An analytical method for determining organic iodine in seaweed iodized salt using an amino acid analyzer

Abstract

The present invention provides an analytical method for determining organic iodine in seaweed iodized salt using an amino acid analyzer. A edible salt sample to be tested is added to a mixed solvent of methanol and ethanol (4:1), subjected to ultrasonic extraction, the extract is concentrated by nitrogen blowing and then redissolved, and 3,5-diiodo-L-tyrosine (DIT) and 3-iodo-L-tyrosine (MIT) in the seaweed iodized salt are determined using an amino acid analyzer. Within the concentration range of 0 to 200 μg / mL, the linear equation correlation coefficients of the two organic iodine are both greater than 0.999. At three concentration levels of high, medium and low, the recovery rate of DIT is 91.7% to 113.3%, with an RSD of 1.8% to 8.8%, and the recovery rate of MIT is 93.3% to 107.7%, with an RSD of 2.7% to 6.4%. This method has the advantages of simple operation, high accuracy and good precision, and is suitable for determining organic iodine in seaweed iodized salt.

Description

Technical Field

[0001] The invention belongs to the technical field of food safety detection and relates to an analytical method for detecting seaweed iodine in edible salt. Background Art

[0002] Iodine, known as the "element of life" and the "element of wisdom," is essential for the intellectual development of infants, young children, and adolescents, as well as for normal human metabolism. Humans cannot synthesize iodine naturally and must ingest it externally. According to the World Health Organization, most inland areas of my country are iodine-deficient. To combat the harmful effects of iodine deficiency, my country promulgated the "Regulations on the Administration of Iodized Salt to Eliminate the Hazards of Iodine Deficiency" in 1994, implementing comprehensive prevention and control measures, primarily through the long-term supply of iodized salt. GB 26878-2011, "National Food Safety Standard for Iodine Content in Edible Salt," specifies that iodine fortifiers include potassium iodate, potassium iodide, and seaweed iodine. In 2018, my country promulgated "GB 1903.39-2018, National Food Safety Standard for Food Nutrition Fortifier Seaweed Iodine," listing seaweed iodine as an edible iodine supplement, a recognition of its nutritional value. Compared to inorganic iodine (potassium iodate and potassium iodide), seaweed iodine exhibits greater thermal stability and safety. As people pay more and more attention to the health concept of natural food, more new seaweed iodized salts that meet market needs are produced and sold.

[0003] Currently, labels on seaweed iodized salt in my country only indicate the total iodine content, lacking specific information on the organic iodine content. The existing legal testing method, GB5009.42-2016, uses a strong oxidant to completely oxidize the three iodine additives, destroying the amino acids in the seaweed iodine. There is no dedicated testing method to determine the seaweed iodine in seaweed iodized salt. Due to its complex preparation process, seaweed iodized salt is generally more expensive than inorganic iodized salt. This may lead some companies to illegally profit by packaging ordinary inorganic iodized salt as seaweed iodized salt. Reported methods for testing iodine content in table salt include inductively coupled plasma mass spectrometry, redox titration, spectrophotometry, ion-selective electrodes, and chromatography. These methods primarily focus on detecting the total or inorganic iodine content in table salt and are similarly unable to accurately determine the seaweed iodine content or verify the authenticity of seaweed iodized salt.

[0004] Research by Han Lijun and others has identified 3-iodo-L-tyrosine and 3,5-diiodo-L-tyrosine as two common organic iodine compounds found in seaweed. The main structural component of these compounds is iodine-containing tyrosine, and the proportion of free amino acids in seaweed extracts is greater than that of iodine-containing amino acids. Currently, iodized seaweed salt on the market is primarily prepared by spraying seaweed iodine extract onto ordinary table salt. Therefore, a method is needed to separate the free and iodine-containing amino acids in seaweed iodine additives and seaweed iodized salt.

[0005] The amino acid analyzer is an ion exchange chromatograph based on cation exchange column separation, post-column ninhydrin derivatization, and photometric determination. However, the total composition of seaweed iodized salt is complex. In addition to iodinated amino acids such as 3,5-diiodo-L-tyrosine and 3,5-diiodotyrosine, it also includes at least 17 free amino acids such as tyrosine, histidine, phenylalanine, and lysine. The content of iodinated amino acids is generally low. In current detection methods, due to the inability to separate or insufficient sensitivity, it has been impossible to achieve effective qualitative and quantitative detection of amino acids other than 3,5-diiodo-L-tyrosine and 3,5-diiodotyrosine. The present invention uses amino acid analysis to determine seaweed iodized salt on the market, establishing a detection method that is simple to operate, highly accurate, and has good precision. Summary of the Invention

[0006] The object of the present invention is to provide an analytical method for determining organic iodine in seaweed iodized salt, which has a satisfactory detection limit and / or quantification limit.

