HPLC-ICP-MS联用检测转基因大豆中的硒形态

本文研究建立了高效液相色谱-电感耦合等离子体质谱(HPLC-ICP-MS)联用检测转基因大豆中的硒酸盐(Se VI)、亚硒酸盐(Se IV)、硒代蛋氨酸(selenomethionine,Se Met)、硒代胱氨酸(selenocystine,Se Cys2)和硒代乙硫氨酸(selenoethionine,Se Et)的方法。探讨了色谱柱、流动相及其酸度对分离效果的影响,使用10 mmol/L的柠檬酸溶液(p H值4.5)作流动相,Hamilton PRP X-100色谱柱分离,碰撞反应池技术消除40Ar40Ar+和40Ar2H2+等多原子离子干扰,ICP-MS检测82Se同位素,在21 min内可完全分离检测5种硒形态。探讨了不同提取方法的提取效果,优化了提取条件,采用蛋白酶提取,针对美国进口的转基因大豆样品进行加标回收实验,结果表明采用蛋白酶提取,Se IV和Se VI的回收率在100%左右,Se Met的回收率在92.6%~109.3%之间,Se Cys2和Se Et的回收率为81.2%~95.9%。该方法可完全满足转基因大豆中的硒形态定量分析鉴定。     

高效液相色谱-电感耦合等离子体质谱法测定富硒食用菌中的4种硒形态

目的建立富硒食用菌中硒代胱氨酸(Se Cys)、4价硒(Se(IV))、硒代蛋氨酸(Se Met)、6价硒(Se(VI))4种硒形态的高效液相色谱-电感耦合等离子体质谱(high performance liquid chromatography-inductively coupled plasma-mass spectrometry,HPLC-ICP-MS)的分析方法。方法以Hamilton PRP-X100为色谱分离柱,以40mmol/L磷酸氢二铵(pH 6.0)为流动相,对富硒食用菌中4种硒形态进行分离,利用电感耦合等离子体质谱法对质量数为77的硒同位素(~77Se)进行定量检测,样品提取采用蛋白酶(来源于地衣芽孢杆菌)为提取剂,60℃水浴提取1 h。结果 4种硒形态在0~100μg/L浓度范围内线性良好,相关系数r~2大于0.997,检出限在2μg/L以下。结论该方法适用于富硒食用菌中硒的形态分析。     

超声辅助酶法提取-高效液相色谱-电感耦合等离子体质谱联用分析食品中无机硒和硒氨基酸6种硒形态

建立了超声辅助酶法提取-高效液相色谱-电感耦合等离子体质谱联用同时测定食品中无机硒和硒氨基酸等6种硒形态的新方法。对影响硒形态分离及提取的诸因素如提取方式、流动相pH、浓度、甲醇含量、流速和柱温等进行详细考察和优化,同时利用碰撞反应池,有效地消除了复合离子的干扰,提高了检测灵敏度。该方法在9.50 min内使亚硒酸盐(Se(IV))、硒酸盐(Se(VI))、硒胱氨酸(SeCys_2)、硒蛋氨酸(SeMet)、甲基硒代半胱氨酸(MeSeCys)和硒代乙硫氨酸(SeEt)等6种目标分析物完全达到基线分离,各物质的检出限为(0.012~0.60)μg Se/L,线性范围达3~4个数量级,相对标准偏差(RSDs,n=7)为4.0%~8.2%;将该方法应用于食品中硒形态的分析,其加标回收率为81.6%~108.6%。方法具有操作简单方便、快速高效、灵敏度高和环境友好等优点,可满足食品中硒形态定量分析     

