Doris Kuehnelt

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 terrestrial organisms. IV. Green plants and lichens from an old arsenic smelter site in Austria

Two lichens and 12 green plants growing at a former arsenic roasting facility in Austria were analyzed for total arsenic by ICP–MS, and for 12 arsenic compounds (arsenous acid, arsenic acid, dimethylarsinic acid, methylarsonic acid, arsenobetaine, arsenocholine, trimethylarsine oxide, the tetramethylarsonium cation and four arsenoriboses) by HPLC–ICP–MS. Total arsenic concentrations were in the range of 0.27 mg As (kg dry mass)−1 (Vaccinium vitis idaea) to 8.45 mg As (kg dry mass)−1 (Equisetum pratense). Arsenic compounds were extracted with two different extractants [water or methanol/water (9:1)]. Extraction yields achieved with water [7% (Alectoria ochroleuca) to 71% (Equisetum pratense)] were higher than those with methanol/water (9:1) [4% (Alectoria ochroleuca) to 22% (Deschampsia cespitosa)]. The differences were caused mainly by better extraction of inorganic arsenic (green plants) and an arsenoribose (lichens) by water. Inorganic arsenic was detected in all extracts. Dimethylarsinic acid was identified in nine green plants. One of the lichens (Alectoria ochroleuca) contained traces of methylarsonic acid, and this compound was also detected in nine of the green plants. Arsenobetaine was a major arsenic compound in extracts of the lichens, but except for traces in the grass Deschampsia cespitosa, it was not detected in the green plants. In contrast to arsenobetaine, trimethylarsine oxide was found in all samples. The tetramethylarsonium cation was identified in the lichen Alectoria ochroleuca and in four green plants. With the exception of the needles of the tree Larix decidua the arsenoribose (2′R)-dimethyl[1-O-(2′,3′-dihydroxypropyl)-5-deoxy-β-D-ribofuranos-5-yl]arsine oxide was identified at the low μg kg−1 level or as a trace in all plants investigated. In the lichens an unknown arsenic compound, which did not match any of the standard compounds available, was also detected. Arsenocholine and three of the arsenoriboses were not detected in the samples. Copyright

Arsenic speciation in freshwater organisms from the river Danube in Hungary

Total arsenic and arsenic species were determined in a range of freshwater samples (sediment, water, algae, plants, sponge, mussels, frog and fish species), collected in June 2004 from the river Danube in Hungary. Total arsenic concentrations were measured by ICPMS and arsenic species were measured in aqueous extracts of the samples by ion-exchange HPLC-ICPMS. In order to separately determine the efficiency of the extraction method and the column recovery, total arsenic concentrations in the extracts were obtained in three ways: (i) ICPMS determination after acid digestion; (ii) flow injection analysis performed directly on the extract; (iii) the sum of arsenic species eluting from the HPLC column. Extraction efficiencies were low (range 10-64%, mean 36%), but column recovery was acceptable (generally >80%) except for the fish samples, where substantial, currently unexplained, losses were observed. The dominating arsenic species in the extracts of freshwater algae were arsenosugars, whereas arsenate [As(V)] was present only as a minor constituent. On the other hand, plant extracts contained only inorganic arsenic, except for two samples which contained trace amounts of dimethylarsinate (DMA) and the tetramethylarsonium cation (TETRA). The oxo-arsenosugar-phosphate (ca. 35% of extractable arsenic) and the oxo-arsenosugar-glycerol (ca. 20%) as well as their thio-analogues (1-10%) were found in the mussel extracts, while arsenobetaine (AB) was present as a minor species only. In general, fish extracts contained only traces of arsenobetaine, and the oxo-arsenosugar-phosphate was the major arsenic compound. In addition, samples of white bream contained thio-arsenosugar-phosphate; this is the first report of a thio-arsenical in a fish sample. The frog presented an interesting arsenic speciation pattern because in addition to the major species, arsenite [As(III)] (30%) and the tetramethylarsonium cation (35%), all three intermediate methylation products, methylarsonate (MA), dimethylarsinate and trimethylarsine oxide (TMAO), and arsenate were also present. Collectively, the data indicate that arsenobetaine, the major arsenical in marine animals, is virtually absent in the freshwater animals investigated, and this represents the major difference in arsenic speciation between the two groups of organisms.

Thio arsenosugars identified as natural constituents of mussels by liquid chromatography-mass spectrometry

Two novel thio arsenosugars have been identified by liquid chromatography-mass spectrometry as significant arsenic constituents in samples of mussels.

