Introduction

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^–: “IO^3– + 6e + 6H⁺ = I^– + 3H₂O.” The UV response to I^– is nearly 10 times more than that of IO₃^–, thus amplifying the signal of IO^3– (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.

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.

Materials and methods

Materials

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.

Equipment and chromatographic conditions

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.

Operating conditions optimization of online electrochemical reduction system

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.

Sample preparation

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.

Statistical analysis

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.

Results

Operating conditions optimization of online electrochemical reduction system

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.

Calibration of method

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^–, CO₃^2–, SO₄^2–, SO₃^2–, 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).

Analysis of iodized salt, seawater, and human urine samples

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.

Discussion

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.

Conclusion

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.