This research was supported by Basic Science Research program (Project number: 2016R1A2B4013467) through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT &#38; Future Planning 2016 and also supported by Korea Basic Science Institute Research Program (Project number: C36707).

1. Synthesis of DMMTAV
<ol>
	<li>Chemical preparation and molar ratio mixing of DMAV, Na2S, and H2SO4 
	NOTE: DMAV:Na2S:H2SO4 = 1:1.6:1.6
	<ol>
		<li>Dissolve 5.24 g of DMAV in 40 mL deionized and N2-purged (purged for at least 30 min) water in a 50 mL centrifuge tube.</li>
		<li>Prepare Na2S reagent by dissolving 14.41 g of Na2S&#183;9H2O in 50 mL deionized and N2-purged water in a 250 mL flask.</li>
		<li>Prepare H2SO4 reagent by adding 3.3 mL of concentrated sulfuric acid (96%) to 40 mL of deionized and N2-purged water in a 50 mL centrifuge tube. 
		NOTE: Final molar ratio of DMAV:Na2S:H2SO4 = 1:1.6:1.6 1,3,4,8,15.</li>
		<li>Add the prepared 40 mL of DMAV solution (step 1.1.1) to the 50 mL of Na2S solution contained in the 250 mL flask (step 1.1.2). Rinse the tube containing DMAV with 10 mL of purged water and add this to the 250 mL flask as well.</li>
		<li>Close the flask with a three-hole rubber stopper fitted with glass tubes. Use the glass tubes for N2 gas inflow, outflow, and H2SO4 solution inlet, respectively. Immediately after closing the flask, allow N2 gas flow into the flask. 
		NOTE: Gas pressure should be maintained to flow over the surface of the reaction solution without splashing.</li>
		<li>Connect acid resistant tubing to the glass tube of the H2SO4 solution inlet with a 50 mL syringe to add the prepared 40 mL of H2SO4 solution (step 1.1.3). Add 40 mL of H2SO4 solution, slowly and in a stepwise manner. 
		Caution: Upon adding sulfuric acid, white fumes will be generated; use a well-ventilated fume hood.</li>
		<li>Monitor color change of the reaction mixture in the flask while adding sulfuric acid at regular intervals. Maintain an interval of 4 - 5 mL drops of H2SO4; the mixture should be a white cloudy solution. 
		NOTE: Instantaneous yellow precipitation might appear due to rapid addition of concentrated sulfuric acid.</li>
		<li>Ensure the reaction solution has been standing for 1 h since the start of step 1.1.4.</li>
	</ol>
	</li>
	<li>DMMTAV extraction using liquid-liquid extraction method
	<ol>
		<li>After 1 h, pour the reaction solution into a separatory funnel containing around 200 mL of diethyl ether.</li>
		<li>Shake the funnel for 5 - 10 min, releasing gas several times by switching the stopcock. 
		NOTE: Synthesized DMMTAV will be transferred to the upper layer of diethyl ether (0.713 g&#183;mL-1). 
		Caution: Diethyl ether gas might be harmful; use a well-ventilated fume hood.</li>
		<li>Collect the reaction solution in a beaker, and separately collect the diethyl ether containing DMMTAV in a bottle. Place the reaction solution back in the same separatory funnel and add about 200 mL of fresh diethyl ether for reshaking. Repeat steps 1.2.2 - 1.2.3 three times.</li>
		<li>Pour the collected diethyl ether from step 1.2.3 into the same separatory funnel again, and add about 100 mL N2-purged deionized water. Shake for 5 - 10 min, and discard the N2-purged deionized water and a few mL of diethyl ether for purity purposes. Collect the remaining diethyl ether in a glass petri dish (minimum inner diameter of 160 mm and minimum height of 50 mm).</li>
		<li>Transfer the glass petri dish into a N2 atmosphere glove box to prevent species transformation. 
		Caution: Ensure the solvent is not pulled into the vacuum pump through the outlet of the pass box.</li>
		<li>Dry until a white precipitate of dimethylmonothioarsinate (crystalized DMMTAV) is formed on the glass petri dish. 
		NOTE: The protocol can be paused here.</li>
	</ol>
	</li>
	<li>Verification of synthesized DMMTAV and storage
	<ol>
		<li>Take the white precipitate of crystalized DMMTAV, and measure and record its total weight. 
		Caution: Use a well ventilated fume hood or glove box to prevent inhalation of hydrogen sulfide gas.</li>
		<li>Dissolve crystalized DMMTAV in 50 mL of N2-purged deionized water, and filter the yellow precipitate through a 0.2-&#181;m syringe filter.</li>
		<li>Assume the total As of used DMAV is converted into DMMTAV, i.e.&#8776;9,649 mg As&#183;L-1. Dilute DMMTAV stock solution to &#8776;1 mg&#183;L-1 and &#8776;40 &#181;g&#183;L-1 for verification analysis using Electrospray ionization Mass Spectromtery (ESI-MS) and HPLC-ICP-MS, respectively.</li>
		<li>Analyze m/z of DMMTAV using ESI-MS11,16 and fragments at m/z 155 in the positive-ion mode or at m/z 153 in the negative-ion mode (Table 1). 
		NOTE: See reference values of m/z (Table 1).</li>
		<li>Analyze chromatogram of DMMTAV in the stock solution using HPLC-ICP-MS11,16,17 with appropriate eluent conditions and confirm a major peak is found at the retention time described in the literature. 
		NOTE: Purity of synthesized DMMTAV should be calculated using the analysis results from step 1.3.5.</li>
		<li>Analyze the total arsenic concentration using ICP-MS after acid digestion11 and calculate the true DMMTAV concentration in synthesized DMMTAV stock solution using dilution factors and purity, as in the following equation: 
		Analyzed total As concentration (&#181;g&#183;L-1) &#183; Dilution factor &#183; Purity (%) = True DMMTAV concentration in DMMTAV stock solution (&#181;g&#183;L-1)</li>
		<li>Store DMMTAV stock solution at 4 &#176;C in the dark for further quantitative speciation analysis18.</li>
	</ol>
	</li>
</ol>
2. Synthesis of DMDTAV
<ol>
	<li>Chemical preparation and molar ratio mixing of DMAV , Na2S, and H2SO4 
	NOTE: DMAV:Na2S:H2SO4 = 1:7.5:7.5
	<ol>
		<li>Dissolve 1.38 g of DMAV in 40 mL deionized water in a 50 mL centrifuge tube.</li>
		<li>Prepare Na2S reagent by dissolving 18.01 g of Na2S&#183;9H2O in a 50 mL deionized water in a 250 mL flask.</li>
		<li>Prepare H2SO4 reagent by adding 4 mL of concentrated sulfuric acid (96%) to 40 mL of deionized water contained in a 50 mL centrifuge tube. 
		NOTE: Final molar ratio of DMAV:Na2S:H2SO4 = 1:7.5:7.51,3,4,8,15.</li>
		<li>Add the prepared 40 mL of DMAV solution (step 2.1.1) to the 50 mL of Na2S solution contained in the 250 mL flask (step 2.1.2). Rinse the tube containing DMAV with 10 mL of deionized water and add this to the 250 mL flask as well.</li>
		<li>Add the prepared 40 mL of H2SO4 solution (step 2.1.3), slowly and in a stepwise manner, into the flask.</li>
		<li>Monitor the color change of the reaction mixture in the flask while adding sulfuric acid at regular intervals. Maintain an interval of 4 - 5 mL drops of H2SO4; the mixture should be a white/yellow cloudy solution. 
		NOTE: Instantaneous yellow precipitation might appear due to a rapid addition of concentrated sulfuric acid. 
		Caution: Upon adding sulfuric acid, white fumes will be generated; use a well ventilated fume hood.</li>
		<li>Maintain the reaction solution in the flask overnight without covering. 
		NOTE: The protocol can be paused here.</li>
	</ol>
	</li>
	<li>DMDTAV extraction using solid-phase extraction (SPE) method
	<ol>
		<li>After the overnight standing reaction, filter the reaction solution using a C18 syringe type silica-based SPE in order to trap synthesized DMDTAV on the resin. 
		Caution: Use a well-ventilated fume hood.</li>
		<li>Prepare 10 mM ammonium acetate (pH 6.3) by dissolving 0.77 g ammonium acetate in 1 L of deionized water. Elute a sufficient volume of 10 mM ammonium acetate through the C18 syringe (step 2.2.1) to extract the adsorbed DMDTAV. Collect the filtered ammonium acetate in a glass Petri dish (minimum inner diameter of 160 mm and minimum height of 50 mm).</li>
		<li>Transfer the glass petri dish into a N2 atmosphere glove box to prevent species transformation. 
		Caution: Ensure solvent is not pulled into the vacuum pump through the outlet of the pass box.</li>
		<li>Dry until a white precipitate of dimethyldithioarsinate is formed (crystalized DMDTAV) on the glass petri dish. 
		NOTE: The protocol can be paused here.</li>
	</ol>
	</li>
	<li>Verification of synthesized DMDTAV and storage
	<ol>
		<li>Take the white precipitate of crystalized DMDTAV, and measure its total weight, recording the measurement. 
		Caution: Use a well-ventilated fume hood or glove box to prevent inhalation of hydrogen sulfide gas.</li>
		<li>Dissolve crystalized DMDTAV in 50 mL of N2-purged deionized water in a 50 mL centrifuge tube, and filter any precipitate through a 0.2-&#181;m syringe filter.</li>
		<li>Assume that the total As of used DMAV is converted into DMDTAV, i.e.&#8776;2,539 mg As&#183;L-1. Dilute DMDTAV stock solution to &#8776;1&#160;mg&#183;L-1 and &#8776;40 &#181;g&#183;L-1 for verification analysis using ESI-MS and HPLC-ICP-MS, respectively.</li>
		<li>Analyze m/z of DMDTAV using ESI-MS11,16 and fragments at m/z 171 in the positive-ion mode or at m/z 169 in the negative-ion mode (Table 1). 
		NOTE: See reference values of m/z (Table 1).</li>
		<li>Analyze the chromatogram of DMDTAV in the stock solution using HPLC-ICP-MS11,16,17 with appropriate eluent conditions and confirm that a peak is found at the retention time described in the literature. 
		NOTE: Purity of synthesized DMDTAV should be calculated using the analysis results from step 2.3.5.</li>
		<li>Analyze the total arsenic concentration using ICP-MS after acid digestion11 and calculate the true DMDTAV concentration in synthesized DMDTAV stock solution using dilution factors and purity as the following equation: 
		Analyzed total As concentration (&#181;g&#183;L-1) &#183; Dilution factor &#183; Purity (%) = True DMDTAV concentration of DMDTAV in stock solution (&#181;g&#183;L-1)</li>
		<li>Store DMDTAV stock solution at 4 &#176;C in the dark for further quantitative speciation analysis18.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