[0007] An amino acid analysis method for determining organic iodine in edible salt comprises:

[0008] S1: mixing the edible salt sample to be tested with a solvent to obtain a mixed solution, wherein the solvent is one or a mixture of methanol and ethanol;

[0009] S2: subjecting the mixed solution obtained in S1 to ultrasonic-assisted extraction, filtering the obtained mixed solution, and collecting the filtrate;

[0010] S3: The filter residue obtained in S2 is washed with a solvent, and the resulting mixed solution is subjected to ultrasonic-assisted extraction, filtered, and the filtrate is collected; the filtrates obtained in S2 and S3 are combined, blown to near dryness with nitrogen, and the concentrate is diluted to 1 mL with sample diluent, and then filtered through a 0.22 μm filter membrane to obtain the sample solution to be tested;

[0011] In a particular embodiment, the 0.22 μm filter membrane is a polyethersulfone filter membrane.

[0012] The ultrasonic extraction time in S2 and S3 is 10 to 30 minutes under the conditions of 40KHz ultrasonic frequency and 30°C temperature;

[0013] S4: The sample obtained in S3 is quantitatively detected using an amino acid analyzer. The quantitative method is the external standard method. A standard curve is drawn with the response area of ​​each substance on the amino acid analyzer as the ordinate and the concentration of the standard solution as the abscissa. A linear equation and correlation coefficient are obtained to calculate the contents of 3,5-diiodo-L-tyrosine and 3-iodo-L-tyrosine in the edible salt sample to be tested. The liquid chromatography conditions for amino acid analysis are as follows:

[0014] Chromatographic column: strong acid ion exchange resin Na filled column;

[0015] Mobile phase A: 0.12 mol / L sodium citrate in 7% methanol, pH 3.45;

[0016] Mobile phase B: 0.2 mol / L sodium citrate in water, pH 10.85;

[0017] Mobile phase D: 0.5 mol / L sodium hydroxide in 0.02% EDTA aqueous solution;

[0018] Ninhydrin reaction solution: Take ninhydrin hydrate, phenol, and ascorbic acid, add 30% lauryl alcohol polyoxyethylene ether solution, potassium sodium buffer, and anhydrous methanol to dissolve; the mass ratio of the ninhydrin, phenol, and ascorbic acid is 100:10:1, the volume ratio of the potassium sodium buffer to anhydrous methanol is 4:6, and the mass volume concentration of the ninhydrin hydrate in the potassium sodium buffer and anhydrous methanol mixed solution is 2%;

[0019] Eluent flow rate: 0.45 mL / min,

[0020] Ninhydrin reaction solution flow rate: 0.25 mL / min;

[0021] Detection wavelength: 570nm;

[0022] Injection volume: 50 μL;

[0023] Separation column temperature: 65°C;

[0024] Reactor temperature: 135°C;

[0025] Gradient elution, the gradient elution program is as follows:

[0026]

[0027] Furthermore, the solvent in S1 is a mixture of methanol and ethanol, and the volume ratio of methanol to ethanol is 4:1.

[0028] Furthermore, the weight of the edible salt sample to be tested in S1: the volume of the solvent = 1g: 10ml to 2g: 1ml

[0029] Furthermore, the weight of the edible salt sample to be tested of the seaweed iodized salt described in S1: the volume of the solvent = 1g:1ml to 1g:5ml;

[0030] Furthermore, the weight of the edible salt sample to be tested of the seaweed iodized salt described in S1: the volume of the solvent = 1 g: 1 ml.

[0031] Furthermore, the sample diluent in S3 is an aqueous solution of 0.12 mol / L sodium citrate, pH 2.20.

[0032] In a specific embodiment, the sample diluent is prepared as follows: 11.8 g of trisodium citrate dihydrate and 6.0 g of citric acid are weighed, added to 900 mL of water, dissolved, the pH is adjusted to 2.20 with concentrated hydrochloric acid, the volume is made up to 1000 mL with water, filtered through a 0.45 μm aqueous microporous filter membrane, and 0.1 mL of caprylic acid is added for later use.

[0033] Furthermore, the specification of the chromatographic column in S4 is 4.6 mm×150 mm.

