Characterization of hot particles in surface soil around the Chernobyl NPP

An ICP-AES method for the analysis of trace amounts of lanthanides and yttrium in sodium or magnesium diuranate samples (yellow cake) and other beneficiation product generated during the uranium extraction process (hydrometallurgy) from its ores is described. Most of the matrix elements are removed by an initial oxalate precipitation of lanthanides using calcium as carrier. A solvent extraction procedure using a mixture of mono 2-ethylhexyl dihydrogen phosphate (H₂MEHP) and bis (2-ethylhexyl) hydrogen phosphate (HDEHP) is used for the removal of calcium, iron and the occluded uranium. A combination of oxalate precipitation and solvent extraction procedure is applied for the selective separation and preconcentration of traces of lanthanides from yellow cake and iron cake samples. The solvent extraction procedure is directly applied for the separation of lanthanides from the uranium leach liquor and lime cake. The accuracy of the method is checked by analyzing synthetic mixture containing known amounts of traces of lanthanides and also by comparing with another standard separation procedure like ion exchange method, and the recovery was better than 95%. The method is rapid, simple, accurate and suitable for the separation of lanthanides from uranium, iron and calcium rich materials. The precision of the method is characterized by an RSD of 2 to 4%.     

Quantification of the metal distribution in metallothioneins of the human liver by HPLC coupled with ICP-AES

Fractions containing metallothioneins (MT’s), extracted from the liver cytosol of humans, were analysed to determine the complete distribution pattern of the metals copper, cadmium and zinc. Samples of cirrhotic livers which had come from organs removed during transplantation were examined for differences in the trace-element binding pattern. After the extraction of supernatants from the tissue samples, membrane ultrafiltration of the cytosolic solution was carried out to separate all high-molecular proteins with molecular weights >100 kDa. This procedure retains the metal content of the MT’s in its initial form, in contrast to the often-used heat treatment of samples, which changes the copper distribution significantly. The MT’s themself were isolated using size exclusion and anion exchange chromatography. Their metal content was determined simultaneously on-line by combination with an ICP-AES as element detector. Calibration of the procedure was performed by means of a column by-pass-injection of elemental standards into the separation system. The MT content in the samples was calculated using the determined metal concentrations and the generally accepted metal/protein ratios for Cu (12:1), Cd (7:1) and Zn (7:1). These values were compared with values resulting from a ¹⁰⁹Cd-saturation-assay. When various liver samples of different pathogenesis were compared, the highest level of Cu-MT was found in primary biliary cirrhosis.     

Speciation analysis of trace elements in the brains of individuals with Alzheimer's disease with special emphasis on metallothioneins

A speciation analysis of protein-bound elements in the cytosol of human brain was achieved by size exclusion chromatographical separation of the biomolecules and on-line detection of the metal profiles in the eluate by hyphenated inductively coupled plasma-mass spectrometry. Post-mortem samples from Alzheimer's disease brains and from brains of a control group were investigated to elucidate changes in the trace element distribution during the pathological process. Special attention was paid to the metallothioneins (MT) - cysteine-rich, metal-binding proteins of low molecular weight, existing in several isoforms. The isoform MT-3 is found especially in the brain and has a growth inhibition function on neurons. The MT peaks were identified in the element profiles. For this purpose, the metal binding capability and the heat stability of MT were taken into consideration. For verification, a comparison with pure MT-3 was carried out and further biochemical and analytical methods were applied to the fractions of the chromatographical run. A comparison between Alzheimer's disease and control brains showed a significant difference concerning the MT-1/-2 and MT-3 metal levels, leading to the assumption that there were oxidative processes having taken place in the Alzheimer's brain samples.     

