Zdenka Šlejkovec

Arsenobetaine and other arsenic compounds in the National Research Council of Canada Certified Reference Materials DORM 1 and DORM 2

A silica-based cation-exchange column was used to determine the arsenic compounds in the National Research Council of Canada (NRCC) CRMs DORM 1 and DORM 2 (Dogfish Muscle). With a 20 mM aqueous pyridine mobile phase at pH 3.0, the concentration of arsenobetaine was only 10.7 mg kg–1 As in the extract of DORM 1. When the same extract was chromatographed on an anion-exchange column, 15.9±0.3 mg kg–1 As (arsenobetaine) were found. The calibration for arsenobetaine was linear from 0.5 µg dm–1 As to 10 mg dm–3 As. When the extracts were diluted with water the cation-exchange results approached the anion-exchange results. The multi-element capabilities of ICP-MS allowed the simultaneous monitoring of arsenic and alkali metals. Sodium and potassium were found to co-elute with arsenobetaine. When aqueous solutions of arsenobetaine with 250 mg dm–3 Na were chromatographed, the signal obtained for arsenobetaine was only 60% of the signal without sodium in the solution. When the pH of the 20 mM aqueous pyridine mobile phase was lowered, the alkali metals were separated from arsenobetaine and the results obtained from cation-exchange chromatography were not significantly different from the anion-exchange results. Because DORM 1 is no longer available, the arsenic compounds in DORM 2 were determined. No significant difference was found for the concentration of arsenobetaine (15.6±0.7 mg kg–1 As for DORM 1; 16.0±0.7 mg kg–1 As for DORM 2). The concentrations of the minor arsenic compounds (dimethylarsinic acid, arsenocholine, the tetramethylarsonium cation and an unknown arsenic compound) in DORM 2 were only half the concentrations in DORM 1.

Arsenic Compounds in Higher Fungi

In 50 mushroom species (56 samples) from Slovenia, Switzerland, Brazil, Sweden, The Netherlands and USA, total arsenic was determined by radiochemical neutron activation analysis (RNAA). Arsenic concentrations ranged from 0.1 to 30u2009μgu2009g−1 (dry mass). Arsenic compounds were determined in methanol extracts from the mushrooms by HPLC–ICP–MS. The aim of the study was not only to quantify arsenic compounds in mushrooms but also to uncover trends relating the methylating ability of a mushroom to its taxonomic or evolutionary status. n n n nThe main arsenic compound found in many mushrooms (various puffballs, Agaricales and Aphyllophorales) was arsenobetaine. Arsenate [As(V)] was the main arsenic species in Laccaria fraterna and Entoloma rhodopolium and arsenite [As(III)] in Tricholoma sulphureum. A mixture of arsenite and arsenate was present in Amanita caesarea. Dimethylarsinic acid (DMA) and methylarsonic acid were present in many mushrooms, but generally as minor components. In Laccaria laccata, Leucocoprinus badhamii and Volvariella volvacea, DMA was the major metabolite. Arsenocholine (AC) and the tetramethylarsonium ion were present in a few species, generally at low concentrations, except for Sparassis crispa, in which AC was the main compound. Tri- methylarsine oxide was not found in any of the mushrooms. In some species small amounts of unknown compounds were also present. The possible taxonomic significance of the metabolite patterns and the predominance of arsenobetaine in more advanced fungal types are discussed.

Determination of arsenic compounds in reference materials by HPLC-(UV)-HG-AFS

Arsenic compounds were determined in six reference materials of biological origin. None of them has yet been certified for arsenic compounds but some are in the process of certification; for most of these reference materials indicative literature values are available. Eight commonly used arsenic standards were used for quantification using a recently developed hyphenated speciation system comprising high performance liquid chromatography (HPLC) and atomic fluorescence spectrometry (AFS), interfaced via a UV-photoreactor and a hydride generation (HG) unit. Absolute detection limits were ca. 0.2 and 0.4 ng As for separation on anion and cation exchange columns, respectively. Our results agree well with indicative literature values which were generated by different authors using various separation and detection methods. The HPLC-(UV)-HG-AFS system validated in this way is suitable for quantification of eight arsenic compounds. Moreover, the system is capable of separation of at least six more compounds in the mentioned reference materials, of which two could be attributed to arsenosugars (OH and phosphodiester form) but due to the lack of standards, quantification was not possible. For accurate and extensive speciation analysis the availability of certified reference materials and standards for arsenic compounds should be promoted.

