Trypsin Digestion Pretreatment of Kelp Samples and the Iodine Speciation Analysis of Iodate, Iodide, MIT, and DIT by HPLC-ICP-MS

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Introduction
As an essential trace element for human life activities, iodine mainly exists in the iodide or iodate in nature, and a small part exists in the organic iodine in seaweed [1]. Iodine can only be obtained from the outside world, and the iodine content in food is extremely low. Te resulting iodine defciency causes goiter and afects the intelligence and physical development of the human body [1][2][3]. Te bioavailability and toxicity of diferent forms of iodine are diferent, and the toxicity of iodate is more signifcant than iodide. So far, potassium iodide and potassium iodate are the main iodine additives in the iodized salt. Excessive consumption fails to supplement iodine and leads to adverse reactions such as iodine poisoning [4]. Organic iodine is an ideal scientifc iodine supplement product, which can be directly absorbed by the human body, not afected by the absorption of iodine due to insufcient protein intake. Terefore, the speciation analysis of iodine in kelp is essential for producing iodized table salt.
Due to the complex and diverse forms of iodine, the characteristics of instability and easy sublimation greatly increase the limitation of the determination of iodine species. Kelp, as a biological sample, has a complex matrix, so it is hugely challenging for speciation analysis of trace and ultratrace iodine. At present, there are the following measurement methods of ultraviolet spectrophotometry (U.V.) [5], capillary electrophoresis (C.E.) [6,7], liquid chromatography and inductively coupled plasma mass spectrometry (LC-ICP-MS) [2,4,[8][9][10][11][12], and ion chromatography (I.C.) [13], and so on, for the determination of total and speciation of iodine. Nunes et al. [5] used a U.V. spectrophotometer to measure iodine in kelp, which is simple but easily afected by substances such as protein. Sun et al. [6] successfully separated IO 3 − , I − , MIT, and DIT by UV electrophoresis. Xu et al. [7] completed the separation of various iodine forms using surfactant-coated multiwalled carbon nanotubes as the stationary phase in electrophoresis. However, the separation conditions were complicated and challenging to operate. Liquid chromatography coupled with inductively coupled plasma mass spectrometry (HPLC-ICP-MS) has high sensitivity, low detection limit, and low negative interference by other ions [2,4,[8][9][10][11][12] and is able to separate various iodine species and simultaneously measure iodine content. Terefore, this paper selects the HPLC-ICP-MS combined method for iodine speciation analysis and determination.
Before sample determination, it is necessary to extract the iodine in kelp into the solution. At present, the common pretreatment methods include dry alkali incineration [5], ionic surfactant extraction [14], ultrasonic microwaveassisted extraction [6,10,15], alkali digestion [4,12], and enzymatic hydrolysis [10,16]. Wang et al. [14] treated kelp samples using microwave assistance with zwitterionic surfactants as extractants, and I − , MIT, and DIT could be measured. However, the surfactants are expensive and the high temperature of microwaves can destroy partial iodine species. Alkaline digestion is mostly used as a pretreatment method for the total iodine content determination in kelp samples and cannot extract organic iodine in the samples. Romaris-Hortas et al. [10] comparatively studied the enzymatic hydrolysis properties of cellulase, α-amylase, β-glucose, lipase, pepsin, pancreatin, and mixed enzymes on kelp under ultrasound, and experimental data showed that the highest content of organic iodine (MIT and DIT) was obtained by trypsin extraction. In this work, we systematically compared the enzymatic hydrolysis performance of trypsin, pancreatin, protamex, and neutral protease. Finally, we frst establish a pretreatment method for kelp samples using trypsin. Analysis method of iodine speciation (IO 3 − , I − , MIT, and DIT) was established by using liquid chromatography coupled with inductively coupled plasma mass spectrometry (HPLC-ICP-MS), and it was implemented in kelp samples.

