Enzyme-assisted extraction of arsenic species from plant material

Investigations regarding the transfer and metabolism of arsenic species in plants require mild extraction conditions to conserve the original composition of arsenic species. Beside the use of water or water/methanol for extraction of arsenic species from plant samples, enzymes can assist this procedure by digestion of cellulose and other constituents of cell walls, resulting in a faster, more efficient extraction technique which preserves the arsenic species. The investigations presented here were focused on the stability of certain arsenic species in enzymatic solutions, optimal conditions for their chromatographic separation and detection namely by means of ion chromatography–inductively coupled plasma mass spectrometry and improvements with respect to extraction efficiency. With commercially available enzymes and enzyme mixtures, the digestion rate of soluble starch as model cellulose was determined using high-performance anion exchange chromatography–pulsed amperometric detection analysis of glucose as the major digestion product. The most effective digestion rate (80% within 4?h) was obtained with Viscozyme®. This enzyme mixture was applied to extracted arsenic species from algae and terrestrial plant materials. Qualitative and quantitative differences in the results between enzyme-assisted and water extractions were obtained and discussed. The results show that the application of enzymes in mild extraction protocols should be evaluated as an additional step for the identification of As-metabolics in organisms. Careful selection of suitable enzyme mixtures can overcome the disadvantage that extraction efficiency is very organism-specific.     

Extraction of antimony and arsenic from fresh and freeze-dried plant samples as determined by HG-AAS

Six extraction media (acetic acid, EDTA, tetrabutylammonium hydroxide, NaOH, MeOH/H2O, acetonitrile/H2O) were tested for their ability to extract antimony (Sb) and arsenic (As) from freeze-dried poplar leaves, pine shoots and spruce shoots, as well as from a peat matrix. Additionally, the extraction efficiency of Sb and As in fresh and freeze-dried elder leaves and poplar leaves was compared. Total concentrations of Sb and As of aliquots (approximately 220 mg) of the freeze-dried samples were analysed by flow injection hydride generation atomic absorption spectrometry (FI-HG-AAS) after open vessel digestion with adequate mixtures of nitric, sulfuric, hydrochloric, and perchloric acid. Three reference materials GBW 07602 Bush Branches and Leaves, GBW 07604 Poplar Leaves, and SRM 1575 Pine Needles were analysed with every batch of samples to ensure the accuracy and precision of the applied analytical procedures. The use of hydrofluoric acid in the digestion mixture leads to distinctly lower As values (down to 40%) than actual concentrations in the investigated plant materials. Extraction efficiencies were generally low and lower for Sb than for As. Solutions of 0.66 mol L(-1) NaOH liberated highest amounts of Sb with approximately 10% for poplar leaves, and approximately 19% each for pine shoots and spruce shoots. Distinctly higher concentrations of As in NaOH extracts of poplar leaves (22%), pine shoots (32%), and spruce shoots (36%) were quantified. Extraction experiments resulted in yields of 7-9% from fresh elder and poplar leaves, respectively, and 8-13% for freeze-dried samples for Sb. The corresponding values for As were 10-35% for the fresh material and 7-37% for the freeze-dried samples.     

Extraction of antimony and arsenic from fresh and freeze-dried plant samples as determined by HG-AAS

Six extraction media (acetic acid, EDTA, tetrabutylammonium hydroxide, NaOH, MeOH/H₂O, acetonitrile/H₂O) were tested for their ability to extract antimony (Sb) and arsenic (As) from freeze-dried poplar leaves, pine shoots and spruce shoots, as well as from a peat matrix. Additionally, the extraction efficiency of Sb and As in fresh and freeze-dried elder leaves and poplar leaves was compared. Total concentrations of Sb and As of aliquots (～220 mg) of the freeze-dried samples were analysed by flow injection hydride generation atomic absorption spectrometry (FI-HG-AAS) after open vessel digestion with adequate mixtures of nitric, sulfuric, hydrochloric, and perchloric acid. Three reference materials GBW 07602 Bush Branches and Leaves, GBW 07604 Poplar Leaves, and SRM 1575 Pine Needles were analysed with every batch of samples to ensure the accuracy and precision of the applied analytical procedures. The use of hydrofluoric acid in the digestion mixture leads to distinctly lower As values (down to 40%) than actual concentrations in the investigated plant materials. Extraction efficiencies were generally low and lower for Sb than for As. Solutions of 0.66 mol L^–1 NaOH liberated highest amounts of Sb with ～10% for poplar leaves, and ～19% each for pine shoots and spruce shoots. Distinctly higher concentrations of As in NaOH extracts of poplar leaves (22%), pine shoots (32%), and spruce shoots (36%) were quantified. Extraction experiments resulted in yields of 7–9% from fresh elder and poplar leaves, respectively, and 8–13% for freeze-dried samples for Sb. The corresponding values for As were 10–35% for the fresh material and 7–37% for the freeze-dried samples.     

