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Critical assessment of hexavalent chromium species from different solid environmental, industrial and food matrices 

Séby F.,Vacchina V.

Trends in Analytical Chemistry, 2018, in press


The accurate determination of Cr(VI), the most hazardous form of chromium, is an analytical challenge because of its facility to be reduced during all steps of the analytical procedure. This review presents a synthesis of the available studies for the analysis of Cr species in different solid samples (soils/wastes, working atmosphere, industrial and food samples). A particular attention has been paid on Cr(III) and Cr(VI) interconversions that can occur during extraction and efforts made to avoid these reactions. Although the use of NaOH/Na 2 CO 3 solutions with heating at 90-95°C is the more widespread procedure, species transformation can still occur and several studies suggest that speciated isotope dilution (SID) could be a suitable tool for correction of these interconversions.

Quantification of SeMet and SeCys in biological fluids and tissues by liquid chromatography coupled to inductively coupled plasma mass spectrometry (HPLC-ICP MS) 

Vacchina, V., Bierla, K., Spuznar, J., Lobinski, R. 

Methods in Molecular Biology, 2018, 1661, 153-162


Selenium (Se) is an element readily absorbed during the intestinal tract and distributed in the body. In biological fluids, tissues, and animal products, Se is known to be present mainly in the form of a selenoamino-acid (selenomethionine (SeMet) or selenocysteine (SeCys)). Both amino-acids have different biological activities which justifies their discrimination. Here, we describe the method allowing the simultaneous determination of SeMet and SeCys in blood/plasma, animal tissues, milk, and eggs by two-dimensional Liquid Chromatography coupled to Inductively Coupled Plasma Mass Spectrometry (2D HPLC-ICP MS).

Total selenium quantification in biological samples by inductively coupled plasma mass spectrometry (ICP-MS) 

Vacchina, V., Dumont, J. 

Methods in Molecular Biology, 2018, 1661, 145-152


Selenium (Se) is an element readily absorbed during the intestinal tract, distributed in the body by means of blood and excreted mainly by urine or feces. Here, we describe the method allowing the determination of the total Se content in biological tissues and fluids by Inductively Coupled Plasma Mass Spectrometry (ICP MS).


Optimisation of selective alkaline extraction for Cr(VI) determination in dairy and cereal products by HPIC–ICPMS using an experimental design

Hernandez F., Séby F., Millour S., Noël L., Guérin T.

Food Chemistry, 2017, 113, 981-988


This study presents the optimisation through an experimental design then the validation of a method to determine Cr(VI) in certain foodstuffs by high-performance ionic chromatography with inductively coupled plasma mass spectrometry. This method is free from interferences possibly associated with chloride and organic or inorganic carbon. Analytical performances assessed by the accuracy profile method were satisfactory in terms of linearity, specificity, sensitivity, accuracy, repeatability and intermediate precision. Limits of quantification ranged from 0.6 μ−1 in dairy products to 0.8 μ−1 in cereal products. The method was applied to the determination of Cr(VI) in dairy and cereal products from different brands and origins. Despite the method's very high sensitivity, Cr(VI) was not found in all the studied samples. This confirms the results of the most recent studies using an on-line speciation method, and invalidates older studies that found traces of Cr(VI) in food by using a less specific off-line speciation method.

Optimisation of selective alkaline extraction for Cr(VI) determination in dairy and cereal products by HPIC–ICPMS using an experimental design

Hernandez F., Séby F., Millour S., Noël L., Guérin T.

Food Chemistry, 2017, 113, 981-988


This study presents the optimisation through an experimental design then the validation of a method to determine Cr(VI) in certain foodstuffs by high-performance ionic chromatography with inductively coupled plasma mass spectrometry. This method is free from interferences possibly associated with chloride and organic or inorganic carbon. Analytical performances assessed by the accuracy profile method were satisfactory in terms of linearity, specificity, sensitivity, accuracy, repeatability and intermediate precision. Limits of quantification ranged from 0.6 μ−1 in dairy products to 0.8 μ−1 in cereal products. The method was applied to the determination of Cr(VI) in dairy and cereal products from different brands and origins. Despite the method's very high sensitivity, Cr(VI) was not found in all the studied samples. This confirms the results of the most recent studies using an on-line speciation method, and invalidates older studies that found traces of Cr(VI) in food by using a less specific off-line speciation method.

Screening of TiO2 and Au nanoparticles in cosmetics and determination of elemental impurities by multiple techniques (DLS, SP-ICP-MS, ICP-MS and ICP-OES)

De la Calle I., Menta M., Klein M., Séby F.

Talanta, 2017, 171, 291-306


Cosmetics are part of the daily life of most of the people. Thus, a complete characterization of the products we applied in our skin is necessary. In this work, an analytical investigation of a wide variety of cosmetics from the point of view of total element content and metallic nanoparticles (NPs) has been performed. Firstly, we analyzed the total element content by ICP-MS and ICP-OES after acid digestion as an assessment of the presence of metal impurities. Prohibited elements in cosmetics, according to the European Commission regulation No 1223/2009, were not detected, and only elements mentioned in the label were found (e.g. Al, Fe, Ti and Si). Secondly, a screening of the presence of NPs has been performed by Dynamic Light Scattering (DLS) and Single Particle Inductively-Coupled Plasma Mass Spectrometry (SP-ICP-MS). Two sample preparation procedures were applied. The first protocol consisted in the preparation of suspensions in 0.1% w/v SDS and the second based on defatting with hexane followed by resuspension in water. DLS was employed as a routine method for a fast analysis of NPs, but this technique showed limitations due to the lack of specificity. SP-ICP-MS analyses were then performed, first as a screening techni que to evaluate the presence of TiO2 and Au NPs in cosmetics suspensions prepared in SDS; and second, when a positive answer was obtained about the presence of NPs from the screening, SP-ICP-MS was used for particle size determination. Results showed that only TiO2 NPs were present in two sunscreens, one anti-wrinkle day cream, one lip balm protector labeled as ‘nano’ and in one brand of toothpaste not labeled as ‘nano’. Sizes obtained for both sample preparations were compared and ranged from 30 to 120 nm in most of the samples.

Optimization and validation of the methods for the total mercury and methylmercury determination in breast milk

V. Vacchina, F. Séby, R. Chekri, J. Verdeil, J. Dumont, M. Hulin, V. Sirot, J-L. Volatier, R. Serreau, A. Rousseau, T. Simon, T. Guérin.

Talanta, 2017, 167, 404-410


The objective of the work was to develop and validate methods for the total Hg and methylmercury (MeHg) in breast milk that could be further used to obtain first data on chemical contamination of French breast milk. For total Hg determination, the potential of two techniques, namely Advanced Mercury Analyzer (AMA) and ICP MS, was compared. For MeHg determination, ICP MS detection associated to a quantification by isotopic dilution was used and the potential of a preliminary separation by gas or liquid chromatography was evaluated and discussed. The optimization studies have shown that AMA for total Hg determination and HPLC – ID – ICP MS, after a preconcentration step by freeze-drying, for MeHg quantification were the most relevant methods to use for epidemiologic studies. The figures of merit for both methods were evaluated by means of accuracy profiles in terms of limits of quantification (1.82 and 1.35 μg Hg/kg dry weight, corresponding to 0.22 and 0.16 μg Hg/kg wet basis for total Hg and MeHg, respectively), repeatability (2–11% and 3–8% for total Hg and MeHg respectively), intermediate precision reproducibility (4–12% and 4–8% for total Hg and MeHg respectively) and trueness bias (−0.1–9% and −4–0% for total Hg and MeHg respectively). The methods were then applied to 180 breast milk samples. Total Hg concentrations ranged from < LD to 16.9 μg Hg/kg wet basis (< LD to 142 μg Hg/kg dry weight) and the MeHg contents from < LD to 0.43 μg Hg/kg wet basis (< LD to 3.67 μg Hg/kg dry weight).

Rapid ion-exchange matrix removal for a decrease of detection limits in the analysis of salt-rich reservoir waters for fluorobenzoic acids by liquid chromatography coupled with tandem mass spectrometry

Kubica P., Vacchina V., Wasilewski T., Reynaud S., Szpunar J., Lobinski R.

Analytical and Bioanalytical Chemistry, 2017, 409, 871-879


A matrix removal procedure with ion-exchange resin prior to analysis for 18 fluorinated benzoic acids (FBAs) tracers in saline ( > 25% salt) reservoir water was optimized. The elimination of > 98% of salt and the simultaneous matrix sample cleanup allowed the direct analysis using the supernatant by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). This resulted in a gain in detection limits for most of the tracers in comparison with the reference method (direct analysis after minimum required dilution). The limits of detection (LODs) were in the range of 0.01–0.15 ng/ml and compared to other studies the developed method provided comparable limits of detection and advantage of simplified and shorter sample preparation. The presented method offers a considerable gain in simplicity and analysis time. Recoveries for all the tracers reached 80–100%, except for 2-FBA and 2,6-dFBA for which they were ca. 60%. The low recoveries were corrected by the use of five isotopically labeled internal standards. The method was validated by the analysis of spiked samples and by an independent comparison of the results with those obtained by solid-phase extraction LC-MS/MS method.


Development of an alternative analytical methodology to monitor industrial degreasing baths by dynamic light scattering

Menta M., Frayret J., Grassl B., Gleyzes C., Castetbon A., Potin-Gautier M.

