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PubMed1986

Detection and speciation of common cell culture mycoplasmas by an enzyme-linked immunosorbent assay with biotin-avidin amplification and microporous membrane solid phase.

An enzyme-linked immunosorbent assay (ELISA) was developed in order to serve in detecting and speciating mycoplasmas isolated from cell cultures. Its main features included a biotin-streptavidin amplification step and a solid phase consisting of a microporous membrane. Cell samples in the form of suspensions were applied to nitrocellulose or ion exchange membranes immobilized in commercially-available microtiter, multiwell manifolds. The blocking buffer contained 1% purified alpha-casein. The primary antibodies were monoclonal and the polyclonal secondary antibody was biotinylated. The enzyme utilized was streptavidin-horseradish peroxidase. The substrate-dye complex consisted of either 4-chloro-1-naphthol and hydrogen peroxide or ortho phenylene diamine (OPD) and hydrogen peroxide. The presence of homologous antiserum in the reaction sequence gave clearly visible, colored reactions on the membrane when 50 ul with approximately 10(5) or more cfu/ml were present. This new biotin-avidin microporous membrane (BAMM-ELISA) test can be used both to detect mycoplasmas and to speciate them. The BAMM-ELISA is simple, rapid, sensitive, specific and economical. As such, it has potential for aiding in the control of mycoplasma contamination in cell culture, and could prove useful in clinical diagnostic applications as well.

Animals↗
PubMed1994

Radiotracer method in the study of environmental speciation and migration of contaminants.

Principles, advantages, and limitations of the use of radiotracer method for the analysis of speciation and migration of contaminants in the environment are briefly discussed. Several recent examples of use in the author's laboratory are given: development of the separation method for methylmercury and inorganic mercury in hair, analysis of the speciation of cadmium in soil solutions, study of the interaction of 137Cs and 58Co with suspended sediments in river water, and determination of input data for mathematical modeling of radiocesium migration in a small river.

Radioactive Pollutants↗
PubMed2002

Speciation of mercury, tin, and lead compounds by gas chromatography with microwave-induced plasma and atomic-emission detection (GC-MIP-AED).

Because of their high toxicity and widespread distribution, the reliable selective quantification of alkyl and aryl species containing mercury, tin, or lead has been one of the goals of speciation analysis in recent years. Since becoming commercially available, GC-MIP-AED has been one of the most-used tools in this work. In this paper, the value and limitations of GC-MIP-AED for the speciation of Hg, Sn, and Pb compounds in environmental samples are reviewed and compared with the analytical characteristics of other hyphenated GC-based techniques. Because quantification of Hg, Sn, and Pb species by GC techniques normally requires complex sample preparation involving several steps, the effect of sample-preparation methods on the accuracy and precision of the results is discussed. Finally, we describe the current status of a rapid, low-cost GC-MIP-PED system specifically designed for routine quantification of Hg, Sn, and Pb species in environmental control laboratories.

Journal Article↗
PubMed2001

Improving selenium extraction by sequential enzymatic processes for Se-speciation of selenium-enriched Agaricus bisporus.

Sample preparation methods based on the use of proteolytic and cell wall digesting enzymes for the speciation analysis of selenized mushroom were investigated. The sample (Agaricus bisporus; 160 microg total Se per g sample) was grown on compost supplemented with selenized yeast. Experiments were carried out to elucidate the possible role of the cell wall digesting enzymes--Lysing enzyme and Driselase--in the improvement of extraction efficiency with and without inhibiting proteolysis during cell wall digestion. A 3-step procedure applying Lysing enzyme and pronase gave the highest extraction efficiency (89%); however, the best species recovery was achieved by a one-step proteolytic procedure. All the procedures of selenium speciation were controlled by independent ICP-AES analysis measuring the total amount of selenium.

Agaricus↗
PubMed2002

Overview of the methodology of nuclear analytical techniques for speciation studies of trace elements in the biological and environmental sciences.

