Analysis method for determining organic iodine in algae iodized salt by amino acid analyzer

Abstract

The invention provides an analysis method for determining organic iodine in algae iodized salt by using an amino acid analyzer. The method comprises the following steps: adding an edible salt sample to be detected into a mixed solvent of methanol and ethanol (4: 1), carrying out ultrasonic extraction, carrying out nitrogen blowing concentration on an extracting solution, redissolving, and determining 3, 5-diiodo-L-tyrosine (DIT) and 3-iodo-L-tyrosine (MIT) in the seaweed iodized salt by using an amino acid analyzer. In the concentration range of 0-200 [mu] g / mL, the linear equation correlation coefficients of the two organic iodine are both greater than 0.999. Under the three concentration levels of high, medium and low, the recovery rate of DIT is 91.7%-113.3%, the RSD is 1.8%-8.8%, the recovery rate of MIT is 93.3%-107.7%, and the RSD is 2.7%-6.4%. The method has the advantages of simplicity and convenience in operation, high accuracy and good precision, and is suitable for determining the organic iodine in the algae 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 "element of wisdom", is an indispensable element for the intellectual development of infants and adolescents and the normal metabolism of the human body. Humans cannot synthesize iodine by themselves and must ingest it from the outside. According to the standards of the World Health Organization, most of my country's inland areas are iodine-deficient areas. In order to eliminate the hazards of iodine deficiency, my country promulgated the "Regulations on the Management of Iodine-Fortified Salt to Eliminate the Hazards of Iodine Deficiency" in 1994, and adopted comprehensive prevention and control measures mainly based on the long-term supply of iodized salt. GB 26878-2011 "National Food Safety Standard for Iodine Content of Edible Salt" stipulates that iodine fortifiers include potassium iodate, potassium iodide and seaweed iodine. In 2018, my country promulgated the "GB 1903.39-2018 National Food Safety Standard Food Nutrition Fortifier Seaweed Iodine", which included seaweed iodine as an edible iodine supplement material, which is an affirmation of the nutritional value of seaweed iodine. Compared with inorganic iodine (potassium iodate, potassium iodide), seaweed iodine has better thermal stability and better 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] At present, the labels of seaweed iodized salt in my country only show the total iodine content, and lack the specific content of organic iodine. The existing statutory detection method GB5009.42-2016 uses a strong oxidant to completely oxidize the three iodine additives. The amino acids in the seaweed iodine are all destroyed, and there is a lack of exclusive detection methods to determine the seaweed iodine in seaweed iodized salt. Due to the complex preparation process, the price of seaweed iodized salt is generally higher than that of inorganic iodized salt, which may cause some companies to package ordinary inorganic iodized salt as seaweed iodized salt to obtain high illegal profits. The reported detection methods for iodine content in table salt include inductively coupled plasma mass spectrometry, redox titration, spectrophotometry, ion selective electrode method, chromatography, etc. These methods mostly focus on detecting the total iodine or inorganic iodine content in table salt. They are also unable to accurately determine the seaweed iodine content, nor can they distinguish the authenticity of seaweed iodized salt.

[0004] The research of Han Lijun and others determined that 3-iodo-L-tyrosine and 3,5-diiodo-L-tyrosine are two common organic iodine in seaweed. The main structure of the compound is iodine-containing tyrosine, and the proportion of free amino acids in seaweed extract is greater than that of iodine-containing amino acids. The preparation process of seaweed iodized salt on the market is mainly to spray seaweed iodine extract on ordinary table salt. Therefore, it is necessary to develop a method for separating free amino acids and iodine-containing amino acids in seaweed iodized salt 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 iodine 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, lysine, etc., and the content of iodine-containing amino acids is generally low. In the current detection method, due to the inability to separate or insufficient sensitivity, it has not been possible to achieve effective qualitative and quantitative detection of 3,5-diiodo-L-tyrosine and 3,5-diiodotyrosine. The present invention adopts amino acid analysis to determine the seaweed iodized salt on the market, and establishes a detection method that is simple to operate, high in accuracy, and good in 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 quantitative limit.

