A method for detecting nitrite

Abstract

The present invention relates to a method for detecting nitrite, belonging to the technical field of detection and analysis. The method is that under acidic conditions, 3,3'-dimethoxy-5,5'-dimethylbenzidine reacts with nitrite to change the color of the solution. Qualitative analysis can be carried out by the naked eye, or semi-quantitative analysis can be performed by comparing the reaction solution with a nitrite concentration colorimetric card, or quantitative analysis can be carried out by measuring the maximum absorbance of the reaction solution with a spectrophotometer. The chromogenic molecule used in the method of the present invention is 3,3'-dimethoxy-5,5'-dimethylbenzidine molecule, which is a TMB derivative, reducing the potential toxicity and carcinogenic and mutagenic risks during the detection process. At the same time, it has high specificity and sensitivity to nitrite, and the color development is stable, capable of meeting the detection requirements of a wide range of samples. In addition, the method of the present invention is simple to operate, and the reagents used are safe, easy to transport and store, and are suitable for home detection of food in daily life.

Description

Technical Field

[0001] The present invention belongs to the technical field of detection and analysis, and particularly relates to a method for detecting nitrite. Background Art

[0002] Nitrite is an industrial salt, which is widely used as a preservative, antibacterial agent and color fixative in food processing. A small amount of nitrite intake does not affect human health, but excessive intake (>0.06 mg / kg / day) will cause two major hazards to the human body: one is that nitrite binds to hemoglobin, which will reduce the oxygen-carrying capacity of hemoglobin in the blood, and then lead to anoxic poisoning of the human body; the other is that excessive nitrite will react with amine substances in the human body to form carcinogenic nitrosamines, increasing the risk of cancer. Therefore, accurate quantitative analysis of nitrite in food is a major guarantee for ensuring food safety and safeguarding the lives and health of the masses.

[0003] So far, there have been many classic methods for detecting nitrite, including chromatography, electrochemistry, fluorescence analysis, capillary electrophoresis, etc. However, the above technologies have relatively high thresholds and require professional technicians for analysis, so they are usually only limited to central laboratories. In recent years, the increasingly complex food supply chain has greatly increased food safety risks and regulatory difficulties, and conventional laboratory detection technologies are also difficult to meet the monitoring needs of existing foods. Therefore, new technologies that are easy to operate, fast in analysis, and suitable for on-site detection of nitrite will play an increasingly important role in food safety supervision.

[0004] Colorimetry is one of the more ideal signal readout technologies for developing on-site detection technologies. The nitrite colorimetry based on Griess reagent has also been determined as one of the national standard methods (GB 5009.33-2016) for nitrite detection and is widely used in the determination of nitrite in food. However, this technology uses toxic sulfanilic acid and N-(1-Naphthyl)ethylenediamine dihydrochloride during the detection process, so this method poses potential hazards to both operators and the environment. Therefore, developing a relatively safe and stable chromogenic molecule and constructing a new technology that is simple, fast, and capable of realizing colorimetric detection of nitrite in food still has important significance for ensuring food safety. Summary of the Invention

[0005] In view of the above deficiencies, the present invention provides a new method for detecting nitrite. Under acidic conditions, 3,3'-dimethoxy-5,5'-dimethylbenzidine reacts with nitrite to change the color of the solution. Thus, qualitative analysis can be carried out by the naked eye, or the reaction solution can be compared with a nitrite concentration colorimetric card for semi-quantitative analysis, or quantitative analysis can be carried out by measuring the maximum absorbance of the reaction solution with a spectrophotometer. The chromogenic molecule used in the method of the present invention is the newly synthesized 3,3'-dimethoxy-5,5'-dimethylbenzidine molecule, which has high specificity and sensitivity to nitrite and stable color development, and can meet the detection requirements of a wide range of samples. In addition, since the method provided by the present invention is simple to operate and the reagents used are safe, easy to transport and store, it is suitable for home detection of food in daily life.

[0006] The technical solution of the present invention:

[0007] The first technical problem to be solved by the present invention is to point out the use of 3,3'-dimethoxy-5,5'-dimethylbenzidine in the colorimetric detection of nitrite.