[0034] Furthermore, the preparation method of the mobile phase A described in S4 is: the preparation method of the mobile phase A described in S4 is: take trisodium citrate dihydrate (dihydrate) and citric acid, add water to dissolve, add anhydrous methanol and water, adjust the pH of the concentrated hydrochloric acid solution to 3.45, and then make up the volume with water. The concentration of sodium citrate is 0.12 mol / L. After filtering through a 0.45 μm aqueous phase microporous filter membrane, add caprylic acid for later use; the mass ratio of the trisodium citrate dihydrate and citric acid is 59:30, and the volume ratio of anhydrous methanol to water is 7:93.

[0035] In a particular embodiment, trisodium citrate dihydrate (dihydrate) and citric acid are dissolved in water, anhydrous methanol is added, and the volume is made up to 900 mL. After the concentrated hydrochloric acid solution is adjusted to pH 3.45, the volume is made up to 1000 mL with water, and the sodium citrate concentration is 0.12 mol / L. After filtering through a 0.45 μm aqueous microporous filter membrane, caprylic acid is added for later use. The mass ratio of trisodium citrate dihydrate to citric acid is 59:30, and the volume ratio of anhydrous methanol to water is 70 ml / 930 ml.

[0036] Furthermore, the preparation method of mobile phase B described in S4 is: take trisodium citrate dihydrate and boric acid, dilute the volume with water to a sodium citrate concentration of 0.2 mol / L, adjust the pH to 10.85 with 50% sodium hydroxide solution, filter through a 0.45 μm aqueous phase microporous filter membrane, and then add caprylic acid for later use; the mass ratio of trisodium citrate dihydrate to boric acid is 98:25.

[0037] Furthermore, the mobile phase D in S4 is prepared as follows: 20 g of sodium hydroxide and 0.2 g of ethylenediaminetetraacetic acid are dissolved in water and the volume is fixed to 1000 ml.

[0038] Furthermore, the preparation method of the ninhydrin reaction solution described in S4 is: take hydrated ninhydrin, phenol, and ascorbic acid, add 30% lauryl alcohol polyoxyethylene ether solution, potassium sodium buffer solution, and anhydrous methanol to dissolve; the mass ratio of the ninhydrin, phenol, and ascorbic acid is 100:10:1.

[0039] In the method of the present invention, a seaweed iodine salt sample is mixed with a solvent, and organic iodine is completely dissolved in a methanol-ethanol (4:1) solvent by ultrasound.

[0040] In the method of the present invention, when the ratio of the weight (g) of seaweed iodized salt to the volume (ml) of the solution is 1:1, the seaweed iodine in the salt can be completely extracted through ultrasonic extraction for 20 minutes.

[0041] In the method of the present invention, a conventional 58-minute gradient analysis method is used on an amino acid analyzer. Histidine and the two organic iodine amino acids completely overlap, so the gradient optimization method is adjusted to 68 minutes. 3-iodotyrosine is completely separated from histidine, but 3,5-diiodotyrosine is still contained in the histidine and cannot be separated. The elution program is further optimized to further separate histidine from the two organic iodine amino acids. By increasing the proportion of mobile phase B, a short-time gradient elution program of only 35 minutes is established (23 minutes is the target component peak cutoff time) for separating histidine from the two organic iodine amino acids, completely achieving baseline separation, shortening the analysis time, and other amino acids do not interfere with the analysis of the two organic iodine amino acids. The gradient elution program is shown in the table below:

[0042]

[0043] The beneficial effects of the present invention are as follows:

[0044] The present invention provides an amino acid analysis method for determining organic iodine in seaweed iodized salt. A mixed solvent of methanol and ethanol (1:4) is added to an edible salt sample and ultrasonically extracted. The extract is concentrated by nitrogen purging and then redissolved. An amino acid analyzer is used to determine 3,5-diiodo-L-tyrosine (DIT) and 3-iodo-L-tyrosine (MIT) in the seaweed iodized salt. Within a concentration range of 0 to 200 μg / mL, the linear correlation coefficients for the two organic iodine compounds are both greater than 0.999. At three concentration levels (high, medium, and low), the recoveries of DIT ranged from 91.7% to 113.3% with RSDs of 1.8% to 8.8%, and the recoveries of MIT ranged from 93.3% to 107.7% with RSDs of 2.7% to 6.4%. This method offers the advantages of simple operation, high accuracy, and good precision, and is suitable for determining organic iodine in seaweed iodized salt. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 Separation of 17 amino acids and mixed standard of organoiodine amino acids by conventional method;

[0046] Figure 2 Separation of a mixed standard of 17 amino acids and organoiodine amino acids by the optimized method. DETAILED DESCRIPTION

[0047] The present invention is further explained below with reference to the following examples, but the examples do not limit the present invention in any form.