Journal of Cleaner Production, 2016, 113, 981-988.


In the metal finishing or aeronautical industries, efficient degreasing of metallic alloy surfaces is an essential step. This step was first performed with halogenated solvents. Legislation has involved since 1999 their replacement with green cleaners due to the hazardous effects of solvents on human health and on environment. Aqueous cleaners, commonly called detergents, are widely used as an alternative. The monitoring of such degreasing baths is usually realized by alkaline titration or conductimetry. However these techniques do not provide significant information on the state of degreasing baths. In this study a new method is proposed using dynamic light scattering (DLS). This technique can evaluate the size of the micelles formed in the degreasing bath. The free and "full of oil" micelles can be identified and quantified due to their different sizes. The contamination rate and the loss of detergent all along the use of the bath can be determined with these new indicators. After a validation step with prepared degreasing solutions, this method has allowed monitoring precisely industrial degreasing baths, reducing the frequency of the baths replacement, the volume of aqueous effluents and optimizing baths management.


Beryllium in exhaled breath condensate as a biomarker of occupational exposure in primary production aluminium plant

Hulo S., Radauceanu A., Chérot-Kornobis N., Howsam M., Vacchina V., De Broucker V., Rousset D., Grzebyk M., Dziurla M., Sobaszek A., Edme J.L.

International Journal of Hygiene and Environmental Health, 2016, 219, 40-47


Low beryllium exposure can induce pulmonary granulomatosis, so called berylliosis. For occupational health monitoring, it is more relevant to assess the internal dose of Be received by the lungs than urinary or atmospheric Be. Exhaled breath condensate (EBC) is a matrix collected non-invasively that derives from the airway lining fluid. EBC beryllium (Be) levels were evaluated as a marker of occupational exposure in a primary aluminum production plant. Methods: We collected urine and EBC from controls and workers recently exposed to beryllium in the pot room and the anode repair sectors, and calculated a cumulative beryllium exposure index (CBEI) summing the number of years of employment in each task and multiplying by the estimated average beryllium exposure for the task. Concentrations of beryllium and aluminum were measured in EBC (Be-EBC and Al-EBC) and in urine (Be-U and Al-U) by ICP-MS. Results and conclusion: We have shown that it was possible to measure Be and Al in workers' EBC. Compared with controls and after adjustment for smoking status, levels of Be-EBC and Al-EBC were higher in pot room workers and exposed subjects, respectively. Due to its relationship with CBEI, but not with Be-U, it appears that Be-EBC could be a promising marker of occupational exposure and provide additional toxicokinetic information in occupational health studies.

Current trends and challenges in sample preparation for metallic nanoparticles analysis in daily products and environmental samples: A review

De la Calle I., Menta M., Séby F.

Spectrochimica Acta – Part B Atomic Spectroscopy, 2016, 126, 66-96


Due to the increasing use of nanoparticles (NPs) in consumer products, it becomes necessary to develop different strategies for their detection, identification, characterization and quantification in a wide variety of samples. Since the analysis of NPs in consumer products and environmental samples is particularly troublesome, a detailed description of challenges and limitations is given here. This review mainly focuses on sample preparation procedures applied for the mostly used techniques for metallic and metal oxide NPs characterization in consumer products and most outstanding publications of biological and environmental samples (from 2006 to 2015). We summarize the procedures applied for total metal content, extraction/separation and/or preconcentration of NPs from the matrix, separation of metallic NPs from their ions or from larger particles and NPs' size fractionation. Sample preparation procedures specifically for microscopy are also described. Selected applications in cosmetics, food, other consumer products, biological tissues and environmental samples are presented. Advantages and inconveniences of those procedures are considered. Moreover, selected simplified schemes for NPs sample preparation, as well as usual techniques applied are included. Finally, promising directions for further investigations are discussed.


Analyses de formes chimiques et de nanoparticules dans les échantillons d’eau : méthodes analytiques, préconcentration et validation

Séby F., Dumont J., Gleyzes C., Menta M., Vacchina V., Bueno M.

Revue des Sciences de l’Eau, 2015, 28, 27-32.


Il est maintenant reconnu que la mesure de la concentration totale d’un élément ne permet pas d’obtenir d’information fiable sur son impact environnemental et sa toxicité, ces éléments pouvant être présents sous différentes formes chimiques ou à l’état de nanoparticules (NPs). Il est alors nécessaire de faire appel à des techniques de fractionnement en taille pour les NPs ou à des analyses de spéciation permettant d'identifier et doser les différentes formes chimiques des éléments. Cette approche nécessite de disposer de protocoles d’échantillonnage, de conservation et de préparation d’échantillon stricts qui ne modifient pas la répartition des formes chimiques, notamment. Étant donné les nouvelles exigences des normes, cette approche nécessite également des outils analytiques de plus en plus précis, sensibles et robustes.

Le couplage de techniques séparatives basées sur la chromatographie (liquide ou gazeuse) ou le fractionnement de flux, d’une part, et la spectrométrie de masse à plasma induit (ICP MS), d’autre part, présente un réel potentiel pour ces analyses. Ces approches sont détaillées d’une manière générale en termes de potentiels et de performances analytiques et des applications sont présentées pour différents éléments (As, Hg, Sn, Cr ou Sb). Au travers de ces différents exemples, sont particulièrement abordés les principaux outils de validation, tels que les apports de la dilution isotopique (analyse du méthylmercure dans les eaux naturelles) et l’utilisation de méthodes alternatives (exemple de la spéciation de Sb), les techniques de préconcentration pour abaisser les limites de quantification en réponse à la réglementation (cas du Cr(VI)) et la caractérisation physico-chimique des colloïdes nanoparticulaires dans les eaux naturelles.

Cr(VI) speciation in foods by HPLC-ICP MS: investigation of Cr(VI)/food interactions by size exclusion and Cr(VI) determination and stability by ion-exchange on-line separations

Vacchina V., De la Calle I., Séby F.

Analytical and Bioanalytical Chemistry, 2015, 407, 3831-3839.


A method has been developed for the specific and sensitive determination of Cr(VI) in foods. First, the interactions between Cr(VI) and the matrices were investigated by size-exclusion HPLC-ICP-MS (SEC-ICP-MS). Evidence was found for the complexation of Cr(VI) potentially present with the ligands. For quantification of Cr(VI), the method was based on an alkaline extraction (NH4OH solution at pH 11.5) followed by Cr(VI) determination by anion-exchange HPLC-ICP-MS. Analytical performances of the method were satisfactory in terms of linearity, specificity, accuracy, repeatability, and intermediate precision. Detection limits ranged from 1 to 10 μg/kg, depending on the matrices investigated. The method was then applied for the determination of Cr(VI) in several products (dairy products, flour, chocolate, vegetables, fruits, meat, fish, eggs, and beverages) from different brands and origins. Cr(VI) was found in none of the samples investigated. To further investigate the reason for this absence, a stability study of spiked Cr(VI) was therefore conducted. A semi-skimmed cow milk was selected for this study. Cr(VI) was shown to be unstable in this matrix with a degradation rate increasing with the temperature.                          

Thiomonas sp. CB2 is able to degrade urea and promote toxic metal precipitation in acid mine drainage waters supplemented with urea

Farasin J., Andres J., Casiot C., Barbe V., Faerber J., Halter D., Heintz D., Koechler S., Lièvremont D., Lugan R., Marchal M., Plewniak F., Séby F., Bertin P.N., Arsène-Ploetze F.

Frontiers in Microbiology, 2015, 6, doi: 10.3389/fmicb.2015.00993


The acid mine drainage (AMD) in Carnoulès (France) is characterized by the presence of toxic metals such as arsenic. Several bacterial strains belonging to the Thiomonas genus, which were isolated from this AMD, are able to withstand these conditions. Their genomes carry several genomic islands (GEIs), which are known to be potentially advantageous in some particular ecological niches. This study focused on the role of the "urea island" present in the Thiomonas CB2 strain, which carry the genes involved in urea degradation processes. First, genomic comparisons showed that the genome of Thiomonas sp. CB2, which is able to degrade urea, contains a urea genomic island which is incomplete in the genome of other strains showing no urease activity. The urease activity of Thiomonas sp. CB2 enabled this bacterium to maintain a neutral pH in cell cultures in vitro and prevented the occurrence of cell death during the growth of the bacterium in a chemically defined medium. In AMD water supplemented with urea, the degradation of urea promotes iron, aluminum and arsenic precipitation. Our data show that ureC was expressed in situ, which suggests that the ability to degrade urea may be expressed in some Thiomonas strains in AMD, and that this urease activity may contribute to their survival in contaminated environments.

Apport du couplage ETV-ICP/AES pour les analyses de spéciation dans le solide : cas de l’arsenic et du mercure

Frayret J., Gleyzes C.

Spectra Analyse 2015, 302, 49-56.


