Updated estimates of the selenomethionine content of NIST wheat reference materials by GC–IDMS

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     

Selenomethionine content of candidate reference materials.

Selenium has been identified as an antioxidant of importance in the diet. Accurate determination of its chemical forms depends on the availability of suitable reference materials (RMs). Two candidate reference materials for determination of selenomethionine (Semet) in food-related materials, a standard wheat gluten sample (NIST RM 8418 Wheat Gluten) and a commercial selenium enriched yeast, have been examined by use of a gas chromatography-isotope dilution mass spectrometry (IDMS) procedure, after treatment of the matrix with 0.1 mol L(-1) hydrochloric acid containing stannous chloride, addition of CNBr, and extraction with chloroform. This procedure results in cleavage of the CH3Se group to form volatile CH3SeCN. Addition of isotopically enriched 74Semet to an analytical sample enables estimation of the naturally occurring protein-bound 80Semet by IDMS without a protein-digestion process. We found that the Wheat Gluten RM contains a significant amount of Semet as a portion of its assigned value of 2.58 microg Se(total g(-1). Commercial selenium yeast tablets are labeled as containing an elevated level of "organic selenium", usually as Semet. The sample we investigated contained 210 microg Se(total) g(-1) sample as determined separately by IDMS, measuring elemental selenium after digestion. 73% of this total (153 +/- 21 microg Se(semet) g(-1); n = 23) was present as Semet. Thus, these two materials contain significant amounts of their total selenium content as Semet and would be good candidates for further study and characterization as reference materials for determining this important food component. The CNBr reaction used will also enable the determination of Se-(methyl)selenocysteine, the biological role of which is of recent interest. In addition to matrix RMs for Semet, it is important to have standard materials of the pure substance. We have examined a sample of a candidate standard material of selenomethionine being prepared by the USP. It was confirmed that this material is pure selenomethionine.     

Selenomethionine content of candidate reference materials

Selenium has been identified as an antioxidant of importance in the diet. Accurate determination of its chemical forms depends on the availability of suitable reference materials (RMs). Two candidate reference materials for determination of selenomethionine (Semet) in food-related materials, a standard wheat gluten sample (NIST RM 8418 Wheat Gluten) and a commercial selenium enriched yeast, have been examined by use of a gas chromatography–isotope dilution mass spectrometry (IDMS) procedure, after treatment of the matrix with 0.1 mol L^–1 hydrochloric acid containing stannous chloride, addition of CNBr, and extraction with chloroform. This procedure results in cleavage of the CH₃Se group to form volatile CH₃SeCN. Addition of isotopically enriched ⁷⁴Semet to an analytical sample enables estimation of the naturally occurring protein-bound ⁸⁰Semet by IDMS without a protein-digestion process. We found that the Wheat Gluten RM contains a significant amount of Semet as a portion of its assigned value of 2.58 μg Se_total g^–1. Commercial selenium yeast tablets are labeled as containing an elevated level of “organic selenium”, usually as Semet. The sample we investigated contained 210 μg Se_total g^–1 sample as determined separately by IDMS, measuring elemental selenium after digestion. 73% of this total (153±21 μg Se_Semet g^–1; n = 23) was present as Semet. Thus, these two materials contain significant amounts of their total selenium content as Semet and would be good candidates for further study and characterization as reference materials for determining this important food component. The CNBr reaction used will also enable the determination of Se-(methyl)selenocysteine, the biological role of which is of recent interest. In addition to matrix RMs for Semet, it is important to have standard materials of the pure substance. We have examined a sample of a candidate standard material of selenomethionine being prepared by the USP. It was confirmed that this material is pure selenomethionine. Received: 13 December 2000 / Revised: 5 March 2001 / Accepted: 12 March 2001     

Determination of selenium compounds in urine by high-performance liquid chromatography--inductively coupled plasma mass spectrometry

