Florence Pannier

Speciation analysis of selenium in garlic by two-dimensional high-performance liquid chromatography with parallel inductively coupled plasma mass spectrometric and electrospray tandem mass spectrometric detection.

Speciation of selenium in garlic harvested in naturally seleniferous soil was investigated. The sample was leached with water and the aqueous extract was fractionated by preparative size-exclusion chromatography. Selenium was found in only one (low-molecular mass) fraction. The chromatographic purity of the fraction was verified by reversed-phase chromatography with inductively coupled mass spectrometric detection. Again, one major signal accounting for more than 95% of the total selenium was observed. The heartcut fraction containing this compound produced an intense peak in an electrospray mass spectrum with the selenium pattern centered at m/z 313 (80Se). Protonated molecular ions corresponding to the four Se isotopes gave rich fragmentation patterns by collision induced dissociation that allowed the identification of the selenium species to be γ-glutamyl-Se-methylselenocysteine without the need for an authentic standard.

Speciation of seleno compounds in yeast aqueous extracts by three-dimensional liquid chromatography with inductively coupled plasma mass spectrometric and electrospray mass spectrometric detection

A three-dimensional liquid chromatographic purification protocol based on sequential size-exclusion, anion-exchange and cation-exchange separation mechanisms was developed for the mapping of seleno compounds in aqueous yeast extracts. The method allowed the demonstration of the presence of more than 30 different seleno compounds. Semi-preparative size-exclusion and anion-exchange chromatography were optimized for maximum resolution using electrospray-compatible buffers in order to purify the compounds for mass spectrometric analysis. Molecular masses were attributed to many of the compounds on the basis of the selenium isotopic pattern in the electrospray mass spectra and of the collision-induced fragmentation patterns. Limitations preventing the ultimate identification of the selenium species detected are discussed.

Selenate bioaccumulation and toxicity in Chlamydomonas reinhardtii: Influence of ambient sulphate ion concentration

The aim of the present study was to investigate selenate toxicity in the unicellular green algae Chlamydomonas reinhardtii as a function of sulphate ion concentration and the relationship with intracellular bioaccumulation. The toxicity of selenate was evaluated by measuring the effect of different selenate concentrations on algal growth during a 96h exposure period. A non-linear regression according to the Hill model was used to describe the dose-effect relationship and estimate the effect concentrations (EC) of selenate. EC(50) values of 0.40[0.24-0.52]micromolL(-1) and of 3.10[1.65-4.86]micromolL(-1) of ambient selenate were obtained, at 8 and 80micromolL(-1) of sulphate ions in the medium, respectively. For non-toxic and low-level ambient selenate concentrations, bioaccumulation in presence of 80micromolL(-1) was one tenth that of 8micromolL(-1) of sulphate ions. When expressed as intracellular selenium burden, EC(50) values determined at 8 and 80micromolL(-1) of sulphate ions were not significantly different (126 and 67nmolSe.10(9)cells(-1), respectively). In conclusion, toxicity appeared to be correlated to selenate bioaccumulation which suggests that toxicity must be linked to intracellular selenium accumulation that is directly dependent on ambient sulphate ions that may compete with selenate for transport sites.

Quantitative analysis of volatile selenium metabolites in normal urine by headspace solid phase microextraction gas chromatography-inductively coupled plasma mass spectrometry.

The combination of headspace-solid phase microextraction (HS-SPME) and gas chromatography-inductively coupled plasma mass spectrometry (GC-ICPMS) was evaluated for the determination of volatile selenium metabolites in normal urine samples, i.e. without selenium supplementation. HS-SPME operating conditions were optimised and a sampling time of 10 min was found to be suitable for simultaneous extraction of dimethylselenide (DMSe) and dimethyldiselenide (DMDSe). The amount of DMSe and DMDSe extracted onto fibre coating was calculated in clean matrix, i.e. Milli-Q water, on the basis of depletion experiments. When applied to normal urine samples, the developed method allowed the detection of four volatile selenium containing species, among which DMSe and DMDSe could be quantified by standard additions.