Le couplage ETV-ICP/AES représente une alternative simple et rapide à mettre en oeuvre pour l’analyse directe totale d’éléments trace dans le solide par rapport à des méthodes nécessitant une mise en solution pouvant entraîner des facteurs de dilution, des biais et des contaminations. Ce couplage peut aussi être utilisé pour obtenir des informations de spéciation directement dans la matrice solide. Cette information peut être obtenue par séparation des différents composés grâce à leur température d’évaporation. Les étapes de préparation de l’échantillon généralement utilisées sont ainsi réduites au minimum avec uniquement une étape de broyage et d’homogénéisation, limitant ainsi les risques de modification de la spéciation et permettant une analyse simple et rapide. Une modification et une optimisation des conditions d’analyses par ETV et par ICP/AES permettent d’obtenir des informations sur les différentes formes chimiques de l’arsenic et du mercure.


Use of dried blood spots and inductively coupled plasma mass spectrometry for multi-element detection in blood

Vacchina V., Huin V., Hulo S., Cuny D., Broly F., Renom G., Perini J.-M.

Journal of Trace Elements in Medecine and Biology, 2014, 28, 255-259.


The paper describes the development of an inductively coupled plasma mass spectrometry (ICP MS) method for multitrace element determination in dried blood spots (DBSs). The analytical conditions were optimized using SeronormTM L-3 and L-1 Certified Reference Materials. The best results were obtained by sampling blood drops on a decontaminated PVDF filter membrane. After drying under metal-free conditions, the DBSs underwent acidic digestion and were analyzed with ICP MS. The method was then validated for As, Cd, Cu, Pb, Mo, Se and Zn. Using a matrix-matched calibration curve, the recovery levels ranged from 96% to 117%. The repeatability and reproducibility were generally below 15%. Limits of quantification ranging from 0.5 to 50 μg/L. In order to investigate the analytical procedure under real sampling conditions, the results obtained from DBSs and liquid blood aliquots (less subject to contamination) from two adult subjects were compared.


Zinc fate in animals husbandry systems

Romeo A., Vacchina V., Legros S., Doelsch E.,

Metallomics, 2014, 6, 1999-2009


Zinc (Zn) is considered in animal production systems as both an essential nutrient and a possible pollutant. While it is generally supplemented at low levels in animal diets, with less than 200 mg kg-1 in complete feeds, it is under scrutiny due to potential accumulation in the environment. This explains why international regulations limit maximum supplementation levels in animal feeds in a stricter way. This article gives an overview of the current knowledge on the fate of zinc in animal production systems, from animal diets to animal wastes. Some analytical methods can be used for the quantification and qualification of Zn chemical forms: X-ray crystallography, electrospray tandem mass spectrometry, separation techniques, hyphenated techniques... Analysis of chelated forms issued from complex matrices, like hydrolysed proteins, remains difficult, and the speciation of Zn in diluted carriers (premix and feed) is a challenge. Our understanding of Zn absorption has made progress with recent research on ZnT/Zip families and metallothioneins. However, fine-tuned approaches towards the nutritional and metabolic interactions for Zn supplementation in farm conditions still require further studies. The speciation of zinc in pig manure and poultry litter has been a priority as monogastric animals are usually raised under intensive conditions and fed with high quantities of trace minerals, leading to high animal density and elevated quantities of zinc from animal wastes.



Metagenomic insights into microbial metabolism affecting arsenic dispersion in Mediterranean marine sediments

Plewniak F., Koechler S., Navet B., Dugat-Bony E., Bouchez O., Peyret P., Séby F., Battaglia-Brunet F., Bertin P.N.

Molecular Ecology, 2013, 22, 4870-4883.


Microorganisms dwelling in sediments have a crucial role in biogeochemical cycles and are expected to have a strong influence on the cycle of arsenic, a metalloid responsible for severe water pollution and presenting major health risks for human populations. We present here a metagenomic study of the sediment from two harbours on the Mediterranean French coast, l'Estaque and St Mandrier. The first site is highly polluted with arsenic and heavy metals, while the arsenic concentration in the second site is below toxicity levels. The goal of this study was to elucidate the potential impact of the microbial community on the chemical parameters observed in complementary geochemical studies performed on the same sites. The metagenomic sequences, along with those from four publicly available metagenomes used as control data sets, were analysed with the RAMMCAP workflow. The resulting functional profiles were compared to determine the over-represented Gene Ontology categories in the metagenomes of interest. Categories related to arsenic resistance and dissimilatory sulphate reduction were over-represented in l'Estaque. More importantly, despite very similar profiles, the identification of specific sequence markers for sulphate-reducing bacteria and sulphur-oxidizing bacteria showed that sulphate reduction was significantly more associated with l'Estaque than with St Mandrier. We propose that biotic sulphate reduction, arsenate reduction and fermentation may together explain the higher mobility of arsenic observed in l'Estaque in previous physico-chemical studies of this site. This study also demonstrates that it is possible to draw sound conclusions from comparing complex and similar unassembled metagenomes at the functional level, even with very low sequence coverage.


Comparative efficiency of a new organic selenium source vs. seleno-yeast and mineral selenium sources on muscle selenium enrichment and selenium digestibility in broiler chickens

Briens M., Mercier Y., Rouffineau F., Vacchina V., Geraert P.A.

British Journal of Nutrition, 2013, 110, 617-624.


Two experiments were conducted on broiler chickens to compare the effect of a new organic Se source, 2-hydroxy-4-methylselenobutanoic acid (HMSeBA; SO), with two practical Se additives, sodium selenite (SS) and Se yeast (SY). The relative bioavailability of the different Se sources was compared on muscle (pectoralis major) total Se, selenomethionine (SeMet) and selenocysteine (SeCys) concentrations and apparent digestibility of total Se (ADSe). In the first experiment, from day (d) 0 to d21, Se sources were tested at different supplied levels and compared with an unsupplemented diet (NC). No significant effects were observed on growth performance during the experimental period. However, the different Se sources and levels improved muscle Se concentration compared with the NC, with a significant source effect in the following order: SS < SY < SO (P< 0·05). Seleno-amino acids speciation results for NC, SY and SO at 0·3 mg Se/kg feed indicated that muscle Se was only present as SeMet or SeCys, showing a full conversion of Se by the bird. The second experiment (d0-d24) compared SS, SY or SO at 0·3 mg Se/kg feed. The ADSe measurements carried out between d20 and d23 were 24, 46 and 49 % for SS, SY and SO, respectively, with significant differences between the organic and mineral Se sources (P< 0·05). These results confirmed the higher bioavailability of organic Se sources compared with the mineral source and demonstrated a significantly better efficiency of HMSeBA compared with SY for muscle Se enrichment.

Determination of Zn-, Cu- and Mn-glycinate complexes in feed samples and in-vitro and in-vivo assays to assess their bioaccessibility in feed samples

Vacchina V., Ionescu C., Oguey S., Lobinski R.

Talanta, 2013, 113, 14-18.


A method was developed for the quantification of Zn-, Cu- and Mn-glycinates in supplemented feed samples. The coupling of capillary electrophoresis (CE) with ICP MS detection after purification of the extract by ultrafiltration was shown to be efficient for the quantitative recovery of glycinates. The method developed was then applied to evaluate the bioaccessibility of glycinates using a sequential enzymolysis approach. The data obtained indicated a strong bioaccessibility of each element (79-94%). A new complex was also found to be formed during the digestion process. Bioavailability was then evaluated by analyzing plasma samples of horses supplemented with glycinates-rich feed. Intact glycinates could not be detected in plasma samples but a Cu-containing molecule was found more abundant after CuGly treatment.


Arsenic in marina sediments from the Mediterranean coast: speciation in the solid phase and occurrence of thioarsenates

Mamindy-Pajany Y., Bataillard P., Séby F. , Crouzet C., Moulin A., Guezennec A.G., Hurel C., Marmier N., Battaglia-Brunet F.

Soil and Sediment Contamination, 2013, 22, 984-1002


Arsenic speciation was determined in marina sediments of the French Mediterranean coast. The sediment from L′Estaque marina was highly impacted by both metallurgical activities and by the commercial port of Marseille, in contrast to the sediment from St. Mandrier marina that was less polluted. In the solid phases, As(III) was the dominant species in the L′Estaque sediment, whereas As(V) was the main form in the St. Mandrier sediment, with total As concentrations in the range 160-350 mg/kg and 17-20 mg/kg, respectively. In both sites, arsenic was the major trace element detected in interstitial water, its concentration reaching values higher than 1 mg/L in the L′Estaque sediment. Sulfate-reduction seemed to be more active in the L′Estaque site, probably because of the availability of easily biodegradable organic matter. This condition favored the formation of thioarsenates that were quantified here for the first time in a polluted marine sediment. Thioarsenates represented 69 ± 24% of total dissolved As in the 15-60 cm depth sediment layer. Our results suggest that bacterial activity may significantly contribute to increased arsenic solubility and mobility in harbor sediments, where crude oil input stimulates sulfate-reducers.



Speciation of antimony in injectable drugs used for leishmaniasis (Glucantime) by HPLC-ICP MS and DPP

Séby F., Gleyzes C., Grosso O., Plau B., Donard O.F.X.

Analytical and Bioanalytical Chemistry, 2012, 404, 2939-2948.