The developed protocol has clarified critical steps that previous studies1,3,4,8,15 omitted or abbreviated, which may have led to difficulties with or failure during DMMTAV and DMDTAV synthesis. As DMMTAV is oxidation-sensitive1,5, chemical reagents for its synthesis were prepared using N2-purged deionized water (steps 1.1.1 - 1.1.3) to prevent the possible retardation of DMAV thiolation and the oxidation of DMMTAV. DMMTAV prepared using this protocol had a purity of 92%. The reference DMDTAV extraction method featured silica-based C18 column extraction with ammonium acetate or phosphate buffer solution as an eluent,3,4,7,8 with the purity of the thus prepared DMDTAV varying depending on the column compartments used, which is not described in references studies3,4,8. In contrast, the use of disposable SPE in the developed protocol (step 2.2.1) allowed the extraction of 88% pure DMDTAV. In addition, previous methods of crystallizing DMDTAV referenced only a freeze-drying procedure20, whereas in this protocol, simple drying of the extracted DMDTAV solution in an atmosphere of N2 inside the glove box (step 2.2.3) produced a white residue of crystalized dimethyldithioarsinate.
Identity verification of the synthesized DMMTAV and DMDTAV is a critical step for determining ideal standard chemical. In our previous work16 based on this protocol, major fragments at m/z 155 in the positive-ion mode and m/z 169 in the negative-ion mode were detected by ESI-MS analysis of as-synthesized DMMTAV and DMDTAV, respectively. The latter fragment was ascribed to [CH3]2AsS(=S)S]- (i.e., [M-H]-), in good agreement with previous results5,17,20,22, whereas the fragment at m/z 155 was assigned to [CH3]2As(=S)(OH) + H]+ (i.e., [M+H]+), again, in agreement with references13,14,17,19,21,24. Since ESI-MS analysis cannot be used as the only verification method, major peaks in the chromatograms of DMMTAV and DMDTAV stock solutions obtained by HPLC-ICP-MS were compared to those reported by Li et al.17 (Figure 1). Although DMAIII is known as an intermediate produced in the initial stage of DMMTAV formation1,7,8,25, it exhibits low stability, disappearing within 70 min19 and therefore is not detectable by this procedure (Figure 1).
Another goal of this study was to suggest storage conditions for DMMTAV and DMDTAV stock solutions with fewer impurities to prevent sulfide-to-oxide conversion and thus achieve greater stability18. The storage conditions used here (i.e., 4 &#730;C in the dark) allowed the preservation of the synthesized DMMTAV and DMDTAV as major species in stock solutions (Figure 2) for four weeks, with no drastic decomposition observed even after 13 weeks. Although the solvent pH (i.e., that of deionized water) may affect the transformation of species during storage due to the presence of native10 and excess sulfide species originating from chemical reagents such as HS- or H2S, the stock solutions maintained neutral pH without significant transformation of the species contained therein (Figure 2). Therefore, stock solutions could be stored at 4 &#730;C in the dark for 13 weeks prior to quantitative speciation analysis.
In this study, the reference molar ratio mixing method of synthesizing DMMTAV and DMDTAV 1,3,4,8,15 was modified to produce stable DMMTAV and DMDTAV stock solutions for HPLC-ICP-MS quantitative analysis. Due to a lack of critical step details, including not only chemical reagent preparation, extraction, and crystallization steps, but also the storage conditions of DMMTAV and DMDTAV, stock solutions had to be modified and optimized. Therefore, stock solutions prepared using this protocol are sufficiently applicable for quantitative analysis of DMMTAV and DMDTAV for environmental monitoring purposes.