Analysis of metallothionein isoforms by capillary electrophoresis: optimisation of protein separation conditions using micellar electrokinetic capillary chromatography

Capillary electrophoresis (CE) techniques have been successfully applied to the separation of metallothionein (MT) isoforms and have proved to be rapid, practical and economical. Study of a variety of different electrolytes and capillaries has shown that electrolyte buffer composition and capillary wall surface modifications can have considerable influence on isoform separation and resolution. Ionic surfactants such as sodium dodecyl sulphate (SDS) form micelles at elevated concentrations and the partitioning of molecules between the hydrophobic micelle phase and the aqueous phase and their resulting migration in an electric field is the basis of the technique known as micellar electrokinetic capillary chromatography (MECC). In the present work, we have used sheep and rabbit MT to optimise MECC conditions for analysis of MT isoforms. Capillaries of 57 cm gave much better separations than shorter columns although analysis times were increased to about 12 min. Changing the buffer and SDS concentration or the pH affected the selectivity of isoform separation and up to 5 isoforms in sheep MT and 6 in rabbit MT were completely or partially resolved. Comparing different diameter capillaries we conclude that 25 μm I.D. columns give better separations than 50 or 75 μm I.D. columns although sensitivity is reduced by a factor of about 3 and 5, respectively. Using our MECC conditions, columns coated with C₁ or C₁₈ hydrophobic material were not found to be useful in improving MT separation or resolution although further evaluation of these columns is in progress. Analysis of sheep liver extracts using optimised conditions showed the expression of at least 4 MT isoforms in response to Zn injection and 3 of these forms were evident in extracts from untreated sheep. We therefore conclude that MECC is a suitable method for MT isoform analysis.     

A novel approach to Ca-Sr separation in the determination of90Sr using inorganic exchangers: An application to environmental samples

A procedure for⁹⁰Sr determination in calcium rich samples is presented. It is based on the precipitation of calcium oxalate in homogeneous solution and under controlled conditions to minimize the coprecipitation of strontium. The latter is subsequently separated as carbonate and radiochemical purification is completed by ion exchange chromatography on two inorganic exchangers: PRTD (partially reduced tin dioxide) and CUCR (copper chromate). The procedure was applied to environmental samples such as ashed sediment, fish and vegetable and results are reported.     

Production of Metallothionein Polyclonal Antibodies Using Chickens as Model

The production of polyclonal antibodies (pAbs) against metallothioneins (MT) has been done in mammals. In this work, we describe a model where pAbs against rat liver MT were produced in chickens. Liver MT-1 and MT-2 isoforms isolated from rats were used as immunogens. MT was purified by exclusion chromatography and MT isoforms isolated by ionic exchange chromatography. Chickens were immunized with each isoform emulsified with Freund adjuvant over 6 weeks. MT-pAbs obtained from egg yolk were purified by ammonium sulfate precipitation followed by thiophilic interaction chromatography. MT-pAbs were characterized by ELISA, SDS-PAGE electrophoresis, and Western blot assays. Results showed significant titers (1:1,000) of MT-1 and MT-2 IgY in the eggs collected 30 days after the first immunization as determined by a direct ELISA assay; results also show a cross-reaction between MT-1 and MT-2 isoforms: however, the Abs obtained did not react with other non-MT proteins in hepatic homogenates. Sensitivity assays showed that MT-pAbs detected MT-1 and MT-2 at nanogram levels. These data suggest that chickens are an alternative model for producing pAbs against mammal high-homology proteins such as MT.     

Application of accelerated solvent extraction to the investigation of saikosaponins from the roots of Bupleurum falcatum

Accelerated solvent extraction (ASE) was applied to the extraction of saikosaponin a, saikosaponin c and saikosaponin d from the roots of Bupleurum falcatum. Main extraction parameters such as the extraction solvents, extraction temperature and static extraction time were investigated and optimized. The optimized procedure employed 70% methanol as extraction solvent, 120°C of extraction temperature, 10 min of static extraction time, 60% of flush volume and the extraction recoveries of the three compounds were near to 100% with one extraction cycle. The extracted samples were analyzed by HPLC with UV detector. The HPLC conditions were as follows: Hypersil ODS2 (4.6 mm×250 mm, 5 μm) column, acetonitrile and water as mobile phase, flow rate of 1.0 mL/min, UV detection wavelength of 204 nm and injection volume of 20 μL. Compared with the traditional methods including heat‐reflux extraction and ultrasonic‐assisted extraction, the proposed ASE method was more efficient and faster to be operated. The results indicated that ASE was an alternative method for extracting saikosaponins from the roots of B. falcatum.     

