Ryszard Lobinski

Guidelines for terms related to chemical speciation and fractionation of elements. Definitions, structural aspects, and methodological approaches (IUPAC Recommendations 2000)

This paper presents definitions of concepts related to speciation of elements, more particularly speciation analysis and chemical species. Fractionation is distinguished from speciation analysis, and a general outline of fractionation procedures is given. We propose a categorization of species according to isotopic composition of the element, its oxidation and electronic states, and its complex and molecular structure. Examples are given of methodological approaches used for speciation analysis. A synopsis of the methodology of dynamic speciation analysis is also presented.

Hyphenated Techniques for Elemental Speciation in Biological Systems

he recognition of the fact thatin environmental chemistry,occupational health, nutrition,and medicine the chemical, biologi-cal, and toxicological properties ofan element are critically dependenton the form in which the element oc-curs in the sample has spurred rapiddevelopment of an area of analyticalchemistry referred to as speciationanalysis.

Determination of rare earth elements in wine by inductively coupled plasma mass spectrometry using a microconcentric nebulizer

A method for the direct determination of REEs in undiluted wine by ICP-MS was developed. The performance of a microconcentric nebulizer (MCN) was optimized regarding the nebulizer gas flow rate and sample uptake rate for all the REEs in terms of signal intensity and signal-to-noise ratio. The effect of ethanol on the sensitivity was also studied. Possible spectral interferences were identified and are discussed in detail. The MCN was shown to reduce the sample consumption to 30–50 µl min–1 and to decrease the matrix signal suppression by a factor of 2–5 in comparison with a de Galan nebulizer. Polyatomic interferences were found to be negligible except for those of BaO on 151Eu, 153Eu and 155Gd and of 139La16O on 155Gd. Data obtained with three different ICP-MS instruments were compared. Several wines from different vineyards in France, California and Australia were analysed for REEs. The REE concentration patterns showed inter-continental and inter-regional variations, whereas no difference was observed for different vintages from the same vineyard over a period of several years.

A Common Highly Conserved Cadmium Detoxification Mechanism from Bacteria to Humans HEAVY METAL TOLERANCE CONFERRED BY THE ATP-BINDING CASSETTE (ABC) TRANSPORTER SpHMT1 REQUIRES GLUTATHIONE BUT NOT METAL-CHELATING PHYTOCHELATIN PEPTIDES

Cadmium poses a significant threat to human health due to its toxicity. In mammals and in bakers yeast, cadmium is detoxified by ATP-binding cassette transporters after conjugation to glutathione. In fission yeast, phytochelatins constitute the co-substrate with cadmium for the transporter SpHMT1. In plants, a detoxification mechanism similar to the one in fission yeast is supposed, but the molecular nature of the transporter is still lacking. To investigate further the relationship between SpHMT1 and its co-substrate, we overexpressed the transporter in a Schizosaccharomyces pombe strain deleted for the phytochelatin synthase gene and heterologously in Saccharomyces cerevisiae and in Escherichia coli. In all organisms, overexpression of SpHMT1 conferred a markedly enhanced tolerance to cadmium but not to Sb(III), AgNO3, As(III), As(V), CuSO4, or HgCl2. Abolishment of the catalytic activity by expression of SpHMT1K623M mutant suppressed the cadmium tolerance phenotype independently of the presence of phytochelatins. Depletion of the glutathione pool inhibited the SpHMT1 activity but not that of AtHMA4, a P-type ATPase, indicating that GSH is necessary for the SpHMT1-mediated cadmium resistance. In E. coli, SpHMT1 was targeted to the periplasmic membrane and led to an increased amount of cadmium in the periplasm. These results demonstrate that SpHMT1 confers cadmium tolerance in the absence of phytochelatins but depending on the presence of GSH and ATP. Our results challenge the dogma of the two separate cadmium detoxification pathways and demonstrate that a common highly conserved mechanism has been selected during the evolution from bacteria to humans.

Multiplexed Determination of Protein Biomarkers Using Metal-Tagged Antibodies and Size Exclusion Chromatography−Inductively Coupled Plasma Mass Spectrometry

A liquid-phase immunoassay was developed for the simultaneous determination of five cancer biomarker proteins: alpha-fetoprotein (AFP), human chorionic gonadotropin (hCG), carcinoembryonic antigen (CEA), ovarian tumor antigen (CA125/MUC16), and gastrointestinal tumor antigen (CA19-9). The method was based on the incubation of a serum (or tissue cytosol) with five antibodies, each labeled with a different lanthanide (Pr(3+), Eu(3+), Gd(3+), Ho(3+), and Tb(3+), respectively) followed by the specific determination of the immunocomplex formed by size exclusion chromatography with inductively coupled plasma mass spectrometric detection (SEC-ICPMS). The sensitivity of the method was comparable with that attainable by enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay with the advantages of multiplexed analysis capacity, virtually no sample preparation, and sample amount consumption, ca. 3 times lower than an ELISA test. The method was validated for the analysis of the proteins in human serum and proved to be able to discriminate ovary and uterus tumor tissue samples from those of healthy subjects.

