Subsequent Elution Research Articles

N-(2-(2-pyridyl)ethyl)chitosan: Synthesis, characterization and sorption properties.

The method of producing N-2-(2-pyridyl)ethylchitosan (PE-chitosan) with substitution degrees (DS) up to 1.2 has been developed using the "synthesis in gel" approach for direct addition reaction between 2-vynilpyridine and chitosan. Investigation of sorption properties has revealed significantly higher affinity of pyridylethyl fragments to Pt(IV)) and Pd(II) ions compared to the unsubstituted amino groups of chitosan. The maximum sorption capacities of PE-chitosan in 0.1M HCl solution were estimated as 5.56mmol/g for Au(III), 3.67mmol/g for Pd(II), and 2.75mmol/g for Pt(IV). Sorption capacities of PE-chitosan for transition metal ions at pH 4-8 were 1.5-2.6 higher than those of chitosan with the highest values attained for Cu(II) and Ag(I) ions - 1.50mmol/g and 1.53mmol/g, respectively. The PE-chitosan application for preconcentration of Au(III) with subsequent elution with HCl/thiourea mixtures was proved to be efficient for atomic absorption spectroscopy analysis of multi-component solutions with low gold content.

Room-temperature fluorescence spectroscopy of monohydroxy metabolites of polycyclic aromatic hydrocarbons on octadecyl extraction membranes

Urine analysis of monohydroxy metabolites is recognized as an accurate assessment of human exposure to polycyclic aromatic hydrocarbons. Despite the sophisticated arsenal of analytical tools, monitoring of monohydroxy metabolites via simple, cost effective and direct methods of analysis still remains a challenge. This article evaluates the analytical potential of solid-phase extraction room-temperature fluorescence spectroscopy for the problem at hand. Extraction membranes serve the dual purpose of sample pre-concentration and solid substrate for RTF measurements. The potential of our proposition is demonstrated with the analysis of 2-hydroxy-fluorene, 1-hydroxy-pyrene, 3-hydroxy-benzo[a]pyrene and 9-hydroxy-phenanthrene in synthetic urine samples. Signal reproducibility is improved with the aid of a sample holder specifically designed for the manual optimization of luminescence signals. Background correction of solid substrates is carried out with the aid of Asymmetric Least Squares. Recovery values for the studied metabolites varied from 99.0±1.2% (3-hydroxy-benzo[a]pyrene) to 99.9±0.05% (1-hydroxy-pyrene). With only 10mL of urine sample, the limits of detection varied from 57pgmL−1 (2-hydroxy-fluorene) to 2pgmL−1 (1-hydroxy-pyrene). Additional figures of merit include a simple experimental procedure for routine screening of numerous samples and compatibility with portable instrumentation for field analysis. Because of the non-destructive nature of fluorescence measurements, membranes can be brought to the lab for subsequent elution and confirmation of compounds via high-resolution techniques.

The best practice for preparation of samples from FTA®cards for diagnosis of blood borne infections using African trypanosomes as a model system

BackgroundDiagnosis of blood borne infectious diseases relies primarily on the detection of the causative agent in the blood sample. Molecular techniques offer sensitive and specific tools for this although considerable difficulties exist when using these approaches in the field environment. In large scale epidemiological studies, FTA®cards are becoming increasingly popular for the rapid collection and archiving of a large number of samples. However, there are some difficulties in the downstream processing of these cards which is essential for the accurate diagnosis of infection. Here we describe recommendations for the best practice approach for sample processing from FTA®cards for the molecular diagnosis of trypanosomiasis using PCR.ResultsA comparison of five techniques was made. Detection from directly applied whole blood was less sensitive (35.6%) than whole blood which was subsequently eluted from the cards using Chelex®100 (56.4%). Better apparent sensitivity was achieved when blood was lysed prior to application on the FTA cards (73.3%) although this was not significant. This did not improve with subsequent elution using Chelex®100 (73.3%) and was not significantly different from direct DNA extraction from blood in the field (68.3%).ConclusionsBased on these results, the degree of effort required for each of these techniques and the difficulty of DNA extraction under field conditions, we recommend that blood is transferred onto FTA cards whole followed by elution in Chelex®100 as the best approach.

