Determination of selenomethionine, selenocysteine, and inorganic selenium in eggs by HPLC–inductively coupled plasma mass spectrometry

A method for the simultaneous determination of selenomethionine (SeMet), selenocysteine (SeCys), and selenite [Se(IV)] in chicken eggs was developed. A sample preparation protocol including defatting, protein denaturation, and carbamidomethylation was optimized in order to achieve complete protein digestion and to avoid SeCys losses. Quantification was carried out by reversed-phase HPLC–inductively coupled plasma mass spectrometry (ICP MS) after quantitative isolation of the selenium-containing fraction by size-exclusion liquid chromatography. The detection limits were 0.06, 0.003, and 0.01 μg g⁻¹ (dry weight) for SeCys, Se(IV) and SeMet, respectively, and the precision was 5–10%. The end products of carbamidomethylation of the different selenium species were identified for the first time by electrospray QTOF MS after custom-designed 2D HPLC purification. Differences in selenium speciation in egg yolk and white were highlighted, the yolk containing more SeCys and the white more SeMet. An insight into selenium bioaccessibility in eggs was obtained by digestion with simulated gastric and gastrointestinal juices and size-exclusion HPLC-ICP MS.     

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).     

Column-switching system for selenium speciation by coupling reversed-phase and ion-exchange high-performance liquid chromatography with microwave-assisted digestion-hydride generation-atomic fluorescence spectrometry

Speciation of selenocysteine (SeCys), selenomethionine (SeMet), selenoethionine (SeET), selenite (Se(IV)) and selenate (Se(VI)) has been accomplished using high-performance liquid chromatography, with the aid of an anion exchange column and a reversed-phase column, both connected through a six-port switching valve. On-line microwave-assisted digestion and hydride generation steps were performed prior to the atomic fluorescence detection. The elution of the seleno amino acids was accomplished in the reversed-phased column using water as mobile phase. Selenite and selenate were separated in the anion exchange column, using gradient elution with an acetate buffer. The separation of the five selenium compounds took place in 15 min. The detection limits obtained ranged between 0.6 and 0.9 microg l(-1). Values of r>0.998 were obtained for linear fit graphs. A commercial available urine sample was analyzed, in which SeCys and Se(IV) were quantified.     

Direct amino acid analysis method for speciation of selenoamino acids using high-performance anion-exchange chromatography coupled with integrated pulsed amperometric detection

Speciation analysis of selenomethylcysteine (SeMeCys), selenomethionine (SeMet) and selenocystine (SeCys) has been performed using a direct amino acid analysis method with high-performance anion-exchange chromatography (HPAEC) coupled with integrated pulsed amperometric detection (IPAD). Three selenoamino acids could be baseline-separated from 19 amino acids using gradient elution conditions for amino acids and determined under new six-potential waveform. Detection limits for SeMeCys, SeMet and SeCys were 0.25, 1 and 20 microg/L (25 microL injection, 10 times of the baseline noise), respectively. The relative standard deviations (RSDs) of 200 microg/L SeMeCys, SeMet and SeCys were 3.1, 4.1 and 2.8%, respectively (n=9, 25 microL injection). The proposed method has been applied for determination of selenoamino acids in extracts of garlic and selenious yeast granule samples. No selenoamino acids were found in garlic. Both SeMet and SeCys were detected in selenious yeast tablet with the content of 45 and 129 microg Se/g, respectively. Selenoamino acids standards were spiked in garlic and yeast granule samples and the recovery ranged from 90 to 106%.     

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。     

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。     

Speciation analysis of selenium in rice samples by using capillary electrophoresis-inductively coupled plasma mass spectrometry

An enzyme-assisted extraction used to extract all species of selenium in rice sample and a sensitive analytical method for the determination of ultratrace Se(VI), Se(IV), SeCys₂ (selenocystine) and SeMet (selenomethionine) with capillary electrophoresis-inductively coupled plasma mass spectrometry were firstly described in this study. The extraction method is simple, effective and can be used to extract trace selenium compounds in rice with high extraction efficiency and no altering its species. The analytical method has a detection limit of 0.1-0.9 ng Se/mL, and can be used to determine trace Se(VI), Se(IV), SeCys₂ and SeMet in rice directly without any derivatization and pre-concentration. With the help of above methods, we have successfully determined Se(VI), Se(IV), SeCys₂ and SeMet in selenium-enriched rice within 18 min with a recovery of 90-103% and a RSD (relative standard deviation, n = 6) of 3-7%. Our results indicated that selenium-enriched rice contained only one species of selenium, SeMet, and its concentration is in range of 0.136-0.143 μg Se/g dried weight. The proposed method providing a realistic approach for the nutritional and toxical evaluation of different selenium compounds in nutritional supplements.     

