An evaluation of M2+ interference correction approaches associated with As and Se in ICP-MS using a multi-day dataset along with ICP-MS/MS/HR-ICP-MS based analysis and hierarchical modeling as a means of assessing bias in fortified drinking waters and single component matrices.

Three 1 2 mass oriented rare earth element (REE) M2+ correction approaches (fixed factor, a dual internal standard, and an in-sample) are evaluated for use in an ICP-MS environmental method update. The multi-variant-based evaluation includes analyzing the same 19 REE-fortified matrices on eight different days over a two-month period using two instrument tunes. These REE-fortified matrices were also analyzed using HR-ICP-MS and ICP-MS/MS to estimate the reference value for use in the principal component analysis (PCA) and hierarchical modeling evaluation. A fixed factor is unable to compensate for matrix and mass dependent drift and because of this it generates the largest across matrix, tune, and day 95th percent confidence bounds for the REE corrections on both As (1.1 ppb) and Se (23 ppb) using samples fortified with 100 ppb Nd, Sm & Gd. The PCA analysis indicated that M2+ ions cluster together across matrix, tune and day better than M1+ and these tighter correlations are reflected in reduced 95th percentile confidence bounds for dual M2+ internal standards (M2+; As = 0.3 ppb; Se = 5.4 ppb; n = 704) relative to M1+ internal standards (M1+; As = 0.6 ppb; Se = 12.0 ppb; n = 1056). The use of an in-sample M2+ correction produced comparable 95th percent confidence bounds (As = 0.2 ppb; Se = 3.4 ppb; n = 352) relative to the M2+ internal standard approaches. Finally, the hierarchical modeling indicated M2+ ions as internal standards tend to minimize the across day variability induced by cone changes and the daily reoccurring matrix shifts in the M2+/M1+ ratio associated with 250 ppm matrices of Na, Ca, and Mg. This internal standard driven reduction in variability can be beneficial in compliance monitoring methods.

Analytical considerations associated with implementing M2+ correction factors to address false positives on As and Se within U.S. EPA method 200.8.

Rare earth elements (REE) can produce M2+ ions in ICP-MS and 150Nd2+, 150Sm2+, and 156Gd2+ can produce false positives on 75As and 78Se. Alternative instrumental tuning conditions, that utilize lower He flows within the collision cell, reduce these false positives by a factor of 2 (to 0.8 ppb As and 19 ppb Se in solutions containing 50 ppb Nd and Gd) with comparable 16O35Cl+ reduction (<100 ppt false 51V in 0.4% HCl) and Se sensitivity (DL < 1 ppb). Further reduction of these false positives is achieved by estimating the M2+ correction factors and utilizing them in the interference-correction software. Approaches to estimating the M2+ correction factor were evaluated with an emphasis on techniques that tolerate daily variability in end-user backgrounds and their ability to reduce the initial and ongoing purity requirements associated with the rare earth standards used to estimate the M2+ correction factor. The direct elemental and polyatomic overlaps associated with unit-mass approaches tend to overcorrect as non-rare-earth signals as small as 30 cps at the unit mass can induce bias relative to the <300 cps signals associated with the M2+ from a 50 ppb REE standard solution. Alternatively, shifting the M2+ estimate to a half mass (i.e., m/z 71.5: 143Nd2+) eliminates the direct overlap source of bias and allows the unit mass signal to approach 150000 cps before it bleeds over on the 1/2 mass because of abundance sensitivity limitations. The performance of the half-mass approach was evaluated in reagent water and regional tap waters fortified with Nd, Sm, and Gd at 2 ppb and 50 ppb. In addition, a half-mass in-sample approach was also evaluated. This approach was found to be beneficial relative to the external or fixed-factor half-mass approach as it could compensate for instrument drift and matrix-induced shifts in the M2+ factors. Finally, all results were evaluated relative to the As and Se concentrations determined using an ICP-QQQ in mass shift mode and a high-resolution ICP-MS.

Estimating Inorganic Arsenic Exposure from U.S. Rice and Total Water Intakes.

