation, and chemotaxis of polymorphonucleated leukocyte (PMNL) cells. The primary sequences and the bioassay results suggest that these toxins constitute members of a new sub-class of mastoparan toxins, directly involved in the occurrence of inflammatory processes after wasp stinging.     

Kinetics and Mechanism of Ni(II) Chelation in Model and Real Solutions of Xylem Sap of Quercus ilex

The kinetics of formation and dissociation of Ni(II) complexes with oxalic and citric acids was studied by cyclic voltammetry in model solutions of xylem sap of Q. ilex (the dominant tree growing on serpentine soils of Northeast Portugal) using representative concentrations, pH and ionic strength. The role of magnesium on complex formation was analyzed from solutions where Mg is present at concentration levels found in the xylem sap of Q. ilex growing on both nonserpentine and serpentine soils. Kinetics studies were also done in diluted solutions of real xylem sap samples, spiked with increasing amounts of magnesium. The values obtained for the apparent rate constants were those anticipated by the proposed model. To test the validity of the methodology and mechanisms, formation rate constants, k_f (M^?1 s^?1) of Ni(II) complexes with citrate and oxalate were evaluated that compare with the values from Eigen mechanism.     

Study of the diclofenac/phospholipid interactions with liposomes and monolayers

The interaction of diclofenac sodium (SD) with soya phosphatidylcholine (SPC) has been studied with floating Langmuir monolayers and liposomes. SD was either introduced into the subphase of SPC monolayers or co-spread with SPC on an aqueous subphase. In both cases, SD caused the surface pressure isotherm to become more expanded, thus demonstrating the affinity between SD and SPC. The incorporation of SD caused SPC liposomes to have a decreased diameter according to light scattering experiments. When SPC liposomes were injected into an aqueous subphase, their destruction yielding surface-active monomers could be monitored by changes in surface pressure. SD-loaded liposomes displayed a much faster kinetics when the surface density of surface-active monomers was plotted against time, with rate constants increasing significantly with the SD concentration. The kinetic profile can be quantitatively analyzed by plotting ln[1 - (gamma/gamma infinity)] versus t1/2.     

A Layer‐by‐Layer Film of Chitosan in a Taste Sensor Application

Chitosan is alternated with sulfonated polystyrene (PSS) to build layer‐by‐layer (LBL) films that are used as sensing units in an electronic tongue. Using impedance spectroscopy as the principle method of detection, an array using chitosan/PSS LBL film and a bare gold electrode as the sensing units was capable of distinguishing the basic tastes – salty, sweet, bitter, and sour – to a concentration below the human threshold. The suitability of chitosan as a sensing material was confirmed by using this sensor to distinguish red wines according to their vintage, vineyard, and brands.

PCA Plot for red wine samples obtained from impedance measurements at 1 kHz for the sensor array comprising a 3‐bilayer chitosan/PSS film and a bare gold electrode.     

Determination of manganese and zinc in powdered chocolate samples by slurry sampling using sequential multi-element flame atomic absorption spectrometry

In the present work, a slurry sampling flame atomic absorption spectrometric method to determine directly manganese and zinc in powdered chocolate samples is proposed. The optimization step was performed using univariate methodology involving the following factors: nature and concentration of the acid solution, sonication time, and particle size. The established conditions led to the use of a sample mass of 150 mg, 2.0 mol L^− 1 nitric acid solution, sonication time of 15 min, and a slurry volume of 50 mL. This method allows the determination of manganese and zinc with detection limit of 52 and 61 ng g^− 1, respectively, and a precision expressed as relative standard deviation (RSD) of 2.6% and 3.2% (both, n = 10) for contents of manganese and zinc of 52.4 and 100.0 μg g^− 1, respectively. The proposed method was applied for determination of manganese and zinc in five powdered chocolate samples. In these, the manganese content varied from 42.8 to 52.7 and from 88.6 to 102.4 μg g^− 1 of zinc. The analytical results were compared with the results obtained by analysis of these samples after digestion using open vessel and acid bomb digestion procedures and determination using FS-FAAS. The statistical comparison by t-test (95% confidence level) showed no significant difference between these results.     

Synthesis of amberlite XAD-2-PC resin for preconcentration and determination of trace elements in food samples by flame atomic absorption spectrometry

A new chelating sorbent has been developed using Amberlite XAD-2 resin anchored with pyrocatechol through –N=C– group. This sorbent, characterised by elemental analysis and infrared (IR) spectra, was used as packing for the minicolumn in an on-line system preconcentration system for cadmium, cobalt, copper and nickel determination. Metal ions were sorbed in the minicolumn, from which it could be eluted directly to the nebulizer–burner system of the flame atomic absorption spectrometer (FAAS). Elution of all metals from minicolumn can be made with 0.50 mol L^− 1 HCl or HNO₃. The enrichment factors obtained were 16 (Cd), 24 (Co), 15 (Cu) and 19 (Ni), for 60 s preconcentration time, and 39 (Cd), 69 (Co), 36 (Cu) and 41 (Ni), if used 180 s preconcentration time. Under the optimum conditions, the proposed procedure allowed the determination of cadmium, cobalt, copper and nickel with detection limits of 0.31, 0.32, 0.39 and 1.64 μg L^− 1, respectively, when used preconcentration periods of 180 s. The accuracy of the developed procedure was sufficient and evaluated by the analysis of the certified reference materials NIST 1515 apple leaves and NIST 1570a spinach leaves. The method was applied to the analysis of food samples (spinach, black tea and rice flour).     

