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This work represents the first systematic speciation study of selenium (Se) in plasma from subjects participating in a pilot study for a cancer prevention trial (PRECISE). This involved supplementation of elderly British and Danish individuals with selenised yeast for 6 months and 5 years, respectively, at 100, 200, and 300 μg Se/day or placebo. Speciation data was obtained for male plasma using HPLC-ICP-MS and HPLC-ESI-MS/MS. With the proposed strategy, approximately 1.5 mL of plasma was needed to determine total Se concentration and the fractionation of Se in high molecular weight (HMW) and low molecular weight (LMW) pools, and for quantification and identification of small Se species. For the first time, Se-methyl-selenocysteine (MSC) and methyl-2-acetamido-2deoxy1-seleno-β-D-galactopyranoside (Selenosugar-1) were structurally confirmed in plasma after supplementation with selenised yeast within the studied range. Determination of selenomethionine (SeMet) incorporated non-specifically into albumin (SeALB) was achieved by HPLC-ICP-MS after hydrolysis. By subtracting this SeMet concentration from the total Se in the HMW pool, the concentration of Se incorporated into selenoproteins was calculated. Results from the speciation analysis of the free Se metabolite fraction (5% of total plasma Se) suggest a significant increase in the percentage of Se (as SeMet plus Selenosugar-1) of up to 80% of the total Se in the LMW fraction after 6 months of supplementation. The Se distribution in the HMW fraction reflects a significant increase in SeALB with Se depletion from selenoproteins, which occurs most significantly at doses of over 100 μg Se/day after 5 years. The results of this work will inform future trial design. Graphical abstract.

A new anion-exchange chromatographic separation method was used for the simultaneous speciation analysis of selenoamino acids and the more ubiquitous inorganic selenium oxyanions, selenite and selenate. For quantification, this separation was coupled to inductively coupled plasma-mass spectrometry to achieve an instrumental detection limit of 5 ng Se L(-1) for all species. This chromatographic method was also coupled to electrospray tandem mass spectrometry to observe the negative ion mode fragmentation of selenomethionine and one of its oxidation products. Low detection limits were achieved, which were similar to those obtained using inductively coupled plasma-mass spectrometry. An extensive preconcentration and cleanup procedure using cation-exchange solid-phase extraction was developed for the identification and quantification of trace levels of selenomethionine in environmental samples. Preconcentration factors of up to five were observed for selenomethionine, which in addition to the removal of high concentrations of sulphate and chloride from industrial process waters, allowed for an unambiguous analysis that would have been impossible otherwise. Following these methods, selenomethionine was identified at an original concentration of 3.2 ng Se L(-1) in samples of effluent collected at a coal-fired power plant's biological remediation site. It is the first time that this species has been identified in the environment, outside of a biological entity. Additionally, oxidation products of selenomethionine were identified in river water and laboratory algal culture samples. High-resolution mass spectrometry was employed to postulate the chemical structures of these species.

Determination of selenomethionine in selenized yeast by HPLC–ICP MS has been revisited with the focus on recovery of this amino acid during the proteolytic digestion and chromatography steps. Recovery of the extracted selenium from an anion-exchange column was 100% but selenomethionine quantified by the method of standard additions accounted only for 67% of the selenium injected. Analysis (by size-exclusion LC–ICP MS) of the eluate collected before and after the selenomethionine peak showed the presence of oxidized selenomethionine (ca. 3%) and selenomethionine likely to be unspecifically associated with the biological matrix continuum (ca. 11%). This finding was validated by two-dimensional LC–ICP MS using a different elution order, i.e. size-exclusion anion-exchange. The approach developed enabled demonstration that more than 80% of selenium in the selenized yeast is actually present in the form of selenomethionine and suggests that many results reported elsewhere for the concentration of this vital amino acid in selenized yeast may be negatively biased. The research also provided insight into speciation of selenium in the solid residue after proteolytic extraction but the additional amount of selenomethionine recovered was negligible (

Values of the total selenium and selenomethionine (Semet) content of four wheat-based reference materials have been obtained by gas chromatography-stable isotope dilution mass spectrometry methods. The total Se method is an established one, and the results obtained with it are consistent with previously-assigned values. The Semet method (previously reported by our laboratory) is based on reaction with CNBr. Our data indicate that the four wheat samples (wheat gluten, durum wheat, hard red spring wheat, and soft winter wheat), though having a 30-fold range in total Se content, all have about 45% of their total Se values in the form of selenomethionine. Investigation of the CNBr-based method suggests that additional experiments are needed to verify that all selenomethionine in the wheat samples is accounted for, but also indicates that the values obtained are within 15% of the true values. As the form in which Se occurs in foods and dietary supplements is important from a nutritional perspective, adding information about Se speciation to total Se values in appropriate reference materials makes these materials more valuable in relevant analytical work.

