Bente Gammelgaard

Selenium speciation in human urine samples by LC- and CE- ICP-MS—separation and identification of selenosugars

Human urine samples were analysed by a reversed-phase chromatographic system and an ion-pair chromatographic system. The chromatographic system was connected to the ICP-MS either by a microconcentric nebulizer (MCN) in combination with a cyclonic spraychamber or by a modified direct injection nebulizer (MDIN). The sensitivity of the latter was better than the sensitivity of the MCN, which on the other hand was more robust for the analysis of samples with high concentrations of dissolved solids. Urine sample composition did not seem to change when urine was exposed to evaporation under nitrogen at ambient temperature and methanol extraction. A pre-concentration factor of 10 was achieved with this procedure. On occasions when a pre-concentration factor of 100 was obtained by lyophilsation and methanol extraction, at least 10 selenium compounds were separated in the urine sample. Urine samples were collected from two healthy volunteers who had been supplied with 1000 µg and 2000 µg of selenium, respectively, in the form of selenized yeast. When samples were spiked with 8 different standards, only two standards co-eluted with compounds in urine in both chromatographic systems: the major urinary metabolite Se-methyl-N-acetylgalactosamine and Se-methyl-N-acetylglucosamine. The presence of Se-methyl-N-acetylglucosamine in urine was verified by co-migration with the standard in capillary electrophoresis after fractionation by preparative reversed-phase chromatography. Se-methyl-N-acetylglucosamine is only a minor metabolite as its concentration was less than 2% of the concentration of Se-methyl-N-acetylgalactosamine. The presence of this metabolite in urine has, to our knowledge, not been suggested before. Trimethylselenonium, selenomethionine, Se-methylselenocysteine, Se-methylselenomethionine and selenocystamine were not detected in these samples.

Permeation of chromium salts through human skin in vitro.

Chromium permealation studies were performed on full thickness human skin in diffusion cells. AH samples were analysed for the total chromium content by graphite furnace Zeeman‐corrected atomic absorption spectrometry. Some samples were analysed by an ion chromatographic method permitting the simultaneous determination of Cr(VI) and Cr(III) as well. The amounts of chromium found in all skin layers were significantly higher when potassium dichromate was applied lo the skin compared with chromium chloride or chromium nitrate. Chromium could only detected in the recipient phase after application of the dichromate solution. Chromium skin levels increased with increasing concentrations of applied chromium salts up to 0.034 M Cr. The amount of chromium in recipient phase and skin layers increased with increasing pH when the applied solution contained potassium dichromate. This was ascribed to a decreased skin barrier function of the skin. The amount of chromium found in all skin layers after application of chromium chloride decreased with increasing pH due to lower solubility of the salt. The % of chromium found in the recipient phase us chromium VI)increased with increasing total chromium concentration indicating a limned reduction ability of the skin in vitro.

Determination of selenium in urine by inductively coupled plasma mass spectrometry: interferences and optimization

The aim of this study was to develop a method for selenium determination in urine and examine the influence of sensitivity enhancement reagents, instrument parameters, internal standards and the salt content of the urine matrix on the determination. Several carbon-containing solutions (methanol, ethanol, propanol, butanol, glycerol, acetonitrile and acetic acid) were examined for their sensitivity enhancement effect. Enhancement factors up to six were obtained and were dependent on the nebulizer gas flow and rf power. There was no important difference in the enhancement effects of these solutes when the nebulizer gas flow rate was optimized for each solute. The influences of sample uptake rate, nebulizer flow rate and rf power were examined in multivariate experiments. The nebulizer gas flow rate and rf power were found to be interdependent, but the sample pump flow rate was independent of the other parameters. The sensitivities of different selenium species (selenite, selenate, selenomethionine and trimethylselenonium iodide) were equal during the experiments in different enhancement solutes and when analysed with the optimized parameter settings. The influence of the urine matrix and different salts on four selenium isotopes were examined. It was concluded that only 82 Se was usable for quantitative determination in urine as the blank at mass 82 was close to zero. The blank values at masses 76, 77 and 78 varied considerably and differently with different salts and salt concentrations. Yttrium, indium and gallium were compared for their use as internal standards. There was no difference in the efficiency of these internal standards and they were all usable. The accuracy of the method was determined using the NIST SRM 2670 Toxic Metals in Freeze-dried Urine (certified value 0.030±0.008 mg l –1 ). The result was 29.4±1.0 µg l –1 (n=8) and the precision was 3.4%. In synthetic urine diluted 1+1, the limit of detection was 0.9 µg l –1 and the limit of determination was 2.2 µg l –1 .