HPLC-ICP-MS在线联用分析食品中无机硒和硒氨基酸的形态

以亚硒酸盐(Se(Ⅳ))、硒酸盐(Se(Ⅵ))、硒胱氨酸(Se Cys_2)、硒蛋氨酸(Se Met)、甲基硒代半胱氨酸(Me Se Cys)和硒代乙硫氨酸(Se Et)为硒形态的目标分析物,采用Dionex Ion Pac AS11色谱柱(250 mm×4.0 mm)为分离柱,通过优化流动相的p H、浓度、甲醇含量、流速和色谱柱的柱温等因素对六种目标硒形态分离及不同提取方法对目标分析物提取效率的影响,建立了高效液相色谱-电感耦合等离子体质谱(HPLC-ICP-MS)在线联用同时测定食品中无机硒和硒氨基酸的分析方法。Me Se Cys、Se Met、Se Et、Se(Ⅳ)、Se Cys_2和Se(Ⅵ)的检出限分别为0.25、0.20、0.35、0.15、0.30、0.15μg Se/L;Me Se Cys和Se Cys_2的线性范围为2.0~2500μg Se/L,Se(Ⅳ)和Se(Ⅵ)线性范围为1.2~1200μg Se/L,Se Met和Se Et的线性范围分别是1.5~1500μg Se/L和2.2~2200μg Se/L,各硒形态的线性相关系数均不少于0.9997。将该方法应用于食品中硒形态的分析,其加标回收的回收率为80.8%~106.7%,相对标准偏差(RSDs,n=3)为4.7%~9.6%。方法具有操作简单、方便快速、灵敏度高和环境友好等优点,可满足食品中硒形态定量分析。     

高效液相色谱-电感耦合等离子体质谱法检测富硒苹果中5种硒形态

目的建立高效液相色谱-电感耦合等离子体质谱联用技术(high performance liquid chromatographyinductively coupled plasma mass spectrometry,HPLC-ICP-MS)测定富硒苹果中5种硒形态的方法。方法对样品提取方法、流动相的类型、浓度、p H值等条件进行考察及确定,采用柠檬酸超声提取的方式处理样品,流动相为浓度为5 mmol/L、p H=5.0的柠檬酸溶液。选用Hamiltion PRP-X100阴离子分析柱,色谱进样量为100μL,质谱采用碰撞池模式进行测定。结果本方法在15 min内可以完全分离5种硒形态,硒代胱氨酸(Se Cys2)、甲基硒代半胱氨酸(Me Se Cys)、亚硒酸根(Se(Ⅳ))、硒代蛋氨酸(Se Met)、硒酸根(Se(Ⅵ))的检出限分别为0.6、0.7、1、0.9、1μg/L,样品加标回收率范围为82.1%~98.3%,相对标准偏差为1.6%~3.8%。结论本方法可以简单、快速、准确地测定富硒苹果中硒的5种形态。     

高效液相色谱-电感耦合等离子质谱法分析富硒茶叶中硒的形态

建立富硒茶叶中硒代胱氨酸（SeCys2）、甲基-硒代半胱氨酸（SeMC）、亚硒酸根（Se（IV））、硒代蛋氨酸（SeMet）、硒酸根（Se（VI））5?种硒化合物的高效液相色谱-电感耦合等离子体质谱的分离检测方法。方法利用Hamilton?PRP-X100色谱柱对富硒茶叶中5?种硒化合物进行分离,利用电感耦合等离子体质谱对硒元素进行检测,外标法定量测定5?种硒化合物的含量。研究采用蛋白酶K溶液为提取剂,超声提取60?min,以柠檬酸溶液为流动相,梯度洗脱,5?种硒化合物在15?min内得到了有效分离。5?种硒化合物标准曲线的线性相关系数不小于0.999?8,检出限为0.13～1.09?μg/L。该前处理方法样品加标回收率在91.8%～109.4%之间。方法操作简便、分离效果好、检出限低,适用于富硒茶叶中硒的形态分析。     