Multivariate optimisation of the experimental conditions for determination of three methylxanthines by reversed-phase high-performance liquid chromatography

Caffeine (1,3,7-trimethylxanthine), theobromine (3,7-dimethylxanthine) and theophylline (1,3-dimethylxanthine) are the most important naturally occurring methylxanthines. Caffeine is a constituent of coffee and other beverage and included in many medicines. Theobromine and theophylline are formed as metabolites of caffeine in humans, and are also present in tea, cocoa and chocolate products.

In order to improve the chromatographic resolution (R_s) with a good analysis time, experimental designs were applied for multivariate optimisation of the experimental conditions of an isocratic reversed-phase high-performance liquid chromatographic (RP-HPLC) method used for the simultaneous determination of caffeine, theobromine and theophylline. The optimisation process was carried out in two steps using full three-level factorial designs. The factors optimised were: flow rate and mobile phase composition. Optimal conditions for the separation of the three methylxanthines were obtained using a mixture of water/ethanol/acetic acid (75:24:1%, v/v/v) as mobile phase and a flow rate of 1.0 mL min⁻¹. The RP-HPLC/UV method was validated in terms of limit of detection (LOD), limit of quantitation (LOQ), linearity, recovery and the precision, calculated as relative standard deviation (R.S.D.). In these conditions, the LOD was 0.10 μg L⁻¹ for caffeine, 0.07 μg L⁻¹ for theobromine and 0.06 μg L⁻¹ for theophylline. The proposed method is fast, requires no extraction step or derivatization and was suitable for quantification of these methylxanthines in coffee, tea and human urine samples.     

Quantitative separation of zinc traces from cadmium matrices by solid-phase extraction with polyurethane foam

A system for separation of zinc traces from large amounts of cadmium is proposed in this paper. It is based on the solid-phase extraction of the zinc in the form of thiocyanate complexes by the polyurethane foam. The following parameters were studied: effect of pH and of the thiocyanate concentration on the zinc extraction, shaking time required for quantitative extraction, amount of PU foam necessary for complete extraction, conditions for the separation of zinc from cadmium, influence of other cations and anions on the zinc sorption by PU foam, and required conditions for back extraction of zinc from the PU foam. The results show that zinc traces can be separated from large amounts of cadmium at pH 3.0±0.50, with the range of thiocyanate concentration from 0.15 to 0.20 mol l⁻¹, and the shaking time of 5 min. The back extraction of zinc can be done by shaking it with water for 10 min. Calcium, barium, strontium, magnesium, aluminum, nickel and iron(II) are efficiently separated. Iron(III), copper(II) and cobalt(II) are extracted simultaneously with zinc, but the iron reduction with ascorbic acid and the use of citrate to mask copper(II) and cobalt(II) increase the selectivity of the zinc extraction. The anions nitrate, chloride, sulfate, acetate, thiosulphate, tartarate, oxalate, fluoride, citrate, and carbonate do not affect the zinc extraction. Phosphate and EDTA must be absent. The method proposed was applied to determine zinc in cadmium salts using 4-(2-pyridylazo)-resorcinol (PAR) as a spectrophotometric reagent. The result achieved did not show significant difference in the accuracy and precision (95% confidence level) with those obtained by ICP–AES analysis.     

Development of a rapid and specific assay for detection of busulfan in human plasma by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry

A sensitive and specific assay for detection of busulfan in human plasma was developed. The assay is based on rapid isolation of busulfan by liquid-liquid extraction with ethyl acetate, and detection by high-performance liquid chromatography with electrospray ionization and tandem mass spectrometry. 1,6-Bis(methanesulfonyloxy)hexane, a synthesized analogue of busulfan, was used as the internal standard (IS). The acquisition was performed in the multiple reaction monitoring mode; busulfan and the IS were detected with no interferences from plasma matrix. The method was linear over the range 5-2500 ng mL(-1), with r2 > 0.99 and a run time of only 3.5 min. The intra- and inter-assay precisions were in the ranges 2.1-11.9% and 3.2-10.1%, respectively, and the intra- and inter-assay accuracies were 92.2-107.6% and 94.7-104.1%, respectively. The absolute recoveries were 82.0% (20 ng mL(-1)), 90.6% (1000 ng mL(-1)) and 80.0% (2000 ng mL(-1)) for busulfan, and 89.1% for the IS (1000 ng mL(-1)). The limits of detection and quantification were 2 and 5 ng mL(-1), respectively. The validated method was successfully applied to analyze plasma samples obtained from six adults receiving doses of 1 mg kg(-1) in a conditioning regimen prior to bone marrow transplantation. A marked intra-patient variation in busulfan concentrations during the steady state was observed, which limits the application of pharmacokinetic modeling and suggests that continuous therapeutic monitoring is necessary for adequate individualized dosing. In this regard, the present assay brings important advantages relative to other methods described in the literature, i.e., it is highly specific and simple to perform, with a rapid chromatographic run time (3.5 min), and the whole procedure can be completed in 4-5 h, which would permit dose corrections after the third dose allowing earlier and better dosing adjustments towards the target level of busulfan.