Uncertainty estimation for the determination of Fe, Pb and Zn in natural water samples by SPE-ICP-OES

In this paper we estimate measurement of uncertainty for determination of Fe, Pb and Zn in natural water samples by solid-phase extraction and inductively coupled plasma optical emission spectrometry (SPE-ICP-OES). The procedure is based on the retention of analytes in the form of 8-hydroxyquinoline (8-HQ) complexes on a mini column of XAD-4 resin and subsequent elution with nitric acid. The influence of various analytical parameters including the amount of solid phase, pH, elution factors (concentration and volume of eluting solution), volume of sample solution, and amount of ligand on the extraction efficiency of analytes was investigated. To estimate the uncertainty of analytical result obtained, we propose assessing trueness by employing spiked sample. Two kinds of bias (proportional bias and constant bias) estimated. We applied Nested design for calculating proportional bias and Youden method to calculate the constant bias. The results we obtained for proportional bias are calculated from spiked sample...

Raman mapping of pharmaceuticals

Raman spectroscopy may be implemented through a microscope to provide fine scale axial and lateral chemical maps. The molecular structure of many drugs makes Raman spectroscopy particularly well suited to the investigation of pharmaceutical systems. Chemometric methods currently used to assess bulk Raman spectroscopic data are typically applied to Raman mapping data from pharmaceuticals; few reports exist where the spatial information inherent to a mapped dataset is used for the calculation of chemical maps. Both univariate and multivariate methods have been applied to Raman mapping data to determine the distribution of active pharmaceutical ingredients (APIs) in tablets, solid dispersions for increased solubility and controlled release devices. The ability to axially (depth) profile using Raman mapping has been used in studies of API penetration through membranes, cellular uptake of drug delivery liposomes, and initial API distribution and subsequent elution from coatings of medical devices. New instrumental developments will increase the efficiency of Raman mapping and lead to greater utilisation of Raman mapping for analyses of pharmaceutical systems.

Poster Session 1: Drug Delivery

The in vitro loading and subsequent elution of topotecan (used to treat a variety of cancers) from drug-eluting beads (DEBs) was assessed, while evaluating the effects of selected physical attributes (size and compressibility)

Production of carrier free 188Re radioisotope generator based on aluminum tungstate matrix

Improved radionuclide generator include a substantially insoluble salt of a radioactive parent which may be directly packed in column for subsequent elution of the daughter radionuclide. An improved 188Re generator was prepared by reacting a radioactive tungsten (188W) as parent radionuclide incorporated with aluminum chloride to obtain an insoluble radioactive aluminum tungstate matrix. The investigated matrix was characterized on the basis of the chemical composition, IR, thermal analysis and mechanical stabilities. The factors affecting the elution performance were studied such as influence of pH, molar ratio and drying temperature. From the obtained data, the molar ratio W:Al was 1.5:1 at pH = 4, the matrix dried at 105 °C for 2 h. Chromatographic and multichannel analysis has been currently used to investigate the radiochemical and radionuclidic purity respectively on eluted 188Re. An elution yield more than 80%, with radiochemical purity 10−4% of the column. The Al+3 and W contents value were about 2 and 3 μg/mL eluate. The obtained data approved the stability of the prepared generator and its suitability for medical application.

Co-purified gelatinases alter the stability and biological activities of human plasma fibronectin preparations.

