Arsenic Speciation in Urine and Blood Reference Materials

Acute and chronic exposure to arsenic is a growing problem in the industrialized world. Arsenic is a potent carcinogen and toxin in humans. In the body, arsenic is metabolized to produce several species, including inorganic forms, such as trivalent (As^III) and pentavalent (As^V), and the methylated metabolites such as monomethylarsonic acid, (MMA^V), and dimethylarsinic acid (DMA^V), in addition to arsenobetaine (AsB) which is ingested and excreted from the body in the same form. Each of these species has been reported to possess a specific but different degree of toxicity. Thus, not only is the measurement of total As required, but also quantification of the individual metabolites is necessary to evaluate the toxicity and risk assessment of this element. There are a large number of reference materials that are used to validate methodology for the analysis of As in blood and urine, but they are limited to total As concentrations. In this study, the speciation of five arsenic metabolites is reported in blood and urine from commercial available control materials certified for total arsenic levels. The separation was performed with an anion exchange column using inductively coupled plasma mass spectrometry as a detector. Baseline separation was achieved for As^III, As^V, MMA^V, DMA^V, and AsB, allowing us to quantify all five species. Excellent agreement between the total arsenic levels and the sum of the speciated As levels was obtained.     

Enzymatic digestion and chromatographic analysis of arsenic species released from proteins

A method combining gel filtration chromatography (GFC), protease digestion, and ion pair chromatography with inductively coupled plasma mass spectrometry detection was developed for the determination of arsenic species bound to proteins. The method was first established by examining the interactions of two model proteins, metallothionein (MT) and hemoglobin, with three reactive trivalent arsenic species. It was then successfully applied to the speciation of arsenic in red blood cells of rats. Inorganic arsenite (iAs^III), monomethylarsonous acid (MMA^III), and dimethylarsinous acid (DMA^III) were efficiently released from the proteins by protease digestion at pH 8.0, with the recovery ranging from 93% to 106%. There was no oxidation of iAs^III or MMA^III during the protease digestion process. Up to 61% DMA^III (the least stable arsenic species) was unchanged, and the rest was oxidized to the pentavalent dimethylarsinic acid (DMA^V). The arsenic species in the red blood cells of control rats was present as DMA^III complex with hemoglobin. The method enabling the determination of the specific arsenic species that bind to cellular proteins is potentially useful for studying arsenic distribution, metabolism, and toxicity.     

HPLC-ICP-MS method development to monitor arsenic speciation changes by human gut microbiota

Inorganic arsenic (iAs) has been classified as a type 1 carcinogen and has also been linked to several noncancerous health effects. Prior to 1995, the As^V methylation pathway was generally considered to be a detoxification pathway, but cellular and animal studies involving MMA^III (mono metyl arsonous acid) and DMA^III (dimethyl arsinous acid) have indicated that their toxicities meet or exceed that of iAs, suggesting an activation process. In addition, thiolated arsenic metabolites were observed in urine after oral exposure of inorganic arsenic in some studies, for which the toxicological profile was not yet fully characterized in human cells. Studies have revealed that microorganisms from the gut environment are important contributors to arsenic speciation changes. This presystemic metabolism necessitates the development of protocols that enable the detection of not only inorganic arsenic species, but also pentavalent and trivalent methylated, thiolated arsenicals in a gastrointestinal environment. We aim to study the biotransformation of arsenic (As) using a Simulator of the Human Intestinal Microbial Ecosystem (SHIME). To be able to analyze the arsenicals resulting from biotransformation reactions occurring in this system, a method using liquid chromatography hyphenated to an inductively coupled plasma mass spectrometer (HPLC‐ICP‐MS) was developed. A Hamilton PRP‐X100 anion exchange column was used. The method allowed separation, identification and quantification of As^III(arsenite), As^V(arsenate), DMA^V(dimethylarsinicacid), MMA^V(monomethylarsonicacid) and MMMTA (monomethylmonothioarsenate). Attempts to optimize the same method for also separating MMA^III and DMA^III did not succeed. These compounds could be successfully separated using a method based on the use of a Zorbax C₁₈ column. The properties of the column, buffer strength, pH and polar nature of mobile phase were monitored and changed to optimize the developed methods. Linearity, sensitivity, precision, accuracy and resolution of both methods were checked. The combination of the two methods allowed successful quantification of arsenic species in suspensions sampled in vitro from the SHIME reactor or in vivo from the human colon and feces. Copyright © 2011 John Wiley & Sons, Ltd.     

