# New Method for Simultaneous Arsenic and Selenium Speciation Analysis in Seafood and Onion Samples
## 2 Results and Discussion
### Speciation Analysis of Water-Soluble Arsenic and Selenium by HPLC-ICP-MS  
The applicability of the new method was successfully performed to simultaneous analysis of five arsenic and four selenium species, AsB, As(III), As(V), DMA, MMA, Se-cystine, Se-Met, Se(IV), and Se(VI), in onions and seafood samples. Sample chromatograms from the shrimp and onion analyses are presented in Figures 3 and 4, and the quantitative results from the determination of inorganic and organic arsenic and selenium species are shown in tables (Table 1). Figure 4: The overlapped chromatograms of As and Se species in green bio onions. Figure 3: The overlapped chromatograms of As and Se species in red shrimp. It is known that dietary seafood are considered to be the most important source of human exposure to As contamination. Moreover, the biotoxicity and bioavailability of As strongly depend on its chemical species, in the same way that forms of arsenite and arsenate are known to be the most toxic forms, which are categorized to a class 1, no threshold carcinogen, while organic species, for example, methylarsonic acid (MMA), dimethylarsinic acid (DMA), and arsenobetaine (AsB), are much less toxic to marine organisms and humans [19]. In this study for all seafood samples the inorganic arsenic concentration was below LOD. In contrast to inorganic forms, organic arsenic species were found in the seafood samples. The predominant form of the arsenic species was arsenobetaine in the range 2.221-20.69 mg\(\cdot\)kg\({}^{-1}\). The highest concentration was found in Argentine red shrimp, and the lowest, in white shrimp. In the case of speciation analysis of As in shellfish, similar to that in this study, the highest concentration was obtained of AsB forms. For shellfish, where the clam samples dominate, the range of AsB was 7.878-25.817 mg\(\cdot\)kg\({}^{-1}\), which was analyzed in extract after acid mineralization [19]. In this study, in the case of clams, AsB was determined at a similar concentration level, but it should be emphasized that the sample was extracted with water and not with acids. For this reason, obtained results are in accordance with the statement that AsB as a polar molecule can be easily extracted with aqueous solution [41] Moreover, water can be helpful to effectively extract hydrophilic arsenicals while keeping their chemical integrity [33]. On the other hand, the results for AsB in water-soluble extract of seaweeds were much lower (range 0.017-0.357 mg\(\cdot\)kg\({}^{-1}\)), but it should be emphasized that, contrary to our research, toxic forms iAs (the highest was above 50 mg\(\cdot\)kg\({}^{-1}\)) were determined in most algae, and in all seaweeds, the DMA form was determined in the range (0.049-2.924 mg\(\cdot\)kg\({}^{-1}\)) [33], which was also higher compared to this study. [PERSON] et al. (2020) [42] conducted the speciation analysis in edible tissues of mitten crabs and obtained that AsB was the predominant form among the six arsenic species (in the range 0.015-0.042 mg\(\cdot\)kg\({}^{-1}\)), and the ranking was as follows: AsB \(>\) MMA \(>\) As(III) \(>\) AsC \(>\) DMA \(>\) As(V). For seafood, the order is as follows: AsB \(>\) MMA \(>\) DMA \(>\) As(III) \(=\) As(V), but with much higher concentration of the AsB form. Taking into consideration the toxicity of arsenic species, the categories based on the International Agency for Research on Cancer (IARC) classification of the carcinogenicity of arsenic species were suggested. First is toxic inorganic arsenic fraction; second, AsB, which is established as nontoxic; and the last, leftover arsenicals, which may contain arsenosugars and other nonwater-extractable, lipophilic compounds. Results obtained in this study are in accordance with the finding that seafood is considered safe, owing to the benign nature of AsB that predominates and the low levels of iAs [33]. In the case of onion, inorganic arsenic was quantified in all samples, where only the As(III) form was found at the level below 0.2 mg\(\cdot\)kg\({}^{-1}\). According to the [PERSON] et al.