[0048] The present invention discloses an analytical method for organic iodine in edible salt, particularly an amino acid analysis method. Those skilled in the art can learn from the content of the present invention, combine analytical chemistry principles, and appropriately improve parameters or similar principle methods to achieve the above. It should be particularly noted that all similar replacements and modifications are obvious to those skilled in the art and are considered to be included within the scope of the present invention. The application of the present invention has been preferably described by way of example, and relevant personnel can make appropriate changes to the methods and applications described herein without departing from the content, spirit, and scope of the present invention, and the present invention can be implemented and applied by modification.

[0049] The present invention is further described below with reference to examples, but the examples are not intended to limit the scope of protection of the present invention.

[0050] The reagents and instruments used in the embodiments of the present disclosure are all commercially available, and the main materials and reagents meet the relevant standards, such as trisodium citrate (dihydrate), citric acid, sodium hydroxide, boric acid, ethylenediaminetetraacetic acid, potassium acetate, sodium acetate, lauryl polyoxyethylene ether, caprylic acid, ninhydrin, phenol, and hydrochloric acid: Sinopharm Chemical Reagent Co., Ltd.; methanol: Merck, Germany; glacial acetic acid: Shanghai McLean Biochemical Technology Co., Ltd.; 3,5-diiodo-L-tyrosine (DIT) standard: purity ≥98%, sourced from SIGMA; 3-iodo-L-tyrosine (MIT) standard: purity ≥99%, sourced from SIGMA; 17 kinds of amino acid mixed standard solution (2.5 μmol / mL): Safecam (Beijing) Scientific Instrument Co., Ltd.

[0051] Example 1 Selection of extraction solution

[0052] Table salt is primarily composed of sodium chloride. When dissolving samples in aqueous solutions, the high ion content in the aqueous solution can damage the amino acid analysis column and affect the sensitivity of the amino acid analyzer. Therefore, the solubility of 3,5-diiodo-L-tyrosine and 3-iodo-L-tyrosine in water, methanol, ethanol, isopropanol, and ethyl acetate was investigated. The results showed that 3,5-diiodo-L-tyrosine was nearly insoluble in water, methanol, ethanol, isopropanol, and ethyl acetate. 3-iodo-L-tyrosine was slightly soluble in water, slightly soluble in methanol, slightly soluble in ethanol, and nearly insoluble in isopropanol and ethyl acetate. After further sonication of the above-mentioned solubility sample solutions at 30°C for 15 minutes, 3,5-diiodo-L-tyrosine was soluble only in ethanol. Therefore, considering the solubility of the two organic iodine compounds in the four solvents, a mixture of methanol and ethanol was selected as the extraction solvent. The solubility of the two organic iodine compounds in various methanol-ethanol ratios was further verified.

[0053] Ultrasonic dissolution of 10 mg of organic iodine in 4 different mixed solvents (10 ml each)

[0054]

[0055] Conclusion: The experiment found that 10 mg of the two organic iodine compounds could be completely dissolved in methanol-ethanol (4:1) solvent after 15 minutes of ultrasonic treatment. Taking into account the ultrasonic dissolution time and solvent volume of the two organic iodine compounds in four different mixed solvents, methanol-ethanol (4:1) was selected as the extraction solvent for organic iodine.

[0056] Example 2 Optimization of sample sampling amount, pretreatment solvent volume and extraction times

[0057] (1) Weigh 1g, 5g, 10g, 20g, and 30g of seaweed iodized salt samples, respectively, and add 10ml of methanol-ethanol (4:1) to a 100ml beaker and mix well. Ultrasonic extraction was performed at an ultrasonic frequency of 40KHz and a temperature of 30℃ for 30 minutes. The extract was filtered into a centrifuge tube, and the residue was washed with 5ml of a methanol-ethanol (4:1) mixed solvent. The combined washings were nitrogen-purged in a 30℃ water bath until nearly dry. The concentrate was diluted to 1ml with sample diluent, filtered with a 0.22μm filter membrane, and then analyzed by amino acid analyzer.

[0058] The sample diluent was prepared as follows: 11.8 g of trisodium citrate dihydrate and 6.0 g of citric acid were weighed, 10.4 mL of 37% hydrochloric acid solution was measured, appropriate amount of water was added to dissolve, the volume was made up to 1000 mL with water, the pH was adjusted to 2.20 with concentrated hydrochloric acid, and 0.1 mL of octanoic acid was added after filtering through a 0.45 μm aqueous microporous filter membrane.

