Sulfur-Containing Arsenical Mistaken for Dimethylarsinous Acid [DMA

Jul 17, 2004 - The shared identity between the products of the two synthetic methods has never been questioned or proven. Here, we characterize and id...
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Chem. Res. Toxicol. 2004, 17, 1086-1091

Sulfur-Containing Arsenical Mistaken for Dimethylarsinous Acid [DMA(III)] and Identified as a Natural Metabolite in Urine: Major Implications for Studies on Arsenic Metabolism and Toxicity Helle R. Hansen, Andrea Raab, Marcel Jaspars, Bruce F. Milne, and Jo¨rg Feldmann* Department of Chemistry, College of Physical Sciences, University of Aberdeen, Meston Walk, Old Aberdeen AB24 3UE, United Kingdom Received January 8, 2004

It is vital that methylated trivalent arsenicals [MA(III) and DMA(III)] are described and characterized unequivocally due to their high toxicity. Two different ways of generating the methylated trivalent arsenicals have been practicedsreduction of the methylated pentavalent arsenical either by the sodium-metabisulfite (Na2S2O5)/sodium thiosulfate (Na2S2O3) reagent (method A) or by KI, H2SO4, and SO2 (method B). The shared identity between the products of the two synthetic methods has never been questioned or proven. Here, we characterize and identify the arsenic species formed when reducing DMA(V) by method A or B. Dimethylarsinous acid [DMA(III)] was formed when reducing DMA(V) by method B, but DMA(III) was not the main product of the reaction by method A. The product was revealed by HPLC-ICP-MS coupled simultaneously to HPLC-ES-MS and ES-Q-TOF-MS to have the molecular formula C2H7OSAs. The structure was further confirmed by 1H NMR, and ab initio tautomeric energy calculations showed it to be present as Me2As(dS)OH (dimethylarsinothioic acid). Dimethylarsinothioic acid was also identified as a metabolite in urine and in wool extract from sheep naturally consuming large amounts of arsenosugars (35 mg of As daily) through their major food source, seaweed.

Introduction In recent years, the trivalent methylated arsenicals [MA(III)1 and DMA(III)] have received particular attention, since they are likely candidates for the carcinogenic effects of arsenic. Arsenite was long believed to be the most toxic form of arsenic, but studies in cell cultures have revealed that MA(III) and DMA(III) are at least as geno- (1, 2) and cytotoxic (3-6) as arsenite and are also potent inhibitors of a number of enzymes (7-10). The attention of the toxicologists was drawn to MA(III) and DMA(III), because they were both identified in urine from people exposed to extremely high levels of arsenic in their drinking water. In the studies concerning the identification of MA(III) and DMA(III) in urine samples, the identification has been purely based on chromatographic coelution with standards using mostly ICP-MS as an arsenic specific detector for HPLC, and no molecular information was obtained (11-16). The MA(III) and DMA(III) standards used in these studies were generated by two different methods, which are described here for DMA(III). In one method (method A), the DMA(V) is reduced to DMA(III) using sodium-metabisulfite (Na2S2O5)/ sodium thiosulfate (Na2S2O3) as a reducing agent (17). The second method (method B) involves the initial synthesis of the DMAI by treatment of (CH3)2AsO2Na * To whom correspondence should be addressed. E-mail: [email protected]. 1 Abbreviations: MA(III), methylarsonous acid; DMAI, dimethylarsinous iodide; DMA(III), dimethylarsinous acid; DMA(V), dimethylarsinic acid.

with KI, H2SO4, and SO2 followed by purification by distillation (18). The identity of DMAI has been confirmed by 1H NMR (19), but neither the hydrolyzed product of method B (referred to from here on as product B) nor the product of method A (product A) have been sufficiently characterized by NMR or MS. Most toxicity tests on the methylated trivalent arsenicals have been carried out using MA(III) and DMA(III) synthesized by method B, whereas identification of the compounds in biological samples has used either method A or method B. In light of the high toxicity and the importance of risk assessments of the methylated trivalent arsenicals, it is vital that these species are described and characterized unequivocally. In this study, we are the first to fully characterize the arsenic metabolite in urine coeluting with the “DMA(III)” standards.

