A chip-based supersonic microfluidic nebulizer for efficient sample introduction into inductively coupled plasma - Mass spectrometry.

As the use of microfluidic chips for handling biological samples is increasing, so is the need for combining them with powerful analytical techniques for metal determination such as inductively-coupled plasma mass spectrometry (ICP-MS). So far, coupling a microfluidic chip to an ICP-MS has been demonstrated mainly through the use of conventional pneumatic micro-flow nebulizers. However, disadvantages associated with the use of such nebulizers entail dead volume issues and liquid suction exerted on the outlet channel of the chip. Herein, we propose that a microfluidic chip, bearing a pneumatic nozzle for liquid nebulization, has the potential to advance metal determination in chip-based ICP-MS. More specifically, we demonstrate for the first time that the coupling of a chip-based supersonic microfluidic nebulizer (chip-µf-Neb) to an ICP-MS can be conveniently achieved through the use of a spray chamber with a laminar flow makeup gas. Operation of the combined system was evaluated at low liquid flow rates across 0.5-20 µL min-1, while nebulization and makeup argon (Ar) gas flow rates were optimized with respect to maximizing indium (In) sensitivity and minimizing oxide formation; a maximum sensitivity of 40000 cps (µg L-1)-1 was achieved at 10 µL min-1. The system was further evaluated for its performance in single-particle analysis, featuring a transport efficiency of 46% for Ag nanoparticles. Finally, the capabilities for conducting single-cell analysis were demonstrated with the detection of 80Se and 75As in individual Chlamydomas reinhardtii cells, which were previously incubated in 20 µM of selenate and 300 µM of arsenate, respectively. Efficient operation at low liquid flow rates along with the absence of self-aspiration render this nebulizer a promising tool for combining the powerful field of microfluidics with metal quantitation by means of ICP-MS.

Metals in tissues of seabass and seabream reared in sites with oxic and anoxic substrata and risk assessment for consumers.

Twenty-eight metals and elements were measured in the muscle, liver, gills, bone and intestine of farmed seabass and gilthead seabream from four Mediterranean fish farms. The influence of fish species and the effect of environmental conditions on the metal accumulation in fish tissues was investigated. Most concentrations were lower in muscle and higher in liver and bone than in other body tissues. Seabass accumulates more elements in its tissues than seabream. Fish reared in coarse, oxic sites accumulate more elements with higher concentrations in muscle, bone and intestine and with lower concentrations in liver and gills than fish reared in silty, anoxic sites. This may be attributed to feed type and sediment properties. According to the metal pollution index, hazard quotient, selenium health benefit values, carcinogenic risk of arsenic, maximum safe consumption and the permitted limits, the consumption of both farmed species should be considered as safe for human health.

Gender-specific distribution of selenium to serum selenoproteins: associations with total selenium levels, age, smoking, body mass index, and physical activity.

The epidemiology of selenium (Se) is mainly based on the determination of total serum selenium levels (TSe) which by many aspects is an inadequate marker of Se status. In this study we applied a recently developed LC-ICP-MS method, for the determination of the selenium content of the three main serum selenium-containing proteins, in a subcohort of the ATTICA study. This enables us to investigate whether the selenium distribution to selenoproteins may correlate with demographic (age, gender) and lifestyle variables (smoking, physical activity) that are crucial for the development of chronic diseases. A sub-sample from the ATTICA Study, consisted of 236 males (40 ± 11 years) and 163 females (38 ± 12 years), was selected. The selenium content of glutathione peroxidase (GPx-3), selenoprotein P (SelP) and selenoalbumin (SeAlb) was determined in serum by LC-ICP/MS method. We found that 26% of TSe is found in GPx-3, 61% in SelP and 13% in SeAlb. We have assessed the different ratios of selenoproteins' selenium content (Se-GPX-3/Se-SelP, Se-GPX-3/Se-SeAlb, Se-SelP/Se-SeAlb), showing that people with similar TSe may have different distribution of this selenium to selenoproteins. Total selenium levels and gender are the variables that mostly affect selenium distribution to selenoproteins while age, smoking, physical activity and BMI do not significantly influence selenium distribution. In conclusion, the simultaneous determination of the selenium content of serum selenium-containing selenoproteins is necessary for a thorough estimation of selenium status. The ratio of the Se content between selenoproteins may be proven a novel, valid marker of selenium status.

Metals in benthic macrofauna and biogeochemical factors affecting their trophic transfer to wild fish around fish farm cages.