Comparison of extraction techniques of robenidine from poultry feed samples

In this paper, effectiveness of six different commonly applied extraction techniques for the determination of robenidine in poultry feed has been compared. The sample preparation techniques included shaking, Soxhlet, Soxtec, ultrasonically assisted extraction, microwave – assisted extraction and accelerated solvent extraction. Comparison of these techniques was done with respect to the recovery extraction, temperature and time, reproducibility and solvent consumption. Every single extract was subjected to clean – up using aluminium oxide column (Pasteur pipette filled with 1 g of aluminium oxide), from which robenidine was eluted with 10 ml of methanol. The eluate from the clean-up column was collected in a volumetric flask, and finally it was analysed by HPLC–DAD–MS. In general, all extraction techniques were capable of isolating of robenidine from poultry feed, but the recovery obtained using modern extraction techniques was higher than that obtained using conventional techniques.

In particular, accelerated solvent extraction was more superior to other techniques, which highlights the advantages of this sample preparation technique. However, in routine analysis, shaking and ultrasonically assisted extraction is still the preferred method for the solution of robenidine and other coccidiostatics.     

Comparison of supercritical fluid extraction and liquid-liquid extraction for isolation of selected pesticides stored in freeze-dried water samples

Summary The stability of freeze-dried water samples spiked with eight agrochemicals (atrazine, simazine, linuron, carbaryl, propanil, fenitrothion, parathion and fenamiphos) were examined to evaluate their suitability as candidate reference materials for their determination in water samples. In addition, two different extraction procedures, liquid-liquid and supercritical fluid extraction, were compared for the isolation and trace enrichment of target analytes from freeze-dried water samples. Final analytical determinations were by gas chromatography-nitrogen phosphorus detection and electronic impact mass spectrometry, and by liquid chromatography-diode array detection. The whole methodology developed in this paper permitted the determination of pesticides spiked in water at levels varying from 0.03 to 6.9 g L^–1.     

Pretreatment procedures for characterization of arsenic and selenium species in complex samples utilizing coupled techniques with mass spectrometric detection

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.     

Hemimicelle capped functionalized carbon nanotubes-based nanosized solid-phase extraction of arsenic from environmental water samples

The end functionalization of CNTs can introduce oxygen-containing negatively functional groups such as -COOH, -OH, or -CO on their surface site. If cationic surfactant such as cetyltrimethylammonium chloride (CTAC) was added to the functionalized CNTs, then interactions such as hydrophobic and ionic may lead to formation of hemimicelle/admicelle aggregates on the CNTs, a new kind of adsorbents, namely, the hemimicelle capped CMMWCNTs, is obtained. The application of the hemimicelle capped carbon nanotubes-based nanosized solid-phase extraction (SPE) adsorbents in environmental analysis is reported for the first time using arsenic as model target. The effect of adsorption and desorption conditions for arsenic including the amount of surfactant, initial pH of sample solution, the ultrasonic time of sample solution, the amount of electrolyte, flow rate, eluent and its amount were investigated and optimized prior to its determination by atomic fluorescence spectrophotometry (AFS). Arsenic can be quantitatively retained on the hemimicelle capped CMMWCNTs at pH 5-6 from sample volume up to 500 mL and then eluted completely with 2 mol L⁻¹ HNO₃ in the presence of 10 mg L⁻¹ CTAC. The method detection limit for arsenic determination with AFS detection was 2 ng L⁻¹, and the relative standard deviation (RSD, n = 11) was 5.3% at the 0.5 μg L⁻¹ level. The recoveries of arsenic in the spiked environmental water samples ranged from 94% to 104.29% with 500 mL of water sample. The proposed method has been applied successfully to the analysis of arsenic in aqueous environmental samples, which demonstrates the hemimicelle capped CMMWCNTs can be an excellent SPE adsorbents for arsenic pretreatment and enrichment from real water samples.     