Fibronectin (FN) is an important cell adhesion molecule that is used widely to characterize cell behavior. Preparations of FN purified from human plasma by gelatin-Sepharose affinity chromatography typically also contain gelatin-binding gelatinases that may cleave FN, reduce its stability and alter its biological activities. Available methods for separating gelatinases from FN are resource demanding. Therefore, our objective was to devise a time- and cost-efficient protocol for purification of gelatinase-free FN. Experiments tested the elution profiles for FN and gelatinases from gelatin-Sepharose using a concentration range (1-7%) of dimethyl sulfoxide (DMSO) and 4 m urea as eluants. Subsequently, we explored the sequential application of those eluants for differential elution of gelatinases and FN using a single affinity column. Finally, experiments characterized the stability of purified FN with or without contaminating gelatinases, as well as the effects of FN degradation on cell attachment and migration. Assay optimization demonstrated that pre-elution with 3% DMSO efficiently eliminated gelatinases but not FN from gelatin-Sepharose, whereas subsequent elution with 4 m urea released FN. Sequential elutions with DMSO and urea produced gelatinase-free FN, which was more stable than FN eluted by urea only. Fibronectin degradation did not affect human gingival fibroblast attachment, but increased cell migration significantly. The present experiments devised a time- and cost-efficient protocol for eliminating gelatinases during purification of human plasma FN. Gelatinase-free FN preparations had greater stability, which may be essential for experiments because FN fragments have altered biological activities compared with intact FN.

Sequential Enrichment of Sulfated Glycans by Strong Anion-Exchange Chromatography Prior to Mass Spectrometric Measurements

Structural characterization of sulfated glycans through mass spectrometry (MS) has been often limited by their low abundance in biological materials and inefficient ionization in the positive-ion mode. Here, we describe a microscale method for sequentially enriching sulfated glycans according to their degree of sulfation. This method is based on modifying the binding ability of strong anion-exchange material through the use of different sodium acetate concentrations, thus enabling fairly selective binding and a subsequent elution of different glycans according to their degree of sulfation. Before this enrichment, the negative charge on the sialic acid, which is commonly associated with such glycans, was eliminated through permethylation that is used to enhance the positive-ion mode matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-MS) signal for all glycans. This enrichment approach minimizes competitive ionization between sulfated and neutral glycans, as well as that between sulfated species with a different degree of sulfation. The described method was initially optimized using sulfated oligosaccharide standards, while its potential has been verified for the sulfated N-glycans originated from the bovine thyroid-stimulating hormone (bTSH), a glycoprotein possessing mono- and disulfated N-glycans. This enhancement of the MALDI-MS signal facilitates analysis of some otherwise undetected components.

Production and characterization of a milk-clotting enzyme from Aspergillus oryzae MTCC 5341

Microbial milk-clotting enzymes are valued as calf rennet substitutes in the cheese industry. Aspergillus oryzae MTCC 5341 was identified to produce the highest milk-clotting activity during screening of 16 fungal strains. Solid state fermentation using wheat bran along with 4% defatted soy flour and 2% skim milk powder as substrate was optimal for growth of A. oryzae and production of the enzyme. Nearly 40,000 U/g bran of milk-clotting activity was present at the end of 120 h. The enzyme could be recovered by percolating the bran with 0.1 M sodium chloride for 60 min at 4 degrees Celsius. The decolorized enzyme preparation had high ratio of milk clotting to proteolytic activity. Affinity precipitation with alginate and subsequent elution with 0.5 M sodium chloride containing 0.2 M CaCl(2) resulted in an enzyme preparation with specific activity of 3,500 U/mg and 72% yield. Optimum pH and temperature for activity of the enzyme were characterized as 6.3 and 55 degrees Celsius, respectively. Milk-clotting enzyme showed differential degree of hydrolysis on casein components. High ratio of milk clotting to proteolytic activity coupled with low thermal stability strengthens the potential usefulness of milk-clotting enzyme of A. oryzae MTCC 5341 as a substitute for calf rennet in cheese manufacturing.