Since dimethylarsinic acid (DMAV) has been demonstrated to exhibit both acute toxicity and genotoxicity due to undergoing methylation and thiolation upon ingestion1,2, the metabolic pathway of arsenic thiolation has been intensively studied both in vitro and in vivo3,4 as well as in environmental media (e.g., landfill leachate)5,6. Previous studies have found both reduced and thiolated analogs of DMAV in living cells, for example, dimethylarsinous acid (DMAIII), dimethylmonothioarsinic acid (DMMTAV), and dimethyldithioarsinic acid (DMDTAV)7,8,9, with dimethylated thioarsenicals such as DMMTAV exhibiting greater toxicity than other known inorganic or organic arsenicals10. The abundance of highly toxic thioarsenicals has serious environmental implications, since they may pose a risk to humans and the environment under highly sulfidic conditions11. However, the mechanisms of DMMTAV and DMDTAV (trans)formation and their fates in environmental media still require further study. Thus, the quantitative analysis of thioarsenicals is required to improve understanding of the environmental effects of DMMTAV and DMDTAV.
Although standard chemicals are the key requirement for quantitative analysis, the standards of DMMTAV and DMDTAV are difficult to obtain by replicating previous studies, owing to the high risk of species transformation into other species and unstandardized synthesis procedures12. Moreover, the methods referenced have limitations that may lead to practical difficulties in synthesizing the standard chemicals and performing quantitative analysis. DMMTAV and DMDTAV are commonly prepared by mixing DMAV, Na2S, and H2SO4 in a certain molar ratio1 or bubbling H2S gas through a solution of DMAV 13,14. The bubbling method features substitution of oxygen by sulfur using a direct supply of H2S gas, which, is highly toxic and difficult to control for an inexperienced user. Conversely, the above mixing method1, widely used for the qualitative analysis of DMMTAV and DMDTAV in environmental sudies5,6,12, features the thiolation of DMAV with H2S generated by mixing Na2S and H2SO4 and produces DMMTAV and DMDTAV, allowing easier stoichiometric control to produce target chemicals, as compared to the direct use of H2S gas.
The reference mixing method procedures1,3,4,8,15 mentioned in this study exhibit limitations in some of their critical experimental steps, which might lead to experimental failure. For example, the details of specific solvent (i.e., deionized water) preparation and the extraction and crystallization of the synthesized arsenicals are over-abbreviated or not described in sufficient detail. Such dispersed and limited information on procedural steps might lead to the inconsistent formation of thioarsenicals and unreliable quantification analysis. Therefore, the modified protocol developed herein describes the synthesis of DMMTAV and DMDTAV stock solutions with quantitative species separation analysis.

