Metal-metallothioneins like proteins investigation by heteroatom-tagged proteomics in two different snails as possible sentinel organisms of metal contamination in freshwater ecosystems

Metal speciation analysis in MLPs was carried out in two snails, Marisa cornuarietis and Pomacea bridgesi, in order to investigate them as possible sentinel organisms of heavy metal contamination. To carry out this study snails born in a non-contaminated environment were divided into two groups: a control group and a contaminated one with cadmium administered for 40 days. Subsequently, we investigated the speciation of the induced MLPs in exposed animals in relation to controls. In order to obtain the MLP fraction, cytosols from both snail species where subjected to size-exclusion fractionation, monitoring on-line the metal signal (Cd, Cu and Zn) by ICP-MS while protein elution was followed by on-line UV detection. MLP fraction was then separated by anion-exchange (AE)-FPLC using optimal chromatographic conditions for the separation of the different MLP isoforms in both snail species. Specific detection of separated metalloforms was carried out again by the hyphenation of the AE chromatographic system with ICP-MS. The determination of the amount of metal bound to MLPs was carried out by post-column isotope dilution analysis ICP-MS, finding that the snail M. cornuarietis accumulated higher concentrations of cadmium than P. bridgesi. Thus this first snail could therefore be a better candidate sentinel organism of pollution in natural waters. Identification and characterization of the isoforms separated in M. cornuarietis was carried out for the entire or intact isoforms by MALDI-TOF and then conventional triptic digestion was also carried out to identify the nature of the formed peptides. The presence identification of a MLP isoform of relatively low molecular weight in M. cornuarietis is reported.     

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.     

Study of phytochelatins and other related thiols as complexing biomolecules of As and Cd in wild type and genetically modified Brassica juncea plants

The accumulation of As and Cd in Brassica juncea plants and the formation of complexes of these elements with bioligands such as glutathione and/or phytochelatins (PCs) is studied. The genetic manipulation of these plants to induce higher As and Cd accumulation has been achieved by overexpressing the genes encoding for gamma-glutamyl cysteine synthetase (gamma-ECS) and glutathione synthetase (GS). These two enzymes are responsible for glutathione (GSH) formation in plants, which is the first step in the production of PCs. The biomass produced in both the wild type and the genetically modified plants, has been evaluated. Additionally, the total Cd and As concentration accumulated in the plant tissues was measured by inductively coupled plasma mass spectrometry (ICP-MS) after extraction. Speciation studies on the extracts were conducted using size exclusion liquid chromatography (SEC) coupled online with ICP-MS to monitor As, Cd and S. For further purification of the As fractions, reversed phase high performance liquid chromatography (RP-HPLC) was used. Structural elucidation of the PCs and other thiols, as well as their complexes with As and Cd, was performed by electrospray-quadrupole-time-of-flight (ESI-Q-TOF). In both the Cd and As exposed plants it was possible to observe the presence of oxidized PC2 ([M + H]+, m/z 538), GS-PC2(-Glu) ([M + H]+, m/z 716) as well as reduced GSH ([M + H]+, m/z 308) and oxidized glutathione (GSSG) ([M + H]+, m/z 613). However, only the GS plants exhibited the presence of As(GS)3 complex ([M + H]+, m/z 994) that was further confirmed by MS/MS. This species is reported for the first time in B. juncea plant tissues.     

Multielement trace-element speciation in metal-biomolecules by chromatography coupled with ICP-MS

The state-of-the-art of multi-element speciation in biological materials by hybrid techniques with inductively coupled plasma-mass spectrometric (ICP-MS) detection is critically reviewed with special attention to multi-metal speciation in metallothioneins (MT) and metallothionein-like proteins (MLP). The most common separation techniques used for the purpose include conventional size-exclusion chromatography (SEC) (with off-line detection) and anion-exchange (AE) or reversed-phase (RP) HPLC coupled on-line to ICP-MS. Advantages and limitations of the different commercial element-selective ICP-MS detectors, including the quadrupole (Q), double focusing (DF), and time-of-flight (TOF) mass analysers, are discussed. Procedures reported for speciation analysis, by use of hybrid techniques, for multi-metal speciation in mussel MLP and rabbit liver MT are illustrated with special emphasis on work performed in the authors' laboratory.     