Benthic macroinvertebrates and wild fish aggregating in the vicinity of four Mediterranean fish farms were sampled. Concentrations of metals and other elements were measured in macrofaunal taxa and in fish tissues (muscle, liver, gills, bone, gonad, stomach, intestine, and stomach content). Biological and geochemical characteristics play an important role in metal accumulation in benthic invertebrates, and consequently in metal transfer to higher trophic levels. Macroinvertebrates accumulated lower concentrations of most metals and elements than their respective sediment, except As, P, Na, Zn and Cd. Elemental concentrations of benthic organisms increased with increasing sediment metal content, except Cd, and with % silt, refractory organic matter and chlorophyll-a of sediment due to the influence of sediment geochemistry on metal bioavailability. Tolerant species were found to accumulate higher concentrations of most metals and elements, except for Cd, than equilibrium species. The ecological and morphological characteristics of the benthic invertebrates can affect the bioaccumulation of metals and elements in macrobenthos. Hg and P were found to increase their concentrations from zoobenthos to wild fish aggregating around fish cages feeding on macrofauna.

Heavy metals, trace elements and sediment geochemistry at four Mediterranean fish farms.

Trace element concentrations in sediment were investigated at four fish farms in the Eastern Mediterranean Sea. Fish farms effects were negligible beyond 25-50 m from the edge of the cages. Based on elemental distribution, sediments from the farms were separated into coarse oxidized and silty reduced ones. Fish feed is richer in P, Zn and Cd than reference and impacted stations. Comparison among impacted stations and the respective reference stations shows that, in anoxic sediments, all elements had higher concentrations at the impacted stations than at reference stations while in oxic sediments, many elemental concentrations were lower at impacted stations than at reference stations. The behavior of elements and therefore their distribution is affected by changes in sediment grain size, organic content and redox regime. Elements in sediments around fish farms can be clustered into five groups according to these environmental variables. In silty and anoxic sediments, element concentrations were higher than in coarse and oxic ones. Several approaches were used to assess potential sediment toxicity (enrichment factors, geoaccumulation indices, contamination factors) as well as to assess the potential danger to aquatic life (Sediment Quality Guidelines, SQG). Cu, Zn and Fe can cause from threshold to extreme effects on aquatic life in anoxic, fine-grained sediments and As can cause threshold effects in all types of sediment around fish farms. Other elements (Cr, Pb, Mn) can also cause unwanted effects when compounded with elevated background levels.

Using nanoelectrospray ion mobility spectrometry (GEMMA) to determine the size and relative molecular mass of proteins and protein assemblies: a comparison with MALLS and QELS.

The determination of protein assembly size and relative molecular mass is currently of great importance in biochemical analysis. In particular, the technique of nanoelectrospray (nES) with a gas-phase electrophoretic mobility molecular analyzer (GEMMA) has received increased attention for such measurements. However, in order for the GEMMA technique to gain broader acceptance in protein analysis, it must be further evaluated and compared with other established bioanalytical techniques. In the present study, nES-GEMMA was evaluated for the analysis of a set of protein and protein complexes involved in the Sec and the bacterial type III secretion pathway of enteropathogenic Escherichia coli bacteria. The same set of proteins, isolated and purified using standard biochemical protocols, were also analyzed using multi-angle laser light scattering (MALLS) and quasi-elastic light scattering (QELS), following size exclusion chromatography. This allowed for direct comparisons between the three techniques. It was found that nES-GEMMA, in comparison to the more established MALLS and QELS techniques, offers several complementary advantages. It requires considerably less amount of material, i.e., nanogram vs. milligram amounts, and time per sample analysis, i.e., few minutes vs. tens of minutes. Whereas the determined size and relative molecular mass are similar between the compared methods, the electrophoretic diameters determined using nES-GEMMA seem to be systematically smaller compared to the hydrodynamic diameter derived by QELS. Some of the GEMMA technique disadvantages include its narrow dynamic range, limited by the fact that at elevated protein concentrations there is increased potential for the occurrence of nES-induced oligomers. Thus, it is preferred to analyze dilute protein solutions because non-specific oligomers are less likely to occur whereas biospecific oligomers remain detected. To further understand the formation of nES-oligomers, the effect of buffer concentration on their formation was evaluated. Also, nES-GEMMA is not compatible with all the buffers commonly used with MALLS and QELS. Overall, however, the nES-GEMMA technique shows promise as a high-throughput proteomics/protein structure tool.

Dietary habits of Greek adults and serum total selenium concentration: the ATTICA study.

