1Key Laboratory of Natural Medicines of the Changbai Mountain, Ministry of Education, Yanbian University College of Pharmacy, Yanji, P.R. China;
2Sinovac Biotech Co. Ltd., Beijing, P.R. China
We developed an ultrasensitive and highly selective method to quantify iodate (IO3–) in iodized salt and biological samples. In the high-performance liquid chromatography (HPLC) system, IO3– was injected with sampler and reduced to iodide (I–) online using a Coulomb detector (CD) as a pre-column reactor, retained by C18 column with the help of ion pairs, and then determined by an amperometric detector (AD). For analysis of IO3–, several parameters and reactive conditions of Coulomb detector, including applied potential, pH value, and salt concentration, were optimized to obtain the best reduction efficiency. The optimized HPLC–CD-AD method was found to be linear over a wide range of IO3– concentration (0.05–0.25 μg/mL) with appreciable recovery rates (86.90–94.70%) of quality controls at excellent detection limits (100 pg) and acceptable variability. The analysis of IO3– in actual samples, such as urine of volunteers, iodized salt, and seawater, discovered that the method has high selectivity and little interference. It can be concluded that the proposed method was ultrasensitive and highly selective in the trace detection of IO3.
Key words: iodate, electrochemical reduction, high-performance liquid chromatography (HPLC), amperometric -detector, Coulomb detector
*Corresponding Authors: Gao Li, Key Laboratory of Natural Medicines of the Changbai Mountain, Ministry of -Education, Yanbian University College of Pharmacy, Yanji, P.R. China. Email: [email protected]. Tao Wang, Sinovac -Biotech Co. Ltd., P.R. China. Email: [email protected]
Received: 16 October 2022; Accepted: 18 May 2023; Published: 1 July 2023
© 2023 Codon Publications
This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0). License (http://creativecommons.org/licenses/by-nc-sa/4.0/)
Iodine is one of the trace elements required by the human body. For the synthesis of human thyroid hormones, iodine is essential and affects human growth and development, especially the growth and development of the brain. Lack of iodine causes large thyroid gland and decreased intelligence (Delange and Lecomte, 2000). China is one of the major countries with iodine deficiency disorders. Fortunately, in the past few decades, the problem of iodine deficiency has been gradually solved by common use of iodized salt. However, in some economically developed areas of China, people have taken in excess of iodine to cause iodine-induced hyperthyroidism (Delange et al., 1999). Both iodine deficiency and excess of iodine lead to changes in the morphology and functioning of the thyroid, causing various disorders. Therefore, it is necessary to find an accurate and precise determination method of iodine for quality control of iodized salt and monitoring human urine samples.
Nowadays, salt products in Chinese markets are “iodized” by the addition of potassium iodate (KIO3). Thus, it is important to analyze iodate (IO3–) in salt and urine samples of patients.
In the literature, many analytical methods have been developed for determining IO3–, including spectrophotometry (Ford and Johnson, 1991), volumetric methods (Edmonds and Morita, 1998), mass spectrometry (Barnett et al., 1999; Shin et al., 1996), electrochemical analysis (Shabani et al., 1999), atomic absorption spectroscopy, ion chromatography (Kumar et al., 2000), and high--performance liquid chromatography (HPLC; Li et al., 2001). Among the above-mentioned methods, ion chromatography is undoubtedly the most attractive method of choice. At the very beginning, ion chromatography equipped with conductivity detector was used to determine iodide (I–) in seawater, urine, and other natural product samples, and then it was developed to a more sensitive stage, in which electrochemical detector (ED) was used.
Commercial ion chromatographs with various detectors are used to analyze various inorganic ions. However, some challenge still prevailed. First, high price hinders the application of ion chromatograph brand machine equipped with ion chromatography column in any China laboratory. Second, large amounts of matrix ions (chloride, sulfate, and other inorganic ions) impede the determination of target analysts by disturbing IO3– signals. Furthermore, with the development and fusion of liquid chromatography, boundaries between conventional ion chromatography and other HPLC techniques have blurred. For example, reversed-phase liquid chromatography, through the addition of ion-pairing reagents, can retain and separate periodate, IO3–, and I– very well in C8 or C18 column without using a dedicated ion exchange chromatography column (Huang et al., 2013; Sajonz, 2006). Ion chromatography columns are expensive and have a short lifetime. The iodine method is cumbersome to operate and cannot achieve online automated analysis; therefore, it is necessary to develop highly sensitive, inexpensive, and automatic instrumental analysis methods.