Determination of Cd,Hg, Pb and Se in sediments slurries by isotopic dilution calibration ICP-MS after chemical vapor generation using an on-line system or retention in an electrothermal vaporizer treated with iridium

A method for the determination of Cd, Hg, Pb and Se in sediments reference materials by slurry sampling chemical vapor generation (CVG) using isotopic dilution (ID) calibration and detection by inductively coupled plasma mass spectrometry (ICP-MS) is proposed. Two different systems were used for the investigation: an on-line flow injection system (FI-CVG-ICP-MS) and an off-line system with in situ trapping electrothermal vaporization (CVG-ETV-ICP-MS). About 100 mg of the reference material, ground to a particle size ≤50 μm, was mixed with acid solutions (aqua regia, HF and HCl) in an ultrasonic bath. The enriched isotopes ¹¹¹Cd, ¹⁹⁸Hg, ²⁰⁶Pb and ⁷⁷Se were then added to the slurry in an adequate amount in order to produce an altered isotopic ratio close to 1. For the on-line system, a standing time for the slurry of 12 h before measurement was required, while for the batch system, no standing time is needed to obtain accurate results. The conditions for the formation of the analyte vapor were optimized for the evaluated systems. The following altered isotope ratios were measured: ¹¹¹Cd/¹¹⁴Cd, ¹⁹⁸Hg/¹⁹⁹Hg, ²⁰⁶Pb/²⁰⁸Pb e ⁷⁷Se/⁸²Se. The obtained detection limits in the on-line system, in μg g⁻¹, were: Cd: 0.15; Hg: 0.09; Pb: 6.0 and Se: 0.03. Similar detection limits were obtained with the system that uses the ETV: 0.21 for Hg, 6.0 for Pb and 0.06 μg g⁻¹ for Se. No signal for Cd was obtained in this system. One estuarine, two marine and two river certified sediments were analyzed to check the accuracy. The obtained values by both systems were generally in agreement with the certified concentrations, according to the t-test for a confidence level of 95%, demonstrating that isotope equilibration was attained in the slurries submitted to a chemical vapor generation procedure and detection by ICP-MS. The relative standard deviations were lower than 10%, adequate for slurry analysis. The almost quantitative analytes extractions to the aqueous phase of the slurry must favor equilibration of the added enriched isotope with the isotope in the sample, allowing the use of isotopic dilution calibration for slurry analysis.     

Use of elemental and molecular-mass spectrometry to assess the toxicological effects of inorganic mercury in the mouse Mus musculus

The biochemical response of mice (Mus musculus) to acute subcutaneous inorganic-mercury exposure was assessed over a 14-day period by analyzing cytosolic extracts of the liver, the kidneys, and blood plasma. Integrated metallomic and metabolomic approaches using elemental and molecular-mass spectrometry were used to obtain comprehensive insight into the toxicological effects of mercury regarding its distribution and possible perturbation of metabolic pathways. The metallomic approach involved the use of size-exclusion chromatography (SEC) coupled with multiaffinity chromatography inductively coupled plasma-mass spectrometry (ICP-MS) and isotopic-dilution analysis. The metabolomic approach involved the direct infusion of polar and lipophilic tissue extracts into a mass spectrometer (DIMS) in the positive and negative acquisition mode (ESI+and ESI?). The use of SEC–ICP-MS enabled us to detect changes in the metalloproteome in the liver and the kidneys during the exposure period, and revealed that interactions between Hg and endogenous Cu and Zn adversely affected the homeostasis of these essential metals. The detection of an Hg–Se detoxification product in mouse plasma substantiated the known interaction between Hg and Se in mammals. Use of DIMS in conjunction with partial-least-squares discriminant analysis (PLS-DA) uncovered time-dependent changes of endogenous metabolites over time, corroborated by histopathology investigation of specific mouse tissues. The perturbations of endogenous metabolic profiles were explained in terms of the adverse effect of mercury on energy metabolism (e.g. glycolysis, Krebs cycle), the degradation of membrane phospholipids (apoptosis), and increased levels of specific lipids in plasma. In summary, use of an SEC–ICP-MS-based metallomics approach in conjunction with molecular-mass-spectrometry-based metabolomics is revealed as a promising strategy to more comprehensively investigate the toxicological effects of harmful environmental pollutants and xenobiotics. ?      