Meglumine antimonate is the active of Glucantime ® used for the treatment of leishmaniasis, a tropical disease caused by parasitic protozoa, and it is estimated that 12 million people worldwide are affected. This drug mainly contains Sb(V) under the form of an organic complex with N-methylglucamine (NMG). During the synthesis of this molecule, traces of Sb(III) may be present, also probably complexed. Due to the fact that Sb(III) is considered more toxic than Sb(V), it is important to evaluate the Sb(III) concentration in the drug samples. In the literature, very different concentrations for residual concentrations of Sb (III) in the drug ampoules are found. Therefore, to have a true insight of antimony speciation, two independent analytical methods were developed in this work. We used an anion exchange method coupled with inductively coupled plasma mass spectrometry (ICP-MS) which was crossreferenced with an electrochemistry method (differential pulse polarography (DPP)) that could be used for routine analysis on the production site. To obtain Sb species in detectable forms, the complexes between Sb species and NMG need to be broken. This was obtained by diluting samples in hydrochloric acid in deaerated conditions to avoid Sb redox reactions. For the two analytical methods, the HCl concentration was optimized to obtain simultaneously a complete destruction of the complexes as well as limited redox reactions for Sb(V) and Sb(III) released species. For high-performance liquid chromatography (HPLC)-ICP-MS, a dilution with 5 M HCl gives the better results. The side reaction is an oxidation of Sb(III) which can be limited by the removal of oxygen. When DPP is used, the major problem is the reduction of Sb(V) which is present in high amount in the samples. Working with 0.6 M HCl allows this problem to be minimized. When applied to different lots of Glucantime®, Sb(III) concentration values are in good agreement for the two analytical methods, with, for HPLC-ICP-MS, the advantage of the simultaneous detection of both Sb redox species.

Les méthodologies analytiques pour les analyses de spéciation

Séby F., Dumont J., Vacchina V.

Spectra Analyse, 2012, 287, 49-59.


Cet article présente les techniques analytiques disponibles pour l’analyse de spéciation des éléments trace. Il est maintenant reconnu que la mesure de la concentration totale d’un élément ne permet pas d’obtenir d’information fiable sur son impact environnemental et sa toxicité. Seules les analyses de spéciation dont le principe est d’identifier et de doser les différentes formes chimiques des éléments permettent d’accéder à cette information. Cette approche nécessite de disposer de protocoles d’échantillonnage, de conservation et de préparation d’échantillon stricts qui ne modifient pas la répartition des formes chimiques. Ces protocoles sont détaillés dans cet article ainsi que les techniques instrumentales disponibles et leurs applications. Ces techniques sont la plupart du temps basées sur l’association en ligne d’une méthode séparative (méthodes chromatographiques) et d’une détection par spectrométrie atomique. Les principaux outils de validation et d’amélioration de la fiabilité des résultats (la dilution isotopique) sont détaillés.

Determination of organotin compounds in urine samples using GC-ICP-MS

Dumont J., Frery N., Vandentorren S., Séby F.

Agilent speciation handbook 2nd edition. 2012, 82-84.

Selenium exposure in subjects living in areas with high selenium concentrated drinking water: results of a French integrated exposure assessment survey

Barron E., Migeot V., Séby F., Ingrand I., Potin-Gautier M., Legube B., Rabouan S.

Environmental International, 2012, 40, 155-161


Selenium is an essential element which can be toxic if ingested in excessive quantities. The main human exposure is food. In addition, intake may be boosted by consumption drinking water containing unusual high selenium concentration. Objective: We measured the individual selenium level of people exposed to selenium concentration in drinking water greater than the maximum recommended limit which is 10μg/L. Methods: We carried out a prospective cohort study on 80 adults (40 exposed subjects i.e. living in the involved area and 40 non-exposed ones i.e. living elsewhere) in western France. We used three different approaches: (1) direct measurement of ingested selenium by the duplicate portion method, (2) dietary reconstitution with a food frequency questionnaire (FFQ) and (3) evaluation of the individual selenium status by measuring the selenium content in toenail clippings. Analyses were performed by inductively coupled plasma-mass spectrometry. The association between toenail selenium concentration and area of residence was analyzed using linear regression with repeated measurements. Results: We estimated selenium intake from FFQ at 64 ± 14μg/day for exposed subjects as opposed to 52 ± 14μg/day for the non-exposed ones. On the basis of 305 duplicate diet samples, average intake was estimated at 64 ± 26μg/day for exposed subjects. Area of residence (p = 0.0030) and smoking (p = 0.0054) were independently associated with toenail selenium concentration. Conclusion: Whatever method used for estimating selenium intake, the selenium level in this studied area with high selenium concentrated drinking water is much lower than in seleniferous areas.

Development of an analytical method to monitor industrial degreasing and rinsing baths

Menta M., Frayret J., Gleyzes C., Castetbon A., Potin-Gautier M.

Journal of Cleaner Production, 2012, 20, 161–169


In the metal finishing or aeronautical industries, efficient degreasing of metallic alloy surfaces is an essential step. This step was first performed with halogenated solvents. Legislation has recently required their replacement with green cleaners due to the hazardous effects of the solvents on human health and on the environment. The industrial partners of the study chose to use aqueous cleaners, commonly called detergents, as an alternative. The monitoring of these baths is important to evaluate degreasing power on soiled alloy pieces. This study is focused on the evaluation of typical analytical tools recommended by providers (alkaline titration and electrical conductivity measurements) to perform degreasing bath monitoring. The results demonstrate that these techniques do not provide significant information on the state of the degreasing bath if they are used alone. Indeed, investigations performed on prepared samples simulating a degreasing bath demonstrate that alkaline titration is mainly representative of the oil in solution, whereas electrical conductivity measurements are influenced by oil and surfactants or detergents. Detergents are surfactants that possess adapted characteristics for detergency. From these results, an alternative methodology has been developed to monitor industrial samples, and conductivity measurements were used as age indicators for the rinsing baths.

Evaluation of compost and mixture of compost and activated carbon as biofilter media for the treatment of indoor air pollution

Ondarts M., Hort C., Sochard S., Platel V., Moynault L., Séby F.

Environment Technology, 2012, 33, 273-284.


Indoor air pollution (IAP), defined by a lot of pollutants at low concentrations (μg m-3), is recognized as a major environmental health issue. In order to remove this pollution, biofiltration was investigated in this study. Two biofilters packed with compost and a mixture of compost and activated carbon (AC) were compared during the treatment of an influent with characteristics close to those of IAP. Very high removal efficiencies (RE) were achieved for the two biofilters (RE more than 90% for butyl acetate, butanol, formaldehyde, limonene, toluene and undecane at mass loading from 6-24 mg m-3 h-1 and 19 s empty bed retention time). The fact that high RE of hydrophobic compounds (undecane and limonene) were achieved, along with the results of an abiotic sorption study, lead us to suggest a mechanism including adsorption followed by biodegradation at the interface of the biofilm where microorganisms tend to concentrate near the available substrate. Both chemical reactions with the packing materials and biological degradation led to average RE greater than 91.4% for nitrogen dioxide. It was observed that adding AC to compost had significant effects. First, its buffering capacity led to shorter acclimation duration and more stable operation efficiencies than for the compost biofilter. Secondly, the only compound which was not removed by the compost biofilter, trichloroethylene, was strongly adsorbed by the compost/AC biofilter. Finally, the concentration profile along the two biofilters demonstrated that adding of AC could lead to a reduction of the retention time required to reach the maximal RE.


Evaluation of hexavalent chromium extraction method EPA method 3060A for soils using XANES spectroscopy

Malherbe J., Isaure M.-P., Séby F., Watson Russell P., Rodriguez-Gonzalez P., Stutzman P.E., Davis C.W., Maurizio C., Unceta N., Sieber J.R., Long S. E., Donard O.F.X.

Environmental Science and Technology, 2011, 45, 10492-10500


Hexavalent chromium (Cr(VI)) occurrence in soils is generally determined using an extraction step to transfer it to the liquid phase where it is more easily detected and quantified. In this work, the performance of the most common extraction procedure (EPA Method 3060A) using NaOH-Na2CO3 solutions is evaluated using X-ray absorption near edge structure spectroscopy (XANES), which enables the quantification of Cr(VI) directly in the solid state. Results obtained with both methods were compared for three solid samples with different matrices: a soil containing chromite ore processing residue (COPR), a loamy soil, and a paint sludge. Results showed that Cr(VI) contents determined by the two methods differ significantly, and that the EPA Method 3060A procedure underestimated the Cr(VI) content in all studied samples. The underestimation is particularly pronounced for COPR. Low extraction yield for EPA Method 3060A was found to be the main reason. The Cr(VI) present in COPR was found to be more concentrated in magnetic phases. This work provides new XANES analyses of SRM 2701 and its extraction residues for the purpose of benchmarking EPA 3060A performance.

Unsuspected diversity of arsenite-oxidizing bacteria as revealed by widespread distribution of the aoxB gene in prokaryotes

Heinrich-Salmeron A., Cordi A., Brochier-Armanet C., Halter D., Pagnout C., Abbaszadeh-Fard E., Montaut D., Séby F., Bertin P.N., Bauda P., Arsene-Ploetze F.

Applied and Environmental Microbiology, 2011, 77, 4685-4692


In this study, new strains were isolated from an environment with elevated arsenic levels, Sainte-Marieaux-Mines (France), and the diversity of aoxB genes encoding the arsenite oxidase large subunit was investigated. The distribution of bacterial aoxB genes is wider than what was previously thought. AoxB subfamilies characterized by specific signatures were identified. An exhaustive analysis of AoxB sequences from this study and from public databases shows that horizontal gene transfer has likely played a role in the spreading of aoxB in prokaryotic communities.

