Boric acid-assisted anion-exchange chromatography for separating arsenic species

doi:10.1016/j.aca.2004.09.057

Analytica Chimica Acta

Volume 526, Issue 1, 15 November 2004, Pages 69-76

https://doi.org/10.1016/j.aca.2004.09.057 Get rights and content

Abstract

Separation of 11 arsenic species on a single column was achieved by anion-exchange high performance liquid chromatography (HPLC) and selective detection was provided by inductively coupled plasma mass spectrometry (ICPMS). Boric acid was used to selectively modify the retention of arsenosugars. The borate–arsenosugar complexation improved the separation of the following 11 arsenic compounds: tetramethylarsonium ion (TMI), arsenobetaine (AsB), arsenate, arsenite, dimethylarsinic acid (DMA), monomethylarsonic acid (MMA), arsenosugars 1–4, and an unidentified arsenic compound. The optimised mobile phase composition was 10 mM ammonium phosphate, 30 mM boric acid at pH 8.5. Rice and rice cereal extracts were analyzed using this method revealing the presence of arsenite, arsenate, DMA, and an unidentified arsenic species that has the same retention time as that of arsenosugar 2. Relative standard deviation of retention time from analyses of arsenic in different sample matrices, Fucus serratus, rice, rice cereal, oyster, dogfish, and mushroom extracts, was less than 1.0%, demonstrating the robustness of the separation method.

Introduction

Arsenic is one of the oldest known poisons. Chronic exposure to high doses of inorganic arsenic has been linked to skin and various internal cancers, cardiovascular, and neurological effects [1]. The effects of exposure to chronic low doses are still under investigation. The primary method of arsenic exposure in humans is drinking contaminated water and consuming foods high in arsenic. A challenge with determining the toxicity of arsenic in drinking water and food samples is that arsenic can exist in a variety of different forms, each possessing their own level of toxicity [2].

Arsenite (AsIII, As(OH)₃) and arsenate (AsV, AsO(OH)₃) are both highly toxic forms of inorganic arsenic found in water and food samples. Arsenobetaine (AsB, (CH₃)₃As⁺CH₂COO⁻), however, possesses negligible toxicity and is commonly found in fish and crustaceans. Other arsenic species with intermediate levels of acute toxicity include monomethylarsonic acid (MMA, (CH₃)AsO(OH)₂), dimethylarsinic acid (DMA, (CH₃)₂AsO(OH)), and tetramethylarsonium ion (TMI, (CH₃)₄As⁺). A family of arsenic species found primarily in marine plant species is arsenosugars. Arsenosugars 1–4 (see Scheme 1) are the most common corresponding to glycerol–ribose (1), phosphate–ribose (2), sulfonate–ribose (3), and sulfate–ribose (4) structures, respectively [3], [4]. While the acute toxicities of these arsenosugars are believed to be relatively low, reports of DMA as a metabolite have caused concern [5], [6], [7], [8]. Progress is being made toward the characterization of arsenosugar metabolites in urine [5], [6], [7], [8] as well as their toxicity on mammalian cells [9]. Also, work has been done to investigate the chemical stability of arsenosugars during digestion with simulated gastric fluid [10].

While much research has focused on characterizing arsenic species in marine foods, there has been limited research on the distribution of arsenic in terrestrial foods, such as rice [11], [12], yam [12], carrots [13], [14], and apples [15]. Contaminated ground water and the historic use of arsenic-containing pesticides have increased the risk of arsenic contamination in rice [16], [17]. Second to seafood, rice products were found to be a major dietary source of arsenic in the United States [18], [19] and Europe [20]. Uncooked rice was also found to contain higher concentrations of inorganic arsenic compared to seafoods [18].

The large variety of arsenic species and the fact that the arsenic distribution in many foods has yet to be studied give rise to the need to develop sensitive and robust methods for determining the various arsenic species in a variety of different food samples. The main difficulty in developing such methods is separating the numerous arsenic species in a single, simple chromatographic separation. Often more than one chromatographic technique is needed to separate all of the anionic, cationic, and neutral arsenic species present in a sample prior to detection. The use of multiple HPLC columns with different stationary phases has been reported [21], [22], [23], [24], [25], [26]. While the use of multiple columns offers the highest degree of separating power for resolving all arsenic species within a sample, the added equipment and complexity make it less practical for characterizing many samples quickly. Often the use of a single column to separate the most common arsenic compounds mentioned above is desirable.

