Chromatography-hydride Generation-atomic Fluorescence Spectrometry Research Articles

Determination of Arsenic in the Rainbow Trout Muscle and Rice Samples

The content of arsenic was determined in the rainbow trout muscle (<I>Oncorhynchus mykiss) </I> and in rice samples. The method of atomic absorption spectrometry with hydride generation (HG-AAS) in graphite furnace was used for total arsenic determination. Arsenic speciation was done using the high performance liquid chromatography-hydride generation-atomic fluorescence spectrometry (HPLC-HG-AFS). The rainbow trout muscle was extracted by ultra pure deionised water; trifluoroacetic acid was used for the extraction of rice samples. Total arsenic values ranged from 60.90 μg/kg to 310.00 μg/kg in rice samples and from 0.72 mg/kg to 2.23 mg/kg in rainbow trout muscle. An inorganic trivalent arsenic As(III) was determined as the main species in rice and non-toxic arsenobetaine in the samples of rainbow trout.

Glutathione-Dependent Reduction of Arsenate by Glycogen Phosphorylase—A Reaction Coupled to Glycogenolysis

Arsenate (As(V)) is reduced in the body to the more toxic arsenite (As(III)). We have shown that two enzymes catalyzing phosphorolytic cleavage of their substrates, namely purine nucleoside phosphorylase and glyceraldehyde-3-phosphate dehydrogenase, can reduce As(V) in presence of an appropriate thiol and their substrates. Another phosphorolytic enzyme that may also reduce As(V) is glycogen phosphorylase (GP). With inorganic phosphate (P(i)), GP catalyzes the breakdown of glycogen to glucose-1-phosphate; however, it also accepts As(V). Testing the hypothesis that GP can reduce As(V), we incubated As(V) with the phosphorylated GPa or the dephosphorylated GPb purified from rabbit muscle and quantified the As(III) formed from As(V) by high-performance liquid chromatography-hydride generation-atomic fluorescence spectrometry. In the presence of adenosine monophosphate (AMP), glycogen, and glutathione (GSH), both GP forms reduced As(V) at rates increasing with enzyme and As(V) concentrations. The As(V) reductase activity of GPa was 10-fold higher than that of GPb. However, incubating GPb with GP kinase and ATP (that converts GPb to GPa) increased As(V) reduction by phosphorylase up to the rate produced by GPa incubated under the same conditions. High concentration of inorganic sulfate, which activates GPb like phosphorylation, also promoted reduction of As(V) by GPb. As(V) reduction by GPa (like As(V) reduction in rats) required GSH. It also required glycogen (substrate for GP) and was stimulated by AMP (allosteric activator of GP) even at low micromolar concentrations. P(i), substrate for GP competing with As(V), inhibited As(III) formation moderately at physiological concentrations. Glucose-1-phosphate, the product of GP-catalyzed glycogenolysis, also decreased As(V) reduction. Summarizing, GP is the third phosphorolytic enzyme identified capable of reducing As(V) in vitro. For reducing As(V) by GP, GSH and glycogen are indispensable, suggesting that the reduction is linked to glycogenolysis. While its in vivo significance remains to be tested, further characterization of the GP-catalyzed As(V) reduction is presented in the adjoining paper.

Glutathione-Dependent Reduction of Arsenate by Glycogen Phosphorylase—Responsiveness to Endogenous and Xenobiotic Inhibitors