Intake and excretion of disodium monomethylarsonate in horses: a speciation study

Capillary electrophoresis coupled to inductively coupled plasma mass spectrometry was used in a speciation study on disodium monomethylarsonate (DS-MMA^V) and its metabolites in horses, to which the drug was administered by intramuscular injection on five consecutive days at a single arsenic dosage of 270 mg day⁻¹. Samples of urine, whole blood, plasma, and mane hair were analyzed before, during, and after drug administration. The data show that blood clearing and urinary excretion of MMA is a fast process following first-order kinetics with biological half-lives of about 38 h and 44 h for urine and plasma, respectively. In the time period of 9 days studied, the only metabolite detected in urine was dimethylarsinic acid (DMA^V), which 4 days after the last drug administration accounted for up to 75% of the total excreted arsenic species. This shows, for the first time, that biomethylation of MMA^V to DMA^V is the principal metabolic pathway of this drug in horses. Although DS-MMA^V was administered only during a short 5-day period, an up to six fold increase of arsenic could be measured in the newly grown mane hair.     

Speciation of arsenic in body fluids

Inorganic arsenic is metabolized by consecutive reduction and methylation reactions to dimethylated arsenic (DMA), and then excreted into the urine mostly in the form of DMA. Therefore, arsenic metabolites in the body fluids and organs/tissues are present in the form of inorganic (arsenite and arsenate) and methylated arsenics (MMA and DMA). Although pentavalent arsenics can be present mostly in the form of free ions, trivalent ones may be present more in the forms conjugated with thiol groups of glutathione (GSH) or proteins. Arsenic in the body fluids (plasma, bile and urine) is present in the soluble forms and can be speciated on ion exchange columns by HPLC with on-line detection by an inductively coupled argon plasma-mass spectrometer (ICP-MS). Free forms of arsenite, arsenate, and monomethylarsonous, monomethylarsonic, dimethylarsinous and dimethylarsinic acids in the body fluids have been demonstrated to be speciated simultaneously within 10 min or so on both anion and cation exchange columns together with arsenobetaine (AsB) and arsenocholine (AsC). Trivalent arsenics conjugated with GSH were eluted in intact forms on an anion exchange column but were liberated into free forms on a cation exchange column. Thus, free and GSH-conjugated arsenic metabolites in the bile and urine have been speciated simultaneously on ion exchange columns by HPLC-ICP-MS.     

Hydride generation activity of arsenosugars and thioarsenicals

The major arsenosugar compounds have been reported to be hydride-generation-active, however to a lesser extent in comparison with the inorganic arsenicals. We report here for the first time the identity and quantity of the volatile arsenicals generated by As-sugar-SO₃, As-sugar-SO₄, dimethylarsinoyl acetic acid and dimethylarsinoyl ethanol. Only one major volatile compound was identified for all four compounds studied: dimethylarsine. This means that the As–C bond to the longer carbon chain was cleaved during the hydride-generation process. Theoretical calculations at the RHF/6-31G(d,p) ab initio level confirm that this As–C bond is much weaker than the As–CH₃ bonds. Furthermore, it was revealed that the sulphur analogue of dimethylarsinic acid (DMAS ) is hydride-generation-active at pH 7 in contrast to dimethylarsinic acid, despite the fact that arsenic is also pentavalent. This has been substantiated by the calculation of the change in susceptibility of the arsenic towards nucleophilic attack when oxygen is replaced by sulphur. Hence, DMAS can easily be mistaken for a trivalent arsenic species.     

Speciation of arsenic trioxide metabolites in blood cells and plasma of a patient with acute promyelocytic leukemia

Arsenic trioxide (As₂O₃) has been widely accepted as the second-best choice for the treatment of relapsed and refractory acute promyelocytic leukemia (APL) patients. However, a few studies have been conducted on a detailed speciation of As₂O₃ metabolites in blood samples of patients. To clarify the speciation of arsenic, the blood samples were collected at various time points from a patient with APL after remission induction therapy and during consolidation therapy. The total amounts of arsenic in blood cells and plasma, and the plasma concentrations of inorganic arsenic and methylated metabolites were determined by inductively coupled plasma mass spectrometry (ICP-MS) and high-performance liquid chromatography/ICP-MS, respectively. The total amounts of arsenic in the blood cells were 4–10 times higher than those in plasma. Among all arsenic metabolites, the pentavalent arsenate (As^V) in plasma was more readily eliminated. During the drug-withdrawal period, the initial plasma concentrations of trivalent arsenic (As^III) declined more rapidly than those of methylarsonic acid and dimethlyarsinic acid, which are known as the major methylated metabolites of As^III. On the other hand, during the consecutive administration in the consolidation therapy period, the plasma concentrations of total arsenic and arsenic metabolites increased with time. In conclusion, these results may support the idea that methylated metabolites of As₂O₃ contribute to the efficacy of arsenic in APL patients. These results also suggest that detailed studies on the pharmacokinetics as well as the pharmacodynamics of As₂O₃ in the blood cells from APL patients should be carried out to provide an effective treatment protocol. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Presented at the 4th International Conference on Trace Element Speciation in Biomedical, Nutritional and Environmental Sciences, 25–29 May 2008, Munich-Neuherberg, Germany.     