Structural changes in metallothionein isoforms revealed by capillary electrophoresis and Brdicka reaction

Metallothionein (MT) as a potential cancer marker is at the center of interest and its properties, functions and behavior under various conditions is intensively studied. In the present study, two major mammalian MT isoforms (MT‐1 and MT‐2) were separated using capillary electrophoresis (CE) coupled with UV detector in order to describe their basic behavior. Under the optimized conditions, the separation of both isoforms was enabled as well as estimation of detection limits as subunits and units of ng per μL for MT‐2 and MT‐1, respectively. Further, the effects of thermal treatment and the presence of denaturing agent such as urea on MT‐1 and MT‐2 isoforms were studied by CE‐UV. Thermal treatment caused an increase in the signals of both isoforms. A new parameter called precipitation rate has been defined based on this finding. This parameter can be expressed as a slope of the linear regression of the time dependency curve recalculated on the MT concentration. The thermal precipitation rate for MT‐1 and MT‐2 was determined as 1.1 and 0.9 ng of MT/min, respectively. The chemical precipitation rate calculated from the linear regression for both isoforms provided the same value of 0.25 ng of MT/min. The results were confirmed by manual spectrometric measurements and by differential pulse voltammetry Brdicka reaction. Based on these results, a model of MT behavior under the conditions studied was suggested.     

Detection of the anabolic steroid methylestosterone in hair by HPLC-EIA

Summary An analytical method to detect the illegal application of the anabolic steroid methyltestosterone (MT) in cattle by hair analysis was developed. The time course of the incorporation of this orally active xenobiotic steroid into growing hair and the duration of its possible detection by hair analysis were measured. Female veal calves were fed with 35 μg MT per kg body weight, twice daily, for 10 days. Before, during and after the treatment, hair samples were obtained and analyzed for MT residues. An appropriate method to extract MT from hair was developed. The extraction procedure consisted of liquid-liquid and solid-phase extraction and was followed by an essential high performance liquid chromatography (HPLC) step for further purification of the extracts. Final quantitfication was done with a specific enzyme immunoassay (EIA). MT residues could be detected in hair from day 4 (approximately 5 ng MT/g hair) of the experiment up to day 94 (approaximately 0.5 ng MT/g). Our results suggest that hair analysis may be a powerful means to detect and track the illegal use of anabolic steroids. Presented at the 21^st ISC held in Stuttgart, Germany, 15^th–20^th September, 1996     

Comparison of several extraction techniques for multiclass analysis of veterinary drugs in eggs using ultra-high pressure liquid chromatography-tandem mass spectrometry

This study compared four extraction methods for the simultaneous determination of tetracyclines, macrolides, quinolones, sulphonamides and anthelmintics (including benzimidazoles and avermectins) in eggs by ultra-high pressure liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS). Solvent extraction, solid-phase extraction (SPE), matrix solid-phase dispersion (MSPD) and modified QuEChERS procedure were compared in terms of recovery and number of veterinary drugs extracted. The solvent extraction procedure with a clean-up step provided better results than the other tested procedures. The QuEChERS procedure was simpler and faster, but extracted fewer compounds than solvent extraction. MSPD did not extract tetracyclines and quinolones, whereas macrolides and tetracyclines were not extracted when SPE was applied. The solvent extraction procedure was validated, obtaining recoveries ranging from 60% (sulfaquinoxaline) to 119% (levamisole) with repeatability values (expressed as relative standard deviations, RSDs) lower than 20% at two concentration levels (10 and 100 μg kg⁻¹), except for erythromycin, emamectin and ivermectin that showed RSD values close to 25% at 10 μg kg⁻¹. Limits of quantification (LOQs) were always equal or lower than 5 μg kg⁻¹. Finally the method was applied to egg samples, and erythromycin, enrofloxacin, difloxacin, thiabendazole, emamectin and fenbendazole were detected in four samples.     