[0008] Further, the structural formula of the 3,3'-dimethoxy-5,5'-dimethylbenzidine is shown in Formula I:

[0009]

[0010] Further, the 3,3'-dimethoxy-5,5'-dimethylbenzidine is used as a chromogenic substrate for the detection of nitrite.

[0011] The second technical problem to be solved by the present invention is to provide a chromogenic solution for the detection of nitrite, and the substrate in the chromogenic solution is 3,3'-dimethoxy-5,5'-dimethylbenzidine.

[0012] Further, the pH range of the chromogenic solution is 2.0 - 4.0; preferably the pH is 2.6 - 3.2; more preferably 2.8, 3.0, 3.2.

[0013] The third technical problem to be solved by the present invention is to provide a qualitative detection method for nitrite. The qualitative detection method is as follows: first, extract nitrite from the test substance to obtain an extract, and then mix the extract with the chromogenic solution. If the color of the mixed solution changes after standing, it indicates that the test substance contains nitrite, thereby realizing the qualitative analysis of nitrite; wherein, the substrate in the chromogenic solution is 3,3'-dimethoxy-5,5'-dimethylbenzidine.

[0014] Further, the color developing solution comprises a 3,3'-dimethoxy-5,5'-dimethylbenzidine solution and a disodium hydrogen phosphate-citric acid buffer solution, and the concentration of 3,3'-dimethoxy-5,5'-dimethylbenzidine in the color developing solution is 0.4 - 0.7 mM.

[0015] Further, the pH range of the color developing solution is 2.0 - 4.0; preferably the pH is 2.6 - 3.2; more preferably it is 2.8, 3.0, or 3.2.

[0016] Further, the volume ratio of the extraction solution to the color developing solution is 1:4 - 1:6, preferably 1:4.

[0017] Further, the standing time is 10 - 90 minutes, preferably 15 - 30 minutes.

[0018] Further, the method for extracting nitrite in the analyte is a common method in the prior art.

[0019] The fourth technical problem to be solved by the present invention is to provide a semi-quantitative detection method for nitrite. The semi-quantitative detection method is as follows: First, extract nitrite in the analyte to obtain an extraction solution, then mix the extraction solution and the color developing solution, and after standing, compare the color development result with the corresponding value on the nitrite concentration color comparison card to semi-quantitatively analyze nitrite; wherein, the substrate in the color developing solution is 3,3'-dimethoxy-5,5'-dimethylbenzidine.

[0020] Further, the color developing solution is composed of a 3,3'-dimethoxy-5,5'-dimethylbenzidine solution and a disodium hydrogen phosphate-citric acid buffer solution, the concentration of 3,3'-dimethoxy-5,5'-dimethylbenzidine in the color developing solution is 0.4 - 0.7 mM, preferably 0.5 mM.

[0021] Further, the pH range of the color developing solution is 2.6 - 3.2, preferably 3.0.

[0022] Further, the volume ratio of the extraction solution to the color developing solution is 1:4 - 1:6, preferably 1:4.

[0023] Further, the standing time is 15 - 30 minutes, preferably 15 minutes.

[0024] Further, the method for extracting nitrite in the analyte is a common method in the prior art; the nitrite concentration color comparison card is a common color comparison card in the prior art.

[0025] The fifth technical problem to be solved by the present invention is to provide a method for quantitatively detecting nitrite. The quantitative detection method is as follows: First, extract nitrite from the analyte to obtain an extraction solution, then mix the extraction solution with a chromogenic solution, let it stand for the chromogenic solution to develop color, and then calculate the content of nitrite in the analyte through the following formula:

[0026]

[0027] In the formula, X refers to the content of nitrite in the analyte (calculated as NaNO2, unit: mg / kg), V refers to the total volume of the extraction solution (mL), c refers to the concentration of nitrite in the mixed solution (μM), V1 refers to the volume of the extraction solution taken for mixing with the chromogenic solution (mL), V2 refers to the volume of the chromogenic solution taken (mL), and m refers to the mass of the analyte (g).