液相色谱-双通道原子荧光检测联用法同时测定砷和硒的形态

建立了一种利用高效液相色谱-双通道原子荧光检测联用同时进行砷和硒形态分析的方法。以10 mmol/L NH4H2PO4溶液(pH 5.6)(添加2.5%(体积分数)的甲醇)为流动相,在12 min内同时分离了三价砷(As(III))、一甲基砷(MMA)、二甲基砷(DMA)、五价砷(As(V))、硒代胱氨酸(SeCys)、硒代蛋氨酸(SeMet)和四价硒［Se(IV)］等化合物。As(III)、DMA、MMA、As(V)、SeCys、SeMet和Se(IV)的检出限分别为1,3,2,3,4,18和3 μg/L (进样量为200 μL),5次测定的相对标准偏差为1.9%～6.1%(As 100 μg/L, Se 300 μg/L)。应用该方法对人体尿样及硒酵母片中砷和硒的形态进行了分析,目标物在尿样中的加标回收率为83%～108%,在硒酵母片中的加标回收率为88%～105%。实验结果表明,该方法可用于尿样及药品中砷和硒形态的日常分析。该方法减少了样品的分析时间和试剂用量,降低了工作强度,提高了工作效率。     

Selenium speciation by high-performance anion-exchange chromatography-post-column UV irradiation coupled with atomic fluorescence spectrometry

A technique for the speciation of selenomethylcysteine (SeMeCys), selenocystine (SeCys), selenite [Se(IV)] and selenomethionine (SeMet) was established in this paper using high-performance anion-exchange chromatography coupled with atomic fluorescence spectrometry (HPAEC-AFS). Analytes were separated on an AminoPac PA10 column and then digested by on-line ultraviolet (UV) irradiation, which destroyed organic compound structure. Hydride generation was used as an available sample introduction technique for atomic fluorescence detection. The detection limits of four compounds were 1-5 microg/L (250 microL injection, 10 times of the baseline noise). The relative standard deviations (RSDs), calculated from seven consecutive injections of 100 microg/L standard mixtures, were from 2 to 4%. Selenious yeast tablet, which had been proposed as selenium supplement, and human urine collected from a volunteer were analyzed. Good spiked recoveries from 86 to 103% were obtained.     

Selenium speciation in cow milk obtained after supplementation with different selenium forms to the cow feed using liquid chromatography coupled with hydride generation-atomic fluorescence spectrometry

The purpose of this paper is to develop an easy and quick on-line selenium speciation method (LC-UV-HG-AFS) in cow milk obtained after different supplementation to cow feed. This study focuses on selenium speciation in cow milk after the use of different selenium species (organic selenium as selenised yeast and inorganic selenium as sodium selenite) in the supplementation of forages. Separation was carried out on a μBondapack C₁₈ column with the positively charged ion-pairing agent tetraethylammonium chloride in the mobile phase. The optimization of pre-reduction conditions was carried out; this step was done with UV irradiation and a heating block to improve the reduction of the different Se-compounds. Variables such as exposure time, hydrochloric acid concentration and temperature were studied. The detection limits for SeCyst₂, Se(IV), SeMet and Se(VI) were 0.4, 0.5, 0.9 and 1.0 μg l⁻¹, respectively. The proposed method was applied to cow milk samples. The milk samples obtained after an organic supplementation of feeding as selenised yeast present three species of selenium, SeCyst2, Se(IV) and SeMet, while only SeCyst2 and Se(IV) are present in milk samples obtained after an inorganic supplementation of feeding.     

