Speciation of Selenium in Selenium-Enriched Foods by High-Performance Liquid Chromatography-Inductively Coupled Plasma-Tandem Mass Spectrometry
by
Yue Luo
1, 
Gang Chen
1,
Xiuqing Deng
1,
Hanqing Cai
1,
Xueheng Fu
1,
Fujian Xu
2,*,
Xiaonian Xiao
3,
Yumeng Huo
4 and
Jin Luo
1,* 1
Sichuan Analysis and Testing Service Center, Chengdu 610023, China
2
School of Chemistry and Environment, Southwest Minzu University, Chengdu 610041, China
3
OAI Sino-German United Research Institute, Nanchang University, Nanchang 330047, China
4
National Anti-Drug Laboratory Beijing Regional Center, Beijing 100164, China
*
Authors to whom correspondence should be addressed.
Separations 2022, 9(9), 242; https://doi.org/10.3390/separations9090242
Submission received: 20 July 2022
/
Revised: 18 August 2022
/
Accepted: 29 August 2022
/
Published: 3 September 2022
(This article belongs to the Special Issue Applications of Chromatography Technology)
Abstract
:Herein, a method was established for the speciation of six selenium species by high performance liquid chromatography-inductively coupled plasma-tandem mass spectrometry (HPLC-ICP-MS/MS). The factors affecting separation were carefully investigated, including ionic strength, pH, and methanol content. Six species of selenium could be completely separated within 20 min, under the mobile phase of 25 mM citric acid in pH = 4.0 containing 2% methanol. The detection limits of selenite (Se(IV)), selenate (Se(VI)), selenomethionine (SeMet), selenocystine (SeCys2), methylselenocysteine (MeSeCys), and selenoethionine (SeEt) were 0.04, 0.02, 0.05, 0.02, 0.03, and 0.15 ng mL−1, respectively. To verify the practicality of this method, the analysis of selenium-enriched foods such as selenium-enriched spring water, selenium-enriched salts, and selenium-enriched tea were conducted, and recovery of 93.7–105% was achieved with RSD < 5%, revealing the high practical utility of the proposed method.
1. Introduction
Selenium is considered a key element and essential nutrient for human health [1], and plays an important metabolic role in various enzymes such as glutathione peroxidase, iodothyronine deiodinase, and thioredoxin reductase [2]. The World Health Organization (WHO) recommends that the daily intake of selenium for adults is 50–70 μg/d [3]. A lack of selenium in the human body could lead to Kashin–Beck disease, lung damage, liver insufficiency, and other diseases [4]. While selenium supplementation can enhance the antioxidant capacity through synthetic cytoplasmic enzymes, an excessive intake of selenium is harmful, and its toxicity and bioavailability depend on the chemical form and concentration [5]. The distribution of selenium is very uneven in China, and people in selenium-deficient areas face the risk of insufficient selenium intake [6]. Moreover, selenium mainly exists in the form of inorganic selenium in soil and water. Considering the double-edged sword properties of selenium, the production and transportation of selenium-enriched food is one of the best ways to solve this problem [7]. In recent years, the selenium-enriched food industry in China has been in a period of rapid development, and a large number of selenium-enriched foods have appeared on the market. Among them, selenium-enriched drinking water, selenium-enriched tea, and selenium-enriched salt are favored because of their relatively affordable prices, convenience, and high frequency of consumption [8]. In order to understand the authenticity of the selenium content in food, accurate selenium speciation in selenium-enriched foods in urgent demand.