Combinatorial peptide ligand libraries for urine proteome analysis: Investigation of different elution systems

Proteome treatments with peptide libraries in view of reducing high-abundance proteins and increasing the concentration of rare species involve the adsorption on solid-phase material. Subsequent elution of captured proteins may not be fully effective except when sequences of eluting agents are used. The standard way utilized up to the present has been a three- to four-step, sequential elution system consisting of various agents mixed together such as urea, thiourea, CHAPS, sodium chloride, citric or acetic acid and some polar solvents such as ACN and isopropanol. Elution sequences produce distinct fractions adding to the burden of having to analyze all of them. An alternative, highly effective, single elution to reduce the workload is here reported for the first time, namely elution in boiling 10% SDS added with 3% DTE. This single step elutes almost quantitatively the adsorbed proteins, thus ensuring, for all practical purposes, a full recovery. This high efficiency is believed to be due to the fact that the SDS micelles bury the polypeptide chains within their hydrophobic core, thus shielding them from the surroundings and impeding accidental adsorption to surfaces. Suggestions for selecting the best method to eliminate the excess of SDS for further protein analysis are also evaluated. The merits and limits of this novel system are assessed and discussed.

Comparison of off- and in-line solid-phase extraction for enhancing sensitivity in capillary electrophoresis using ochratoxin as a model compound

This paper proposes and compares two approaches based on off- and in-line solid-phase extraction (SPE), intended to enhance sensitivity in capillary electrophoresis with ultraviolet detection (CE-UV) using as a model the determination of ochratoxin A (OA) in river water samples. In the off-line SPE mode, the reversed-phase sorbent (octadecilsylane, C(18)) selectively retains the target analyte (OA) and allows large volumes of the sample (70 mL) to be introduced and subsequently eluted in a small volume (0.1 mL) of an appropriate solution. In the in-line SPE mode, a custom-made microcartridge is inserted near the inlet of the capillary, which is filled with the same C(18) sorbent. This solid phase selectively retains OA present in a sample volume as low as approximately 640 microL for subsequent elution with ca. 135 nL of an appropriate eluent. The limit of detection (LOD) obtained with the in-line SPE method was 1 ng L(-1), which is 3 orders of magnitude lower than that obtained with CE-UV and roughly 1 order lower than that provided by the off-line SPE-CE-UV method.

Ultrananocrystalline Diamond Thin Films Functionalized with Therapeutically Active Collagen Networks

The fabrication of biologically amenable interfaces in medicine bridges translational technologies with their surrounding biological environment. Functionalized nanomaterials catalyze this coalescence through the creation of biomimetic and active substrates upon which a spectrum of therapeutic elements can be delivered to adherent cells to address biomolecular processes in cancer, inflammation, etc. Here, we demonstrate the robust functionalization of ultrananocrystalline diamond (UNCD) with type I collagen and dexamethasone (Dex), an anti-inflammatory drug, to fabricate a hybrid therapeutically active substrate for localized drug delivery. UNCD oxidation coupled with a pH-mediated collagen adsorption process generated a comprehensive interface between the two materials, and subsequent Dex integration, activity, and elution were confirmed through inflammatory gene expression assays. These studies confer a translational relevance to the biofunctionalized UNCD in its role as an active therapeutic network for potent regulation of cellular activity toward applications in nanomedicine.

On-line solid phase extraction system using 1,10-phenanthroline immobilized on surfactant coated alumina for the flame atomic absorption spectrometric determination of copper and cadmium

An on-line solid phase extraction (SPE) preconcentration system coupled to flame atomic absorption spectrometer (FAAS) was developed for determination of copper and cadmium at μg L −1 level. The method is based on the on-line retention of copper and cadmium on a microcolumn of alumina modified with sodium dodecyl sulfate (SDS) and 1,10-phenanthroline and subsequent elution with ethanol and determination by FAAS. The effect of chemical and flow variables that could affect the performance of the system was investigated. The relative standard deviation ( n = 6) at 20 μg L −1 level for copper and cadmium were 1.4 and 2.2% and the corresponding limits of detection (based on 3σ) were 0.04 and 0.14 μg L −1, respectively. The method was successfully applied to determination of copper and cadmium in human hair and water samples.