BACKGROUND: Among nonoccupationally exposed U.S. residents, drinking water and diet are considered primary exposure pathways for inorganic arsenic (iAs). In drinking water, iAs is the primary form of arsenic (As), while dietary As speciation techniques are used to differentiate iAs from less toxic arsenicals in food matrices. OBJECTIVES: Our goal was to estimate the distribution of iAs exposure rates from drinking water intakes and rice consumption in the U.S. population and ethnic- and age-based subpopulations. METHODS: The distribution of iAs in drinking water was estimated by population, weighting the iAs concentrations for each drinking water utility in the Second Six-Year Review data set. To estimate the distribution of iAs concentrations in rice ingested by U.S. consumers, 54 grain-specific, production-weighted composites of rice obtained from U.S. mills were extracted and speciated using both a quantitative dilute nitric acid extraction and speciation (DNAS) and an in vitro gastrointestinal assay to provide an upper bound and bioaccessible estimates, respectively. Daily drinking water intake and rice consumption rate distributions were developed using data from the What We Eat in America (WWEIA) study. RESULTS: Using these data sets, the Stochastic Human Exposure and Dose Simulation (SHEDS) model estimated mean iAs exposures from drinking water and rice were 4.2 µg/day and 1.4 µg/day, respectively, for the entire U.S. population. The Tribal, Asian, and Pacific population exhibited the highest mean daily exposure of iAs from cooked rice (2.8 µg/day); the mean exposure rate for children between ages 1 and 2 years in this population is 0.104 µg/kg body weight (BW)/day. CONCLUSIONS: An average consumer drinking 1.5 L of water daily that contains between 2 and 3 ng iAs/mL is exposed to approximately the same amount of iAs as a mean Tribal, Asian, and Pacific consumer is exposed to from rice. https://doi.org/10.1289/EHP418. BACKGROUND: Among nonoccupationally exposed U.S. residents, drinking water and diet are considered primary exposure pathways for inorganic arsenic (iAs). In drinking water, iAs is the primary form of arsenic (As), while dietary As speciation techniques are used to differentiate iAs from less toxic arsenicals in food matrices. OBJECTIVES: Our goal was to estimate the distribution of iAs exposure rates from drinking water intakes and rice consumption in the U.S. population and ethnic- and age-based subpopulations. METHODS: The distribution of iAs in drinking water was estimated by population, weighting the iAs concentrations for each drinking water utility in the Second Six-Year Review data set. To estimate the distribution of iAs concentrations in rice ingested by U.S. consumers, 54 grain-specific, production-weighted composites of rice obtained from U.S. mills were extracted and speciated using both a quantitative dilute nitric acid extraction and speciation (DNAS) and an in vitro gastrointestinal assay to provide an upper bound and bioaccessible estimates, respectively. Daily drinking water intake and rice consumption rate distributions were developed using data from the What We Eat in America (WWEIA) study. RESULTS: Using these data sets, the Stochastic Human Exposure and Dose Simulation (SHEDS) model estimated mean iAs exposures from drinking water and rice were [Formula: see text] and [Formula: see text], respectively, for the entire U.S. population. The Tribal, Asian, and Pacific population exhibited the highest mean daily exposure of iAs from cooked rice ([Formula: see text]); the mean exposure rate for children between ages 1 and 2 years in this population is [Formula: see text] body weight (BW)/day. CONCLUSIONS: An average consumer drinking 1.5 L of water daily that contains between 2 and [Formula: see text] is exposed to approximately the same amount of iAs as a mean Tribal, Asian, and Pacific consumer is exposed to from rice. https://doi.org/10.1289/EHP418.

Speciation and preservation of Cr VI and Cr III in finished drinking water matrixes using collision cell ion chromatography-inductively coupled plasma-mass spectrometry.

The polyatomic background at the major isotope of Cr was evaluated as a function of collision cell gas flow rate using three different mobile phases. The stability of Cr VIwas evaluated as a function of solution pH by fortifying samples with enriched 53Cr VI. The 53Cr VI recovery was > or =95% at pH 7.8, but by pH 6.2 a significant amount of the enriched spike was recovered as 53Cr III-EDTA. The instability of Cr III at pHs above 7.8 required that Cr III be chelated prior to speciation. The concentration of EDTA was optimized by evaluating Cr III recoveries in six finished drinking waters, a U.S. Geological Survey reference water, and a challenge water fortified with competing matrix cations. If the Cr III was allowed to precipitate (characteristic of shipping a sample without field preservation), acceptable recoveries of Cr III required the sample to be heated to 70 degrees C in 10 mM EDTA to quantitatively produce the Cr III-EDTA complex. The overall preservation/speciation approach was evaluated using an enriched 53Cr VI and natural Cr III in seven drinking water matrixes over a 46 day period. A linear least squares analysis was performed on each water, and the corresponding P-values were estimated. The method detection limits for Cr III-EDTA and Cr VI were 0.06 and 0.1 pglL, respectively.

Comparative oxidation state specific analysis of arsenic species by high-performance liquid chromatography-inductively coupled plasma-mass spectrometry and hydride generation-cryotrapping-atomic absorption spectrometry.