食品中硒元素形态分析的研究进展

硒是人体必需的微量元素,具有丰富的化学形态,分为无机硒和有机硒,不同硒形态对人体的生物活性不同。无机硒主要包括硒酸Se(Ⅵ)、亚硒酸Se(Ⅳ),有机硒主要包括硒蛋白、硒核酸和硒多糖等大分子硒,及硒代胱氨酸(selenocystine,SeCys2)、硒代蛋氨酸(selenomethinonine,SeMet)、甲基硒代半胱氨酸(methyl selenocysteine,MeSeCys)、硒代乙硫氨酸(selenoethionine,SeEt)等小分子硒化物。无机硒不易吸收,可能对人体造成危害,有机硒易吸收,适当摄取对人体有多方面益处。准确测定食品中不同硒形态及具体含量,对人体按需摄入硒元素具有重要指导意义。本文总结了近年来食品行业中硒形态检测研究涉及的领域,包括果蔬、水产、药食同源的中药、茶叶、谷物等方面的研究,常用的提取、分离、分析技术,重点介绍了高效液相色谱-电感耦合等离子体质谱联用技术和液相色谱-氢化物发生-原子荧光光谱联用技术在不同食品中检测硒形态的应用情况,并展望了未来硒形态分析的前景。     

不同培养条件下杏鲍菇对硒的耐受性与硒形态分析

为了明确不同培养条件下杏鲍菇对外源硒的耐受性,分别在固体培养基、液体发酵培养基和栽培基质中添加不同量的亚硒酸钠,并检测杏鲍菇子实体中富集硒的存在形态。结果表明:固体培养条件下,硒浓度低于160 mg/L时,对菌丝的生长没有显著影响;液体培养条件下,4 mg/L的硒元素即可对杏鲍菇菌丝体的生长产生显著抑制;栽培模式下,基质中补充10～600 mg/kg的硒元素,不会影响杏鲍菇菌丝体的生长,且子实体中的硒含量会随基质中硒浓度的增加而增加。子实体硒形态分析表明,富集硒以硒代蛋氨酸、甲基硒代半胱氨酸、硒代胱氨酸和亚硒酸盐[Se (IV)]四种形式存在,其中硒代蛋氨酸是硒与氨基酸的主要结合形式。     

HPLC-ICP-MS法测定富硒小麦中硒的形态

建立高效液相色谱-电感耦合等离子体质谱联用(high performance liquid chromatography-inductively coupled plasma mass spectrometry,HPLC-ICP-MS)检测富硒小麦中4种硒形态分析方法。使用微波辅助酶萃取富硒小麦中亚硒酸盐(SeIV)、硒酸盐(SeVI)、硒代蛋氨酸(SeMet)和硒代胱氨酸(SeCys2),采用Hamilton PRP X-100色谱柱,以6 mmol/L柠檬酸为流动相,pH=5.0,在9 min内可完全分离4种硒形态。SeCys2、SeIV、SeMet、SeVI的检出限分别为0.23、0.15、0.30、0.16μg/L,线性相关系数(r2)均大于0.999。以富硒小麦为基体进行加标回收试验,SeCys2、SeIV、SeMet、SeVI的回收率在93.7%～105.2%之间,相对标准偏差为2.2%～6.6%(n=6)。方法简单快速,具有良好的精密度和准确度,适用于富硒小麦中硒形态分析。     

HPLC-ICPMS联用法测定富硒酵母粉中砷和硒的形态分析

文章采用高效液相色谱-电感耦合等离子体质谱仪测定砷和硒的形态。以硒同位素的自然丰度比为参考值排除了质谱的离子干扰,并使用单元素实验确定目标谱峰的保留时间;优化了流动相的比例、盐浓度、pH值和流速条件,最终选择pH为5.7的2.5%甲醇+15 mmol/L的磷酸二氢铵+1 mmol/L的四丁基溴化铵混合溶液为流动相,实验采用Hamilton PRP-Ⅹ100阴离子交换柱,此条件下可以把砷和硒的7种形态全分离,并符合分离度要求。实验表明,三价砷As(Ⅲ),二甲基砷DMA,一甲基砷MMA,五价砷As(Ⅴ),硒代胱氨酸SeCys,硒代蛋氨酸Se Met和四价硒Se(Ⅳ)都具有良好的线性和较低的检出限,回收率为81%～109%。将该方法应用于不同种类的富硒酵母样品分析测试,结果令人满意。     