PURPOSE: The trace element selenium is an essential micronutrient for human health, and its low levels in serum are implicated in the pathogenesis of several chronic diseases. The determination of total serum selenium levels may contribute to the assessment of the health status of all populations. Since the serum selenium levels are highly affected by diet, we assessed its association with the dietary habits of Greek adults. METHODS: Serum selenium levels were determined with inductively coupled plasma mass spectrometry in a cohort of 506 participants (men: 296, women: 210) aged 18-75 from the ATTICA study. Food consumption was evaluated with a validated food-frequency questionnaire. RESULTS: Evaluation of the relationship between serum total selenium with major food groups and beverages by multi-adjusted analysis revealed that serum selenium was positively correlated with the consumption of red meat (2.37 ± 0.91, p = 0.01) while the consumption of other selenium-containing foods (i.e., fish, cereals, dairy products, vegetables) did not demonstrate such a relationship. Moreover, principal component analysis revealed that the adoption of a vegetarian type of diet is inversely correlated with total selenium (-3.94 ± 2.28, p = 0.08). CONCLUSIONS: Among the dietary habits that were examined, red meat seems to be the major determinant of serum selenium in Greek adults.

Characterisation of horse dander allergen glycoproteins using amino acid and glycan structure analyses. a mass spectrometric method for glycan chain analysis of glycoproteins separated by two-dimensional electrophoresis.

Separation of horse dander allergens using two-dimensional PAGE resulted in the identification of 16 proteins that react with allergic patient sera. A sensitive method has been developed for analysing the structures of the glycan chains of individual glycoprotein allergens transferred to blots following two-dimensional PAGE, and has allowed the structural identification of the glycan chains of the most abundant isoforms of Equ c 1, a glycosylated horse dander major allergen. The method involves separation of the allergens by two-dimensional PAGE, transfer to polyvinylidene difluoride membranes, release of the glycan chains using peptide N-glycosidase F, permethylation and mass spectrometric analysis of the derivatised glycans. The amino acid compositions of the 16 horse dander allergens separated by two-dimensional PAGE have been determined, allowing the identification of the various isoforms of Equ c 1. These results also confirmed that the two non-glycosylated major allergens, Equ c 2.0101 and Equ c 2.0102, belong to the lipocalin family, and support the idea that these two allergens are most probably isoforms of the same protein. The glycan structures identified using the mass spectrometric method are common biantennary and triantennary glycan chains. These carbohydrate moieties may have a role in the binding of IgE; however, it is more likely that the overall glycoprotein structure involving both the glycan and protein moieties, rather than the structure of the glycan chains alone, is responsible for eliciting allergic responses.

Identification of arsenosugars at the picogram level using nanoelectrospray quadrupole time-of-flight mass spectrometry.

State-of-the-art analytical methods for arsenic speciation rely typically on the availability of standards of defined structure, limiting the applicability of such methods to the determination of known compounds. Our previous high-energy tandem mass spectrometric studies demonstrated the strength of mass spectrometry for generating structurally diagnostic ions that allow for the identification of arsenic-containing ribofuranosides (arsenosugars) without the use of standards. We now report a more widely applicable and more sensitive approach, using negative-ion nano-electrospray low-energy tandem mass spectrometry for the generation of structurally useful product ions that allow for identification of arsenosugars at the picogram level. In the negative-ion mode, numerous product ions, suitable for characterizing naturally occurring dimethylated arsenosugars, were generated in high abundance. Application of the method to an algal extract unequivocally demonstrated the presence of a single dimethylated arsenosugar. In the positive-ion mode, characteristic tandem mass spectra were obtained for four trimethylarsonioribosides, allowing their identification without the need for standards. Overall it was demonstrated that nano-ES-MS/MS techniques can be used for characterizing arsenosugars on a routine basis, a necessary requirement for assessing potential health risks associated with consuming foods containing elevated levels of arsenosugars and for improving our understanding of arsenic biochemistry.

Mass spectrometric determination of the sites of O-glycan attachment with low picomolar sensitivity.

A sensitive protocol for unambiguously and positively identifying O-glycosylation sites in glycopeptides is described, based on beta-elimination of the glycan chain(s) using NH4OH. On glycan elimination, NH3 is incorporated into the amino acid residue(s) to which the glycan(s) had been attached, to yield a modified amino acid residue having a distinct mass. Electrospray ionization collision-induced dissociation tandem mass spectrometry allows the released, modified peptide to be sequenced and the site(s) of the modified amino acid residue(s) to be identified. The protocol has been optimized using a series of structurally related O-glycopeptides, and standard conditions are recommended for handling unknowns. We demonstrate that site determination can be achieved using as little as 1 pmol of starting material.

Liquid chromatography and mass spectrometry for the speciation of arsenic animal feed additives.