The formation of methylarsonous acid (MAsIII) and dimethylarsinous acid (DMAsIII) in the course of inorganic arsenic (iAs) metabolism plays an important role in the adverse effects of chronic exposure to iAs. High-performance liquid chromatography-inductively coupled plasma-mass spectrometry (HPLC-ICP-MS) and hydride generation-cryotrapping-atomic absorption spectrometry (HG-CT-AAS) have been frequently used for the analysis of MAsIII and DMAsIII in biological samples. While HG-CT-AAS has consistently detected MAsIII and DMAsIII, HPLC-ICP-MS analyses have provided inconsistent and contradictory results. This study compares the capacities of both methods to detect and quantify MAsIII and DMAsIII in an in vitro methylation system consisting of recombinant human arsenic (+3 oxidation state) methyltransferase (AS3MT), S-adenosylmethionine as a methyl donor, a non-thiol reductant tris(2-carboxyethyl)phosphine, and arsenite (iAsIII) or MAsIII as substrate. The results show that reversed-phase HPLC-ICP-MS can identify and quantify MAsIII and DMAsIII in aqueous mixtures of biologically relevant arsenical standards. However, HPLC separation of the in vitro methylation mixture resulted in significant losses of MAsIII, and particularly DMAsIII with total arsenic recoveries below 25%. Further analyses showed that MAsIII and DMAsIII bind to AS3MT or interact with other components of the methylation mixture, forming complexes that do not elute from the column. Oxidation of the mixture with H2O2 which converted trivalent arsenicals to their pentavalent analogs prior to HPLC separation increased total arsenic recoveries to ~95%. In contrast, HG-CT-AAS analysis found large quantities of methylated trivalent arsenicals in mixtures incubated with either iAsIII or MAsIII and provided high (>72%) arsenic recoveries. These data suggest that an HPLC-based analysis of biological samples can underestimate MAsIII and DMAsIII concentrations and that controlling for arsenic species recovery is essential to avoid artifacts.

An in vitro assessment of bioaccessibility of arsenicals in rice and the use of this estimate within a probabilistic exposure model.

In this study, an in vitro synthetic gastrointestinal extraction protocol was used to estimate bioaccessibility of different arsenicals present in 17 rice samples of various grain types that were collected across the United States. The across matrix average for total arsenic was 209 ng/g±153 (\[xmacr]±2σ). The bioaccessibility estimate produced an across matrix average of 61%±19 (\[xmacr]±2σ). The across matrix average concentrations of inorganic arsenic (iAs) and dimethylarsinic acid (DMA) were 81 ng/g±67.7 and 41 ng/g±58.1 (\[xmacr]±2σ), respectively. This distribution of iAs concentrations in rice was combined with the distribution of consumption patterns (from WWEIA) in a Stochastic Human Exposure and Dose Simulator model to estimate population-based exposures. The mean consumption rate for the population as a whole was 15.7 g per day resulting in a 0.98 µg iAs per day exposure. The mean consumption rate for children 1-2 years old was 7 g per day resulting in a 0.48 µg iAs per day exposure. Presystemic biotransformation of DMA in rice was examined using an in vitro assay containing the anaerobic microbiota of mouse cecum. This assay indicated that DMA extracted from the rice was converted to dimethylthioarsinic acid, although a second oxygen-sulfur exchange to produce DMDTA was not observed.

Preabsorptive metabolism of sodium arsenate by anaerobic microbiota of mouse cecum forms a variety of methylated and thiolated arsenicals.

The conventional scheme for arsenic methylation accounts for methylated oxyarsenical production but not for thioarsenical formation. Here, we report that in vitro anaerobic microbiota of mouse cecum converts arsenate into oxy- and thio- arsenicals. Besides methylarsonic acid (MMA(V)), arsenate was transformed into six unique metabolites: mono-, di-, and trithio-arsenic acid, monomethyldithio- and monomethyltrithio-arsonic acid, and dimethyldithioarsonic acid. Thioarsenicals were found in soluble and particulate fractions of reaction mixtures, suggesting interactions with anaerobic microbiota. Metabolism of ingested arsenate to oxy- and thio-arsenicals before absorption across the gastrointestinal barrier could affect bioavailability, systemic distribution, and resulting toxicity.

Arsenic metabolism by human gut microbiota upon in vitro digestion of contaminated soils.