Recent achievements in speciation studies of trace elements in the biological and environmental sciences by nuclear analytical techniques, mainly molecular activation analysis, position-sensitive spectrometry with a variety of exciting sources, and synchronous radiation-based analytical techniques (although radioisotope or enriched stable isotope-based speciation techniques are also used), particularly in our laboratory, are outlined. In this paper the merits and drawbacks of the nuclear analytical techniques are discussed, as are reagent blanks, contamination, and artifacts.

Animals↗
PubMed2002

Multidimensional approaches in biochemical speciation analysis.

An understanding of the mechanisms controlling the essentiality and toxicity of trace elements in biological systems at the molecular level depends critically on the possibility of the identification, characterization, and quantification of chemical forms of these elements involved in life processes. Hyphenated techniques based on the combination of (electro)chromatography with ICP MS have become a routine tool for the analysis for metallospecies present in biological tissues. Finer analytical information on the true (down to individual species) speciation of trace elements in living organisms can be obtained by adding additional dimensions to the separation and detection steps, consisting of a sequential use of different HPLC separation mechanisms and capillary electrophoresis at the separation level, and of the use of electrospray MS, including collision induced dissociation MS, on the detection level. The value of the instrumental analytical data is decisively enhanced by the complementary use of molecular biology approaches involving gene identification, cloning and in vitro reproduction of the metal-controlled processes. A brief summary of the recent progress in biochemical speciation analysis is presented in the context of the latest research carried out in the authors' laboratory.

Biochemistry↗
PubMed2002

Trace element speciation in largemouth bass ( Micropterus salmoides) from a fly ash settling basin by liquid chromatography-ICP-MS.

Analytical techniques used to examine the chemical speciation of multiple trace elements are important for the investigation of biological systems. Size exclusion chromatography (SEC) coupled to ICP-MS was used to investigate the speciation of Se, As, Cu, Cd and Zn in tissue extracts from a largemouth bass (Micropterus salmoides) collected from a coal fly ash basin and results were compared to a largemouth bass collected at a reference site. Using a Biosil SEC column, with an effective separation range of 100-7 KDa, Cu, Zn and Cd were shown to be bound to metallothionein (MT) in the liver, gill and, to a lesser extent, gonad tissue extract. In liver, muscle and gill of the ash basin bass, Se was predominantly present as low molecular weight species. Only in the gonad extract was the major fraction of Se associated with high molecular weight species. For the liver and gill extracts, further SEC-ICP-MS on a column with an effective separation range of 7000-500 Da was performed, but Se species still eluted near the total volume of the column suggesting a low molecular weight organic or inorganic species. Ion chromatography (IC)-ICP-MS using an AS7 column and HNO(3) gradient elution indicated that the Se and As species in the liver and gill extracts had similar retention times but these retention times did not correspond to retention times for As(III), As(V), dimethylarsenate, arsenobetaine, Se(IV), Se(VI), seleno-methionine, or seleno-cystine.

Animals↗
PubMed2002

Electrothermal vaporisation ICP-mass spectrometry (ETV-ICP-MS) for the determination and speciation of trace elements in solid samples - A review of real-life applications from the author's lab.

