RESUMO
A method for arsenic speciation in shark, shrimp, squid, oyster and scallop using liquid chromatography coupled to inductively coupled plasma triple quadrupole mass spectrometry (LC-ICP-MS/MS) was proposed. Suitable sensitivity and selectivity by LC-ICP-MS/MS were obtained using 10â¯mmolâ¯L-1 (NH4)2HPO4 diluted in 1% methanol (pH 8.65) as mobile phase. Recoveries from 90 to 104% for arsenobetaine (AsB), arsenite [As(III)], dimethylarsinic acid (DMA), monomethylarsonic acid (MMA) and arsenate [As(V)] were obtained for all samples. A certificated reference material was also analyzed and the sum of As species was in agreement with the total As concentration. Limits of quantification (LOQ) for AsB, As(III), DMA, MMA, and As(V) were 6, 30, 6, 12 and 26â¯ngâ¯g-1, respectively. Higher concentration of AsB was found in all seafood, while As(III) and DMA were found only in oyster. Arsenate was found in squid and scallops, and MMA was below the LOQ in all samples.
Assuntos
Arsênio/análise , Alimentos Marinhos/análise , Arseniatos/análise , Arsenicais/análise , Arsenitos/análise , Ácido Cacodílico/análise , Cromatografia Líquida , Estudos de Viabilidade , Sensibilidade e Especificidade , Espectrometria de Massas em TandemRESUMO
Trace metal analysis has been long regarded as one of the principle tasks in areas of chemical analysis. At the early stage of instrumental development, total concentration was assessed in a variety of samples, yielding results, among others, for environmental, biological, and clinical samples. With the power of newer analytical techniques, such as inductively coupled plasma mass spectrometry (ICP-MS), accurate quantitative results can now be obtained at ultra-trace levels not only for metals, but also for metalloids and several non-metals. Even though the importance of trace elements in many biological processes is widely accepted, the elucidation of their biological pathways, understanding specific biological functions, or possible toxicological aspects is still a challenge and a driving force to further develop analytical methodology. Over the past decades, the scientific interest has moved from total element determination to include speciation analysis, which provides quantitative information of one or more individual element species in a sample. More recently, metallomics has been introduced as a more expanded concept, in which the global role of all metal/metalloids in a given system is considered. Owing to the multi-elemental focus of metallomics research, the use of ICP-MS becomes indispensable. Furthermore, considering the biological role of metals/metalloids and the use of elements as internal or external molecular tags, epigenetics should be considered as an important emerging application for metallomics studies and approaches. Among a variety of epigenetic factors, essential nutrients, but also environmental toxins, have been shown to affect DNA methylation, modification of histone proteins, and RNA interference, all of them being implicated in cancer, cardiovascular disease, and several inherited conditions. Recent studies suggest that epigenetics may be a critical pathway by which metals produce health effects. In this Trends article, the basic epigenetic concepts are introduced, followed by the early applications of ICP-MS classified as: (i) detection of (31)P as a natural element tag for DNA, (ii) analysis of DNA adducts with metal-based drugs, (iii) element species as epigenetic factors.
Assuntos
Epigênese Genética , Metais/análise , Animais , Arsênio/análise , DNA/química , Humanos , Preparações Farmacêuticas/química , Fósforo/análise , Selênio/análiseRESUMO
In this work, different analytical speciation schemes have been used to study the reduction of Cr(VI) by a chromate-resistant strain of filamentous fungi Ed8 (Aspergillus sp), indigenous to contaminated industrial wastes. As demonstrated previously, this strain has the capability to reduce chromate present in the growth medium without its accumulation in the biomass, yet the reduced chromium end-products have not been characterized. Liquid growth medium, initially containing 50 mg L(-1) Cr(VI), was analyzed for Cr(III)/Cr(VI) and for total Cr at different time intervals (0-24 h) after inoculation with fungi. Three hyphenated procedures, based on the Cr(III)-EDTA formation and species separation by anion-exchange or ion-pairing reversed-phase chromatography with ICP-MS or DAD detection were used. The results obtained for Cr(VI) in each case were consistent, demonstrating efficient reduction of chromate during 24 h of Ed8 growth. However, pre-column complexation with EDTA did not ensure complete recovery of the reduced forms of chromium in the above procedures. An alternative speciation scheme, based on extraction of Cr(VI)-benzyltributylammonium bromide (BTAB) ion pairs into chloroform and subsequent determination of residual chromium by ICP-MS has provided evidence on the effective conversion of chromate into reduced chromium species in the growth medium. The results indicate the feasibility of using Ed8 strain for chromate bioremediation purposes. Analytically it can be concluded that speciation of chromium in biological systems should not be limited to its two most common oxidation states, because the actual reduced chromium species are not converted quantitatively to Cr(III)-EDTA.