Development of a method for very low level selenium determination in water soluble protein and peptide fractions, obtained after various separation procedures, is presented. A hydride generation atomic fluorescence spectrometry (HG-AFS) detection system was optimised and the influence of Cu(II), Sb(V), As(III) and HNO3 interferences in the measurement of Se by HG-AFS was investigated. A destruction procedure using HNO3 and H2O2 was also optimised and the average recovery of the digestion of a solution of selenomethioneine was 92 +/- 4% (n=14). Combination of this digestion with the detection system gave reliable results. Accuracy was tested by comparison with two independent methods. A very low detection limit (DL) of 0.2 ng/g of measuring solution was achieved. The whole procedure from weighing to measuring was performed in the same Teflon tube. The addition of HNO3 to the fractions before long term storage at -20 degrees C was necessary to prevent adsorption on the test tubes. Selenium was measured in water soluble protein and peptide fractions obtained after extraction, and Sephadex G-75 chromatography performed on liver samples from: i) hens exposed to As2O3, ii) hens fed with a high fat feed and iii) the certified reference material dogfish liver (CRM DOLT-2). Because of the very low DL we were able to observe the Se distribution in chromatographic fractions of samples of organisms which were not exposed to excess amounts of Se. The presence of selenium associated with metallothioneins was observed.

In this work, the use of methanesulfonic acid for protein hydrolysis is proposed for evaluation of Se-methionine in yeast, Brazil nuts, and possibly other selenium-rich biological samples. The hydrolysis was carried out by heating the sample with 4 mol L(-1) acid at reflux for 8 h. Two chromatographic techniques (size-exclusion and ion-pairing) coupled with ICP-MS detection were used to compare the release of Se-methionine from proteins by enzymatic (proteinase K, protease XIV) and acid hydrolyses. A more efficient liberation of Se-methionine was observed by acid hydrolysis. For quantification, the sample extracts were introduced onto a C8 Alltima column, and the separation was achieved with a mobile phase containing 5 mmol L(-1) hexanesulfonic acid in citrate buffer (pH 4.5)/methanol (95:5). The results obtained by standard addition showed 816+/-17 micro g g(-1) and 36.2+/-1.5 micro g g(-1) of selenium in the form of Se-methionine in yeast and nuts, respectively (65% and 75% of total selenium).

The distribution and metabolism of an inorganic selenium (Se) compound and a selenoamino acid in quails were evaluated by speciation with inductively coupled plasma mass spectrometry (ICP-MS) and a stable isotope. Quails were orally administered stable isotope [(77)Se]-labeled selenite and selenomethionine (SeMet) at the nutritional dose of 10 μg Se/bird. Then, the quails were dissected 3, 9, and 24 h after the administration to examine the metabolic pathway and the time-dependent change of Se. The concentrations of exogenous Se in all the organs and tissues of the SeMet-administered group were significantly higher than those of the selenite-administered group 3 h after the administration. This suggested that SeMet was more rapidly and/or efficiently incorporated into the quail body than selenite. A Se-containing protein in the serum was detected only in the SeMet-administered quails, but not in the selenite-administered quails. The major urinary Se metabolite, i.e., Se-methylseleno-N-acetyl-galactosamine (selenosugar), was detected in the quail serum after the administration of both selenite and SeMet. The endogenous amount of Se-methylated selenosugar (MeSeSug) in the serum of quails seemed to be larger than that of the rodents. We conclude that the metabolic pathway of Se in quails was the same as that in rodents, but the metabolic capacity for Se seemed to be larger in quails than in rodents.

Effects of the two most widespread sample preparation techniques on the d,l-enantiomer ratio of extracted selenomethionine were monitored through the analysis of the certified reference material selenium-enriched yeast and the isolated protein fraction of high selenium monkeypot nut. The extracted selenomethionine (SeMet) fractions were orthogonally cleaned up with anion exchange chromatography before carrying out the enantiomer-specific detection to increase the robustness and the efficiency of the subsequent o-phthal-aldehyde and n-isobutyril-cysteine-based derivatisation process and reversed phase-high-performance liquid chromatography-inductively coupled plasma mass spectroscopy (ICP-MS) detection. The two techniques, namely methanesulphonic acid (MSA) based digestion and proteolytic digestion with protease XIV, resulted in significantly different ratio of d,l-selenomethionine with the final results of 2.2–2.7% and 0.5–0.6% of d-SeMet, respectively. The study revealed significant differences in the ICP-MS-related sensitivity of the derivatised selenomethionine enantiomers, which calls attention to the quantification of this selenoamino acid after MSA hydrolysis.