[0028] Further, the concentration of nitrite in the mixed solution is calculated through the following formula: A = 0.0146c + 0.0104. In the formula, A refers to the maximum absorbance of the mixed solution, which is measured by a spectrophotometer.

[0029] Further, the chromogenic solution is composed of 3,3'-dimethoxy-5,5'-dimethylbenzidine solution and disodium hydrogen phosphate-citric acid buffer solution. The concentration of 3,3'-dimethoxy-5,5'-dimethylbenzidine in the chromogenic solution is 0.4 - 0.7 mM, preferably 0.5 mM.

[0030] Further, the pH of the chromogenic solution is 2.6 - 3.2, preferably 3.0;

[0031] Further, the volume ratio of the extraction solution to the chromogenic solution is 1:4 - 1:6, preferably 1:4;

[0032] Further, the standing time is 15 - 30 minutes, preferably 15 minutes;

[0033] Further, the method for extracting nitrite from the analyte is a general method in the prior art.

[0034] The sixth technical problem to be solved by the present invention is to provide a nitrite detection kit. The detection kit includes a chromogenic solution and a colorimetric tube. The substrate in the chromogenic solution is 3,3'-dimethoxy-5,5'-dimethylbenzidine.

[0035] Advantages of the present invention:

[0036] 1. In the nitrite detection method of the present invention, 3,3'-dimethoxy-5,5'-dimethylbenzidine used is a newly synthesized TMB derivative molecule, which reduces the potential toxicity and carcinogenic and mutagenic risks during the detection process.

[0037] 2. In the method of the present invention, 3,3'-dimethoxy-5,5'-dimethylbenzidine used has high specificity and sensitivity to nitrite under acidic conditions. It can detect nitrite as low as 0.2 μM (detection limit is 0.2 μM) using a spectrophotometer, and can quantify nitrite as low as 0.5 μM (quantification limit is 0.5 μM). Moreover, its color development is stable, and qualitative or semi-quantitative analysis can be carried out with the naked eye.

[0038] 3. Since the method provided by the present invention does not require the assistance of large-scale instrument equipment, does not require excessive support conditions, is simple to operate, and the reagents used are easy to transport and store, it is suitable for home detection of food in daily life. Description of the Drawings

[0039] Figure 1 It is the synthetic route diagram of 3,3'-dimethoxy-5,5'-dimethylbenzidine in Example 1.

[0040] Figure 2 It is the color development result of detecting 50 μM sodium nitrite with 0.5 mM 3,3'-dimethoxy-5,5'-dimethylbenzidine in Example 1.

[0041] Figure 3 It is the test result of the stability of the color development solution for nitrite detection in Example 2.

[0042] Figure 4 It is the color development result of detecting 25 μM sodium nitrite with 0.25 mM 3,3'-dimethoxy-5,5'-dimethylbenzidine under different pH conditions in Example 3.

[0043] Figure 5 It is the color development result of detecting 25 μM sodium nitrite with 3,3'-dimethoxy-5,5'-dimethylbenzidine at different concentrations in Example 4.

[0044] Figure 6 It is the color development result of detecting different ions by the method of the present invention in Example 5.

[0045] Figure 7 It is the color development result of detecting the coexistence of nitrite ions and other ions by the method of the present invention in Example 6.

[0046] Figure 8 It is the schematic diagram of the nitrite detection process in food samples of the embodiments of the present invention.

[0047] Figure 9 It is the color development result of detecting nitrite with 3,3',5,5'-tetramethylbenzidine (TMB) in Comparative Example 1.

[0048] Figure 10It is the synthetic route diagram of 3,3',5,5'-tetramethoxybenzidine in Comparative Example 2.

