**2. Sample preparation**

Sample preparation step is crucial in every analysis where analytes are present at very low concentration levels. In the speciation analysis, there is also another difficulty that should be overcome. An important requirement for reliable speciation is to retain the concentration and structure of the original chemical forms in the sample. In general, aquatic samples such as rain, ground- or surface water, tap and drinking water, seawater, and soil solutions do not require any pretreatment procedures other than filtration through 0.45 μm filter.

Extraction of selenium species from the solid samples with the highest recovery is quite challenging. According to Peachey et al. [14], selection of the extraction method, which provides high extraction efficiency while preventing the integrity of selenium species, is essential for the accurate measurement of its species. The most used method can be divided into three main groups:


Since selenoaminoacids are water-soluble, extraction with hot water is extensively used [14–17]. However, the efficiency of water extraction from yeast was only 10% [17]. To release bounded selenoaminoacids, enzymatic or acidic hydrolysis was necessary [18–20]. The addition of methanesulfonic acid was used for selenomethionine extraction from yeast when heated under reflux [21]. Casiot et al. [22] reported that extraction of selenium species from yeast with water and ethanol led only to 10–20% recoveries of selenium and not allowed to extract

selenomethionine. The addition of pectinolytic enzymes released additional 20% of selenomethionine, while the addition of dodecyl sulfate solution allowed solubilization of a selenoprotein that accounted for 30% of total selenium. On the other hand, using tetramethylammonium hydroxide solubilized the sample completely, but the extracted selenium species were entirely degraded to selenomethionine and inorganic selenium [22]. It should be noticed that this type of extraction strictly depends on the choice of an enzyme, pH of the extraction solution, as well as temperature and time of the extraction. The most commonly used enzymes for this purpose are proteinase K or proteolytic enzymes (protease XIV), which were used for water-insoluble selenium fraction in many complicated matrices [23]. To reduce the extraction time, also ultrasonic hydrolysis can be used as the breakdown of Se-containing proteins (peptide bonds) into selenoaminoacids occurs [24]. The in vitro digestion with gastric juice was also used for selenium extraction from fish samples [25]. This procedure allowed SeMet determination, but the whole process takes few hours.

The sequential procedure developed by Chassaigne et al. [26] consisted of three steps: first, Tris-HCl buffer was used for extraction of water-soluble fractions, then also Tris-HCl with the addition of SDS for solubilization of protein fraction, and, finally, concentrated HNO3 was used for dissolving of the remaining solid residue. A similar three-step procedure was used for extraction of selenium from mushrooms with 89% extraction efficiency [27]. It should be noticed that sequential treatment was also applied for dietary supplements [28, 29].

Cuderman et al. [30] examined different extraction media to identify selenium species in buckwheat. The optimal extraction efficiencies were obtained by hydrolysis with HCl, followed by breaking the cells with liquid nitrogen and then enzymatic hydrolysis with protease XIV. Ammonium acetate [31], sodium hydroxide [32], and enzymatic hydrolysis with pronase E [33] have been proposed for extraction of selenium species from green onion leaves.

#### **3. Total selenium determination**

Determination of the total selenium content is still the first step of its analysis. This procedure requires that organic forms must be transferred into inorganic selenium that is usually achieved with digestion using strong mineral acids or UV irradiation after addition of hydrogen peroxide [12, 34]. For this purpose, fluorometry, electrochemical detectors, atomic absorption spectrometry (AAS), atomic fluorescence spectrometry (AFS), mass spectrometry (MS), and neutron activation analysis have been used (**Table 2**).

Hydride generation coupled to AAS or AFS detectors is specific for Se(IV) determination, where these species are selectively reduced to volatile SeH2, usually by sodium tetrahydroborate in hydrochloric medium. This technique can be applied for the determination of total inorganic selenium (e.g., sum of selenite and selenate) after quantitative reduction. The content of Se(VI) is then obtained by the difference between two determinations. This technique can be fully automatic by connection with flow injection analysis system. The advantage of such system is minimum sample and reagent consumption as well as short time of single run.

Stripeikis et al. [44] determined selenite and selenate in drinking water using fully automatic online separation/pre-concentration system coupled to electrochemical atomic spectrometry. Preconcentration of both selenium forms was carried out onto a microcolumn packed with an anionic resin (Dowex 1X8) that was placed in the robotic arm of the autosampling device. Selenite and selenate were then sequentially eluted with HCl at concentration of 0.1 and 4 mol L<sup>−</sup><sup>1</sup> ,

*Chromatographic Analysis of Selenium Species DOI: http://dx.doi.org/10.5772/intechopen.87053*


#### **Table 2.**

*Analytical methods for the determination of total selenium.*

respectively. The interference of large amounts of chloride ions during selenium atomization was prevented by using iridium as a permanent chemical modifier.