Selenium Biofortification: Roles,Mechanisms, Responses and Prospects

The trace element selenium (Se) is a crucial element for many living organisms, including soil microorganisms, plants and animals, including humans. Generally, in Nature Se is taken up in the living cells of microorganisms, plants, animals and humans in several inorganic forms such as selenate, selenite, elemental Se and selenide. These forms are converted to organic forms by biological process, mostly as the two selenoamino acids selenocysteine (SeCys) and selenomethionine (SeMet). The biological systems of plants, animals and humans can fix these amino acids into Se-containing proteins by a modest replacement of methionine with SeMet. While the form SeCys is usually present in the active site of enzymes, which is essential for catalytic activity. Within human cells, organic forms of Se are significant for the accurate functioning of the immune and reproductive systems, the thyroid and the brain, and to enzyme activity within cells. Humans ingest Se through plant and animal foods rich in the element. The concentration of Se in foodstuffs depends on the presence of available forms of Se in soils and its uptake and accumulation by plants and herbivorous animals. Therefore, improving the availability of Se to plants is, therefore, a potential pathway to overcoming human Se deficiencies. Among these prospective pathways, the Se-biofortification of plants has already been established as a pioneering approach for producing Se-enriched agricultural products. To achieve this desirable aim of Se-biofortification, molecular breeding and genetic engineering in combination with novel agronomic and edaphic management approaches should be combined. This current review summarizes the roles, responses, prospects and mechanisms of Se in human nutrition. It also elaborates how biofortification is a plausible approach to resolving Se-deficiency in humans and other animals.     

Separation and determination of seleno amino acids using gas chromatography hyphenated with inductively coupled plasma mass spectrometry after hollow fiber liquid phase microextraction

A new derivatization–extraction method for preconcentration of seleno amino acids using hollow fiber liquid phase microextraction (HF‐LPME) was developed for the separation and determination of seleno amino acids in biological samples by gas chromatography–inductively coupled plasma mass spectrometry (GC–ICP‐MS). Derivatization was performed with ethyl chloroformate (ECF) to improve the volatility of seleno amino acids. Parameters influencing microextraction, including extraction solvent, pH of sample solution, extraction time, stirring speed, and inorganic salt concentration have been investigated. Under the optimal conditions, the limits of detection (LODs) obtained for Se‐methyl‐selenocysteine (SeMeCys), selenomethionine (SeMet), and selenoethionine (SeEth) were 23, 15, and 11 ng Se l⁻¹, respectively. The relative standard deviations (RSDs) were 14.6%, 16.4%, and 19.4% for SeMeCys, SeMet, and SeEth (c = 1.0 ng ml⁻¹, n = 7), respectively, and the RSDs for SeMeCys, SeMet could be improved obviously if SeEth was utilized as the internal standard. The proposed method was applied for the determination of seleno amino acids in extracts of garlic, cabbage, and mushroom samples, and the recoveries for the spiked samples were in the range of 96.8–108% and 93.4–115% with and without the use of SeEth as internal standard. The developed method was also applied to the analysis of SeMet in a certified reference material of SELM‐1 yeast and the determined value is in good agreement with the certified value. Copyright © 2008 John Wiley & Sons, Ltd.     

Organic and inorganic selenium speciation in environmental and biological samples by nanometer-sized materials packed dual-column separation/preconcentration on-line coupled with ICP-MS