Cu(II) Mediates Kinetically Distinct, Non-amyloidogenic Aggregation of Amyloid-β Peptides

Cu(II) ions are implicated in the pathogenesis of Alzheimer disease by influencing the aggregation of the amyloid-β (Aβ) peptide. Elucidating the underlying Cu(II)-induced Aβ aggregation is paramount for understanding the role of Cu(II) in the pathology of Alzheimer disease. The aim of this study was to characterize the qualitative and quantitative influence of Cu(II) on the extracellular aggregation mechanism and aggregate morphology of Aβ1–40 using spectroscopic, microelectrophoretic, mass spectrometric, and ultrastructural techniques. We found that the Cu(II):Aβ ratio in solution has a major influence on (i) the aggregation kinetics/mechanism of Aβ, because three different kinetic scenarios were observed depending on the Cu(II):Aβ ratio, (ii) the metal:peptide stoichiometry in the aggregates, which increased to 1.4 at supra-equimolar Cu(II):Aβ ratio; and (iii) the morphology of the aggregates, which shifted from fibrillar to non-fibrillar at increasing Cu(II):Aβ ratios. We observed dynamic morphological changes of the aggregates, and that the formation of spherical aggregates appeared to be a common morphological end point independent on the Cu(II) concentration. Experiments with Aβ1–42 were compatible with the conclusions for Aβ1–40 even though the low solubility of Aβ1–42 precluded examination under the same conditions as for the Aβ1–40. Experiments with Aβ1–16 and Aβ1–28 showed that other parts than the Cu(II)-binding His residues were important for Cu(II)-induced Aβ aggregation. Based on this study we propose three mechanistic models for the Cu(II)-induced aggregation of Aβ1–40 depending on the Cu(II):Aβ ratio, and identify key reaction steps that may be feasible targets for preventing Cu(II)-associated aggregation or toxicity in Alzheimer disease.

Separation and identification of Se-methylselenogalactosamine—a new metabolite in basal human urine—by HPLC-ICP-MS and CE-nano-ESI-(MS)2

Three minor metabolites were isolated from human urine. Two of these were identified by nano electrospray ionisation mass spectrometry (nESI-MS) as Se-methylseleno-N-acetylglucosamine and Se-methylselenogalactosamine, respectively. A human urine pool was lyophilised and reconstituted in methanol prior to fractionation by preparative reversed phase HPLC. In addition to the major urinary metabolite, Se-methylseleno-N-acetylgalactosamine, more than seven minor metabolites were separated by this system and detected by ICP-MS. Three of the metabolite fractions were isolated, re-chromatographed in the reversed phase system and further purified in different separation systems before analysis by nESI-MS. By CE-nESI-MS analysis of one of the fractions, the characteristic selenium pattern was recognized around m/z 285 and (MS)2 fragmentation resulted in a fragments at m/z 267, 173 and 155, respectively. It was not possible to identify this selenium compound on basis of the available data. The selenium compound in the second fraction showed co-elution with a Se-methylseleno-N-acetylglucosamine standard. The identity of this compound was verified by nESI-MS after further purification by size exclusion chromatography. The third fraction was further purified by ion-pair and anion exchange chromatography, reconstituted and subjected to CE-nESI-MS. The m/z of the compound was 258 and (MS)2 resulted in a fragment at m/z 162, corresponding to loss of methylselenium. This indicated that the structure of the compound was Se-methylselenogalactosamine. To verify the identity of the compound, the Se-methylselenogalactosamine and the Se-methylselenoglucosamine were prepared by hydrolysis of the corresponding N-acetylhexosamines. The mass spectra of these standards were identical and also identical to the mass spectra of the purified urine compound. The urine selenium compound co-eluted with Se-methylselenogalactosamine in a reversed phase chromatographic system able to separate Se-methylselenogalactosamine and Se-methylselenoglucosamine. Analysis of basal urine samples from volunteers who had not been supplemented with selenium showed the presence of Se-methylselenogalactosamine when only traces of the metabolite Se-methylseleno-N-acetylgalactosamine, which is the major metabolite in urine after selenium supplementation was present. Hence, this new metabolite may be the main metabolite in basal urine.

Capillaries modified by noncovalent anionic polymer adsorption for capillary zone electrophoresis, micellar electrokinetic capillary chromatography and capillary electrophoresis mass spectrometry.