[0007] The 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 of methanol and ethanol or a mixture of the two;

[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 filtrate residue obtained from S2 is washed with a solvent, the obtained mixed solution is subjected to ultrasonic-assisted extraction, filtered, and the filtrate is collected; the filtrates obtained from S2 and S3 are combined, blown to near dryness with nitrogen, the concentrated solution is fixed to 1 mL with a sample diluent, and filtered through a 0.22 μm filter membrane to obtain a sample solution to be tested;

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

[0012] In S2 and S3, the ultrasonic frequency is 40KHz, the temperature is 30°C, and the ultrasonic extraction time is 10 to 30 minutes;

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

[0014] Chromatographic column: strongly acidic ion exchange resin Na filled column;

[0015] Mobile phase A: 0.12 mol / L sodium citrate in 7% methanol aqueous solution, 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, phenol, and ascorbic acid, add 30% lauryl alcohol polyoxyethylene ether solution, potassium sodium buffer solution, and anhydrous methanol to dissolve; the mass ratio of ninhydrin, phenol, and ascorbic acid is 100:10:1, the volume ratio of the potassium sodium buffer solution to anhydrous methanol is 4:6, and the mass volume concentration of the ninhydrin in the mixed solution of potassium sodium buffer solution and anhydrous methanol 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] Further, the weight of the edible salt sample to be tested in S1: the volume of the solvent = 1g: 10ml to 2g: 1ml

[0029] Further, 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 preparation method is: weigh 11.8g of trisodium citrate dihydrate and 6.0g of citric acid, add water to 900mL, dissolve, adjust the pH to 2.20 with concentrated hydrochloric acid, make up to 1000mL with water, filter through a 0.45μm aqueous phase microporous filter membrane, add 0.1mL of caprylic acid 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 to volume with water, the concentration of sodium citrate is 0.12 mol / L, filter through a 0.45μm aqueous phase microporous filter membrane, and add caprylic acid 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 7:93.

[0035] In a particular embodiment: trisodium citrate dihydrate (dihydrate) and citric acid are taken, water is added to dissolve, anhydrous methanol is added, water is added to 900 mL, concentrated hydrochloric acid solution is adjusted to pH 3.45, and then the volume is made up to 1000 mL with water, the concentration of sodium citrate is 0.12 mol / L, and caprylic acid is added after filtering through a 0.45 μm aqueous phase microporous filter membrane for standby 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 in S4 is: take trisodium citrate dihydrate and boric acid, dilute 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 add caprylic acid for later use; the mass ratio of trisodium citrate dihydrate to boric acid is 98:25.

[0037] Furthermore, the preparation method of the mobile phase D in S4 is: take 20g of sodium hydroxide and 0.2g of ethylenediaminetetraacetic acid, dissolve them in water and make up to 1000ml.

[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, 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 iodized 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 two organic iodine amino acids completely overlap, so the gradient optimization method is adjusted to 68 minutes, 3-iodotyrosine and histidine are completely separated, but 3,5-diiodotyrosine is still contained in histidine and cannot be separated. The elution program is further optimized to further separate histidine and 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 peak cutoff time of the target component), which is used to separate histidine and the two organic iodine amino acids, completely achieving baseline separation, shortening the analysis time, and other amino acids have no interference with the analysis of the two organic iodine amino acids. The gradient elution program is shown in the following table:

[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 (1:4) of methanol and ethanol is added to an edible salt sample, and ultrasonic extraction is performed. The extract is concentrated by nitrogen blowing and then redissolved. An amino acid analyzer is used to determine 3,5-diiodo-L-tyrosine (DIT) and 3-iodo-L-tyrosine (MIT) in seaweed iodized salt. Within the concentration range of 0 to 200 μg / mL, the linear equation correlation coefficients of the two organic iodine are greater than 0.999. At three concentration levels of high, medium and low, the recovery rate of DIT is 91.7% to 113.3%, the RSD is 1.8% to 8.8%, and the recovery rate of MIT is 93.3% to 107.7%, and the RSD is 2.7% to 6.4%. The method has 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 Mixed standard diagram of 17 amino acids separated by conventional method and organoiodine amino acids;

[0046] Figure 2 Mixture of 17 amino acids and organo-iodine amino acids separated by optimized method. DETAILED DESCRIPTION

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

[0048] The present invention discloses an analysis method for organic iodine in edible salt, in particular 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. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included within the scope of the present invention. The application of the present invention has been well described by embodiments, 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 change to achieve and apply the present invention.