A novel, fast, and cheap nonchromatographic method for direct speciation of dissolved inorganic and organic selenium species in environmental and biological samples was developed by flow injection (FI) dual-column preconcentration/separation on-line coupled with ICP-MS determination. In the developed technique, the first column packed with nanometer-sized Al(2)O(3) could selectively adsorb the inorganic selenium [Se(IV), Se(VI)], and the retained inorganic selenium could be eluted by 0.2 mol l(-1) NaOH, while the organic Se [selenocystine (SeCys(2)) and selenomethionine (Se-Met)] was not retained. On the other hand, the second column packed with mesoporous TiO(2) chemically modified by dimercaptosuccinic acid (DMSA) could selectively adsorb Se(IV) and SeCys(2) and barely adsorb Se(VI) and Se-Met. When the sample solution was passed through the column 1, separation of inorganic selenium and organic selenium could be achieved first. Then, the effluent from column 1 was successively introduced into the column 2 and the speciation of organic selenium could be attained due to the different adsorption behaviors of Se-Met and SeCys(2) on DMSA modified TiO(2). After that, the eluent from column 1 contained Se(IV), and Se(VI) was adjusted to desired pH and injected into column 2, and the speciation of Se(IV) and Se(VI) could also be realized thanks to their different retention on column 2. The parameters affecting the separation were investigated systematically and the optimal separation conditions were established. The detection limits obtained for Se(IV), Se(VI), Se-Met and SeCys(2) were 45-210 ng l(-1) with precisions of 3.6-9.7%. The proposed method has been successfully applied for the speciation of dissolved inorganic and organic selenium in environmental and biological samples. In order to validate the methodology, the developed method was also applied to the speciation of selenium in certified reference material of SELM-1 yeast, and the determined values were in good agreement with the certified values.     

Sensitivities of selenite, selenate, selenomethionine and trimethylselenonium ion in aqueous solution and in blood plasma-ETAAS compared with ICP-MS

Aqueous solutions and blood plasma spiked with selenite (Se(IV)), selenate (Se(VI)), selenomethionine (SeMet) or trimethylselenonium (TMSe) iodide were analyzed by Zeeman-corrected electrothermal atomic absorption spectrometry (ETAAS) using palladium as a chemical modifier, and by inductively coupled plasma mass spectrometry (ICP-MS). Using ETAAS, the sensitivities for Se(IV), SeMet and TMSe in aqueous solution were similar, whereas the sensitivity of Se(VI) was 63% of that value. In blood plasma, the ETAAS sensitivities of Se(IV) and Se(VI) were equal, whereas the sensitivities of SeMet and TMSe were 87 and 56%, respectively, of that value. In contrast, the ICP-MS sensitivities obtained for Se(VI), TMSe and SeMet were between 96 and 98% of that obtained for Se(IV) in aqueous solution and in blood plasma. It is concluded, that ICP-MS is superior to ETAAS as the problem of differences in sensitivity of the selenium species when using ETAAS are not prevalent when using the ICP-MS technique.     

Retention study of inorganic and organic selenium compounds on a silica-based reversed phase column with mixed ion-pairing reagents

Summary Separation of seven inorganic and organic selenium compounds, namely selenic acid [Se(VI)], selenous acid [Se(IV)], trimethylselenonium iodide (TMSe⁺), selenocystine (SeCys), selenomethionine (SeMet), selenoethionine (Seet), and selenocystamine (SeCM), has been performed on a LiChrosorb C 18 column by using mixed ion-pair reagents; 1-butanesulfonic acid and tetramethylammonium hydroxide. Flame atomic absorption spectrometry (FAAS) was used as an element-specific detector. The retention behaviors of selenium compounds in terms of several chromatographic parameters, such as pH of the mobile phase, the concentrations of ion-pair reagents, and the content of organic modifier (methanol) were investigated. It was found that the separation of both inorganic and organic selenium compounds can be achieved within 12 min with a mobile phase of 10 mM 1-butanesulfonic acid −4 mM tetramethylammonium hydroxide −4 mM malonic acid −0.05% methanol adjusted to pH 4.5 at a flow rate of 1.0 mL min⁻¹. The results obtained in this study showed that the use of mixed ion-pair reagents is very useful to improve the separation of selenium compounds. The applicability of this technique for the speciation of selenium compounds in real samples was demonstrated by the determination of selenium compounds in a selenium nutritional supplement. The results were found to be in good agreement with those obtained by ion-exchange HPLC-ICP-MS.     

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.     