Methylated arsenic species in estuarine porewaters

Inorganic arsenic, monomethylarsenic and dimethylarsenic species have been observed in samples of sediment porewater collected from the Tamar Estuary in South-West England. Porewater samples were collected using in situ dialysis. The arsenic species were separated by hydride generation and concentrated by liquid nitrogen trapping, prior to analysis by directly coupled gas chromatography-atomic absorption spectroscopy. The predominant dissolved arsenic species present was inorganic arsenic (5-62 m?g dm^?3). However, this is the first time significant concentrations of methylated arsenic species have been quantified in estuarine porewaters (0.04–0.70 m?g dm^?3), accounting for between 1 and 4% of the total dissolved arsenic. The presence of methylated arsenic compounds in porewaters is attributed to in situ environmental methylation, although the possibility of methylated arsenic species being derived from biological debris cannot be excluded.     

Arsenic Speciation in Marine Sediments: Effects of Redox Potential and Reducing conditions

Abstract

The speciation of arsenic in the environment is among others controlled by reduction, methylation and oxidation processes and therefore influenced by the prevailing redox conditions. In this study we have analyzed sediments taken from La Coruña estuary in the north west of Spain. Inorganic (trivalent and pentavalent) and the organic (MMA and DMA) arsenic speciation is related to Eh, Fe and Mn load. The various of the arsenic species concentration and other parameters was analyzed at different depths in some of the sampling points. Low arsenic concentrations (1–10 μg·g^–1) were found. In spite of oxidising conditions (Eh values between 31–96 mV), most of the samples showed a higher As(V) percentage than As (III). Principal component analysis was made to see a sample groups and the results showed that speciation depends on reducing conditions (Eh and Mn).     

Redox substoichiometric determination of arsenic in biological materials by neutron activation analysis

A redox substoichiometry is proposed for an accurate and precise determination of arsenic. This method is based on the substoichiometric oxidation of trivalent arsenic to pentavalent with potassium bromate or ceric sulfate followed by the separation of these species by thionalide extraction of trivalent arsenic. It was applied to neutron activation analysis of arsenic in the NBS SRM Orchard Leaves and the Shark Powder. The results were obtained with an excellent accuracy and precision.     

The separation of molybdenum valencies by paper chromatography part II1

The preparation of Mo^VI oxinate and a purported Mo^V oxine complex is described. The latter was analysed, and a number of its properties, including separation from the Mo^VI compound by paper chromatography, were studied.The preparation of a solution containing trivalent molybdenum and the separation of this valency from the pentavalent state by chromatography upon cellulose is also described.Suggestions as to the stability of thiocyanate and oxine compounds of Mo^III were made from the results of some extraction experiments performed upon trivalXnt molybdenum solutions.     

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 the interaction between arsenic species and thiols via electrospray ionization tandem mass spectrometry

Arsenic species have been known to participate in a number of chemical and biological reactions, including oxidation-reduction reactions, acid-base reactions, covalent interactions, and methylation-demethylation reactions because of the element's multiple and interconvertible oxidation states. Little is known about the structure or bonding behavior between arsenic species and thiolcontaining biomolecules. Therefore, a better understanding of the bonding behavior and detailed information on the molecular structure for arsenic-thiol complexes is needed. As a result, we have investigated the interaction between arsenic species (arsenate (As^V), arsenite (As^III), monomethylarsonic acid (MMA^V), and dimethylarsinic acid (DMA^V)) with biomolecules containing thiol groups (glutathione and cysteine) by electrospray ionization mass spectrometry (ESI-MS). These compounds were dissolved in methanol/water solution and introduced into the MS instrument in order to elucidate the direct bonding behavior of thiol group of biomolecules with arsenic species. In addition, further detailed structural information on this complex was obtained by collision-induced dissociation (CID) measurements.In each mass spectrum for mixture solutions between arsenic species and thiol compounds, various peaks such as protonated arsenic-thiol complexes, protonated noncomplexed thiol compounds, sodium bound cluster ions, and proton bound cluster ions were observed. In these mass spectra, the arsenic complexes were formed by interaction with thiol groups on the cysteine residues. These arsenic-thiol complexes produced a variety of fragment ions by cleavage of chemical bonds, and by interaction of other binding site on thiol compounds in tandem mass spectrometry experiments.     