Chemotrapping-atomic fluorescence spectrometric method as a field method for volatile arsenic in natural gas

Volatile arsenic compounds in natural gas, existing in the form of trimethylarsine (TMAs), have been determined using gas cryo-trapping gas chromatography coupled to inductively coupled plasma-mass spectrometry (CT-GC-ICP-MS). The results from a number of different gas wells revealed a huge concentration spread ranging from below the detection limit of 0.2 up to 1800 microg/m(3) TMAs (as As) in the gas. Due to the toxicity and corrosive nature of these arsines, they need near real time monitoring via a method that can easily be implemented on site, i.e. during gas exploitation. Here, we introduce a novel method which utilises silver nitrate impregnated silica gel tubes for quantitative chemotrapping of trimethylarsine (TMAs) from a natural gas matrix. Subsequent elution with hot nitric acid followed by online photo-oxidation hydride generation atomic fluorescence spectrometry (HG-AFS) is used for the determination of TMAs gas standards in nitrogen and natural gas samples, respectively. The chemotrapping method was validated using CT-GC-ICP-MS as a reference method. The recovery of arsenic from nitrogen or natural gas matrix ranged from 85 to 113% for a range of 20 to 2000 ng As. Trapping efficiency was >98%, from the methods LOD of 20 ng to 4.8 microg (absolute amount As) with sample sizes of 0.02 and 2 L gas. Method performance was established by comparing the results obtained for eight natural gas samples containing between 1 and 140 microg As/m(3) with those achieved by the reference method (CT-GC-ICP-MS).

A New Integrated Membrane Filtration and Chromatographic Device

To improve protein separation, a novel integrated device combining membrane filtration and chromatography has been developed. The device basically consists of a hollow fiber filtration module whose shell side is filled with chromatographic resin beads. However, there is an essentially impermeable coated zone near the hollow fiber module outlet. The integrated device enjoys the advantages of both membrane filtration and chromatography; it also allows one to load the chromatographic media directly from the fermentation broth or lysate and separate the adsorbed proteins through the subsequent elution step in a cyclic process. Interfacial polymerization was carried out to coat the bottom section of the hollow fiber membrane; the rest of the hollow fiber membrane remained unaffected. Myoglobin (Mb) and alpha-lactalbumin (alpha-LA) were primarily used as model proteins in a binary mixture; binary mixtures of Mb and bovine serum albumin (BSA) were also investigated. Separation behaviors of binary protein mixtures were studied in devices having either an ultrafiltration (UF) or a microfiltration (MF) membrane. Experimental results show that the breakthrough time and the protein loading capacities were dramatically improved after introducing the impermeable coating in both UF and MF modules. For a synthetic yeast fermentation broth feed, four loading-washing-elution-reequilibration-based cyclic runs for separation of Mb and alpha-LA were performed in the device using a MF membrane with a coated zone without cleaning in between. The Mb and alpha-LA elution profiles for the four consecutive runs were almost superimposable. Due to lower transmembrane flux in this device plus the periodical washing-elution during the chromatographic separation, fouling was not a problem, unlike in conventional microfiltration.

Development of a Countergradient Parking System for Gradient Liquid Chromatography with Online Biochemical Detection of Serine Protease Inhibitors

A gradient HPLC approach in combination with a countergradient system for online biochemical detection (BCD) to screen for inhibitors of serine proteases is described. For gradient separations, this novel countergradient system was developed to produce a biocompatible constant solvent composition in the BCD. The countergradient system is based on retaining complete gradients in an additional preparative HPLC column, followed by subsequent and reversible elution to the separation column effluent. Major advantages compared with existing countergradient systems are that no additional LC pumps are needed and enhanced stability. The developed countergradient system was systematically characterized applying different gradient programs. Inhibitors eluting in a postcolumn continuous flow analysis interfere with the enzymatic release of fluorescent 7-amino-4-methylcoumarin (AMC) from an AMC-labeled peptide. The inhibitory activity of eluting substances is sensitively detected as the degree of reduced fluorescence intensity. This biochemical detection system (BCD) for proteases was validated with three known inhibitors of the benzamidine type. Their IC 50 values were in good accordance with the results of conventional plate reader assays. Finally, a small library of protease inhibitors was successfully screened with the combination of the BCD and the countergradient system.