Rabbit muscle glycogen phosphorylase-a (GPa) reduces arsenate (As(V)) to the more toxic arsenite (As(III)) in a glutathione (GSH)-dependent fashion. To determine whether reduction of As(V) by GPa is countered by compounds known to inhibit GP-catalyzed glycogenolysis, the effects of thiol reagents, endogenous compounds (glucose, ATP, ADP) as well as nonspecific glycogen phosphorylase inhibitors (GPIs; caffeine, quercetin, flavopiridol [FP]), and specific GPIs (1,4-dideoxy-1,4-imino-D-arabinitol [DAB], BAY U6751, CP320626) were tested on reduction of As(V) by rabbit muscle GPa in the presence of glycogen (substrate), AMP (activator), and GSH, and the As(III) formed from As(V) was quantified by high-performance liquid chromatography-hydride generation-atomic fluorescence spectrometry. The As(V)-reducing activity of GPa was moderately sensitive to thiol reagents. Glucose above 5mM and ADP or ATP at physiological levels diminished GPa-catalyzed As(V) reduction. All GPIs inhibited As(V) reduction by GPa in a concentration-dependent fashion; however, their effects were differentially affected by glucose (10mM) or AMP (200microM instead of 25microM), known modulators of the action of some GPIs on the GP-catalyzed glycogenolysis. Inhibition of As(V) reduction by DAB and quercetin was not influenced by glucose or AMP. Glucose that potentiates the inhibitory effects of caffeine, BAY U6751, and CP320626 on the glycogenolytic activity of GPa also enhanced the inhibitory effects of these GPIs on GPa-catalyzed As(V) reduction. AMP at high concentration alleviated the inhibition by BAY U6751 and CP320626 (whose antagonistic effect on GP-catalyzed glycogen breakdown is also AMP sensitive), whereas the inhibition in As(V) reduction by FP or caffeine was little affected by AMP. Thus, GPIs inhibit both the glycogenolytic and As(V)-reducing activities of GP, supporting that the latter is coupled to glycogenolysis. It was also shown that a GPa-rich extract of rat liver contained GSH-dependent As(V)-reducing activity that was inhibited by specific GPIs, suggesting that the liver-type GPa can also catalyze reduction of As(V).

Analytical speciation as a tool to assess arsenic behaviour in soils polluted by mining

A study is performed to evaluate the occurrence of arsenic in polluted soils using acidic extractions and liquid chromatography-hydride generation-atomic fluorescence spectrometry (LC-HG-AFS) for speciation analysis. Seven soil samples were collected in an abandoned area polluted by mining in the Eastern Pyrenees (Spain), and two uncontaminated soils were taken for reference purposes. Moreover, the total arsenic content is evaluated in two different sieved fractions in order to obtain information on the possible particle-size-dependent association of arsenic with soil components. Soil samples were extracted with both phosphoric and ascorbic acids and the stabilities of the extracted species were studied. The arsenic species were determined by LC-HG-AFS. In addition, the ability of soil grinding to effect species change is also assessed. Arsenite and arsenate were found in the polluted soils, but only arsenate was found in the unpolluted soils. The quality of the results was assessed through a mass balance calculation and by analysing two soil Certified Reference Materials. Valuable information regarding arsenic occurrence in the studied soils is obtained from the speciation results. The presence of arsenite in the extracts can be attributed to arsenopyrite residues, whereas the presence of arsenate indicates release from weathered material.

Preservation procedures for arsenic speciation in a stream affected by acid mine drainage in southwestern Spain

A preservation study has been performed for arsenic speciation in surface freshwaters affected by acid mine drainage (AMD), a pollution source characterized by low pH and high metallic content. Two sample preservation procedures described in the literature were attempted using opaque glass containers and refrigeration: i) addition of 0.25 mol L(-1) EDTA to the samples, which maintained the stability of the arsenic species for 3 h; and ii) in situ sample clean-up with a cationic exchange resin, in order to reduce the metallic load, which resulted in a partial co-adsorption of arsenic onto Fe precipitates. A new proposed method was also tried: sample acidification with 6 mol L(-1) HCl followed by in situ clean-up with a cationic exchange resin, which allowed a longer preservation time of at least 48 h. The proposed method was successfully applied to water samples with high arsenic content, taken from the Aguas Agrias Stream (Odiel River Basin, SW Spain), which is severely affected by AMD that originates at the nearby polymetallic sulfide mine of Tharsis. The speciation results obtained by liquid chromatography-hydride generation-atomic fluorescence spectrometry (HPLC-HG-AFS) indicated that during the summer the main arsenic species was As(V) at the hundred microg L(-1) level, followed by DMA (dimethyl arsenic) and As(III) below the ten microg L(-1) level. In winter, As(V) and As(III) increased at least fivefold, whereas the DMA was not detected.