For selenium speciation analysis, the most commonly used method is chromatography coupled with atomic spectrometry [9]. Typically, selenium compounds in different forms are separated by high performance liquid chromatography (HPLC) [10], gas chromatography (GC) [11], and capillary electrophoresis (CE) [12], and the quantitative detection is performed by atomic absorption spectroscopy (AAS) [10], atomic fluorescence spectroscopy (AFS) [13], atomic emission spectroscopy (AES) [14], and inductively coupled plasma mass spectrometry (ICP-MS) [15]. Among them, HPLC-ICP-MS has been widely used in the field of speciation analysis for its advantage of a high separation ability, low detection limit, wide dynamic linear range, and good analytical precision [16,17]. There are six isotopes of selenium, namely 74Se, 76Se, 77Se, 78Se, 80Se, and 82Se, of which 80Se is the most abundant (49.96%). However, 80Se, 78Se, and 82Se are seriously interfered by 40Ar40Ar+, 40Ar38Ar+, and 40Ar42Ca+. The presence of these interferences affects the detection limit and accuracy of Se detection by ICP-MS [18]. To deal with the spectral overlap affecting the determination of Se, the triple quadrupole ICP–MS/MS setup is used, which consists of a reaction cell located between two quadrupole mass analyzers [19,20]. ICP–MS/MS allows for removing the interference ions with different m/z ratios from the target nuclide with the first quadrupole, the second quadrupole then selects one of the possible reaction products formed after the reaction of the cell for the analysis of the target element [21]. Thus, a rapid and accurate method for the speciation of Se(IV), Se(VI), selenocystine (SeCys2), methylselenocysteine (MeSeCys), selenomethionine (SeMet), and selenoethionine (SeEt) by HPLC-ICP-MS/MS was established. The detection limit, precision, accuracy, and recovery rate of the standard addition were carefully investigated. The proposed method was further explored for the real sample analysis, including selenium-enriched drinking water, selenium-enriched tea extract, and selenium-enriched salts, with good analytical results obtained.
2. Experimental
2.1. Chemicals and Instruments
The reagents used in this work are at least analytical grade reagents. Ultrapure water (18.2 MΩ cm at 25 °C) was used throughout the experiment. Se(IV) and Se(VI) stock solutions (1000 mg L−1) were prepared from high-purity Na2SeO3 and Na2SeO4 from Sigma Reagent Company (Shanghai, China). High-purity SeCys2, MeSeCys, SeMet, and SeEt were purchased from Shanghai McLean Biochemical Technology Company (Shanghai, China). Chromatographically pure methanol and sodium citrate were purchased from Chengdu Kelong Chemical Company (Chengdu, China). Argon (99.999%) was purchased from Chengdu Jinli Gas Company (Chengdu, China). Oxygen (99.999%) was purchased from Chengdu Jinnengda Gas Company (Chengdu, China). Selenium-enriched drinking water, selenium-enriched tea, and selenium-enriched salt were purchased from Chinese shopping websites (https://www.taobao.com/ and https://www.jd.com/, accessed on 1 September 2022) and local markets (Chengdu, China). The 0.22 μm PP housing nylon filter was purchased from Tianjin Keyilong Experimental Equipment Company (Tianjin, China).
2.2. Chromatographic Separation
Chromatographic separation was performed using a Vanquish liquid chromatography system (Thermo Fisher Scientific, Waltham, MA, USA), equipped with a binary high-performance liquid chromatography pump. Various forms of selenium were separated on a Hamilton PRP-X100 (10 μm, 4.1 × 250 mm) anion exchange column (Hamilton, Reno, NV, USA). Each mobile phase was filtered through a 0.22 μm filter and degassed prior to use. The detection of selenium was performed using a Thermo Fisher iCAP TQ (Thermo Fisher Scientific, Waltham, MA, USA).
2.3. Instrument Conditions
The instrument operating conditions are shown in Table 1. ICP-MS/MS adopts the O2 reaction cell mode, where the first quadrupole (Q1) allows for the passage of 80Se, the second quadrupole (Q2) is the collision reaction cell of 80Se and O2, and the third quadrupole (Q3) allows 80Se16O to pass through and generate the signal. HPLC and ICP-MS/MS are connected by a PEEK tube, and the chromatographic injection valve is used as the trigger signal for MS.
2.4. Extraction of Selenium for Chromatographic Speciation
The selenium-enriched drinking water did not need to be enriched and derivatized, and was shaken evenly and filtered through a filter for measurement.
The selenium-enriched salt was put in a vacuum drying oven at 50 °C overnight. Then, 2.0 g of sample was dissolved in freshly washed glassware containing 50 mL of water. After being completely dissolved, it was filtered through a filter and then measured.
The selenium-enriched tea leaves were placed in a vacuum drying oven at 50 °C overnight. About 5.0 g of the sample was added into 50 mL of boiling water. After 30 min, it was taken out and cooled to room temperature. It was centrifuged at 4000 r/min for 10 min and the supernatant was collected. We added 50 mL of boiling water to the residue and repeated the extraction once. The supernatants from the two extractions were combined and cooled to room temperature.