Fractionation metallothionein-like proteins in mussels with on line metal detection by high performance liquid chromatography-inductively coupled plasma-optical emission spectrometry

A rapid method for the determination of Al, Ba, Cu, Fe, Mn, Sr and Zn binding metallothionein-like proteins (MLPs) in mussels (Mytilus galloprovincialis) by inductively coupled plasma-optical emission spectrometry was developed. The method uses a short column (8 mm × 75 mm) anion exchange high performance liquid chromatography (HPLC) with inductively coupled plasma-optical emission spectrometric (ICP-OES) detection. Working in isocratic mode (75 mM Tris-HCl at pH 7.4, flow rate at 0.8 ml min⁻¹), two major MLPs isoforms (MLP-1 and MLP-2) can be separated in ten minutes. The distribution of basal metals binding MLPs was assessed by on line HPLC and ICP-OES, while the basal contents of metals binding MLPs were obtained after off line HPLC and ICP-OES (collection of the two major fractions and multi-element determination by ICP-OES). The calculated LODs were 81, 6, 10, 13, 30, 9 and 123 ng g⁻¹ for Al, Ba, Cu, Fe, Mn, Sr and Zn binding MLP-1 and MLP-2, respectively. The repeatability of the over-all method (five different mussel cytosols prepared from the same mussel sample and subjected to the off line HPLC-ICP-OES procedure twice) was from 11.2 for Cu to 16.2% for Zn. The method was finally applied to different raft mussels from Ría de Arousa estuary in order to know basal levels of elements binding MLPs.     

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.     

Determination of free iodide in human serum: Separation from other I-species and quantification in serum pools and individual samples

A method for the determination of free iodide in human serum was developed. For this purpose iodide from pooled serum samples was separated from the organic manner by SEC. The iodide fraction subsequently was freezedried and analyzed by ion chromatography for quantification. Investigations for recovery and precision were carried out and were found to show sufficient results. For quality assurance ICP-MS was taken additionally as an total I-detector [1], using native and iodide-spiked serum samples. The iodide results of ICP-MS as well as those of IC were well corresponding. Iodine containing SEC-fractions from iodide-spiked samples showed no increased I-values except that in the iodide fractions, proving that there was no iodide conversion into other I-species (and vice versa) during the whole procedure.Free iodide from two serum pools of different healthy persons was determined as 2.25 and 2.43 g I^–/L, respectively. The values are related to total iodine levels determined by ICP-MS. For comparative reasons a table of individual iodine and iodide values is presented.Abbreviations IC ion chromatography - ICP-MS inductively coupled plasma mass spectrometry - LPLC low pressure liquid chromatography - PED pulsed electrochemical detector - SEC size exclusion chromatography - RT retention time     

Simultaneous determination of arsenic, selenium and antimony species using HPLC/ICP-MS

A new method for the simultaneous separation and determination of four arsenic species [As(III), As(V), monomethylarsonic acid and dimethylarsinic acid], three selenium species [Se(IV), Se(VI) and selenomethionine] as well as Sb(III) and Sb(V) is presented. The speciation was achieved by on-line coupling of anion exchange high-performance liquid chromatography (HPLC) with inductively coupled plasma mass spectrometry (ICP-MS). Chromatographic parameters such as the composition and pH of the mobile phase were optimised. Limits of detection are below 4.5 μg L^–1 (as element) for Sb(III) and the selenium species and below 0.5 μg L^–1 for the other species. Precisions of retention times were better than 2% RSD and of peak areas better than 8% RSD for all the species investigated.     