The use of electrothermal vaporisation (ETV) from a graphite furnace as a means of sample introduction in inductively coupled plasma mass spectrometry (ICP-MS) permits the direct analysis of solid samples. A multi-step furnace temperature programme is used to separate the vaporisation of the target element(s) and of the matrix components from one another. Sometimes, a chemical modifier is used to enable a higher thermal pre-treatment temperature, by avoiding premature analyte losses (stabilisation) or promoting the selective volatilisation of matrix components. In almost all instances, accurate results can be obtained via external calibration or single standard addition using an aqueous standard solution. Absolute limits of detection are typically ~1 pg, which corresponds to 1 ng/g for a typical sample mass of 1 mg. Real-life applications carried out in the author's lab are used to illustrate the utility of this approach. These applications aim at trace element determination in industrial and environmental materials. The industrial materials analysed include different types of plastics - Carilon, polyethylene, poly(ethyleneterephtalate) and polyamide - and photo- and thermographic materials. As samples from environmental origin, plant material, animal tissue and sediments were investigated. Some applications aimed at a multi-element determination, while in other, the content of a single, but often challenging, element (e.g., Si or S) had to be measured. ETV-ICP-MS was also used in elemental speciation studies. Separation of Se-containing proteins was accomplished using polyacrylamide gel electrophoresis (PAGE). Subsequent quantification of the Se content in the protein spots was carried out using ETV-ICP-MS. As the volatilisation of methylmercury and inorganic mercury could be separated from one another with respect to time, no chromatographic or electrophoretic separation procedure was required, but ETV-ICP-MS as such sufficed for Hg speciation in fish tissue.

Mass Spectrometry↗
PubMed2003

Preparation, homogeneity and stability studies of a candidate LRM for Se speciation.

A laboratory reference material (LRM) was prepared from Brazil nuts (Bertholletia excelsa) for quality control (QC) purposes of selenium speciation. The preparation of this LRM led through the usual operation steps applied during routine reference material production from biota samples-preparation of the raw material, homogenisation, storage design, checking of homogeneity, microbiological status and possible irradiation effects, and monitoring the species stability vs time at different storage temperatures. The selenium speciation studies to check species stability were carried out on a HPLC-UV-HG-AFS measurement set-up. Special attention was paid to the correct identification of selenium species by applying independent HPLC separation techniques (ion-pairing and anion-exchange chromatography). The concentration of selenomethionine (SeMet) and total Se content were quantified (79.9 microg g(-1) (calculated as Se) and 82.9 microg g(-1), respectively). The homogeneity and stability of this candidate reference material passed the relevant tests recommended by Bureau Communautaire de Référence (BCR).

Journal Article↗
PubMed2003

Speciation of Co(II), Co(III), and Cu(II) in ethylenediamine solutions by capillary electrophoresis.

A capillary electrophoretic (CE) method for the speciation of Co(II), Co(III), and Cu(II) in electroless copper-plating baths containing ethylenediamine (En) has been developed. The method is based on the selective pre-capillary derivatization of Co(II) with 1,10-phenanthroline (Phen) followed by CE separation of stable [CoPhen(3)](2+), [CoEn(3)](3+), and [CuEn(2)](2+) chelates. The proposed derivatization procedure protects Co(II) from oxidation by dissolved oxygen and enables rapid determination of all three metal species within a single run. The optimized separations were carried out in a fused silica capillary (57 cmx75-microm I.D.) filled with an ethylenediamine sulfate electrolyte (20 mmol L(-1) H(2)SO(4), pH 7.0 with En, applied voltage +30 kV) using direct UV detection at 214 nm. The detection limits for a signal-to-noise ratio of 3 and 10 s, hydrodynamic injections were 5x10(-6) mol L(-1) for Cu(II), 1x10(-6) mol L(-1) for Co(III), and 4x10(-7) mol L(-1) for Co(II). Application of the method to the speciation of Co(II), Co(III), and Cu(II) in copper-plating bath samples is also demonstrated.

Journal Article↗
PubMed2004

Mercury speciation in thawed out and refrozen fish samples by gas chromatography coupled to inductively coupled plasma mass spectrometry and atomic fluorescence spectroscopy.