Experimental Section Caution: Inorganic arsenic is classified as a human carcinogen, and appropriate precautions should be taken in the handling of its different species. Reagents. DMA(V) [(CH3)2AsVO(OH)] was obtained from Strem (United States). Potassium iodide (KI), sodium metabisulfite (Na2S2O5), sodium thiosulfate (Na2S2O3), sulfuric acid (85%), ammonia (25%), formic acid (98-100%), and ammonium carbonate were all AnalR and were obtained from BDH (United Kingdom); ultrapure acetic acid was from Fluka; D2O was from Aldrich; and SO2 was from Sigma (United States). All stock solutions described below were kept at 4 °C, apart from DMAI, which was stored at -20 °C. Standards. The reduction of DMA(V) by metabisulfite (Na2S2O5) and sodium thiosulfate (Na2S2O3) medium (method

10.1021/tx049978q CCC: $27.50 © 2004 American Chemical Society Published on Web 07/17/2004

Arsenical Mistaken for Dimethylarsinous Acid A) was done according to the instructions of Reay and Asher who reduced arsenate to arsenite (17). DMAI was prepared by the method of Burrows and Turner (18) (method B). The yellow oil separated during synthesis was collected and dried over Na2SO4. The aqueous phase was also collected, and to prevent oxidation, both phases were stored under argon. DMA(III) (product B) stock solutions were prepared from the yellow oil shortly before use (maximum 2 h) from the synthesized compound by diluting with deionized water or methanol under nitrogen. Additionally, a solution of DMA(V) was purged with gaseous H2S for 2 h (method C). Samples. Urine and wool samples were obtained from a primitive breed of wild sheep, which live on the beaches of North Ronaldsay (Orkney Islands, United Kingdom) and naturally consume large amounts of arsenosugars through their major food sourcesseaweed. The urine samples were collected from 12 North Ronaldsay ewes fed one meal of a mixture of Laminaria hyperborea and Laminaria digitata (3-5 kg) once a day for 5 days. The samples were collected twice a day, transferred to polyethylene tubes, and stored without any addition of chemicals in a refrigerator (4 °C) until analysis (20). The urine samples were analyzed untreated and only diluted to suitable concentration with water (1-3 mg/L). The wool was collected from 22 North Ronaldsay sheep and stored in dry plastic bags. Before analysis, it was cleaned with hexane and extracted with water under boiling (21). Separation of Arsenic Compounds by HPLC-ICP-MS and HPLC-ES-MS. Chromatographic conditions used in specific experiments are given in the relevant figure captions. Additional tests (without a figure) were carried out using a Hamilton PRP-X100 (250 mm × 4.6 mm) with 1.0 mL/min, 30 mM phosphate buffer (pH 5) containing 20% methanol [earlier used by Gailer et al. (22)] and a reverse phase column (Waters S5 ODS2 250 mm × 4.1 mm, 0.5 mL/min, 100% methanol). An HP1100 HPLC system (Agilent Technologies, United States) with a cooled autosampler was used throughout the experiments. The autosampler was cooled to 4 °C, and the column was kept at an ambient temperature. Postcolumn, the flow was split in a ratio of 1:4 (one part into the ICP-MS, four parts into the ES-MS) using a microsplitter (Upchurch, United Kingdom). The conditions for the ICP-MS (ICP-MS 7500, Agilent Technologies) and the ES-MS (Agilent HP1100 series LC-MSD) used throughout the study are as described elsewhere (23). ES-Q-TOF-MS. A Mariner (AppliedBiosystems, United States) fitted with an ES-spray-head and a syringe pump was used in the positive ionization mode to obtain accurate masses for the species. A mixture of water and acetonitrile (1:1) with 1% acetic acid was used as the carrier. The capillary voltage was set to 4000 V with a nozzle voltage of 100 or 300 V. Nitrogen (purity 99.8%) was used as curtain gas (∼1 L/min) and nebulizer gas (∼0.5 L/min). The resolution and sensitivity of the instrument were optimized using a peptide mixture (angiotensin, bradykinin, and neurotensin). The signal of DMA(V) was used for internal calibration. 1H NMR. A Varian Unity Inova 400 MHz NMR instrument was used for the 1H NMR experiments. A standard presaturation pulse sequence was used. The samples were referenced to added methanol at 3.34 ppm in D2O/H2O. Tautomeric Energies for the Me2As(dS)OH/Me2As(dO)SH Pair. All calculations were carried out with the GAMESSUS [14 Jan 2003 (R2) build] (24). The polarized double-ζ 6-31G(d,p) basis set was employed for all atoms. Initially, geometry optimizations and subsequent single point energy calculations were carried out in the gas phase using the restricted Hartree-Fock (RHF) level of theory. As electron correlation was expected to be important due to the electronic configuration of the As and S atoms, this was repeated using both density functional theory with the “hybrid” B3LYP functional [DFT(B3LYP)] and with post-HF second-order perturbation theory (MP2). The effects of aqueous solvation were investigated by repeating the sequence of geometry optimizations and single point energy calculations with the solvent