Kocot et al. [36] proposed a dispersive micro-solid-phase extraction with graphene as a solid adsorbent and ammonium pyrrolidine dithiocarbamate as a chelating agent for Se(IV) in analysis of inorganic selenium by the energy-dispersive X-ray fluorescence spectrometry; the concentration of Se(VI) was calculated as the difference between the concentration of selenite after and before reduction of selenate.

Due to the high selectivity and sensitivity, wide linear range, as well as multielement and multi-isotope detection, inductively coupled plasma mass spectrometry (ICP-MS) is a great tool for selenium analysis. However, some difficulties can be found when conventional ICP-MS is used for this purpose. To avoid the spectra interferences with 80Se isotope (the largest natural abundance of 49.6%), 82Se and 77Se are often monitored. The use of collision/reaction cell, operating with hydrogen gas, lowers argon dimer interferences. This technique was applied by Reyes et al. to determine the selenium in biological reference materials and serum samples [45] and yeast [46].

### **4. Separation techniques in selenium speciation analysis**

The occurrence of selenium at very low concentration levels as well as the dependence of its toxicity on the form in which it occurs resulted in the need for reliable analytical procedures for identification and quantification of its species. The coupling of separation of selenium species with sensitive detection has become a powerful technique for Se speciation. Liquid chromatography operating in different modes is the most used analytical technique for this purpose; however electrophoresis and gas chromatography also were used in selenium analysis. That is why these two methods will be also described in this chapter, which will mainly focus on liquid chromatography.

#### **4.1 Gas chromatography**

Selenium species can be divided into volatile and nonvolatile compounds. The first group of compounds can be directly analyzed using gas chromatography,

for example, dimethylselenide was determined in human breath after ingestion of Se-enriched selenite [47]. For other nonvolatile compounds, derivatization is required.

In the literature many derivatization procedures can be found. Pelaez et al. [48] tested two methods of derivatization of selenomethionine and selenomethionine in selenium supplements. The first method consisted of esterification of the carboxylic acid group using propan-2-ol and then acylation of amino group. The second described procedure used ethanol-ethyl chloroformated for one step derivatization. Then detection using ICP-MS was performed.

In general, many types of detectors were coupled with GC for speciation analysis of selenium. Yang et al. [49] successfully used ICP detection for the determination of selenium in yeast. Organoselenium species in plants were determined using GC-MIP-AES detection [50]. Because of the requirement of the derivatization, GC is not so widely used in selenium speciation analysis in comparison to liquid chromatography.

#### **4.2 Capillary electrophoresis**

Due to the high resolving power, capillary electrophoresis (CE) has a potential to be used in speciation analysis as an alternative or complementary technique to HPLC. CE coupled with ICP-MS was used in the analysis of selenium in yeast extract after SEC separation [51]. The obtained limit of detection for organic and inorganic species was in range 7–18 μg L<sup>−</sup><sup>1</sup> . The main difficulty in such separation can be big sizes of selenopolypeptides resulting in their slow migration. In such case, the predigestion of selenoprotein fraction is recommended.

It should be highlighted that gel electrophoresis offers better resolution than liquid chromatography in the analysis of high molecular weight selenoproteins, which was used in such analysis by Chassaigne et al. [26], and in Se-enriched yeast analysis as well by Chery et al. in blood sample analysis [52].

#### **4.3 High-performance liquid chromatography**

#### *4.3.1 Size-exclusion chromatography*

Separation in size-exclusion chromatography (SEC) strictly depends on the size of separated analytes. That is why this technique is widely used as a preliminary step to sample purification or to separate selenoproteins from the matrix. Ayouni [53] observed high molecular weight selenium compounds in the extract of dietary supplements after separation by SEC. Conjunction of SEC with ICP-MS was used for analysis of the products of enzymatic digestion of selenoproteins fraction [54, 55]. Size-exclusion chromatography was also used in selenium analysis in human plasma [56] and extracts from rats' internal organs [57].

#### *4.3.2 Ion-exchange chromatography*

Anion-exchange chromatography has been mainly employed in selenium speciation analysis [58–63]. Mobile phases used in this mode usually contain small content of organic modifier (e.g., 2–5% methanol) and buffered salt solution (e.g., acetate, phosphate, and citrate). During the separation process, the equilibria between the charged solute ion and the oppositely charged surface of the stationary phase are established. The separation is achieved based on the differences of the strength of such interaction between analytes. Anion-exchange

#### *Chromatographic Analysis of Selenium Species DOI: http://dx.doi.org/10.5772/intechopen.87053*

chromatography was used in the selenium speciation analysis in garlic, sunflowers, and radish sprouts [64]. In addition to well-known compounds like SeMet or MeSeCys, several unidentified signals were obtained. The application of high-resolution mass spectrometry enabled identification of additional selenocompounds as inorganic metabolites, such as deamino-hydroxy-seleno-homolanthionine, *N*-acetylcysteine-selenomethionine, methylseleno-pentose-hexose, methylselenoglutathione, 2,3-dihydroxy-propionyl selenocysteine-cysteine, methyltio-selenoglutathione, 2,3-dihydroxy propionyl-seleno-lanthionine, and two Se-containing compounds with proposed molecular formula C10H18N2O6Se and C10H13N5O3Se.