An HPLC/ICPMS study of the stability of selenosugars in human urine: implications for quantification, sample handling, and storage

We report a study with HPLC/ICPMS on the long-term stability of the major selenium metabolite in human urine, namely methyl 2-acetamido-2-deoxy-1-seleno-β-D-galactopyranoside (selenosugar 1). Three separate experiments were performed of 4–28 weeks duration and incorporating various storage conditions: room temperature, 4 °C, −20 °C, −80 °C, lyophilisation, deoxygenation, or addition of a bactericide (NaN3). Triplicate samples of urine or water, spiked with selenosugar 1 at 200 μg Se L−1, were processed in each case. Selenosugar 1 was stable in water under all investigated conditions. For the urine samples, no significant degradation (<2%) was observed after 17 weeks frozen storage at −80 °C, or after lyophilisation and frozen storage at −20 °C, whereas small quantities of degradation products (ca. 3%) were recorded for frozen storage of wet samples at −20 °C. At 4 °C, the selenosugar was essentially unchanged after storage for up to 2 weeks, but clear losses were observed thereafter ranging up to 75% loss after 28 weeks. Several decomposition products were detected by HPLC/ICPMS, one of which was identified as dimethyl diselenide. Although present as only a trace constituent in the urine, dimethyl diselenide was recorded as a large HPLC peak, presumably because of a marked vapour enhancement effect due to more efficient transfer of volatile analytes to the plasma. In addition, total Se analyses revealed that Se was lost from the solutions during storage/handling, presumably as volatile species. Qualitative analysis of volatile species using head space sampling with solid phase microextraction followed by GC/MIP-AES and GC/MS revealed the presence of dimethyl selenide and dimethyl diselenide, based on comparison with standard compounds, and indicated the presence of dimethyl selenylsulfide based on comparison with literature data. The stability of selenosugar 1 and its isomer methyl 2-acetamido-2-deoxy-1-seleno-β-D-glucopyranoside (selenosugar 2), which occurs as a minor species in urine, was also investigated under room temperature storage in the presence and absence of light. Although both species were moderately stable when stored in the dark, their degradation was rapid in the light with clear losses recorded within three days. The work indicates that urine samples should be cooled immediately after collection, and that they may be stored at 4 °C (often the easiest way) for up to 2 weeks before analysis with no appreciable loss of selenosugar. For longer-term storage, urine samples should be kept at −80 °C or, when such facilities are not available, at −20 °C after lyophilisation. The study has also revealed potential quantification problems in Se speciation analysis resulting from different responses for Se species during ICPMS analysis.

Speciation of arsenic in plants by HPLC-HG-AFS: extraction optimisation on CRM materials and application to cultivated samples

A recently developed method for the determination of arsenic species (arsenite, arsenate, monomethylarsonate, MMAA, and dimethylarsinate, DMAA) has been applied to the study of arsenic speciation in plants. This method uses ion-exchange liquid chromatography coupled on-line to atomic fluorescence spectrometry through continuous hydride generation. Various extraction procedures have been studied in detail using three plant certified reference materials. None of the procedures tested revealed fully satisfying results with all kinds of plant samples; microwave assisted extraction with 0.3 mol dm-3 orthophosphoric acid was found to be the most convenient for dealing with terrestrial plants. Species stability appears good. This method was applied to real world cultivated plant parts. Arsenate appears to predominate in soils, roots and leaves; unidentified species (probably arsenosugars) play an important role (60%) in rice fruits. Carrot was found to be the most contaminated edible plant part, containing 1 mg kg-1 essentially as arsenate species. MMAA was detected in all soils and some plant parts especially shallots at low levels, whereas DMAA was found only in one soil sample and in hot pepper leaves. Arsenite is a minor component of all soils; it is also present in some plant parts at low levels. However, no evident relationships were found between As speciation in the various plant parts and much more detailed studies will be necessary to elucidate As behaviour in plants.

