# New Method for Simultaneous Arsenic and Selenium Speciation Analysis in Seafood and Onion Samples
## 3 Materials and Methods
### Final Method  
Summarizing all optimization steps, the chosen analytical columns, eluent composition, and its pH were suitable to separate efficiently speciation forms of arsenic and selenium with significantly short RTs and acceptable signal-to-noise ratio. Unfortunately, the optimized speciation was not appropriate for speciation of chromium and antimony forms. Below, the characterization of speciation method of arsenic and selenium is presented. An anion exchange column Dionex IonPac AS22 analytical column (Thermo, USA) containing an alkanol quaternary ammonium ion and a cation-anion exchange analytical column and a Dionex IonPac CG5A analytical guard column (Thermo, USA) containing a fully sulfonated latex for cation exchange as the first layer and a fully aminated layer for anion exchange as the second layer were used. The total analysis time was set to 12 min. The mobile phase gradient elution was set to 0-2 min 100% mobile phase A, 2-4 min 100 \(\rightarrow\) 0% mobile phase A and 0 \(\rightarrow\) 100% mobile phase B, 4 min-10 min 100% mobile phase B, and 10-12 min 100% A. The mobile phases were aqueous solutions of 1b mmol-L\({}^{-1}\) NH4 NO3 (mobile phase A) and 75 mmol-L NH4 NO3 (mobile phase B), both at pH 9.0, adjusted by NH4 OH. Methanol (1%) was added to the mobile phases to enhance the signal response for arsenic [31]. The oven temperature was set to 40 \({}^{\circ}\)C with a flow rate of 1.0 mL min\({}^{-1}\), and the injection volume was set to 100 \(\upmu\)L. All analytes were quantified on the basis of external calibrations constructed using standards prepared on the day of the analysis. Calibration curves for the As species (AsB, As(III), DMA, MMA, and As(V)) and Se species (Se-Met, Se-cystine, Se(IV), and Se(VI)) were constructed at concentrations of 0.1, 0.25, 1.0, 5, 10, 25, and 100 \(\upmu\)g\(\cdot\)L\({}^{-1}\). Analytes as speciation forms of arsenic and selenium were identified by matching their retention times with those of standards. Using optimized chromatographic conditions, the retention times for all arsenic species were in the range from 2.4 min to 9.7 min, and the order of elution was AsB, As(III), DMA, MMA, and As(V). The retention times for all selenium species were in the range from 2.7 min to 10.1 min, and the order of elution was Se-Met, Se-cystine, Se(IV), and Se(VI). The accuracy of new speciation method was evaluated by analysis of the certified reference material Tuna Fish Tissue BCR-627. It is worth mentioning that there is a lack of Certified Reference Materials (CRMs), which are able to simultaneously determine all the forms of arsenic, which were separated by new speciation method. The main aim was to compare the arsenic speciation forms AsB and DMA. The obtained recovery for dimethylarsinic acid was 97 \(\pm\) 5% and 98 \(\pm\) 3% for arsenobetaine. The obtained results confirmed that nothing is held in the analytical column, suggesting that both the separation and detection elements of the analytical system were working correctly. The characteristics parameters of new method are presented in Table 3.