Selenium species, selenite, selenate, selenomethionine (Semet), seneloethionine (Seet) and trimethylselenonium ion (TmSe) were separated in aqueous solution using a gel-permeation (polyvinyl alcohol-based resin) GS-220 column by eluting with 25 mM tetramethylammonium hydroxide and 25 mM malonic acid at pH 7.9. The GS-220 column coupled with inductively coupled plasma mass spectrometry was used for the separation, identification, and quantification of selenium compounds present in certified reference material (CRM) No. 18 human urine from the National Institute for Environmental Studies in Japan (NIES). Spiking of the authentic standard to the urine and use of a silica-based LC-SCX cation-exchange column validated the peak of selenium compounds. High concentrations of chloride and bromide in the urine eluted from the GS-220 column formed molecular ions 40Ar37Cl+ and 81Br1H+ in the plasma, and these molecular ions created additional peaks in the chromatograms when 77Se and 82Se isotopes were monitored respectively. Thus, both the isotopes were selected concurrently for signal monitoring to eliminate the interfering signals. On the LC-SCX column, chloride and bromide were eluted with selenate and complicated its determination, but the peak of TmSe was baseline separated from rest of the Se compounds. Two unknown Se compounds were detected in both the columns. An additional Se compound having the same retention time as that of Semet was detected on the LC-SCX column. Peaks of selenite, selenate, TmSe and unknown selenium compounds in the urine were baseline separated on the GS-220 column, and were free from interferences. Therefore, the GS-220 column was used for the determination of selenium compounds in NIES CRM No. 18. Unknown Se compounds were the predominant selenium species followed by selenite, TmSe and selenate. The estimated value of TmSe as Se, by the standard additions method using the GS-220 column, was 3.42 +/- 0.17 microg l(-1) and was in good agreement with the LC-SCX value [3.38 +/- 0.21 (n=5) microg l(-1)].     

Determination of selenium species in plant leaves by HPLC-UV-HG-AFS

The purpose of this work was the development of a method for the determination of Se compounds in leaves of plants. Water-soluble Se compounds were extracted from samples by water. Enzymatic hydrolysis with the non-specific enzyme protease XIV was used for the release of Se compounds bound to proteins. Separation of Se species was made by ion exchange chromatography, using an anion exchange column for Se^IV, Se^VI and selenomethionine (SeMet), and a cation exchange column for selenomethylselenocysteine (SeMeSeCys) and selenocystine (SeCys₂). Columns were connected “on line” to a hydride generation atomic fluorescence spectrometer (HG-AFS) using a UV lamp between the separation and detection system. The repeatability of the results obtained by the developed method was under 15% (R.S.D.) for all Se species; the detection limit was 2-10 ng Se/g of supernatant. The accuracy was checked by comparison with some literature data for reference materials since there were no suitable certified reference materials available. The method was used for the determination of Se compounds in chicory (Cichorium intybus L.) leaves from plants which were cultivated aeroponically with elevated concentrations of Na₂SeO₄ for different periods. Se accumulated efficiently in chicory leaves; up to 480 μg/g after 41 days of exposure, mostly (64%) as Se^VI, i.e. in the form of Se added. Beside inorganic Se, in the extracts from enzyme hydrolysis we also found SeMet (4.2-8.4%) and SeMeSeCys (<DL−0.7%). Some unidentified peaks were also observed in the chromatograms of plant extracts.     

Determination of selenoamino acids by gas chromatography-mass spectrometry

The application of the recently introduced ethylchloroformate derivatization method for the separation and determination of selenomethionine and selenocystein in selenium-enriched yeast and yeast-free tablets by means of a gas chromatography-mass spectrometry (GC-MS) system has been studied. The efficiency of three methods for the extraction of selenomethionine from the tablets were compared. Total selenium content of the same tablets were measured using inductively coupled plasma (ICP)-MS and it was found that in the selenized yeast tablets about 80% of the total selenium is present as selenomethionine. The results were in agreement with the values in the labels and with the literature. The accuracy of the total selenium analysis was controlled by the analysis of a reference material.     