Arsenic Compounds in Terrestrial Organisms I: Collybia maculata, Collybia butyracea and Amanita muscaria from Arsenic Smelter Sites in Austria

Three mushroom species from two old arsenic smelter sites in Austria were analyzed for arsenic compounds. The total arsenic concentrations were determined by ICP–MS. Collybia maculata contained 30.0 mg, Collybia butyracea 10.9 mg and Amanita muscaria 21.9 mg As kg−1 dry mass. The arsenic compounds extracted with methanol/water (9:1) from the dried mushroom powders were separated by HPLC on anion-exchange and reversed-phase columns and detected by ICP-MS using a hydraulic high-pressure nebulizer. In Collybia maculata almost all arsenic is present as arsenobetaine. Collybia butyracea contained mainly arsenobetaine (8.8 mg As kg−1 dry mass) and dimethylarsinic acid (1.9 mg As kg−1). Amanita muscaria contained arsenobetaine (15.1 mg As kg−1), traces of arsenite, dimethylarsinic acid and arsenate, and surprisingly arsenocholine (2.6 mg As kg−1) and a tetramethylarsonium salt (0.8 mg As kg−1).

Bacterial degradation of arsenobetaine via dimethylarsinoylacetate.

Microorganisms from Mytilus edulis (marine mussel) degraded arsenobetaine, with the formation of trimethylarsine oxide, dimethylarsinate and methylarsonate. Four bacterial isolates from these mixed-cultures were shown by HPLC/hydride generation-atomic fluorescence spectroscopy (HPLC/HG-AFS) analysis to degrade arsenobetaine to dimethylarsinate in pure culture; there was no evidence of trimethylarsine oxide formation. Two of the isolates ( Paenibacillus sp. strain 13943 and Pseudomonas sp. strain 13944) were shown by HPLC/inductively coupled plasma-mass spectrometry (HPLC/ICPMS) analysis to degrade arsenobetaine by initial cleavage of a methyl-arsenic bond to form dimethylarsinoylacetate, with subsequent cleavage of the carboxymethyl-arsenic bond to yield dimethylarsinate. Arsenobetaine biodegradation by pure cultures was biphasic, with dimethylarsinoylacetate accumulating in culture supernatants during the culture growth phase and its removal accompanying dimethylarsinate formation during a carbon-limited stationary phase. The Paenibacillus sp. also converted exogenously supplied dimethylarsinoylacetate to dimethylarsinate only under carbon-limited conditions. Lysed-cell extracts of the Paenibacillus sp. showed constitutive expression of enzyme(s) capable of arsenobetaine degradation through methyl-arsenic and carboxymethyl-arsenic bond cleavage. The work establishes the capability of particular bacteria to cleave both types of arsenic-carbon bonds of arsenobetaine and demonstrates that mixed-community functioning is not an obligate requirement for arsenobetaine biodegradation.

Retention behavior of inorganic and organic selenium compounds on a silica-based strong-cation-exchange column with an inductively coupled plasma mass spectrometer as selenium-specific detector

Abstract The retention behavior of eight selenium compounds (selenous acid, selenic acid, selenocystine, selenohomocystine, selenomethionine, selenoethionine, trimethylselenonium iodide, and dimethyl(3-amino-3-carboxy-1-propyl)selenonium iodide) with aqueous solutions of pyridine (20 mmol/l) in the pH range 2.0–5.7 on a Supelcosil LC-SCX cation-exchange column was investigated. An inductively coupled plasma mass spectrometer was employed as the selenium-specific detector. To increase the nebulization efficiency, the Meinhard concentric glass nebulizer was replaced by a hydraulic high-pressure nebulizer. At pH 5.0, seven selenium compounds could be separated within 400 s, but selenohomocystine and selenomethionine had the same retention time. Selenomethionine can be separated from selenohomocystine with an aqueous solution of pyridine (20 mmol/l) adjusted with formic acid to pH 2.0. At 1 ng Se ml−1, the relative standard deviations (n=5) of the signal area for the eight selenium compounds ranged from 7 to 11%, and at 50 ng Se ml−1 from 0.6 to 2.6%.

Arsenic Compounds in Terrestrial Organisms II: Arsenocholine in the Mushroom Amanita muscaria

Arsenic compounds were identified and quantified in the mushroom Amanita muscaria, collected close to a facility that had roasted arsenic ores. The powdered dried mushrooms were extracted with methanol/water (9:1), the extracts were concentrated and the concentrates were dissolved in water. The resulting solutions were chromatographed on anion-exchange, cation-exchange and reversed- phase columns. Arsenic was detected on-line with an ICP–MS detector equipped with a hydraulic high-pressure nebulizer. Arsenite, arsenate, dimethylarsinic acid and the tetramethylarsonium cation were minor arsenic compounds (∼2% each of the total 22 mg kg−1 dry mass), and arsenobetaine, arsenocholine (∼15% each) and several unidentified arsenic compounds (∼60%) were the major arsenic compounds in Amanita muscaria. The presence of arsenocholine (detected for the first time in a terrestrial sample) was ascertained by matching retention times in the anion-exchange, cation- exchange and reversed-phase chromatograms with the retention time of synthetic arsenocholine bromide and chromatographing extracts spiked with arsenocholine bromide.