BACKGROUND: Speciation analysis is essential when evaluating risks from arsenic (As) exposure. In an oral exposure scenario, the importance of presystemic metabolism by gut microorganisms has been evidenced with in vivo animal models and in vitro experiments with animal microbiota. However, it is unclear whether human microbiota display similar As metabolism, especially when present in a contaminated matrix. OBJECTIVES: We evaluated the metabolic potency of in vitro cultured human colon microbiota toward inorganic As (iAs) and As-contaminated soils. METHODS: A colon microbial community was cultured in a dynamic model of the human gut. These colon microbiota were incubated with iAs and with As-contaminated urban soils. We determined As speciation analysis using high-performance liquid chromatography coupled with inductively coupled plasma mass spectrometry. RESULTS: We found a high degree of methylation for colon digests both of iAs (10 microg methylarsenical/g biomass/hr) and of As-contaminated soils (up to 28 microg/g biomass/hr). Besides the formation of monomethylarsonic acid (MMA(V)), we detected the highly toxic monomethylarsonous acid (MMA(III)). Moreover, this is the first description of microbial thiolation leading to monomethylmonothioarsonic acid (MMMTA(V)). MMMTA(V), the toxicokinetic properties of which are not well known, was in many cases a major metabolite. CONCLUSIONS: Presystemic As metabolism is a significant process in the human body. Toxicokinetic studies aiming to completely elucidate the As metabolic pathway would therefore benefit from incorporating the metabolic potency of human gut microbiota. This will result in more accurate risk characterization associated with As exposures.

Disruption of the arsenic (+3 oxidation state) methyltransferase gene in the mouse alters the phenotype for methylation of arsenic and affects distribution and retention of orally administered arsenate.

The arsenic (+3 oxidation state) methyltransferase (As3mt) gene encodes a 43 kDa protein that catalyzes methylation of inorganic arsenic. Altered expression of AS3MT in cultured human cells controls arsenic methylation phenotypes, suggesting a critical role in arsenic metabolism. Because methylated arsenicals mediate some toxic or carcinogenic effects linked to inorganic arsenic exposure, studies of the fate and effects of arsenicals in mice which cannot methylate arsenic could be instructive. This study compared retention and distribution of arsenic in As3mt knockout mice and in wild-type C57BL/6 mice in which expression of the As3mt gene is normal. Male and female mice of either genotype received an oral dose of 0.5 mg of arsenic as arsenate per kg containing [(73)As]-arsenate. Mice were radioassayed for up to 96 h after dosing; tissues were collected at 2 and 24 h after dosing. At 2 and 24 h after dosing, livers of As3mt knockouts contained a greater proportion of inorganic and monomethylated arsenic than did livers of C57BL/6 mice. A similar predominance of inorganic and monomethylated arsenic was found in the urine of As3mt knockouts. At 24 h after dosing, As3mt knockouts retained significantly higher percentages of arsenic dose in liver, kidneys, urinary bladder, lungs, heart, and carcass than did C57BL/6 mice. Whole body clearance of [(73)As] in As3mt knockouts was substantially slower than in C57BL/6 mice. At 24 h after dosing, As3mt knockouts retained about 50% and C57BL/6 mice about 6% of the dose. After 96 h, As3mt knockouts retained about 20% and C57BL/6 mice retained less than 2% of the dose. These data confirm a central role for As3mt in the metabolism of inorganic arsenic and indicate that phenotypes for arsenic retention and distribution are markedly affected by the null genotype for arsenic methylation, indicating a close linkage between the metabolism and retention of arsenicals.

Exploring the in vitro formation of trimethylarsine sulfide from dimethylthioarsinic acid in anaerobic microflora of mouse cecum using HPLC-ICP-MS and HPLC-ESI-MS.

Although metabolism of arsenicals to form methylated oxoarsenical species has been extensively studied, less is known about the formation of thiolated arsenical species that have recently been detected as urinary metabolites. Indeed, their presence suggests that the metabolism of ingested arsenic is more complex than previously thought. Recent reports have shown that thiolated arsenicals can be produced by the anaerobic microflora of the mouse cecum, suggesting that metabolism prior to systemic absorption may be a significant determinant of the pattern and extent of exposure to various arsenic-containing species. Here, we examined the metabolism of 34S labeled dimethylthioarsinic acid (34S-DMTA(V)) by the anaerobic microflora of the mouse cecum using HPLC-ICP-MS and HPLC-ESI-MS/MS to monitor for the presence of various oxo- and thioarsenicals. The use of isotopically enriched 34S-DMTA(V) made it possible to differentiate among potential metabolic pathways for production of the trimethylarsine sulfide (TMAS(V)). Upon in vitro incubation in an assay containing anaerobic microflora of mouse cecum, 34S-DMTA(V) underwent several transformations. Labile 34S was exchanged with more abundant 32S to produce 32S-DMTA(V), a thiol group was added to yield DMDTA(V), and a methyl group was added to yield 34S-TMAS(V). Because incubation of 34S-DMTA(V) resulted in the formation of 34S-TMAS(V), the pathway for its formation must preserve the arsenic-sulfur bond. The alternative metabolic pathway postulated for formation of TMAS(V) from dimethylarsinic acid (DMA(V)) would proceed via a dimethylarsinous acid (DMA(III)) intermediate and would necessitate the loss of 34S label. Structural confirmation of the metabolic product was achieved using HPLC-ESI-MS/MS. The data presented support the direct methylation of DMTA(V) to TMAS(V). Additionally, the detection of isotopically pure 34S-TMAS(V) raises questions about the sulfur exchange properties of TMAS(V) in the cecum material. Therefore, 34S-TMAS(V) was incubated and the exchange was monitored with respect to time. The data suggest that the As-S bond associated with TMAS(V) is less labile than the As-S bond associated with DMTA(V).