Our previous study reported a novel technique to determine IO3– in table salt by HPLC–ultraviolet (UV) detector after online electrochemical reduction to I–. IO3– undergoes reduction on the electrodes of Coulomb detector (CD) to produce I–: “IO3– + 6e + 6H+ = I– + 3H2O.” The UV response to I– is nearly 10 times more than that of IO3–, thus amplifying the signal of IO3– (Wang et al., 2015).
In this study, we improved the above-mentioned method of online electrochemical reduction. An ultrasensitive result for quantification of IO3– was obtained by selecting amperometric detector (AD) to detect the resulting I–. The sensitivity of amperometric detector is higher by two orders of magnitude than that of ultraviolet detector.
A schematic diagram of the method is illustrated in Figure 1. A Coulomb detector (Eicom, Japan) was linked on pre-column as an electrochemical reactor, and a high-voltage switching six-way valve (Shiseido, Japan) prevented the cell and electrode of Coulomb detector from high back pressure of column. The samples containing IO3– were injected by auto-sampler and transferred into Coulomb detector through mobile phase 1. In the reactor, IO3– was changed into I– by electrochemical reduction at electrode, and the resulting I– was collected in the peek loop.
Figure 1. Schematic diagram of HPLC–CD-AD system for determining IO3–.
The process would take 30 s to get ready for the next stage. At the next stage, the high-voltage six-way valve was immediately switched to INJECT position, and pump 2 transferred mobile phase 2 to inject I– from the opposite end of peek loop to C18 column for subsequent isolation and analysis. Finally, amperometric detector was used to detect I–, and ultrasensitive signal was obtained.
Potassium iodate and potassium iodide (KI) were purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Potassium dihydrogen phosphate (KH2PO3), tetrabutylammonium chloride, and ethylenediaminetetracetic acid disodium (EDTA-2Na) were obtained from Fluka (Milwaukee, WI). Deionized water was purified through a water purification system (Millipore Milli-Q, Millipore, Bedford, MA). Methanol was of HPLC gradient grade and purchased from Mallinckrodt Baker (Deventer, the Netherlands). Different iodized salt samples were purchased from supermarkets in Beijing, China. Seawater samples were obtained from Bohai Seashore in Tianjin, China. Human urine samples were provided by volunteers. All chemicals were used without further purification.
In this study, the HPLC equipment consisted of two 3001 high-pressure pumps (Shiseido, Japan) equipped with a 3010 degasser, a HTEC-500 Coulomb detector (Eicom, Japan), a HTEC-500 amperometric detector (Eicom, Japan), a high-voltage switching six-way valve (Shiseido, Japan), and a 3004 column oven (Shiseido, Japan). A MG II C18 column (5 μm, Φ 4.6 × 250 mm; Shiseido, Japan) was used for analysis of I–. The operating conditions of HPLC system are given in Table 1.
Table 1. Operating conditions for HPLC system.
|Mobile phase of reduction||200 mmol L–1 phosphate buffer solution (pH 1.0)|
|Flow rate||0.2 mL•min–1|
|Applied potential of CD||–2,000 mV|
|Peek loop volume||200 μL|
|Switch internal time||30 s|
|Mobile phase of I– detection||Aqueous solution (20 mmol/L KH2PO3, 10 mmol/L tetrabutylammonium chloride, 1 mmol/L EDTA-2Na, pH 4.5): methanol = 90:10|
|Stationary phase||MG II C18|
|Flow rate||0.3 mL•min–1|
|Applied potential of amperometric detector||+50 mV|
Clearly, the reduction efficiency of IO3– in Coulomb detector is critical to the sensitivity and accuracy of determination. Reduction efficiency (RE) was calculated by using the following equation:
Reduction efficiency (RE) = (Amount of producing I–/amount of total IO3–) × 100%
In order to obtain high reduction efficiency, four operating factors of condition that could influence reduction efficiency, including applied potential, pH, salt concentration, and flow rate of mobile phase 1, were investigated in detail.
We prepared 1 ug•mL–1 of I– and IO3– standard solutions by diluting their stock solutions (100 ug mL–1) with water (Watsons, HK). Then IO3–1 was analyzed by HPLC–CD-AD system in the form of I– with different conditions listed in Table 1.
Samples of Sseawater, iodized salt, and human urine samples were diluted and filtered through a 0.22-μm filter membrane for detection under the above-mentioned chromatographic conditions.
For methodological validation, the data were calculated as mean and relative standard deviation (RSD). Statistical differences between groups were assessed using Student’s t-test. For measurements, P < 0.05 was considered statistically significant between groups.