Arsenic speciation patterns in freshwater fish

Muscle of 16 freshwater fish (9 different species belonging to 4 different families) was analysed for arsenic species using HPLC separation (anion and cation exchange) followed by on-line UV-decomposition, hydride generation and AFS detection. The main arsenic compounds found in the extracts were arsenobetaine (AsB), which accounted for 92-100% of extractable arsenic in species of salmonids (Salmo marmoratus, Oncorhynchus mykiss, Salmo trutta m. fario), and dimethylarsinic acid (DMAA), which accounted for 75% of extractable arsenic in burbot (Lota lota). AsB was also found in lower concentrations in almost all other fish species analysed (Silurus glanis, L. lota, Barbus barbus, Rutilus pigus virgo, Chondrostoma nasus). Arsenite (As(III)) and trimethylarsine oxide (TMAO) were detected in low concentrations in some representatives of Cyprinidae only (R. pigus virgo, C. nasus). Except in salmonids, an unknown cationic compound was present in most of the samples in relatively low concentrations. Cluster analysis of the generated data seems to indicate that there is a correlation between fish family and the arsenic speciation pattern. This is especially clear for the salmonids which show a completely separate cluster and thus a very distinct arsenic speciation pattern.

Ion-exchange separation of eight arsenic compounds by high-performance liquid chromatography–UV decomposition–hydride generation–atomic fluorescence spectrometry and stability tests for food treatment procedures

A novel separation for cationic arsenic compounds on a polymer-based cation-exchange column was developed using an ion-pairing reagent (3-carboxy-4-hydroxybenzenesulphonic acid) in the mobile phase. An existing anion-exchange separation was used for anionic arsenic compounds. Combining both separation techniques, eight environmentally important arsenic compounds can be determined using on-line decomposition in a UV reactor prior to hydride generation (HG) and atomic fluorescence spectrometry (AFS). The method was applied for testing the stability of arsenic compounds (in aqueous media) related to food treatment procedures. Boiling and microwave treatment gave no degradation, whereas gamma-irradiation and dry heating resulted in partial decomposition of several arsenic compounds. No health hazards are to be expected when these data are extrapolated to commercial or domestic food treatment procedures.

Preliminary study on the determination of selenium compounds in some selenium-accumulating mushrooms

Using various chromatographic techniques (size exclusion, anion exchange, and cation exchange) combined with several detectors (neutron activation analysis and atomic fluorescence spectrometry), an attempt was made to characterize selenium compounds in some edible, selenium-accumulating mushrooms (Albatrellus pes-caprae and Boletus edulis).The mushrooms contained mostly low-molecular-weight (6 kDa) selenium compounds. After proteolysis, only a small fraction of the extractable selenium could be identified as selenite (3.0–9.2%, Albatrellus pes-caprae), selenocystine (minor, Albatrellus pes-caprae; 7.5%, Boletus edulis), or selenomethionine (1.0%, Boletus edulis), leaving the form of the bulk still to be elucidated.

Underestimation of the total arsenic concentration by hydride generation techniques as a consequence of the incomplete mineralization of arsenobetaine in acid digestion procedures

Abstract Individual arsenobetaine (AsB) decomposition products obtained with five different acid digestion procedures (nitric acid/200°C/10 or 30xa0min, nitric acid/sulphuric acid/300°C/10 or 30xa0min and nitric acid/sulphuric acid/hydrogen peroxide/300°C/30xa0min) were determined by high-performance liquid chromatography-(ultraviolet digestion)-hydride generation atomic fluorescence spectrometry (HPLC-(UV)-HGAFS). It was found that AsB was converted to mainly inorganic arsenic, trimethylarsine oxide (TMAO) and dimethylarsinic acid (DMAA), depending on acid digestion procedure; the “stronger” the digestion procedure the more mineralization occurred. The sensitivity of the flow injection-hydride generation atomic fluorescence spectrometry (FI-HGAFS) for each of the decomposition products was also measured which allowed us to calculate the FI-HGAFS response, and thus the total arsenic concentration using arsenate as a quantification standard. Total arsenic concentrations calculated were between 56 and 100% of the initially present AsB, depending on acid digestion procedure. This was completely in accordance with total arsenic concentrations measured so that underestimation of the total arsenic concentration in AsB digests measured by the FI-HGAFS can be fully explained in terms of incomplete mineralization and the lower response of the partly degraded products.