[0049] Figure 11 It is the color development result of 3,3',5,5'-tetramethoxybenzidine for detecting nitrite in Comparative Example 2. Specific Embodiments

[0050] The nitrite detection method disclosed by the present invention is as follows: under acidic conditions, 3,3'-dimethoxy-5,5'-dimethylbenzidine reacts with nitrite to change the color of the solution. Thus, qualitative analysis can be carried out with the naked eye, or the reaction solution can be compared with a nitrite concentration colorimetric card for semi-quantitative analysis, or quantitative analysis can be carried out by measuring the maximum absorbance of the reaction solution with a spectrophotometer. The chromogenic molecule used in the method of the present invention is the newly synthesized 3,3'-dimethoxy-5,5'-dimethylbenzidine molecule, which has high specificity and sensitivity to nitrite, and the color development is stable, and can meet the detection requirements of a wide range of samples. In addition, since the method provided by the present invention is simple to operate, the reagents used are safe, easy to transport and store, so it is suitable for home detection of food in daily life.

[0051] The present invention will be further described below through examples. It should be noted that the given examples should not be construed as limiting the protection scope of the present invention. Some non-essential improvements and adjustments made by those skilled in the art to the present invention based on the content of the present invention should still fall within the protection scope of the present invention.

[0052] It should be noted that: 1) 3,3'-dimethoxy-5,5'-dimethylbenzidine in Example 1 and 3,3',5,5'-tetramethoxybenzidine in Comparative Example 2 are two TMB derivative molecules synthesized by the applicant of the present invention; 2) the detection kit in the examples is a nitrite detection kit prepared according to the method of the present invention; 3) Comparative Examples 3-32 were detected by the second method (Griess reagent colorimetry) in the national standard method for the determination of nitrite (GB 5009.33-2016), and the detection results were compared with the detection results of Examples 8-37; 4) Examples 8-37 and Comparative Examples 3-32 judged whether the nitrite content measured exceeded the standard according to the national limit standards for nitrite in food (GB 2762-2022, GB 2760-2014).

[0053] Example 1

[0054] (1) Preparation of 3,3'-dimethoxy-5,5'-dimethylbenzidine: As Figure 1As shown in the figure, first, p-bromoaniline (2) is generated through the substitution reaction of 2-methoxy-6-methylaniline (1). After 2 is esterified with bis(pinacolato)diboron, the esterification product (3) undergoes a Suzuki–Miyaura reaction with the brominated product (2) to generate 3,3'-dimethoxy-5,5'-dimethylbenzidine. The 3,3'-dimethoxy-5,5'-dimethylbenzidine in the following examples is prepared by this method.

[0055] (2) Detection of nitrite by 3,3'-dimethoxy-5,5'-dimethylbenzidine: Prepare sodium nitrite solution (5 mM, with pure water as the solvent), 3,3'-dimethoxy-5,5'-dimethylbenzidine solution (50 mM, with DMSO as the solvent), and disodium hydrogen phosphate-citric acid buffer solution (pH = 2.5, with pure water as the solvent) respectively. Take a colorimetric tube and add 1960 μL of disodium hydrogen phosphate-citric acid buffer solution and 20 μL of 3,3'-dimethoxy-5,5'-dimethylbenzidine solution in sequence, mix well, then add 20 μL of sodium nitrite solution, mix well, start timing, and measure the absorbance of the reaction solution at 454 nm by a spectrophotometer at 1, 3, 5, 10, 20, 30, 45, and 60 minutes respectively. The results are shown in Figure 2 .

[0056] Example 2

[0057] In the present invention, the nitrite detection color-developing solution is prepared by the following method: Prepare a 0.5 mL of 50 mM 3,3'-dimethoxy-5,5'-dimethylbenzidine solution with DMSO as the solvent, and prepare a 49.5 mL of disodium hydrogen phosphate-citric acid buffer solution with a pH of 2.8 with pure water as the solvent. Then mix the two evenly to obtain 50 mL of colorless color-developing solution. The concentration of 3,3'-dimethoxy-5,5'-dimethylbenzidine in the obtained color-developing solution is 0.5 mM. Keep the prepared color-developing solution in the dark and at low temperature. Take out the color-developing solution every other day for stability testing. Take a colorimetric tube and add 1980 μL of color-developing solution and 20 μL of the national standard substance solution of sodium nitrite for food detection (200 μg / mL) in sequence, mix well, and measure the absorbance of the reaction solution at 454 nm by a spectrophotometer after reacting for 15 minutes. The results are shown in Figure 3 . It can be seen from Figure 3 that the prepared nitrite detection color-developing solution can be stably stored within one month.