2.1.2.
Instrumentation. An iCAP Q ICP-MS (Termo Scientifc, USA) was used for quantitative analysis of iodine speciation. In this study, a Termo U3000 HPLC system was successfully used to separate iodine species with an injection volume of 20 μL. Separation of iodine species was achieved using an IonPac AS-14 anion column (4 mm × 250 mm, Termo Scientifc, USA). Te pH value was measured by a high-precision pH meter (WTW PH-7310, Shanghai Precision Scientifc Instruments Co., Ltd., China) with an uncertainty of ±0.003.

Sample Preparation.
Te kelp samples were rinsed twice with deionized water and dried in an oven at 40°C. Grind dried kelp to powder, seal, and store in a cool and dry place for later use. 0.04 g of trypsin and 0.2 g of dried kelp samples were weighed and sonicated in 20 mL of NaH 2 PO 4 /NaOH (pH � 8) bufer solution at 50°C for 10 h [9,16]. Te obtained solution was centrifuged and fltered repeatedly, and the volume was fxed in a 50 mL volumetric fask and then stored at low temperature and protected away from light. All solutions were fltered through 0.45 μm microporous membranes before use.

Experimental Conditions.
Te operating conditions of the HPLC-ICP-MS are shown in Table 1.

Sample Analysis and Determination
. Te sample to be tested was diluted and injected into the sampling system with an injection needle. Under optimal instrument operating conditions, a gradient elution method with 100 mmol·L −1 (NH 4 ) 2 CO 3 (pH � 9) as mobile phase at a fow rate of 0.8 mL·min −1 from 0 to 1500 s and a fow rate of 1.2 mL·min −1 from 1500 s to 3300 s was used.

Pretreatment Method Establishment.
Diferent biological enzymes' biological activities vary, and the corresponding active sites of enzymatic hydrolysis are various, resulting in diferent peptide chain structures and lengths obtained by enzymatic hydrolysis. Considering that 3-iodo-L-tyrosine (MIT) and 3,5-diiodo-L-tyrosine (DIT) are obtained by enzymatic hydrolysis of kelp protein, the experiment mainly explored hydrolytic characteristics of trypsin, pancreatin, protamex, and neutral protease.
When the solid-liquid ratio was 1 : 20, and the amount of enzyme added was 0.2%, ultrasonication was performed at pH � 7-8 and 50°C for 10 h, respectively. Te obtained solution was centrifuged and fltered and then brought to volume by deionized water in a 50 mL volumetric fask to be measured. Te contents of diverse iodine species measured after treatment with diferent enzymes are shown in Table 2. In addition, the chromatogram of the hydrolysis efects of the four enzymes is shown in Figure 1.
It can be seen from Figure 1 that the four enzymes all showed a certain enzymatic hydrolysis efect on the protein of kelp. Te experimental results show that the hydrolysis efect of neutral protease and pancreatin is poor and that only iodide can be obtained. Both trypsin and protamex can hydrolyze I − and MIT. However, the content of two kinds of iodine in kelp hydrolyzed by trypsin was higher than that of protamex. It can be seen from Table 3 that after adding I − and MIT to the kelp samples and enzymatic hydrolysis, the recovery rates of standard additions are all between 98% and 103%. Hence, trypsin was selected for the pretreatment of kelp samples due to its best hydrolysis efect on the protein of kelp without damaging the iodine form.

Optimization of Enzyme Dosage.
Te enzyme concentration is a key parameter afecting the extraction concentration of organic iodine (MIT and DIT). In order to explore the efect of enzyme solvent concentration on the hydrolysis of kelp protein, 0.02, 0.04, and 0.10 g of trypsin were weighed and dissolved in 10.0 mL of pH � 8 bufer (0.2 mol·L −1 / 0.2 mol·L −1 sodium dihydrogen phosphate/sodium hydroxide), and a blank control group was set. Te trypsin concentrations of the bufer were 0, 2.0, 4.0, and 10.0 mg·mL −1 , respectively. From the data in Figure 2 and Table 4, it can be seen that there was no organic iodine     leaching when no trypsin was involved in the hydrolysis, indicating that the bufer would not extract MIT and DIT from the kelp under ultrasonic assistance. When the trypsin was 0∼0.04 g, with the increase of enzyme concentration, the degree of hydrolysis increased, and the concentration of iodide and monoiodothyronine increased. When the trypsin was more than 0.04 g, the concentrations of iodine and monoiodothyronine decreased to varying degrees. It was speculated that due to the excessive concentration of enzyme, kelp protein was hydrolyzed into iodine amino acids and then continued to hydrolyze, resulting in the decrease of monoiodothyronine.