Different sub-sampling procedures were applied for the determination of mercury species (as total mercury Hg, methylmercury MeHg+ and inorganic mercury Hg2+) in frozen fish meat. Analyses were carried out by two different techniques. After the sample material was pre-treated by microwave digestion, atomic fluorescence spectroscopy (AFS) was used for the determination of total Hg. Speciation analysis was performed according to the following procedure: dissolution of sample material in tetramethylammonium hydroxide (TMAH), derivatisation with sodium tetraethylborate (NaBEt4), extraction into isooctane and measurement with gas chromatography inductively coupled plasma mass spectrometry (GC-ICPMS) for the identification and quantification of methylmercury (MeHg+) and inorganic mercury (Hg2+). The concentration range of total Hg measured in the shark fillets is between 0.9 and 3.6 microg g(-1) thawed out shark fillet. Speciation analysis leads to > or =94% Hg present as MeHg+. Homogeneity, storage conditions and stability of analytical species and sample materials have great influence on analytical results. Sub-sampling of half-frozen/partly thawed out fish and analysis lead to significantly different concentrations, which are on average a factor of two lower.

Animals↗
PubMed2004

Preparation of trout liver microsomes for iron speciation in P-450 enzymes by AE-FPLC with ICP-(ORS)MS detection.

Cytochromes P-450 are members of a superfamily of hemoproteins involved in the oxidative metabolism of various physiological and xenobiotic compounds in eukaryotes and prokaryotes. The multiplicity of this group of enzymes has been widely studied by chromatographic techniques, mainly high-performance liquid chromatography (HPLC). Because these enzymes are membrane-bound proteins, sample preparation for chromatographic separation of P-450 enzymes requires a solubilization step. The sample-preparation procedures are critical, because detergents affect not only the efficiency of protein solubilization but also their further chromatographic resolution. Trout liver microsomes have been taken here as a model sample to investigate iron speciation in cytochrome P-450. Trouts were treated intraperitoneally with beta-naphthoflavone, a potent inducer of some P-450 enzymes, and a microsomal suspension containing 7.4+/-0.1 nmol mL(-1) P-450 enzymes was obtained by ultracentrifugation. Lubrol PX was selected as detergent for solubilization, resulting in about 90% solubilization recovery. The solubilized cytochromes P-450 were further separated by AE-FPLC, with UV detection, or coupled to ICP-MS with an octapole reaction system, ICP-(ORS)MS (monitoring Fe signals at masses 54, 56, and 57). A sampling procedure and chromatographic conditions are developed and were successfully applied to iron speciation in trout liver P-450 enzymes. ICP-(ORS)MS detection of P-450 enzymes is Fe-specific and so will give accurate information on the prosthetic group of the protein, which can constitute an advantageous alternative to classical methods for detection of these hemoproteins.

Animals↗
PubMed2005

Speciation analysis of inorganic antimony in soil using HPLC-ID-ICP-MS.

Speciation analysis of Sb(III) and Sb(V) in a soil sample was performed through extraction and on-line isotope dilution concentration determination after a chromatographic separation. The total Sb concentration found in a through traffic contaminated soil sample was (4.17 microg g(-1), 0.3 microg g(-1) SD, n=6). It was determined using ICP-MS after soil digestion using the sodium peroxide sintering method. The optimized extraction procedure for speciation analysis was carried out using 100 mmol L(-1) citric acid at pH 2.08 by applying an ultrasonic bath for 45 min at room temperature. The effects of citric acid concentration (0-500 mmol L(-1)), pH (1-6), and temperature (30-60 degrees C) on inorganic antimony species distribution in the examined sample were studied and optimized. The separation of Sb(III) and Sb(V) was achieved using an anion exchange column (PRP-X100) and 10 mmol L(-1) EDTA and 1 mmol L(-1) phthalic acid at pH 4.5 as a mobile phase. The eluent from the HPLC was mixed with an enriched (94.2%) (123)Sb spike solution that was pumped by a peristaltic pump with a constant flow rate (0.5 mL min(-1)) in a three-way valve. The blend passed directly to the Conikal nebulizer of the ICP-MS. By using the above extraction procedure and methodology, 43.2% Sb(V) (2.9% RSD, n=3) and 6.0% Sb(III) (1.3% RSD, n=3) of total Sb found in the sample could be detected. The detection limits achieved by the proposed method were 20 ng L(-1) and 65 ng L(-1) for Sb(V) and Sb(III), respectively. The precision, evaluated by using RSD with 100 ng L(-1) calibration solutions, was 2.7% and 3.2% (n=6) for Sb(V) and Sb(III), respectively, in aqueous solutions.