Chem. Res. Toxicol., Vol. 17, No. 8, 2004 1087 Table 1. Suggested Structures of Molecular and Fragment Ions Measured by ES-MSa fragmentor voltage (V) species

m/z

DMA(V) [As]+ 75 [AsdO]+, [MeAsH]+ 91 + [As(OH)2] 109 [Me2AsdO]+ 121 [Me2As(OH)2]+ 139 [Me4As2(O)2OH]+ 259 [OH(Me)2AsOHOAs(Me)2OH]+ 277 [AsdO]+, [MeAsH]+ [MeAsOH]+ [Me2AsdO]+ [Me2AsOH2]+ [Me2As(OH)2]+ [Me2As(OMe)OH]+ [Me2As(OMe)2]+

100

2 100 12 8

product B (in methanol) 91 107 121 123 139 3 153 100 167 57

[AsdO]+, [MeAsH]+ [AsdS]+ [H2AsS]+, [Asd34S]+ [Me2AsdO]+ ? [Me2AsdS]+ [Me2As(OH)2]+ [Me2As(SH)OH]+ ?

product A 91 107 109 121 127 137 139 155 177

[AsdO]+, [MeAsH]+ [AsdS]+ [H2AsdS]+ [Me2AsdO]+ [Me2AsdS]+ [Me2As(SH)OH]+ [Me2As(SH)2]+

product C 91 107 109 121 137 155 171

19

26 14 54 100 44

15 58 100

200

100 9 20 21

100 15 34 9

400 7 100

100 7

18

43 100 29 19

100 14

9

16 100 33 9 16

100 35

a The abundance of each ion is given as a percent of the dominant ion for each applied fragmentor voltage.

environment approximated using the polarizable continuum method (PCM) (25). This technique was only available in combination with the RHF and DFT levels of theory; therefore, MP2:PCM calculations could not be carried out. However, because of the good agreement of the MP2 and DFT(B3LYP) gas phase results, it was felt that the solvated DFT results were sufficiently reliable.

Results Analysis of Standards by ES-MS (Flow Injection). DMA(V), the products of methods A, B, and C (products A, B, and C) were analyzed by flow injection ES-MS (0.1% formic acid). By altering the fragmentor voltage, molecular ions (100 V) and fragment ions (200 and 400 V) can be observed. The suggested structures of molecular and fragment ions measured (Supporting Information) are summarized in Table 1. The MS of DMA(V) showed a dominant m/z 139 [M + H]+ (100 V), and increasing the fragmentor voltage increased the dominance of m/z 109, m/z 121 (200 V), and m/z 91 (400 V). The molecular ion and reported fragment ions of DMA(V) confirm an earlier report (26). Product B (DMAI) was very prone to oxidation, and its MS was identical to that of DMA(V) in aqueous solvent. However, when dissolving DMAI in methanol and using methanol as the eluent, it was possible to detect m/z 123 ([M + H]+ ion) at a fragmentor voltage of 200 V only, indication of the difficulty in ionizing this compound. Methanol forms an adduct with DMA(V) to give m/z 153 and m/z 167 (Table 1). The MS of product A and product C showed a distinctive differ-