Determination of selenomethionine by high performance liquid chromatography-direct hydride generation-atomic absorption spectrometry

A simple, reliable, trace determination of selenomethionine (Semet) based on a direct hydride generation atomic absorption spectrometric method was developed using sodium tetrahydroborate (0.3% in 0.2% NaOH) and hydrochloric acid (3 M). The method excluded any chemical pretreatment prior to hydride generation (HG). The optimized HG system was successfully coupled with the HPLC system. The detection limit (3σ of blank; n=5), reproducibility (R.S.D. of three successive analyses/day, performed on three different days), and repeatability (R.S.D. of three successive analyses) of the method were 1.08 ng ml⁻¹, 9.8% for 9.04 ng ml⁻¹ and 2.1–9.5% for 30.0–1.27 ng ml⁻¹ Semet as Se (standards prepared in Milli-Q water). Calibration graph was linear up to 30 ng ml⁻¹. This HPLC-HG-AAS method is very promising and successfully determined Semet (spiked) in human urine.     

Speciation of selenium(IV) and selenium(VI) in environmental samples by the combination of graphite furnace atomic absorption spectrometric determination and solid phase extraction on Diaion HP-2MG

A simple solid phase extraction procedure for speciation of selenium(IV) and selenium(VI) in environmental samples has been proposed prior to graphite furnace atomic absorption spectrometry. The method is based on the solid phase extraction of the selenium(IV)-ammonium pyrrolidine dithiocarbamate (APDC) chelate on the Diaion HP-2MG. After reduction of Se(VI) by heating the samples in the microwave oven with 4 mol l⁻¹ HCl, the system was applied to the total selenium. Se(VI) was calculated as the difference between the total selenium content and Se(IV) content. The experimental parameters, pH, amounts of reagents, eluent type and sample volume were optimized. The recoveries of analytes were found greater than 95%. No appreciable matrix effects were observed. The adsorption capacity of sorbent was 5.20 mg g⁻¹ Se (IV). The detection limit of Se (IV) (3sigma, n = 11) is 0.010 μg l⁻¹. The preconcentration factor for the presented system was 100. The proposed method was applied to the speciation of selenium(IV), selenium(VI) and determination of total selenium in natural waters and microwave digested soil, garlic, onion, rice, wheat and hazelnut samples harvested various locations in Turkey with satisfactory results. In order to verify the accuracy of the method, certified reference materials (NIST SRM 2711 Montana Soil, NIST SRM 1568a Rice Flour and NIST SRM 8418 Wheat Gluten) were analyzed and the results obtained were in good agreement with the certified values. The relative errors and relative standard deviations were below 6 and 10%, respectively.     

Determination of Ga, Ge, As, Se and Sb in fly ash samples by ultrasonic slurry sampling electrothermal vaporization inductively coupled plasma mass spectrometry

Ultrasonic slurry sampling electrothermal vaporization inductively coupled plasma mass spectrometry (USS-ETV-ICP-MS) has been applied to determine Ga, Ge, As, Se and Sb in fly ash samples. The influences of instrument operating conditions and slurry preparation on the ion signals were reported. Pd and ascorbic acid were used as the mixed modifiers to enhance the ion signals. This method has been applied to determine Ga, Ge, As, Se and Sb in NIST SRM 1633a and 1633b coal fly ash reference materials and a fly ash sample collected locally. Since the sensitivities of the elements studied in slurry solution and aqueous solution were different slightly, analyte addition technique was used for the determination of Ga, Ge, As, Se and Sb in these samples. The As and Se analysis results of the reference materials agreed with the certified values. The results for which no certified value was available were also found to be in good agreement between the ETV-ICP-MS results and the reference values. The reference value was obtained by digesting the samples and analyzing the digested sample solutions by pneumatic nebulization Dynamic Reaction Cell™ (DRC) ICP-MS. The method detection limits estimated from analyte addition curves were about 0.23, 0.13, 0.17, 0.25 and 0.11 μg g⁻¹ for Ga, Ge, As, Se and Sb, respectively, in original fly ash samples.     