Determination of 5 natural selenium species in selenium-enriched bamboo shoots using LC-ICP-MS

A method for simultaneous determination of the selenium species methylselenocysteine (MeSeCys), selenocysteine (SeCys₂), selenite (SeIV), selenomethionine (SeMet), and selenates (SeVI) in natural selenium-enriched bamboo shoots was developed. Samples were extracted using sonication with a solution of pepsin. Different selenium species were separated using an anion exchange column with isocratic elution and a citric acid mobile phase at pH 4.7. Separated selenium species were detected as ⁸⁰Se using inductively coupled plasma mass spectrometry (ICPMS) in dynamic reaction cell (DRC) gas mode. The method achieved acceptable quantitative recoveries of 73.9 to 113.4% with relative standard deviations of <5%. The limits of detection were 1, 2, 2, 3, and 3 μg/kg (fresh weight) for SeCys₂, Se, MeSeCys, SeMet, and Se, respectively. The method was simple, rapid, accurate, and useful for determination of selenium species in natural selenium enriched bamboo shoots.     

Selenium speciation studies in Se-enriched chives (Allium schoenoprasum) by HPLC-ICP–MS

In this study, three liquid chromatographic techniques were employed to better understand selenium species distribution in Chives (Allium schoenoprasum) separately grown in three different supplementation media Se(IV), Se(VI), and SeMet. The highest selenium accumulation up to 700 μg Se g⁻¹ was observed in the case of the Se(VI)-enriched samples on the basis of total selenium measurements. Size-exclusion chromatography (SEC-HPLC) was performed for investigation of selenium containing proteins in chives. For speciation of selenium containing amino acids, reversed-phase ion-pairing chromatography (RP-IP-HPLC) and for enantiomeric separations, a crown ether column was used. In all three cases online detection with inductively coupled plasma mass spectrometry was performed for selenium specific detection. Two extractions (perchloric acid–ethanol and enzymatic) were carried out on chive samples. Speciation analysis on the chives grown in three different media revealed that selenium distribution among different forms of amino acids in the sample strongly depends on the type of enrichment employed. Enrichment with Se(VI) leads to accumulation of selenium in inorganic forms, while in case of Se(IV) and SeMet-enriched samples, methyl-selenocysteine and selenocystine were found to be present. Not surprisingly, chiral speciation revealed the presence of the l-enantiomeric forms of selenoamino acids in the sample. The major enantiomer found in the perchloric acid–ethanol extracts was l-MeSeCys, while in the enzymatic extracts l-SeMet was also detected.     

Effect of selenium and reducing agents on in vitro immunoglobulin M synthesis by bovine lymphocytes

A series of experiments was conducted to evaluate the effects of inorganic and organic forms of Se with or without reducing agents on in vitro IgM production by bovine lymphocytes. Peripheral mononuclear cells were isolated from nonlactating Jersey cows fed a diet with adequate Se. Cells were stimulated with pokeweed mitogen and, in addition, were cultured with various Se compounds at a concentration of 100 ng Se/ml. Mercaptoethanol (50 microM) and glutathione (1 mM) were included in cultures of cells stimulated by pokeweed mitogen with and without inorganic Se. Sodium selenite was less effective than selenomethionine and selenocystine in augmenting pokeweed mitogen-induced Ig synthesis. The addition of mercaptoethanol to pokeweed mitogen-stimulated control cultures enhanced in vitro IgM production, whereas the addition of glutathione had a negligible effect, but addition of either in combination with sodium selenite dramatically depressed IgM production. These results suggest that Se in inorganic or organic forms enhances B-cell function in vitro.     