Methylated Phenylarsenical Metabolites Discovered in Chicken Liver

We report the discovery of three toxicologically relevant methylated phenylarsenical metabolites in the liver of chickens fed 3‐nitro‐4‐hydroxyphenylarsonic acid (ROX), a feed additive in poultry production that is still in use in several countries. Methyl‐3‐nitro‐4‐hydroxyphenylarsonic acid (methyl‐ROX), methyl‐3‐amino‐4‐hydroxyphenylarsonic acid (methyl‐3‐AHPAA), and methyl‐3‐acetamido‐4‐hydroxyphenylarsonic acid (or methyl‐N ‐acetyl‐ROX, methyl‐N ‐AHPAA) were identified in such chicken livers, and the concentration of methyl‐ROX was as high as 90 μg kg⁻¹, even after a five‐day clearance period. The formation of these newly discovered methylated metabolites from reactions involving trivalent phenylarsonous acid substrates, S‐adenosylmethionine, and the arsenic (+3 oxidation state) methyltransferase enzyme As3MT suggests that these compounds are formed by addition of a methyl group to a trivalent phenylarsenical substrate in an enzymatic process. The IC₅₀ values of the trivalent phenylarsenical compounds were 300–30 000 times lower than those of the pentavalent phenylarsenicals.     

Development of mass spectrometric methods for detecting arsenic-glutathione complexes

It has been suggested recently that arsenic-glutathione (As-GSH) complexes play an important role in the methylation of arsenic. The present study describes the development of high-performance liquid chromatography (HPLC)-electrospray tandem mass spectrometry (ES-MS/MS), operated in the selected reaction monitoring (SRM) mode, and HPLC-inductively coupled plasma mass spectrometry (ICP-MS) methods suitable for the sensitive and selective identification of four As-GSH complexes. Method optimization was carried out using a series of synthetically prepared standards, i.e., three As-GSH species containing trivalent arsenic: tri(glutamyl-cysteinyl-glycinyl)trithio-arsenite (ATG), di(glutamyl-cysteinyl-glycinyl)methyl-dithio-arsonite (MADG), and (ã-glutamyl-cysteinyl-glycinyl) dimethyl-thio-arsinite (DMAG), as well as one As-GSH species containing pentavalent As: dimethylthioarsinic acid-glutathione (DMTA^V-GSH). The collision induced dissociation behavior of these compounds was investigated in detail to identify optimum SRM transitions for each complex. Both methods were based on reversed-phase chromatography using gradient elution with methanol, formic acid, and water as solvents. The amount of methanol that was used with this HPLC method (up to 12% vol/vol) was compatible with ICP-MS, without the need of a specially adapted interface. Subsequently, these analytical methods were applied to carry out a preliminary investigation about the role of As-GSH complexes in the methylation of arsenite by methylcobalamin (CH₃B₁₂) in the presence of glutathione (GSH). For the first time, the complexes ATG, MADG, and trace amounts of DMAG were detected as products of this reaction.     

Speciation of arsenic in a contaminated soil by solvent extraction

Soil collected from a disused cattle dip in northern New South Wales was studied with the aim of developing an inexpensive, yet effective method for quantitative determination of arsenic(III), arsenic(V) and total organic arsenic in a contaminated soil. Hydrochloric acid extractions were used as a method for removal of the arsenic from the soil in a form suitable for speciation. It was found that the extraction efficiency varied with the ratio of soil to acid, and the concentration of the acid. Arsenic(III), as arsenic trichloride, was selectively extracted into chloroform from a solution highly concentrated in hydrochloric acid. This was followed by back-extraction of the arsenic into water. Total inorganic arsenic was determined in a similar manner after the reduction of arsenic(V) to the trivalent state with potassium iodide. Arsenic(V) was determined by the difference between the results for arsenic(III) and total inorganic arsenic. All analyses for the various arsenic species were performed by hydride generation-atomic absorption spectroscopy; concentrations of total arsenic in the soil were confirmed using X-ray fluorescence spectrometry. It was found that all the arsenic in the soil was present as inorganic arsenic in the pentavalent state. This reflects the ability of arsenic to interchange between species, since the original species in cattle dipping solution is arsenic(III).     