Bioaccumulation of metals and induction of metallo‐ thioneins in selected tissues of common carp (Cyprinus carpio L.) co‐exposed to cadmium,mercury and lead

The concentrations of mercury (Hg), cadmium (Cd) and lead (Pb) at various exposure periods were determined in the gill, kidney, liver and muscle of common carp (Cyprinus carpio L.) co‐exposed to 1.0 µg ml^?1 each of Cd²⁺, Hg²⁺ and Pb²⁺ for up to 10 days. Metallothionein fractions (MTs) in these organs were characterized using the hyphenated technique of size‐exclusion chromatography (SEC) and inductively coupled plasma mass spectrometry (ICP‐MS). After 10 days of exposure, maximum toxic metal concentrations of Hg, Cd and Pb were 10.7 (gill), 0.145 (kidney) and 0.112 µg g^?1_dryweight (gill), respectively. The pattern of accumulation of Hg and Pb was in the order gill > kidney > liver > muscle. In the case of Cd, accumulation was in the order kidney > gill > liver > muscle. Cd and Hg binding MTs were significantly induced in the gill, kidney and liver of all the exposure groups in comparison with the control group (p < 0.05), and the amounts of them increased with the longer exposure time. Despite the higher intracellular Hg concentration and the stronger Hg? SH binding affinity, the amount of Cd‐binding MTs was much higher than that of Hg‐binding MTs. The results indicate that MT synthesis in these organs was clearly metal‐specific. MTs in gill may be used as a bio‐marker to detect the metal pollution caused by Hg and Cd. Zinc and copper binding MTs in the organs of the exposed fish were also increased. This may be due to the MTs' important role in the homeostatic regulation of essential metals and their protective role against the acute toxicity of non‐essential metals. Even though there was considerable accumulation of lead in the organs of the exposed fish, Pb‐binding MT synthesis was non‐significant. Copyright © 2006 John Wiley & Sons, Ltd.     

Speciation analysis of platinum antitumoral drugs in impacted tissues

Chemical compounds containing platinum have been employed since 1978 as drugs to beat certain type of tumours. Nevertheless, besides of their exceptional antitumoral properties, these drugs also have important deleterious side effects, such as, nephrotoxicity and ototoxicity.A study of Pt accumulation and a speciation analysis has been performed by ICP-MS in samples from kidney and inner ear in a controlled population of Wistar rats treated with, either, cisplatin, carboplatin or oxaliplatin. The results on Pt accumulation point out to drug structure and not only to Pt content as the responsible for the alteration of organ functionality.Speciation studies in the samples from kidney and inner ear were performed coupling two-dimensional liquid chromatography (2D-LC) to ICP-MS. Size exclusion (SEC) and anion exchange fast protein liquid chromatography (FPLC) was employed for 2D orthogonal separation. After these separations, free drug peaks were not observed in any of the samples.The binding of Pt to biomolecules was demonstrated by SEC and, independently of the drug used, Pt eluted as two main bands with molecular weights of 12 kDa and 25-65 kDa for inner ear samples, and as two different bands with 20 kDa and 50-60 kDa in the samples from kidney. However, the relative band intensity presented important differences for the three drugs. Using the same chromatographic conditions, it was shown that a metallothionein (MT) standard eluted in the same position as some of the cytosolic Pt-biomolecules.High Pt-containing fractions eluting from the SEC column were analysed by anion exchange FPLC after a preconcentration step. Among the different preconcentration methods tested, sample focusing on the head of the FPLC column shows main advantages. In this way, the separation by 2D chromatography of the high molecular Pt-species has been considerably improved.     