Supercritical fluid extraction and gas chromatography analysis of arsenic species from solid matrices

A method in combination with derivatization-supercritical fluid extraction(SFE) and gas chromatography(GC) for the speciation and quantitative determination of dimethylarsinate(DMA), monomethylarsonate(MMA) and inorganic arsenic in solid matrices was investigated. Thioglycolic acid methyl ester(TGM) and thioglycolic acid ethyl ester(TGE) were evaluated as derivatization reagents. The effects of pressure, temperature, flow rate of supercritical CO_2, extraction time, modifier and microemulsion on the efficiency of extraction were systematically investigated. The procedure was applied to the analysis of real soil and sediment samples. Results showed that TGE was more effective for arsenic speciation as a derivatization reagent. Modifying supercritical CO_2 with methanol can greatly improve the extraction efficiency. Further, the addition of microemulsion containing surfactant Triton X-100 can further enhance recoveries of arsenic species. The optimum extraction conditions were 100 ℃, 30 MPa, 10 min static and 25 min dynamic extraction with 5%(v/v) methanol, and surfactant modified supercritical CO_2. Detection limits in solid matrices were 0.15, 0.3 and 1.2 mg/kg for DMA, MMA and inorganic arsenic,respectively. The method was validated by the recovery data. The resulting method was fast, easy to perform and selective in the extraction and detection of various arsenic species in solid matrices.     

Preliminary studies on arsenic species in some environmental samples

Arsenic compounds have been determined in some environmental samples from the German Environmental Specimen Bank (ESB) (marine mussels, freshwater mussel and fish, sea-gull eggs) and certified reference materials (DORM-1, DOLT-1, NBS Oyster Tissue) after separation by open column cation and anion exchange chromatography by two different methods of total arsenic determination in separated fractions (instrumental neutron activation analysis or hydride generation atomic absorption spectrometry). Arsenobetaine has been identified as the major species in all the different materials.     

A systematic study on extraction of total arsenic from down-scaled sample sizes of plant tissues and implications for arsenic species analysis

An easily feasible, species-conserving and inexpensive protocol for the extraction of total arsenic and arsenic species from terrestrial plants was designed and applied to the investigation of accumulation and metabolization of arsenite (As(III)), arsenate (As(V)), monomethylarsonate (MMA(V)), and dimethylarsinate (DMA(V)) by the model plant Tropaeolum majus. In contrast to existing extraction methods hazardous additives and elaborate procedures to enhance the extraction yields were omitted. The proposed protocol is suited to down-scale the sample sizes used for the extractions and to promote a compartmentally resolved analysis of the arsenic distribution within individual leaves, leaf stalks, and stems instead of the conventional extraction of pooled samples. In a two-step extraction, the high extraction efficiencies (85-92%) for arsenic achieved by phosphate buffer from larger amounts (200mg) of homogenized leaf material in a one-step extraction, could be enhanced to 94-100% in a second extraction step. A strong dependence of the arsenic extractability on the type of arsenic species accumulated in the tissue as well as on the type of the tissue (leaf, leaf stalk, stem) was found. For the extraction of 5mm long segments cut from individual leaves without previous homogenization of the plant parts yields between 75 and 93% depending on arsenic species prevailing in the cells were obtained using 1 or 10mM phosphate buffer. The total extraction and analysis protocol was validated using a standard reference material as well as by spiking experiments. The arsenic species analysis by IC/ICPMS revealed a number of nine unidentified metabolites in the plant extracts in addition to the species MMA(V), DMA(V), As(III), and As(V) that were provided to the plants during their growth phase.     

Reactive supercritical fluid extraction and chromatography of arsenic species

Reactive supercritical fluid extraction has been used for the speciation of organic (DMA and MMA) and inorganic (As(III) and As(V)) arsenic compounds in solid samples. Derivatization with thioglycolic acid methylester (TGM) was performed in supercritical carbon dioxide. Different extraction conditions have been tested. The arsenic derivatives have been analyzed by GC. A capillary-SFC method was evaluated for the analysis of the TGM derivatives and compared with GC.Dedicated to Professor Dr. Dieter Klockow on the occasion of his 60th birthday     

Preliminary evaluation of new polymer matrix for solid-phase extraction of nonylphenol from water samples

Molecularly imprinted (MIP) and blank polymers with affinity for nonylphenol were designed using computational modelling. Chromatographic tests demonstrated higher affinity of imprinted polymers towards the template nonylphenol as compared with blank polymers. The performance of both polymers in solid-phase extraction was however very similar. Both blank and imprinted polymers appeared to be suitable for the removal and pre-concentration of nonylphenol from contaminated water samples with 99% efficiency of the recovery. The commercial resins PH(EC) (Biotage) and C18 (Varian) tested in the same conditions used for comparative purposes had recovery rate <84%. The polymer capacity for nonylphenol was 231 mg g⁻¹ for blank and 228 mg g⁻¹ for MIP. The synthesised materials can have significance for sample pre-concentration and environmental analysis of this class of compounds.     

Determination of arsenic species in marine samples by HPLC-ICP-MS.