<table><tbody><tr><td>Cacodylic acid</td><td>Sigma-Aldrich</td><td>20835-10G-F</td><td></td></tr><tr><td>Sodium sulfide nonahydrate</td><td>Sigma-Aldrich</td><td>S2006-500G</td><td></td></tr><tr><td>Sulfuric acid 96%</td><td>J.T.Baker</td><td>0000011478</td><td></td></tr><tr><td>Ammonium acetate</td><td>Sigma-Aldrich</td><td>A7262-500G</td><td></td></tr><tr><td>Formic acid 98%</td><td>Wako Pure Chemical Industries, Ltd.</td><td>066-00461</td><td></td></tr><tr><td>Diethyl ether (Extra Pure)</td><td>Junsei Chemical</td><td>33475-0380</td><td></td></tr><tr><td>Adapter cap for 60 mL Bond Elut catridges</td><td>Agilent Technologies</td><td>12131004</td><td>Syringe type of SPE</td></tr><tr><td>Bond Elut C18 cartridge</td><td>Agilent Technologies</td><td>14256031</td><td>Syringe type of SPE</td></tr><tr><td>HyPURITY C-18</td><td>Thermo Scientific</td><td>22105-254630</td><td>5 um, 125 x 4.6 mm</td></tr><tr><td>Glovebox</td><td>Chungae-chun, Rep. of Korea</td><td></td><td>Customized&amp;nbsp;</td></tr><tr><td>Agilent 1260 Infinity Bio-inert LC</td><td>Agilent Technologies</td><td>DEAB600252, DEACH00245</td><td></td></tr><tr><td>Agilent Technologies 7700 Series ICP-MS</td><td>Agilent Technologies</td><td>JP12031510</td><td></td></tr><tr><td>Finnigan LCQ Deca XP MAX Mass Spectrometer System</td><td>Thermo Electron Corporation</td><td>LDM10627</td><td></td></tr></tbody></table>

preparation of dmmtav and dmdtav using dmav for environmental applications: synthesis, purification, and confirmation

Dimethylated thioarsenicals such as dimethylmonothioarsinic acid (DMMTAV) and dimethyldithioarsinic acid (DMDTAV), which are produced by the metabolic pathway of dimethylarsinic acid (DMAV) thiolation, have been recently found in the environment as well as human organs. DMMTAV and DMDTAV can be quantified to determine the ecological effects of dimethylated thioarsenicals and their stability in environmental media. The synthesis method for these compounds is unstandardized, making replicating previous studies challenging. Furthermore, there is a lack of information about storage techniques, including storage of compounds without species transformation. Moreover, because only limited information about synthesis methods is available, there may be experimental difficulties in synthesizing standard chemicals and performing quantitative analysis. The protocol presented herein provides a practically modified synthesis method for the dimethylated thioarsenicals, DMMTAV and DMDTAV, and will help in the quantification of species separation analysis using high performance liquid chromatography in conjunction with inductively coupled plasma mass spectrometry (HPLC-ICP-MS). The experimental steps of this procedure were modified by focusing on the preparation of chemical reagents, filtration methods, and storage.

Since DMMTAV has been mistakenly prepared by the DMAIII synthesis method19, verification of synthesized DMMTAV and DMDTAV is a critical step for synthesis and extraction and determining the ideal standard chemical materials. Synthesized chemicals can be verified by the peak of DMMTAV (MW 154 g&#183;mol-1) and DMDTAV (MW 170 g&#183;mol-1) mass-to-charge ratio (m/z) using either the positive or negative ion mode of electrospray ionization-mass spectrometer (ESI-MS) through real-time injection. The reference values of m/z are listed in Table 119. Additional verification of the successful synthesis of DMMTAV and DMDTAV was conducted by comparing the species separation analysis results of the retention time (RT) of major peaks to reference data using HPLC-ICP-MS. Figure 1 shows similar RT of major and minor peaks, DMAV and DMMTAV (Figure 1a) or DMDTAV (Figure 1b), using 1.0 mL&#183;min-1 of 5 mM formic acid as an eluent and a C18 Liquid Chromatography (LC) column as described in17. Note that the RT of the major peaks may vary depending on the instrumental and eluent conditions, and which LC column is used. Species included in the stock solutions of DMMTAV and DMDTAV should be examined prior to every analysis, although this protocol suggests storage conditions of 4 &#730;C in the dark, which maintains synthesized DMMTAV and DMDTAV with, respectively, 2.2% and 5.8% transformation during the 13 weeks of analysis (Figure 2).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56603/56603fig1.jpg" /> 
Figure 1: HPLC-ICP-MS chromatogram of the synthesized DMMTAV and DMDTAV. 1: DMAV, 2: DMDTAV, and 3: DMMTAV were measured as major peaks at 3.8 min, 5.9 min, and 8.0 min in each stock solutions of a: DMMTAV and b: DMDTAV, which were corresponded to those reported by Li et al. in 201017. Instrumental conditions of ICP-MS were 1550 V RF power and 50 &#181;L injection volume. A C18 column was used with 5 mM formic acid as an eluent17. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56603/56603fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56603/56603fig2.jpg" /> 
Figure 2: Stability of DMMTAV and DMDTAV at 4 &#730;C in the dark. Percentage changes of As species distribution in each of the stock solutions DMMTAV (a) and DMDTAV (b) for 13 weeks. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56603/56603fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Synthesized stock solution&#160;</td>
			<td>ion mode</td>
			<td>Fragments&#160;</td>
			<td>m/z</td>
			<td>References</td>
		</tr>
		<tr>
			<td rowspan="7">DMMTAV</td>
			<td rowspan="4">Positive</td>
			<td>[Me2As(SH)OH]+</td>
			<td>155</td>
			<td>13,14,19,17,21,24</td>
		</tr>
		<tr>
			<td>[Me2As(OH)2]+</td>
			<td>139</td>
			<td>19,17</td>
		</tr>
		<tr>
			<td>[Me2AsS]+</td>
			<td>137</td>
			<td>13,19,17,24</td>
		</tr>
		<tr>
			<td>[Me2As(S)SAsMe2]+</td>
			<td>275</td>
			<td>13</td>
		</tr>
		<tr>
			<td rowspan="3">Negative</td>
			<td>[Me2AsOS]-</td>
			<td>153</td>
			<td>17,5,23</td>
		</tr>
		<tr>
			<td>[MeAsSO]-</td>
			<td>138</td>
			<td>17,5</td>
		</tr>
		<tr>
			<td>[AsSO]-</td>
			<td>123</td>
			<td>17,5</td>
		</tr>
		<tr>
			<td rowspan="6">DMDTAV</td>
			<td rowspan="3">Positive</td>
			<td>[Me2As(SH)2]+</td>
			<td>171</td>
			<td>19,17</td>
		</tr>
		<tr>
			<td>[Me2As(SH)OH]+</td>
			<td>155</td>
			<td>19</td>
		</tr>
		<tr>
			<td>[Me2AsS]+</td>
			<td>137</td>
			<td>19,17</td>
		</tr>
		<tr>
			<td rowspan="3">Negative</td>
			<td>[Me2AsS2]-</td>
			<td>169</td>
			<td>20,17,5,22</td>
		</tr>
		<tr>
			<td>[MeAsS2]-</td>
			<td>154</td>
			<td>20,17,5</td>
		</tr>
		<tr>
			<td>[AsS2]-</td>
			<td>139</td>
			<td>17,5,22</td>
		</tr>
	</tbody>
</table>
Table 1: Suggested structure of synthesized DMMTAV and DMDTAV, and minor fragment ions by positive and/or negative ion mode of ESI-MS. The list of DMMTAV, DMDTAV, and minor fragments m/z measured by ESI-MS was reproduced from literature5,13,14,17,19,20,21,22,23,24. Note that m/z peaks may vary with instrumental conditions and/or matrices of the stock solutions.

