体积排阻色谱与电感耦合等离子体质谱联用技术在富硒金针菇硒多糖形态分析中的应用研究

提取含硒7.97μg/g的富硒金针菇硒多糖,采用体积排阻色谱与电感耦合等离子体质谱(SE-HPLC-ICP-MS)联用技术进行在线分离分析,确定富硒金针菇中至少含有三种硒多糖,其分子量分别为4.27 kDa1、.868 kDa和1.54 kDa,含硒量分别占可溶性硒多糖中硒含量的14.95%、40.39%和44.66%。进一步研究证明:在金针菇的富硒培养过程中,硒不仅参与了生物大分子的合成,而且在富硒金针菇体内存在大量的具有生物活性的硒多糖。从分子生物学角度对新的补硒资源和生物活性因子进行有益的探索,为研究人类硒源的生物安全性和高效可利用性提供科学的理论依据。     

HPLC-ICP-MS测定中药中砷的形态

报道了高效液相色谱-电感耦合等离子体质谱(HPLC-ICP-MS)联用技术测定中药中砷的形态.采用阴离子交换柱,以含0.2 mmol/L乙二胺四乙酸(EDTA)和2 mmol/L NaH2PO4的水溶液为流动相,pH 6.0,流速为1.0 mL/min,成功分离了亚砷酸(AsⅢ)、砷酸(AsⅤ)、甲基砷(MMA)和二甲基砷(DMA).检出限分别为0.67 μg/L (AsⅢ),0.85 μg/L (DMA),0.43 μg/L (MMA),0.70 μg/L (AsⅤ).中药样品经过(1 1)甲醇水溶液超声提取,离心、过滤、氮气吹干甲醇,超纯水定容.样品加标平均萃取回收率分别为: 92.8% (AsⅢ),108% (DMA),104% (MMA),101% (AsⅤ),RSD (n=7)均小于10%.     

HPLC-ICP-MS测定中药中砷的形态

报道了高效液相色谱-电感耦合等离子体质谱（HPLC—ICP-MS）联用技术测定中药中砷的形态，采用阴离子交换柱，以0．2mmol／L EDTA和2mmol／L NaH2PO4的溶液为流动相，pH6．0，流速为1．0mL／min，成功分离了亚砷酸（AsⅢ）、砷酸（AsV）、甲基砷（MMA）和二甲基砷（DMA）。检出限分别为0．67μg／L（AsⅢ），0．85μg／L（DMA），0．43μg／L（MMA），0．70μg／L（AsV）。中药样品经过（1＋1）甲醇和水的溶液超声提取，离心、过滤、氮气吹干甲醇，超纯水定容。样品加标平均萃取回收率分别为：92．8％（AsⅢ），108％（DMA），104％（MMA），101％（AsV），相对标准偏差（RsD，n=7）均小于10％。     

Evaluation of different sample extraction strategies for selenium determination in selenium-enriched plants (Alliumsativum and Brassicajuncea) and Se speciation by HPLC-ICP-MS

Several sample extraction techniques have been evaluated in order to obtain highest selenium (Se) extraction efficiency in two types of selenium-enriched plants (Allium sativum and Brassica juncea). Three extracting solutions have been studied for this purpose: 0.1 M HCl, 25 mM ammonium acetate buffer (pH 5.6) and protease in aqueous solution. In each case, the effect of the ultrasonic probe during extraction was also evaluated. Selenium extraction yields were calculated based on the ICP-MS determination of the total selenium content in the corresponding extracts and in the plant tissue after its microwave digestion. The action of ultrasounds allowed the reduction on the extraction time while maintaining good Se recoveries (which ranged from 75 to 120% of the total Se in the plant). The accuracy of total Se determination was controlled by analyzing a reference material (aquatic plant, BCR-670). On the other hand, speciation studies of the extracts were carried out by using ion-pairing reversed phase and size exclusion/ion exchange (Shodex Asshipak) liquid chromatographic columns. The two separation mechanisms were suitable to isolate the main extractable Se species which were identified as Se-methyl selenocysteine and Se-methionine in both systems. The extracts of both plants (A. Sativum and B. juncea) exhibited also the presence of several unknown Se-species.     