[0049] The present invention is further described below by way of examples, but the examples are not intended to limit the scope of protection of the present invention in any way.

[0050] The reagents and instruments used in the embodiments of the present disclosure are all commercially available, and the main materials and reagents all meet the relevant standards, such as trisodium citrate (dihydrate), citric acid, sodium hydroxide, boric acid, ethylenediaminetetraacetic acid, potassium acetate, sodium acetate, lauryl alcohol polyoxyethylene ether, caprylic acid, ninhydrin, phenol, 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%, source SIGMA; 3-iodo-L-tyrosine (MIT) standard: purity ≥99%, source SIGMA; 17 kinds of amino acid mixed standard solution (2.5μmol / mL): Secham (Beijing) Scientific Instrument Co., Ltd.

[0051] Example 1 Selection of extraction solution

[0052] The main component of table salt is sodium chloride. When the sample is dissolved in an aqueous solution, the ion content in the aqueous solution of the sample is high, which will 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 respectively. The results showed that 3,5-diiodo-L-tyrosine was almost 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 almost insoluble in isopropanol and ethyl acetate. After the above solubility sample solution was further ultrasonicated at 30°C for 15 minutes, 3,5-diiodo-L-tyrosine was only dissolved in ethanol. Therefore, considering the solubility of the two organic iodine in the four solvents, a mixed solvent of methanol and ethanol was selected as the extraction solvent, and the solubility of the two organic iodine in methanol-ethanol solutions of different proportions 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 can be completely dissolved in methanol-ethanol (4:1) solvent after 15 minutes of ultrasonic treatment. Considering the ultrasonic dissolution time and solvent volume of the two organic iodine in 4 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, add 10ml of methanol-ethanol (4:1) to a 100mL beaker, and mix well. Ultrasonic extraction was performed for 30 minutes at an ultrasonic frequency of 40KHz and a temperature of 30°C. The extract was filtered into a centrifuge tube, and the residue was washed with 5ml of a methanol-ethanol (4:1) mixed solvent. The washings were combined and blown to near dryness with nitrogen in a 30°C water bath. The concentrate was diluted to 1mL with the sample diluent, filtered with a 0.22μm filter membrane, and then determined by an 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 after filtering through a 0.45 μm aqueous microporous filter membrane, 0.1 mL of caprylic acid was added for later use.

[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] It can be seen from the measurement results that with the increase of sample amount, the concentration of organic iodine measured in the sample solution obtained by sampling 1g to 10g in the same volume of extraction solvent increases exponentially, while the concentration of organic iodine measured in the sample solution obtained by sampling 10g to 30g in the same volume of extraction solvent does not increase exponentially. When the sample amount increases, the extraction solvent cannot fully contact the sample and cannot fully extract the organic iodine. Through experimental comparison, when the sample weight is 10g, the response value of organic iodine in the sample solution is moderate, and the concentration of organic iodine determination is stable, so 10g is determined as the sample weight.

[0063] (2) Weigh 10g of seaweed iodized salt sample into a 100mL beaker, add different volumes of methanol-ethanol (4:1) mixed solvent and mix well. Ultrasonic extraction was performed for 30 minutes at an ultrasonic frequency of 40KHz and a temperature of 30°C. 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°C water bath. The concentrate was diluted to 1mL with sample diluent, filtered with a 0.22μm filter membrane, and then measured by an 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 methanol-ethanol (4:1) mixed solvent was selected as the extraction solvent for organic iodine.

[0067] (3) Take the above-mentioned residue of the test sample 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 washing liquid and place it in a 30℃ water bath and blow it with nitrogen until it is almost dry. Use diluent to make the concentrated solution dilute to 1mL and then pass it through a 0.22μm filter membrane. After being determined by an 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) in a 100mL beaker, add 10ml of methanol-ethanol (4:1) and mix well. Ultrasonic extraction was performed for 10 minutes, 20 minutes, and 30 minutes at an ultrasonic frequency of 40KHz and a temperature of 30°C. The extract was filtered into a centrifuge tube, and the residue was washed with 5ml of a mixed solvent of methanol-ethanol (4:1). The combined washings were placed in a 30°C water bath and blown to near dryness with nitrogen. The concentrated solution was fixed to 1mL with a diluent and then filtered through a 0.22μm filter membrane for determination by an amino acid analyzer. The contents of the two organic iodine 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 is low, and the concentrations measured under 20 minutes and 30 minutes are basically the same. Considering the time cost, the ultrasonic frequency of 40KHz and the temperature of 30℃ for 20 minutes are selected as the ultrasonic extraction parameters.