New preservation method for inorganic arsenic speciation in acid mine drainage samples

A new preservation method has been proposed for the speciation of As(III) and As(V) in acid mine drainage (AMD) samples, characterised by low pH and high metallic content. Samples were taken from a polymetallic sulphides mining area in the province of Huelva (SW Spain), under exploitation until the 1960s for its Cu, Pb and Zn sulphides. The abandoned mine works and the numerous waste rocks heaps produce AMD with high As content, an aqueous pollution source for the nearby streams. Short-term (from few hours to 1 week) preservation of the two inorganic arsenic species was studied, trying different containers (polyethylene, glass), presence or absence of light, temperatures (ambient, refrigerated, frozen), preserving agents and procedures (EDTA, HCl or AcH acids, cation-exchange resin). The speciation results obtained by liquid chromatography-hydride generation-atomic fluorescence spectrometry (HPLC-HG-AFS) indicated a rapid conversion of the samples with most of the preservation procedures reported in the literature after 3 h after sample collection. A promising method for arsenic preservation has been developed in this work, which maintains the arsenic species distribution in the original samples for a longer time. It consists in the use of opaque glass containers, acidification of the samples with HCl and in situ cleanup with cationic exchange resin, which allowed to preserve the samples for As speciation for at least 48 h.

Liquid chromatography–hydride generation–atomic fluorescence spectrometry hybridation for antimony speciation in environmental samples

Atomic fluorescence spectrometry was used as an element-specific detector in hybridation with liquid chromatography (LC) and hydride generation for the speciation of Sb(III), Sb(V) and trimethylantimony dichloride (TMSbCl 2). The three species were poorly resolved in a single chromatogram but good results were obtained by anion-exchange chromatography, using a mobile phase with 20 mM EDTA and 8 mM hydrogenphthalate to separate Sb(III) and Sb(V) and 1 mM carbonate at pH 10 to separate Sb(V) and TMSbCl 2. Calibration graphs were linear between 2 and 100 μg l −1. Detection limits were 0.9, 0.5 and 0.7 μg l −1 for Sb(III), Sb(V) and TMSbCl 2, respectively. The method was applied to the speciation of antimony in environmental samples.

Simultaneous speciation analysis of Sb(III), Sb(V) and (CH 3) 3SbCl 2 by high performance liquid chromatography-hydride generation-atomic fluorescence spectrometry detection (HPLC-HG-AFS): Application to antimony speciation in sea water

This paper presents an improvement for the simultaneous separation of Sb(V), Sb(III) and (CH 3) 3SbCl 2 species by high performance liquid chromatography (HPLC) and its detection by hydride generation-atomic fluorescence spectrometry (HG-AFS). The separation was performed on an anion exchange column PRP-X100 using a gradient elution program between EDTA/KHP (potasium hydrogen phtalate) as first mobile phase and phosphate solutions solution as the second one. The chromatographic separation and the HG-AFS parameters were optimized by experimental design. The best results were obtained by using an elution program with 20 mmol l −1 EDTA + 2 mmol l −1 KHP solution at pH 4.5, during 1.15 min, then change to 50 mmol l −1 (NH 4) 2HPO 4 solution at pH 8.3, switching back after 4.0 min to the first mobile phase, until 5 min, with a constant flow rate of 1.5 ml min −1. Retention time of Sb(V), Sb(III) and trimethylantimony species were 1.22, 2.31 and 3.45 min and the detection limits were 0.13; 0.07 and 0.13 μg l −1, respectively. Studies on the stability of this antimony species in sea water samples on the function of the elapsed time of storage in refrigerator at 4 °C was performed employing the optimized method. Results revealed that Sb(III) is easily oxidized within some hours to Sb(V) in sea water stored at 4 °C. However, when the sea water was immediately mixed with EDTA no oxidation of Sb(III) was observed up to 1 week of storage. The proposed methodology was then applied to the antimony speciation in sea water samples.