Watch this Scientific Journal Video about Preparation of DMMTAV and DMDTAV Using DMAV for Environmental Applications: Synthesis, Purification, and Confirmation at JoVE.com

Preparation of DMMTAV and DMDTAV Using DMAV for Environmental Applications: Synthesis, Purification, and Confirmation

This article presents modified experimental protocols for dimethylmonothioarsinic acid (DMMTAV) and dimethyldithioarsinic acid (DMDTAV) synthesis, inducing dimethylarsinic acid (DMAV) thiolation through mixing of DMAV, Na2S, and H2SO4. The modified protocol provides an experimental guideline, thereby overcoming limitations of the synthesis steps that could have caused experimental failures in quantitative analysis.

Preparation of DMMTAV and DMDTAV Using DMAV for Environmental Applications: Synthesis, Purification, and Confirmation

Dimethylated thioarsenicals such as dimethylmonothioarsinic acid (DMMTAV) and dimethyldithioarsinic acid (DMDTAV), which are produced by the metabolic ...

preparation-dmmtav-dmdtav-using-dmav-for-environmental-applications

Nanyang Technological University

Research

JoVE Journal

Environment

5.5K Views.  Korea Basic Science Institute, Seoul Center. Dit artikel presenteert een gewijzigde experimentele protocollen voor dimethylmonothioarsinic zuur (DMMTAV) en dimethyldithioarsinic zuur (DMDTAV) synthese, dimethylarsinic zuur (DMAV) thiolation door het mengen van DMAV inducerende , Na2S en H2zo4. Het gewijzigde protocol biedt een experimentele richtsnoer, waardoor het overwinnen van beperkingen van de synthese stappen die hebben geleid experim...

Video: Preparation of DMMTAV and DMDTAV Using DMAV for Environmental Applications: Synthesis, Purification, and Confirmation

Controlling the Size, Shape and Stability of Supramolecular Polymers in Water

For aqueous based supramolecular polymers, the simultaneous control over shape, size and stability is very difficult1. At the same time, the ability to do so is highly important in view of a number of applications in functional soft matter including electronics, biomedical engineering, and sensors. In the past, successful strategies to control the size and shape of supramolecular polymers typically focused on the use of templates2,3, end cappers4 or selective solvent techniques5. 
Here we disclose a strategy based on self-assembling discotic amphiphiles that leads to the control over stack length and shape of ordered, chiral columnar aggregates. By balancing electrostatic repulsive interactions on the hydrophilic rim and attractive non-covalent forces within the hydrophobic core of the polymerizing building block, we manage to create small and discrete spherical objects6,7. Increasing the salt concentration to screen the charges induces a sphere-to-rod transition. Intriguingly, this transition is expressed in an increase of cooperativity in the temperature-dependent self-assembly mechanism, and more stable aggregates are obtained. 
For our study we select a benzene-1,3,5-tricarboxamide (BTA) core connected to a hydrophilic metal chelate via a hydrophobic, fluorinated L-phenylalanine based spacer (Scheme 1). The metal chelate selected is a Gd(III)-DTPA complex that contains two overall remaining charges per complex and necessarily two counter ions. The one-dimensional growth of the aggregate is directed by &pi;-&pi; stacking and intermolecular hydrogen bonding. However, the electrostatic, repulsive forces that arise from the charges on the Gd(III)-DTPA complex start limiting the one-dimensional growth of the BTA-based discotic once a certain size is reached. At millimolar concentrations the formed aggregate has a spherical shape and a diameter of around 5 nm as inferred from 1H-NMR spectroscopy, small angle X-ray scattering, and cryogenic transmission electron microscopy (cryo-TEM). The strength of the electrostatic repulsive interactions between molecules can be reduced by increasing the salt concentration of the buffered solutions. This screening of the charges induces a transition from spherical aggregates into elongated rods with a length &gt; 25 nm. Cryo-TEM allows to visualise the changes in shape and size. In addition, CD spectroscopy permits to derive the mechanistic details of the self-assembly processes before and after the addition of salt. Importantly, the cooperativity -a key feature that dictates the physical properties of the produced supramolecular polymers- increases dramatically upon screening the electrostatic interactions. This increase in cooperativity results in a significant increase in the molecular weight of the formed supramolecular polymers in water.

The goal of this experiment is to determine and control the size, shape and stability of self-assembled discotic amphiphiles in water. For aqueous based supramolecular polymers such level of control is very difficult. We apply a strategy using both repulsive and attractive interactions. The experimental techniques applied to characterize this system are broadly applicable.

For aqueous based supramolecular polymers, the simultaneous control over shape, size and stability is very difficult1. At the same time, the ability to do ...