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Figure 1. Chromatographic [PRP-X100 anion exchange column (150 mm × 4.1 mm)] dependence of the retention time of product A (1) and product C (2) on the pH of the mobile phase (ICP-MS detection); eluent, 30 mM acetic acid mobile phase, pH 5.3, or 30 mM ammonium carbonate, pH 8 or 9.4; flow rate, 1 mL/min.

ence from the MS of either DMA(V) or product B. Both spectra showed several masses including m/z 155 (100 V). When increasing the fragmentor voltage, m/z 91, 107, 109, 121, and 137 appeared. The spectrum of product C showed only an additional m/z 171 (100 V) presumably created by a double sulfur exchange on DMA(V). Analysis of Standards by HPLC Coupled Simultaneously to ICP-MS and ES-MS. To reveal the identity of products A, B, and C, HPLC was coupled simultaneously to ICP-MS and ES-MS. The arsenic signal (m/z 75 ICP-MS) of product A or C was separated from DMA(V) (which eluted much earlier under all tested conditions) but showed identical retention times when changing the pH of the mobile phase on an anion exchange column (Figure 1). The ES-MS data revealed that only m/z 137, 139, and 155 (100 V) showed peaks at the same retention time as m/z 75, and other masses observed in their MS (100 V) (Supporting Information) cannot be fragments of the eluting arsenic species. A coelution of m/z 34 (34S), m/z 48 (32S16O), and the arsenic peak (m/z 75) using ICP-MS (not shown), together with ES-MS fragment ions m/z 137 [(CH3)2AsS+] and m/z 107 (AsS+) (200 V, Table 1) suggest [in agreement with a recent study (27)] that the compound contains sulfur. The main m/z 171 of product C observed by flow injection (ESMS) did not show up in the arsenic peak when injected on the anion column, and only when using a short precolumn (Hamilton PRP-X100) with 20 mM ammonium carbonate, pH 9.4, as a mobile phase was it possible to elute it as a different peak from m/z 155 (Supporting Information). Several approaches were used to elute product B as a well-defined peak from the strong anion exchange column, but the compound always appeared as a tailing of the DMA(V) peak (Figure 2). However, when injecting product B dissolved in methanol, a coelution between m/z 75 (ICP-MS) and m/z 123 (ES-MS) did show up in the solvent front from the anion exchange column (not shown). Product B dissolved in methanol was then injected on a reverse phase column using 100% methanol as the eluent. This less polar eluent increased the solubility and the ionization of product B on the ES source and made it possible to detect on m/z 123 (200 V) as a coeluting peak with m/z 75 (ICP-MS) (data not shown). The main m/z 155 of product A was not present in the MS of product B, but it was the main arsenical in the aqueous phase collected from the synthesis of DMAI

Hansen et al.

Figure 2. Anion exchange chromatogram [PRP-X100 anion exchange column (150 mm × 4.1 mm), 1 mL/min 30 mM ammonium carbonate, pH 8)] of product B detected by ICP-MS (m/z 75) and by ES-MS (m/z 123, 139) using the scan mode.

Figure 3. Anion exchange chromatogram [weak anion exchange column with DEAE functional group (DAEA TSK 75 mm × 7.5 mm) 1 mL/min 10 mM citric acid, pH 2.0)] of product A (1), product B (2), and DMA(V) (3) on the ICP-MS (m/z 75) (A) and on the ES-MS (m/z given in figure) (B).

(method B) when analyzed under similar chromatographic conditions as Figure 2 (not shown). To be able to compare our chromatographic results with those of other studies, we analyzed products A and B by similar conditions as Suzuki et al. [weak anion exchange column with DEAE as functional group, 1 mL/ min flow, and 15 mM citric acid buffer (pH 2.0)] (16, 28). We used, however, a shorter column and a more diluted citric acid buffer of 10 mM (pH 2.0). DMA(V) showed a peak (m/z 139) with a retention time of 2.6 min, product A showed a peak with a retention time of 3.1 min (m/z 155), and product B (dissolved in methanol) showed no m/z 123 peak but two m/z 139 peaks, one at the same retention time to DMA(V) and one at 3.1 min (Figure 3). The first peak of product B can be assigned to DMA(V), and the second peak can be assigned to DMA(III) as

