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A LC-DRC-ICP-MS method for the simultaneous detection of selenium and Sulfur in the selenotrisulfides selenocysteineglutathione (Cys-Se-SG) and selenodiglutathione (GS-Se-SG) is described. Both sulfur and selenium are reacted with oxygen in the dynamic reaction cell (DRC) and detected as oxides. The selenotrisulfides were separated applying a 30 rnin gradient liquid chromatographic (LC) method with a formic acid/methanol eluent. The detection limits for sulfur (as (SO+)-S-32-O-16) and selenium (as (SeO+)-Se-80-O-16) in the chromatographic system were 4.0 and 0.2 mu g L-1 (100 and 5 ng in absolute mass units), respectively. S/Se elemental ratios in the 1.9-2.1 range, close to the theoretical value of 2.0, was found for both selenotrisulfides along with accurate (

Three sample introduction systems were compared for their efficiency in introducing a variety of biologically relevant selenium species in organic solutions with the purpose of mimicking the effluent from a chromatographic system. The sample introduction systems consisted of a microconcentric nebuliser (MCN) in combination with a membrane desolvator ( MD), a MCN in combination with a cyclonic spray chamber ( CS) and a direct injection nebuliser ( DIN), respectively. Thirteen selenium standards diluted in 50% methanol were introduced in each system at a flow rate of 50 mu l min(-1). Except for the selenite and methaneseleninic acid standards that were lost in the MCN-MD system, similar normalized response factors were achieved for the standards regardless of the sample introduction system. When trimethylselenonium and selenate standards were continuously infused in a linear methanol gradient from 10 to 90%, the signal was almost unaffected when the MCN-MD was used as interface. The signals from the MCN-CS introduction system were almost unaffected in methanol concentrations between 10 and 40%, after which they slowly declined. The signal from the DIN showed a plateau in signal intensity at methanol concentrations between 35 and 70%. Detection limits in flow injection analysis, expressed as the concentration that would result in a signal equivalent to three times the peak to peak noise of the baseline, were estimated to be 200, 150 and 100 ng Se L-1 for the MCN-CS, MCN-MD and DIN, respectively. The independence of the signal from the methanol concentration in the eluent allowed quanti. cation of human selenoprotein P isoforms by gradient microbore reversed phase chromatography (mu RPC) on the basis of an internal trimethylselenonium standard. The MCN-MD system was applied for mu RPC analysis of Se-77 enriched yeast samples and more than 30 selenium containing compounds were separated using a linear gradient from 5% to 95% methanol, some of the selenium species eluting

Ultra performance liquid chromatography is a new development of the HPLC separation technique that allows separations on column materials at high pressures up to 10(8) Pa using particle diameters of 1.7 mu m. This increases the efficiency, the resolution and the speed of the separation. Four aqueous selenium standards were separated within 1.2 min on a 1.00 id x 50 mm reversed phase column in an ion-pair chromatographic system using a flow rate of 200 mu L min(-1). Hence, analysis times could be reduced to 1/10 compared with ordinary HPLC for aqueous standards. The precision and detection limits were comparable to values obtained by HPLC. Detection limits were better than 0.4 mu g Se L-1. A urine sample was analysed on a 1.0 id x 100 mm column within 5 min using a flow rate of 100 mu L min(-1). The improved separation efficiency, owing to the use of 1.7 mu m column particles, allowed the use of short columns. Hence, analysis times could be halved without loss of separation efficiency in this biological sample

Two selenium metabolites, Se-methylseleno-N-acetylgalactosamine (SeGal-N-Ac) and Se-methyl-selenogalactosamine (SeGal-NH2), were quantified in human urine by LC-DRC-ICP-MS. Urine samples were analysed after 1+1 dilution in a reversed phase chromatographic system using an eluent consisting of 200 mM ammonium acetate and 5% methanol with a pH of 9.25 and quantified by standard addition. Samples were collected from 8 volunteers before and after 5 days ingestion of 100 mu g Se day(-1) in form of selenized yeast. The average concentration of (SeGal-NH2) before and after selenium intake was 1.4 and 1.9 mu g Se L-1, respectively, while the average concentration of Se-Gal-N-Ac increased from 2.6 to 11.6 mu g Se L-1 before and after selenium consumption. Detection limits calculated on basis of three times the standard deviation on peak areas of 2 mu g Se L-1 solutions were 0.1 mu g Se L-1 for SeGal-NH2 and 0.2 mu g Se L-1 for SeGal-N-Ac based on peak areas and monitoring Se-80. The precision expressed as the relative standard deviation (n=6) at the 2 mu g Se L-1 level was 3.1 and 1.7% for SeGal-NH2 and SeGal-N-Ac, respectively, while the corresponding values were 1.0 and 0.7% at the 10 mu g Se L-1 level. Linearity in urine matrix was examined in the range 0.5-100 mu g Se L-1 and correlation coefficients better than 0.999 were obtained. As the cationic compound SeGal-NH3+ may be confounded with the trimethylselenonium ion (TMSe+), urine samples were also analysed in a cation exchange chromatographic system in which SeGal-NH3+ was separated from the trimethylselenonium ion. None of the samples contained TMSe+ in detectable amounts. Three sample introduction systems were compared-a microconcentric nebuliser in combination with a cyclonic spray chamber (MCN), a direct injection nebuliser (DIN) and an ultrasonic nebuliser (USN). The MCN was most suitable for this purpose

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 mug and 2000 mug 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

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, Semethylseleno-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-methy

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 Se-80 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

Selenomethionine (SeMet) was oxidized by heating an acidic solution with hydrogen peroxide. Samples were taken before and during the oxidation process. The oxidation products were separated by cation exchange chromatography followed by ICP-MS detection to identify the selenium containing compounds as well as electrospray ionization MS detection to determine the masses of the degradation products. Furthermore, the samples were analyzed by Se-77-NMR. The first appearing degradation product was selenomethionine selenoxide, which was converted via the deaminated selenoxide to methane seleninic acid and selenite. (C) 2003 Elsevier Science B.V. All rights reserved.

