Selenomethionine Extraction from Selenized Yeast: an LC-MS Study of the Acid Hydrolysis of a Synthetic Selenopeptide

A synthetically prepared seleno-peptide (AHPDVLTVXLQMLDDGR) was used as a model system for the acid hydrolysis of selenized yeast proteins. The seleno-peptide is a tryptic peptide of a heat shock protein 104 from Saccharomyces cerevisiae, was subjected to acid hydrolysis using methanesulfonic acid over a time period of 8 hours. Aliquots of the solution were sub-sampled at predetermined time intervals and the peptide fragments characterized by reversed phase LC MSⁿ. Similarly, the appearance of amino acid residues in the solution was monitored. It was found that after about 8 hours the synthetic peptide completely hydrolyzed. The use of a selenopeptide as a model for hydrolysis of selenized yeast hydrolysis was validated by comparing the decomposition time profile of the synthetic peptide with that of a selenized yeast sample. The rate of hydrolysis was identical in both systems, suggesting that the employed acid hydrolysis yields to the complete decomposition of the Se containing proteins in yeast and consequently to the liberation of selenomethionine.     

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.     

An improved HPLC method for the investigation of L‐selenomethionine metabolism in rat gut contents

Selenomethionine (SeMet) is a widely used nutritional supplement that has potential benefit for people living in selenium‐deficient areas. Previous research has shown that selenium administered as SeMet undergoes significant enterohepatic recycling which may involve the gut microflora. In order to investigate this we have developed a simple method for the quantitation of l ‐SeMet in rat gut content suspensions prepared from jejunum, ileum, caecum and colon. After incubation of l ‐SeMet with gut content suspensions, samples were deproteinized with sulfosalicylic acid and derivatized with o‐phthaldialdehyde (OPA) and N‐acetyl‐l ‐cysteine (NAC). Mass spectrometry confirmed the formation of a 1:1:1 derivative of l ‐SeMet with OPA and NAC. Samples were analysed by reversed‐phase high‐performance liquid chromatography with fluorescence detection. The assay was linear in the concentration range 0.5–100 µg/mL (r² = 0.9992) with a limit of detection of 0.025 µg/mL (signal‐to‐noise ratio of 5). Intra‐day and inter‐day accuracies were 91.1–92.8 and 91.7–95.5%, respectively with corresponding precisions as relative standard deviation of <5%. Incubation of l ‐SeMet with gut content suspensions from different parts of the rat intestine showed that l ‐SeMet metabolism occurs mainly in the caecum. Copyright © 2009 John Wiley & Sons, Ltd.     

Electrochemical oxidation of selenocystine and selenomethionine

Electrochemical oxidation of selenocystine (SeCys) and selenomethionine (SeMet), on a gold electrode was studied by cyclic voltammetry (CV), rotating disk electrode technique (RDE) and chronocoulometry (CC). In 0.2 mol/L HAc-NaAc (pH = 3.90) supporting electrolyte, anodic peak I potential of SeCys and SeMet was 810 mV and 638 mV, respectively, and this electrode process was diffused controlled. The electrochemical oxidation process of SeCys, in which six electron-transfers were involved, yielded selenocystine selenoxide. The electrochemical oxidation process of SeMet, in which two electron-transfers were involved, yielded selemethionine selenoxide.     

Determination of the absolute configuration of selenomethionine from antarctic krill by RP-HPLC/ICP-MS using chiral derivatization agents

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 µg 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.     