Engineering

Production of Disulfide-stabilized Transmembrane Peptide Complexes for Structural Studies

Physical interactions among the lipid-embedded alpha-helical domains of membrane proteins play a crucial role in folding and assembly of membrane protein complexes and in dynamic processes such as transmembrane (TM) signaling and regulation of cell-surface protein levels. Understanding the structural features driving the association of particular sequences requires sophisticated biophysical and biochemical analyses of TM peptide complexes. However, the extreme hydrophobicity of TM domains makes them very difficult to manipulate using standard peptide chemistry techniques, and production of suitable study material often proves prohibitively challenging. Identifying conditions under which peptides can adopt stable helical conformations and form complexes spontaneously adds a further level of difficulty. Here we present a procedure for the production of homo- or hetero-dimeric TM peptide complexes from materials that are expressed in E. coli, thus allowing incorporation of stable isotope labels for nuclear magnetic resonance (NMR) or non-natural amino acids for other applications relatively inexpensively. The key innovation in this method is that TM complexes are produced and purified as covalently associated (disulfide-crosslinked) assemblies that can form stable, stoichiometric and homogeneous structures when reconstituted into detergent, lipid or other membrane-mimetic materials. We also present carefully optimized procedures for expression and purification that are equally applicable whether producing single TM domains or crosslinked complexes and provide advice for adapting these methods to new TM sequences.

Biophysical and biochemical studies of interactions among membrane-embedded protein domains face many technical challenges, the first of which is obtaining appropriate study material. This article describes a protocol for producing and purifying disulfide-stabilized transmembrane peptide complexes that are suitable for structural analysis by solution nuclear magnetic resonance (NMR) and other analytical applications.

Physical  interactions among the lipid-embedded alpha-helical domains of membrane  proteins play a crucial role in folding and assembly of membrane ...

Chemistry

Compact Quantum Dots for Single-molecule Imaging

Single-molecule imaging is an important tool for understanding the mechanisms of biomolecular function and for visualizing the spatial and temporal heterogeneity of molecular behaviors that underlie cellular biology 1-4. To image an individual molecule of interest, it is typically conjugated to a fluorescent tag (dye, protein, bead, or quantum dot) and observed with epifluorescence or total internal reflection fluorescence (TIRF) microscopy. While dyes and fluorescent proteins have been the mainstay of fluorescence imaging for decades, their fluorescence is unstable under high photon fluxes necessary to observe individual molecules, yielding only a few seconds of observation before complete loss of signal. Latex beads and dye-labeled beads provide improved signal stability but at the expense of drastically larger hydrodynamic size, which can deleteriously alter the diffusion and behavior of the molecule under study.
Quantum dots (QDs) offer a balance between these two problematic regimes. These nanoparticles are composed of semiconductor materials and can be engineered with a hydrodynamically compact size with exceptional resistance to photodegradation 5. Thus in recent years QDs have been instrumental in enabling long-term observation of complex macromolecular behavior on the single molecule level. However these particles have still been found to exhibit impaired diffusion in crowded molecular environments such as the cellular cytoplasm and the neuronal synaptic cleft, where their sizes are still too large 4,6,7.
Recently we have engineered the cores and surface coatings of QDs for minimized hydrodynamic size, while balancing offsets to colloidal stability, photostability, brightness, and nonspecific binding that have hindered the utility of compact QDs in the past 8,9. The goal of this article is to demonstrate the synthesis, modification, and characterization of these optimized nanocrystals, composed of an alloyed HgxCd1-xSe core coated with an insulating CdyZn1-yS shell, further coated with a multidentate polymer ligand modified with short polyethylene glycol (PEG) chains (Figure 1). Compared with conventional CdSe nanocrystals, HgxCd1-xSe alloys offer greater quantum yields of fluorescence, fluorescence at red and near-infrared wavelengths for enhanced signal-to-noise in cells, and excitation at non-cytotoxic visible wavelengths. Multidentate polymer coatings bind to the nanocrystal surface in a closed and flat conformation to minimize hydrodynamic size, and PEG neutralizes the surface charge to minimize nonspecific binding to cells and biomolecules. The end result is a brightly fluorescent nanocrystal with emission between 550-800 nm and a total hydrodynamic size near 12 nm. This is in the same size range as many soluble globular proteins in cells, and substantially smaller than conventional PEGylated QDs (25-35 nm).

We describe the preparation of colloidal quantum dots with minimized hydrodynamic size for single-molecule fluorescence imaging. Compared to conventional quantum dots, these nanoparticles are similar in size to globular proteins and are optimized for single-molecule brightness, stability against photodegradation, and resistance to nonspecific binding to proteins and cells.

Single-molecule imaging is an important tool for  understanding the mechanisms of biomolecular function and for visualizing the  spatial and temporal ...

Biochemical Measurement of Neonatal Hypoxia

Neonatal hypoxia ischemia is characterized by inadequate blood perfusion of a tissue or a systemic lack of oxygen. This condition is thought to cause/exacerbate well documented neonatal disorders including neurological impairment 1-3. Decreased adenosine triphosphate production occurs due to a lack of oxidative phosphorylation. To compensate for this energy deprived state molecules containing high energy phosphate bonds are degraded 2. This leads to increased levels of adenosine which is subsequently degraded to inosine, hypoxanthine, xanthine, and finally to uric acid. The final two steps in this degradation process are performed by xanthine oxidoreductase. This enzyme exists in the form of xanthine dehydrogenase under normoxic conditions but is converted to xanthine oxidase (XO) under hypoxia-reperfusion circumstances 4, 5. Unlike xanthine dehydrogenase, XO generates hydrogen peroxide as a byproduct of purine degradation 4, 6. This hydrogen peroxide in combination with other reactive oxygen species (ROS) produced during hypoxia, oxidizes uric acid to form allantoin and reacts with lipid membranes to generate malondialdehyde (MDA) 7-9. Most mammals, humans exempted, possess the enzyme uricase, which converts uric acid to allantoin. In humans, however, allantoin can only be formed by ROS-mediated oxidation of uric acid. Because of this, allantoin is considered to be a marker of oxidative stress in humans, but not in the mammals that have uricase.
We describe methods employing high pressure liquid chromatography (HPLC) and gas chromatography mass spectrometry (GCMS) to measure biochemical markers of neonatal hypoxia ischemia. Human blood is used for most tests. Animal blood may also be used while recognizing the potential for uricase-generated allantoin. Purine metabolites were linked to hypoxia as early as 1963 and the reliability of hypoxanthine, xanthine, and uric acid as biochemical indicators of neonatal hypoxia was validated by several investigators 10-13. The HPLC method used for the quantification of purine compounds is fast, reliable, and reproducible. The GC/MS method used for the quantification of allantoin, a relatively new marker of oxidative stress, was adapted from Gruber et al 7. This method avoids certain artifacts and requires low volumes of sample. Methods used for synthesis of MMDA were described elsewhere 14, 15. GC/MS based quantification of MDA was adapted from Paroni et al. and Cighetti et al. 16, 17. Xanthine oxidase activity was measured by HPLC by quantifying the conversion of pterin to isoxanthopterin 18. This approach proved to be sufficiently sensitive and reproducible.