Finding a single set of chromatographic conditions in which most species are separated and elute within a reasonable time can also be difficult due to the variety of structures and properties that arsenic species possess. One option is to use mobile phase gradients that increase the mobile phase strength with time. This technique can give better peak shapes and facilitate faster separations than isocratic elution. However, gradients require re-equilibration time between runs and can suffer from poor mobile phase buffering capacity and low ionic strength in the initial stages of the separation, depending on the conditions. If the sample matrix is sufficiently concentrated to cause a local change in the mobile phase pH or ionic strength, shifts in retention time can result [3], [27], [28], [29].

The aim of this paper is to demonstrate a simple and robust method for separating as many commonly found arsenic species as possible under isocratic conditions. Our method involves the use of a polymer-based strong anion-exchange column (PRP-X100) to separate arsenic compounds based on their affinity for positively charged trimethylammonium exchange sites covalently bonded to a packed polymeric stationary phase. Previous studies [3], [4], [21], [30] have reported difficulty in separating arsenite, arsenobetaine, and arsenosugar 1 while maintaining the separation of other species using anion-exchange chromatography.

Our solution has been to selectively increase the retention of arsenosugars through the use of boric acid. Boric acid has been used previously to improve capillary electrophoresis separations of carbohydrates by increasing their anionic behaviour [31], [32], [33]. Boric acid reacts with (preferably cis-) 1,2-diols under alkaline conditions to form a negatively charged complex [31], [32], [33].

Here, the negatively charged complex interacts more strongly with the positively charged exchange sites resulting in longer retention times. The introduction of boric acid to the mobile phase resulted in the improved separation of arsenosugars. The boric acid-assisted ion exchange chromatography enabled the separation of 11 arsenic compounds, including four arsenosugars and an unidentified arsenic species in a brown algae Fucus serratus sample. The optimised separation conditions were used to determine the arsenic species in rice and rice cereal extracts. Arsenic species in F. serratus, oyster, dogfish, and mushroom extracts were also separated to examine the sample matrix effect on retention time.

Section snippets

Reagents and solutions

All solutions were prepared in OmniSolv^® HPLC grade water (EM Science, Gibbstown, NJ, USA). Mobile phases were prepared by dissolving the individual components in water, adjusting the pH with ammonium hydroxide (Fisher Scientific, Fair Lawn NJ, USA) and filtering through a 0.22 μm filter. Assurance grade ammonium orthophosphate was obtained from BDH (Toronto, Ont., Canada). ACS grade boric acid was obtained from Anachemia (Champlain, NY, USA).

Sodium m-arsenite, sodium arsenate, and cacodylic

Results and discussion

Efficient separation is essential to the determination of 11 arsenic species. We have accomplished this goal by introducing boric acid complexation to assist the separation of arsenosugars and by optimising the separation parameters.

Acknowledgements

This work was supported by the Natural Sciences and Engineering Research Council of Canada. The authors would like to thank Dr. Kevin A. Francesconi (Karl-Franzens University, Graz, Austria) for providing the secondary standards of arsenosugars that were extracted and purified from F. serratus.

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Boric acid-assisted anion-exchange chromatography for separating arsenic species

Abstract

Introduction

Section snippets

Reagents and solutions

Results and discussion

Acknowledgements

Appl. Organomet. Chem.

J. Anal. At. Spectrom.

Fresen. J. Anal. Chem.

Clin. Chem.

Clin. Chem.

Clin. Chem.

J. Anal. At. Spectrom.

Appl. Organometal. Chem.

Analyst

J. Anal. At. Spectrom.

Human Ecol. Risk Assess.

Analyst

Analyst

Analyst

Environ. Sci. Technol.

Anal. Bioanal. Chem.

Food Chem. Toxicol.

Food Add. Contam.

Eur. Food Res. Technol.

Anal. Chim. Acta

Analyst

J. Anal. At. Spectrom.