Comparative study of the instrumental couplings of high performance liquid chromatography with microwave-assisted digestion hydride generation atomic fluorescence spectrometry and inductively coupled plasma mass spectrometry for chiral speciation of selenomethionine in breast and formula milk

A comparison of chiral separation and analysis of selenomethionine in breast and formula milk, using high performance liquid chromatography (HPLC) on a glycopeptide teicoplanin-based chiral stationary phase (Chirobiotic T), coupled to atomic fluorescence spectrometry (AFS) and inductively coupled plasma (ICP) MS detectors has been performed. The coupling HPLC-microwave-assisted digestion hydride generation requires on-line post-column analytes treatment, and a severe sample clean-up for fat and proteins elimination using centrifugation and ultrafiltration. Underivatized -selenomethionine enantiomers were completely resolved in 10 min using unbuffered water mobile phase at 1 ml min⁻¹ flow. Good selectivity and sensitivities (detection limits 3.1 and 3.5 ng ml⁻¹ as Se for - and -selenomethionine, respectively) were obtained, and method robustness and simplicity, together to the low cost of AFS detector, makes it suitable for infant milk routine analysis. HPLC–ICP-MS coupling exhibits very low detection limits (0.9 ng ml⁻¹, as Se) for each -selenomethionine enantiomers, but the method suffers from matrix influence, that produces a poor S/N ratio and low reliability.

The methods were applied to breast and formula milk samples with recoveries of 80% of the total selenium presence, which is attributable to the existence of other unknown species. -Selenomethionine was the only isomer present in breast milk, but a 30% of -selenomethionine was also detected in formula milk.     

Enzymatic probe sonication extraction of Se in animal-based food samples: a new perspective on sample preparation for total and Se speciation analysis

This paper describes a fast, simple and novel extraction method for total selenium and selenium species determination in food samples. Parameters influencing extraction, such as sonication time, extracting media, temperature, sample mass, ultrasound amplitude and sample/enzyme mass ratio were investigated. The enzymatic hydrolysis proposed, enhanced by probe sonication, allowed the quantitative extraction of selenium in chicken muscle, liver, kidney and feed (97, 93, 95 and 102%, respectively) in 2 min, maintaining the original Se-species integrity. Total Se content of the samples was determined using inductively coupled plasma mass spectrometry. Se-species were identified and quantified using high-performance liquid chromatography in conjunction with inductively coupled plasma mass spectrometry. Chromatographic analyses were carried out under two chromatographic conditions and led to the identification of SeMet in all samples. The accuracy of the proposed method was assessed using certified reference materials as well as microwave digestion. Potential advantages of the proposed method over traditional hydrolysis are speed, simplicity and safety of the procedure.     

Speciation of arsenic in ground water samples: A comparative study of CE-UV, HG-AAS and LC-ICP-MS

The performance of capillary electrophoresis-ultraviolet detector (CE-UV), hydride generation-atomic absorption spectrometry (HG-AAS) and liquid chromatography-inductively coupled plasma mass spectrometry (LC-ICP-MS) have been compared for the speciation of arsenic (As) in groundwater samples. Two inorganic As species, arsenite (As^III), arsenate (As^V) and one organo species dimethyl arsenic acid (DMA) were mainly considered for this study as these are known to be predominant in water. Under optimal analytical conditions, limits of detection (LD) ranging from 0.10 (As^III, AsT) to 0.19 (DMA) μg/l for HG-AAS, 100 (As^III, DMA) to 500 (As^V) μg/l for CE-UV and 0.1 (DMA, MMA) to 0.2 (As^III, As^V) μg/l for LC-ICP-MS, allowed the determination of the above three species present in these samples. Results obtained by all the three methods are well correlated (r² = 0.996*** for total As) with the precision of <5% R.S.D. except CE-UV. The effect of interfering ions (e.g. Fe²⁺, Fe³⁺, SO₄²⁻ and Cl⁻) commonly found in ground water on separation and estimation of As species were studied and corrected for. Spike recovery was tested and found to be 80-110% at 0.5 μg/l As standard except CE-UV where only 50% of the analyte was recovered. Comparison of these results shows that LC-ICP-MS is the best choice for routine analysis of As species in ground water samples.     