A method is described to measure biochemical markers of neonatal hypoxia-ischemia. The approach utilizes high pressure liquid chromatography (HPLC) and Gas Chromatography Mass Spectrometry (GC/MS).

Neonatal hypoxia ischemia is characterized by inadequate blood perfusion of a tissue or a systemic lack of oxygen. This condition is thought to ...

Medicine

Preparation and In Vitro Characterization of Dendrimer-based Contrast Agents for Magnetic Resonance Imaging

Paramagnetic complexes of gadolinium(III) with acyclic or macrocyclic chelates are the most commonly used contrast agents (CAs) for magnetic resonance imaging (MRI). Their purpose is to enhance the relaxation rate of water protons in tissue, thus increasing the MR image contrast and the specificity of the MRI measurements. Current clinically approved contrast agents are low molecular weight molecules that are rapidly cleared from the body. The use of dendrimers as carriers of paramagnetic chelators can play an important role in the future development of more efficient MRI contrast agents. Specifically, the increase in local concentration of the paramagnetic species results in a higher signal contrast. Furthermore, this CA provides a longer tissue retention time due to its high molecular weight and size. Here, we demonstrate a convenient procedure for the preparation of macromolecular MRI contrast agents based on poly(amidoamine) (PAMAM) dendrimers with monomacrocyclic DOTA-type chelators (DOTA &#8211; 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate). The chelating unit was appended by exploiting the reactivity of the isothiocyanate (NCS) group towards the amine surface groups of the PAMAM dendrimer to form thiourea bridges. Dendrimeric products were purified and analyzed by means of nuclear magnetic resonance spectroscopy, mass spectrometry, and elemental analysis. Finally, high resolution MR images were recorded and the signal contrasts obtained from the prepared dendrimeric and the commercially available monomeric agents were compared.

Preparation and In Vitro Characterization of Dendrimer-based Contrast Agents for Magnetic Resonance Imaging

This protocol describes the preparation and characterization of a dendrimeric magnetic resonance imaging (MRI) contrast agent that carries cyclen-based macrocyclic chelates coordinating paramagnetic gadolinium ions. In a series of MRI experiments in vitro, this agent produced an amplified MRI signal when compared to the commercially available monomeric analogue.

Paramagnetic complexes of gadolinium(III) with acyclic or macrocyclic chelates are the most commonly used contrast agents (CAs) for magnetic resonance ...

Continuous Flow Chemistry: Reaction of Diphenyldiazomethane with p-Nitrobenzoic Acid

Continuous flow technology has been identified as instrumental for its environmental and economic advantages leveraging superior mixing, heat transfer and cost savings through the &#34;scaling out&#34; strategy as opposed to the traditional &#34;scaling up&#34;. Herein, we report the reaction of diphenyldiazomethane with p-nitrobenzoic acid in both batch and flow modes. To effectively transfer the reaction from batch to flow mode, it is essential to first conduct the reaction in batch. As a consequence, the reaction of diphenyldiazomethane was first studied in batch as a function of temperature, reaction time, and concentration to obtain kinetic information and process parameters. The glass flow reactor set-up is described and combines two types of reaction modules with &#34;mixing&#34; and &#34;linear&#34; microstructures. Finally, the reaction of diphenyldiazomethane with p-nitrobenzoic acid was successfully conducted in the flow reactor, with up to 95% conversion of the diphenyldiazomethane in 11 min. This proof of concept reaction aims to provide insight for scientists to consider flow technology&#39;s competitiveness, sustainability, and versatility in their research.

Continuous Flow Chemistry: Reaction of Diphenyldiazomethane with p-Nitrobenzoic Acid

Flow chemistry carries environmental and economic advantages by leveraging superior mixing, heat transfer and cost benefits. Herein, we provide a blueprint to transfer chemical processes from batch to flow mode. The reaction of diphenyldiazomethane (DDM) with p-nitrobenzoic acid, conducted in batch and flow, was chosen for proof of concept.

Continuous flow technology has been identified as instrumental for its environmental and economic advantages leveraging superior mixing, heat transfer and ...

Fabricating Reactive Surfaces with Brush-like and Crosslinked Films of Azlactone-Functionalized Block Co-Polymers

In this paper, fabrication methods that generate novel surfaces using the azlactone-based block co-polymer, poly (glycidyl methacrylate)-block-poly (vinyl dimethyl azlactone) (PGMA-b-PVDMA), are presented. Due to the high reactivity of azlactone groups towards amine, thiol, and hydroxyl groups, PGMA-b-PVDMA surfaces can be modified with secondary molecules to create chemically or biologically functionalized interfaces for a variety of applications. Previous reports of patterned PGMA-b-PVDMA interfaces have used traditional top-down patterning techniques that generate non-uniform films and poorly controlled background chemistries. Here, we describe customized patterning techniques that enable precise deposition of highly uniform PGMA-b-PVDMA films in backgrounds that are chemically inert or that have biomolecule-repellent properties. Importantly, these methods are designed to deposit PGMA-b-PVDMA films in a manner that completely preserves azlactone functionality through each processing step. Patterned films show well-controlled thicknesses that correspond to polymer brushes (~90 nm) or to highly crosslinked structures (~1-10 &#956;m). Brush patterns are generated using either the parylene lift-off or interface directed assembly methods described and are useful for precise modulation of overall chemical surface reactivity by adjusting either the PGMA-b-PVDMA pattern density or the length of the VDMA block. In contrast, the thick, crosslinked PGMA-b-PVDMA patterns are obtained using a customized micro-contact printing technique and offer the benefit of higher loading or capture of secondary material due to higher surface area to volume ratios. Detailed experimental steps, critical film characterizations, and trouble-shooting guides for each fabrication method are discussed.