Ultrasound extraction and thin layer chromatography-flame ionization detection analysis of the lipid fraction in marine mucilage samples

This paper reports an analytical procedure based on ultrasound to extract lipids in marine mucilage samples. The experimental conditions of the ultrasound procedure (solvent and time) were identified by a FT-IR study performed on different standard samples of lipids and of a standard humic sample, before and after the sonication treatment. This study showed that diethyl ether was a more suitable solvent than methanol for the ultrasonic extraction of lipids from environmental samples because it allowed to minimize the possible oxidative modifications of lipids due to the acoustic cavitation phenomena. The optimized conditions were applied to the extraction of total lipid amount in marine mucilage samples and TLC-flame ionization detection analysis was used to identify the relevant lipid sub-fractions present in samples.     

Rapid method for the determination of organochlorine pesticides and PCBs in fish muscle samples by microwave-assisted extraction and analysis of extracts by GC-ECD

A procedure for the multiresidue determination of organochlorine pesticides and polychlorinated biphenyls in fish muscle samples has been developed. The method is based on the microwave-assisted extraction (MAE) of food samples from an acetonitrile-water (95 + 5, v/v) mixture followed by SPE cleanup of the extracts and analysis by GC with an electron capture detector. MAE operational parameters, such as the extraction solvent, temperature, and time, were optimized with respect to the extraction efficiency of the target compounds from food samples with 10-13% fat content. The chosen extraction technique allows reduction of the solvent consumption and extraction time when compared with methods already used. Acetonitrile is a good extraction solvent for low-fat matrixes (2-20% fat content), such as fish samples, because it does not significantly dissolve the highly polar proteins, salts, and sugars commonly found in food and gives high recoveries of a wide polarity range of analytes. For purification, SPE using LC-Florisil was shown to be sufficient for the removal of coextracted substances. Recoveries > 78% with RSD values < 15% were obtained for all compounds under the selected conditions. Method quantification limits were in the 5-10 microg/kg range. The method was applied to the analysis of samples of herring (Clupea harengus) purchased at the local fish market. The method is rapid and reliable for the determination of organochlorine analytes in fish muscle.     

Determination of fluoroquinolones in bovine milk samples using a pipette‐tip SPE step based on multiwalled carbon nanotubes prior to CE separation

A simple CE–UV method was developed for the simultaneous determination of ciprofloxacin, norfloxacin, and ofloxacin in milk samples. The optimum separation was obtained using a 20 mM ammonium dihydrogenphosphate solution with 2 mM cetyltrimethylammonium bromide at pH 3.0 as the BGE. Satisfactory resolution for structurally very similar analytes, like norfloxacin and ciprofloxacin, was achieved without including any organic solvent. Milk samples were prepared using a simple/extraction procedure based on acidic protein precipitation followed by an SPE step using only 5 mg of multiwalled carbon nanotubes as the sorbent material. The LODs for the three compounds were between 7.5 and 11.6 μg/L and the RSDs for the peak areas were between 2.6 and 4.9%. The complete method was applied to spiked real milk samples with satisfactory recoveries for all analytes (84–106%).     

Separation of metallothionein isoforms of mouse liver cytosol by capillary zone electrophoresis

Metallothionein (MT) isoforms of mouse liver cytosol were separated by capillary zone electrophoresis (CZE) using a polyacrylamide-coated tube at neutral pH, samples prepared from non-treated, heat-treated, and ethanol-precipitated specimens were compared. The liver was homogenized in three kinds of media, 0.25 M sucrose containing 100 mM Tris-HCl buffer at pH 7.4 (BS), BS containing 1% ascorbic acid (BS-C), and BS containing 5 mM beta-mercaptoethanol (BS-M). Mouse liver was used 24 h after subcutaneous injection of 50 mg Zn kg(-1). In the non-treated specimen of the cytosol fraction, the MT-2 isoform was separated in all three media, while the MT-1 isoform was difficult to identify. In the ethanol-precipitated specimen, MT isoforms were separated well using either BS or BS-C. However, when BS-M was used, a small MT-2 peak was obtained the MT-1 peak could not be identified. MT-1 isoform in the heat-treated specimen was difficult to identify. In contrast, MT-2 isoform was separated well in all three kinds of media. In the non-treated specimen of the control liver cytosol, the MT-2 isoform was detected using all three media, the MT-1 peak was undetected. Based on these results, MT isoforms can be detected in the crude cytosol fraction of liver using CZE combined with a polyacrylamide-coated tube at neutral pH.     