Investigation of the stability of selenoproteins during storage of human serum by size-exclusion LC-ICP-MS

The stability of selenium-containing proteins in human serum was investigated by size-exclusion chromatography-ICP-MS under non-denaturating conditions. The peak characteristic to glutathione peroxidase (GSHPx) (tetramer) was the most affected; peaks of the GSHPx monomer and of selenite were found to appear with time. No trace of selenite was present in fresh samples, which suggest that the presence of this species in serum may be an artifact of storage. The mass balance of selenium species indicates that some of selenium is lost as a function of the storage time.     

Study of the lipophilicity of selenium species

In this work the lipophilicity of different selenium species occurring in environmental matrices and food, Se(IV), Se(VI), selenomethionine (Se-Met), selenocystamine (Se-CM), selenocystine (Se-Cyst), and dimethyl diselenide (CH₃)₂Se₂, was investigated in the octanol–water system, using the shaking flask method and detection with inductively coupled plasma–atomic emission spectrometry (ICP–AES), in order to assess their environmental fate and tendency to bioaccumulate. Polarography was also used for the electrochemically active Se species, Se(IV), Se-Cyst, Se-CM and (CH₃)₂Se₂, and the results were compared with those measured by ICP–AES. Furthermore, the influence of pH was studied by determining the logarithm of the distribution coefficient, log D, at three pH values, 5, 7, and 9, as was the impact of the marine environment on the lipophilicity profile of the six investigated Se species. The results were compared with those estimated approximately by use of PrologD software, based on the Ghose-Crippen log P (P: partition coefficient) calculation system, the only system which incorporates values—even though approximate—for the atom type of Se. Finally, from our experimental data an indicative value of the Se–Se fragment for log P prediction, for use in drug design, was estimated.     

硒酵母中有机硒及硒代氨基酸含量的测定方法

报道了人工培养硒酵母中有机硒及硒代胱氨酸（ＳｅＣｙｓ）和硒代蛋氨酸（ＳｅＭｅｔ）含量的测定方法。采用透析处理法使硒酵母中的无机硒和有机硒得以分离,并采用催化分光光度法测定了硒酵母中有机硒的含量;采用氨基酸自动分析仪测定了硒酵母中ＳｅＣｙｓ和ＳｅＭｅｔ的含量。     

Approach for rapid extraction and speciation of mercury using a microtip ultrasonic probe followed by LC-ICP-MS

A fast method for mercury extraction from biological samples based on the use of HCl leaching plus different enzymatic hydrolysis (with and without mercury complexing agents), and the use of focussed ultrasounds (2-mm microtip) is here proposed. Total mercury content in several biological samples was determined by FI-ICP-MS using a carrier solution consisting of 0.1% (v/v) HCl, 0.1% (v/v) 2-mercaptoethanol, to avoid memory effect, and 0.15% (w/v) KCl. For mercury speciation a RP18 chromatographic column coupled to ICP-MS was used. A mobile phase consisting of 0.1% (v/v) formic acid, 0.1% (v/v) HFBA, 2% (v/v) methanol, and 0.02% (w/v) mM l-cysteine at pH 2.1 was used for chromatographic separation of the mercury species in the sample extracts. Extraction procedures were validated by using 50 mg of tuna fish tissue CRM-463 (2.85 ± 0.16 mg kg⁻¹ for methylmercury). The recoveries obtained were 99 ± 3% and 93 ± 1% after acid leaching (HCl 7 M) and enzymatic extraction (15 mg protease type XIV in 2.5% (v/v) 2-mercaptoethanol), respectively. The optimal sonication conditions (5 min of exposure time and 40% of ultrasound amplitude) were applied to 5 mg of CRM-463 (88 ± 5%), 5 mg of mussel tissue (81 ± 11%) and to 2 mg of zebra fish embryos (90 ± 10%) obtaining good recoveries in all cases. Methylmecury was found to be the most abundant Hg specie in all samples. The developed method is simple and rapid (5 min sample treatment); it is suitable for very small samples and does not alter the original form of the mercury species. Thus, it is of special interest in those cases in which validation of the results may often be hampered by lack of sample availability.     