[0059] The contents of the two organic iodine in sample solutions with different sampling amounts are shown in the table below.

[0060] Concentration determination of samples with different sampling volumes

[0061]

[0062] The results show that as the sample size increases, the organic iodine concentration measured in the sample solution obtained from a sample size of 1g to 10g in the same volume of extraction solvent increases exponentially, while the organic iodine concentration measured in the sample solution obtained from a sample size of 10g to 30g in the same volume of extraction solvent does not increase exponentially. As the sample size increases, the extraction solvent cannot fully contact the sample and cannot fully extract the organic iodine. Through experimental comparison, when the sample size is 10g, the organic iodine response value in the sample solution is moderate, and the measured organic iodine concentration is stable, so 10g is determined to be the sample size.

[0063] (2) Weigh 10g of seaweed iodine salt sample into a 100mL beaker and add different volumes of methanol-ethanol (4:1) mixed solvent to mix well. Ultrasonic extraction was performed at an ultrasonic frequency of 40KHz and a temperature of 30℃ for 30 minutes. The extract was filtered into a centrifuge tube and the residue was washed with 5ml of methanol-ethanol (4:1) mixed solvent. The washings were combined and blown to near dryness with nitrogen in a 30℃ water bath. The concentrate was diluted to 1mL with sample diluent, filtered with a 0.22μm filter membrane, and then measured by amino acid analyzer. The contents of the two organic iodine in the sample solution under different volumes of extraction solvent are shown in the table below.

[0064] Extraction efficiency of different extraction solvent volumes

[0065]

[0066] Conclusion: The extraction effects of 10ml, 20ml and 50ml are basically the same. Considering the impact of organic solvents on the environment and the extraction time cost, 10ml of methanol-ethanol (4:1) mixed solvent was selected as the extraction solvent for organic iodine.

[0067] (3) Take the above-mentioned test sample residue after filtration and washing, place it in a 100ml beaker, add 10ml of methanol-ethanol (4:1) mixed solvent, and ultrasonically extract for 30 minutes at an ultrasonic frequency of 40KHz and a temperature of 30℃. Filter the extract into a centrifuge tube, and wash the residue with 5ml of methanol-ethanol (4:1) mixed solvent. Combine the washings and place them in a 30℃ water bath and blow nitrogen until almost dry. Use diluent to make the concentrate volume 1mL and pass it through a 0.22μm filter membrane. After determination by amino acid analyzer, no 3,5-diiodo-L-tyrosine and 3-iodo-L-tyrosine were detected.

[0068] Conclusion: Ultrasonic extraction once and residue washing once can meet the extraction requirements.

[0069] Example 3 Parameter optimization of ultrasound-assisted extraction

[0070] Weigh 10g of seaweed iodized salt sample (accurate to 0.001g) into a 100mL beaker and add 10ml of methanol-ethanol (4:1) to mix thoroughly. Ultrasonic extraction was performed at 40kHz and 30°C for 10, 20, and 30 minutes, respectively. The extract was filtered into a centrifuge tube, and the residue was washed with 5ml of a 4:1 methanol-ethanol mixture. The combined washings were placed in a 30°C water bath and nitrogen purged until nearly dry. The concentrate was diluted to 1ml with diluent, filtered through a 0.22μm filter, and analyzed by an amino acid analyzer. The contents of the two organic iodine compounds in the samples at different extraction times are shown in the table below.

[0071] Extraction efficiency at different ultrasonic times

[0072]

[0073] Conclusion: The concentration of the sample measured under ultrasonic treatment for 10 minutes was lower, while the concentrations measured under 20 minutes and 30 minutes were basically the same. Considering the time cost, the ultrasonic frequency of 40KHz and the temperature of 30℃ for 20 minutes were selected as the ultrasonic extraction parameters.

[0074] Example 4 Verification of the Reconstitution Volume of the Extract

[0075] Because the seaweed iodine content in seaweed iodized salt is low and mostly free amino acids, to improve the response of iodine-containing amino acids in the amino acid analyzer, a small amount of diluent was used for reconstitution. The response of sample solutions with 1 ml and 2 ml of reconstitution solvent was also examined. The experiment found that after vortexing for one minute, the concentrate completely dissolved in both 1 ml and 2 ml of diluent. The contents of the two organic iodine compounds in the sample solutions using different volumes of extraction solvent are shown in the table below.