Mass spectrometric investigations of the kinetic stability of chromium and copper complexes with humic substances by isotope-labelling experiments

Isotope-labelling exchange experiments were carried out to investigate the kinetic stability of Cr(III) complexes with humic substances (HS). To compare the results with those of an ion, not expected to form kinetically stable HS complexes with respect to its electron configuration, Cu(II) was investigated under the same conditions. HS solutions of different origin were therefore spiked with ⁵³Cr(III) or ⁶⁵Cu(II) after saturation of HS with chromium and copper of natural isotopic composition. In fractions of metal/HS complexes with different molecular weight, obtained by ultrafiltration and HPLC/ICP-MS using size exclusion chromatography (SEC), respectively, the isotope ratios of chromium and copper were determined by ICP and thermal ionisation mass spectrometry. Distinct differences in the isotopic composition of chromium were found in the permeate of the ultrafiltration compared with the corresponding unseparated solution, which indicates kinetically stable Cr(III)/HS complexes. On the other hand, the copper isotopic composition was identical in the permeate and the unseparated solution, which shows that a total exchange of Cu²⁺ ions took place between free and HS complexed copper ions. The SEC/ ICP-MS experiments also resulted in a different isotopic distribution of chromium in the chromatographically separated complexes whereas the copper complexes, separated by SEC, showed identical isotopic composition. The kinetic stability of Cr(III)/HS complexes could be explained by the d³ electron configuration of Cr³⁺ ions, a fact which is well known from classical Cr(III) complexes, and influences substantially the mobility of this heavy metal in the environment.     

Investigation of mercury-containing proteins by enriched stable isotopic tracer and size-exclusion chromatography hyphenated to inductively coupled plasma-isotope dilution mass spectrometry

In order to investigate trace mercury-containing proteins in maternal rat and their offspring, a method of enriched stable isotopic tracer (¹⁹⁶Hg and ¹⁹⁸Hg) combined with size-exclusion chromatography (SEC) coupled to inductively coupled plasma-isotope dilution mass spectrometry (ICP-IDMS) was developed. Prior to the analysis, ¹⁹⁶Hg- and ¹⁹⁸Hg-enriched methylmercury was administrated to the pregnant rats. Then the mercury-containing proteins in serum and brain cytosol of the dam and pup rats were separated by size-exclusion columns and the mercury was detected by ICP-MS. The ICP-MS spectrogram of the tracing samples showed significantly elevated ¹⁹⁶Hg and ¹⁹⁸Hg isotopic signals compared with the natural ones, indicating that the detection sensitivity could be increased by the tracer method. The contents of mercury in chromatographic fractions of the dam and pup rat brain cytosol were quantitatively estimated by post-column reverse ID-ICP-MS. The quantitative speciation differences of mercury in brain cytosol between the dam and pup rats were observed, indicating that such studies could be useful for toxicological estimation. Additionally, the isotopic ratio measurement of ¹⁹⁸Hg/²⁰²Hg in the tracing samples could be used to identify the artifact mercury species caused in the analytical procedure. The study demonstrates that the tracer method combined with high-performance liquid chromatography (HPLC)-ICP-IDMS could provide reliably qualitative and quantitative information on mercury-containing proteins in organisms.     

Mass spectrometric investigations of the kinetic stability of chromium and copper complexes with humic substances by isotope-labelling experiments