Multielemental speciation of As, Se, Sb and Te by HPLC-ICP-MS

An anion exchange HPLC-ICP-MS procedure allowing the simultaneous multielemental speciation analysis of arsenic, selenium, antimony and tellurium has been developed. Four arsenic species (As^III, As^V, monomethylarsonic acid and dimethylarsinic acid), two selenium species (Se^IV and Se^VI) may be determined in a single run as well as one antimony (Sb^V) and one tellurium species (Te^VI). Alternatively Sb and/or Te may be used as internal standards for As and Se speciation studies. Optimisation of ICP-MS conditions led to satisfactory relative (0.01 (Sb^V) to 1.8 (Se^VI) ng ml⁻¹) and absolute detection limits (1–180 pg). Reproducibility ranged from 3.1 to 5.6% and the linearity was verified in the 0–200 ng ml⁻¹ range.     

Determination of selenocysteine and selenomethionine in edible animal tissues by 2D size-exclusion reversed-phase HPLC-ICP MS following carbamidomethylation and proteolytic extraction

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).     

Determination of trimethylselenonium iodide, selenomethionine, selenious acid, and selenic acid using high-performance liquid chromatography with on-line detection by inductively coupled plasma mass spectrometry or flame atomic absorption spectrometry

An analytical method has been developed for the determination of selenious acid, selenic acid, trimethylselenonium ion, and selenomethionine. The four selenium compounds were separated by HPLC on a column (25 cm×4 mm I.D.) of the anion-exchanger ESA Anion III with a mobile phase (1.5 ml/min) of 0.0055 M ammonium citrate (pH 5.5). Detection was carried out using an on-line inductively coupled plasma mass spectrometer (ICP-MS) or a flame atomic absorption spectrometer (FAAS) as the selenium-specific detector. The chromatographic parameters and the chemical factors affecting the separation of the selenium species were optimized. The four selenium compounds could be separated within 8 minutes. The detection limits of the coupled HPLC–FAAS system were approximately 1 mg Se/l for each compound (100 μl injection), estimated as three times the base-line noise of the chromatograms. More powerful selenium detection was achieved with an ICP-MS. Selenium was measured at m/z 78. To increase the nebulization efficiency, the Meinhard concentric glass nebulizer was replaced by an ultrasonic nebulizer. The ICP-MS signal intensity was increased with the ultrasonic nebulization by a factor of 7 times for selenious acid and 24 to 31 times for trimethylselenonium ion, selenomethionine, and selenic acid compared to that with the Meinhard nebulization. The detection limits achieved by the HPLC–ICP-MS with the ultrasonic nebulization were 0.08 μg Se/l for trimethylselenonium ion, 0.34 μg Se/l for selenious acid, 0.18 μg Se/l for selenomethionine, and 0.07 μg Se/l for selenic acid, respectively.     

In vitro translation with [<Superscript>34</Superscript>S]-labeled methionine,selenomethionine, and telluromethionine

Heteroisotope and heteroatom tagging with [³⁴S]-enriched methionine (Met), selenomethionine (SeMet), and telluromethionine (TeMet) was applied to in vitro translation. Green fluorescent protein (GFP) and JNK stimulatory phosphatase-1 (JSP-1) genes were translated with wheat germ extract (WGE) in the presence of Met derivatives. GFPs containing Met derivatives were subjected to HPLC coupled with treble detection, i.e., a photodiode array detector, a fluorescence detector, and an inductively coupled plasma mass spectrometer (ICP-MS). The activities of JSP-1-containing Met derivatives were also measured. GFP and JSP-1 containing [³⁴S]-Met and SeMet showed comparable fluorescence intensities and enzyme activities to those containing naturally occurring Met. TeMet was unstable and decomposed in WGE, whereas SeMet was stable throughout the experimental period. Thus, although Te was the most sensitive to ICP-MS detection among S, Se, and Te, TeMet was less incorporated into the proteins than Met and SeMet. Finally, the potential of heteroisotope and heteroatom tagging of desired proteins in in vitro translation followed by ICP-MS detection was discussed. Figure TeMet was less incorporated into GFP than Met and SeMet due to its instability in WGE     