Determination of Arsenic in Algae – Results of an Interlaboratory Trial: Determination of Arsenic Species in the Water-Soluble Fraction

Seven algae samples, five purchased from food stores and two reference algae (BCR 279 Sea Lettuce) were distributed as blind samples to 13 laboratories from which five labs attempted a full characterisation of the water-soluble fraction with respect to their arsenic species. The extraction efficiency is largely dependant on the algae and varied from 3% to 96%. Besides inorganic arsenic (mainly as As^(V)) DMA^(V) and, in particular, several arsenosugars were identified in all samples. From the five labs, three labs gave agreeable results in respect of the arsenic species identification and its quantification, although different chromatographic methods were used. Different Hijiki samples seem to contain largely different arsenic concentration (67–113 mg As/kg) which may also have an influence on the distribution of inorganic arsenic and arsenosugars.     

Non-chromatographic hydride generation atomic spectrometric techniques for the speciation analysis of arsenic,antimony, selenium,and tellurium in water samples—a review

Hydride generation (HG) coupled with AAS, ICP–AES, and AFS techniques for the speciation analysis of As, Sb, Se, and Te in environmental water samples is reviewed. Careful control of experimental conditions, offline/online sample pretreatment methods employing batch, continuous and flow-injection techniques, and cryogenic trapping of hydrides enable the determination of various species of hydride-forming elements without the use of chromatographic separation. Other non-chromatographic approaches include solvent extraction, ion exchange, and selective retention by microorganisms. Sample pretreatment, pH dependency of HG, and control of NaBH₄/HCl concentration facilitate the determination of As(III), As(V), monomethylarsonate (MMA), and dimethylarsinate (DMA) species. Inorganic species of arsenic are dominant in terrestrial waters, whereas inorganic and methylated species are reported in seawater. Selenium and tellurium speciation analysis is based on the hydrides generation only from the tetravalent state. Se(IV) and Se(VI) are the inorganic selenium species mostly reported in environmental samples, whereas speciation of tellurium is rarely reported. Antimony speciation analysis is based on the slow kinetics of hydride formation from the pentavalent state and is mainly reported in seawater samples.     

Oxidation State Specific Generation of Arsines from Methylated Arsenicals Based on L- Cysteine Treatment in Buffered Media for Speciation Analysis by Hydride Generation - Automated Cryotrapping - Gas Chromatography-Atomic Absorption Spectrometry with the Multiatomizer

An automated system for hydride generation - cryotrapping- gas chromatography - atomic absorption spectrometry with the multiatomizer is described. Arsines are preconcentrated and separated in a Chromosorb filled U-tube. An automated cryotrapping unit, employing nitrogen gas formed upon heating in the detection phase for the displacement of the cooling liquid nitrogen, has been developed. The conditions for separation of arsines in a Chromosorb filled U-tube have been optimized. A complete separation of signals from arsine, methylarsine, dimethylarsine, and trimethylarsine has been achieved within a 60 s reading window. The limits of detection for methylated arsenicals tested were 4 ng l(-1). Selective hydride generation is applied for the oxidation state specific speciation analysis of inorganic and methylated arsenicals. The arsines are generated either exclusively from trivalent or from both tri- and pentavalent inorganic and methylated arsenicals depending on the presence of L-cysteine as a prereductant and/or reaction modifier. A TRIS buffer reaction medium is proposed to overcome narrow optimum concentration range observed for the L-cysteine modified reaction in HCl medium. The system provides uniform peak area sensitivity for all As species. Consequently, the calibration with a single form of As is possible. This method permits a high-throughput speciation analysis of metabolites of inorganic arsenic in relatively complex biological matrices such as cell culture systems without sample pretreatment, thus preserving the distribution of tri- and pentavalent species.     

Differential determination of arsenic(III) and total arsenic using flow injection on-line separation and preconcentration for graphite furnace atomic absorption spectrometry

Arsenic(III) can be quantitatively extracted using sodium diethyldithiocarbamate (NaDDTC) as the complexing agent and C18 reversed phase packing as the column material for solid phase extraction. Arsenic(V) must be reduced to its trivalent oxidation state prior to extraction. A mixture of sodium sulphite, hydrochloric acid, sodium thiosulphate and potassium iodide was found to be optimum for on-line reduction. When the sorbent extraction is carried out without and with the addition of the reduction mixture, arsenic(III) and total arsenic can be determined sequentially by graphite furnace atomic absorption spectrometry with detection limits (3 σ) of 0.32 ng for As(III) and 0.43 ng for total arsenic. A 7.6-fold enhancement in peak area compared to direct injection of 40 μl samples was obtained after 60 s preconcentration. Results obtained for sea water standard reference materials, using aqueous standards for calibration, agree well with certified values. A precision of 5.5% RSD was obtained for total arsenic in a sea water sample (1.65 As). Results obtained for synthetic mixtures of trivalent and pentavalent arsenic agreed well with expected values.