Electrochemical Oxidation of l-selenomethionine and Se-methylseleno-l-cysteine at a Thiol-Compound-Modified Gold Electrode: Its Application in a Flow-Through Voltammetric Sensor

A flow-electrolytic cell that consists of a bare gold wire or of different thiol-compound-modified gold electrodes (such as 2,4-thiazolidinedione, 2-mercapto-5-thiazoline, 2-mercaptothiazoline, l-cysteine, thioglycolic acid) was designed to be used in a voltammetric detector to identify l-selenomethionine and Se-methylseleno-l-cysteine using high-performance liquid chromatography. Both l-selenomethionine and Se-methylseleno-l-cysteine are more efficiently electrochemically oxidized on a thiol/gold than on a bare gold electrode. For the DC mode, and for measurements with suitable experimental parameters, a linear concentration from 10 to 1600 ng·mL−1 was found. The limits of quantification for l-selenomethionine and Se-methylseleno-l-cysteine were below 10 ng·mL−1. The method can be applied to the quantitative determination of l-selenomethionine and Se-methylseleno-l-cysteine in commercial selenium-containing supplement products. Findings using high-performance liquid chromatography with a flow-through voltammetric detector and ultraviolet detector are comparable.


Introduction
Rezvanfar et al. [1] reported that the immunomodulator drug (IMOD) they tested was an electromagnetically-treated, selenium (Se)-based, herbal medicine with antioxidant properties and that it prevented the complications of polycystic ovary. The effects of bacteriostasis in vitro of Se-enriched herbal medicine on Escherichia coli and Salmonella have been studied [2,3]. Therefore, it is used to prepare a high-energy composite peptide selenoprotein nutrient solution [4]. Selenium is an essential component of several antioxidant enzymes in animal taxonomic groups, and exposure to too much or too little Se can negatively affect an organism [5]. It has been reported that the concentrations for toxicity, bioavailability and reactivity of Se depend on its chemical forms and concentration. Medicinal plants enriched with anticarcinogenic Se compounds can be used to improve public health. Se in plants exists predominantly in organic forms, such as selenoamino acids (selenocysteine, selenocysteine, selenomethionine and Se-methylselenocysteine) and in trace quantities in inorganic Se species. Hence, accurate and precise analytical methodologies for determining Se are required to obtain the correct chemical forms and concentrations [6][7][8].

Determining L-Selenomethionine Using Cyclic Voltammetry
Cyclic voltammetry (CV) was done in phosphate buffer (pH 2.23-6.57), acetate buffer (pH 4.18), Britton and Robison buffer (pH 2.46-10.45) and lithium perchlorate (LiClO 4 ) solutions as supporting electrolytes for a gold electrode. CV potentials ranged from −0.4 V to +1.5 Vat a scan rate of 25 mV·s −1 . The CV data were calculated using SP-150, Bio-Logic SAS and EC-Lab ® software. The standard stock solution was prepared by dissolving the appropriate amount of L-selenomethionine in methanol.

Preparing Thiol-Modified-Au Electrodes
The gold wire electrode was cleaned then placed in the tube containing thiol compound solution, deoxygenated by purging with nitrogen for 5 min. The different thiol-modified-Au electrodes were electrolytically plated with a thiol compound (2-mercapto-5-thiazoline, 2-mercaptothiazoline, L-cysteine, thioglycolic acid or 2,4-thiazolidinedione) from 10 mL of 0.1 M acetate buffer (pH 4.23), 0.01 to 1.6 mM thiol solution, respectively, and the potential was cycled between 0.0 V and 1.5 V (versus an Ag/AgCl reference electrode) with a scan rate of 25 mV·s −1 until the CVs showed no further change.

Construction of a Voltammetric Sensor for Liquid Chromatography
The flow cell was designed with the following electrodes: an Ag/AgCl/0.1 M KCl reference electrode, a stainless steel auxiliary electrode and a modified thiol/Au (length: 8 cm; i.d.: 0.3 mm) as the working electrode for detecting L-selenomethionine and Se-methylseleno-L-cysteine. The amperometric detection was achieved in a homemade flow-through cell prepared in our laboratory as previously described [37]. A flow-through electrolysis cell was used for DC-mode amperometric detection. Reversed-phase HPLC was done on a column (250 mm × 4.6 mm) (LiChroCart ® ; Merck, Darmstadt, Germany) eluted with methanol and water (10:90 v/v) containing 1.0 mM of KH 2 PO 4 (pH 3.89) as the mobile phase, at a flow rate of 1.0 mL·min −1 . It was examined using an ultraviolet (UV) detector set at 248 nm. The electrochemical detector was operated between +0.6 V and 1.3 V for gold and thiol-modified-Au electrodes.