An HPLC/ICPMS study of the stability of selenosugars in human urine: implications for quantification, sample handling, and storage

We report a study with HPLC/ICPMS on the long-term stability of the major selenium metabolite in human urine, namely methyl 2-acetamido-2-deoxy-1-seleno-β-D-galactopyranoside (selenosugar 1). Three separate experiments were performed of 4–28 weeks duration and incorporating various storage conditions: room temperature, 4 °C, −20 °C, −80 °C, lyophilisation, deoxygenation, or addition of a bactericide (NaN3). Triplicate samples of urine or water, spiked with selenosugar 1 at 200 μg Se L−1, were processed in each case. Selenosugar 1 was stable in water under all investigated conditions. For the urine samples, no significant degradation (<2%) was observed after 17 weeks frozen storage at −80 °C, or after lyophilisation and frozen storage at −20 °C, whereas small quantities of degradation products (ca. 3%) were recorded for frozen storage of wet samples at −20 °C. At 4 °C, the selenosugar was essentially unchanged after storage for up to 2 weeks, but clear losses were observed thereafter ranging up to 75% loss after 28 weeks. Several decomposition products were detected by HPLC/ICPMS, one of which was identified as dimethyl diselenide. Although present as only a trace constituent in the urine, dimethyl diselenide was recorded as a large HPLC peak, presumably because of a marked vapour enhancement effect due to more efficient transfer of volatile analytes to the plasma. In addition, total Se analyses revealed that Se was lost from the solutions during storage/handling, presumably as volatile species. Qualitative analysis of volatile species using head space sampling with solid phase microextraction followed by GC/MIP-AES and GC/MS revealed the presence of dimethyl selenide and dimethyl diselenide, based on comparison with standard compounds, and indicated the presence of dimethyl selenylsulfide based on comparison with literature data. The stability of selenosugar 1 and its isomer methyl 2-acetamido-2-deoxy-1-seleno-β-D-glucopyranoside (selenosugar 2), which occurs as a minor species in urine, was also investigated under room temperature storage in the presence and absence of light. Although both species were moderately stable when stored in the dark, their degradation was rapid in the light with clear losses recorded within three days. The work indicates that urine samples should be cooled immediately after collection, and that they may be stored at 4 °C (often the easiest way) for up to 2 weeks before analysis with no appreciable loss of selenosugar. For longer-term storage, urine samples should be kept at −80 °C or, when such facilities are not available, at −20 °C after lyophilisation. The study has also revealed potential quantification problems in Se speciation analysis resulting from different responses for Se species during ICPMS analysis.

Concurrent quantitative HPLC-mass spectrometry profiling of small selenium species in human serum and urine after ingestion of selenium supplements.

Selenium metabolic patterns in the human body originating from five distinct selenium dietary sources, selenate, selenite, selenomethionine (SeMet), methylselenocysteine (MeSeCys) and selenized yeast, were investigated by performing concurrent HPLC-mass spectrometric analysis of human serum and urine. Total selenium and selenium species time profiles were generated by sampling and analyzing serum and urine from volunteers treated with selenium supplements, up to 5 and 24h following ingestion, respectively. We found that an increase in total serum selenium levels, accompanied by elevated selenium urinary excretion, was the common pattern for all treatments, except for that of selenite supplementation. Selenosugar 1 was a universal serum metabolite in all treatments, indicating that ingested selenium is favorably metabolized to the sugar. Except for selenite and selenized yeast ingestion, these patterns were reflected in the urine time series of the different treatments. Selenosugar 1 was the major selenium species present in urine in all treatments except for the selenate treatment, accounting for about 80% of the identified excreted species within 24h of ingestion. Furthermore, the urinary metabolite trimethylselenonium ion (TMSe) was detected for the first time in human background serum by using HPLC coupled to elemental and molecular mass spectrometry. The concurrent monitoring of non-protein selenium species in both body fluids provides the relation between bioavailability and excretion of the individual ingested species and of their metabolic products, while the combined use of elemental and molecular mass spectrometry enables the accurate quantitation of structurally confirmed species. This successfully applied approach is anticipated to be a useful tool for more extensive future studies into human selenium metabolism.