Taxonomic and functional prokaryote diversity in mildly arsenic-contaminated sediments

Halter D., Cordi A., Gribaldo S., Gallien S., Goulhen-Chollet F., Heinrich-Salmeron A., Carapito C., Pagnout C., Montaut D., Séby F., Van Dorsselaer A., Schaeffer C., Bertin P.N., Bauda P., Arsene-Ploetze F.

Research in Microbiology, 2011, 162, 878-887.


Arsenic-resistant prokaryote diversity is far from being exhaustively explored. In this study, the arsenic-adapted prokaryotic community present in a moderately arsenic-contaminated site near Sainte-Marie-aux-Mines (France) was characterized, using metaproteomic and 16S rRNA-encoding gene amplification. High prokaryotic diversity was observed, with a majority of Proteobacteria, Acidobacteria and Bacteroidetes, and a large archaeal community comprising Euryarchaeaota and Thaumarchaeota. Metaproteomic analysis revealed that Proteobacteria, Planctomycetes and Cyanobacteria are among the active bacteria in this ecosystem. Taken together, these results highlight the unsuspected high diversity of the arsenic-adapted prokaryotic community, with some phyla never having been described in highly arsenic-exposed sites.


Chromium speciation in solid matrices and regulation: a review

Unceta N., Séby F., Malherbe J., Donard O.F.X.

Analytical Bioanalytical Chemistry, 2010, 397, 1097-1111


In recent years, the extensive use of chromium in industrial processes has led to the promotion of several directives and recommendations by the European Union, that try to limit and regulate the presence of Cr(VI) in the environment and to protect industrial workers using chromium and end-users of manufactured products. As a consequence, new standard methods and analytical procedures have been published at the EU level for Cr(VI) determination in soil, sludge, sediment, and similar waste materials, workplace atmospheres, cement, packaging materials, industrially produced samples, and corrosion-protection layers on some components of vehicles and electrical and electronic equipment. The objective of this article is to summarize the different directives and recommendations and to critically review the currently existing standard methods and the methods published in the literature for chromium speciation in the above mentioned solid matrices, putting the emphasis on the different extraction procedures which have been developed for each matrix. Particular attention has been paid to Cr(III) and Cr (VI) inter-conversions that can occur during extraction and efforts to minimize these unwanted reactions. Although the use of NaOH-Na2CO3 solutions with hot plate extraction seems to be the more widespread procedure, species transformation can still occur and several studies suggest that speciated isotope-dilution mass spectrometry (SIDMS) could be a suitable tool for correction of these interconversions. Besides, recent studies have proved the role of Cr(III) in chromium toxicology. As a consequence, the authors suggest an update of standard methods in the near future.

Simultaneous speciation of selenomethionine and 2-hydroxy-4-methylselenobutanoic acid by HPLC – ICP MS in biological samples

Vacchina V., Moutet M., Yadan J.C., de Baene F., Kudla B., Lobinski R.

Journal of Chromatography B, 2010, 878, 1178-1180


An analytical method was developed for the simultaneous speciation of selenomethionine (SeMet) and 2-hydroxy-4-methylselenobutanoic acid (NutraSelen®), a new SeMet precursor. The compounds could be baseline resolved by ion-pairing reversed-phase HPLC using ICP MS detection. Detection limits of 1 ng mL-1 (Se content) could be reached. SELM-1 reference material was used to validate the SeMet measurement. Additionally, the quantification of NutraSelen® was validated by standard addition together with checking the Se mass balance. The procedure developed was then applied to the monitoring of the conversion of NutraSelen® into SeMet by yeast.

Characterization of metal glycinate complexes by electrospray Q-TOF-MS/MS and their determination by capillary electrophoresis – ICP MS : application to premix samples

Vacchina V., Oguey S., Ionescu C., Bravo D., Lobinski R.

Analytical Bioanalytical Chemistry, 2010, 398, 435-449.


A method was developed for the determination of metal complexes with glycine (glycinates, [M(Gly)x(H2O)y(SO4)z]n, where M denotes Zn, Cu, Mn and Fe) in premix samples used for the preparation of animal feeds enriched in essential trace elements. The method was based on the extraction of the glycinates with 10 mM ammonium acetate (pH 7.4) followed by their determination using capillary electrophoresis with ICP MS detection. The stability of the glycinates in solution was verified by electrospray TOF-MS. Each supplement was shown to be a mixture of complexes, with polymerization degrees ranging from n=1 to n=4 (depending on the metal), that were fully or partially dehydrated. The metal glycine complex moiety was found to be preserved during capillary electrophoresis. The detection limits, calculated as three times the standard deviation of the blank plus the blank, were between 0.05 and 0.2 μg mL-1 (as the metal), and the calibration curves were linear, allowing the analysis of premix samples. Repeatability for glycinate standards was below 12%, and analytical precision was typically within 15%.

Chromium speciation in cement extracts and airborne particulates using HPLC coupled with ICP-MS

Mc Sheehy S., Séby F., Nash M.

Brazilian Journal of Analytical Chemistry, 2010, 1, 25-28


A HPLC-ICP-MS method was developed to analyze trivalent and hexavalent chromium in cement extracts and airborne particulates. Method validation was performed using a NIST Certified Reference Material (CRM-545) and agreement between the measured and certified values was achieved. The determined method detection limits (MDLs) were 0.10 μg/kg for CrVI and 0.64 μg/kg for CrIII. The determined limits of quantification (LOQs) were 0.35 μg/kg for CrVI and 2.13 μg/kg for CrIII. The developed methodology allowed for sensitive and accurate chromium speciation in real samples of cement and dust extracts.

ICP-MS: Couplage plasma induit par haute fréquence – spectrométrie de masse

Paucot H., Potin-Gautier M.

Techniques de l’Ingénieur, 2010, P2720v2, 1-12


Aujourd’hui, l’intérêt du couplage plasma induit par haute fréquence/ spectrométrie de masse (ICP-MS) ne laisse aucun doute. En effet, ce type d’équipement, du fait entre autres de sa facilité de mise en œuvre, d’un nombre limité d’interférences chimiques et spectrales, des qualités de sa source, est devenu très présent dans les laboratoires, même s’il n’offre pas encore à ce jour de limites de détection très basses. Parmi les appareillages, citons notamment le filtre quadripolaire, les secteurs magnétique et électrostatique, le temps de vol… leurs principes de fonctionnement et leurs performances analytiques sont détaillés dans cet article.


Sensitivity improvement in ICP MS analysis of fuels and light petroleum matrices using a microflow nebulizer and heated spray chamber sample introduction

Caumette G., Lienemann C.-P., Merdrignac I., Paucot H., Bouyssiere B., Lobinski R.

Talanta, 2009, 80, 1039-1043


Reasons for signal suppression during the analysis of light petroleum matrices by inductively coupled plasma mass spectrometry (ICP MS) were examined. A decrease of the ionization efficiency of the plasma was found to be the principal factor responsible for this loss of sensitivity. Consequently, an interface based on a total consumption micronebulizer and a heated spray chamber was constructed to alleviate this problem. A method based on flow-injection ICP MS using this interface was developed for the direct multielement analysis of undiluted fuels (gasoline, kerosene) and gas condensates offering an increase in sensitivity by at least a factor of 3-4 in comparison with the existing setups.

The case for re-evaluating the upper limit value for selenium in drinking waters in Europe

Barron E., Migeot V., Rabouan S., Potin-Gautier M., Séby F., Hartemann P., Levi Y., Legube B.

Journal of Water and Health, 2009, 7, 630-641


Selenium is an essential trace element for life, which can be toxic for humans when intakes reach a certain amount. Therefore, since the margin between healthy intake and toxic intake is narrow, the selenium concentration of tap water is a parameter that must be monitored because of its potential for increased intake. The present work gives an overview of the different approaches used to calculate safe limits for selenium. As recommended by WHO, the guidelines for drinking water form the basis of national legislated standards for drinking water. Before setting a maximum acceptable level in drinking water, it is necessary to take into account the total intake of selenium in both food and beverage. The limit value of 10 μgl-1 for drinking water laid down in the European regulations for all countries should be adapted depending on geographic area, as previously recommended by WHO.

Etude de l’évolution des formes chimiques des métaux dans des sédiments marins dragués stockés à terre

Séby F., Benoit-Bonnemason C., Tessier E., Alzieu C., Aqua J.L., Sannier L., Donard O.F.X.

PARALIA, 2009, 2, s3.1-s3.12


Le stockage à terre des sédiments est un traitement qui a été envisagé dans le cadre du projet SEDI.MAR.D 83 dans le but de diminuer la charge polluante des sédiments marins dragués. Ce traitement serait susceptible d’entraîner, par vieillissement naturel, des transformations physico-chimiques et/ou biologiques au sein de la matrice sédimentaire qui pourraient modifier sa toxicité. Ce travail a porté sur le devenir des métaux et de leurs formes chimiques lors du vieillissement des sédiments.