Application to Commercial Selenium Supplement Drugs
A set of standard solutions was produced by diluting aliquots of the stock solutions with methanol to 10 mL in calibrated flasks. Taking into account the L-selenomethionine or Se-methylseleno-L-cysteine content of the selenium supplement drug samples (approximately 0.008-0.02 g), the latter were accurately weighed in a 55-mL polyfluoroalkoxy bottle diluted to about 10 mL of methanol:water (1:1, v/v), put into a microwave (MARS 6 System; CEM Corp., Matthews, NC, USA), dissolved and then centrifuged. The supernatant was transferred into a 10-mL calibrated flask. An aliquot of the solution was filtered through a 0.45-µm membrane filter before it was analyzed using HPLC. A chromatograph was obtained using 20 mL of the prepared standard solution under the operating conditions described above.

Voltammetric Behavior of L-Selenomethionine on Au Electrodes
The anodic peak potential and current of L-selenomethionine in 0.1 M acetate buffer (pH 4.23) of the bare gold electrode were (0.823 V, 6.67 µA) and (1.11 V, 9.  Acid-based equilibrium directly affected the electroactive species; changes in peak current, potential and pH can also occur because of chemical reactions [22,23]. L-selenomethionine was protonized in the pH < 2.1 region because of acid-catalysis ( Figure 2A). In the pH 2.2-4.0 region, the first current peak was almost constant, but when the pH was >3.2, the current increased with increasing pH values: the highest peak was at pH 4.0. In the pH 5-7 region, the unprotonized form of L-selenomethionine underwent uncatalyzed changes that were similar to the isoelectric points of cysteine and L-methionine at pH 5.05 and pH 5.74, respectively. The peak potential decreased with increasing pH values, and the shift was less positive ( Figure 2B). The two approximately linear portions (plotted for (pK −1) > pH > (pK +1)) intersect at a pH value corresponding to pK. Based on L-selenomethionine pKa values in acid (pK 2.15) and alkaline (pK 8.94), different species (protonized and unprotonized) might be present in aqueous solutions. The peak currents are higher at pH 2.5 and pH 4.2, and at pH 4.2, the peak potential was the lowest within the acidic range. Because acidic solution is suitable for HPLC column analysis, all of the following experiments were done at pH 4.2. However, in inorganic redox systems, there are no proton transfers (only electron state transitions); thus, the electron transition was not significantly affected by changes in pH. Acid-based equilibrium directly affected the electroactive species; changes in peak current, potential and pH can also occur because of chemical reactions [22,23]. L-selenomethionine was protonized in the pH < 2.1 region because of acid-catalysis ( Figure 2A). In the pH 2.2-4.0 region, the first current peak was almost constant, but when the pH was >3.2, the current increased with increasing pH values: the highest peak was at pH 4.0. In the pH 5-7 region, the unprotonized form of L-selenomethionine underwent uncatalyzed changes that were similar to the isoelectric points of cysteine and L-methionine at pH 5.05 and pH 5.74, respectively. The peak potential decreased with increasing pH values, and the shift was less positive ( Figure 2B). The two approximately linear portions (plotted for (pK −1) > pH > (pK +1)) intersect at a pH value corresponding to pK. Based on L-selenomethionine pKa values in acid (pK 2.15) and alkaline (pK 8.94), different species (protonized and unprotonized) might be present in aqueous solutions. The peak currents are higher at pH 2.5 and pH 4.2, and at pH 4.2, the peak potential was the lowest within the acidic range. Because acidic solution is suitable for HPLC column analysis, all of the following experiments were done at pH 4.2. However, in inorganic redox systems, there are no proton transfers (only electron state transitions); thus, the electron transition was not significantly affected by changes in pH.