Arsenic speciation and identification of monomethylarsonous acid and monomethylthioarsonic acid in a complex matrix.

Anion-exchange chromatography was utilized for speciation of arsenite (As(III)), arsenate (As(V)), dimethylarsinic acid (DMA(V)), monomethylarsonic acid (MMA(V)), monomethylarsonous acid (MMA(III)), and the new As species monomethylthioarsonic acid (MMTA), using inductively coupled plasma mass spectrometric (ICPMS) detection. MMA(III) and MMTA were identified for the first time in freeze-dried carrot samples that were collected over 25 years ago as part of a joint U.S. EPA, U.S. FDA, and USDA study on trace elements in agricultural crops. The discovery of MMA(III) and MMTA in terrestrial foods necessitated the analytical characterization of synthetic standards of both species, which were used for standard addition in carrot extracts. The negative ion mode, high-resolution electrospray mass spectrometry (HR-ESI-MS) data produced molecular ions of m/z 122.9418 and 154.9152 for MMA(III) and MMTA, respectively. However, ESI-MS was not sensitive enough to directly identify MMA(III) and MMTA in the carrot extracts. Therefore, to further substantiate the identification of MMA(III) and MMTA, two additional separations using an Ion-120 column were developed using the more sensitive ICPMS detection. The first separation used 20 mM tetramethylammonium hydroxide at pH 12.2 with MMA(III) eluting in less than 7 min. In the second separation, MMTA eluted at 11.2 min by utilizing 40 mM ammonium carbonate at pH 9.0. Oxidation of MMA(III) and MMTA to MMA(V) with hydrogen peroxide was observed for standards and carrot extracts alike. Several samples of carrots collected from local markets in 2006 were also analyzed and found to contain low levels of inorganic arsenic species.

Complementary molecular and elemental detection of speciated thioarsenicals using ESI-MS in combination with a xenon-based collision-cell ICP-MS with application to fortified NIST freeze-dried urine.

The simultaneous detection of arsenic and sulfur in thioarsenicals was achieved using xenon-based collision-cell inductively coupled plasma (ICP) mass spectrometry (MS) in combination with high-performance liquid chromatography. In an attempt to minimize the (16)O(16)O(+) interference at m/z 32, both sample introduction and collision-cell experimental parameters were optimized. Low flow rates (0.25 mL/min) and a high methanol concentration (8%) in the mobile phase produced a fourfold decrease in the m/z 32 background. A plasma sampling depth change from 3 to 7 mm produced a twofold decrease in background at m/z 32, with a corresponding fourfold increase in the signal associated with a high ionization surrogate for sulfur. The quadrupole bias and the octopole bias were used as a kinetic energy discriminator between background and analyte ions, but a variety of tuning conditions produced similar (less than twofold change) detection limits for sulfur ((32)S). A 34-fold improvement in the (32)S detection limit was achieved using xenon instead of helium as a collision gas. The optimized xenon-based collision cell ICP mass spectrometer was then used with electrospray ionization MS to provide elemental and molecular-based information for the analysis of a fortified sample of NIST freeze-dried urine. The 3sigma detection limits, based on peak height for dimethylthioarsinic acid (DMTA) and trimethylarsine sulfide (TMAS), were 15 and 12 ng/g, respectively. Finally, the peak area reproducibilities (percentage relative standard deviation) of a 5-ppm fortified sample of NIST freeze dried urine for DMTA and TMAS were 7.4 and 5.4%, respectively.

Tissue dosimetry, metabolism and excretion of pentavalent and trivalent dimethylated arsenic in mice after oral administration.