[0058] Example 3

[0059] Prepare sodium nitrite solution (5 mM, with pure water as the solvent), 3,3'-dimethoxy-5,5'-dimethylbenzidine solution (50 mM, with DMSO as the solvent), and prepare disodium hydrogen phosphate-citric acid buffer solution with pH gradients of 2.2, 2.4, 2.6, 2.8, 3.0, 3.2, 3.4, 3.6 (with pure water as the solvent). Take 8 colorimetric tubes and add 1980 μL of the above-mentioned disodium hydrogen phosphate-citric acid buffer solution with different pH gradients respectively, then add 10 μL of 3,3'-dimethoxy-5,5'-dimethylbenzidine solution respectively, mix well, and then add 10 μL of sodium nitrite solution respectively, mix well, and start timing for 15 minutes. After 15 minutes, measure the absorbance of the reaction solution at 454 nm through a spectrophotometer, and the results are shown in Figure 4 . It can be seen from Figure 4 that the absorbance value of the reaction solution is the largest when the pH is 3.0. Therefore, pH 3.0 is selected as the optimal acidic condition for nitrite detection.

[0060] Example 4

[0061] Prepare sodium nitrite solution (5 mM, with pure water as the solvent), 3,3'-dimethoxy-5,5'-dimethylbenzidine solution (50 mM, with DMSO as the solvent), and disodium hydrogen phosphate-citric acid buffer solution (pH = 3.0, with pure water as the solvent) respectively. Take 8 colorimetric tubes and add 1990 μL, 1986 μL, 1982 μL, 1978 μL, 1974 μL, 1970 μL, 1966 μL, 1962 μL of disodium hydrogen phosphate-citric acid buffer solution respectively, then add 0, 4 μL, 8 μL, 12 μL, 16 μL, 20 μL, 24 μL, 28 μL of 3,3'-dimethoxy-5,5'-dimethylbenzidine solution respectively, mix well, and then add 10 μL of sodium nitrite solution respectively, mix well, and start timing for 15 minutes. After 15 minutes, measure the absorbance of the reaction solution at 454 nm through a spectrophotometer, and the results are shown in Figure 5 . It can be seen from Figure 5 that as the concentration of 3,3'-dimethoxy-5,5'-dimethylbenzidine increases, the absorbance of the reaction solution gradually increases. When the concentration of 3,3'-dimethoxy-5,5'-dimethylbenzidine reaches 0.5 mM, the absorbance basically no longer increases. Therefore, 0.5 mM is selected as the optimal substrate concentration for nitrite detection.

[0062] In the present invention, the final concentration c” (mM) of 3,3'-dimethoxy-5,5'-dimethylbenzidine is calculated by the following formula:

[0063]

[0064] In the formula, c' refers to the concentration (mM) of the 3,3'-dimethoxy-5,5'-dimethylbenzidine stock solution, V' refers to the volume (mL) of the 3,3'-dimethoxy-5,5'-dimethylbenzidine stock solution taken, and V" refers to the total volume (mL) of the solution. The 3,3'-dimethoxy-5,5'-dimethylbenzidine stock solution is prepared with DMSO as the solvent, and its concentration c' (mM) is calculated by the following formula:

[0065]

[0066] In the formula, m' refers to the mass (mg) of the 3,3'-dimethoxy-5,5'-dimethylbenzidine taken, M refers to the relative molecular mass (g / mol) of 3,3'-dimethoxy-5,5'-dimethylbenzidine, and V''' refers to the total volume (mL) of the stock solution.