Differential role of cathepsins B and L in autophagy-associated cell death induced by arsenic trioxide in U87 human glioblastoma cells.

Abstract Arsenic trioxide (arsenite) was the first chemotherapeutic drug to be described and is now being rediscovered in cancer treatment, including glioblastoma multiforme. Arsenite toxicity triggers autophagy in cancer cells, although final stages of the process involve executive caspases, suggesting an interplay between autophagic and apoptotic pathways that awaits to be explained at a molecular level. We evaluated the contribution of the lysosomal cathepsins (Cat) L and B, which are upregulated in glioblastomas, in the mechanism of arsenite toxicity in human glioblastoma cells. Arsenite treatment induced autophagosome formation and permeabilization of mitochondria, followed by caspase 3/7-mediated apoptosis. The autophagy inhibitor 3-methyladenine protected from arsenite toxicity, whereas bafilomycin A1 did not. Furthermore, arsenite significantly decreased CatB levels and selectively inhibited its cellular and recombinant protein activity, while not affecting CatL. However, downregulation of CatL greatly enhanced apoptosis by arsenite. Our results show that arsenite toxicity involves a complex interplay between autophagy and apoptosis in human glioblastoma cells and is associated with inhibition of CatB, and that this toxicity is highly exacerbated by simultaneous CatL inhibition. The latter points to a synergy that could be used in clinical treatment to lower the therapeutic dose, thus avoiding the toxic side effects of arsenite in glioblastoma management.

Extraction of arsenic compounds from lichens

Different extraction procedures were applied to improve the extraction efficiency of arsenic compounds from lichens. Two lichen species were chosen from an arsenic-contaminated environment: epiphytic Hypogymnia physodes (L.) Nyl. and terricolous Cladonia rei Schaer. Samples were extracted with water at temperatures of 20, 60 and 90 degrees C, using mixtures of methanol/water (9:1, 1:1 and 1:9), Tris buffer and acetone and the extracts speciated. Water and Tris buffer showed the best extraction efficiency of all extractants used; however, the extraction efficiency was still less than 23%. Since a major fraction of arsenic appeared to be associated with trapped soil particles, a sequential extraction procedure originally designed for soils (extraction steps: (1) 0.05 mol l(-1) (NH(4))(2)SO(4); (2) 0.05 mol l(-1) (NH)(4)H(2)PO(4); (3) 0.2 mol l(-1) NH(4)-oxalate buffer, pH 3.25; (4) mixture of 0.2 mol l(-1) NH(4)-oxalate buffer and 0.1 mol l(-1) ascorbic acid, pH 3.25; (5) 0.5 mol l(-1) KOH) was applied and found to remove 45% of the total arsenic from H. physodes and 83% from C. rei. The lipid-soluble fraction of arsenic was estimated by k(0)-INAA analysis of diethylether extracts and was found to be negligible. An HPLC-UV-HGAFS system was used to determine the arsenic compounds extracted. In both lichen species, arsenous acid, arsenic acid, monomethylarsonic acid, dimethylarsinic acid, arsenobetaine, trimethylarsine oxide and glycerol-ribose were detected. In addition, phosphate-ribose was found in H. physodes.

A mathematical model for optimisation of flow injection-hydride generation atomic fluorescence spectrometry

Abstract A mathematical model was developed to predict signal attentuation and signal broadening in flow injection-hydride generation atomic fluorescence spectrometry owing to gas–liquid separation in order to optimise the performance with regard to sensitivity and throughput. Experimental data for arsenic measurements matched well with the model so that theoretical optimisation of various mutually dependent instrumental parameters (sample loop volume, carrier solution flow rate, GLS headspace and purge gas flow rate) was possible. A minimal GLS headspace and a maximal carrier solution flow rate resulted in the highest sensitivity and throughput for the system set-up under study.