Optimization of Enzymatic Hydrolysis: pH.
Te pH value is the main parameter afecting the enzymatic hydrolysis process. Te change of pH will afect the stability of the protein structure and has a signifcant efect on the enzymatic hydrolysis efect. Trypsin has a suitable pH. When the pH is not reached, the trypsin activity is not high and the contact with the substrate is less. Trypsin is easily inactivated at pH 8.0 or more and the substrate cannot be digested. Above this pH, the structure of trypsin was destroyed, the activity decreased, and the degree of hydrolysis decreased. Terefore, in this experiment, bufers (0.2 mol·L −1 / 0.2 mol·L −1 sodium dihydrogen phosphate/sodium hydroxide) with pH of 6.0, 7.0, and 8.0 were prepared. Te same quality of kelp samples and 0.04 g trypsin was weighed to explore the efect of pH on trypsin hydrolysis. According to the experimental data, the histogram is shown in Figure 3. When the bufer pH � 8.0, the trypsin activity is the highest, indicating that the enzyme is in sufcient contact with the substrate kelp sample, and the kelp protein can be fully hydrolyzed, so the released iodine content is the highest.

Optimization of Enzymolysis Time.
When using trypsin to hydrolyze kelp protein, it is necessary to determine the appropriate enzymatic hydrolysis time. Too short time will make inadequate enzymolysis; too long time will not only cause excessive hydrolysis of kelp protein but also unnecessary resource waste. In this experiment, 0.2 g kelp sample and 0.04 g trypsin were weighed to explore the efect of enzymolysis time. In order to destroy the cell membrane and wall of seaweeds more efciently and rapidly and promote the direct contact between trypsin and intracellular structures, ultrasound was used as an auxiliary means in the hydrolysis process. According to the data of Table 5, the histogram can be drawn as shown in Figure 4. Te total iodine amount of enzymatic hydrolysis increases with the prolongation of time in the range of 6-10 h. When the enzymolysis time was more than 10 h, the total iodine did not change signifcantly and decreased slightly. It is speculated that monoiodothyronine is not stable in this case. Prolonging the hydrolysis time may cause the decomposition of iodine amino acids, and too long time will also cause the loss of iodide. Terefore, the enzymatic hydrolysis time was controlled at 10 h to ensure the highest content of iodide and monoiodide.