T cell responses to orbital antigens in thyroid-associated ophthalmopathy.

Thyroid-associated ophthalmopathy (TAO) is most likely to be a T cell-mediated disease, in which cytokines released in the extraocular muscles activate fibroblasts, increasing glycosaminoglycan production. The nature of the orbital antigen recognized by the infiltrating T cells is unclear, although it is possible that there is cross-reactivity between this and a thyroid autoantigen to explain the close association with thyroid autoimmunity. We have tested the ability of human and porcine eye muscle antigen preparations to stimulate proliferation of circulating T cells from healthy subjects and patients with TAO or Graves' disease without clinical TAO. Occasional responses were seen, particularly after depletion of CD8+ T cells, and two out of 10 TAO patients responded to eye muscle proteins of 25-50 kD after fractionation of antigens on gels and subsequent elution. There was no disease-specific response of T cells to R1, R14, D1 and 1D3, recombinant proteins identified from screening an eye muscle cDNA library with sera from patients with autoimmune thyroid disease. We have also found that interferon-gamma (IFN-gamma) production by T cells from TAO patients was not stimulated by eye muscle membrane antigens or by 1D3. These results suggest that the frequency of circulating T cells responding to eye muscle antigens in TAO is low, and that several candidate orbital antigens, including the 64-kD protein 1D3, are unlikely to be important T cell autoantigens in this condition.

Dimethylamine-Modified Waste Paper for the Recovery of Precious Metals

Waste newsprint paper was modified with dimethylamine (DMA) to obtain a tertiary amine type adsorption gel called DMA-paper gel. This new derivative was investigated for adsorption, from hydrochloric acid medium, of gold, palladium, and platinum as well as some base metals. The gel exhibited selectivity only for precious metals with a remarkably high capacity for Au(III), i.e., 4.6 mol/kg dry gel and comparable capacities, i.e., 2.1 and 0.9 mol/kg for Pd(II) and Pt(IV), respectively. Also, Au(III) was reduced to the elemental form during adsorption. Furthermore, column adsorption and subsequent elution of the adsorbed metal ions by acidic thiourea revealed encouraging recoveries (approximately 90%), thus enhancing the scope of the gel for effective preconcentration, separation, and recovery of precious metals. The effectiveness of recovery of precious metals from real industrial liquor was also tested, and it showed highly encouraging results with respect to the stability of the gel in the harsh medium, and selectivity for the targeted metal ions in the presence of excess of other metal ions.

SIMAC (Sequential Elution from IMAC), a Phosphoproteomics Strategy for the Rapid Separation of Monophosphorylated from Multiply Phosphorylated Peptides

The complete analysis of phosphoproteomes has been hampered by the lack of methods for efficient purification, detection, and characterization of phosphorylated peptides from complex biological samples. Despite several strategies for affinity enrichment of phosphorylated peptides prior to mass spectrometric analysis, such as immobilized metal affinity chromatography or titanium dioxide, the coverage of the phosphoproteome of a given sample is limited. Here we report a simple and rapid strategy, SIMAC (sequential elution from IMAC), for sequential separation of monophosphorylated peptides and multiply phosphorylated peptides from highly complex biological samples. This allows individual analysis of the two pools of phosphorylated peptides using mass spectrometric parameters differentially optimized for their unique properties. We compared the phosphoproteome identified from 120 mug of human mesenchymal stem cells using SIMAC and an optimized titanium dioxide chromatographic method. More than double the total number of identified phosphorylation sites was obtained with SIMAC, primarily from a 3-fold increase in recovery of multiply phosphorylated peptides.