Isotope-labelling exchange experiments were carried out to investigate the kinetic stability of Cr(III) complexes with humic substances (HS). To compare the results with those of an ion, not expected to form kinetically stable HS complexes with respect to its electron configuration, Cu(II) was investigated under the same conditions. HS solutions of different origin were therefore spiked with ⁵³Cr(III) or ⁶⁵Cu(II) after saturation of HS with chromium and copper of natural isotopic composition. In fractions of metal/HS complexes with different molecular weight, obtained by ultrafiltration and HPLC/ICP-MS using size exclusion chromatography (SEC), respectively, the isotope ratios of chromium and copper were determined by ICP and thermal ionisation mass spectrometry. Distinct differences in the isotopic composition of chromium were found in the permeate of the ultrafiltration compared with the corresponding unseparated solution, which indicates kinetically stable Cr(III)/HS complexes. On the other hand, the copper isotopic composition was identical in the permeate and the unseparated solution, which shows that a total exchange of Cu²⁺ ions took place between free and HS complexed copper ions. The SEC/ ICP-MS experiments also resulted in a different isotopic distribution of chromium in the chromatographically separated complexes whereas the copper complexes, separated by SEC, showed identical isotopic composition. The kinetic stability of Cr(III)/HS complexes could be explained by the d³ electron configuration of Cr³⁺ ions, a fact which is well known from classical Cr(III) complexes, and influences substantially the mobility of this heavy metal in the environment. Received: 7 December 1998 / Revised: 25 March 1999 / Accepted: 27 March 1999     

Analysis of bioaccessible concentration of trace elements in plant based edible materials by INAA and ICPMS methods

The total metal concentration and bioaccessible concentration of Cr, Mn, Fe, Cu, Zn, Se in Momordica charantia, Asparagus racemosus, Terminalia arjuna and Syzyzium cumini were measured by instrumental neutron activation analysis and by inductively coupled plasma mass spectrometry analysis (ICP-MS). The bioaccessible concentrations were determined in the gastrointestinal digest obtained after treating dried powdered samples sequentially in gastric and intestinal fluid of porcine origin at physiological conditions. The bioaccessible concentration of Fe was in the range of 58–67 mg kg^?1, Mn was 10.2–14.6 mg kg^?1, Cu was 3.7–4.8 mg kg^?1 and Zn was 10.6–18.4 mg kg^?1, were within the safety limits set for vegetable food stuff set by Joint FAO/WHO. The bioaccessibility of Zn, an essential element, was high (40–50 %) in M. charantia and in S. cumini. In addition, the total metal contents and bioaccessible concentration of Ni, Se, Cd and Pb in these samples were measured by ICP-MS. The total Cd content in S. cumini (2.6 ± 0.2 mg kg^?1) and its bioaccessible concentration (0.6 mg kg^?1) were strikingly high as compared to the other samples. Though total Hg contents were determined by ICP-MS, but their bioaccessible concentrations were below the detection limit (0.036 mg kg^?1).     

A new methodology involving stable isotope tracer to compare simultaneously short- and long-term selenium mobility in soils

A better understanding of Se fate in soils is required for different environmental issues, such as radioactive waste management or soil fertilization procedures. In these contexts, the mobility and speciation of Se have to be studied at both short and long terms after Se inputs. Here, we present a new methodology to monitor simultaneously the reactivity of added (isotopic enriched tracers) and ambient Se at trace level in soils by high-performance liquid chromatography inductively coupled plasma mass spectrometry (ICP-MS) following specific extractions. To do so, the collision/reaction cell of the ICP-MS instrument and the interference corrections were optimized to measure reliably the four major Se isotopes. To exemplify the method capabilities, the behaviors of added ⁷⁷Se(IV) and ambient Se were followed up in two soils submitted to an ageing process during 3 months. The solid/liquid distribution of added Se reached a steady state after 1 month while its speciation and distribution among soil solid phases were still changing after 3 months. The results clearly demonstrate that slow processes are involved in Se retention and transformation in soils. The usual short-term experiments (<1 month) performed after Se addition are thus not suitable for long-term risk assessment. Interestingly, the behavior of added Se tended to that of ambient Se, suggesting that ambient Se would be useful to infer the fate of Se input over long time scales.