Improved sample treatment for the determination of insoluble soap in sewage sludge samples by liquid chromatography with fluorescence detection

A new selective and sensitive method for the determination of insoluble fatty acid salts (soap) in sewage sludge samples is proposed. The method involves a clean up of sample with petroleum ether, the conversion of calcium and magnesium insoluble salts into soluble potassium salts, potassium salts extraction with methanol, and a derivatization procedure previous to the liquid chromatography with fluorescence detection (LC-FLD) analysis. Three different extraction techniques (Soxhlet, microwave-assisted extraction and ultrasounds) were compared and microwave-assisted extraction (MAE) was selected as appropriate for our purpose. This allowed to reduce the extraction time and solvent waste (50 mL of methanol in contrast with 250 mL for Soxhlet procedure). The absence of matrix effect was demonstrated with two standards (C_13:0 and C_17:0) that are not commercials and neither of them has been detected in sewage sludge samples. Therefore, it was possible to evaluate the matrix effect since both standards have similar environmental behaviour (adsorption and precipitation) to commercial soaps (C_10:0-C_18:0). The method was successfully applied to samples from different sources and consequently, with different composition.     

Physico-chemical characterization of metal binding proteins using HPLC-ICP-MS, HPLC-MA-AAS and electrospray-MS

A size exclusion HPLC method was developed and interfaced with ICP-MS detection for determining the metal profiles of commercially available rabbit liver metallothioneins (MT) and metallothionein-like proteins (MLP) extracted from fresh water mussels and hemolyzed osprey blood. The redox state of the cysteine residues was indirectly evaluated via a cadmium saturation approach in the presence or absence of a reducing agent, followed by HPLC-microatomization (MA)-AAS and HPLC-ICP-MS analyses. An electrospray-MS protocol was also developed to accurately measure the molecular weight of rabbit MT isoform II. Nanogram quantities of Cd-MT/MLP were poorly chromatographed on silica based supports. A copolymeric styrene-divinylbenzene size exclusion support provided a symmetrical peak (rabbit MT standard) and linear HPLC-MA-AAS calibration curves [r=0.9988; from the LOD (27 ng, as protein) to about 300×LOD], indicating negligible losses of Cd during the chromatography of trace quantities. Co-injection of Cd²⁺ saturated samples with beta-mercaptoethanol (BMSH) was essential to repress Cd²⁺-support interactions which otherwise induced an undesirable metalaffinity retention mechanism. In the presence of added Cd²⁺, 22 mmol/L BMSH did not significantly compete for Cd²⁺ specifically bound to MT, while preventing non-specific binding to non-thiolic complexing sites. Crude mussel and osprey blood MLP extracts (in cold, deoxygenated Tris-HCl buffer) were obtained by ultracentrifugation (145,000 g) and thermocoagulation/centrifugation, respectively. Incubation with BMSH was prerequisite to obtain a maximum saturation of mussel and osprey blood MLP by Cd²⁺, even for samples conserved (–80° C) in the presence of BMSH (22 mmol/L). These observations indicated that a major proportion of the cysteine residues present in these MLP were oxidized. The assumption of a fully reduced MT/MLP pool binding metals in a definite stoichiometry has been the basis of several quantitative metal binding assays involving the saturation of the thiolic complexing sites with a metallic marker (Ag+, Cd²⁺, or Hg²⁺). Since thiolic agents may interfere, the metal saturation protocols do not include a reducing step to ensure that all cysteines in a MT/MLP extract are available for co-ordination. Given that variations in the redox state of crude MT/MLP extracts may compromise the accuracy of metal saturation assays, it is concluded that the preparation of reference samples certified for total metallothionein content would be desirable.     