Arsenical Mistaken for Dimethylarsinous Acid

Chem. Res. Toxicol., Vol. 17, No. 8, 2004 1089 Table 2. Energy Difference (∆E) between Me2As(dS)OH and Me2As(dO)SH and the Boltzmann Population Ratio at 300 K method

∆E (kJ/mol)

ratio SH:OH

RHFa DFT (B3LYP)a MP2a RHF:PCMb DFT (B3LYP):PCMb

-86.8 -57.6 -53.0 -91.5 -61.1

1:1.3 × 1015 1:1.1 × 1010 1:1.7 × 109 1:1.9 × 1015 1:1.4 × 1010

a Calculations carried out in the gas phase. b Calculations carried out with aqueous solvation.

Figure 4. Time-dependent generation of product A measured by NMR. The peak areas (% of total peak area) of product A, DMA(V), and product B are shown as a function of time (min).

Figure 5. Possible tautomeric forms of product A (C2H7OSAs).

oxidation of the trivalent arsenical during ionization or detection by the ES-MS can explain the detection of m/z 139. m/z 155 was not detected in the second peak of product B suggesting that the product is not identical to product A despite their coelution. Analysis by NMR and ES-q-TOF-MS. 1H NMR data confirmed the shared identity between product A (2.05 ppm) and product C (2.03 ppm), and its chemical shift value [closer to that of DMA(V) (2.11 ppm) than product B (1.32 ppm)] suggests that the compound is a pentavalent arsenical. The reaction of method A was followed by 1H NMR analysis from the addition of the reagent to DMA(V) (time 0 min). The reaction was followed for 8 h (Figure 4). Product A was the main product of the reaction, and its concentration increased rapidly within the first 70 min after which the concentration leveled off. Product B was a byproduct of the reaction (7%, t ) 0) and disappeared completely within 70 min. Molecular masses obtained on the ES-Q-TOF-MS also showed a similarity between product A (153.941) and product C (153.948), and the data were accurate enough to confirm the molecular formula of product A as C2H7OSAs (153.950). Tautomeric Energies for the Me2As(dS)OH/Me2As(dO)SH Pair. Two tautomeric forms of product A were theoretically possible: Me2As(dS)OH/Me2As(dO)SH (Figure 5). To identify the population ratio of this pair, the energy differences were determined using theoretical modeling at various levels of theory, with and without solvation (Table 2). The large negative energy differences (∆E > -50 kJ/mol) obtained indicate that the OH form would be considerably more highly populated than the SH form in a system at thermodynamic equilibrium. This was verified by calculation of the ratio of Boltzmann factors (Table 2) for the two species, which indicated that for an aqueous solution in which only OH and SH forms were present population ratios of the order of 1:1 × 1010 (as in Table 2) (10) in favor of Me2As(dS)OH might be expected; thus, compound A was characterized as Me2As(dS)OH [dimethylarsinothioic acid, Figure 5(1)].

Figure 6. Anion exchange chromatograms [PRP-X100 anion exchange column (150 mm × 4.6 mm), 1 mL/min 20 mM ammonium carbonate)] of a urine sample and wool extract (solid line) and the samples spiked with 1 mg/L dimethylarsinothioic acid (dotted line) detected by ICP-MS (m/z 75) (A) and by ESMS (m/z 155) using the scan mode (B). Peak 1 consists mainly of DMA(V) and dimethylarsinoyl acetic acid, and peak 3 consists of 2-dimethylarsinothioyl acetic acid.