Dimethylarsinic acid (DMA(V)) is a rat bladder carcinogen and the major urinary metabolite of administered inorganic arsenic in most mammals. This study examined the disposition of pentavalent and trivalent dimethylated arsenic in mice after acute oral administration. Adult female mice were administered [(14)C]-DMA(V) (0.6 or 60 mg As/kg) and sacrificed serially over 24 h. Tissues and excreta were collected for analysis of radioactivity. Other mice were administered unlabeled DMA(V) (0.6 or 60 mg As/kg) or dimethylarsinous acid (DMA(III)) (0.6 mg As/kg) and sacrificed at 2 or 24 h. Tissues (2 h) and urine (24 h) were collected and analyzed for arsenicals. Absorption, distribution and excretion of [(14)C]-DMA(V) were rapid, as radioactivity was detected in tissues and urine at 0.25 h. For low dose DMA(V) mice, there was a greater fractional absorption of DMA(V) and significantly greater tissue concentrations of radioactivity at several time points. Radioactivity distributed greatest to the liver (1-2% of dose) and declined to less than 0.05% in all tissues examined at 24 h. Urinary excretion of radioactivity was significantly greater in the 0.6 mg As/kg DMA(V) group. Conversely, fecal excretion of radioactivity was significantly greater in the high dose group. Urinary metabolites of DMA(V) included DMA(III), trimethylarsine oxide (TMAO), dimethylthioarsinic acid and trimethylarsine sulfide. Urinary metabolites of DMA(III) included TMAO, dimethylthioarsinic acid and trimethylarsine sulfide. DMA(V) was also excreted by DMA(III)-treated mice, showing its sensitivity to oxidation. TMAO was detected in tissues of the high dose DMA(V) group. The low acute toxicity of DMA(V) in the mouse appears to be due in part to its minimal retention and rapid elimination.

Tissue distribution and urinary excretion of dimethylated arsenic and its metabolites in dimethylarsinic acid- or arsenate-treated rats.

Adult female Fisher 344 rats received drinking water containing 0, 4, 40, 100, or 200 parts per million of dimethylarsinic acid or 100 parts per million of arsenate for 14 days. Urine was collected during the last 24 h of exposure. Tissues were then taken for analysis of dimethylated and trimethylated arsenicals; urines were analyzed for these arsenicals and their thiolated derivatives. In dimethylarsinic acid-treated rats, highest concentrations of dimethylated arsenic were found in blood. In lung, liver, and kidney, concentrations of dimethylated arsenic exceeded those of trimethylated species; in urinary bladder and urine, trimethylated arsenic predominated. Dimethylthioarsinic acid and trimethylarsine sulfide were present in urine of dimethylarsinic acid-treated rats. Concentrations of dimethylated arsenicals were similar in most tissues of dimethylarsinic acid- and arsenate-treated rats, including urinary bladder which is the target for dimethylarsinic acid-induced carcinogenesis in the rat. Mean concentration of dimethylated arsenic was significantly higher (P<0.05) in urine of dimethylarsinic acid-treated rats than in arsenate-treated rats, suggesting a difference between treatment groups in the flux of dimethylated arsenic through urinary bladder. Concentrations of trimethylated arsenic concentrations were consistently higher in dimethylarsinic acid-treated rats than in arsenate-treated rats; these differences were significant (P<0.05) in liver, urinary bladder, and urine. Concentrations of dimethylthioarsinic acid and trimethylarsine sulfide were higher in urine from dimethylarsinic acid-treated rats than from arsenate-treated rats. Dimethylarsinic acid is extensively metabolized in the rat, yielding significant concentrations of trimethylated species and of thiolated derivatives. One or more of these metabolites could be the species causing alterations of cellular function that lead to tumors in the urinary bladder.

Dimethylthioarsinic anhydride: a standard for arsenic speciation.

Dimethylthioarsinic acid (DMTA(V)) has recently been identified in biological, dietary and environmental matrices. The relevance of this compound to the toxicity of arsenic in humans is unknown and further exposure assessment and metabolic studies are difficult to conduct because of the unavailability of a well characterized standard. The synthesis of DMTA(V) was accomplished by the reaction of dimethylarsinic acid (DMA(V)) with hydrogen sulfide. The initial reaction product produced is DMTA(V) but multiple products over the course of the reaction are also observed. Therefore, a chromatographic separation was developed to monitor the reaction progress via LC-ICP-MS. In this synthesis, conversion of DMA(V) to DMTA(V) was not taken to completion to avoid the production of side products. The product was isolated from the starting material by standard organic techniques. Single crystal diffraction demonstrated that solid DMTA(V) is present in the form of the oxygen-bridged dimethylthioarsinic anhydride. Dissolution of the anhydride in water produces the acid form of DMTA(V) and the aqueous phase DMTA(V) provided a characteristic molecular ion of m/z 155 by LC-ESI-MS. The synthesis and isolation of dimethylthioarsinic anhydride provides a stable crystalline standard suitable for identification, toxicological study and exposure assessment of dimethylthioarsinic acid.