[0076] Extraction efficiency of different extraction solvent volumes

[0077]

[0078] Conclusion: When 1 ml of diluent is used as the reconstitution solvent, the response value of the sample solution is multiples of that of 2 ml of diluted sample solution. In order to improve the sensitivity of the test, 1 ml is selected as the volume of the reconstitution solvent.

[0079] Example 5 Selection of polytetrafluoroethylene filter

[0080] After solvent extraction and concentration, samples may contain insoluble residues, which can easily clog the chromatographic column and instrument tubing, potentially damaging the column and amino acid analyzer. Therefore, a filter is necessary to purify the sample. Common laboratory filters include polyethersulfone, nylon, and polytetrafluoroethylene. Because the amino acid analysis system is aqueous and the reconstitution solvent is also aqueous, a polyethersulfone filter was selected to investigate its adsorption of two organic iodine compounds. The simultaneously processed sample solutions were filtered through a polyethersulfone filter before injection. The results are shown in the table below.

[0081] The results showed that polyethersulfone filter had little adsorption effect on organic iodine, so polyethersulfone filter was used to purify the sample.

[0082]

[0083] Example 6 Selection and Verification of Elution Conditions for Amino Acid Analysis Column

[0084] The main structure of organic iodine in seaweed iodized salt is amino acid. The mobile phase system of the 18 amino acid determination method was used to prepare a mixed standard solution of 18 amino acids and two organic iodine amino acids for testing.

[0085] Mobile phase (A): Weigh 11.8 g of trisodium citrate dihydrate (dihydrate) and 6.0 g of citric acid and dissolve in water. Add 70 mL of anhydrous methanol and make up to 900 mL with water. Adjust the pH to 3.45 with concentrated hydrochloric acid, then dilute to 1000 mL with water. Filter through a 0.45 μm aqueous microporous membrane, then add 0.1 mL of octanoic acid and set aside.

[0086] Mobile phase (B): Weigh 19.6 g of trisodium citrate dihydrate, 3.1 g of sodium hydroxide, and 5.0 g of boric acid, and dilute to 1000 mL with water. Adjust the pH to 10.85 with 50% sodium hydroxide solution. Filter through a 0.45 μm aqueous microporous membrane, then add 0.1 mL of octanoic acid and set aside.

[0087] Mobile phase (D) is potassium-sodium buffer D: Take 20 g of sodium hydroxide and 0.2 g of ethylenediaminetetraacetic acid, dissolve them in water and make up to 1000 mL.

[0088] Ninhydrin reaction solution: weigh 20 g of hydrated ninhydrin, 2 g of phenol, and 0.2 g of ascorbic acid, add 1 mL of 30% lauryl alcohol polyoxyethylene ether solution, 400 mL of potassium sodium buffer, and 600 mL of anhydrous methanol, and dissolve by ultrasonication.

[0089] After the ninhydrin reaction solution is prepared, it is placed on the instrument and flows into the chromatographic column along the instrument flow path for post-column derivatization reaction.

[0090] Liquid chromatography conditions are:

[0091] The chromatographic column was a strongly acidic ion exchange resin Na-filled column (4.6 mm × 150 mm);

[0092] The eluent flow rate was 0.45 mL / min.

[0093] The flow rate of ninhydrin reaction solution was 0.25 mL / min;

[0094] Detection wavelength: 570nm and 440nm;

[0095] Injection volume: 50 μL;

[0096] Separation column temperature: 65°C;

[0097] Reactor temperature: 135°C.

[0098] The conventional gradient elution program for separating 17 amino acids was used for determination (i.e., the "gradient elution program before optimization" in the table below). The results showed that histidine (retention time 37.14 min) completely encapsulated 3,5-diiodo-L-tyrosine and 3-iodo-L-tyrosine ( Figure 1 ).

[0099] Since the purpose of this invention is to determine the iodine-containing amino acids in seaweed iodized salt, the chromatographic gradient and run time are further optimized according to the peak time of each amino acid and the iodine-containing amino acid, and the interference of other amino acids needs to be eliminated. The optimization comparison is shown in the following table:

[0100]

[0101]

[0102] Gradient program optimization results

[0103]

[0104] Conclusion: By increasing the starting ratio of mobile phase B and adjusting the gradient, the optimized chromatographic conditions were obtained. 3,5-diiodo-L-tyrosine and 3-iodo-L-tyrosine can be detected simultaneously with good peak shape and good separation. It is suitable for the detection of two organic iodine. Other amino acids do not interfere with the determination of the two iodine-containing amino acids. Figure 2 ).