[0067] Example 5

[0068] Ion selectivity. Under the optimal reaction conditions (reaction time 15 minutes, pH 3.0, 3,3'-dimethoxy-5,5'-dimethylbenzidine concentration 0.5 mM), the selectivity of the method of the present invention for nitrite ions was verified. NO2 - was compared with 17 other inorganic ions (Fe 3+ , K + , Ca 2+ , Mg 2+ , Na + , Al 3+ , Zn 2+ , Cu 2+ , Ni 2+ , Mn 2+ , NO3 - , SO4 2- , PO4 3- , I - , F - , Cl - , Br - ). The above ion solutions (5 mM, with pure water as the solvent), 3,3'-dimethoxy-5,5'-dimethylbenzidine solution (50 mM, with DMSO as the solvent), and disodium hydrogen phosphate-citric acid buffer solution (pH = 3.0, with pure water as the solvent) were respectively prepared. 18 colorimetric tubes were taken and 1972 μL of disodium hydrogen phosphate-citric acid buffer solution and 20 μL of 3,3'-dimethoxy-5,5'-dimethylbenzidine solution were added respectively, mixed well, and then 8 μL of the above ion solutions were added respectively, mixed well, and timed for 15 minutes. After 15 minutes, the absorbance of the reaction solution at 454 nm was measured by a spectrophotometer. The results are shown in Figure 6 . From Figure 6It can be seen that the presence of ions other than nitrite ions does not cause a significant change in the absorbance value of the solution. Therefore, the method of the present invention has high selectivity for nitrite and does not respond to other ions.

[0069] Example 6

[0070] Ion interference. Under the optimal reaction conditions (reaction time 15 minutes, pH 3.0, 3,3'-dimethoxy-5,5'-dimethylbenzidine concentration 0.5 mM), the anti-ion interference of the method of the present invention was verified. The above inorganic ions (K + , Ca 2+ , Mg 2+ , Na + , Al 3+ , Zn 2+ , Cu 2+ , Ni 2+ , Mn 2+ , NO3 - , SO4 2- , PO4 3- , I - , F - , Cl - , Br - ) were selected as interfering ions. The above ion solutions (100 mM, with pure water as the solvent), NaNO2 solution (5 mM, with pure water as the solvent), 3,3'-dimethoxy-5,5'-dimethylbenzidine solution (50 mM, with DMSO as the solvent), and disodium hydrogen phosphate-citric acid buffer (pH = 3.0, with pure water as the solvent) were prepared separately. One colorimetric tube was taken as the blank control group, and 1972 μL of disodium hydrogen phosphate-citric acid buffer and 8 μL of NaNO2 solution were added successively and mixed well; then 16 colorimetric tubes were taken and 1952 μL of disodium hydrogen phosphate-citric acid buffer, 8 μL of NaNO2 solution, and 20 μL of the above 16 ion solutions were added respectively and mixed well. Finally, 20 μL of 3,3'-dimethoxy-5,5'-dimethylbenzidine solution was added to each of the 17 colorimetric tubes and mixed well, and the time was counted for 15 minutes. After 15 minutes, the absorbance of the reaction solution at 454 nm was measured by a spectrophotometer, and the results are shown in Figure 7 . It can be seen from Figure 7 that the coexistence of the remaining ions (1 mM) does not cause obvious interference to the detection of nitrite ions. In addition, Fe 3+ (≤0.5 mM) can coexist with nitrite ions without interfering with the determination under the masking of EDTA. Therefore, this detection method has good anti-ion interference performance.

[0071] Example 7

[0072] Accuracy of nitrite detection. Prepare 3,3'-dimethoxy-5,5'-dimethylbenzidine solution (50 mM, with DMSO as the solvent) and disodium hydrogen phosphate-citric acid buffer solution (pH = 3.0, with pure water as the solvent). Take a colorimetric tube and sequentially add 1960 μL of disodium hydrogen phosphate-citric acid buffer solution and 20 μL of 3,3'-dimethoxy-5,5'-dimethylbenzidine solution, mix well, then add 20 μL of the national standard substance solution of sodium nitrite for food detection (200 μg / mL), mix well. After reacting for 15 minutes, measure the absorbance of the reaction solution at 454 nm using a spectrophotometer, and quantitatively analyze the concentration of sodium nitrite therein. The results are shown in Table 1. As can be seen from Table 1, the method of the present invention has a relatively high accuracy in detecting nitrite.

[0073] Table 1 Test results of the method of the present invention for detecting the national standard substance solution in Example 7

[0074]

[0075] The operation procedures for qualitative, semi-quantitative, and quantitative detection of nitrite in food samples by the method of the present invention are as Figure 8 shown.