High-performance liquid chromatography-ultrasonic nebulizer high-power nitrogen microwave-induced plasma mass spectrometry, real-time on-line coupling for selenium speciation analysis

The coupling of a high-power nitrogen (N2) microwave-induced plasma (MIP) mass spectrometry--(MS) (1.3 kW) with high-performance liquid chromatography, connected with concentric nebulizer (CN), ultrasonic nebulizer (USN) and a hydride generation (HG) systems, for the optimization and determination of selenium compounds, has been carried out. The MIP-MS system fulfils the ideal requirement being an on-line real-time chromatographic detector for Se speciation analysis. Interchanging of MIP-MS system fabricated nebulizer (concentric) with an ultrasonic nebulizer increases about 3.4-12 (peak height) and 6.5-10 (peak area) times ion signals for the selenium compounds. The detection limits for selenate, selenite, trimethylselenonium ion (TmSe), selenomethionine (Semet) and selenoethionine (Seet) (in Milli-Q-water) obtained with the optimized HPLC-USN-N2MIP-MS system are 0.11, 0.14, 0.09, 0.14 and 0.10 microg L(-1), respectively, about 12-48 times lower than the HPLC-CN-MIP-MS and 1.5-4.4 (peak height) times lower compared to the HPLC-CN-inductively coupled plasma (ICP)-MS coupling. Considering peak area, the repeatability (R.S.D. for three successive analyses) and intermediate precision (R.S.D. for three successive analyses performed on three different days), achieved for five Se compounds are 0.8-5.6, and 1.1-5.9%, comparable with the HPLC-CN-ICP-MS, HPLC-HG-MIP-MS and HPLC-CN-MIP-MS systems. The combined HPLC-USN-N2MIP-MS has been adequately applied for the determination of Se compounds in certified National Institute for Environmental studies human urine CRM No. 18. The results reasonably agree with the HPLC-CN-ICP-MS values. This encouraging combination may be an alternative ion source of mass spectrometry for coming generation in regard to the selenium speciation analysis.     

Slurry sampling electrothermal vaporization inductively coupled plasma mass spectrometry for the determination of As and Se in soil and sludge

Slurry sampling electrothermal vaporization (ETV) inductively coupled plasma mass spectrometry (ICP-MS) has been applied to determine As and Se in soil and sludge samples. The influences of instrument operating conditions and slurry preparation on the ion signals were reported. Pd and ascorbic acid were used as mixed modifiers to enhance the ion signals. The effectiveness of ETV sample introduction technique for alleviating various spectral interferences in ICP-MS analysis has been demonstrated. This method has been applied to determine As and Se in NIST SRM 2709 San Joaquin soil reference material and NIST SRM 2781 domestic sludge reference material and a farmland soil sample collected locally. Since the sensitivities of As and Se in slurry solution and aqueous solution were different, analyte addition technique was used to determine As and Se in these samples. The As and Se analysis results of the reference materials agreed with the certified values. The precision between sample replicates was better than 5% for all determinations. The method detection limit estimated from analyte addition curves was about 0.03 and 0.02 μg g⁻¹ for As and Se, respectively, in original soil and sludge samples.     

Determination of selenium in nutritional supplements and shampoos by flow injection-hydride generation-atomic fluorescence spectrometry

A method for the determination of Se in pharmaceutical samples (nutritional supplements and shampoos) is proposed. The method involves two steps: (1) digestion of the samples and reduction of all forms of Se to Se(IV), which is complete in only 10 min by the use of a focused microwave digestor; and (2) continuous derivatisation (hydride formation) and spectrometry detection by atomic fluorescence. The method can be applied over a wide range of concentrations (0.3-1300 ng ml(-1) of Se) with good repeatability (RSD values lower than 4.6%). The method has been applied successfully to a reference material, and two different types of pharmaceuticals (namely, five different nutritional supplements-with Se present as sodium selenite and Se-methionine-and two shampoos, with selenium sulphide), in agreements with the certified and nominal values, respectively. Yields ranged between 86.5 and 104.8%, and good precision (RSD values lower than 4.2%) were obtained in all instances.     