International audience; 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). © 2008 Springer-Verlag.

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.

Updated estimates of the selenomethionine content of four NIST wheat reference materials have been obtained by use of a revised gas chromatography-stable-isotope dilution mass spectrometric method. The revised method makes use of digestion with methanesulfonic acid, which enables more complete recovery of endogenous selenomethionine than was previously achieved by overnight denaturing treatment in 0.1 mol L(-1) HCl. The NIST wheat reference materials each contain approximately 55% of their total Se content as selenomethionine. Information about forms of Se in reference materials adds value to these materials in Se speciation studies. Estimates of selenomethionine content are also provided for other wheat samples, including several grown under conditions of exposure to high Se levels. These samples also contain approximately 55% of their total Se content as selenomethionine. The consistent level of 55% of total selenium occurring in the form of selenomethionine when the total selenium content varies by a factor of 500 is suggestive of an active mechanism of incorporation of selenium into wheat grain. Figure Selenomethionine content of wheat samples.

To obtain quantitative information on human metabolism of selenium, we have performed selenium speciation analysis by HPLC/ICPMS on samples of human urine from one volunteer over a 48-hour period after ingestion of selenium (1.0 mg) as sodium selenite, L-selenomethionine, or DL-selenomethionine. The three separate experiments were performed in duplicate. Normal background urine from the volunteer contained total selenium concentrations of 8-30 microg Se/L (n=22) but, depending on the chromatographic conditions, only about 30-70% could be quantified by HPLC/ICPMS. The major species in background urine were two selenosugars, namely methyl-2-acetamido-2-deoxy-1-seleno-beta-D-galactopyranoside (selenosugar 1) and its deacylated analog methyl-2-amino-2-deoxy-1-seleno-beta-D-galactopyranoside (selenosugar 3). Selenium was rapidly excreted after ingestion of the selenium compounds: the peak concentrations (approximately 250-400 microg Se/L, normalized concentrations) were recorded within 5-9 hours, and concentrations had returned to close to background levels within 48 hours, by which time 25-40% of the ingested selenium, depending on the species ingested, had been accounted for in the urine. In all experiments, the major metabolite was selenosugar 1, constituting either approximately 80% of the total selenium excreted over the first 24 hours after ingestion of selenite or L-selenomethionine or approximately 65% after ingestion of DL-selenomethionine. Selenite was not present at significant levels (1 microg Se/L) in any of the samples; selenomethionine was present in only trace amounts (approximately 1 microg/L, equivalent to less than 0.5% of the total Se) following ingestion of L-selenomethionine, but it constituted about 20% of the excreted selenium (first 24 hours) after ingestion of DL-selenomethionine, presumably because the D form was not efficiently metabolized. Trimethylselenonium ion, a commonly reported urine metabolite, could not be detected (1 microg/L) in the urine samples after ingestion of selenite or selenomethionine. Cytotoxicity studies on selenosugar 1 and its glucosamine isomer (selenosugar 2, methyl-2-acetamido-2-deoxy-1-seleno-beta-D-glucosopyranoside) were performed with HepG2 cells derived from human hepatocarcinoma, and these showed that both compounds had low toxicity (about 1000-fold less toxic than sodium selenite). The results support earlier studies showing that selenosugar 1 is the major urinary metabolite after increased selenium intake, and they suggest that previously accepted pathways for human metabolism of selenium involving trimethylselenonium ion as the excretionary end product may need to be re-evaluated.

A fast and sensitive method was developed for the determination of the absolute configuration of selenomethionine. The enantiomers of selenomethionine were converted into diastereomeric isoindole derivatives by reaction with o-phthaldialdehyde and N-isobutyryl-L-cysteine. This easy-to-handle reaction proceeds quantitatively in a few minutes at room temperature. Separation and detection of the diastereomers was achieved by reversed-phase high-performance liquid chromatography-inductively coupled plasma-mass spectrometry (RP-HPLC/ICP-MS) using a conventional C18 reversed-phase column. Detection limits of about 4 microg L(-1) were obtained. The method was applied to the determination of the configuration of selenomethionine extracted from antarctic krill, which turned out to possess the L-configuration.

A method was developed allowing the separation, detection and identification of Se species extracted from yeast supplements during simulated digestion processes. The in vitro gastric and intestinal digests were studied for their Se compounds by successive high-performance liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICP-MS) and high-performance liquid chromatography-electrospray tandem mass spectrometry (HPLC-ES-MS-MS) analyses. The conditions for the separation were chosen as to be compatible with both ICP-MS and ES-MS-MS detection. HPLC-ICP-MS was used to screen the extracts for their Se content. By means of HPLC-ES-MS-MS, the compounds extracted were identified on-line according to their retention time, m/ z of the molecular ion and the presence of typical product ions. From these results, it was clear that the main compound extracted by both gastric and intestinal fluid was Se-methionine, which was also the main Se compound extracted by proteolytic digestion from the yeast supplements. Two other minor compounds could be identified as Se-cystine and Se(O)-methionine, a degradation product of Se-methionine.