Identification of Dimethylarsinothioic Acid in Urine and Wool Extract. Samples had been obtained from sheep feeding on seaweed containing natural high levels of arsenic in the form of arsenosugars (74 mg As/ kg dry mass) as described elsewhere (20). The anion exchange chromatogram of urine samples showed three main arsenic peaks, and the wool extract showed two main arsenic peaks (Figure 6A). Peaks 1 and 3 in the urine sample have previously been characterized by HPLC-ICP-MS and HPLC-ES-MS as DMA(V), dimethylarsinoyl acetic acid, and 2-dimethylarsinothioyl acetic acid (26, 27). In this study, we focus on peak 2. Peak 2 in the wool extract of the same sheep was characterized as DMA(III) in an earlier study, based on the chromatographic retention time and pH-dependent hydride generation (HPLC-HG-ICP-MS) (21). Spiking the urine sample and wool extract with dimethylarsinothioic acid [Me2As(dS)OH, product A] showed a coelution between the standard and peak 2 (Figure 6A). When HPLC was coupled simultaneously to ICP-MS and ES-MS, the main signal observed at the same retention time in the ESMS as m/z 75 in the ICP-MS was m/z 155 (Figure 6B). The retention time of peaks u1 and u2 (m/z 155, ES-MS) did not match the retention time of any arsenic peak (m/z 75) and hence does not contain arsenic.

Discussion The ES-MS results revealed distinctive differences (Table 1) between product A (m/z 155) and product B (m/z 123), whereas product A showed similarities to product C (Figure 1). A coelution of sulfur [m/z 34 (34S), m/z 48 (32S16O)] and the arsenic peak (m/z 75) by ICP-MS together with ES-MS fragment ions m/z 137 [(CH3)2AsS+]

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and m/z 107 (AsS+) (Table 1) suggest [in agreement with a recent paper (27)] that products A and C contain sulfur. Thio-arsenicals were in the 1930s (29-32) commonly synthesized by treatment of arsenicals with H2S, and product C can be expected to contain sulfur. Product A has recently been suggested to contain sulfur, and on the background of its oxidation product [DMA(V)], it is believed to be a trivalent compound (33). In agreement, we observed that the stability of product A was low at room temperature and within a few days the compound would degrade or oxidize to DMA(V). The compound showed, however, good stability when stored in a fridge (4 °C). The 1H NMR data obtained in this study, however, suggest that product A is pentavalent as the chemical shift value of the methyl group is more similar to DMA(V) (2.11 ppm) than a trivalent arsenical (1.32 ppm). The accuracy of the ES-Q-TOF-MS (153.941) together with the results of theoretical modeling reveals that product A has the molecular formula C2H7OSAs (153.950) and is present in the tautomeric form of Me2As(dS)OH [dimethylarsinothioic acid, Figure 5(1)]. Following the reaction of method A by 1H NMR, analysis revealed that dimethylarsinothioic acid was the main product of the reaction and product B was only a byproduct (7%), which rapidly decomposed (Figure 4). Compound B was generally very unstable in aqueous solution. It was not stable enough to elute from the strong anion exchange column and oxidized before and during the separation resulting in tailing of the DMA(V) peak (Figure 2). Only by using a less polar eluent (methanol) was the solubility and ionization of product B increased sufficiently to measure on the ES source (m/z 123). All MS data on product B together with its low chemical shift value (1.32) by 1H NMR confirm that product B is identical to DMA(III). None of the measurements on product B suggests that it contains sulfur and that the fragment ion m/z 123 should be assigned (CH3)AsSH+ rather than (CH3)2AsOH2+. All measurements by HPLC-ICP-MS and by flow injection ES-MS (at variable fragmentor voltage) confirm that product B does oxidize into DMA(V). The molecular ion of DMA(III) [(CH3)2AsOH2+] has to our knowledge not previously been reported, presumably due to its extreme instability in solvation and/or in the electrospray source as well as the difficulties in ionizing the compound using ES-MS. The biological relevance of dimethylarsinothioic acid is ratified by the identification of this compound as a natural metabolite in urine (Figure 6). The occurrence of dimethylarsinothioic acid in the wool extract is presumably the result of the extraction process. The extracted DMA(V) (the main arsenic species in the wool) may react with the extracted thiol-containing amino acids as the compound increases with the length of the extraction time and temperature (21). We have previously mistaken dimethylarsinothioic acid in wool extract for DMA(III) (21). The identification was based on chromatographic retention time and pH-dependent hydride generation (HPLC-HG-ICP-MS). The actual DMA(III) standard used was synthesized by method A, and its identity was never proven. We later tested the volatilization of dimethylarsinothioic acid during HG-GC-ICP-MS (not shown) and found that its hydride (Me2AsH) was volatile at pH 1 and pH 7. The quality of forming volatile hydrides at pH 7 has until recently only been attributed to trivalent arsenicals, a feature commonly used for the separation between pentavalent and trivalent arsenic