[0105] Example 7 Verification of the linear range, correlation coefficient, detection limit and quantification limit of 3,5-diiodo-L-tyrosine and 3-iodo-L-tyrosine

[0106] GB 1903.39-2018 "National Food Safety Standard Food Nutrient Fortifier Seaweed Iodine" stipulates that seaweed iodine is a food additive made from kelp through a special process.

[0107] 3,5-Diiodo-L-tyrosine and 3-iodo-L-tyrosine are two iodotyrosines with confirmed morphologies and structures found in seaweed. Both are tyrosine derivatives. Tyrosine is a primary amine amino acid. Upon high-temperature reaction with ninhydrin, primary amines produce a bluish-purple substance with a maximum absorption wavelength of 570 nm. Under specific amino acid analyzer conditions, the response of 3,5-diiodo-L-tyrosine and 3-iodo-L-tyrosine at 570 nm is higher than that at 440 nm, so 570 nm was selected as the detection wavelength. Both organic iodine compounds exhibited good peak shapes and excellent resolution. A standard curve was plotted using the peak area of ​​each component as the ordinate and the concentration of the standard solution as the abscissa, yielding a linear equation and correlation coefficient.

[0108] The results showed that the correlation coefficients of the linear equations of the two organic iodine compounds were greater than 0.999 in the concentration range of 0-200 μg / mL, indicating a good linear relationship.

[0109] Two organic iodine standard solutions were added to blank samples, and the chromatographic responses of the standard solutions were calculated. When the instrument signal-to-noise ratio (S / N) was 3, the method detection limits were determined to be 1.6 μg / kg for 3,5-diiodo-L-tyrosine and 0.9 μg / kg for 3-iodo-L-tyrosine. When the instrument signal-to-noise ratio (S / N) was 10, the method quantification limits were determined to be 6.0 μg / kg for 3,5-diiodo-L-tyrosine and 3.0 μg / kg for 3-iodo-L-tyrosine.

[0110]

[0111] Example 8 Recovery and Precision of Addition of 3,5-Diiodo-L-Tyrosine and 3-Iodo-L-Tyrosine

[0112] Refer to GB / T 27404-2008 for precision. The precision of the method was evaluated by measuring the recoveries and relative standard deviations (RSDs) of spiked samples at the limit of quantification (LOQ), 10 times the LOQ, and 50 times the LOQ. The spiked samples for 3,5-diiodo-L-tyrosine were spiked at 6.0 μg / kg, 60.0 μg / kg, and 300.0 μg / kg; the spiked samples for 3-iodo-L-tyrosine were spiked at 3.0 μg / kg, 30.0 μg / kg, and 150.0 μg / kg.

[0113] The results showed that at spike levels of 6.0 μg / kg, 60.0 μg / kg, and 300.0 μg / kg, the method had recoveries of 91.7% to 113.3%, with RSDs of 1.8% to 8.8%. At spike levels of 3.0 μg / kg, 30.0 μg / kg, and 150.0 μg / kg, the method had recoveries of 93.3% to 107.7%, with RSDs of 2.7% to 6.4%. These methods met the requirements of GB / T 27404-2008 and demonstrated good precision, meeting routine testing requirements.

[0114] Recovery and precision of 3,5-diiodo-L-tyrosine (n=6)

[0115]

[0116]

[0117] Recovery and precision of 3-iodo-L-tyrosine (n=6)

[0118]

[0119] Example 9 Sample Determination

[0120] The above method was used to determine the common seaweed iodized salt and ordinary iodized salt on the market. At the same time, the total iodine content of seaweed iodized salt from different origins was determined using the "GB5009.42-2016 National Food Safety Standard - Determination of Salt Indicators". The results are shown in the table Seaweed iodized salt determination results

[0121]

[0122]

[0123] Inorganic iodine salt determination results

[0124]

[0125] From the above results, it can be seen that the total iodine content in commercially available seaweed iodized salt meets the national standard requirements (18-33 mg / kg), iodine mainly exists in the form of inorganic iodine, the content of iodine-containing amino acids is generally low, and no iodine-containing amino acids are detected in ordinary iodized salt. The above method can be used as a test method to distinguish seaweed iodized salt from ordinary iodized salt.