Selenium speciation analysis in a sediment using strong anion exchange and reversed phase chromatography coupled with inductively coupled plasma-mass spectrometry

An analytical procedure for selenium speciation of analysis of selenourea (SeU), selenoethionine (SeE), selenomethionine (SeM), Se(VI), Se(IV), dimethylselenide (dMeSe) and dimethyldiselenide (dMedSe) was developed, based on two complementary liquid chromatography (LC) techniques coupled with inductively coupled plasma-mass spectrometry (ICP-MS). Specifically, strong anion exchange (SAX) chromatography coupled with ICP-MS was used for the separation and quantification of all the earlier mentioned Se compounds, except for the two methyl selenides, which could be separated and determined by reversed phase chromatography coupled with ICP-MS. This procedure was applied to a soil sample from the warm springs area of Thermopyles (Greece). For leaching the Se species from the soil sample, four extraction methods, using water at ambient temperature, hot water, methanol and 0.5 M HCl, were tested for their efficiency of extracting the different Se species. The speciation results obtained by the LC-ICP-MS methods were compared with those obtained by voltammetric techniques. The determination of total selenium in the sample was achieved by graphite furnace atomic absorption spectrometry, as well as by ICP-atomic emission spectrometry, after suitable digestion of the sediment sample.     

Geographical Discrimination of Honeysuckle (Lonicera japonica Thunb.) from China by Characterization of the Stable Isotope Ratio and Multielemental Analysis

A total of 117 honeysuckle (Lonicera japonica Thunb.) samples from four major regions of production in China, including Fengqiu in Henan, Pingyi in Shandong, Julu in Hebei, and Xiushan in Chongqing, were analyzed to determine their geographical origin. δ¹³C, δ¹⁵N, and δ¹⁸O values were determined by isotope ratio mass spectrometry (IRMS), and the contents of 18 elements (Fe, Mn, Cu, Zn, K, Ca, Mg, Pb, Cd, Cr, As, Hg, Se, Sr, Ni, Co, B, and Mo) were measured by inductively coupled plasma optical emission spectrometry (ICP-OES) and inductively coupled plasma mass spectrometry (ICP-MS). Multivariate statistical analysis by analysis of variance (ANOVA), principal component analysis (PCA), and linear discriminant analysis (LDA) were performed. The results showed that there were very significant differences in the stable isotope ratios and elemental concentrations in honeysuckle based on geographical origin, with plants from each region having a unique fingerprint. Discriminant functions were established to distinguish the origin of honeysuckle using suitable indicators including Cd, Cr, As, Hg, Se, Co, Ni, Sr, Fe, δ¹³C, δ¹⁵N, and δ¹⁸O. Cross-validated cases of 95.7% were correctly classified.     

Comparison of no gas and He/H2 cell modes used for reduction of isobaric interferences in selenium speciation by ion chromatography with inductively coupled plasma mass spectrometry

Inductively coupled plasma mass spectrometry (ICP-MS) used for the detection of most common selenium isotopes ⁷⁸Se (23.8%, abundance) and ⁸⁰Se (49.6%, abundance) is interfered in the presence of ³⁸Ar⁴⁰Ar⁺ and ⁴⁰Ar⁴⁰Ar⁺ in argon (Ar) gas. To address this issue, ICP-MS with an octopole reaction system (ORS) was explored for the detection of selenite and selenate, which was separated by anion-exchange chromatography. Results indicate that it was possible to detect ⁷⁸Se using no collusion gas, while to detect ⁸⁰Se a H₂ as the collusion/reaction gas was recommended since the background and noise were significantly reduced using H₂ as the gas. The selenium speciation interested was separated on a new column (G3154/101A, Agilent technologies) within 5 min using a mobile phase containing 10 mM NH₄H₂PO₄ and 20 mM NH₄NO₃ at pH 6.5. Linear plots were obtained in a concentration range of 1–200 μg/L with detection limits less than of 0.4 μg/L for ⁸⁰Se (IV) and 0.6 μg/L for ⁸⁰Se (VI) using H₂ as the reaction gas. Finally, the proposed method was used in the determination of selenium in water and soil.