Selenium-enriched fermented milk: A suitable dairy product to improve selenium intake in humans

The suitability of Se-enriched fermented milk as a food product for humans has been studied and sensory features, survival of microorganisms in the presence of selenium and Se bioaccessibility are presented. Sensory features and the number of colonies of microorganisms observed until the 4th week after fermentation were not affected at Se concentration levels below 2 μg g^?1. To investigate bioaccessible selenium-containing compounds in selenized fermented milk, total Se was quantified after in vitro gastrointestinal digestion. Results showed that 76 ± 3% Se was extracted after gastrointestinal digestion and 24 ± 6% remained in the insoluble residue. To identify the main species of bioaccessible Se of low molecular weight (MW) generated during gastrointestinal digestion, liquid chromatography-inductively coupled plasma-mass spectrometry (LC-ICP-MS) was used. Results showed that identified selenocompounds were smaller than 1.5 kDa, with selenocystine and Se-methylselenocysteine being the most abundant compounds present in the extracts.     

Metabolic pathway for selenium in the body: speciation by HPLC-ICP MS with enriched Se

Selenium (Se) is an ultramicro essential nutrient and both inorganic (selenite and selenate) and organic (selenocysteine and selenomethionine) forms of Se can be used as nutritional sources. Metabolic pathways for Se in the body were studied for selenite and selenate, with the use of enriched 82 Se, by speciation with separation by gel filtration HPLC and detection by element-specific mass spectrometry with ionization with inductively coupled argon plasma (HPLC-ICP MS). The concentrations of 82 Se in organs and body fluids and the distributions of their constituents depending on the dose and time after the intravenous administration of 82 Se-selenite and -selenate to rats were determined. Selenite was taken up by red blood cells within several minutes, reduced to selenide by glutathione, and then transported to the plasma, bound selectively to albumin and transferred to the liver. Contrary to selenite, intact selenate was either taken up directly by the liver or excreted into the urine. The 82 Se of selenite origin and that of selenate origin were detected in the forms of the two Se peak materials in the liver, A and B. The former one was methylated to the latter in vivo and in vitro . The latter one was identical with the major urinary metabolite and it was identified as Se-methyl- N -acetyl-selenohexosamine (selenosugar). The chemical species-specific metabolic pathway for Se was explained by the metabolic regulation through selenide as the assumed common intermediate for the inorganic and organic Se sources and as the checkpoint metabolite between utilization for the selenoprotein synthesis and methylation for the excretion of Se.     

Selenium concentration and speciation in biofortified flour and bread: Retention of selenium during grain biofortification,processing and production of Se-enriched food

The retention and speciation of selenium in flour and bread was determined following experimental applications of selenium fertilisers to a high-yielding UK wheat crop. Flour and bread were produced using standard commercial practices. Total selenium was measured using inductively coupled plasma-mass spectrometry (ICP-MS) and the profile of selenium species in the flour and bread were determined using high performance liquid chromatography (HPLC) ICP-MS. The selenium concentration of flour ranged from 30 ng/g in white flour and 35 ng/g in wholemeal flour from untreated plots up to >1800 ng/g in white and >2200 ng/g in wholemeal flour processed from grain treated with selenium (as selenate) at the highest application rate of 100 g/ha. The relationship between the amount of selenium applied to the crop and the amount of selenium in flour and bread was approximately linear, indicating minimal loss of Se during grain processing and bread production. On average, application of selenium at 10 g/ha increased total selenium in white and wholemeal bread by 155 and 185 ng/g, respectively, equivalent to 6.4 and 7.1 μg selenium per average slice of white and wholemeal bread, respectively. Selenomethionine accounted for 65–87% of total extractable selenium species in Se-enriched flour and bread; selenocysteine, Se-methylselenocysteine selenite and selenate were also detected. Controlled agronomic biofortification of wheat crops for flour and bread production could provide an appropriate strategy to increase the intake of bioavailable selenium.     