3. Results and Discussion
3.1. The Effect of Methanol in the Mobile Phase
Hamilton PRP-X100 is an anion exchange column that has strongly basic quaternary ammonium groups. The addition of a small amount of methanol to the mobile phase can change the elution capacity of the mobile phase and make it easier to control the retention time of the selenoamino acids. Therefore, the concentration of methanol added to the mobile phase should be optimized. It can be seen from Figure 1A that the retention times of MeSeCys, SeMet, and SeEt decreased with the increase in methanol, which was due to the decrease in the interaction between the alkyl chain of the selenoamino acid molecule and the column. The fact that the retention time of SeCys2 did not change significantly may be due to the weak interaction of SeCys2 with the column under these conditions. In addition, the retention times of both Se(IV) and Se(VI) increased, possibly because the elution power of the mobile phase was reduced. It can be seen from Figure 1B that adding a small amount of methanol to the mobile phase effectively increased the signal intensity of the selenium. The signal enhancement can be attributed to the charge transfer reaction between C+ and Se atoms in the central channel of the plasma, which induced the increment of the ionization degree for hard-to-ionize elements (~33% for Se), and therefore improved the signal intensities of Se [22,23]. Considering the influence of retention time and signal intensity, 2% methanol concentration was selected.
3.2. Influence of Ionic Strength in Mobile Phase
In order to control the retention time of the selenium compounds, the ionic strength of the mobile phase was discussed, and the results are shown in Figure 2. The retention times of Se(IV) and Se(VI) decreased with the increase of ionic strength (Figure 2A). This is because of the interaction between Se(IV) and Se(VI), and the quaternary ammonium group in the column decreased with the ionic strength increase. The retention times of the other selenoamino acids was affected a little by the ionic strength. As Figure 2B shows, the ionic strength had little effect on the peak area of the Se species. Under comprehensive considerations, the subsequent experiments were performed at 25 mM ionic strength.
3.3. Effect of PH
The pKa of the six Se species was across a broad range. Therefore, the pH of the mobile phase could determine the charge and subsequent retention behavior of these compounds. As shown in Figure 3A, SeCys2, MeSeCys, and SeEt did not have a significant change in retention time. This maybe because the ionization of the three species did not change at this pH. After the pH reached 6.0, SeEt carried more negative charges, which enhanced the interaction with the column, resulting in an increase in retention time. The retention behavior of Se(IV) and Se(VI) was controlled by pH-controlled protonation of selenium anion and citrate anion of the mobile phase. The pKa1 and pKa2 of Se(IV) were 2.35 and 7.94, respectively, and the pKa2 of Se(VI) was 1.92 [24]. In the studied pH range, Se(VI) was completely deprotonated, and the predominant form of Se(IV) was converted from HSeO3− to SeO32−. Therefore, the dissociation behavior of citric acid determined the retention of Se(VI). With the increase in pH, the more deprotonation of citric acid, and the more competitive it was for Se(VI), which resulted in a shorter retention time. After the pH reached 5.0, the retention time was basically stable. To ensure the resolution and signal strength, pH 4.0 was chosen.
3.4. Analytical Performance and Sample Analysis
Under the best experimental conditions, the mixed standard series of six forms of selenium were determined, and the chromatogram is shown in Figure 4. Taking the concentration of selenium (x) as the abscissa and the area of the chromatographic peak (y) as the ordinate, the standard curves are shown in Figure S1. The results showed that the concentrations of the six forms of selenium had a linear relationship with their chromatographic peak areas within a certain range, and the correlation coefficients (R2) were all greater than 0.995. The limit of detection (LOD) and limit of quantification (LOQ) for this method were calculated at a three-fold signal-to-noise ratio (S/N = 3) and a 10-fold signal-to-noise ratio (S/N = 10). The LOD of the six forms of selenium was between 0.02–0.15 ng mL−1 and the LOQ was between 0.07–0.50 ng mL−1. The regression equations, linear ranges, LOD, and LOQ of the six forms of selenium are shown in Table 2.