[0074] Example 4 Verification of the reconstitution volume of the extract

[0075] Since the added seaweed iodine content in seaweed iodized salt is low and mostly free amino acids, in order to improve the response of iodine-containing amino acids in the amino acid analyzer, a small amount of diluent was selected for re-dissolution, and the response of sample solutions with 1ml and 2ml re-dissolution solvent volumes was examined at the same time. The experiment found that after vortexing for 1 minute, the concentrate was completely dissolved in both 1ml and 2ml diluents. The contents of the two organic iodine in the sample solution under 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 multiple to 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 the sample is extracted and concentrated by solvent, there may be some insoluble residues, which can easily cause blockage of the chromatographic column and instrument pipelines, and damage the chromatographic column and amino acid analyzer. Therefore, a filter is needed to purify the sample. Commonly used filters in the laboratory include polyethersulfone, nylon and polytetrafluoroethylene. Because the amino acid analysis system is water-based and the re-dissolution solvent is also water-based, a polyethersulfone filter is selected to investigate its adsorption effect on two organic iodine. The sample solution treated simultaneously was filtered with a polyethersulfone filter and then injected. The results are shown in the table below.

[0081] The results showed that polyethersulfone filters had little adsorption effect on organic iodine, so polyethersulfone filters were used to purify the samples.

[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 in the determination method of 18 amino acids 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 11.8g of trisodium citrate dihydrate (dihydrate) and 6.0g of citric acid, add appropriate amount of water to dissolve. Add 70mL of anhydrous methanol and add water to 900mL. Adjust the pH to 3.45 with concentrated hydrochloric acid, make up to 1000mL with water, filter through a 0.45μm aqueous microporous filter membrane, add 0.1mL of octanoic acid for later use.

[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 filter membrane, and add 0.1 mL of octanoic acid for later use.

[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 ninhydrin hydrate, 2 g phenol, and 0.2 g 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 preparation, the ninhydrin reaction solution is placed on the instrument and enters the chromatographic column along the instrument flow path for post-column derivatization reaction.

[0090] The liquid chromatography conditions were:

[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 the present invention is to determine the iodine-containing amino acids in seaweed iodized salt, the chromatographic gradient and running 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 initial ratio of mobile phase B and adjusting the gradient to obtain the optimized chromatographic conditions, 3,5-diiodo-L-tyrosine and 3-iodo-L-tyrosine can be detected simultaneously, with good peak shape and good separation, which 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 Nutrition 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 types of iodotyrosine with confirmed morphology and structure in seaweed, and both are derivatives of tyrosine. Tyrosine is a primary amine type amino acid. After reacting with ninhydrin at high temperature, primary amine type amino acids generate blue-purple substances with a maximum absorption wavelength of 570nm. Under specific amino acid analyzer conditions, the response value of 570nm for 3,5-diiodo-L-tyrosine and 3-iodo-L-tyrosine is higher than that of 440nm, so 570nm is selected as the detection wavelength. Both organic iodine have good peak shapes and good separation. The standard curve is drawn with the peak area of ​​each component as the ordinate and the concentration of the standard solution as the abscissa to obtain the linear equation and correlation coefficient.

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

[0109] Two standard solutions of organic iodine were added to the blank sample, and the results were calculated based on the chromatographic response values ​​of the standard solutions. When the instrument signal-to-noise ratio (S / N) was 3, the detection limit of the method was 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 quantification limit of the method was 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 the statement about precision in GB / T 27404-2008. Determine the concentration of the limit of quantitation, 10 times the limit of quantitation, and 50 times the limit of quantitation, and evaluate the precision of the method by the recovery rate and relative standard deviation (RSD) of the analysis results. The concentrations of the spiked samples of 3,5-diiodo-L-tyrosine are 6.0μg / kg, 60.0μg / kg, and 300.0μg / kg; the concentrations of the spiked samples of 3-iodo-L-tyrosine are 3.0μg / kg, 30.0μg / kg, and 150.0μg / kg.