Antimony↗
PubMed2005

An attempt to differentiate HPLC-ICP-MS selenium speciation in natural and selenised Agaricus mushrooms using different species extraction procedures.

Total determination and speciation analysis of Se in commercial and selenised Agaricus mushrooms have been performed to investigate the Se species naturally occurring in non-enriched mushrooms as well as those present in specimens grown in a Se-enriched medium. Mushroom aqueous and enzymatic extracts have been analysed by three complementary chromatographic separation mechanisms (size-exclusion, anion-exchange and reversed-phase) coupled to an inductively coupled plasma mass spectrometer with an octopole reaction system. Post-column isotope dilution analysis has been used on-line with the separations for quantification of the Se species eluted. The 78Se-to-77Se isotope ratio was monitored after adequate corrections for both total determinations and Se species quantitative speciation. The results showed marked differences not only in total Se contents but also in Se species found in the two types of Agaricus mushrooms investigated. Selenomethionine was detected in both of them (free in commercial mushrooms and incorporated into proteins in selenised ones) together with a number of unknown selenocompounds.

Agaricus↗
PubMed2005

Arsenic speciation in water samples containing high levels of copper: removal of copper interference affecting arsine generation by continuous flow solid phase chelation.

A simple continuous flow method is proposed to eliminate copper interference in arsenic speciation by hydride generation, based on the selective retention of this interfering ion in an iminodiacetate chelating resin previous to the hydride generation process. The arsines generated were cold trapped and measured by ICP/OES. The proposed method allows about 98% of the copper present in the samples to be removed. Minor co-retention of As(V) was observed as a result of electrostatic interaction between the arsenate anion and the nitrogen of the iminodiacetate group of the chelating resin Muromac A-1, the charge distribution of which is modified when copper is chelated. The species As(III), MMA and DMA were not retained in the microcolumn, probably because these species are mainly in the molecular form at the working pH value (4.5). In synthetic samples containing 50 microg l(-1) of each arsenic species together with 100 mg l(-1) copper, the recoveries obtained were: As(V) 97.6%, As(III) 100%, MMA 99.8%, and DMA 99.9%. The method was applied to arsenic speciation in river water samples containing high levels of copper.

Journal Article↗
PubMed2005

Speciation of aluminium in tea infusions by use of SEC and FPLC with ICP-OES and ES-MS-MS detection.

Speciation of Al in tea infusions was studied by size exclusion chromatography (SEC) and anion-exchange fast protein liquid chromatography (FPLC). Fractions were collected throughout the chromatographic separations and Al was determined "off line" by inductively coupled plasma optical emission spectroscopy (ICP-OES). Black, green, and red tea samples were investigated. The total concentration of Al in tea infusions was determined by ICP-OES and ranged between 0.5 and 4 mg dm(-3). The pH of tea infusions ranged between 5.3 and 5.5. Data from SEC-ICP-OES analysis indicated that 10-35% of total Al in tea infusions was eluted at a retention volume corresponding to a molecular mass of approximately 3800 Da. The remaining Al was adsorbed on the column resin. The same tea infusions were also analysed by anion-exchange FPLC-ICP-OES. It was found experimentally that the same percentage of total Al as from the SEC column was eluted at a retention volume that corresponded to negatively charged Al-citrate. The remaining Al was adsorbed on the column resin. Identification of Al-binding ligands eluting under the chromatographic peak was performed by electrospray ionisation tandem mass spectrometry (ES-MS-MS) analysis. It was proven that ionic Al species in tea infusions (10-35% of the total Al) corresponded to negatively charged Al-citrate. The remaining species that was adsorbed on the SEC or FPLC columns was most probably bound to phenolic compounds. Speciation of Al in tea with milk or lemon was also studied. Results for tea with milk indicated that Al-citrate was not transformed and that approximately 60% of total Al was transformed into high-molecular-mass Al species. This fraction was subjected to sodium dodecyl sulfonate polyacryl gel electrophoresis (SDS-PAGE). The results indicated that Al was occluded by milk proteins (mostly caseins). When citric acid was added to tea infusions the percentage of negatively charged Al-citrate remained either the same or increased to 40% of total Al.