Optimal Conditions for a Flow-Through Voltammetric Detector
Methanol-water that contained various ratios of 0.1 mM phosphate buffer (pH 3.5-4.2) was prepared. Experiments showed that L-selenomethionine was retained for 3.62 min and that Se-methylseleno-L-cysteine was retained for 2.77 min, after which, baseline separation was recovered. Methanol-water (10:90, v/v) that contained phosphate buffer (pH 3.92) was found to be the best eluent for sensitivity higher than that of other eluents. Therefore, phosphate-buffered solution was chosen for determining L-selenomethionine and Se-methylseleno-L-cysteine. To compare the electroanalytical utility of thiol/Au electrodes, we used liquid chromatography with electrochemical detection (LC-ECD) to measure the L-selenomethionine and Se-methylseleno-L-cysteine on thiol/Au electrodes and bare gold electrodes in methanol-water (10:90, v/v). The peaks of L-selenomethionine and Se-methylseleno-L-cysteine on thiol/Au electrodes are higher than that of the bare gold electrode (Figures 3 and 4). The sensitivity of thioglycolic acid

Optimal Conditions for a Flow-Through Voltammetric Detector
Methanol-water that contained various ratios of 0.1 mM phosphate buffer (pH 3.5-4.2) was prepared. Experiments showed that L-selenomethionine was retained for 3.62 min and that Se-methylseleno-L-cysteine was retained for 2.77 min, after which, baseline separation was recovered. Methanol-water (10:90, v/v) that contained phosphate buffer (pH 3.92) was found to be the best eluent for sensitivity higher than that of other eluents. Therefore, phosphate-buffered solution was chosen for determining L-selenomethionine and Se-methylseleno-L-cysteine. To compare the electroanalytical utility of thiol/Au electrodes, we used liquid chromatography with electrochemical detection (LC-ECD) to measure the L-selenomethionine and Se-methylseleno-L-cysteine on thiol/Au electrodes and bare gold electrodes in methanol-water (10:90, v/v). The peaks of L-selenomethionine and Se-methylseleno-L-cysteine on thiol/Au electrodes are higher than that of the bare gold electrode (Figures 3 and 4). The sensitivity of thioglycolic acid (TGA) is greater than that of the other thiol/Au electrodes. Because the TGA/Au electrode has only one peak, the interference is less than that of the other modified electrodes, and the catalytic effect (the current) is greater (Figure 1). Therefore, we used the TGA/Au electrode to determine L-selenomethionine and Se-methylseleno-L-cysteine in commercial selenium supplements. (TGA) is greater than that of the other thiol/Au electrodes. Because the TGA/Au electrode has only one peak, the interference is less than that of the other modified electrodes, and the catalytic effect (the current) is greater (Figure 1). Therefore, we used the TGA/Au electrode to determine L-selenomethionine and Se-methylseleno-L-cysteine in commercial selenium supplements.  The voltammetric detector was operated at +1.2 V. Using the injection valve, 20 µL of the prepared standard solutions was chromatographed under the operating conditions described (TGA) is greater than that of the other thiol/Au electrodes. Because the TGA/Au electrode has only one peak, the interference is less than that of the other modified electrodes, and the catalytic effect (the current) is greater (Figure 1). Therefore, we used the TGA/Au electrode to determine L-selenomethionine and Se-methylseleno-L-cysteine in commercial selenium supplements.   The voltammetric detector was operated at +1.2 V. Using the injection valve, 20 µL of the prepared standard solutions was chromatographed under the operating conditions described above. The lower limit of quantitative detection in our method was approximately 0.16 ng for L-selenomethionine and 0.07 ng for Se-methylseleno-L-cysteine. The calibration graph plots obtained by plotting the peak area against the concentrations of L-selenomethionine and Se-methylseleno-L-cysteine show good linearity over the range 10-1600 ng·mL −1 and 50-1600 ng·mL −1 , respectively. The regression equations were y = 157x + 4.5 (r = 0.9987) for L-selenomethionine and y = 322x − 7.8 (r = 0.9996) for Se-methylseleno-L-cysteine. Chromatograms were obtained using LC-ECD and LC-ultraviolet detection (UVD) (wavelengths at 248 nm) ( Figure 5A,B). LC-ECD is more specific and sensitive than LC-UVD.