Ce traitement permet d’abaisser la moitié de la concentration initiale du tributylétain (TBT) après deux mois d’entreposage. Cette dégradation du TBT au cours du temps de stockage à terre s’accompagne de la formation d’espèces de l’étain qui sont moins dangereuses. Pour l’arsenic, seules les formes inorganiques As(III) et As(V) ont été détectées. As(III) est la forme majoritaire dans le sédiment fraîchement dragué et s’oxyde en As(V) en fonction de la durée du traitement. Cette oxydation a également été observée pour le chrome, avec apparition de très faibles quantités de chrome hexavalent. En raison de sa grande toxicité, la formation du Cr(VI) est une limite au traitement étudié. Il semble donc préférable de considérer un temps de stockage à terre assez court de façon à dégrader le TBT tout en minimisant la formation de Cr(VI). Dans la mesure où aucun traitement vraiment efficace n’existe pour gérer les sédiments marins contaminés, cette étude montre que le stockage à terre peut être considéré comme une première approche peu coûteuse. Si le degré de pollution est suffisamment abaissé par ce procédé, il est ensuite possible d’envisager une valorisation de ces matériaux dans le domaine des travaux publics par exemple (remblais).


Total selenium and selenomethionine in pharmaceutical yeast tablets : assessment of the state of the art of measurements capabilities through international intercomparison CCQM-P86

Goenega-Infante H., Sturgeon R., Turner J., Hearn R., Sargent M., Maxwell P., Yang L., Barzev A., Pedrero Z., Camara C., Diaz Huerta V., Fernandez Sanchez M. L., Sanz-Medel A., Emese K., Fodor P., Wolf W., Goldschmidt R., Vacchina V., Szpunar J., Valiente L., Huertas R., Labarraque G., Davis C., Zeisler R., Turk G., Rizzio E., Mackay L. G., Myors R. B., Saxby D. L., Askew S., Chao W., Jun W.

Analytical Bioanalytical Chemistry, 2008, 390, 629-642


Results of an international intercomparison study (CCQM-P86) to assess the analytical capabilities of national metrology institutes (NMIs) and selected expert laboratories worldwide to accurately quantitate the mass fraction of selenomethionine (SeMet) and total Se in pharmaceutical tablets of selenised-yeast supplements (produced by Pharma Nord, Denmark) are presented. The study, jointly coordinated by LGC Ltd., UK, and the Institute for National Measurement Standards, National Research Council of Canada (NRCC), was conducted under the auspices of the Comité Consultatif pour la Quantité de Matière (CCQM) Inorganic Analysis Working Group and involved 15 laboratories (from 12 countries), of which ten were NMIs. Apart from a protocol for determination of moisture content and the provision of the certified reference material (CRM) SELM-1 to be used as the quality control sample, no sample preparation/extraction method was prescribed. A variety of approaches was thus used, including single-step and multiple-step enzymatic hydrolysis, enzymatic probe sonication and hydrolysis with methanesulfonic acid for SeMet, as well as microwave-assisted acid digestion and enzymatic probe sonication for total Se. For total Se, detection techniques included inductively coupled plasma (ICP) mass spectrometry (MS) with external calibration, standard additions or isotope dilution MS (IDMS), inductively coupled plasma optical emission spectrometry , flame atomic absorption spectrometry and instrumental neutron activation analysis. For determination of SeMet in the tablets, five NMIs and three academic/institute laboratories (of a total of five) relied upon measurements using IDMS. For species-specific IDMS measurements, an isotopically enriched standard of SeMet (76Se-enriched SeMet) was made available. A novel aspect of this study relies on the approach used to distinguish any errors which arise during analysis of a SeMet calibration solution from those which occur during analysis of the matrix. To help those participants undertaking SeMet analysis to do this, a blind sample in the form of a standard solution of natural abundance SeMet in 0.1 M HCl (with an expected value of 956 mg kg-1 SeMet) was provided. Both high-performance liquid chromatography (HPLC)-ICP-MS or gas chromatography (GC)-ICP-MS and GC-MS techniques were used for quantitation of SeMet. Several advances in analytical methods for determination of SeMet were identified, including the combined use of double IDMS with HPLC-ICP-MS following extraction with methanesulfonic acid and simplified two-step enzymatic hydrolysis with protease/lipase/driselase followed by HPLC-ICP-IDMS, both using a species-specific IDMS approach. Overall, satisfactory agreement amongst participants was achieved; results averaged 337.6 mg kg-1 (n=13, with a standard deviation of 9.7 mg kg-1) and 561.5 mg kg-1(n=11, with a standard deviation of 44.3 mg kg-1) with median values of 337.6 and 575.0 mg kg-1 for total Se and SeMet, respectively. Recovery of SeMet from SELM-1 averaged 95.0% (n=9). The ability of NMIs and expert laboratories worldwide to deliver accurate results for total Se and SeMet in such materials (selensied-yeast tablets containing approximately 300 mg kg-1 Se) with 10% expanded uncertainty was demonstrated. The problems addressed in achieving accurate quantitation of SeMet in this product are representative of those encountered with a wide range of organometallic species in a number of common matrices.

Determination of selenocysteine and selenomethionine in edible animal tissues by 2D size-exclusion reversed-phase HPLC – ICP MS following carbamidomethylation and proteolytic extraction

Bierla K., Dernovics M., Vacchina V., Szpunar J., Bertin G., Lobinski R.

Analytical Bioanalytical Chemistry, 2008, 390, 1789-1798


A method was developed for the simultaneous determination of selenomethionine (SeMet) and selenocysteine (SeCys) in meat (chicken and lamb muscles) and different offal tissues (heart, liver, kidney). The analytical procedure was based on the protein extraction with urea under reducing conditions (dithiothreitol), derivatization of SeCys and SeMet by carbamidomethylation with iodoacetamide (IAM) followed by quantitative proteolysis. The mixture of the derivatized Se-amino acids was purified by size-exclusion liquid chromatography (LC) and analysed by ion-paring reversed-phase HPLC-inductively coupled plasma mass spectroscopy (ICP MS). The quantification of SeCys and SeMet was carried out by the method of standard additions. 77SeMet was used to control the SeMet derivatization efficiency and recovery. The method was validated by the determination of the Se mass balance. The Se-amino acids accounted for 91±8% of the total selenium (mean of 95 samples of seven tissues analysed over a period of 18 months). The method was applied to the discrimination of the contribution of selenoproteins (containing SeCys) and other Se-containing proteins (containing SeMet) in tissues of animals during supplementation studies (dose-effect and tolerance).

Simultaneous derivatization of selenocysteine and selenomethionine in animal blood prior to their specific determination by 2D size-exclusion ion-pairing reversed-phase HPLC – ICP MS

Bierla K., Vacchina V., Szpunar J., Bertin G., Lobinski R.

Journal of Analytical Atomic Spectrometry, 2008, 23, 508-513


A procedure for the simultaneous quantitative carbamidomethylation of selenocysteine (SeCys) and selenomethionine (SeMet) followed by their proteolytic release from blood proteins was developed. The fraction containing both derivatized selenoaminoacids was isolated by size-exclusion chromatography and submitted to ion-pairing HPLC-ICP MS analysis. The limit of detection was ca. 0.02 μ g g-1 (dry mass) for either amino acid. The quantification of SeCys and SeMet was carried out by the method of standard additions. An internal standard of 77Se-labelled SeMet was used to control the derivatization yield and chromatographic recovery. The determination of SeCys was validated by spiking with glutathione peroxidase. An additional proof of validity was achieved by monitoring the selenium mass balance (12 series of analysis over a period of 18 months; the Se amino acids accounted for 92 ± 8% of the total Se). The method was applied to the monitoring of changes in SeCys and SeMet concentrations in lamb blood during supplementation studies (tolerance and dose effect) with selenium-rich yeast.

Methylmercury exposure assessment using dietary and biomarker data among frequent seafood consumers in France – CALYPSO Study

Sirot V., Guérin T., Mauras Y., Garraud H., Volatier J.-L., Leblanc J.-C.

Environmental Research, 2008, 107, 30-38


Background: Seafood is considered by toxicologists as the main source of methylmercury (MeHg), but little data is available concerning contamination of seafood and MeHg status of French frequent consumers. Objectives: The objectives were to assess food exposure and biomarker of exposure of MeHg from a group of frequent consumers of seafood. Methods: Two approaches to exposure assessment were used: the currently used food intake and the biomarker of exposure. A validated food frequency questionnaire (FFQ) was used to assess seafood consumptions for 80 products of 385 frequent consumers aged 18 and over in four French coastal areas. Seafood samples were collected in each region considering preservation methods and supply habits according to a total diet study sampling. Food samples were analyzed for MeHg. Exposure was assessed by combining consumptions with contamination data. Whole blood samples were collected from the volunteers and analyzed for MeHg. Results: The mean dietary exposure to MeHg or weekly intake (WIFFQ) was 1.51±1.17 μg/kg bw/wk. Thirty-five percent of the subjects exceed the Joint Expert Committee on Food Additives (JECFA) provisional tolerable weekly intake (PTWI), whereas the use of the biological results with the JECFA/Environmental Protection Agency (EPA) one-compartment pharmacokinetic model to calculate weekly intake (WIPKM) shows that only 2% of subjects exceed the PTWI. The mean of the individual ratios WIFFQ/WIPKM is 4.3 and the higher the WIFFQ and the blood MeHg level, the lower is the ratio, close to 1-2. Conclusions: These analyses support the assumption that the calculated dose of methylmercury is overestimated with the FFQ-based method used in this study. Since FFQ are commonly used in risk assessments, the overestimate of dose is public health protective and this finding is somewhat reassuring from a public health point of view, especially since the JECFA or EPA have applied uncertainty factors of 3.2 or 3, respectively, to take into account the inter-individual pharmacokinetic variability.