Detection and identification of hydrophilic selenium compounds in selenium-rich yeast by size exclusion–microbore normal-phase HPLC with the on-line ICP–MS and electrospray Q-TOF-MS detection

Normal-phase HPLC and hydrophilic interaction HPLC (HILIC) were investigated for the separation of selenometabolites in a water extract of Se-rich yeast prior to their detection by ICP-MS and identification by electrospray MS/MS. The targeted fraction was a low-abundant fraction co-eluting with salt and sulfur analogues in size-exclusion chromatography which has so far been inaccessible to Se speciation studies. The optimization of the separation conditions resulted in the highest separation efficiency when HILIC was used and elution was carried out isocratically with a low concentration ammonium acetate buffer (1 mM ammonium acetate/10 mM acetic acid) in 80% acetonitrile. Out of 15 peaks observed with the Se-specific ICP-MS detection 12 was identified by electrospray Q-TOF MS/MS (2,3-dihydroxypropionyl (DHP)-Se-methylselenocysteine [M+H]+: 272, Se-methyl-gamma-glutamyl-selenocysteinylglycine dioxide [M+H]+: 402, gamma-glutamyl-Se-methylselenocysteine [M+H]+: 313; isomers of gamma-glutamylselenocystathionine [M+H]+: 400; Se-methyl-selenoglutathione [M+H]+: 370, isomers of N-acetylselenocystathionine [M+H]+: 313, 2,3-DHP-selenohomolanthionine [M+H]+: 373, isomers of 2,3-DHP-selenocystathionine [M+H]+: 359, 2,3-DHP-selenolanthionine [M+H]+: 345 and selenohomolanthionine [M+H]+: 285).

Concentrations and bioavailability of cadmium and lead in cocoa powder and related products.

Concentrations and bioavailability of cadmium (Cd) and lead (Pb) were determined in cocoa powders and related products (beans, liquor, butter) of different geographical origins. Particular attention was paid to the fractionation of these metals, which was investigated by determining the metal fraction soluble in extractant solutions acting selectively with regard to the different classes of ligands. The targeted classes of Cd and Pb species included: water-soluble compounds, polypeptide and polysaccharide complexes, and compounds soluble in simulated gastrointestinal conditions. The bioavailability of Cd and Pb from cocoa powder, liquor and butter was evaluated using a sequential enzymolysis approach. The data obtained as a function of the geographical origin of the samples indicated strong differences not only in terms of the total Cd and Pb concentrations, but also with regard to the bioavailability of these metals. The Cd concentrations in the cocoa powders varied from 94 to 1833 μg kg-1, of which 10-50% was potentially bioavailable. The bioavailability of Pb was generally below 10% and the concentrations measured in the cocoa powders were in the 11-769 μg kg-1 range. Virtually all the Cd and most of Pb were found in the cocoa powder after the pressing of the liquor.

Signal identification in size-exclusion HPLC-ICP-MS chromatograms of plant extracts by electrospray tandem mass spectrometry (ES MS/MS)

Pneumatically-assisted electrospray (ion-spray®) tandem mass spectrometry (ES-MS/MS) was evaluated for the identification of cadmium binding ligands in complexes detected in extracts of plant cell cultures by size-exclusion HPLC (SEHPLC) with ICP-MS detection. The metal-containing fractions were heartcut, purified by reversed-phase chromatography, and analysed by ES-MS/MS. The purity and identity of signals in ES-MS/MS spectra were further confirmed by collision induced dissociation (CID)-MS. The different peaks in SEHPLC-ICP-MS turned out to contain identical ligands at different ratios. On the other hand, a single SEHPLC-ICP-MS chromatographic signal was demonstrated to contain a number of complexes with ligands varying in terms of amino-acid chain length, amino-acid sequence and stoichiometry. Whereas the particular ligands (phytochelatins modified on the C-terminal amino-acid) could be identified, the complexity of equilibria within the isolated fraction prevented the elucidation of the stoichiometry of the complexes formed. The results question the validity of signal identification in SEHPLC-ICP-MS by elution volume matching with standards unless the chromatographic purity of peaks is demonstrated.

Selenopeptide mapping in a selenium-yeast protein digest by parallel nanoHPLC-ICP-MS and nanoHPLC-electrospray-MS/MS after on-line preconcentration

ICP collision cell MS was optimized for the detection and retention-time marking of selenium-containing peptides in nanoHPLC (75 μm column) after on-line 100-fold preconcentration on a capillary (300 μm id) precolumn. The mobile phase composition, gradient and flow rate were chosen to allow electrospray-MS/MS to be successfully run in parallel in identical separation and preconcentration conditions in order to produce two matching sets of chromatograms: an element-specific one and a molecule-specific one. Knowledge of the retention time of a Se-containing peptide of interest allowed efficient data mining in the corresponding ES-MS chromatogram and the identification of minor Se-species. A third chromatogram was run to obtain collision-induced dissociation data for the target peptides. The performance of the method was demonstrated for a comprehensive on-line characterization of a mixture of peptides in a tryptic digest of a Se-containing protein fraction isolated by size-exclusion chromatography from a selenium yeast extract. The method allowed the identification of whole series of Se/S substitutions in individual peptides and, in some cases, sequencing of isomers differing in the position of selenomethionine residues in the amino acid sequence.

Identification of anionic selenium species in Se-rich yeast by electrospray QTOF MS/MS and hybrid linear ion trap/orbitrap MSn.

An analytical approach allowing the identification of unknown selenium metabolites in selenium-rich yeast was described. Anion-exchange HPLC of the Se-metabolome fraction co-eluting with salts in size-exclusion chromatography allowed the separation of nine selenium species (excluding isomers and selenate) as monitored by inductively coupled plasma mass spectrometry (ICP MS). The individual fractions were analyzed by electrospray QTOF MS/MS and hybrid linear ion trap/Orbitrap MSn after sample introduction by reversed-phase nanoHPLC and by hydrophilic interaction LC (HILIC), respectively. Out of the nine detected species, eight were identified on the basis of accurate mass measurements and collision induced dissociation/fragmentation information. Seven Se-species (selenohomolanthionine, γ-Glu-selenocystathionine, 2,3-DHP-selenocystathionine, N-acetyl-selenocystathionine, 2,3-DHP-selenohomolanthionine, Se-methyl-selenoglutathione, and 2,3-DHP-Se-methylselenocysteine) were reported for the first time in Se-rich yeast, five of them have never been reported in any biological sample before.