A simple coating procedure for generation of a high and pH‐independent electroosmotic flow in capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MEKC) is described. The bilayer coating was formed by noncovalent adsorption of the ionic polymers Polybrene and poly(vinylsulfonate) (PVS). A stable dynamic coating was formed when PVS was added to the background electrolyte. Thus, when the PVS concentration in the background electrolyte was optimized for CZE (0.01%), the EOF differed less than 0.3% after 54 runs. The electroosmotic mobility in the coated capillaries was (4.9 ± 0.1)×10–4 cm2V–1s–1 in a pH‐range of 2–10 (ionic strength = 30 mM). When alkaline compounds were used as test substances intracapillary and intercapillary migration time variations (n = 6) were less than 1% relative standard deviation (RSD) and 2% RSD, respectively in the entire pH range. The coating was fairly stable in the presence of sodium dodecyl sulfate, and this made it possible to perform fast MEKC separations at low pH. When neutral compounds were used as test substances, the intracapillary migration time variations(n = 6) were less than 2% RSD in a pH range of 2–9. In addition to fast CZE and MEKC separations at low pH, analysis of the alkaline compounds by CE‐MS was also possible.

Flow injection on-line dilution for multi-element determination in human urine with detection by inductively coupled plasma mass spectrometry

A simple flow injection on-line dilution procedure with detection by inductively coupled plasma mass spectrometry (ICP-MS) was developed for the determination of copper, zinc, arsenic, lead, selenium, nickel and molybdenum in human urine. Matrix effects were minimized by employing a dilution factor of 16.5 with on-line standard addition, and (103)Rh was used as internal standard to compensate for signal fluctuation. The procedure was validated by the analysis of two standard reference materials SRM 2670 (NIST) and Seronormtrade mark Trace Elements in Urine. Recovery experiments were performed by spiking the reference materials as well as artificial urine. The detection limits (mug l(-1)) were 0.12,0.96,0.30,0.09,0.45,0.08,0.09, and the precisions (RSD,%) were 2.6,2.3,3.0,3.7,3.7,4.9,2.8 for Cu, Zn, As, Pb, Se, Ni and Mo, respectively. The procedure was applied to the analysis of 41 human urine samples. No correlations between the concentrations of the elements were observed.

Separation, purification and identification of the major selenium metabolite from human urine by multi-dimensional HPLC-ICP-MS and APCI-MS

When humans are supplied with selenium-containing nutritional preparations, one of the selenium-containing metabolites in urine increases relatively more than the other selenium metabolites. The purpose of this study was to identify this major selenium metabolite. Urine samples from six male volunteers were collected and analysed by ion-pair chromatography with ICP-MS detection for this major selenium metabolite. Samples containing the metabolite were pooled and solid phase extracted to remove ionic substances. The extracted pool was purified and preconcentrated twice by preparative reversed-phase chromatography. The fractions containing the selenium metabolite were collected and further purified by size exclusion chromatography. It was not possible to ionize the selenium metabolite by electrospray ionization mass spectrometry, ESI-MS. Instead, atmospheric pressure chemical ionization, APCI, was applied. The m/z of the selenium metabolite was 300 for the 80Se isotope. MS/MS experiments indicated that the metabolite was a selenosugar, and it is proposed that the selenium metabolite is a Se-methyl-N-acetylselenohexosamine.

Complementary use of molecular and element-specific mass spectrometry for identification of selenium compounds related to human selenium metabolism

The aim of this paper is to give an overview of analytical data on the identification of selenium compounds in biological samples with relevance for selenium metabolism. Only studies applying the combination of element-specific inductively coupled plasma mass spectrometry as well as molecular electrospray mass spectrometry detection have been included. Hence, selenium compounds are only considered identified if molecular mass spectra obtained by analysis of the authentic biological sample have been provided. Selenium compounds identified in selenium-accumulating plants and yeast are included, as extracts from such plants and yeast have been widely used for examination of the cancer-preventive effect of selenium in cell lines, animal models and human intervention trials. Hence, these selenium compounds are available for absorption and further metabolism. Identification of selenium metabolites in simulated gastric and intestinal juice, intestinal epithelial tissue, liver and urine is described. Hence, selenium metabolites identified in relation to absorption, metabolism and excretion are included.

Simultaneous determination of chromium(III) and chromium(VI) in aqueous solutions by ion chromatography and chemiluminescence detection

A method for the simultaneous determination of chromium(iii) and chromium(vi) in a flow system based on chemiluminescence was developed. A Dionex cation-exchange guard column was used to separate chromium(iii) from chromium(vi), and chromium(vi) was reduced by potassium sulfite, whereupon both species were detected by use of the luminol-hydrogen peroxide chemiluminescence system. Linear calibration for both species was established over the concentration range 1-1000 micrograms l-1. The precision at the 20 micrograms l-1 level was 3.5% for chromium(iii) and 3.3% for chromium(vi), respectively. The detection limit was 0.5 micrograms l-1 for both species. Data were in agreement with Zeeman-effect background corrected atomic absorption spectrometry measurements.