Evaluation of the extraction methods for arsenic speciation in rice straw, Oryza sativa L., and analysis by HPLC-HG-AFS

The most important and difficult step during arsenic speciation analysis is to extract the various arsenic species in the original forms from the samples. In this work, a study has been carried out to compare the different extraction methods by using different extraction solvents including water, methanol, water–methanol, water–ethanol and water–acetonitrile. Different techniques (shaking, ultrasonic, Soxhlet and microwave-assisted extraction) were applied during the procedure. A new, nontoxic and effective extraction method for arsenic speciation in the terrestrial plant material, rice straw, with water–ethanol mixture as extraction solvent using microwave-assisted extraction was found to provide good extraction efficiency without internal species conversion. The extraction of arsenic from the rice straw and subsequent determination of individual arsenic species by high performance liquid chromatography-hydride generation-atomic fluorescence spectrometry (HPLC-HG-AFS) is described. By using the proposed extraction method, the total arsenic extraction efficiency was 90.3%. The arsenic species in rice straw were analyzed and four normal chemical forms, AsIII, AsV, MMAV and DMAV, in straw shoots were found, but no MMAV was found in the straw roots.

Speciation of the immediately mobilisable As(III), As(V), MMA and DMA in river sediments by high performance liquid chromatography–hydride generation–atomic fluorescence spectrometry following ultrasonic extraction

In this work, a fast method is developed for the speciation of As(III), As(V), MMA and DMA in the immediately mobilisable fraction of river sediments (i.e. water-soluble and phosphate-exchangeable) by high performance liquid chromatography–hydride generation–atomic fluorescence detection (HPLC–HG–AFD) after extraction using focused ultrasound. The influence of relevant parameters influencing an ion-pairing chromatographic separation following isocratic elution (i.e. amount of MeOH in the mobile phase, ion pair reagent concentration, pH, flow rate) was studied. Focused ultrasound transmitted from an ultrasonic probe provided the same extractable contents as conventional extraction with no changes in the species distribution. The effect of the drying step over extraction of As species was investigated. The following drying procedures were compared: freeze-, oven-, microwave- and air-drying. No influence of the drying operation on the water-extractable fraction was observed. However, freeze- and air-drying yielded significantly higher phosphate-extractable amounts of As(III) and As(V) as compared to oven and microwaves. Detection limits for the As species were in the range 1.3–4.1 ng/g for the water-soluble fraction and 1.6–4.8 ng/g for the phosphate buffer exchangeable fraction. The method was applied to the speciation of immediately mobilisable As(III), As(V), DMA and MMA in 11 sediment samples collected along the beds of the Louro River (southern Galicia, Spain).

Glutathione-dependent reduction of arsenate in human erythrocytes--a process independent of purine nucleoside phosphorylase.