Aluminum↗
PubMed2005

Pressurised solvent extraction for organotin speciation in vegetable matrices.

Because organotin compounds (OTC) are widely used in many fields of activity, they have become an ubiquitous environmental presence. The presence of organotins in the environment impacts upon food safety, making it important to monitor the levels of organotin pesticides in fruits and vegetables. Nevertheless, only a few studies have been published on organotin speciation in plants. The objective of the present study was to evaluate and optimise a specific procedure based on pressurised solvent extraction (PSE) that is suitable for monitoring organotin content in vegetables. In ASE, solvents are used at elevated temperatures and pressures to increase the rate and efficiency of the extraction process. The results from this procedure were compared to those from the technique usually employed, solid/liquid extraction (SLE) performed in an acidic solvent by mechanical shaking. Three extracting solutions were tested-methanol, ethyl acetate and a mixture of methanol and ethyl acetate-and the mixture was found to give the most quantitative results while preserving the speciation. French bean and lettuce leaves as well as potato tubers were used as the plant materials. These vegetables were considered because they are the vegetables consumed in the most quantities in Europe. The study focuses on trisubstituted OTCs, which are the most toxic tin species. The samples were spiked with four trisubstituted organotins: tributyltin (TBT), triphenyltin (TPhT), tricyclohexyltin (TcHexT) and trioctyltin (TOcT). The influence of the pressure and the temperature of the PSE on the quantitativity of the process and on species preservation was evaluated using the experimental design methodology. The optimised PSE allowed detection limits down to 1-2 ng (Sn) g(-1) to be reached. These are higher than those obtained by SLE (0.1-1 ng (Sn) g(-1)). Although the repeatability is similar for both PSE and SLE (2-12% for triorganotin compounds), this appears to be highly time-dependent in the case of SLE. Comparison with SLE confirms that PSE is an interesting tool for vegetable analysis considering the satisfactory OTC preservation and repeatability obtained for a relatively short extraction duration (only 15 min against 2-12 h for SLE).

Environmental Monitoring↗
PubMed2006

Preservation procedures for arsenic speciation in a stream affected by acid mine drainage in southwestern Spain.

A preservation study has been performed for arsenic speciation in surface freshwaters affected by acid mine drainage (AMD), a pollution source characterized by low pH and high metallic content. Two sample preservation procedures described in the literature were attempted using opaque glass containers and refrigeration: i) addition of 0.25 mol L(-1) EDTA to the samples, which maintained the stability of the arsenic species for 3 h; and ii) in situ sample clean-up with a cationic exchange resin, in order to reduce the metallic load, which resulted in a partial co-adsorption of arsenic onto Fe precipitates. A new proposed method was also tried: sample acidification with 6 mol L(-1) HCl followed by in situ clean-up with a cationic exchange resin, which allowed a longer preservation time of at least 48 h. The proposed method was successfully applied to water samples with high arsenic content, taken from the Aguas Agrias Stream (Odiel River Basin, SW Spain), which is severely affected by AMD that originates at the nearby polymetallic sulfide mine of Tharsis. The speciation results obtained by liquid chromatography-hydride generation-atomic fluorescence spectrometry (HPLC-HG-AFS) indicated that during the summer the main arsenic species was As(V) at the hundred microg L(-1) level, followed by DMA (dimethyl arsenic) and As(III) below the ten microg L(-1) level. In winter, As(V) and As(III) increased at least fivefold, whereas the DMA was not detected.

Journal Article↗