Arsenobetaine Solution

Arsenobetaine calibrant is not commercially available and had to be synthesized according to the following scheme  

The solid collected at the end of the first step of the reaction was identified as MesAs CHaCOOEt by NMR in CD3OD. No organic impurity was detected. The second step enabled a solid to be obtained, which was further purified using acetone, and dried under vacuum this solid was stored under dry nitrogen to avoid water contamination. A yield of 87% was achieved. 

The characterization of arsenobetaine was performed using element analysis, H NMR, mass spectrometry, thermogravimetric/thermodifferential analysis and separative methods such as HPLC, GC and CZE. From all the results obtained it was concluded that the maximum amount of water present in the solid (if stored and handled under dry atmosphere) is 1% (w/w). In addition, arsenic impurities represent less than 0.15% (w/w) other impurities can be neglected. Therefore, the arsenobetaine purity of the calibrant obtained is 98.9%. 

A Stock solution was prepared by dissolving (accurate weighing) 29.367 g arsenobetaine in 2000.8 g double deionized water. Then 345.7 g of the solution were taken, poured into a 2 L bottle and completed to 2055.7 g with double deionized water (Milli-Q). This operation was repeated four times in order to obtain five stock solutions. Ten bottles of 2 L capacity were filled with 170.0 g of each stock solution and completed to 2000.0 g with double deionized water. Each bottle was used to fill penicillin-type flasks of 10 mL previously cleaned for at least one night and rinsed with double deionized water. This operation was carried out under a nitrogen flow. 

Arsenic concentrations were determined in the five stock solutions as well as in the 10 final solutions using ICP-AES. An independent method (EDXRF) was used to confirm the values obtained for these latter solutions. Calibrations were performed using calibrants of well defined purity. The mean value of concentrations found in the five stock solution was (1016 16) mg kg which was in good agreement with the calculated value of (1017 6) mg kg The mean As concentrations in the final solutions ranged between (431.6 7.0) mg kg and (435.0 11.4) mg kg [mean of the means for five series of 10 determinations (431.7 4.6) mg kg ], which agrees very well with the calculated value [(432.4 2.6) mg kg j. 

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Lagarde, F., Asfari, Z., Leroy, M.J.F., Demesmay, C., Olle, M., Lamotte, A., Leperchec, R and Maier, E.A. (1999b) Preparation of pure calibrants (arsenobetaine and arseno-choline) for arsenic speciation studies and certification of an arsenobetaine solution (CRM 626). Fresenius f. Anal. Chem., 363, 12. 

At present, only a few CRMs for arsenic species are available, these are from Institute for Reference Materials and Measurements (IRMM) BCR 626 (Arsenobetaine solution), certified 1031 6 mg kg , and BCR 627 (Tuna fish) certified 52 3 pmol kg arsenobetaine, 2.0 ... 

The tuna fish material (CRM 627) was prepared by the Joint Research Centre, Environment Institute, of Ispra (Italy) whereas the arsenobetaine solution (CRM 626) was prepared by the Laboratoire de Chimie Analytique et Minerale in Strasbourg (France) [134]. 

Arsenobetaine is zwitterionic in solution, displaying cationic properties at pH <3.5 (39). The first reported isolation of arsenobetaine was greatly facilitated by its retention on a cation-exchange resin and subsequent elution with dilute aqueous ammonia (48). Arsenocholine, however, remains cationic at high pH and requires forceful conditions (e.g., 6 M HC1) to displace it from cation-exchange resins—a property it shares with TeMA. 

Importantly, neither arsenobetaine nor arsenocholine forms an arsine on treatment with borohydride solutions. Consequently, arsenobetaine and arsenocholine may remain undetected in samples, seawater for example, when arsines are generated and determined in an arsenic speciation analysis. The technique HPLC/ICP-MS is most suitable for the analysis of these (non-arsine-forming) compounds (49). Use of the highly sensitive ICP-MS detector allows application of small quantities of material to the chromatography column, thereby obviating possible sample matrix effects previously observed for arsenobetaine (50). 