HPLC-ICP-MS determination of selenium distribution and speciation in different types of nut

In addition to determination of total selenium in nuts, the element distribution among different fractions (lipid extract, low molecular weight, and protein fractions), and speciation analysis were studied. Improved precision for total selenium determination was observed after elimination of lipids. Because selenium was not detected in any of the lipid extracts obtained from the different types of nuts (ICP-MS), in each determination and/or speciation procedure used in this work lipids were extracted (chloroform-methanol, 2:1) and discarded before analysis. In agreement with previously reported data, high selenium levels were found in Brazil nuts (those purchased without shells contained approximately a quarter the content than those purchased with shells) and significantly lower levels in walnuts, cashews, and pecans nuts. Low-molecular-weight compounds were extracted with perchloric acid (0.4 mol L(-1)) to furnish a fraction containing 3 to 15% of the total selenium in different types of nuts. The proteins were isolated from nut samples by dissolution in 0.1 mol L(-1) sodium hydroxide and subsequent precipitation with acetone. They were then dissolved in phosphate buffer pH 7.5. Analysis of protein fractions focused on selenium in two possible states - weakly and firmly bound to proteins. Results obtained for Brazil nuts by size-exclusion chromatography with on-line ICP-MS detection, in the absence and in the presence of beta-mercaptoethanol, showed that approximately 12% of total selenium was weakly bound to proteins. To obtain information about firmly bound selenium, the protein extracts were hydrolyzed enzymatically with proteinase K. Speciation was performed by means of ion-pairing HPLC-ICP-MS. The primary species found in all types of nuts was Se-methionine (19-25% of total selenium for different types of nuts).     

高效液相色谱-电感耦合等离子质谱分析烟草中硒形态

采用高效液相色谱-电感耦合等离子体质谱(HPLC-ICP-MS)联用技术建立了烟草中硒的形态分析方法。烟草样品采用0.10mol/L HCl超声提取30min,经离心、过膜后引入HPLC-ICP-MS进行分析。使用Hamilton PRP X-100阴离子交换柱,以20mmol/L柠檬酸水溶液为流动相(pH=7.0),流速1.2mL/min,优化条件下实现了6种硒形态的分离。质谱采用He碰撞模式,硒代胱氨酸、亚硒酸根、甲基硒代半胱氨酸、硒酸根、硒脲、硒代蛋氨酸检出限分别为1.20、0.34、1.65、0.13、3.25和0.65ng/mL。该方法操作简单、快速、灵敏度高,精密度好,加标回收率在86.1%~95.8%之间,适用于烟草中硒元素形态分析。     

Studying the distribution pattern of selenium in nut proteins with information obtained from SEC-UV-ICP-MS and CE-ICP-MS

In this work, size exclusion chromatography (SEC) with UV and inductively coupled plasma mass spectrometry (ICP-MS) detection was used to study the association of selenium to proteins present in Brazil nuts (Bertholletia excelsa) under five different extraction conditions. As expected, better solubilization of proteins was observed using 0.05 mol L⁻¹ sodium hydroxide and 1% sodium dodecylsulfate (SDS) in Tris/HCl buffer (0.05 mol L⁻¹, pH 8) as compared to 0.05 mol L⁻¹ HCl, 0.05 mol L⁻¹ Tris/HCl or hot water (60 °C). Due to non-destructive character of Tris-SDS treatment, this was applied for studying molecular weight (MW) distribution patterns of selenium-containing nut proteins. Three different SEC columns were used for obtaining complete MW distribution of selenium: Superdex 75, Superdex Peptide, and Superdex 200 were tested with 50 mmol L⁻¹ Tris buffer (pH 8), 150 mmol L⁻¹ ammonium bicarbonate buffer (pH 7.8), phosphate (pH 7.5), and CAPS (pH 10.0) mobile phases. Using Superdex 200 column, the elution of at least three MW fractions was observed with UV detection (200-10 kDa) and ICP-MS chromatogram showed the co-elution of selenium with the two earlier fractions. The apparent MWs of these selenium-containing fractions were respectively about 107 and 50 kDa, as evaluated from the column calibration. For further characterization of individual selenium species, the defatted nuts were hydrolyzed with proteinase K and analyzed by capillary electrophoresis (CE) with ICP-MS detection. The suitability of CE for the separation of selenite, selenate, selenocystine and selenomethionine in the presence of the nut sample matrix is demonstrated. Complete separation of the above mentioned selenium species was obtained within a migration time of 7 min. In the analysis of nut extracts with CE-ICP-MS, selenium was found to be present mainly as selenomethionine.     