AE-HG-AFS测定长期汞暴露人群补硒后尿中硒的形态

建立了一种利用阴离子交换高效液相色谱与氢化物发生原子荧光光谱联用同时测定四、六价硒及硒代半胱氨酸(SeCys)形态的方法。优化了六价硒的还原条件及仪器检测参数,以不同浓度的柠檬酸铵作为流动相,在10 min内同时分离了四、六价硒及硒代半胱氨酸(SeCys)。采用加标法定量,加标回收率在90%～119%之间,相对标准偏差为1.6%～3.1%(100μg/L),四、六价硒及硒代半胱氨酸(SeCys)的检出限分别为0.32μg/L、0.47μg/L和0.44μg/L(进样量为100μL)。应用该法对长期汞暴露人群补硒后尿中的小分子硒的形态进行了分析,仅检测到硒代半胱氨酸(SeCys)。     

硒代胱氨酸和硒代蛋氨酸电化学检测的比较

对硒代胱氨酸 (SeCys)和硒代蛋氨酸 (SeMet)在银电极上的电化学性质、电极反应机理及检测灵敏度等方面进行比较研究。实验表明 ,两者在pH9.5的硼砂 氢氧化钠介质中 ,于 -0 .62V和 -0 .68V(vs.SCE)处均存在一对氧化还原峰 ,电化学行为相似 ,两者具有良好的线性范围和低检出限 ,SeMet比SeCys具有更高的检测灵敏度。在相同的实验条件下 ,比较了含硫氨基酸与含硒氨基酸的电化学响应 ,含硫氨基酸的检测灵敏度远低于含硒氨基酸     

高效液相色谱-电感耦合等离子体质谱联用检测食品中的五种硒形态

建立了高效液相色谱-电感耦合等离子体质谱(HPLC-ICP-MS)联用检测硒酸盐(SeVI)、亚硒酸盐(SeIV)、硒代蛋氨酸(SeMet)、硒代胱氨酸(SeCys2)和硒代乙硫氨酸(SeEt)的方法。采用Hamilton PRP X-100色谱柱（250 mm×4.6 mm, 5 μm）,使用5 mmol/L的柠檬酸溶液(pH 4.5)作为流动相,电感耦合等离子体质谱(ICP-MS)检测,在21 min内可以完全分离5种硒形态。各形态硒的线性相关系数均大于0.9995, SeVI、SeIV、SeMet、SeCys2、SeEt的检出限分别为0.4、0.4、5.6、0.9、1.2 μg/L。探讨了不同提取方法的提取效果,鲜蘑菇和猪肉样品加标回收实验表明,对水溶性良好的无机硒和硒代蛋氨酸而言,采用柠檬酸溶液提取的效果非常好,SeIV和SeVI的回收率均在100%左右,SeMet的回收率为85.0%～95.3%;用蛋白酶水解提取,SeCys2和SeEt的回收率为79.9%～91.5%。该方法可完全满足食品中这5种硒形态的准确定量分析。     

Sequential photocatalyst-assisted digestion and vapor generation device coupled with anion exchange chromatography and inductively coupled plasma mass spectrometry for speciation analysis of selenium species in biological samples

We have developed an on-line sequential photocatalyst-assisted digestion and vaporization device (SPADVD), which operates through the nano-TiO₂-catalyzed photo-oxidation and reduction of selenium (Se) species, for coupling between anion exchange chromatography (LC) and inductively coupled plasma mass spectrometry (ICP-MS) systems to provide a simple and sensitive hyphenated method for the speciation analysis of Se species without the need for conventional chemical digestion and vaporization techniques. Because our proposed on-line SPADVD allows both organic and inorganic Se species in the column effluent to be converted on-line into volatile Se products, which are then measured directly through ICP-MS, the complexity of the procedure and the probability of contamination arising from the use of additional chemicals are both low. Under the optimized conditions for SPADVD – using 1 g of nano-TiO₂ per liter, at pH 3, and illuminating for 80 s – we found that Se(IV), Se(VI), and selenomethionine (SeMet) were all converted quantitatively into volatile Se products. In addition, because the digestion and vaporization efficiencies of all the tested selenicals were improved when using our proposed on-line LC/SPADVD/ICP-MS system, the detection limits for Se(IV), Se(VI), and SeMet were all in the nanogram-per-liter range (based on 3σ). A series of validation experiments – analysis of neat and spiked extracted samples – indicated that our proposed methods could be applied satisfactorily to the speciation analysis of organic and inorganic Se species in the extracts of Se-enriched supplements.