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.     

Selenomethionine contents of NIST wheat reference materials

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.     

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.     

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.     

Fourier-transform infrared spectroscopic study of the interactions of selenium species with living bacterial cells

A study of the interactions of several selenium species with living bacterial cells was carried out by Fourier-transform infrared (FT-IR) spectroscopy. Bacterial cells consisted of an Escherichia coli strain (K-12) cultivated in a growth medium based on glucose contaminated with selenium species. Equilibrium between the analyte in the solution and the extraction medium was established, and then the effects of selenium species upon the external membrane of the living bacterial cells were characterized by performing FT-IR spectroscopy of whole cells. The presence of the toxicants at various concentrations in the culture medium had an effect on the FT-IR spectra, and the concentration of the selenium species was determined directly in the biomass by FT-IR spectroscopy. The intensity ratios between several absorption lines, which varied as a function of the concentration of the selenium species, were used as the analytical signal.Electronic Supplementary Material Supplementary material is available for this article if you access the article at . A link in the frame on the left on that page takes you directly to the supplementary 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.     

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     

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).     

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     

用离子抑制色谱法分析二（2,2,6,6—四甲基—4—哌啶基）马来酸酯合？…

建立了用离子抑制色谱法分析二（２,２,６,６－四甲基－４－哌啶基）马来酸酯合成反应液的方法。平均回收率为９８．８％,相对标准偏差为０．５６％,测量的平均相对偏差不大于５．０％,方法简单,快速,可用于工艺条件的选择和质量检测。