Assuntos
Aspergillus/metabolismo , Compostos de Cromo/química , Cromatos/química , Cromatos/metabolismo , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia por Troca Iônica/métodos , Cromo/química , Cromo/metabolismo , Compostos de Cromo/metabolismo , Ácido Edético/química , OxirreduçãoRESUMO
Phytochelatins (PCs) were determined in the wild plants, focusing on their relationship with the levels of heavy metals and humic substances (HS) in soil. Ricinus communis and Tithonia diversifolia were collected from several sites in Guanajuato city (Mexico), which had long been the silver and gold mining center. The analysis of PCs in root extracts was carried out by liquid chromatography (derivatization with monobromobimane). Total Ag, Cd, Cu and Pb in plant roots and in soil samples, as well as soil HS were determined. The association of metals with HS in soils was evaluated by size exclusion chromatography (SEC) with UV and ICP-MS detection. The results obtained revealed the induction of PCs in R. communis but not in T. diversifolia. The levels of Cd and Pb in plant roots presented strong positive correlation with PC-2 (r=0.9395, p=0.005; r=0.9573, p=0.003, respectively), indicating that these two metals promote PCs induction in R. communis. On the other hand, the inverse correlation was found between soil HS and metal levels in roots of R. communis (Cu>Pb>Cd>Ag), in agreement with the decreasing affinity of these metals to HS. Importantly, the inverse correlation between soil HS and plant PC-2 was observed (r=-0.7825, p=0.066). These results suggest that metals strongly bound to HS could be less bioavailable to plants, which in turn would limit their role in the induction of PCs. Indeed, the SEC elution profiles showed Pb but not Cd association with HS and the correlation between metal in soil and PC-2 in plant was statistically significant only for Cd (r=0.7857, p=0.064). Based on these results it is proposed that the role of heavy metals in PCs induction would depend on their uptake by R. communis, which apparently is controlled by the association of metals with soil HS. This work provides further evidence on the role of environmental conditions in the accumulation of heavy metals and phytochelatin production in plants.
Assuntos
Asteraceae/efeitos dos fármacos , Substâncias Húmicas , Metais Pesados/toxicidade , Fitoquelatinas/metabolismo , Ricinus/efeitos dos fármacos , Poluentes do Solo/toxicidade , Asteraceae/metabolismo , Substâncias Húmicas/análise , Resíduos Industriais , Metais Pesados/análise , Metais Pesados/metabolismo , México , Mineração , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/metabolismo , Ricinus/metabolismo , Poluentes do Solo/análise , Poluentes do Solo/metabolismoRESUMO
In this work, Lactobacillus casei rhamnosus were obtained from the commercial product of fermented milk and possible antagonistic effect of selenium (as sodium selenite) against cadmium toxicity was studied. The bacteria capability to incorporate Se was demonstrated: after 1 week exposure to Se(IV), its total concentration in the freeze-dried biomass was 405+/-28 microg/g (7.4+/-0.8 microg/g in control). In the presence of Se(IV) and Cd(II), the bacterial growth and cell viability were improved and lipid peroxidation less marked with respect to bacteria exposed to Cd(II) alone. The distribution of Se and Cd in molecular mass fractions of bacteria extracts was investigated by size exclusion chromatography with diode array and ICP-MS detection. The results obtained suggest that the antagonistic effect of Se is due to lower incorporation of cadmium at a high molecular mass (MM<600 kDa). Slightly different distribution of elements in the fractions of MM<40 kDa suggests the formation of new chemical species involving Cd and Se in bacteria exposed to Cd(II)+Se(IV) as compared to those exposed to Cd(II) alone. The study illustrates the high utility of atomic spectrometry to critically inform molecular questions that could be important in the industrial processes based on bacterial activity.