Evaluation of homogeneity of selected reference materials for selenium by cyclic neutron activation analysis

The homogeneity of four reference materials was evaluated for Se by cyclic instrumental neutron activation analysis (CINAA). The relative standard deviation for Se measurements at ppb levels by CINAA was <12% for NIST Wheat Flour (SRM 1567) in 5–10 mg samples, while it was <11% for Chinese Hair (HH–CH-1), <13% for IAEA Animal Muscle (H-4) and 25% for IAEA Animal Blood (A-13) in 50 mg samples. The highest relative subsampling uncertainties were observed in the mass range of samples 50 mg for Chinese Hair, 100 mg for Wheat Flour and Animal Muscle and 300 mg for Animal Blood. The results of a one-way analysis of variance indicate that all reference materials above these mass ranges are adequately homogeneous with respect to Se distribution. Our data suggest that these materials, except Animal Blood, can be used as reference standards for Se in Quality assurance programs well below the sample masses re commended by the issuing agencies.     

Analysis of the selenium species distribution in cow blood by size exclusion liquid chromatography–inductively coupled plasma collision cell mass spectrometry (SEC–ICPccMS)

A method for performing rapid semiquantitative screening of the distribution of Se species in the blood of cows fed with a diet enriched in selenized yeast was optimized. The method was based on direct injection of a blood sample onto a high resolution size exclusion chromatographic column and fractionation of the selenium species. Selenium was detected on-line by ICP-MS with a collision cell. The concentrations of selenized haemoglobin and free selenomethionine were estimated using the chromatogram. The method was applied to a study involving 15 control and 15 treated dairy cows at four different supplementation time points. The increase in the selenomethionine and selenized haemoglobin was a linear function of the total selenium concentration. A threshold value of 600 ng ml(-1) of total Se was established beyond which selenomethionine could not be incorporated into the protein. No inorganic selenium was found to be present. The total selenium in cow blood correlated well with that in milk. The selenium supplementation did not change the protein distribution profiles for other essential elements (Cu, Fe, Mn, Zn).     

Boron analysis in biological material: microwave digestion procedure and determination by different methods

Investigations were made on the determination of boron in biological material. A procedure using the microwave digestion technique was optimised. The analyses were performed by photometry with azomethine-H, fluorimetry with carminic acid, inductively coupled plasma atomic emission spectrometry and inductively coupled plasma mass spectrometry. The matrix interferences, the interferences caused by iron and the detection limits were investigated for these methods. Determinations were performed for plant material, wheat, milk, meat and blood plasma. Recoveries and precision of all methods were studied. To test accuracy and precision, four NBS reference materials were analysed and the results compared with the values in literature. Good recoveries were found with all methods, except the fluorimetric technique. The boron content found in the NBS standard reference materials agreed well with data reported before. Best precision was obtained for the two ICP methods. The azomethine-H method gave good results when material of high boron content was analysed. With the carminic acid method no reliable results were achieved.     

Simultaneous determination of arsenic and selenium species in fish tissues using microwave-assisted enzymatic extraction and ion chromatography-inductively coupled plasma mass spectrometry

A microwave-assisted enzymatic extraction (MAEE) method was developed for the simultaneous extraction of arsenic (As) and selenium (Se) species in fish tissues. The extraction efficiency of total As and Se and the stability of As and Se species were evaluated by analyzing DOLT-3 (dogfish liver). Enzymatic extraction using pronase E/lipase mixture assisted by microwave energy was found to give satisfactory extraction recoveries for As and Se without promoting interspecies conversion. The optimum extraction conditions were found to be 0.2 g of sample, 20 mg pronase E and 5 mg lipase in 10 mL of 50 mM phosphate buffer, pH 7.25 at 37 °C. The total extraction time was 30 min. The speciation analysis was performed by ion chromatography-inductively coupled plasma mass spectrometry (IC-ICP-MS). The accuracy of the developed extraction procedure was verified by analyzing two reference materials, DOLT-3 and BCR-627. The extraction recoveries in those reference materials ranged between 82 and 94% for As and 57 and 97% for Se. The accuracy of arsenic species measurement was tested by the analysis of BCR 627. The proposed method was applied to determine As and Se species in fish tissues purchased from a local fish market. Arsenobetaine (AsB) and selenomethionine (SeMet) were the major species detected in fish tissues. In the analyzed fish extracts, the sum of As species detected was in good agreement with the total As extracted. However, for Se, the sum of its species was lower than the total Se extracted, revealing the presence of Se-containing peptides or proteins.     