Speciation analysis of antimony in marine biota by HPLC-(UV)-HG-AFS: Extraction procedures and stability of antimony species

Speciation analysis of antimony in marine biota is not well documented, and no specific extraction procedure of antimony species from algae and mollusk samples can be found in the literature. This work presents a suitable methodology for the speciation of antimony in marine biota (algae and mollusk samples). The extraction efficiency of total antimony and the stability of Sb(III), Sb(V) and trimethylantimony(V) in different extraction media (water at 25 and 90 degrees C, methanol, EDTA and citric acid) were evaluated by analyzing the algae Macrosystis integrifolia (0.55+/-0.04mugSbg(-1)) and the mollusk Mytilus edulis (0.23+/-0.01mugSbg(-1)). The speciation analysis was performed by anion exchange liquid chromatography (post-column photo-oxidation) and hydride generation atomic fluorescence spectrometry as detection system (HPLC-(UV)-HG-AFS). Results demonstrated that, based on the extraction yield and the stability, EDTA proved to be the best extracting solution for the speciation analysis of antimony in these matrices. The selected procedure was applied to antimony speciation in different algae samples collected from the Chilean coast. Only the inorganic Sb(V) and Sb(III) species were detected in the extracts. In all analyzed algae the sum of total antimony extracted (determined in the extracts after digestion) and the antimony present in the residue was in good agreement with the total antimony concentration determined by HG-AFS. However, in some extracts the sum of antimony species detected was lower than the total extracted, revealing the presence of unknown antimony species, possibly retained on the column or not detected by HPLC-(UV)-HG-AFS. Further work must be carried out to elucidate the identity of these unknown species of antimony.

Advantages of hydride generation interface for selenium speciation in waters by high performance liquid chromatography–inductively coupled plasma mass spectrometry coupling

This paper focuses on the analytical performance improvement of the coupled technique HPLC-ICPMS using on-line collision/reaction cell technology for selenium elemental and speciation analyses at the ng (Se) l(-1) level in aquatic environment. Collision/reaction cell operating parameters were optimised, resulting in selected conditions of 5.5 ml min(-1) H(2) and 0.5 ml min(-1) He mixture. The detection limits obtained were around 5 ng (Se) l(-1) for total analysis, and between 7 and 15 ng (Se) l(-1) depending on the species for speciation analysis. The capability of UV irradiation-hydride generation interfacing to increase detector sensitivity was also evaluated for speciation analysis. The detection limits obtained were in the range 2-8 ng (Se) l(-1) depending on the species. Moreover, such interface allowed to prevent bromine introduction to the ICPMS which is particularly convenient for selenium trace analysis in natural waters as (80)Se is preserved free from BrH interferences. The developed method was validated using certified water with low selenium content (TM Rain 95, NWRI, Canada) and applied to the analysis of different waters.

Field study of time-dependent selenium partitioning in soils using isotopically enriched stable selenite tracer

A better understanding of selenium fate in soils at both short and long time scales is mandatory to consolidate risk assessment models relevant for managing both contamination and soil fertilization issues. The purpose of this study was thus to investigate Se retention processes and their kinetics by monitoring time-dependent distribution/speciation changes of both ambient and freshly added Se, in the form of stable enriched selenite-77, over a 2-years field experiment. This study clearly illustrates the complex reactivity of selenium in soil considering three methodologically defined fractions (i.e. soluble, exchangeable, organic). Time-dependent redistribution of Se-77 within solid-phases having different reactivity could be described as a combination of chemical and diffusion controlled processes leading to its stronger retention. Experimental data and their kinetic modeling evidenced that transfer towards less labile bearing phases are controlled by slow processes limiting the overall sorption of Se in soils. These results were used to estimate time needed for (77)Se to reach the distribution of naturally present selenium which may extend up to several decades. Ambient Se speciation accounted for 60% to 100% of unidentified species as function of soil type whereas (77)Se(IV) remained the more abundant species after 2-years field experiment. Modeling Se in the long-term without taking account these slow sorption kinetics would thus result in underestimation of Se retention. When using models based on Kd distribution coefficient, they should be at least reliant on ambient Se which is supposed to be at equilibrium.

Validation of a reference measurement procedure for the assessment of selenomethionine in nutritional supplements.

The development of a metrological protocol in order to enhance traceability in speciation analysis of selenium is presented. The measurement procedure is applied for the determination of selenomethionine and total selenium in two nutritional supplements, in particular certified reference material to validate the method. Extraction protocols are tested for their capability to leach out total selenium and selenomethionine from the samples. Speciation analysis is realised with the use of HPLC-ICP-MS. Isotope dilution is employed to determine the analytes contents. An uncertainty budget is elaborated with the method recommended by the Guide to the expression of Uncertainty in Measurement (GUM).