Investigation of sequential and enzymatic extraction of arsenic from drinking water distribution solids using ICP-MS.

A sequential extraction approach was utilized to estimate the distribution of arsenite [As(iii)] and arsenate [As(v)] on iron oxide/hydroxide solids obtained from drinking water distribution systems. The arsenic (As) associated with these solids can be segregated into three operationally defined categories (exchangeable, amorphous and crystalline) according to the sequential extraction literature. The exchangeable As, for the six drinking water solids evaluated, was estimated using 10 mM MgCl(2) and 10 mM NaH(2)PO(4) and represented between 5-34% of the total As available from the solid. The amorphously bound As was estimated using 10 mM (NH(4))(2)C(2)O(4) and represented between 57-124% of the As available from the respective solid. Finally, the crystalline bound As was estimated using titanium citrate and this represented less than 1.5% of the As associated with the solids. A synthetic stomach/intestine extraction approach was also applied to the distribution solids. The stomach fluid was found to extract between 0.5-33.3 microg g(-1) As and 120-2,360 microg g(-1) iron (Fe). The As concentrations in the intestine fluid were between 0.02-0.04 microg g(-1) while the Fe concentration ranged from 0.06-0.7 microg g(-1) for the first six drinking water distribution solids. The elevated Fe levels associated with the stomach fluid were found to produce Fe based precipitates when the intestinal treatment was applied. Preliminary observations indicate that most of the aqueous Fe in the stomach fluid is ferric ion and the observed precipitate produced in the intestine fluid is consistent with the decreased solubility of ferric ion at the pH associated with the intestine.

In vitro biotransformation of an arsenosugar by mouse anaerobic cecal microflora and cecal tissue as examined using IC-ICP-MS and LC-ESI-MS/MS.

This investigation examined chemical and microbiological transformations of an arsenosugar by mouse cecum. To mimic the low oxygen environment in the mammalian gastrointestinal tract, reaction mixtures were incubated under anaerobic conditions. An arsenosugar extracted from ribbon kelp, 3-[5'-deoxy-5-(dimethylarsinoyl)-beta-ribofuranosyloxy]-2-hydroxypropanesulfonic acid, As392, was added to reaction mixtures that contained either cecal microflora or cecal tissue homogenate. These reaction mixtures were incubated at 0 or 37 degrees C for up to 48 hours to monitor biotransformation of the arsenosugar. Analysis of the reaction mixtures by IC-ICP-MS and LC-ESI-MS/MS indicated that the arsenosugar was converted primarily (95%) to its sulfur analog in less than 1 h at 37 degrees C. Conversion of As392 to its sulfur analog was much slower at 0 degrees C (21% conversion after 48 h). In reaction mixtures with cecal tissue homogenate, conversion of As392 to its sulfur analog was slower (77% conversion after 48 h at 37 degrees C). A good mass balance was found in all reaction mixtures between the amount of arsenosugar added and the sum of all detected arsenic-containing products. LC-ESI-MS/MS spectra of the sulfur-containing arsenosugar formed in all reaction mixtures containing cecal microflora compared well with those of a synthetic standard. These results suggest that the anaerobic microflora of the gastrointestinal tract can rapidly convert ingested arsenosugars to sulfur analogs. This biotransformation may affect the subsequent absorption, metabolism, and disposition of arsenic present in arsenosugars.

Chromatographic separation and identification of products from the reaction of dimethylarsinic acid with hydrogen sulfide.

The reaction of dimethylarsinic acid (DMAV) with hydrogen sulfide (H2S) is of biological significance and may be implicated in the overall toxicity and carcinogenicity of arsenic. The course of the reaction in aqueous phase was monitored, and an initial product, dimethylthioarsinic acid, was observed by using LC-ICP-MS and LC-ESI-MS. Dimethylarsinous acid was observed as a minor product. A second slower-forming product was identified, and the electrospray mass chromatograms for this species produced ions at m/z 275, 171, and 137 in positive mode. To aid in the identification of this slower-forming product, crystalline standards of sodium dimethyldithioarsinate and dimethylarsino dimethyldithioarsinate were prepared and re-characterized by using improved spectroscopic and structural analysis techniques. An aqueous solution of sodium dimethyldithioarsinate produced a single major chromatographic peak that matched the retention time (7.6 min) of the slower-forming product and contained similar molecular ions at m/z 275, 171, and 137 via LC-ESI-MS. The dimethylarsino dimethyldithioarsinate standard produced four aqueous phase species one of which coeluted with the slower forming product. This coeluting peak also produced the identical ESI-MS ions as the slower-forming product of DMAV + H2S. ESI-MS/MS experiments conducted on sodium dimethyldithioarsinate in deuterated water produced molecular ions at m/z 276, 173, and 137. Subsequent collisionally activated dissociation (CAD) experiments on m/z 276 did not produce a product ion at m/z 173. These data indicate that two different species are present in solution, while NMR data indicate that only dimethyldithioarsinic acid exists in aqueous solutions. This discrepancy was investigated by conducting NMR studies on the acidic solution of sodium dimethyldithioarsinate after taking this solution to dryness. The resolubilized solution produced a proton NMR signal characteristic of dimethylarsino dimethyldithioarsinate. Therefore, it was concluded that the ESI-MS ion at m/z 275 associated with the slowly forming second reaction product and the sodium dimethyldithioarsinate compound is a product of the ESI desolvation process.