Combined speciation analysis by X-ray absorption near-edge structure spectroscopy, ion chromatography, and solid-phase microextraction gas chromatography-mass spectrometry to evaluate biotreatment of concentrated selenium wastewaters

In this study we evaluate the potential of anaerobic granular sludge as an inoculum for the bioremediation of selenium-contaminated waters using species-specific analytical methods. Solid species formed by microbial reduction were investigated using X-ray absorption near-edge structure (XANES) spectroscopy at the selenium K-edge. Furthermore, dissolved selenium species were specifically determined by ion chromatography (IC) and solid-phase microextraction gas chromatography-mass spectrometry (SPME-GC-MS). Least-squares linear combination of the XANES spectra for samples incubated with the highest selenate/selenite concentrations (10(-3) M) show the predominance of elemental selenium and a Se(-I) selenide, such as ferroselite, the thermodynamically most stable iron selenide. In contrast, elemental selenium and Se(-II) selenides are the main species detected at the lower selenate/selenite concentrations. In each repeated fed batch incubation, most aqueous selenite anions were converted into solid selenium species, regardless of the type of electron donor used (acetate or H(2)/CO(2)) and the selenium concentration applied. On the other hand, at higher concentrations of selenate (10(-4) and 10(-3) M), significant amounts of the oxyanion remained unconverted after consecutive incubations. SPME-GC-MS demonstrated selenium alkylation with both electron donors investigated, as dimethyl selenide (DMSe) and dimethyl diselenide (DMDSe). Selenite was even more alkylated in the presence of H(2)/CO(2) (maximum 2156 μg of Se/L of DMSe + DMDSe) as compared to acetate (maximum 50 μg of Se/L). In contrast, selenate was less alkylated using both electron donors (maximum 166 and 3 μg of Se/L, respectively). The high alkylation potential for selenite limits its bioremediation in selenium laden waters involving H(2)/CO(2) as the electron donor despite the fact that nontoxic elemental selenium and thermodynamically stable metal selenide species are formed.     

Assessment of isotope exchange methodology to determine the sorption coefficient and isotopically exchangeable concentration of selenium in soils and sediments

Isotope exchange methodology is invaluable to determine the solution-solid-phase distribution (Kd) and isotopically exchangeable concentration (Evalue) of elements in soils and sediments. This work examined the use of species-specific stable isotope exchange techniques to determine the Kd and E value of selenium (Se), as selenite (SeO3) and selenate (SeO4), in nine soils and sediments varying in concentration and source of Se. High-performance liquid chromatography-inductively coupled plasma-mass spectrometry (HPLC-ICP-MS) was used to quantify the isotope (e.g., 76Se, 78Se, 80Se, and 82Se) concentrations of the soluble Se oxyanions. The two Se oxyanions were detected in the solution phase of all of the soils and sediments. However, upon spiking the suspensions with stable isotope-labeled 78SeO3 and 76SeO4, it was observed that isotope self-exchange was insignificant to the derivation of Se oxyanion Kd and E values during 24 h (and up to 120 h in four of the samples). These results demonstrate that valid determinations of the Evalue of Se necessitate that the Se oxyanions are speciated in solution. This is clearly evident for these soils and sediments where it was observed that the Evalues of SeO3 and SeO4 represented, respectively, 5-97% and 3-95% of the total Se E value.     

富硒大豆低聚肽的制备及其抗疲劳功能的研究

为探究富硒大豆低聚肽对抗运动性疲劳的影响,实验以富硒大豆为原料制备大豆蛋白,并以超滤萃取辅助中性蛋白酶和风味蛋白酶双酶酶解法制取富硒大豆低聚肽,并通过ICP-MS对硒含量进行鉴定,最后通过小鼠运动性疲劳实验综合评估富硒大豆低聚肽的抗疲劳活性.结果显示:富硒大豆低聚肽的总硒含量为(90.03±3.23)μg/kg,主要形...