In order to verify the precision of the method, selenium-enriched drinking water, selenium-enriched salt, and selenium-enriched tea were selected for standard addition and recovery experiments, and three replicates were performed for each concentration. The recovery results are shown in Tables S1–S3. The recovery rates of the six forms of spiked selenium were between 93.7% and 105%, and the RSD was less than 3.52%. The above results show that the proposed method had good precision, meeting the requirement of real sample analysis.
Furthermore, 13 kinds of selenium-enriched drinking water, 5 kinds of selenium-enriched salts, and 12 kinds of selenium-enriched tea leaves on the market were analyzed for selenium speciation. As shown in Table S4 and Figure S2, only one form of selenium, Se (VI), was contained in the selenium-enriched drinking water. However, the distribution of selenium species in selenium-enriched salts was more varied (Table 3 and Figure S3). The organic selenium species contained in them were SeCys2, MeSeCys, and SeEt, with no SeMet, and the inorganic selenium species were Se(IV) and Se(VI). As listed in Table 4 and Figure S4, it is interesting that the selenium-enriched tea showed five forms of selenium, except SeEt. After the determination of the total selenium, it was found that the extraction rate of the tea leaves was between 13.1% and 27.5%. Moreover, there are still many unknown forms of selenium in the selenium-enriched salt and the selenium-enriched tea that need to be further studied and identified.
4. Conclusions
In this study, a sensitive and accurate method for the simultaneous speciation of six selenium species in water, salt, and tea was established by HPLC-ICP-MS/MS. The chromatographic conditions were carefully investigated, Hamilton PRP-X100 was used with 25 mM sodium citrate and 2% methanol (pH = 4.0) as the mobile phases, with 80Se16O as the quantitative isotope. Furthermore, the application of the proposed method to the analysis of selenium-enriched foods was demonstrated to be feasible, and the obtained recovery was 93.7–105% with RSD < 5%. The detection limits of Se(IV), Se(VI), SeMet, SeCys2, MeSeCys, and SeEt were 0.04, 0.02, 0.05, 0.02, 0.03, and 0.15 ng mL−1, respectively. It was found that the selenium-enriched drinking water was all Se (VI), while the selenium-enriched salt contained Se(IV), Se(VI), SeCys2, MeSeCys, and SeEt without SeMet, and no SeEt was detected in the selenium-enriched tea, where SeMet was the majority. In addition, the unidentified Se compounds found in selenium-enriched salt and selenium-enriched tea should be identified by MS in the future.
In this study, a sensitive and accurate method for the simultaneous speciation of six selenium species in water, salt, and tea was established by HPLC-ICP-MS/MS. The chromatographic conditions were carefully investigated, Hamilton PRP-X100 was used with 25 mM sodium citrate and 2% methanol (pH = 4.0) as the mobile phases, with 80Se16O as the quantitative isotope. Furthermore, the application of the proposed method to the analysis of selenium-enriched foods was demonstrated to be feasible, and the obtained recovery was 93.7–105% with RSD < 5%. The detection limits of Se(IV), Se(VI), SeMet, SeCys2, MeSeCys, and SeEt were 0.04, 0.02, 0.05, 0.02, 0.03, and 0.15 ng mL−1, respectively. It was found that the selenium-enriched drinking water was all Se (VI), while the selenium-enriched salt contained Se(IV), Se(VI), SeCys2, MeSeCys, and SeEt without SeMet, and no SeEt was detected in the selenium-enriched tea, where SeMet was the majority. In addition, the unidentified Se compounds found in selenium-enriched salt and selenium-enriched tea should be identified by MS in the future.
Supplementary Materials
The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/separations9090242/s1, Figure S1: Fitted standard curve of six selenium species; Figure S2: Chromatograms of 13 kinds of selenium-enriched drinking water; Figure S3: Chromatograms of five selenium-enriched salts; Figure S4: Chromatograms of 12 kinds of selenium-enriched tea leaves. Table S1: Recovery test results of standard addition to selenium-enriched drinking water; Table S2: Test results of standard addition and recovery of selenium-enriched salts; Table S3: Test results of standard addition recovery of selenium-enriched tea extract; Table S4: Analysis results of selenium speciation in selenium-enriched drinking water.