The effect of applied potential on reduction efficiency was investigated; the results are shown in Figure 2A. More is the negative applied potential, more is the intensive reduction that takes place at electrode. In the present study, eight levels of applied potential were designed, namely –300, –500, –1,000, –1,300, –1,500 –1,800, –1,900, and –2,000 mV. Reduction efficiency with –1,900 and –2,000 mV were 95.61% and 97.44%, respectively. The effect of pH on the reduction efficiency of IO3– is shown in Figure 2C. It was indicated that the reduction efficiency of IO3– were very low, within the range of pH 4.0–7.5. When pH was adjusted within 2.0–1.0, reduction efficiency increased obviously and reached over 95%. Figure 2B shows the effect of flow rate of mobile phase 1 on the reduction efficiency of IO3–. The effect of flow rate on reduction rate was small, but when the flow rate was too large, reduction rate decreased significantly. If the salt concentration was changed in the range of 50–200 mmol•L–1, the reduction efficiency of IO3– increased from 74.3% to 96.5% (Figure 2D). Ions increase the conductivity of the system and, therefore, promote redox reaction.
Figure 2. (A) Effect of applied potential; (B) flow rate of mobile phase of reduction; (C) pH; and (D) salt concentration on reduction deficiency of IO3– (1.0 μg mL–1).
As shown in Table 2, the recovery of the method was from 86.9% to 91.7% and RSD was in the range of 1.1–3.0%. The linearity of the curves, y = 5199651.8x–1538705.4, r2 = 0.9987 and y = 1765541.126x-24738.1938, were in the range of 0.4–1.7 μg/mL and 0.05–0.25 μg/mL, respectively, with a correlation coefficient of 0.999 (Figure 3). Common anions (Cl–, Br–, NO3–, NO2–, CO32–, SO42–, SO32–, and HCO3–) do not interfere with the determination of I– ions, and the detection limit (LOD) was 100 pg (5 ng/mL, 20 μL was injected, S/N = 3) (Figure 4).
Table 2. Method accuracy and reproducibility.
|Samples||Added (µg/mL)||Test (µg/mL)||Recovery (%)||RSD (%)|
RSD: relative standard deviation.
Figure 3. Calibration linearity of method in the range of (A) 0.4–1.7 µg/mL, and (B) 0.05–0.25 µg/mL.
Figure 4. Ultrasensitive and highly selective determination of IO3–: (A) Common anions do not interfere with the determination of I– ions; and (B) detection limit (LOD) (S/N = 3,100 pg).
Iodate in iodized salt was determined by HPLC–CD-AD system (Table 1) and results are given in Table 3. All results were consistent with those obtained by titration (Chinese Pharmacopoeia Commission, 2015; Ministry of Housing and Urban-Rural Development of PR China, 2012). The results obtained for urine samples (samples were filtered with a 0.22-μm filter membrane) showed the presence of IO3– ions in only one sample (Figure 5). The seawater samples were collected from Bohai Bay in Tianjin and filtered with a 0.22-μm filter membrane. The result of IO3– ions determination are shown in Figure 5A.
Table 3. Detection of iodized salt in Chinese market.
|Samples||Results by standard method (mg/kg)||Results by new HPLC–CD-AD (mg/kg)|
Figure 5. Chromatogram of samples: (A) Seawater sample from Bohai Bay; and (B) urine sample of volunteer A.
The Coulomb carbon electrode is more suitable for the analysis of organic matter, rather than inorganic ions. However, because of large adsorption surface area of carbon material, it has high reaction efficiency as a full reaction electrode, so it is indispensable for online reduction and detection of IO3–. In order to improve the reduction efficiency of IO3–, we adjusted the pH value of the mobile phase to a harsher acidity. Fortunately, it was within the tolerance range of the instrument, and the ideal experimental results were obtained.
According From to Faraday’s law of electrolysis, the mass of produced substance at an electrode, because of electrochemical reaction, is proportional to the mass of -electrons transferred to the electrode. IO3– transferred from the bulk solution to the electrode surface was affected by flow rate, diffusion coefficient, and viscosity of mobile phase 1. And the appropriate flow rate of mobile phase 1 is beneficial for diffusion. Therefore, 0.2 mL•min–1 of flow rate was chosen in the present study.
Naturally, the most important influencing factor is the applied voltage of electrode, which is the driving force of electrolytic reaction. As shown in Figure 4, reduction of IO3– was very weak with applied potential of –600–1,100 mV. The amount of I– increased significantly with increase of applied potential from –1,100 to –1,900 mV.
The selected operating conditions in the present work were: 200 mmol•L–1 sodium dihydrogen phosphate (pH 1.0) of mobile phase 1, –1,900 mV of applied potential, and 0.2 mL•min–1 of flow rate. The efficiency of IO3– reduction to I– ion could be more than 95%, which satisfied the needs of high-sensitivity detection.