Selection of Separation Conditions.
In order to obtain the best separation efect, the separation conditions such as type, concentration, pH, and fow rate of the mobile phase were optimized. NH 4 H 2 PO 4 , (NH 4 ) 2 CO 3 [8], and NH 4 NO 3    3 can completely elute these four iodine species and separate them nicely. Te mobile phase concentration greatly infuences the retention time of iodine. And the continuous injection of high-salinity eluent into the ICP-MS system for a long time will lead to the accumulation of salt in the sampling cone, afecting the experiment's accuracy and the instrument's service life. In this work, the elution of 50, 100, and 150 mmol·L −1 (NH 4 ) 2 CO 3 mobile phase was investigated and is shown in Figure 5. It could be seen that the peak of DIT disappears when the mobile phase concentration is low, and when the mobile phase concentration increases to 150 mmol·L −1 , the retention time is advanced, but the area of peaks becomes smaller, which means that the detection sensitivity is reduced. Terefore, the whole iodine species can be measured within 3000 s by using 100 mmol·L −1 (NH 4 ) 2 CO 3 as the mobile phase. Te pH of the mobile phase afects the degree of dissociation and distribution ratio of ions, which accordingly afects the retention time of target components on the column. 2% nitric acid and 5% ammonia were used to adjust the pH of the mobile phase. It was seen from Figure 6 that IO 3 − , I − , MIT, and DIT were well separated when pH of the mobile phase was 9, while the peak overlapped and MITcould not be eluted when pH of the mobile phase was 8 and 10, respectively. A large fow rate can cause an increase in column pressure, which will afect the service life of the column. Te excessively low fow rate will prolong elution time, resulting in excessive peak width. 1.2 mL·min −1 and 1.5 mL·min −1 of fowrates were studied for better separation. It was found that IO 3 − , I − , MIT, and DIT could be eluted and separated well both in two fow rates. However, during the measurement of kelp samples, it was found that the peak shapes of IO 3 − and I − occurred tail dragging, so the fow rate was reduced to 0.8 mL·min −1 . After the complete separation of IO 3 and I − , increasing the fow rate to 1.2 mL·min −1 can appropriately shorten the entire elution time and maintain good peak shape. Terefore, considering the separation efciency, detection sensitivity, and column lifetime, a mobile phase containing 100 mmol·L −1 (NH4) 2 CO 3 at pH 9 and a fow rate of 1.2 mL·min −1 was applied.

Interference Research of Cl − on Iodine Species.
Kelp contains a small amount of sodium chloride. In order to explore the efect of Cl − in iodine speciation analysis, a mixed standard solution of 150 g·L −1 IO 3 − , I − , MIT, and DIT containing 100 mg·L −1 Cl − was prepared under the optimal experimental conditions. Te experimental results have shown that a small amount of Cl − in the sample will not afect the iodine speciation analysis of the kelp, and its chromatogram is shown in Figure 7.

Analytical Performances. Te mixed standard solutions of IO 3
− , I − , MIT, and DIT with linear ranges of 0, 10, 20, 50, 80, 100, and 150 μg·L −1 were determined by HPLC-ICP-MS [8,12]. Table 6 summarizes all the analytical performance characteristics of the optimal method. Te detection limits of IO 3 − , I − , MIT, and DIT were 1.62, 1.02, 1.59, and 2.55 μg·L −1 , respectively. Te 50 μg·L −1 mixed standard solution was measured fve times in parallel, and the precisions of the four iodine species were all between 1.08% and 1.71%. Figure 8 shows that HPLC-ICP-MS can efectively separate four      Figure 9. Te linear range is wide, the linear relationship is reasonable, and the correlation coefcients are all greater than 0.999.

Recovery of Iodine in Kelp.
To verify the accuracy of this method, the kelp samples were spiked and determined. As shown in Table 7, the experimental results show that I − and MIT recovery rates are between 99% and 103%, which shows that the experimental method is accurate and reliable.
3.9. Iodine Content of Kelp. In this paper, the method of ultrasonic-assisted trypsin was used for pretreatment, and the established method of HPLC-ICP-MS was used for determination. Te iodine content in the kelp sample is shown in Figure 10 and Table 8. It can be seen from Table 8 that the kelp sample only contains I − and MIT, which are 7.90 × 10 3 mg·kg −1 and 390.91 mg·kg −1 , respectively, and the organic iodine content accounts for 4.72% of the total iodine.

Conclusions
In this work, the optimum pretreatment conditions of kelp samples using trypsin with ultrasound were explored for the frst time. A technique combining high-performance liquid chromatography and inductively coupled plasma mass spectrometry (HPLC-ICP-MS) was used to establish a sensitive and accurate quantitative analysis speciation method of iodine in kelp. Tis method can achieve the separation and quantitative analysis of four iodine species (IO 3 − , I − , MIT, and DIT) with high accuracy and low detection limit and can be widely used in the analysis and determination of diferent iodine species in kelp and table salt.

Data Availability
Te data used to support the fndings of this study are available from the corresponding authors upon request.

Conflicts of Interest
Te authors declare that they have no conficts of interest.   Journal of Chemistry