Development of analytical procedures for the determination of hexavalent chromium in corrosion preventing coatings used in automotive industry

Séby F., Castetbon A., Ortega R., Guimon C., Niveau F., Barrois-Oudin N., Garraud H., Donard O.F.X.

Analytical and Bioanalytical Chemistry, 2008, 391, 587-597


The European directive 2000/53/EC limits the use of Cr(VI) in vehicle manufacturing. Although a maximum of 2 g of Cr(VI) was authorised per vehicle for corrosion prevention coatings of key components, since July 2007 its use has been prohibited except for some particular applications. Therefore, the objective of this work was to develop direct analytical procedures for Cr(VI) determination in the different steel coatings used for screws. Instead of working directly with screws, the optimisation of the procedures was carried out with metallic plates homogeneously coated to improve the data comparability. Extraction of Cr(VI) from the metallic parts was performed by sonication. Two extraction solutions were tested: a direct water extraction solution used in standard protocols and an ammonium/ammonia buffer solution at pH 8.9. The extracts were further analysed for Cr speciation by high-performance liquid chromatography (HPLC) inductively coupled plasma (ICP) atomic emission spectrometry or HPLC ICP mass spectrometry depending on the concentration level. When possible, the coatings were also directly analysed by solid speciation techniques (X-ray photoelectron spectroscopy, XPS, and X-ray absorption near-edge structure, XANES) for validation of the results. Very good results between the different analytical approaches were obtained for the sample of coating made up of a heated paint containing Zn, Al and Cr when using the extracting buffer solution at pH 8.9. After a repeated four-step extraction procedure on the same portion test, taking into account the depth of the surface layer reached, good agreement with XPS and XANES results was obtained. In contrast, for the coatings composed of an alkaline Zn layer where Cr(VI) and Cr(III) are deposited, only the extraction procedure using water allowed the detection of Cr(VI). To elucidate the Cr(VI) reduction during extraction at pH 8.9, the reactivity of Cr(VI) towards different species of Zn generally present in the coatings (metallic Zn and zinc oxide) was studied. The results showed that metallic Zn rapidly reduces Cr(VI), whereas this reaction is less evident in the presence of zinc oxide. Water was then retained for coatings containing metallic Zn.


Méthodologies analytiques pour la spéciation des métaux dans les produits de la mer dans le cadre d’une approche bénéfice/risque (étude CALIPSO)

Garraud H., Vacchina V., Séby F., Dumont J., Sirot V., Guérin T., Leblanc J.-C.

Annales de Toxicologie Analytique, 2007, XIX, 71-80


In the frame of the CALIPSO study concerning the determination of impregnation levels of some pollutants for seafood consumers, analyses of arsenic, cadmium, mercury and lead were performed on seafood samples and human whole blood. The originality of this approach is partly based on speciation analyses for arsenic and mercury. Analytical methodologies were fully validated and applied to a significative number of samples. Thus, certified reference materials closed to sample matrices were used when possible (As, Cd, Hg and Pb total content, and arsenic and mercury organic species). In case of mercury speciation analyses, the isotopic dilution was used in order to correct analytical artefacts such conversion and reaction yield correction. Accuracy, reproducibility and detection limits were the determined in seafood and whole blood samples.

Techniques de détermination d’espèces chimiques d’éléments (spéciation) dans l’environnement

Séby F., Donard O.F.X.

Techniques de l’Ingénieur, 2007, P3 870v2, 1-24


Cet article présente les techniques analytiques disponibles pour l’analyse de spéciation des éléments traces dans les échantillons environnementaux. Il est maintenant reconnu que la mesure de la concentration totale d’un élément ne permet pas d’obtenir d’information fiable sur son impact environnemental et sa toxicité. Seules les analyses de spéciation dont le principe est d’identifier et de doser les différentes formes chimiques des éléments permettent d’accéder à cette information. Cette approche nécessite de disposer de protocoles d’échantillonnage, de conservation et de préparation d’échantillon stricts qui ne modifient pas la répartition des formes chimiques. Ces protocoles sont détaillés dans cet article ainsi que les techniques instrumentales disponibles. Ces techniques sont la plupart du temps basées sur l’association en ligne d’une méthode séparative (méthodes chromatographiques) et d’une détection par spectrométrie atomique (ICP-MS, AFS). Cet article présente également quelques applications de méthodes permettant une analyse de spéciation directement sur le solide. Leur principal avantage est de ne pas nécessiter une extraction chimique et donc de diminuer le risque de dégradation des espèces. Toutefois, leur manque de sensibilité ne permet pas toujours d’obtenir une information précise. Enfin, dans une dernière partie, les principaux outils de validation (utilisation de matériaux de référence certifiés) et d’amélioration de la fiabilité des résultats (la dilution isotopique) sont détaillés.


Determination of hexavalent chromium in cement by the use of HPLC-ICP-MS, FPLC-ETAAS, spectrophotometry and selective extraction techniques

Scancar J., Milacic R., Séby F., Donard O.F.X.

Journal of Analytical Atomic Spectrometry, 2005, 9, 871-875


Due to the high toxicity of hexavalent Cr, an European directive has recently been issued limiting the content of water-soluble Cr(VI) in cement to a maximal concentration of 2 mg kg-1. In order to fulfil the legislative requirements, it is often necessary to use reducing agents to lower the content of hexavalent Cr in cement. In the present work the abilities of HPLC-ICP-MS, FPLC-ETAAS, spectrophotometry and selective extraction procedure were estimated for the determination of Cr(VI) in aqueous cement extracts, containing high and low Cr(VI) concentrations. After comparison of the analytical performances of the different analytical methods used, water extracts of different cement samples were analysed. A good agreement was obtained for cement samples containing Cr(VI) concentration higher than 6 mg kg-1. Differences between techniques in general did not exceed 10%. With the exception of selective extraction all techniques applied are accurate (recoveries of spiked samples lay between 95-103%) and of adequate sensitivity. The MIBK extraction procedure exhibited limitations in cement samples that were treated with reducing agents due to the gel formation in the organic phase. Because of its accuracy, high sensitivity and the high speed of the analysis, the HPLC-ICP-MS procedure could be recommended as a technique of choice. However, FPLC-ETAAS and spectrophotometry may also be applied for the determination of Cr(VI) in cement samples and cement samples treated with reducing agents.


Partitioning of metal species during an enriched fuel combustion experiment. Speciation in the gaseous and particulate phases

Pavageau M.P., Morin A., Séby F., Guimon C., Krupp E., Pécheyran C., Donard O.F.X.

Environmental Science and Technology, 2004, 38, 2252-2263


Combustion processes are the most important source of metal in the atmosphere and need to be better understood to improve flue gas treatment and health impact studies. This combustion experiment was designed to study metal partitioning and metal speciation in the gaseous and particulate phases. A light fuel oil was enriched with 15 organometallic compounds of the following elements: Pb, Hg, As, Cu, Zn, Cd, Se, Sn, Mn, V, Tl, Ni, Co, Cr, and Sb. The resulting mixture was burnt in a pilot-scale fuel combustion boiler under controlled conditions. After filtration of the particles, the gaseous species were sampled in the stack through a heated sampling tube simultaneously by standardized washing bottles-based sampling techniques and cryogenically. The cryogenic samples were collected at -80 °C for further speciation analysis by LT/GC-ICPMS. Three species of selenium and two of mercury were evidenced as volatile species in the flue gas. Thermodynamic predictions and experiments suggest the following volatile metal species to be present in the flue gas: H2Se, CSSe, CSe2, SeCl2, Hg0, and HgCl2. Quantification of volatile metal species in comparison between cryogenic techniques and the washing bottles-based sampling method is also discussed. Concerning metal partitioning, the results indicated that under these conditions, at least 60% (by weight) of the elements Pb, Sn, Cu, Co, Tl, Mn, V, Cr, Ni, Zn, Cd, and Sb mixed to the fuel were found in the particulate matter. For As and Se, 37 and 17%, respectively, were detected in the particles, and no particulate mercury was found. Direct metal speciation in particles was performed by XPS allowing the determination of the oxidation state of the following elements: Sb(V), Tl(III), Mn(IV), Cd(II), Zn(II), Cr(III), Ni(II), Co(II), V(V), and Cu(II). Water soluble species of inorganic Cr, As, and Se in particulate matter were determined by HPLC/ICP-MS and identified in the oxidation state Cr(III), As(V), and Se(IV).


Methodological advances for selenium speciation analysis in yeast

Ruiz-Encinar J., Sliwka-Kaszynska M., Polatajko A., Vacchina V., Szpunar J.

Analytica Chimica Acta, 2003, 500, 171-183


Advances in analytical methodology for speciation of selenium in selenized-yeast food supplements were discussed on the basis of the recent developments in the authors' laboratory. Particular attention was given to the sample preparation with regard to the fractionation of selenium into different classes of chemical species, the high resolution fractionation of selenium from yeast water extracts by size-exclusion chromatography and characterization of the water soluble protein fraction by combined matrix-assisted laser desorption ionization (MALDI)-time-of-flight mass spectrometry (TOF MS) and electrospray quadrupole-TOF tandem MS. The true speciation of protein-incorporated selenium (down to individual proteins characterized by a unique aminoacid sequence) was discussed using an example of a family of selenium-containing proteins formed in yeast by the substitution of methionine residues by selenomethionine in a salt stress-induced protein.