Arsenic speciation analysis in marine samples was performed using high performance liquid chromatography (HPLC) with ICP-MS detection. The separation of eight arsenic species viz. arsenite (As(III)), monomethylarsonic acid (MMA), dimethylarsinic acid (DMA), arsenate (As(V)), arsenobetaine, trimethylarsine oxide (TMAO), arsenocholine and tetramethylarsonium ion (TeMAs) was achieved on a Shiseido Capcell Pak C18 column by using an isocratic eluent (pH 3.0), in which condition As(III) and MMA were co-eluted. The entire separation was accomplished in 15 min. The detection limits for 8 arsenic species by HPLC/ICP-MS were in the range of 0.02 - 0.10 microg L(-1) based on 3sigma of blank response (n=9). The precision was calculated to be 3.1-7.3% (RSD) for all eight species. The method then successfully applied to several marine samples e.g., oyster, scallop, fish, and shrimps. For the extraction of arsenic species from seafood products, the low power microwave digestion was employed. The extraction efficiency was in the range of 52.9 - 112.3%. Total arsenic concentrations were analyzed by using the microwave acid digestion. The total arsenics in the certified reference materials (DORM-2 and TORT-2) were analyzed and agreed with the certified values. The concentrations of arsenics in marine samples were in the range 6.6 - 35.1 microg g(-1).     

Determination of ultra trace arsenic species in water samples by hydride generation atomic absorption spectrometry after cloud point extraction

Cloud point extraction (CPE) methodology has successfully been employed for the preconcentration of ultra-trace arsenic species in aqueous samples prior to hydride generation atomic absorption spectrometry (HGAAS). As(III) has formed an ion-pairing complex with Pyronine B in presence of sodium dodecyl sulfate (SDS) at pH 10.0 and extracted into the non-ionic surfactant, polyethylene glycol tert-octylphenyl ether (Triton X-114). After phase separation, the surfactant-rich phase was diluted with 2 mL of 1 M HCl and 0.5 mL of 3.0% (w/v) Antifoam A. Under the optimized conditions, a preconcentration factor of 60 and a detection limit of 0.008 μg L⁻¹ with a correlation coefficient of 0.9918 was obtained with a calibration curve in the range of 0.03–4.00 μg L⁻¹. The proposed preconcentration procedure was successfully applied to the determination of As(III) ions in certified standard water samples (TMDA-53.3 and NIST 1643e, a low level fortified standard for trace elements) and some real samples including natural drinking water and tap water samples.     

Assessment of total arsenic and arsenic species stability in alga samples and their aqueous extracts

In order to achieve reliable information on speciation analysis, it is necessary to assess previously the species stability in the sample to analyse. Furthermore, in those cases where the sample treatment for species extraction is time-consuming, an assessment of the species integrity in the extracts is of paramount importance. Thus, the present paper reports total arsenic and arsenic species stability in alga samples (Sargassum fulvellum and Hizikia fusiformis), as well as in their aqueous extracts, which were stored in amber glass and polystyrene containers at different temperatures. Total arsenic determination was carried out by inductively coupled plasma atomic emission spectroscopy (ICP-AES), after sample acid digestion in a microwave oven, while arsenic speciation was conducted by anion exchange high performance liquid chromatography on-line coupled to ICP-AES, with and without sample introduction by hydride generation (HPLC-ICP-AES and HPLC-HG-ICP-AES), after aqueous microwave-assisted extraction. The results obtained for solid alga samples showed that total arsenic (for Hijiki alga) and arsenic species present (As(V) for Hijiki and NIES No. 9 Sargasso) are stable for at least 12 months when samples are stored in polystyrene containers at +20 degrees C. On the other hand, a different behaviour was observed in the stability of total arsenic and As(V) species in aqueous extracts for both samples, being the best storage conditions for Sargasso extracts a temperature of -18 degrees C and polystyrene containers, under which they are stable for at least 15 days, while Hijiki extracts must be stored in polystyrene containers at +4 degrees C in order to ensure the stability for 10 days.     

Separation of arsenic species by capillary electrophoresis with sample-stacking techniques

A simple capillary zone electrophoresis procedure was developed for the separation of arsenic species (AsO(2)(2-), AsO(4)(2-), and dimethylarsinic acid, DMA). Both counter-electroosmotic and co-electroosmotic (EOF) modes were investigated for the separation of arsenic species with direct UV detection at 185 nm using 20 mmol L(-1) sodium phosphate as the electrolyte. The separation selectivity mainly depends on the separation modes and electrolyte pH. Inorganic anions (Cl(-), NO(2)(-), NO(3)(-) and SO(4)(2-)) presented in real samples did not interfere with arsenic speciation in either separation mode. To improve the detection limits, sample-stacking techniques, including large-volume sample stacking (LVSS) and field-amplified sample injection (FASI), were investigated for the preconcentration of As species in co-CZE mode. Less than 1 micromol L(-1) of detection limits for As species were achieved using FASI. The proposed method was demonstrated for the separation and detection of As species in water.     