Hansen et al.

compounds. However, a recent study has shown that pentavalent arsenosugars form volatile analytes (34). The occurrence of dimethylarsinothioic acid in the urine and wool extracts makes one speculate if other studies may have mistaken dimethylarsinothioic acid for DMA(III). The identification of MA(III) and DMA(III) in urine samples all through the literature has been purely based on chromatographic coelution with standards by ICP-MS measurements. The chromatographic conditions mainly applied in this study were similar to the conditions used in a study where a urinary arsenic metabolite in samples of a human being who had consumed arsenosugars was suggested to be DMA(III) (14). The retention time of “DMA(III)” in this study showed a matching retention time to that of dimethylarsinothioic acid on our column, when using the same conditions. DMA(III) in the relevant study was prepared by reducing DMA(V) by method A. We also observed a similar matching retention time between dimethylarsinothioic acid on our column and a DMA(III) peak in the urine of a different study (22) when using similar conditions. DMA(III) showed under these conditions no defined peak but appeared as a broad-tailed DMA(V) peak as shown in Figure 2. In this study, DMA(III) had been synthesized by method B, but dimethylarsinothioic acid could have been present from the aqueous phase as observed in our study. We also simulated a method commonly used in studies on MA(III) and DMA(III) by Suzuki et al. (Figure 3). Our results showed that by this method it is possible to elute and detect DMA(III) as a separate peak by ICP-MS (at m/z 139 in ES-MS, Figure 3), rather than the broad tailing seen on the strong anion exchange column. However, the DMA(III) signal had a similar retention time as dimethylarsinothioic acid, and solely using HPLC-ICP-MS without the parallel use of ES-MS detection would not reveal which of the two structures was present. This coelution may provide the explanation of conflicting results obtained between two similar recent studies where rats were fed a dose of DMA(V) (33, 35, 36). Lu et al. detected DMA(III) as a urinary metabolite by HPLCHGAFS (36); however, Yoshida et al. showed by HPLCLCQ-MS that DMA(III) was not present in the urine but instead a sulfur-containing arsenic metabolite similar to product A (33, 35). An alternative explanation for the disagreement is present in the reality that chromatographic results may only be a product of the HPLC procedure. Lu et al. modified their strong anion exchange columns by dimercaptopropane sulfonate, which may derivatize DMA(III) standard into dimethylarsinothioic acid and hence have the same retention time to the metabolite in the urine. Thus, it cannot be distinguished between DMA(III) and dimethylarsinothioic acid. In conclusion, it has been shown that DMA(III) is extremely unstable and difficult to measure in aqueous solution. The instability together with chromatographic results obtained in this study, including the identification of dimethylarsinothioic acid in samples of urine and wool extract, suggest that dimethylarsinothioic acid may previously have been mistaken for being DMA(III) in natural samples. The arsenothioyl compounds are arsenic metabolites, which have not been proposed in the metabolic pathway before. They may be breakdown products of arseno-protein compounds, and attention should be paid for other As-S compounds. These findings therefore will redirect the attention of toxicologists from trivalent arsenicals to arsenothioyl compounds in the search for

Arsenical Mistaken for Dimethylarsinous Acid

the toxic mode of action of chronic arsenic toxicity and how to monitor arsenic exposure.

Acknowledgment. We thank Silvia Wehmeier, University of Aberdeen, for testing the volatilization of product Aby HG-GC-ICP-MS, BBSRC (1/REI18479), and the University of Aberdeen for financial support. Supporting Information Available: Mass spectra obtained by ES-MS (flow injection) at fragment voltages of 100, 200, and 400 V (fundamental data for Table 1) and HPLC-ESMS chromatograms of product C separated on a Hamilton PRPX100 precolumn with a mobile phase of 20 mM ammonium carbonate, pH 8 and 9.4. This material is available free of charge via the Internet at http://pubs.acs.org.

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