Reduction of arsenate (AsV) to the more toxic arsenite (AsIII) is toxicologically important, yet its mechanism is unknown. To clarify this, AsV reduction was investigated in human red blood cells (RBC), as they possess a simple metabolism. RBC were incubated with AsV in gluconate buffer, and the formed AsIII was quantified by high performance liquid chromatography-hydride generation-atomic fluorescence spectrometry (HPLC-HG-AFS). The observations are compatible with the following conclusions. (1) Human RBC reduce AsV intracellularly, because 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS, inhibitor of the chloride-bicarbonate exchanger, which also mediates phosphate and AsV uptake), as well as chloride and phosphate, countered AsIII formation. (2) Purine nucleoside phosphorylase (PNP), whose AsV reductase activity has been directly demonstrated, cannot be a physiologically relevant AsV reductase, because its inhibitor (BCX-1777) failed to decrease the basal erythrocytic AsV reduction, although it prevented the increase in AsIII formation caused by artificial activation of PNP with inosine and dithiothreitol. (3) The basal (PNP-independent) AsV reduction requires glutathione (GSH), because the GSH depletor diethylmaleate strongly diminished AsIII formation. (4) The erythrocytic AsV reduction apparently depends on NAD(P) supply, because oxidants of NAD(P)H (i.e., pyruvate, ferricyanide, methylene blue, nitrite, tert-butylhydroperoxide, dehydroascorbate, 4-dimethylaminophenol) enhanced AsIII formation from AsV. The oxidant-stimulated AsV reduction is PNP-independent, because BCX-1777 failed to affect it, but is GSH-dependent, because diethylmaleate impaired it. (5) Pyruvate-induced glucose depletion, which causes NAD enrichment in the erythrocytes at the expense of NADH, enhanced AsV reduction. This suggests that the erythrocytic AsV reduction requires both NAD supply and operation of the lower part of the glycolytic pathway starting from glyceraldehyde-3-phosphate dehydrogenase (GAPDH) that, unlike the upper part, remains fed with substrates originating from the degradation of 2,3-bisphosphoglycerate in RBC depleted of glucose by pyruvate. (6) Fluoride, which arrests glycolysis at enolase and thus prevents NAD formation, inhibited AsV reduction in glucose-sufficient RBC, but increased it in glucose-deficient (NAD-enriched) cells, suggesting that the section of glycolysis coupled to AsV reduction lies between GAPDH and enolase. In conclusion, besides the artificial PNP-dependent AsV reduction, human RBC contain a PNP-independent AsV-reducing mechanism. This appears to require the supply of GSH, NAD, and substrate to one or more of the glycolytic enzymes localized between GAPDH and enolase.

New considerations about the separation and quantification of antimony species by ion chromatography–hydride generation atomic fluorescence spectrometry

A new method for the speciation of inorganic [Sb(III) and Sb(V)] and organic (Me 3SbCl 2) antimony species by using a polystyrene–divinylbenzene-based anion-exchange HPLC column (Hamilton PRP-X100) coupled to hydride generation atomic fluorescence spectrometry (HG-AFS) is presented. Several mobile phases were tested for the baseline separation of these three antimony species, investigating in detail experimental parameters such as concentration and pH. The best efficiency and resolution was achieved by using a gradient elution between diammonium tartrate 250 mmol l −1 pH 5.5 (A) and KOH 20 mmol l −1 pH 12 (B). The gradient programme used was 100% B for 1.5 min, decreasing to 0% B in 0.1 min and maintained the elution with 100% A for 5.5 min. Analysis time was less than 7 min. Equilibration of the column with the complexing mobile phase was found to be critical in order to avoid Sb(III) double peak formation. Dilution in diammonium tartrate medium was necessary in order to avoid Sb(III) oxidation at μg l −1 concentration level. Detection limits of 0.06 μg l −1 for Sb(V), 0.09 μg l −1 for Me 3SbCl 2 and 0.04 μg l −1 for Sb(III) as well as repeatability and reproducibility better than 5% R.S.D. ( n = 10) and 9% R.S.D. ( n = 30) (for 1 and 5 μg l −1 of Sb(V) and Sb(III) and 5 and 10 μg l −1 of Me 3SbCl 2) were obtained. Accuracy and recovery studies were carried out by analysing one river freshwater sample and two water certified reference materials. The proposed methodology can be considered reliable and straightforward for antimony speciation in fresh water samples.

Optimization of the extraction for the determination of arsenic species in plant materials by high-performance liquid chromatography coupled with hydride generation atomic fluorescence spectrometry

A study has been conducted for the separation and the determination of arsenic species in plants using high-performance liquid chromatography–hydride generation atomic fluorescence spectrometry with emphasis on sample extraction procedures. Various extraction solvents have been applied to extract arsenic species from plants in order to investigate the uptake, transfer and accumulation processes of arsenic. The method was optimized with respect to the selection of extraction solvent, extraction time and the number of extraction steps. The analytical procedure has been validated by analyzing standard reference material GBW 82301 (peach leaves) and successfully used for the arsenic speciation in plants grown on contaminated soil near an arsenic mine. Inorganic arsenic, especially arsenate (As(V)) appears to be the major component in plants and organic arsenic species of monomethylarsenic acid and dimethylarsenic acid were detected at low concentrations.