A systematic approach to the accurate quantification of selenium in serum selenoalbumin by HPLC-ICP-MS

In this paper, two different methods are for the first time systematically compared for the determination of selenium in human serum selenoalbumin (SeAlb). Firstly, SeAlb was enzymatically hydrolyzed and the resulting selenomethionine (SeMet) was quantified using species-specific isotope dilution (SSID) with reversed phase-HPLC (RP-HPLC) hyphenated to (collision/reaction cell) inductively coupled plasma-quadrupole mass spectrometry (CRC ICP-QMS). In order to assess the enzymatic hydrolysis yield, SeAlb was determined as an intact protein by affinity-HPLC (AF-HPLC) coupled to CRC ICP-QMS. Using this approach, glutathione peroxidase (GPx) and selenoprotein P (SelP) (the two selenoproteins present in serum) were also determined within the same chromatographic run. The levels of selenium associated with SeAlb in three serum materials, namely BCR-637, Seronorm level 1 and Seronorm level 2, obtained using both methods were in a good agreement. Verification of the absence of free SeMet, which interferes with the SeAlb determination (down to the amino acid level), in such materials was addressed by analyzing the fraction of GPx, partially purified by AF-HPLC, using RP-HPLC (GPx only) and size exclusion-HPLC (SE-HPLC) coupled to CRC ICP-QMS. The latter methodology was also used for the investigation of the presence of selenium species other than the selenoproteins in the (AF-HPLC) SelP and SeAlb fractions; the same selenium peaks were detected in both control and BCR-637 serum with a difference in age of ca. 12 years. It is also for the first time that the concentrations of selenium associated with SeAlb, GPx and SelP species in such commercially available serums (only certified or having indicative levels of total selenium content) are reported. Such indicative values can be used for reference purposes in future validation of speciation methods for selenium in human serum and/or inter-laboratory comparisons.     

Methodological advances for selenium speciation analysis in yeast

Advances in analytical methodology for speciation of selenium in selenized-yeast food supplements were discussed on the basis of the recent developments in the authors’ laboratory. Particular attention was given to the sample preparation with regard to the fractionation of selenium into different classes of chemical species, the high resolution fractionation of selenium from yeast water extracts by size-exclusion chromatography and characterization of the water soluble protein fraction by combined matrix-assisted laser desorption ionization (MALDI)-time-of-flight mass spectrometry (TOF MS) and electrospray quadrupole-TOF tandem MS. The true speciation of protein-incorporated selenium (down to individual proteins characterized by a unique aminoacid sequence) was discussed using an example of a family of selenium-containing proteins formed in yeast by the substitution of methionine residues by selenomethionine in a salt stress-induced protein.     

Speciation analysis of selenium enriched green onions (Allium fistulosum) by HPLC-ICP-MS

Green onions (Allium fistulosum) enriched with 10 or 100 μg mL^− 1 Se(IV) or SeMet were analyzed for total selenium and species distribution. Anion and cation exchange chromatographies were applied for the separation of selenium species with mass spectrometric detection. Two different sample preparation methods (NaOH and enzymatic) were compared from the Se extraction efficiency point of view. Total selenium concentration accumulated by the onions reached the 200 μg g^− 1 level expressed for dry weight when applying SeMet at a concentration of 100 μg mL^− 1 as the source of Se. Speciation studies revealed that both in onion bulbs and leaves the predominant form of organic selenium is Se-methyl-selenocysteine (MeSeCys). When Se(IV) was applied for Se-enrichment at a concentration level of 100 μg mL^− 1 both onion leaf and bulb contained a significant amount of inorganic selenium. An unknown compound was also detected.     