Surface fabrication methods for patterned deposition of nanometer thick brushes or micron thick, crosslinked films of an azlactone block co-polymer are reported. Critical experimental steps, representative results, and limitations of each method are discussed. These methods are useful for creating functional interfaces with tailored physical features and tunable surface reactivity.

In this paper, fabrication methods that generate novel surfaces using the azlactone-based block co-polymer, poly (glycidyl methacrylate)-block-poly (vinyl ...

Exploring the Arginine Methylome by Nuclear Magnetic Resonance Spectroscopy

Protein-bound arginine is commonly methylated in many proteins and regulates their function by altering the physicochemical properties, their interaction with other molecules, including other proteins or nucleic acids. This work presents an easily implementable protocol for quantifying arginine and its derivatives, including asymmetric and symmetric dimethylarginine (ADMA and SDMA, respectively) and monomethyl arginine (MMA). Following protein isolation from biological body fluids, tissues, or cell lysates, a simple method for homogenization, precipitation of proteins, and protein hydrolysis is described. Since the hydrolysates contain many other components, such as other amino acids, lipids, and nucleic acids, a purification step using solid-phase extraction (SPE) is essential. SPE can either be performed manually using centrifuges or a pipetting robot. The sensitivity for ADMA using the current protocol is about 100 nmol/L. The upper limit of detection for arginine is 3 mmol/L due to SPE saturation. In summary, this protocol describes a robust method, which spans from biological sample preparation to NMR-based detection, providing valuable hints and pitfalls for successful work when studying the arginine methylome.

The present protocol describes the preparation and quantitative measurement of free and protein-bound arginine and methyl-arginines by 1H-NMR spectroscopy.

Protein-bound arginine is commonly methylated in many proteins and regulates their function by altering the physicochemical properties, their interaction ...

Cancer Research

Evaluation of Oxidative Stress in Biological Samples Using the Thiobarbituric Acid Reactive Substances Assay

Despite its limited analytical specificity and ruggedness, the thiobarbituric acid reactive substances (TBARS) assay has been widely used as a generic metric of lipid peroxidation in biological fluids. It is often considered a good indicator of the levels of oxidative stress within a biological sample, provided that the sample has been properly handled and stored. The assay involves the reaction of lipid peroxidation products, primarily malondialdehyde (MDA), with thiobarbituric acid (TBA), which leads to the formation of MDA-TBA2 adducts called TBARS. TBARS yields a red-pink color that can be measured spectrophotometrically at 532 nm. The TBARS assay is performed under acidic conditions (pH = 4) and at 95 &#176;C. Pure MDA is unstable, but these conditions allow the release of MDA from MDA bis(dimethyl acetal), which is used as the analytical standard in this method. The TBARS assay is a straightforward method that can be completed in about 2 h. Preparation of assay reagents are described in detail here. Budget-conscious researchers can use these reagents for multiple experiments at a low cost rather than buying an expensive TBARS assay kit that only permits construction of a single standard curve (and thus can only be used for one experiment). The applicability of this TBARS assay is shown in human serum, low density lipoproteins, and cell lysates. The assay is consistent and reproducible, and limits of detection of 1.1 &#956;M can be reached. Recommendations for the use and interpretation of the spectrophotometric TBARS assay are provided.

The goal of the thiobarbituric acid reactive substances assay is to assess oxidative stress in biological samples by measuring the production of lipid peroxidation products, primarily malondialdehyde, using visible wavelength spectrophotometry at 532 nm. The method described here can be applied to human serum, cell lysates, and low density lipoproteins.

Despite its limited analytical specificity and ruggedness, the thiobarbituric acid reactive substances (TBARS) assay has been widely used as a generic ...

Trimethylamine Detection in Animal-Derived Medicine by Headspace GC-MS

An Improved Technique for Trimethylamine Detection in Animal-Derived Medicine by Headspace Gas Chromatography-Tandem Quadrupole Mass Spectrometry

Animal-derived medicines have distinctive characteristics and significant curative effects, but most of them have an obvious fishy odor, resulting in the poor compliance of clinical patients. Trimethylamine (TMA) is one of the key fishy odor components in animal-derived medicine. It is difficult to identify TMA accurately using the existing detection method due to the increased pressure in the headspace vial caused by the rapid acid-base reaction after the addition of lye, which causes TMA to escape from the headspace vial, stalling the research progress of the fishy odor of animal-derived medicine. In this study, we proposed a controlled detection method that introduced a paraffin layer as an isolation layer between acid and lye. The rate of TMA production could be effectively controlled by slowly liquefying the paraffin layer through thermostatic furnace heating. This method showed satisfactory linearity, precision experiments, and recoveries with good reproducibility and high sensitivity. It provided technical support for the deodorization of animal-derived medicine.

Author Spotlight: An Improved Technique for Trimethylamine Detection in Animal-Derived Medicine by Headspace Gas Chromatography-Tandem Quadrupole Mass Spectrometry  

Here, a headspace gas chromatography-tandem quadrupole mass spectrometry (HS-GC-MS/MS) method suitable for the determination of trimethylamine (TMA) in animal-derived medicines is described. The protocol includes sample pretreatment, headspace treatment, analysis conditions, methodological validation, and the determination of TMA in animal-derived medicines.

Animal-derived medicines have distinctive characteristics and significant curative effects, but most of them have an obvious fishy odor, resulting in the ...

Preparation of DMMTA^V and DMDTA^V Using DMA^V for Environmental Applications: Synthesis, Purification, and Confirmation

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