Assuntos
Cádmio/toxicidade , Lacticaseibacillus casei/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Selênio/farmacologia , Cromatografia em Gel , Lacticaseibacillus casei/crescimento & desenvolvimento , Lacticaseibacillus casei/metabolismo , Malondialdeído/metabolismo , Espectrometria de Massas/métodos , Espectrofotometria UltravioletaRESUMO
The incorporation of Se to fungi has been studied, focusing on element distribution among different cellular compartments and, in particular, polysaccharide structures contained in cell walls. Se-enriched mycelia of Pleurotus ostreatus were obtained in submerged cultures. The incorporation of selenium from the growth medium to mycelia was observed with the relative distribution between cytosol plus cell membranes fraction (CCM) and cell walls fraction (CW) of about 44 and 56%, respectively. CCM fractions were analyzed by size exclusion chromatography with on-line UV (280 nm) and ICP-MS detection (80Se). The results obtained showed selenium binding to components of different molecular masses (about 24% of total selenium coeluted with the compounds of molecular mass > 10 kDa). A polysaccharide-containing fraction of mycelia was treated alternatively with Tris-HCl at pH 7.5 or with chitinase. Better solubility and increased contribution of low molecular mass compounds were observed in chitinase extracts (UV detection), confirming the degradation of polysacharides by the enzyme. The total area under the ICP-MS chromatogram of chitinase extract was 2 times higher with respect to the area for Tris-HCl extract. Furthermore, the relative contribution of selenium in the low molecular mass fraction (molecular mass < 1 kDa) in chitinase extract was 72% as compared to 45% in Tris-HCl extract (based on peak area measurements with respect to total area under the chromatogram). The results obtained suggest selenium binding to chitin-containing polysaccharide structures in fungi cell walls.
Assuntos
Membrana Celular/química , Parede Celular/química , Citosol/química , Micélio/ultraestrutura , Pleurotus/química , Selênio/análise , Cromatografia em Gel , Micélio/químicaRESUMO
Huitlacoche is the ethnic name of the young fruiting bodies of Ustilago maydis, a common parasite of maize. In Mexico and other Latin American countries, this fungus has been traditionally appreciated as a local delicacy. In this work a metallomics approach was used with the determination of eight elements in huitlacoche by electrothermal atomic absorption spectrometry as one facet of this approach. The results obtained indicated relatively lower concentrations of commonly analyzed metals, as referred to the data reported for other mushroom types. This effect was ascribed to different accessibilities of elements, depending on fungus substrate (lower from plant than from soil). Subcellular fractionation was accomplished by centrifugation of cell homogenates suspended in Tris-HCl buffer. Recoveries of the fractionation procedure were in the range of 71-103%. For six elements (Cr, Cu, Fe, Mn, Ni, and Pb), the mean relative contributions in cytosol, cell walls, and mixed membrane fraction were 50.7, 48.2, and 1.1% respectively. To attain the molecular weight distribution of compounds containing target elements as an additional aspect of the metallomics approach, the fungus extract (1% sodium dodecyl sulfate in Tris-HCl, 30 mmol L(-)(1), pH 7.0) was analyzed by size exclusion chromatography with UV and ICP-MS detection. With spectrophotometric detection (280 nm), the elution of high molecular weight compounds was observed in the form of one peak (MW > 10 kDa), and several lower peaks appeared at higher retention times (MW < 10 kDa). On ICP-MS chromatograms, a coelution of (59)Co, (63)Cu, (57)Fe, (202)Hg, (60)Ni, and (80)Se with the first peak on the UV chromatogram was clearly observed, indicating that a fraction of each element incorporated with high molecular weight compounds (12.7, 19.8, 33.7, 100, 19.4, and 45.8%, respectively, based on the peak area measurements). From a comparison of (80)Se and (33)S chromatograms (for sulfur analysis, the extract was obtained in the absence of SDS), both elements coeluted with the first UV peak, but their lower molecular weight compounds were apparently different. These findings may contribute to a better understanding of the accumulation of elements in mushrooms.