Distribution and speciation of selenium in Lecythis ollaria plant

Selenium has been determined in different parts of the Lecythis ollaria (LO) plant (Venezuela) and the determination of its speciation has been achieved in fruit seeds. The study has been performed using different analytical techniques including inductively coupled plasma atomic emission spectrometry (ICP-AES), differential pulse cathodic stripping voltammetry (DPCSV), instrumental neutron activation analysis (INAA), high performance liquid chromatography (HPLC) and mass spectrometry (MS). Different parts of the plant (leaves, bark, capsules and seeds) were examined as well as the soil where LO was growing. Among different considered parts, seeds show the highest content in selenium (5 g kg⁻¹) which is dependent on the maturation extent of the fruit. In seeds, about half of the total selenium content is soluble in water while the remaining is involved in protein structure. In the aqueous fraction, the prevailing form of selenium appears to be seleno-cystathionine with much lower amounts of Se(VI) and Se(IV).     

An attempt to differentiate HPLC-ICP-MS selenium speciation in natural and selenised Agaricus mushrooms using different species extraction procedures

Total determination and speciation analysis of Se in commercial and selenised Agaricus mushrooms have been performed to investigate the Se species naturally occurring in non-enriched mushrooms as well as those present in specimens grown in a Se-enriched medium. Mushroom aqueous and enzymatic extracts have been analysed by three complementary chromatographic separation mechanisms (size-exclusion, anion-exchange and reversed-phase) coupled to an inductively coupled plasma mass spectrometer with an octopole reaction system. Post-column isotope dilution analysis has been used on-line with the separations for quantification of the Se species eluted. The ⁷⁸Se-to-⁷⁷Se isotope ratio was monitored after adequate corrections for both total determinations and Se species quantitative speciation. The results showed marked differences not only in total Se contents but also in Se species found in the two types of Agaricus mushrooms investigated. Selenomethionine was detected in both of them (free in commercial mushrooms and incorporated into proteins in selenised ones) together with a number of unknown selenocompounds.     

Preconcentration of selenium compounds on a porous graphitic carbon column in view of HPLC-ICP-AES speciation analysis

The retention of organic selenium compounds on a porous graphitic carbon stationary phase was investigated. Different acids were studied as mobile phases to elute selenocystamine, selenoethionine, selenomethionine and selenocystine. Detection was achieved using inductively coupled plasma-atomic emission spectrometry to provide selenium-specific and sensitive detection. The separation of the four species was carried out using methanoic acid. An important on-column preconcentration was obtained when solutes were injected in nitric acid or trifluoroacetic acid (TFA) media. The large injection volume employed (2,500 µL) allowed us to reach low relative detection limits (2–6 µg/L). The method, employing TFA as injection solvent and methanoic acid as the eluent was found to be robust with respect to different matrices spiked with selenocompounds.     

Inorganic selenium speciation in environmental samples using selective electrodeposition coupled with electrothermal atomic absorption spectrometry

Speciation analyses are of increasing interest in the environmental, toxicological and analytical fields, because the toxicity and reactivity of trace elements depend strongly on the chemical forms in which they are present. A simple electrodeposition–electrothermal atomic absorption spectrometry method for speciation analysis of some organic and inorganic selenium species in typical environmental water and agricultural soil samples has been developed. The method is based on the selective reduction of water-soluble Se(IV) and selenocystine (Se–Cys) species by an uncontrolled applied potential (1.8 V) on a mercury-coated electrode. In acidic media (1.0 M HCl solution) the only inorganic selenium species electrodeposited was Se(IV), and, of the water-soluble organic selenium species Se–Cys and Se–Met only Se–Cys was electrodeposited onto the mercury electrode surface. The proposed methodology was successfully applied to the speciation and determination of selenium in a few environmental samples. The spiked recovery value varied between 91% and 99%. The suggested method has been shown to have a characteristic mass (m₀) of 25 pg, a limit of detection (LOD) of 1.0 μg L^− 1 and a relative standard deviation (RSD%) of 3.5% for 6 measurements at a concentration of 100 μg L^− 1 Se(VI).     