Simple procedure for the determination of Cd, Pb, As and Se in biological samples by electrothermal atomic absorption spectrometry using colloidal Pd modifier

Electrothermal atomic absorption spectrometry was used for the determination of Cd, Pb, As and Se in the whole blood, serum, hair reference standard materials and the samples of algae collected at the coastal Estonian regions of Baltik sea. Instead of tedious and time-consuming experimental comparison of various chemical modifiers, theoretical consideration of the problem was applied for choosing the most prospective one (colloidal Pd) for solution of the given task. The experimental data obtained proved correctness of the choice. Pure standard solutions in diluted nitric acid were used for construction of the calibration graphs. The same experimental conditions were applied for each analyte for calibration solutions and all samples studied. In spite of very limited optimization procedures used, all the values obtained agree well with the corresponding reference values. Accuracy of the analysis of the algae samples was checked by recoveries of the spikes that were in the region 91-109%. Detection limits reached are 0.021, 1.2, 0.62 and 1.1 ng ml⁻¹ for Cd, Pb, As and Se, respectively, in digests of biological samples.     

New separation method for organic and inorganic selenium compounds based on anion exchange chromatography followed by inductively coupled plasma mass spectrometry

We describe a new method for separating the organic and inorganic selenocompounds methaneseleninic acid, selenite, selenate, methylselenocysteine, selenocystine as well as both selenomethionine and its oxidized form. The separation is performed on a Hamilton PRP-X100 column. According to the literature, the oxidized form of selenomethionine—which is easily formed—is eluted close to the dead volume when this column is used. The choice of parahydroxybenzoic acid as mobile phase enabled us to elute all of these species after this oxidized form, resulting in better identification and quantification. The factors determining separation (eluent concentration, pH, gradient) were optimized via an experimental design. Application of the method to yeast and commercial tablets showed that the principal Se compound present was selenomethionine, which was also present in its oxidized form.     

Simultaneous determination of total arsenic and total selenium in Chinese medicinal herbs by hydride generation atomic fluorescence spectrometry in tartaric acid medium

By HG-AFS, a new method was proposed for simultaneous determination of total arsenic and total selenium existed in the Chinese medicinal herbs in tartaric acid medium. The effects of analytical conditions and coexisting ions on the fluorescence signal intensity of analytes were investigated. The proposed method was provided with linear response ranges above 22 μg l⁻¹ for As and 44 μg l⁻¹ for Se, and the detection limits of 0.13 and 0.12 μg l⁻¹ were obtained for As and Se respectively. The recoveries of 93.8–96.1% for As and 95.3–99.1% for Se, and the precision of 1.2–3.8% and 2.4–5.3% (R.S.D., n = 8) respectively, were obtained via simultaneous determined four samples of Chinese medicinal herbs and three certified botanic reference materials successfully. The proposed method has the advantages of simple operation, high sensitivity and high efficiency.     

Synthesis of D- and L-selenomethionine double-labeled with deuterium and selenium-82

The synthesis of D- and L-selenomethionine labeled with ?2Se and three deuteriums at Se-methyl group (D- and L-[2H?, ?2Se]selenomethionine) was described. D- And L-[2H?, ?2Se]selenomethionine were prepared by condensation of (R)- and (S)-2-amino-4-bromobutylic acid with lithium [2H?, ?2Se]methaneselenolate, which was prepared from metal (82)Se and [2H?]methyl iodide. The optical purities of D- and L-[2H?, ?2Se]selenomethionine were determined by HPLC with a chiral stationary phase column and were found more than 99% ee. The chemical ionization mass spectra showed that the molecular related ion for N-isobutyloxycarbonyl ethyl ester derivatives of [2H?, ?2Se]selenomethionine did not overlap with the m/z values known from that of non-labeled selenomethionine.