Accuracy and Precision
To test the applicability of the developed TGA/Au electrode, a commercial selenium supplement was analyzed using the standard addition method. Recovery tests were done on commercial selenium supplements to evaluate the reproducibility and accuracy of the proposed LC-ECD and LC-UVD methods. Each sample was diluted with methanol and water, and they were spiked with different concentrations of L-selenomethionine and Se-methylseleno-L-cysteine. The recoveries ranged from 97%-104% (LC-ECD) ( Table 1). Hence, in this study, determining the concentration of selenoamino acids in commercial selenium supplements was done using a standard additions procedure (Table 2). These results agreed with those obtained using an LC-UVD. The sensitivity of LC-ECD was 680-1280-times greater than that of LC-UVD. In Samples 4 and 5, no Se-methylseleno-L-cysteine was detected because it was below the LC-UVD level of detection. Representative LC-ECD chromatograms ( Figure 6A-C) for the selenium supplement sample are comparable to a chromatogram of pure standard. Inorganic selenium has various oxidation states (VI, IV, 0, −II) over a wide range of pH, and it appears primarily as selenite (Se (IV)) and selenate Se (VI)). The electrochemical behavior of inorganic selenium species, e.g., Se (IV) and Se (VI), requires Se (VI) to be reduced to Se (IV) and then done using stripping voltammetric techniques [38]. Sodium selenite is a major component of some commercial supplements, e.g., Twinlab capsules (selenium 250 mcg) and Allergy Research Group selenium solution (selenium 100 mcg), but cannot be determined in our LC-ECD conditions. The diselenide forms cannot be detected

Accuracy and Precision
To test the applicability of the developed TGA/Au electrode, a commercial selenium supplement was analyzed using the standard addition method. Recovery tests were done on commercial selenium supplements to evaluate the reproducibility and accuracy of the proposed LC-ECD and LC-UVD methods. Each sample was diluted with methanol and water, and they were spiked with different concentrations of L-selenomethionine and Se-methylseleno-L-cysteine. The recoveries ranged from 97%-104% (LC-ECD) ( Table 1). Hence, in this study, determining the concentration of selenoamino acids in commercial selenium supplements was done using a standard additions procedure ( Table 2). These results agreed with those obtained using an LC-UVD. The sensitivity of LC-ECD was 680-1280-times greater than that of LC-UVD. In Samples 4 and 5, no Se-methylseleno-L-cysteine was detected because it was below the LC-UVD level of detection. Representative LC-ECD chromatograms ( Figure 6A-C) for the selenium supplement sample are comparable to a chromatogram of pure standard. Inorganic selenium has various oxidation states (VI, IV, 0, −II) over a wide range of pH, and it appears primarily as selenite (Se (IV)) and selenate Se (VI)). The electrochemical behavior of inorganic selenium species, e.g., Se (IV) and Se (VI), requires Se (VI) to be reduced to Se (IV) and then done using stripping voltammetric techniques [38]. Sodium selenite is a major component of some commercial supplements, e.g., Twinlab capsules (selenium 250 mcg) and Allergy Research Group selenium solution (selenium 100 mcg), but cannot be determined in our LC-ECD conditions. The diselenide forms cannot be detected in commercial products either, because they are first cleaved to yield an Se-Au adsorbate, and then, bulk selenocysteine is reduced on the modified electrode [39]. Table 1. Recovery of L-selenomethionine and Se-methylseleno-L-cysteine in spiked commercial selenium supplements using liquid chromatography with electrochemical detection (LC-ECD).

Samples
Added (

Conclusions
We have developed a sensitive, green electrochemical procedure for determining organic selenium using a flow-through voltammetric sensor. It exhibits a good analytical performance using a thiol/Au electrode for the LC-ECD of L-selenomethionine and Se-methylseleno-L-cysteine with a low limit of quantitation, a rapid response, a satisfactory linear range and good stability and selectively. The presented electrode was simultaneously used to determine L-selenomethionine and Se-methylseleno-L-cysteine in a commercial selenium supplement.

Conclusions
We have developed a sensitive, green electrochemical procedure for determining organic selenium using a flow-through voltammetric sensor. It exhibits a good analytical performance using a thiol/Au electrode for the LC-ECD of L-selenomethionine and Se-methylseleno-L-cysteine with a low limit of quantitation, a rapid response, a satisfactory linear range and good stability and selectively. The presented electrode was simultaneously used to determine L-selenomethionine and Se-methylseleno-L-cysteine in a commercial selenium supplement.