MW-assisted extraction was used for the extraction of As species from bsh tissue [28]. Quantitative extraction of As from the spiny dogbsh muscle (DORM-2) CRM was achieved using 5 percent TMAH solution with MW heating at 65°C in a closed-vessel MW system. The extracted As species were separated using both ion-exchange and ion-pair chromatography with ICP-MS detection. The DORM-2 CRM was analyzed for As along with three different varieties of f>sh purchased from a local market. In all samples, most of the As present was in the form of arsenobetaine, a basically nontoxic chemical species. 

The validation has the objective to identify, during the method development process, all sources of error and eliminate them or to quantify their contribution to the total uncertainty of the determination. For hydride generation techniques particular attention must be given to the quantitative transformation of all As species into hydrides (arsenobetaine, arsenosugars etc). Several types of adapted materials must be prepared to test all steps of the process (from simple calibrant solutions or mixtures to spiked fish tissue samples). If they exist CRMs should be used for validating trueness. Laboratory RMs must be prepared for the establishment of control charts when the method is under statistical control [27, 28]. 

E. A. Maier, C. Demesmay, M. Olle, A. Lamotte, F. Lagarde, R. Heimburger, M.J.F. Leroy, Z. Asfari and H. Muntau, The certification of the contents (mass fractions) of arsenobetaine in solution (CRM 626) and of total arsenic, arsenobetaine dimethylarsinic acid in tuna fish tissue (CRM 627), EUR Report, 17889 EN, European Commission, Brussels (1997). 

If lyophilized material is extracted with a methanol/phospate buffer solution and appropriate conditions are chosen the separation of arsenobetaine from MMA, DMA and inorganic arsenic might also be easily possible in human organs with an acceptable yield and detection limits at low [Pg.311]

Light is one of the major causes of instability of organometallic compounds. With the exception of some compounds e.g. arsenobetaine), the metal-carbon bond is generally weak enough to be photodegraded hence solutions containing organometallic compounds are recommended to be kept in the dark in opaque containers. 

The five solutions were found to be stable [except for As(III)] for four months if kept in the dark at +4 °C. Storage in the dark at +40 °C led to the formation of As(III) in some solutions. Arsenobetaine resulting from the degradation of arsenocholine was observed to occur significantly when solutions were stored at +20 °C in daylight but no trace of degradation was detected at +4 °C in the dark. 

The homogeneity was verified by repeated determinations of total As, arsenobetaine and DMA. The total As content was determined by HG-QFAAS after microwave assisted digestion, whereas DMA was determined by HPLC-ICP-MS. The within-bottle homogeneity was assessed by 10 determinations in each of two bottles, and the between-bottle homogeneity was evaluated by two determinations out of each 20 bottles the method uncertainty was evaluated by five replicate determinations of a digest or extract solution. The within- and between-bottle CV ranged from 0.5 to 1.2% for total As, from 2.1 to 5% for arsenobetaine, and from 7.1 to 10.6% for DMA, which was comparable to the method uncertainty (respectively 2.6, 3.1 and 6.8%) therefore, no inhomogeneity was suspected at a level of 0.3 g for total As and 1 g for As species, and the material was considered to be suitable for use as a CRM. 

Fig. 140. Chromatogram obtained in the case of USN-N2-MIP-MS with (1) a solution of arsenic acid (As(V)), methylarsenic acid (MA), dimethylarsenic acid (DMA), arsenobetaine (AB), arsenocholine (AC), trimethylarsine oxide (PMAO), and tetramethylarsonium ion (TMI)...

Figure 1. Chromatogram obtained with a solution of arsenate, arsenite, methylarsonic acid, dimethylarsinic acid, arsenobetaine, and arsenocholine (0.5 ng of As each species) in distilled water on a Supelcosil LC-SAX anion-exchange column (mobile phase 30 mM NH4H2PO4 with 1 % methanol at pH 3.75, injection volume 0.100 cm3, flow rate 1.5 cm3 min-l)...

Solution AsdU), As(V), arsenobetaine, arsenocholine water/ACN/acetic add 5 mM Na dodecyl-benzene-sulfonate... 

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