A number of analytical techniques have been investigated for the determination of arsenic animal feed additive compounds. More specifically, liquid chromatography methods have been developed for the separation of these arsenicals. Micro liquid chromatography, which is a relatively new technique, shows a number of advantages for these analyses, especially when interfaced to mass spectrometers. Improved limits of detection, low solvent consumption, reduced amounts of stationary phase, as well as flow-rates easily accommodated by continuous-flow liquid secondary ion mass spectrometry and direct liquid introduction mass spectrometry, are some of the advantages we have observed when fabricating and using these columns. The off-line combination of micro liquid chromatography and electrothermal atomic absorption spectrometry improves the limit of detection for arsenic and shows potential for on-line coupling. Further use of the methods developed here may allow for a more detailed understanding of the fate and interactions of arylarsenicals in biological and environmental systems.

Speciation of arsenic animal feed additives by microbore high-performance liquid chromatography with inductively coupled plasma mass spectrometry.

Phenylarsonic compounds have been used as poultry and swine feed additives for the purpose of growth promotion and disease prevention. Owing to the lack of suitable analytical methods, however, knowledge of their metabolism, environmental fate and impact remains incomplete. In order to compensate for this, analytical procedures were developed that allow the speciation of arsenic animal feed additives by using microbore high-performance liquid chromatography (microHPLC) coupled on-line with ICP-MS. More specifically, reversed-phase (RP) chromatographic methods were optimised to achieve the separation of various phenylarsonic acids from each other and from the more toxic inorganic arsenic compounds. This mode of chromatography, however, exhibits limitations, especially in the presence of naturally occurring organoarsenic compounds. The application of RP ion-pairing chromatography eliminates such shortcomings by minimising the co-elution of arsenic species. In general, the microHPLC-ICP-MS methods developed in this study provide high selectivity, extremely good sensitivity, low limits of detection (low-ppb or sub-pg amounts of As), require small sample volumes (< 1 microliter), minimise waste and operate most efficiently under low mobile-phase flow rates (15-40 microliters min-1), which are compatible for use with other types of mass spectrometers, e.g., electrospray. Reference materials containing naturally occurring arsenic compounds were spiked with phenylarsonic compounds and then analysed by using the procedures developed in this study.

Investigation of arsine-generating reactions using deuterium-labeled reagents and mass spectrometry.

Mass spectrometry was used to detect transfer of deuterium from labeled reagents to arsines following hydride-generation reactions. The arsine gases liberated from the reactions of arsenite, arsenate, methylarsonic acid, and dimethylarsinic acid with HCl and NaBD(4) in H(2)O, or with DCl and NaBH(4) in D(2)O, were examined. Differences in the mode of deuterium incorporation for the various arsines were detected. These results may help explain some of the observed variations in arsine-generation efficiency for various arsenic compounds present in environmental and biological samples.

Characterization of arsenosugars of biological origin using fast atom bombardment tandem mass spectrometry.

A fast atom bombardment (FAB) mass spectrometric method has been developed for characterizing arsenosugar compounds. These compounds are of particular interest not only because their biochemistry requires further investigation but also because they are present at relatively high concentrations in commercial seaweed food products. FAB was used for the efficient generation of gas-phase ions of the various arsenosugar compounds. Negative-ion detection of such ions was found to be more sensitive than positive-ion detection due to the presence of negatively charged substituents. Negative-ion collision-induced dissociation (CID) tandem mass spectrometry (MS) of the [M - H](-) precursor ions results in the formation of numerous characteristic product ions via charge-remote fragmentation. These product ions provide abundant structural information for arsenosugar characterization. Separation of the arsenosugar-originating precursor ions from the matrix ions, always present in FAB mass spectra, is achievable using an analyzer resolution of 3000. This resolution allows for accurate selection of a precursor ion for subsequent CID experiments. However, by switching to a resolution of 1000, the quality of the tandem mass spectra can be slightly improved. Such an improvement is especially useful when analyzing nanogram amounts of arsenosugars. Furthermore, it was demonstrated that positive-ion tandem MS provides complementary information for the structural characterization of the arsenosugars examined. The mass spectrometric procedures developed in this study were further applied for the characterization an arsenosugar present in a partially purified algal (Sargassum lacerifolium) extract.

Simplex optimization of conditions for the determination of arsenic in environmental samples by using electrothermal atomic absorption spectrometry.

Simplex optimization was used to efficiently delineate the optimum experimental conditions to be used for the electrothermal atomic absorption spectrometric analysis of arsenic in a standard reference material of marine origin. Four experimental variables, were considered: ashing temperature, atomization temperature, modifier concentration, and atomization ramping time. This combination of methods and materials provides a powerful means of rapidly improving the experimental conditions used for analysis of arsenic in a wide variety of samples of environmental origin. Excellent recoveries of arsenic were obtained when using the optimum electrothermal atomic absorption spectrometry conditions to analyze standard solutions of arsenobetaine, arsenocholine and tetramethylarsonium iodide.