Cathodic stripping voltammetric analysis of arsenic species in environmental water samples

A simple, fast and sensitive arsenic speciation method has been developed for environmental water analysis by using differential pulse cathodic stripping voltammetry (CSV) performed on a hanging mercury drop electrode (HMDE). Electroactive As(III) is determined by direct CSV analysis. As(V) is converted to As(III) species first and is subsequently quantified by the concentration difference between total inorganic arsenic and As(III). A new batch-mode As(V) reduction procedure by l-cysteine was developed in this study. The optimized parameters for quantitative As(V) reduction include treatment with 20 mM l-cysteine and 0.03 M HCl for 6 min at 70 °C. Organic arsenic, including monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA), can be decomposed to As(V) through UV photooxidation with peroxydisulfate and quantified through subtracting total inorganic arsenic from the total arsenic. At optimum condition, the detection limits for As(III), As(V), and organic arsenic (MMA and DMA) were all 0.3 μg/L and with the linear range from 2.5 to 190 μg/L. Interference from ions common in natural water (Mn, Fe, Cr, Cd, Ca, Zn, Mg, and phosphate) is minimal. The method was validated by analyzing the NIST 1640 natural water standard reference material and by recovery tests on spiked tap water and groundwater. When applied to on-site analysis of sediment pore water and stream water, the CSV results agree well with those obtained by inductively coupled plasma–mass spectrometry (ICP–MS) and graphite furnace atomic absorption spectrometry (GFAAS) methods.     

A comparison of sequential extraction techniques for determining arsenic fractionation in synthetic mineral mixtures

The sequential extraction methods according to Tessier et al. [1], Borovec et al. [2], Zhang and Moore [3] and Hall et al. [4] have been tested for their suitability for arsenic fractionation in samples of artificially prepared mineral mixtures. Mixtures containing different amounts of As-containing phases were prepared so that their compositions corresponded to weathering products on As-bearing ore deposits. A comparison of different procedures on simple mineral mixtures containing calcium arsenate (CaHAsO₄·H₂O), As-bearing goethite (FeOOH) and arsenopyrite (FeAsS) showed that only the results of the Hall method satisfactorily correspond to the expected arsenic distribution. A detailed verification of the Hall method was subsequently carried out on most complex synthetic mineral mixtures with varying amounts of As-containing kaolinite and carbonate, calcium arsenate, As-bearing goethite and arsenopyrite. The results confirm that the Hall method cannot be fully employed for an accurate As speciation but may be applied for a route identification of As distribution between "labile", "medium-labile" and "residual" forms in heavily polluted soils.     

Direct measurement of lipid-soluble arsenic species in biological samples with HPLC-ICPMS

Lipid-soluble arsenicals (arsenolipids) occur in a wide range of biological samples where they may play a key role in the biosynthesis of organoarsenic compounds from inorganic arsenic. The study of these compounds has been hindered, however, by the lack of a suitable analytical technique able to separate and measure the various lipid species. As a source of arsenolipids, we used 10 crude fish oils from various regions of the world. Total arsenic analyses on the fish oils, performed with ICPMS following acid digestion with microwave-assisted heating, gave concentrations from 4.3 to 10.5 mg As kg(-1). All of the arsenic was soluble in non-polar solvents such as hexane. Analysis of the fish oils for arsenolipids was performed by normal phase HPLC-ICPMS with various mixtures of organic solvents as mobile phases. Inherent problems of instability associated with the introduction of organic solvents to the plasma were overcome by the use of reduced column flow, a chilled spray chamber, and the addition of oxygen directly to the plasma. All ten fish oils appeared to contain the same 4-6 major arsenolipids, but in varying amounts depending on the origin of the fish. Further chromatography with both normal phase and reversed-phase conditions on some of the oils indicated the presence of many more minor arsenolipids. Quantification was achieved by external calibration against triphenylarsine oxide or triphenylarsine sulfide, and the sum of species following HPLC of the oils matched well the total arsenic results (92-107%). The method was applied to samples of food supplements (fish oil capsules) and a packaged food product (cod liver) whereby arsenolipids were measured and found to be significant arsenic constituents. This study represents the first attempt to directly measure intact arsenolipids and, with appropriate sample preparation, may be suitable for quantitative measurement of these arsenicals in a range of biological samples, including foodstuffs.