Investigation of arsenic speciation on drinking water treatment media utilizing automated sequential continuous flow extraction with IC-ICP-MS detection.

Three treatment media, used for the removal of arsenic from drinking water, were sequentially extracted using 10 mM MgCl2(pH 8), 10 mM NaH2PO4(pH 7) followed by 10 mM (NH4)2C2O4(pH 3). The media were extracted using an on-line automated continuous extraction system which allowed the arsenic in each of the extraction fluids to be speciated on-line using IC-ICP-MS. The 10 mM MgCl2 preferentially extracted As(III) from each of the media. The percentage of the arsenic extracted by the MgCl2, relative to a HNO3/H2O2 digestion of the media, ranged from 0.1-2.3% for the three solids. The next sequential extraction fluid, 10 mM NaH2PO4, extracted some of the residual As(III) remaining on each of the media but the predominant species extracted was As(V). The 10 mM NaH2PO4 extracted 15.3 to 42.8% of the total arsenic relative to a total digested concentration for each of the media. The As(III) and As(V) stability studies conducted in these two extraction fluids indicated that conversion between As(III) and As(V) was not significant for the short extraction fluid sample contact time associated with the on-line continuous flow extraction cell. Finally, the 10 mM (NH4)2C2O4 extraction fluid was utilized in an off-line analysis mode because the Fe and As concentrations extracted from the media were not compatible with direct ICP-MS detection. The (NH4)2C2O4 extracted 2.9-29% As(III) for all three media and caused an oxidation of As(III) to As(V) during the extraction period for one of the three media. The sum of the arsenic from each of the three extraction fluids represented 92%, 44% and 53% of the available total arsenic for the three media, respectively. The speciation results for each media were obtained by adding all the speciation results from all three extraction fluids together and the resulting distribution of As(III)/As(V) compared well with the speciation results obtained via XANES.

Comparison of a chemical and enzymatic extraction of arsenic from rice and an assessment of the arsenic absorption from contaminated water by cooked rice.

Rice is a target food for arsenic speciation based analyses because of its relatively high arsenic concentration and per capita consumption rates. Improved speciation data for rice can be helpful in estimating inorganic arsenic exposures in the U.S. and in endemic populations. The inorganic arsenic exposure for cooked rice should include both the arsenic in raw rice plus the arsenic absorbed from the water used to prepare it. The amount of arsenic absorbed from water by rice during preparation was assessed using five different types of rice cooked in both contaminated drinking water and arsenic-free reagent water. The rice samples were extracted using trifluoroacetic acid (TFA) and speciated using IC-ICP-MS. The TFA procedure was able to extract 84-104% of the arsenic (As) from the five different cooked rice samples. Chromatographic recoveries ranged from 99% to 116%. The dimethylarsinic acid (DMA) and inorganic arsenic concentration ranged from 22 to 270 ng of As/g of rice and from 31 to 108 ng of As/g of rice, respectively, for samples cooked in reagent water. The overall recoveries, which relate the sum of the chromatographic species back to the total digested concentration, ranged from 89% to 117%. The absorption of arsenic by rice from the total volume of water [1:1 to 4:1 (water:rice)] used in cooking was between 89% and 105% for two different contaminated drinking water samples. A comparison of the TFA extraction to an enzymatic extraction was made using the five rice samples and NIST 1568a rice flour. The two extraction procedures produced good agreement for inorganic arsenic, DMA, and the overall recovery. Through the use of IC-ESI-MS/ MS with a parent ion of m/z 153 and fragment ions of m/z 138, 123, and 105, the structure dimethylthioarsinic acid was tentatively identified in two of the rice samples using the enzymatic extraction.