International audience; The major selenium compound in an aqueous extract of the most popular mushroom in Eastern Asian countries, shiitake (Lentinula edodes), fortified with selenium (Se) was identified by means of hyphenated techniques, i.e. HPLC-inductively coupled argon plasma mass spectrometry and HPLC-electrospray ionization mass spectrometry (HPLC-ICP MS and HPLC-ESI MS). Sixty-eight per cent of the total Se in the selenized shiitake was extracted with water, and 49.8% of the Se in the water extract was eluted in the high molecular mass fraction (\textgreater40,000 kDa) before incubation at 37°C. After incubation, 40.6% of the Se in the water extract was eluted in a lower molecular mass fraction and the Se eluted in the high molecular mass fraction had decreased to 14.0%, suggesting that the major selenium compound in the water extract was initially in a form bound to macromolecule(s) and was then enzymatically liberated from the macromolecule(s). The retention time of the liberated selenium compound in HPLC-ICP MS matched that of selenomethionine (SeMet), and the masses of molecular and fragment ions detected by HPLC-ESI MS also suggested that the selenium compound was SeMet. The selenized shiitake accumulated Se as SeMet, and SeMet might be bound to the water extractable high molecular mass protein(s). © Springer-Verlag 2004.

This study focused on the detection/identification of possible selenium metabolites in human urine. Organoselenium compounds not commercially unavailable were synthesized and characterized by electrospray mass spectrometry. Separation of selenomethionine, methylselenomethionine, trimethylselonium, selenoethionine, and selenoadenosylmethionine was achieved by ion-pairing HPLC with a mobile phase of 2 mmol L(-1) hexanesulfonic acid, 0.4% acetic acid, 0.2% triethanolamine (pH 2.5), and 5% methanol. The column effluent was introduced on-line to inductively coupled plasma-mass spectrometry for selenium-specific detection ((77)Se and (78)Se). For selenium speciation in urine, solid-phase extraction was carried out using C(18) cartridges modified with hexanesulfonic acid. Selective retention of cationic species was observed from acidified urine (perchloric acid, pH 2.0). After elution with methanol, evaporation, and dissolution in the mobile phase, the sample was introduced to the HPLC-ICP-MS system and the chromatographic peaks were assigned by adding standards. The species identified in urine were selenomethionine, trimethylselonium ion, and selenoadenosylmethionine. The last species was detected for the first time and our results suggest that selenomethionine might enter the metabolic pathway of its sulfur analog in the activated methylation cycle.

To obtain reliable information on speciation analysis it is necessary to previously evaluate the stability of the species in the sample of interest. Furthermore, in those cases in which sample treatment to extract the species is time-consuming, an evaluation of how to maintain species integrity in the extracts is paramount. Thus, the present paper reports the stability of total Se, SeMet and TMSe+ in freeze-dried oyster and in the enzymatic extracts stored in Pyrex and polyethylene containers at different temperatures (-18, 4 and 20 degrees C). Total selenium determinations and Se speciation were carried out by HG-AAS after acid digestion in a microwave oven and by on-line coupling of cation exchange HPLC-ICP-MS after enzymatic hydrolysis, respectively. The results obtained for the freeze-dried sample showed that total Se and the selenium species evaluated are stable for at least 12 months, under all the conditions tested. However, Se species in the enzymatic extracts are only stable for 10 days if stored at 4 degrees C in Pyrex containers. These results show that the extracts do not necessarily have to be analysed just after sample treatment.

The stability of five selenium compounds, selenate, Se(VI), selenourea, SeUr, trimethylselenonium ion, TMSe(+), selenomethionine, SeMet, and selenoethionine, SeEt, at concentrations from 30-60 micro g L(-1) in a pooled human urine, stored in dark at -20 degrees C, 4 degrees C, or ambient temperature (ca. 25 degrees C), without addition of any stabilizing reagent was evaluated. The investigated Se species were determined independently by mixed ion-pair reversed-phase liquid chromatography with inductively coupled plasma mass spectrometric (ICP-MS) detection. The general trend is the lower the temperature used for storage, the higher the stability of Se species, when other conditions such as light, acidity, and container material are kept constant. On the basis of these results it is considered that the storage of urine samples at -20 degrees C for a short-term (within one month) is safe for Se speciation analysis. Long-term storage of urine samples for speciation analysis should, however, be undertaken with caution.