Speciation analysis of arsenic in environmental solids Reference Materials by high-performance liquid chromatography–hydride generation–atomic fluorescence spectrometry following orthophosphoric acid extraction

A method for the determination of arsenic species (arsenite, arsenate, monomethylarsonic acid (MMAA) and dimethylarsinic acid (DMAA)) in environmental solid samples has been studied and applied to three reference materials (river sediment, agricultural soil, sewage sludge) certified for their total As content and to real world samples. The analytical method used was ion-exchange liquid chromatography coupled on-line to atomic fluorescence spectroscopy through hydride generation. It was applied to the determination of As species in orthophosphoric acid extracts of the solids; the efficiency of this extraction procedure was studied in details, as well as the stability of extracts of As-rich ones. Very low detection limits were obtained, ranging from 0.02 to 0.04 mg (As) kg −1 for all species in all matrices. This high sensitivity allows both the analysis of As-poor samples and the dilution of extracts, of As-rich ones, reducing, thus, the possibility of matrix interferences and allowing a drastic improvement of chromatograms obtained when high concentrations of extractant are used. The efficiency of orthophosphoric acid extraction was shown to depend more upon the nature of the material analysed than on acid concentration, excellent (90–100%) recoveries of total As being obtained for the sediment and the sludge reference materials samples whereas yield did not exceed 62% for the soil Reference Materials. Procedures allowing a convenient stability of As species, including As(III), in the extracts are presented. In the three Reference Materials studied As(V) was found to be the main species, As(III) being only a minor component except in sludge where it amounts to ca. one-third of total As; methylated species, MMAA and DMAA, were evidenced only in the sludge sample at low (0.2–0.3 mg (As) kg −1) concentrations. Some applications to “real world” untreated equatorial sediment or agricultural soil samples evidenced similar abundances of As(V) and As(III) as well as the presence of methylated species. On the whole, the method proposed has a very good potential as a routine speciation analysis procedure for As speciation studies in environmental solids.

Determination of selenite, selenate and selenocyanate in waters by ion chromatography-hydride generation-atomic fluorescence spectrometry (IC-HG-AFS)

Dissolved inorganic selenium (Se) speciation in environmental and industrial waters is determined by ion chromatography-hydride generation-atomic fluorescence spectrometry (IC-HG-AFS). The three species selenite (Se(IV)), selenate (Se(VI)) and selenocyanate (SeCN) are separated by anion-exchange chromatography on a Dionex AS-16 column using gradient elution with NaOH (17.5–100 mmol L−1). Prior to HG-AFS detection, selenate is pre-reduced on-line to selenite by reaction with 5.8 mol L−1 HBr at 100 °C. From a 1 mL sample, detection limits of 26, 33 and 34 ng L−1 were achieved for Se(IV), Se(VI) and SeCN, respectively. The reproducibility of standard solutions, as well as of native and spiked real-world samples, was on the order of 5–10%. Spike recoveries for a wide variety of real-world samples are quantitative for all three species. The method was validated by good agreement between the measured speciation patterns for SRM 1643d and two real-world samples with the certified or measured total Se concentrations, respectively. The method was applied to determine Se speciation in a petroleum refinery waste water, where SeCN was the only detected species, and in a gold mine waste water, where smaller fractions of Se(IV) and Se(VI) were encountered besides the major species SeCN.

Speciation of dimethylarsinic acid and monomethylarsonic acid by gas chromatography–mass spectrometry

A GC–MS method has been developed to determine two methylated arsenic species in human urine samples. This GC–MS method was compared with high-performance liquid chromatography–hydride generation–atomic fluorescence spectrometry. The yield of derivatisation for dimethylarsinic acid (DMA) and monomethylarsonic acid (MMA) using thioglycol methylate was measured. The detection limits for DMA and MMA using this GC–MS method are 0.95 and 0.8 ng/cm 3, respectively.