[0113] The results showed that the recoveries of 3,5-diiodo-L-tyrosine were 91.7% to 113.3% and the RSDs were 1.8% to 8.8% at the spiked levels of 6.0μg / kg, 60.0μg / kg and 300.0μg / kg. The recoveries of 3-iodo-L-tyrosine were 93.3% to 107.7% and the RSDs were 2.7% to 6.4% at the spiked levels of 3.0μg / kg, 30.0μg / kg and 150.0μg / kg. The methods all met the requirements of GB / T 27404-2008, had good precision and met the requirements of daily inspection.

[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 for the Determination of Salt Indicators". The results are shown in the table Seaweed iodized salt determination results

[0121]

[0122]

[0123] Inorganic iodine salt test 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 iodine-containing amino acids are not 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 principle of the present invention. These improvements and modifications should also be regarded as 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 one of methanol and ethanol or a mixture of the two; S2: subjecting the mixed solution obtained in S1 to ultrasonic-assisted extraction, filtering the obtained mixed solution, and collecting the filtrate; S3: The filtrate residue obtained from S2 is washed with a solvent, the obtained mixed solution is subjected to ultrasonic-assisted extraction, filtered, and the filtrate is collected; the filtrates obtained from S2 and S3 are combined, blown to near dryness with nitrogen, the concentrated solution is fixed to 1 mL with a sample diluent, and filtered through a 0.22 μm filter membrane to obtain a sample solution to be tested; In S2 and S3, the ultrasonic frequency is 40KHz, the temperature is 30°C, and the ultrasonic extraction time is 10 to 30 minutes; S4: The sample obtained in S3 is quantitatively detected by an amino acid analyzer. The quantitative method is the external standard method. The response area of ​​each substance on the amino acid analyzer is used as the ordinate and the concentration of the standard solution is used as the abscissa to draw a standard curve, and a linear equation and correlation coefficient are obtained. The contents of 3,5-diiodo-L-tyrosine and 3-iodo-L-tyrosine in the edible salt sample to be tested are calculated. The liquid chromatography conditions for amino acid analysis are: Chromatographic column: strongly acidic ion exchange resin Na filled column; Mobile phase A: 0.12 mol / L sodium citrate in 7% methanol aqueous solution, 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, phenol, and ascorbic acid, add 30% lauryl alcohol polyoxyethylene ether solution, potassium sodium buffer solution, and anhydrous methanol to dissolve; the mass ratio of ninhydrin, phenol, and ascorbic acid is 100:10:1, the volume ratio of the potassium sodium buffer solution to anhydrous methanol is 4:6, and the mass volume concentration of the ninhydrin in the potassium sodium buffer solution 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: 570nm; Injection volume: 50 μL; Separation column temperature: 65°C; Reactor temperature: 135°C; Gradient elution, the gradient elution program is as follows:

2. The analysis method according to claim 1, characterized in that The solvent in S1 is a mixture of methanol and ethanol, and the volume ratio of methanol to ethanol is 4:

1.

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

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

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

6. 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.

7. The analysis method according to claim 1, characterized in that The specification of the chromatographic column S4 is 4.6 mm×150 mm.

8. The analysis method according to claim 1, characterized in that The preparation method of 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 concentrated hydrochloric acid solution to 3.45, and then make up to volume with water, the concentration of sodium citrate is 0.12 mol / L, filter through a 0.45μm aqueous phase microporous filter membrane, and add caprylic acid 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 7:

93.

9. The analysis method according to claim 1, characterized in that The preparation method of mobile phase B described in S4 is: take trisodium citrate dihydrate and boric acid, dilute 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 add caprylic acid for later use; the mass ratio of trisodium citrate dihydrate to boric acid is 98:

25.

10. The analysis method according to claim 1, characterized in that The preparation method of the mobile phase D in S4 is as follows: take 20g of sodium hydroxide and 0.2g of ethylenediaminetetraacetic acid, dissolve them in water and then make up to 1000ml.

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