[0076] Examples 8 - 17

[0077] (1) Weigh 10 portions of crushed pickled vegetable samples, each portion being 2 g. Soak the 10 portions of pickled vegetable samples in clear water for 5 minutes, then add 0.5 mL of potassium ferrocyanide solution (sample extract one) at 106 g / L, mix well, and then add 0.5 mL of zinc acetate solution (sample extract two) at 220 g / L, mix well, and let stand for 10 minutes;

[0078] (2) Filter to obtain the filtrate. Use a pipette to suck 0.5 mL of the filtrate into a colorimetric tube, and then add 2.0 mL of the color-developing solution to the colorimetric tube, mix well, and let stand for 15 minutes to make the colorless color-developing solution develop color; in this example and the following examples, the concentration of 3,3'-dimethoxy-5,5'-dimethylbenzidine in the color-developing solution is 0.5 mM, and the pH of the color-developing solution is 2.8.

[0079] (3) After the color development is completed, measure the absorbance at 454 nm using a spectrophotometer. Substitute the measured absorbance into the formula A = 0.0146c + 0.0104 to calculate the concentration c (μM) of nitrite, and then use to calculate the nitrite content in the sample (calculated as NaNO2, with the unit of mg / kg). The results are shown in Table 2.

[0080] Examples 18 - 27

[0081] (1) Weigh 10 portions of the crushed meat sausage samples, 2 g for each portion. Soak the 10 portions of meat sausage samples in clear water for 5 minutes, then add 0.5 mL of 106 g / L potassium ferrocyanide solution (sample extract one), mix well, then add 0.5 mL of 220 g / L zinc acetate solution (sample extract two), mix well, and let stand for 10 minutes;

[0082] (2) Filter to obtain the filtrate. Use a pipette to suck 0.5 mL of the filtrate into a colorimetric tube, then add 2.0 mL of the color-developing solution to the colorimetric tube, mix well, and let stand for 15 minutes to develop the color of the colorless color-developing solution;

[0083] (3) After the color development is completed, measure the absorbance at 454 nm through a spectrophotometer. Substitute the measured absorbance into the formula A = 0.0146c + 0.0104 to calculate the concentration c (μM) of nitrite, and then use to calculate the nitrite content in the sample (calculated as NaNO2, unit: mg / kg). The results are shown in Table 3.

[0084] Examples 28 - 37

[0085] (1) Weigh 10 portions of the crushed canned meat samples, 2 g for each portion. Soak the 10 portions of canned meat samples in clear water for 5 minutes, then add 0.5 mL of 106 g / L potassium ferrocyanide solution (sample extract one), mix well, then add 0.5 mL of 220 g / L zinc acetate solution (sample extract two), mix well, and let stand for 10 minutes;

[0086] (2) Filter to obtain the filtrate. Use a pipette to suck 0.5 mL of the filtrate into a colorimetric tube, then add 2.0 mL of the color-developing solution to the colorimetric tube, mix well, and let stand for 15 minutes to develop the color of the colorless color-developing solution;

[0087] (3) After the color development is completed, measure the absorbance at 454 nm through a spectrophotometer. Substitute the measured absorbance into the formula A = 0.0146c + 0.0104 to calculate the concentration c (μM) of nitrite, and then use to calculate the nitrite content in the sample (calculated as NaNO2, unit: mg / kg). The results are shown in Table 4.

[0088] Comparative Example 1

[0089] This comparative example uses commercially available 3,3',5,5'-tetramethylbenzidine (TMB) to detect nitrite. Sodium nitrite solution (5 mM, with pure water as the solvent), TMB solution (50 mM, with DMSO as the solvent), and disodium hydrogen phosphate-citric acid buffer (pH = 2.5, with pure water as the solvent) were prepared respectively. Take a colorimetric tube, add 1960 μL of disodium hydrogen phosphate-citric acid buffer and 20 μL of TMB solution in sequence, mix well, then add 20 μL of sodium nitrite solution, mix well, start timing, and measure the absorbance of the reaction solution at 448 nm by a spectrophotometer at 1, 3, 5, 10, 20, 30, 45, and 60 minutes respectively. The results are shown in Figure 9 .