Five aqueous standards, selenomethionine (SeMet), methylselenomethionine (MeSeMet), methylselenocysteine (MeSeCys), selenogammaaminobutyric acid (SeGaba) and the trimethylselenonium ion (TMSe), were separated in ion-pairing chromatographic systems based on perfluorinated carboxylic acids in methanol. Two different perfluorinated carboxylic acids, heptafluorobutanoic acid (HFBA) and nonafluoropentanoic acid (NFPA), were used as ion-pairing agents in the separation. The selectivities of the ion-pairing agents were different. The separation was performed on a microbore column, which was connected to the ICP-MS via a laboratory-made direct injection nebuliser. This nebulisation system allowed methanol concentrations of 50% in the eluent when a flow rate of 50 ml min(-1) was used. The detection limits in urine were between 0.8 and 1.7 mug l(-1) corresponding to absolute detection limits of between 2.3 and 5.1 pg. Urine samples from different individuals before and during supplementation with selenomethionine were analysed. Several species were separated in the different urine samples. A major component eluting at the beginning of the chromatogram was predominant in many samples, especially after selenium consumption. This species was not identified and solid phase extraction experiments suggested that it was neutral. When different urine samples were spiked with the available standards, co-elution of species with TMSe, MeSeMet or SeMet was observed in some samples. None of these species were major compounds in urine samples-even after massive consumption of selenium-containing supplements. The selenium species in the urine samples showed a limited stability, as they changed during storage at +4 degreesC as well as -18 degreesC

Urine samples were extracted by benzo-15-crown-5-ether to remove sodium and potassium. More than 90% of the sodium and potassium content of the urine was removed with this extraction. In a cation-exchange system based on oxalic acid at pH 3, chromatography of an untreated urine pool resulted in a large peak in the front together with three small peaks. In the crown ether treated pool at least five signals were obtained. When the eluent was ammonium formate at pH 3, two small signals together with a large signal in the front were obtained in untreated urine, while three more distinct peaks and a peak in front were obtained in the crown ether extracted urine. In both systems, two of the peaks co-eluted with selenomethionine (SeMet) and the trimethylselenonium ion (TMSe). None of the signals co-eluted with either selenocystine or selenoethionine. Urine samples from different individuals showed different concentrations and ratios of the selenium species present. There was no difference in the chromatograms when the urine pool was treated with alpha -glucuronidase or ultrafiltrated through a membrane with a cut-off value of 10 kDa. When the urine pool was analysed by capillary electrophoresis ICP-MS at pH 8.2, four peaks could be separated. One of the peaks co-migrated with SeMet while TMSe did not appear in this system. Crown ether extraction did not improve the separation. Hence, apart from SeMet and TMSe, at least three more unknown selenium-containing species were present in urine

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) x 10(-4) cm2V(-1)s(-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.

A method based on solid phase extraction was developed for the determination of selenoprotein P as selenium in human plasma. Separation of selenoprotein P from other selenium-containing proteins was accomplished by immobilized metal-ion affinity chromatography. The selenium content was subsequently measured by inductively coupled plasma mass spectrometry (ICP-MS) monitoring the Se-82 isotope. Linear response was observed in the concentration range 0.3-70.8 mu g/l selenium as selenoprotein P with a correlation coefficient of 0.9994. The precision expressed as relative standard deviation was better than 2% in this range. The estimated limit of detection was 2 mu g/l and the experimentally verified quantification limit was 5 mu g/l, giving a relative standard deviation less than 2%. (C) 2000 Elsevier Science B.V. All rights reserved

Pulse radiolytic reduction of disulfide bridges in ceruloplasmin yielding RSSR(-) radicals induces a cascade of intramolecular electron transfer (ET) processes. Based on the three-dimensional structure of ceruloplasmin identification of individual kinetically active disulfide groups and type 1 (T1) copper centers, the following is proposed. The first T1 copper(II) ion to be reduced in ceruloplasmin is the blue copper center of domain 6 (T1A) by ET from RSSR(-) of domain 5. The rate constant is 28 +/- 2 s(-1) at 279 K and pH 7.0. T1A is in close covalent contact with the type 3 copper pair and indeed electron equilibration between T1A and the trinuclear copper center in the domain 1-6 interface takes place with a rate constant of 2.9 +/- 0.6 s(-1). The equilibrium constant is 0.17. Following reduction of T1A Cu(II), another ET process takes place between RSSR(-) and T1B copper(II) of domain 4 with a rate constant of 3.9 +/- 0.8. No reoxidation of T1B Cu(I) could be resolved. It appears that the third T1 center (T1C of domain 2) is not participating in intramolecular ET, as it seems to be in a reduced state in the resting enzyme.