Metallothioneins from horse kidney studied by separation with capillary zone electrophoresis below and above the isoelectric points

Capillary zone electrophoresis (CZE) was used to study metallothionein (MT) isoforms and non-MT components in the horse kidney MT preparation produced by Sigma. This technique was found to be well suited for studies of such forms. The non-MTs are heat resistant, they do not bind metal, but comigrate with MT in the various systems generally used for MT isolations. These forms may cause discrepancies between claimed MT content and sample weight as well as confusion in MT identification. The functional metal binding ability in the Sigma sample was measured, and the elution profile from an anion exchange column, commonly used for separation of tissue MTs, was determined in order to ascertain the MT-I isoform content. Optimum conditions for the separation of the MT isoforms have been studied in the polyacrylamide coated capillary at pHs below and above the isoelectric points (pIs) in various buffer systems. Evidence is put forward that MT forms oligomers or aggregates in the metal binding situation at pHs above pI. Our results may indicate that the oligomerized MT-IA form binds additional Cd atoms than the expected number of seven per monomer.     

A dispersive liquid–liquid micellar microextraction for the determination of pharmaceutical compounds in wastewaters using ultra‐high‐performace liquid chromatography with DAD detection

A dispersive liquid–liquid micellar microextraction (DLLMME) method coupled with ultra‐high‐performance liquid chromatography (UHPLC) using Diode Array Detector (DAD) detector was developed for the analysis of five pharmaceutical compounds of different nature in wastewaters. A micellar solution of a surfactant, polidocanol, as extraction solvent (100 μL) and chloroform as dispersive solvent (200 μL) were used to extract and preconcentrate the target analytes. Samples were heated above critical temperature and the cloudy solution was centrifuged. After removing the chloroform, the reduced volume of surfactant was then injected in the UHPLC system. In order to obtain high extraction efficiency, the parameters affecting the liquid‐phase microextraction, such as time and temperature extraction, ionic strength and surfactant and organic solvent volume, were optimized using an experimental design. Under the optimized conditions, this procedure allows enrichment factors of up to 47‐fold. The detection limit of the method ranged from 0.1 to 2.0 µg/L for the different pharmaceuticals. Relative standard deviations were <26% for all compounds. The procedure was applied to samples from final effluent collected from wastewater treatment plants in Las Palmas de Gran Canaria (Spain), and two compounds were measured at 67 and 113 µg/L in one of them. Copyright © 2014 John Wiley & Sons, Ltd.     

两步沉淀法制备耐高温和优异还原性能CeO2材料

分别采用两种沉淀方法制备了CeO2:以传统的氨水为沉淀剂,在氨水沉淀法前引入碳铵沉淀步骤(两步沉淀法)。采用热重-差热(TG-DTA)、傅里叶变换红外(FTIR)、X光电子能谱(XPS)等手段对沉淀及其分解过程进行了研究。结果表明,在两步沉淀法中的第一步,碳酸物种为主要沉淀物种,而在第二步中被氢氧根取代。X射线衍射(XRD)和透射电子显微镜(TEM)结果表明,两步沉淀法生成的沉淀颗粒粒径更大。通过两步沉淀法制备的CeO2与氨水沉淀相比具有更好的抗高温老化性能和还原性能。经过900℃焙烧3 h后,仍然具有25 m2.g-1和0.11 cm3.g-1的比表面和孔容。     

Quantification of the metal distribution in metallothioneins of the human liver by HPLC coupled with ICP-AES

Fractions containing metallothioneins (MT's), extracted from the liver cytosol of humans, were analysed to determine the complete distribution pattern of the metals copper, cadmium and zinc. Samples of cirrhotic livers which had come from organs removed during transplantation were examined for differences in the trace-element binding pattern. After the extraction of supernatants from the tissue samples, membrane ultrafiltration of the cytosolic solution was carried out to separate all high-molecular proteins with molecular weights >100 kDa. This procedure retains the metal content of the MT's in its initial form, in contrast to the often-used heat treatment of samples, which changes the copper distribution significantly. The MT's themself were isolated using size exclusion and anion exchange chromatography. Their metal content was determined simultaneously on-line by combination with an ICP-AES as element detector. Calibration of the procedure was performed by means of a column by-pass-injection of elemental standards into the separation system. The MT content in the samples was calculated using the determined metal concentrations and the generally accepted metal/protein ratios for Cu (12:1), Cd (7:1) and Zn (7:1). These values were compared with values resulting from a 109Cd-saturation-assay. When various liver samples of different pathogenesis were compared, the highest level of Cu-MT was found in primary biliary cirrhosis.