A micellar electrokinetic chromatography approach using diastereomeric derivatization and a volatile surfactant for the enantioselective separation of selenomethionine

A MEKC methodology with UV detection was developed for the enantioselective separation of selenomethionine (SeMet). The use of (+)‐1‐(9‐fluorenyl)ethyl chloroformate (FLEC) as chiral derivatization reagent to form SeMet diastereomers enabled their subsequent separation using ammonium perfluorooctanoate (APFO) as a volatile pseudostationary phase. The effect of APFO concentration and pH, temperature, injection volume, and derivatization conditions (time and FLEC/SeMet ratio) were evaluated in order to select the best separation conditions. A chiral resolution of 4.4 for DL‐SeMet was achieved in less than 6 min using 100 mM APFO at pH 8.5 as electrophoretic buffer. Satisfactory results were obtained in terms of linearity, precision (RSD from 3.4 to 5.1% for migration times and from 1.8 to 4.6% for corrected peak areas), accuracy, and LODs (3.1 × 10^?6 M and 3.7 × 10^?6 M for d and l enantiomers, respectively). The method was successfully applied to the determination of l ‐SeMet in food supplements.     

Multielemental speciation of As, Se, Sb and Te by HPLC-ICP-MS

An anion exchange HPLC-ICP-MS procedure allowing the simultaneous multielemental speciation analysis of arsenic, selenium, antimony and tellurium has been developed. Four arsenic species (As^III, As^V, monomethylarsonic acid and dimethylarsinic acid), two selenium species (Se^IV and Se^VI) may be determined in a single run as well as one antimony (Sb^V) and one tellurium species (Te^VI). Alternatively Sb and/or Te may be used as internal standards for As and Se speciation studies. Optimisation of ICP-MS conditions led to satisfactory relative (0.01 (Sb^V) to 1.8 (Se^VI) ng ml⁻¹) and absolute detection limits (1–180 pg). Reproducibility ranged from 3.1 to 5.6% and the linearity was verified in the 0–200 ng ml⁻¹ range.     

四川兴文地区硒的地球化学特征及生物效应

四川兴文土地质量调查显示:兴文县表层土壤中硒质量分数为0.075~2.540 mg/kg,平均值为0.43 mg/kg,富硒土地面积占调查面积53.25%,合计734.75 km^2。全区大米硒质量分数平均值为0.064 mg/kg,硒含量处于较为富足水平。水稻中的硒元素含量与根系土中硒元素呈明显正相关关系。根系土中水溶态硒和离子交换态硒含量高时,土壤容易产出富硒作物。     

Mössbauer, NMR and ATR-FTIR spectroscopic investigation of degradation in RTV siloxane foams

The combination of experimental techniques allowed for a comprehensive study of aging processes occurring in RTV siloxane foams. ¹¹⁹Sn Mössbauer spectroscopy demonstrated that tin residues are composed of Sn(II) and Sn(IV) species. The 27-year-old foams showed only Sn(IV) species with a quadrupole-splitting parameter larger than that observed for SnO₂. Solid-state ²⁹Si NMR differentiated between the various functional linkages in the foams, and showed no significant change of the di- to trifunctional linkage ratios. High-resolution NMR, on solvent extract of foams, showed the presence of water, catalyst, plasticizer, and some silicone oligomers. ATR-FTIR demonstrated changes near the surface of the foam when aged with water and with the presence of the tin catalyst. Gamma irradiation at a low dose had little effect on compression sets. The main changes observed for artificially aged and aged in service foams were related to the presence of the tin catalyst.