Cation-exchange chromatography was used to analyze selenium-enriched yeast in a human adsorption study [65]. As a mobile phase, pyridinium formate buffer with 3% of methanol was used. This method was suitable for separation of organic selenium species, however not suitable to separate selenite and selenate.

### *4.3.3 Reversed-phase chromatography*

Both, simple reversed-phase [66–68] and ion-pair (IP) reversed-phase chromatography [15, 28, 69], are widely used for analysis of ionic and neutral selenium species. The mobile phases are aqueous with small amount of polar organic solvent (usually methanol or acetonitrile). Because of their hydrophilicity, selenoaminoacids are not retained onto typical reversed-phase columns. The use of ion-pairing reagents as mobile phase additives allows their separation. The ion-pairing reagent is usually an alkyl sulfonate, an alkyl sulfate, or an alkylammonium salt. Its nonpolar chain interacts with hydrophobic stationary phase (e.g., C8 or C18), while ionizable group is neutralized by oppositely charged analyte. Hexanesulfonic acid has been used as anion-pairing reagent in the speciation analysis of selenium in Brazil nuts, using C8 column for separation [70]. Obtaining separation was satisfied for organic compounds but poor for selenite and selenate. For separation of organic and inorganic forms of selenium, tetrabutylammonium acetate was proposed [71]. Also mixed ion-pairing reagents (butanesulfonic and tetramethylammonium hydroxide) were also used to simultaneously separate inorganic and organic species with satisfactory separation efficiencies [72].

New mobile phase additives are still developed, for example, room temperature ionic liquids [73]. Their mechanism of action is based on bilayer formulation onto stationary phase. It gives the possibility of additional interactions between the analyte and the bed, which significantly affects the retention and shape of the recorded signals. The effects of several imidazolium chlorides on the separation of selenium species mixture was described in details [74]. In all cases, SeMeCys was the first species eluted indicating its weak retention in the column, while the retention time of Se(VI) was increasing with the increase of alkyl chain.

#### *4.3.4 Hydrophilic interaction liquid chromatography*

Hydrophilic interaction liquid chromatography (HILIC) is a complementary technique to reversed-phase mode. The separation mechanism is mainly based on the partition of the analyte between the thin water layer adsorbed onto the stationary surface and the eluent, which contain high content of organic solvent usually acetonitrile. It is known that also other interactions such as hydrogen bonding dipole-dipole interactions and electrostatic forces may play an important role in the retention mechanism in HILIC [75]. The governed retention mechanism strictly depends on the type of used stationary phase and the buffer conditions

(content of organic solvent, concentration of salt, and pH). TSKgel Amide-80 stationary phase with covalently bound carbamoyl groups is frequently used in the analysis of selenium in HILIC mode [76, 77]. According to the characterization of amide stationary phase, the achieved separation is not pH dependent [78]. Also zwitterionic and silica stationary phases have been also used in HILIC separation of selenium [79, 80]. It should be highlighted that in selenium separation in HILIC mode methanol is recommended instead of acetonitrile as a main component of mobile phase [79] which is shown in **Figure 1**.

The use of methanol enhances peak intensity, improve the separation of SeMet and SeMeSeCys, and shorten time of the single run. The best separation conditions

**Figure 1.**

*The chromatogram of selenium compounds obtained on silica column using (A) MeOH and (B) ACN in the mobile phase [79]. Reprinted with permission from Elsevier.*

*Chromatographic Analysis of Selenium Species DOI: http://dx.doi.org/10.5772/intechopen.87053*

have been obtained for silica column and mobile phase consisted of 85% of methanol and 8 mM of ammonium acetate. Using the zwitterionic column (ZIC-HILIC) instead of silica stationary phase resulted in recording of very asymmetric peaks.

The potential of two orthogonal chromatographic modes—RP and HILIC—was examined in the analysis of onion leaf extracts [79]. Higher separation efficiency (mainly for inorganic selenium species) and shorter retention times were obtained when HILIC mode was used (**Figure 2**).

**Figure 2.**

*The chromatographic separation of selenium species present in onion leaf extracts in (A) HILIC mode and (B) RP mode. (A) Atlantis HILIC (silica) column, mobile phase: 85% MeOH and 8 mM CH3COONH4, pH 7. (B) Luna C8 column, mobile phase: 99.5% HCOOH and 0.5% ACN [79]. Reprinted with permission from Elsevier.*