Comparison of standard and reaction cell inductively coupled plasma mass spectrometry in the determination of chromium and selenium species by HPLC-ICP-MS

Elemental speciation is becoming a common analytical procedure for geochemical investigations. The various redox species of environmentally relevant metals can have vastly different biogeochemical properties, including sorption, solubility, bioavailability, and toxicity. The use of high performance liquid chromatography (HPLC) coupled to elemental specific detectors, such as inductively coupled plasma mass spectrometry (ICP-MS), has become one of the most important speciation methods employed. This is due to the separation versatility of HPLC and the sensitive and selective detection capabilities of ICP-MS. The current study compares standard mode ICP-MS to recently developed reaction cell (RC) ICP-MS, which has the ability to remove or reduce many common polyatomic interferences that can limit the ability of ICP-MS to quantitate certain analytes in complex matrices. Determination of chromium and selenium redox species is achieved using ion-exchange chromatography with elemental detection by standard and RC-ICP-MS, using various chromium and selenium isotopes. In this study, method performance and detection limits for the various permutations of the method (isotope monitored or ICP-MS detection mode) were found to be comparable and generally less than 1 μg L⁻¹. The method was tested on synthetic laboratory samples, surface water, groundwater, and municipal tap water matrices.     

Arsenic speciation in chinese seaweeds using HPLC-ICP-MS and HPLC-ES-MS

Three common Chinese edible seaweeds, one brown (Laminaria japonica) and two red (Porphyra crispata and Eucheuma denticulatum), were examined for their total arsenic content. The As species were extracted with yields of 76.4, 69.8 and 25.0%, respectively. Anion-exchange and cation-exchange high-performance liquid chromatography (HPLC) in combination with inductively coupled plasma mass spectrometry (ICP-MS) were used for the separation of the different arsenic species in two of the three seaweed extracts (Laminaria and Porphyra). The main arsenic species in the algal extracts are arseno sugars, although it has been shown that the Laminaria seaweed contains significant amounts of dimethylarsinic acid (DMA). HPLC was coupled with electrospray mass spectrometry (ES-MS) for structural confirmation of the arsenic species. The mass spectrometer settings for the arseno sugars were optimised using standards. The conclusions drawn on the basis of HPLC-ICP-MS were confirmed by the HPLC-ES-MS data. The HPLC-ES-MS method is capable of determining both arseno sugars and DMA in the seaweeds. The unknown compounds seen in the HPLC-ICP-MS chromatogram of Laminaria could not be ascribed to trimethylarsenic oxide or tetramethylarsonium ion.     

吸附材料在元素形态分析中的应用

本文评述了相关吸附材料在元素形态分析中的应用及进展。主要讨论了它们在元素价态、游离态／结合态以及无机态／有机态分析中的应用情况。最后,还探讨了纳米金属氧化物材料在形态分析中应用的可行性。共引用文献107篇。     

Precipitate flotation-separation, speciation and hydride generation atomic absorption spectrometric determination of selenium(IV) in food stuffs

A method for flotation and determination of selenium(IV) in foodstuffs using p-chlorophenylthiosemicarbazide (HCPT) was investigated. At pH  2, selenium(IV) forms a 1:1 reddish-brown precipitate with HCPT easily floated using oleic acid (HOL) surfactant. The separated complex was dissolved in 4 M HCl and diluted in 10-ml double-distilled water (DDW). Selenium(IV) content in the eluate was determined by hydride generation atomic absorption spectrometry (HG-AAS) at 196.4 nm using sodium borohydride. The HCPT–Se(IV) complexes formed in absence and presence of oleic acid were characterized by elemental analysis, mass and infrared spectral studies. The mode of chelation between Se(IV) and HCPT is proposed to be through S and N coordination. Interferences, on the flotation process, from various foreign ions were avoided by adding excess HCPT. The proposed flotation methodology was successfully applied to the analysis of selenium in real foodstuffs and natural water spiked with known amounts of Se(IV) with a preconcentration factor of 100 and a detection limit of 20 pg. Application was also extended to separate Se(IV) successfully from Se(VI) in their synthetic mixtures. The separation mechanism is proposed to be due to hydrogen bond formation between the COOH group of HOL and –NH of the HCPT–Se(IV) complex.     

彭泽贝母中微量元素的测定及初级形态分析

采用HNO3-H2O2微波消解体系,电感耦合等离子质谱法(ICP-MS)测定彭泽贝母中Pb、Ca、Fe、Mn、Zn、Cd、Ba、Cr等10种微量元素,初步探究微量元素的存在形态.结果表明:彭泽贝母中含有丰富的人体必需微量元素,可能促进其有效成分作用的发挥.其中Ca在彭泽贝母中含量最高,而Mo含量最低;彭泽贝母中微量元素是以无机态为主、多种形态共存的复杂体系.