[0126] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. An amino acid analysis method for determining organic iodine in edible salt, characterized in that: The method comprises: S1: Mixing the edible salt sample to be tested with a solvent to obtain a mixed solution, wherein the solvent is a mixture of methanol and ethanol, and the volume ratio of methanol to ethanol is 4:1; S2: subjecting the mixed solution obtained in S1 to ultrasonic-assisted extraction, filtering the obtained mixed solution, and collecting the filtrate; S3: The filter residue obtained in S2 is washed with a solvent, and the obtained mixed solution is subjected to ultrasonic-assisted extraction, filtered, and the filtrate is collected; the filtrates obtained in S2 and S3 are combined, blown to near dryness with nitrogen, and the concentrate is made up to 1 mL with sample diluent, and then filtered through a 0.22 μm filter membrane to obtain the sample solution to be tested; In S2 and S3, the ultrasonic frequency is 40 KHz and the temperature is 30°C, and the ultrasonic extraction time is 20 to 30 minutes; S4: The sample obtained in S3 is quantitatively detected using an amino acid analyzer. The quantitative method is the external standard method. A standard curve is drawn with the response area of ​​each substance on the amino acid analyzer as the ordinate and the concentration of the standard solution as the abscissa. A linear equation and correlation coefficient are obtained to calculate the contents of 3,5-diiodo-L-tyrosine and 3-iodo-L-tyrosine in the edible salt sample to be tested. The liquid chromatography conditions for amino acid analysis are as follows: Chromatographic column: strong acid ion exchange resin Na filled column; Mobile phase A: 0.12 mol / L sodium citrate in 7% methanol, pH 3.45; Mobile phase B: 0.2 mol / L sodium citrate in water, pH 10.85; Mobile phase D: 0.5 mol / L sodium hydroxide in 0.02% EDTA aqueous solution; Ninhydrin reaction solution: Take ninhydrin hydrate, phenol, and ascorbic acid, add 30% lauryl alcohol polyoxyethylene ether solution, potassium sodium buffer, and anhydrous methanol to dissolve; the mass ratio of the ninhydrin, phenol, and ascorbic acid is 100:10:1, the volume ratio of the potassium sodium buffer to anhydrous methanol is 4:6, and the mass volume concentration of the ninhydrin hydrate in the potassium sodium buffer and anhydrous methanol mixed solution is 2%; Eluent flow rate: 0.45 mL / min, Ninhydrin reaction solution flow rate: 0.25 mL / min; Detection wavelength: 570 nm; Injection volume: 50 μL; Separation column temperature: 65°C; Reactor temperature: 135°C; Gradient elution, the gradient elution program is as follows: The mobile phase A described in S4 was prepared as follows: trisodium citrate dihydrate and citric acid were dissolved in water, anhydrous methanol and water were added, the pH of the concentrated hydrochloric acid solution was adjusted to 3.45, and the volume was made up with water to a sodium citrate concentration of 0.12 mol / L. After filtering through a 0.45 μm aqueous microporous filter membrane, caprylic acid was added for later use; the mass ratio of trisodium citrate dihydrate to citric acid was 59:30, and the volume ratio of anhydrous methanol to water was 7:93; The mobile phase B described in S4 was prepared as follows: trisodium citrate dihydrate and boric acid were diluted with water to a sodium citrate concentration of 0.2 mol / L, the pH was adjusted to 10.85 with 50% sodium hydroxide solution, and the mixture was filtered through a 0.45 μm aqueous microporous membrane, and then caprylic acid was added for later use; the mass ratio of trisodium citrate dihydrate to boric acid was 98:25; The mobile phase D described in S4 was prepared as follows: 20 g of sodium hydroxide and 0.2 g of ethylenediaminetetraacetic acid were dissolved in water and the volume was adjusted to 1000 ml.

2. The analysis method according to claim 1, characterized in that The weight of the edible salt sample to be tested in S1: the volume of the solvent = 1 g: 10 ml to 2 g: 1 ml.

3. The analysis method according to claim 1, characterized in that The edible salt sample to be tested in S1 is seaweed iodized salt, and the seaweed iodized salt sample weight: solvent volume = 1 g: 1 ml ~ 1 g: 5 ml.

4. The analysis method according to claim 1, characterized in that The edible salt sample to be tested in S1 is seaweed iodized salt, and the seaweed iodized salt sample weight: solvent volume = 1 g: 1 ml.

5. The analysis method according to claim 1, characterized in that The sample diluent S3 is an aqueous solution of 0.12 mol / L sodium citrate, pH 2.

20.

6. The analysis method according to claim 1, characterized in that The specifications of the chromatographic column S4 are 4.6 mm×150 mm.

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