Assuntos
Oligoelementos/análise , Ustilago/química , Fracionamento Celular , Cromatografia em Gel , Espectrometria de Massas , Peso Molecular , Espectrofotometria Atômica , Ustilago/ultraestruturaRESUMO
Research interest in analyzing arsenic and selenium is dictated by their species-dependent behavior in the environment and in living organisms. Different analytical methodologies for known species in relatively simple chemical systems are well established, yet the analysis of complex samples is still a challenge. Owing to the complex matrix and low concentrations of target species that may be chemically labile, suitable pretreatment of the sample becomes a critical step in any speciation procedure. In this paper, the pretreatment procedures used for arsenic and selenium speciation are reviewed with the emphasis on the link between the analytical protocol applied and the biologically-significant information provided by the results obtained. In the first approach, the aim of pretreatment is to convert the original sample into a form that can be analyzed by a coupled (hyphenated) technique, preventing possible losses and/or species interconversion. Common techniques include different leaching and extraction modes, enzymatic hydrolysis, species volatilization, and so on, with or without species preconcentration. On the other hand, if the speciation analysis is performed for elucidation of elemental pathways and specific functions in a living system, more conscious pretreatment and/or fractionation is needed. The macroscopic separation of organs and tissues, isolation of certain types of cells, cell disruption and separation of sub-cellular fractions, as well as isolation of a specific biomolecules become important. Furthermore, to understand molecular mechanisms, the identification of intermediate-often highly instable--metabolites is necessary. Real life applications are reviewed in this work for aquatic samples, soils and sediments, plants, yeast, and urine.
Assuntos
Arsênio/química , Espectrometria de Massas/métodos , Selênio/química , Sedimentos Geológicos/química , Biologia Marinha , Plantas/química , Poluentes do Solo/análise , Poluentes Químicos da Água/análiseRESUMO
On-line hyphenation of size exclusion chromatography (SEC), UV, and inductively coupled plasma mass spectrometry (ICP-MS) was used to study the molecular weight distribution patterns of several elements in Brazil nuts (Bertholletia excelsa). This technique was used for the elemental speciation of different elements of nutritional and toxicological interests such as Mg, Fe, Co, Mo, Ag, Hg, and Pb. Elemental fractionation in Brazil nuts was studied using a Superdex peptide column with resolving capacity in the range of 14 to 0.18 kDa. Three different mobile phases, Tris buffer solution (pH 8.0), phosphate buffer (pH 7.5), and CAPS buffer solution (pH 10.0), were tried for the SEC fractionation. Size exclusion fractionation of all the extracted solutions was performed using a 50 mmol L(-)(1) Tris buffer (pH 8) as the mobile phase at a flow rate of 0.6 mL min(-)(1). Three different extractions, 0.05 mol L(-)(1) NaOH, 0.05 mol L(-)(1) HCl, and hot water at 60 degrees C, were performed, and the association of elements with various molecular weight fractions was evaluated. Total elemental concentrations in the extracted samples were determined and compared with the values obtained after total digestion to calculate the recovery values. Generally, high extraction efficiency was obtained with the NaOH solution as compared with HCl and hot water except in the case of magnesium, for which HCl was found to be a good extractant. Chromatographic elution profiles for these extractions were quite distinct from each other in most cases. Most of the elemental species were found to be associated with high molecular weight fractions. To study the differences obtained during the sample-processing step, the results obtained for nuts with shell were treated differently from those obtained for nuts purchased without shell and were compared.
Assuntos
Bertholletia/química , Metais/análise , Soluções Tampão , Fracionamento Químico , Cromatografia em Gel , Espectrometria de Massas , Peso Molecular , SoluçõesRESUMO
Brazil nuts have been classified as the foodstuffs that contain the highest level of unadulterated selenium, an essential trace element that appears to prevent cancer. To date, characterization of the selenium species in brazil nuts has not yet been investigated. In this work, various sample preparation approaches, including microwave extractions and enzymatic treatments, are examined with the goal of species preservation and subsequent selenium speciation; of these approaches, an enzymatic treatment with Proteinase K proved most effective. High-performance liquid chromatography (HPLC) separation strategies and inductively coupled plasma mass spectrometry (ICP-MS) detection schemes will also be presented. Extracts are evaluated against available standards for the commercially obtainable seleno-amino acids, selenomethionine (SeMet), selenoethionine (SeEt), and selenocystine (SeCys); selenomethionine was demonstrated to be the most abundant of these seleno-amino acids. Further characterization of unidentified selenium-containing peaks is attempted by the employment of several procedures, including electrospray-mass spectrometry (ES-MS). A peptide structure was identified; however, this was considered a tentative proposal due to the large background produced by the extremely complicated brazil nut matrix.