# New Method for Simultaneous Arsenic and Selenium Speciation Analysis in Seafood and Onion Samples
## 2 Results and Discussion  
### Simultaneous Arsenic and Selenium Speciation Analysis  
The As and Se species were completely resolved on use in the Dionex IonPac AS22 analytical column connected to the Dionex IonPac CG5A guard column with gradient elution. We applied the gradient elution to improve the separation of As and Se species. A similar way to achieve full separation of arsenicals was presented in a study by [PERSON] et al. (2002) [29], where the method with anion exchange with nitric acid eluents together with a doubly charged ion-pairing agent was applied to separate anions and cations of arsenicals together in a single chromatographic run. Additionally, the [PERSON] et al. (2003) [30] used the gradient elution for separation the organoarsenicals in one chromatographic run using cation exchange. The goal of present study was to make the simultaneous speciation of arsenic and selenium in one run; that is why we used anion exchange column in combination with an anion-cation exchange guard column, which was not yet applied in speciation analysis of metalloids. The most commonly column for speciation of arsenic is anion exchange chromatography with Hamilton PRP-X100 column [31], which we tested but did not obtain full separation of arsenic and selenium forms (Table 1). Furthermore, [PERSON] et al. (2001) [32] also used anion and cation exchange columns to separate eight arsenicals. In this study, we applied the combination of anion and cation exchange columns for speciation analysis of arsenicals in seafood. It is well known that chromatographic approaches, such as ion-pairing reversed-phase, ion-exchange, ion exclusion, and reversed-phase chromatographies, are reported to facilitate speciation of arsenicals in marine sample extracts. Moreover, it was confirmed that methylated arsenicals and AsSugars were successfully isolated on anion exchange columns, while cation exchange columns were efficient for separation for AsB, AsC, DMA, TMAO, TETRA, and DMAA, while for AsLipids, RP-LC was typically used with a C8 or C18 column [33]. In addition, in the literature was found that arsenobetaine (AsB) at pKa = 2.18 is zwitterionic; that is why, in our case, the use of a bifunctional column was justified [33]. The chromatographic separation obtained for the analytes in standard solutions at the mentioned concentrations are presented in Figures 1 and 2. It is worth noticing that, in the case in speciation analysis of arsenic, the use of separate chromatographic methods is recommended for different groups of arsenicals because it is difficult to achieve efficient separation of anionic and cationic species using a single method. For example, in the study by [PERSON] et al. (2021) [34] the As(III), As(V), DMA, and MMA were separated by the anion exchange method, and AsB was separated on a cation exchange column in aqueous extracts analyzed by HPLC\(-\)ICP-MS, but each column was used separately. Morevoer, the AsB form was separated in 20 min (cation exchange) and As(V) form in 30 min (anion exchange) by HPLC-ICP-MS. Similar to our study, [PERSON] et al. (2001) used anion and cation exchange columns to separate eight arsenicals [32]. They obtained good resolution, but the serious limitation to apply this method was long chromatographic run time of 40 min. Furthermore, in the case of applied ion exclusion chromatography with ICP-MS detection by [PERSON] et al. (2000) for separation of arsenic species, the long chromatographic run times (up to 60 min) was the main disadvantage of this method [35]. In this study, we obtained in one run of not only arsenic species but also selenium species in short chromatographic run time (10 min) with a sufficient resolution, which is to opposite to previous studies, where species of these metals were analyzed separately [36; 37; 38; 39]. Moreover, the additional advantage of this research was the successful separation of the rarely presented organic form of selenium, Se-cystine. Other studies have focused only on total Se content [40].