Because the electrode potential required for the oxidation of iodine ions to iodine element is extremely low, the ampere detector only needs an operating voltage of +50 mV to obtain a strong response signal so that the detection result with low interference and high sensitivity is obtained. Detection limit and quantity limit (LQD) were 9 ng and 20 ng, respectively.
A novel method based on HPLC–CD-AD for obtaining ultrasensitive signal of IO3– by online reduction to I– ion was described. The proposed method was simple and highly sensitive with good precision. In the future, the HPLC–CD-AD system could be used widely for IO3– detection.
The authors declare that they have no conflict of interests.
Gao Li and Tao Wang designed the study. Qingwei Jiang and Tao Wang performed the experiments and collected the data. Qingwei Jiang and Tao Wang interpreted the data and prepared the manuscript. All authors read and approved the final manuscript.
Barnett, D.A., Guevremont, R. and Purves, R.W., 1999. Determination of parts-per-trillion levels of chlorate, bromate, and iodate by electrospray ionization/high-field asymmetric waveform ion mobility spectrometry/mass spectrometry. Applied Spectroscopy 53(11): 1367–1374. 10.1366/0003702991945984
Chinese Pharmacopoeia Commission, 2015. Chinese pharmacopoeia: appendix. 194–195. China Medical Science Press, Beijing, China
Delange, F., Benoist de B. and Alnwick D., 1999. Risks of iodine--induced hyperthyroidism after correction of iodine deficiency by iodized salt. Thyroid 9: 545–556. 10.1089/thy.1999.9.545
Delange, F. and Lecomte, P., 2000. Iodine supplementation: benefits outweigh risks. Drug Safety 22: 89–95. 10.2165/00002018-200022020-00001
Edmonds, J.S. and Morita, M., 1998. The determination of iodine spices in environmental and biological samples. Pure and Applied Chemistry 72: 1567–1584. 10.1351/pac199870081567
Ford, H.C. and Johnson, L.A., 1991. Ascorbic acid interferes with an automated urinary iodide determination based on the ceric--arsenious acid reaction. Clinical Chemistry 37: 759. 10.1093/clinchem/37.5.759
Huang, Z.P., Subhani, Q., Zhu, Z.Y., Guo, W.Q. and Zhu, Y.A., 2013. Single pump cycling-column-switching technique coupled with homemade high exchange capacity columns for the determination of iodate in iodized edible salt by ion chromatography with UV detection. Food Chemistry 139: 144–148. 10.1016/j.foodchem.2013.01.070
Kumar, S.D., Maiti, B. and Mathur, P.K., 2001. Determination of iodate and sulphate in iodized common salt by ion chromatography with conductivity detection. Talanta 53: 701–705. 10.1016/S0039-9140(00)00504-X
Li, H.B., Chen, F. and Xu, X.R., 2001. Determination of iodide in seawater and urine by size exclusion chromatography with iodine–starch complex. Journal of Chromatography A 918: 335–339. 10.1016/S0021-9673(01)00776-2
Liu, L., Li, X., Wang, H., Cao, X. and Ma, W., 2017. Reduction of iodate in iodated salt to iodide during cooking with iodine as measured by an improved HPLC/ICP–MS method. Journal of Nutritional Biochemistry, 42, 95–100. 10.1016/j.jnutbio.2016.12.009
Ministry of Housing and Urban-Rural Development of PR China, 2012. General test method in salt industry – determination of iodide ion. GB/T 13025.7-2012, the Chinese national standard. China Environment Science Press, Beijing, China.
Sajonz, P., 2006. Separation of periodate, iodate and iodide on a C-18 stationary phase. Dependence of the retention on the temperature and solvent composition. Monitoring of an oxidative cleavage reaction. Chromatographia 64: 635–640. 10.1365/s10337-006-0082-9
Shabani A.M.H., Ellis P.S. and Mckelvie I.D., 2011. Spectrophotometric determination of iodate in iodised salt by flow injection analysis. Food Chemistry 129(2): 704–707. 10.1016/j.foodchem.2011.04.077
Shin, H.S., Oh-Shin, Y.S., Kim, J.H., Ryu, J. K., 1996. Trace level determination of iodide, iodine and iodate by gas chromatography--mass spectrometry. Journal of Chromatography A 732(2): 327–333. 10.1016/0021-9673(95)01281-8
Wang, T., Lin, W.M., Dai, X.L., Gao, L.J., Wang, B. and Quan, D.Q., 2015. Determination of iodate by HPLC-UV after on-line electrochemical reduction to iodide. Journal of Chromatographic Science 53(2): 280–284. 10.1093/chromsci/bmu053