Sensitive species-specific monitoring of a new triplatinum anti-cancer drug and its potential related compounds in spiked human plasma by cation-exchange HPLC - ICP MS

Vacchina V., Torti L. Allievi C., Lobinski R.

Journal of Analytical Atomic Spectrometry, 2003, 18, 884-890


A sensitive method was developed for the specific determination of a new triplatinum anti-cancer drug (BBR3464) in spiked human plasma in the presence of potentially related compounds. In order to recover the pharmacologically active and the free-circulating drug, two methods of sample preparation, one based on the removal of the covalently protein-bound platinum by acid precipitation, and the other involving the removal of the covalently and the non-covalently protein-bound platinum by ultrafiltration, were optimized, respectively. The analytical method was optimized using human plasma spiked with the native drug and five potentially related compounds. The compounds were baseline separated by cation-exchange HPLC and detected by ICP-MS coupled on-line via a Meinhard nebulizer fitted with a refrigerated cyclonic spray chamber. A preconcentration step by freeze-drying prior to analysis was developed to improve the detection limits down to the 0.15 ng mL-1 (as Pt) level, the lowest ever reported. The analytical method developed was validated according to the criteria in force in the pharmaceutical industry. The precision and accuracy of calibration curve points and quality control samples were below 15% and 20% for the 1-10 ng mL-1 and 0.2-1 ng mL-1 concentration ranges, respectively.

Chromium speciation by hyphenation of high-performance liquid chromatography to inductively coupled plasma-mass spectrometry – Study of the influence of interfering ions

Séby F., Charles S., Gagean M., Garraud H., Donard O.F.X.

Journal of Analytical Atomic Spectrometry, 2003, 18, 1386-1390


This study presents the development of a speciation method for chromium redox species by HPLC-ICPMS. Chromatographic separation is performed with CG5A-CS5A columns set (Dionex) containing both anionic and cationic groups to retain Cr(VI) and Cr(III), respectively, associated with a nitric acid elution. After optimisation, detection limits lower than 0.5 μg L -1 were obtained for both Cr species. This method is free from interferences possibly linked to chloride and organic or inorganic carbon on the two most abundant chromium isotopes (52Cr+ and 53Cr+). A modification of the chemistry of Cr(III) was, however, observed in the presence of hydrogencarbonate ions. In this medium, for trivalent chromium, three peaks instead of one arc present on the chromatograms. This modification was attributed to the hydrolysis of Cr(III) that occurs at these pH values. The accuracy of the analytical procedure was verified by analysing a certified reference material (BCR-CRM 544) and spiked natural waters. The concentrations found for Cr(VI) were in good agreement with the certified and spiked values. Because of the high reactivity of Cr(III) in alkaline medium, recoveries were not completely quantitative.

Developments of analytical procedures for determination of total chromium by quadrupole ICP-MS and high-resolution ICP-MS, and hexavalent chromium by HPLC-ICP-MS, in different materials used in automotive industry

Séby F., Gagean M., Garraud H., Castetbon A., Donard O.F.X.

Analytical and Bioanalytical Chemistry, 2003, 377, 685-694


A European directive was recently adopted limiting the use of hazardous substances such as Pb, Hg, Cd, and Cr(VI) in vehicle manufacturing. From July 2003 a maximum of 2g Cr(VI) will be authorised per vehicle in corrosion-preventing coatings of key components. As no standardised procedures are available to check if produced vehicles are in agreement with this directive, the objective of this work was to develop analytical procedures for total chromium and Cr(VI) determination in these materials. The first step of this study was to optimise digestion procedures for total chromium determination in plastic and metallic materials by inductively coupled plasma mass spectrometry (ICP-MS). High resolution (HR) ICP-MS was used to examine the influence of polyatomic interferences on the detection of the 52Cr+ and 53Cr+ isotopes. If there was strong interference with m/z 52 for plastic materials, it was possible to use quadrupole ICP-MS for m/z 53 if digestions were performed with HNO3+H2O2. This mixture was also necessary for digestion of chromium from metallic materials. Extraction procedures in alkaline medium (NH4+/NH3 buffer solution at pH 8.9) assisted by sonication were developed for determining Cr(VI) in four different corrosion-preventing coatings by HPLC-ICP-MS. After optimisation and validation with the only solid reference material certified for its Cr(VI) content (BCR 545; welding dusts), the efficiency of this extraction procedure for screw coatings was compared with that described in the EN ISO 3613 standard generally used in routine laboratories. For coatings comprising zinc and aluminium passivated in depth with chromium oxides the extraction procedure developed herein enabled determination of higher Cr(VI) concentrations. This was also observed for the screw covered with a chromium passivant layer on zinc-nickel. For coating comprising a chromium passivant layer on alkaline zinc the standardized extraction procedure was more efficient. In the case of painted metallic plate, because of a reactive matrix towards Cr(VI), its extraction without degradation was difficult to perform.


Sorption behaviour of caesium on a bentonite sample

Hurel C., Marmier N., Séby F., Giffaut E., Bourg A.C.M., Fromage F.

Radiochimica Acta, 2002, 90, 695-698


Sorption of elements like Cs on clay is one of the principal processes delaying their release from deep repositories of nuclear wastes into the environment. The sorption processes taking place between non-purified natural clay material (bentonite) and synthetic groundwater (containing Ca, Mg, Na, K and carbonates) were therefore studied experimentally and modelled for Cs to determine whether thermodynamic computer codes capable of predicting the behaviour of this element in natural systems might be developed. The model used, based on the properties of a pure montmorillonite phase, incorporates the surface reactions for natural major ions and sorbing cations but does not have any adjustable parameters. The weight of each parameters used in the model is assessed. Surface reactions are classified as either major or minor, and a simplified model of Cs sorption that considers only the major processes is proposed. This simplified model might correspond to the less sophisticated thermodynamic model included in coupled geochemistry-transport models.


A critical review of thermodynamic data for selenium species at 25°C

Séby F., Potin-Gautier M., Giffaut E., Borge G., Donard O.F.X.

Chemical Geology, 2001, 171, 173-194


The accuracy of published selenium thermodynamic data was evaluated to obtain a reliable basis for modelling Se migration behaviour in contaminated areas. The Modified Bromley's Methodology was used to correct equilibrium constant to the standard state (zero ionic strength. 25°C and 1 bar pressure). Since redox reactions control the migration of selenium, equilibria between the various Se oxidation states and the associated reactions - acid-base, precipitation, and complexation - were studied. Although pKa values for H2SeO3 and H2SeO4 acid-base reactions are well known, an ambiguity exists for the second dissociation of H2Se. A value of 14 appears to be more suitable than a value close to 11.5. The most adequate standard potentials for the redox reactions of selenium are proposed. Because of the very low solubility of metal-selenides, most of their solubility product constants are derived from combination of formation thermodynamic data selected in the literature or extrapolated from those of the corresponding sulfides. Several values are reported for Se(IV) and Se(VI), but they are not always in agreement. In generally, few values have been determined recently and noticeable differences can be observed between old and recent values. For complexation reactions, there is a lack of data and for many equilibria only one constant is available.

A critical review of thermodynamic data for inorganic tin species

Séby F., Potin-Gautier M., Giffaut E., Donard O.F.X.

Geochimica et Chimica Acta, 2001, 65, 3041-3053


The accuracy of published inorganic tin thermodynamic data was evaluated to obtain a reliable basis for modeling Sn migration in a deep repository of radioactive waste. Hydrolysis, complexation with halide ions or other inorganic compounds, and precipitation reactions of Sn(II) and Sn(IV) were studied. The Guggenheim-Scatchard Specific Interaction Theory was used to correct equilibrium constant to zero ionic strength. Tin(II) can be hydrolysed into SnOH+, Sn(OH)02 and Sn(OH)-3 at low concentration. For higher tin levels, the Sn2(OH)22+ and Sn(OH)42+ polynuclear species are predominant. Stability constants of these equilibria at the standard state were evaluated from data available in the literature and recommended values are proposed. Complexation reactions between tin(II) and halide ions are well known, but the complex species formed are only present in solution when halide concentration is > 10-3 mol L-1 and at pH values < 4. In the presence of sulphides, selenides, or tellurides, the stannous ions from very stable solid phases. Because of the very low solubility of SnO2(s), little information is available in the literature about Sn(IV) hydrolysis, complexation, or precipitation reactions.

Production and use of reference materials for environmental analyses: conclusions of a workshop

De Guillebon B., Pannier F., Séby F., Bennink D., Quevauviller P.

Trends in Analytical Chemistry, 2001, 20, 160-166


Reference materials (RMs) play a vital role for the verification of the quality control of environmental analysis. The increasing requirements imposed to environmental control laboratories call for greater co-operation among RM producers and end-users to better define future production strategies. It is recognized that the present system is unlikely to be able to cope with the increasing demand for certified and non-certified RMs within the forthcoming years. These issues were discussed in the framework of a workshop funded by the M&T programme of the European Commission, which brought together RM producers, certification experts and end-users. This report summarizes the round-table discussions and expert recommendations.