Author Contributions
Conceptualization, Y.L. and F.X.; methodology, F.X. and J.L.; software, Y.L. and F.X.; validation, X.D., X.F. and H.C.; formal analysis, F.X. and J.L.; investigation, X.X. and Y.H.; resources, G.C. and J.L.; data curation, Y.L. and F.X.; writing—original draft preparation, Y.L. and F.X.; writing—review and editing, F.X.; visualization, Y.L. and F.X.; supervision, G.C. and J.L.; project administration, G.C. and J.L.; funding acquisition, G.C. and J.L. All authors have read and agreed to the published version of the manuscript.
Funding
This research was funded by the Science and Technology Department of Sichuan Province, grant number [2021YFN0019].
Institutional Review Board Statement
Not applicable.
Informed Consent Statement
Not applicable.
Data Availability Statement
Not applicable.
Acknowledgments
We acknowledge financial support from the Science and Technology Department of Sichuan Province (no. 2021YFN0019).
Conflicts of Interest
The authors declare no conflict of interest.
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Figure 1.
The effect of methanol in mobile phases on (A) retention time and (B) peak area of SeCys2, MeSeCys, Se(IV), SeMet, SeEt, and Se(VI). SeCys2: 200 ng mL−1, MeSeCys: 100 ng mL−1, Se(IV): 100 ng mL−1, SeMet: 500 ng mL−1, SeEt: 200 ng mL−1, Se(VI): 100 ng mL−1.
Figure 1.
The effect of methanol in mobile phases on (A) retention time and (B) peak area of SeCys2, MeSeCys, Se(IV), SeMet, SeEt, and Se(VI). SeCys2: 200 ng mL−1, MeSeCys: 100 ng mL−1, Se(IV): 100 ng mL−1, SeMet: 500 ng mL−1, SeEt: 200 ng mL−1, Se(VI): 100 ng mL−1.
Figure 2.
The effect of ionic strength in the mobile phase on (A) retention time and (B) peak area of SeCys2, MeSeCys, Se(IV), SeMet, SeEt, and Se(VI). Mobile phase conditions: 2% methanol, pH = 4.0, 1.0 mL min−1.
Figure 2.
The effect of ionic strength in the mobile phase on (A) retention time and (B) peak area of SeCys2, MeSeCys, Se(IV), SeMet, SeEt, and Se(VI). Mobile phase conditions: 2% methanol, pH = 4.0, 1.0 mL min−1.
Figure 3.
The effects of pH on the (A) retention time of Se species and (B) intensity.
Figure 4.
Chromatograms of the standard curves of six selenium species (Hamilton PRP-X100 column; 25 μL injected; mobile phase conditions: 2% methanol, 25 mM Sodium citrate, pH = 4.0, flow rate of 1.0 mL min−1).
Figure 4.
Chromatograms of the standard curves of six selenium species (Hamilton PRP-X100 column; 25 μL injected; mobile phase conditions: 2% methanol, 25 mM Sodium citrate, pH = 4.0, flow rate of 1.0 mL min−1).
Table 1.
Instrumental parameters.
| ICP-MS/MS | |
|---|---|
| Forward power | 1550 W |
| Carrier gas (Ar) flow rate | 1.0 L min−1 |
| Reaction gas (O2) flow rate | 0.3 mL min−1 |
| Isotopes monitored | 80Se (Q1), 80Se16O (Q3) |
| HPLC | |
| Column | Hamilton PRP-X100 (10 μm, 4.1 × 250 mm) |
| Mobile phase | 25 mM Sodium citrate-2% Methanol (pH = 4.0) |
| Flow rate | 1.0 mL min−1 |
| Column temperature | 30 °C |
| Injection volume | 25 μL |
Table 2.
Analytical characteristics of the proposed method.
| Component | Linear Range (ng mL−1) | R2 | LOD (ng mL−1) | LOQ (ng mL−1) |
|---|---|---|---|---|
| SeCys2 | 0.07–100 | 0.995 | 0.02 | 0.07 |
| MeSeCys | 0.07–100 | 0.998 | 0.03 | 0.11 |
| Se(IV) | 0.07–100 | 0.997 | 0.02 | 0.07 |
| SeMet | 0.35–500 | 0.999 | 0.05 | 0.17 |
| SeEt | 0.35–500 | 0.995 | 0.15 | 0.50 |
| Se(VI) | 0.14–200 | 0.998 | 0.04 | 0.14 |
Table 3.
Selenium speciation in selenium-enriched salt (μg kg−1).
| Sample | SeCys2 | MeSeCys | Se (IV) | SeMet | SeEt | Se (VI) |
|---|---|---|---|---|---|---|
| ChongYan iodine-enriched selenium salt | - | - | 17.9 | - | 11.9 | - |
| XinJiang Tianshan snow crystal salt | - | - | - | - | 17.2 | 71.96 |
| LuJing selenium-enriched sea salt | 4.09 | - | 8.57 | - | - | - |
| ZhongYan iodized table salt | 5.26 | 6.33 | 2.63 | - | - | - |
| JiYan selenium-enriched edible salt | 3.77 | - | 2.87 | - | - | - |
Table 4.
Selenium speciation in selenium-enriched tea extract (μg kg−1).
| Sample | SeCys2 | MeSeCys | Se (IV) | SeMet | SeEt | Se (VI) | Total Se | Extraction Rate (%) |
|---|---|---|---|---|---|---|---|---|
| ZiYang selenium-enriched green tea | 0.77 | 1.13 | 3.83 | 6.62 | - | 1.51 | 52.7 | 26.3 |
| EnShi selenium tea (fried green) | 0.66 | 0.67 | 1.95 | 4.18 | - | 0.40 | 32.6 | 24.1 |
| EnShi selenium tea (premium green tea) | 0.41 | 0.82 | - | 1.56 | - | 0.30 | 11.2 | 27.5 |
| EnShi alpine selenium tea | 0.45 | 1.27 | 1.62 | 3.56 | - | 1.45 | 34.4 | 24.3 |
| LiangPing alpine green tea | 0.34 | 0.65 | 0.47 | 1.53 | - | 1.00 | 22.9 | 17.4 |
| BoChuan EnShi selenium tea | 0.36 | - | - | 1.10 | - | 1.67 | 12.1 | 25.9 |
| XiKeXiKe EnShi selenium tea | 0.26 | 1.10 | 0.68 | 2.88 | - | 0.53 | 26.7 | 20.4 |
| JiYe selenium-enriched green tea | 0.38 | 0.77 | 0.99 | 1.67 | - | - | 14.8 | 26.1 |
| ZiYang green tea | 2.13 | 3.53 | 1.96 | 5.90 | - | 6.39 | 87.7 | 22.7 |
| WuHan selenium-enriched tea | 0.67 | - | 1.14 | 4.32 | - | 0.27 | 45.0 | 14.2 |
| Hubei EnShi selenium tea | 0.56 | 0.22 | 0.50 | 1.71 | - | - | 23.5 | 13.1 |
| LvWanjia selenium chrysanthemum | 4.56 | 0.14 | 7.90 | 2.05 | - | 10.52 | 108 | 23.2 |
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Luo, Y.; Chen, G.; Deng, X.; Cai, H.; Fu, X.; Xu, F.; Xiao, X.; Huo, Y.; Luo, J. Speciation of Selenium in Selenium-Enriched Foods by High-Performance Liquid Chromatography-Inductively Coupled Plasma-Tandem Mass Spectrometry. Separations 2022, 9, 242. https://doi.org/10.3390/separations9090242
AMA Style
Luo Y, Chen G, Deng X, Cai H, Fu X, Xu F, Xiao X, Huo Y, Luo J. Speciation of Selenium in Selenium-Enriched Foods by High-Performance Liquid Chromatography-Inductively Coupled Plasma-Tandem Mass Spectrometry. Separations. 2022; 9(9):242. https://doi.org/10.3390/separations9090242
Chicago/Turabian StyleLuo, Yue, Gang Chen, Xiuqing Deng, Hanqing Cai, Xueheng Fu, Fujian Xu, Xiaonian Xiao, Yumeng Huo, and Jin Luo. 2022. "Speciation of Selenium in Selenium-Enriched Foods by High-Performance Liquid Chromatography-Inductively Coupled Plasma-Tandem Mass Spectrometry" Separations 9, no. 9: 242. https://doi.org/10.3390/separations9090242
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