[0090] From the comparison between Figure 2 and Figure 9 , it can be seen that the absorbance of 3,3'-dimethoxy-5,5'-dimethylbenzidine for detecting nitrite tends to be stable after 10 minutes, while the absorbance of 3,3',5,5'-tetramethylbenzidine (TMB) for detecting nitrite gradually decreases after 10 minutes. Therefore, 3,3'-dimethoxy-5,5'-dimethylbenzidine has improved color development stability compared to TMB in detecting nitrite and is more advantageous for on-site detection of nitrite based on semi-quantitative analysis by the naked eye.

[0091] Comparative Example 2

[0092] (1) Preparation of 3,3',5,5'-tetramethoxybenzidine: As Figure 10 shown, first, p-bromoaniline (5) is generated through the substitution reaction of 2,6-dimethoxyaniline (4). After 5 is esterified with bis(pinacolato)diboron, the esterification product (6) undergoes a Suzuki–Miyaura reaction with the brominated product (5) to generate 3,3',5,5'-tetramethoxybenzidine. The 3,3',5,5'-tetramethoxybenzidine in the following comparative examples is all prepared by this method.

[0093] (2) Detection of nitrite by 3,3',5,5'-tetramethoxybenzidine: Sodium nitrite solution (5 mM, with pure water as the solvent), 3,3',5,5'-tetramethoxybenzidine solution (50 mM, with DMSO as the solvent), and disodium hydrogen phosphate-citric acid buffer (pH = 2.5, with pure water as the solvent) were prepared respectively. Take a colorimetric tube, add 1960 μL of disodium hydrogen phosphate-citric acid buffer and 20 μL of 3,3',5,5'-tetramethoxybenzidine solution in sequence, mix well, then add 20 μL of sodium nitrite solution, mix well, start timing, and measure the absorbance of the reaction solution at 451 nm by a spectrophotometer at 1, 3, 5, 10, 20, 30, 45, and 60 minutes respectively. The results are shown in Figure 11 .

[0094] By Figure 2 and Figure 11 Comparing shows that: compared with 3,3'-dimethoxy-5,5'-dimethylbenzidine, the absorbance of 3,3',5,5'-tetramethoxybiphenylamine for detecting nitrite is lower, which is not conducive to visual reading. In addition, it also shows that the sensitivity of 3,3',5,5'-tetramethoxybiphenylamine for detecting nitrite is not as good as that of 3,3'-dimethoxy-5,5'-dimethylbenzidine.

[0095] Comparative Example 3-12

[0096] (1) Use a pipette to separately suck 0.5 mL of the filtrate obtained in Examples 11-20 and add it into a colorimetric tube containing 2.0 mL of Griess reagent, mix well, and let it stand for 15 minutes to make the Griess reagent develop color;

[0097] (2) After the color development is completed, measure the absorbance at 545 nm through a spectrophotometer and conduct quantitative analysis. The results are shown in Table 2.

[0098] The results show that: the detection results of the present invention are similar to those of the national standard method, verifying the accuracy of the detection method described in the present invention.

[0099] Table 2 Detection Results of Experimental Examples 8-17 and Comparative Examples 3-12

[0100]

[0101] Comparative Example 13-22

[0102] (1) Use a pipette to separately suck 0.5 mL of the filtrate obtained in Examples 21-30 and add it into a colorimetric tube containing 2.0 mL of Griess reagent, mix well, and let it stand for 15 minutes to make the Griess reagent develop color;

[0103] (2) After the color development is completed, measure the absorbance at 545 nm through a spectrophotometer and conduct quantitative analysis. The results are shown in Table 3.

[0104] The results show that: the detection results of the present invention are similar to those of the national standard method, verifying the accuracy of the detection method described in the present invention.

[0105] Table 3 Detection Results of Experimental Examples 18-27 and Comparative Examples 13-22

[0106]

[0107]

[0108] Comparative Example 23-32

[0109] (1) Use a pipette to separately aspirate 0.5 mL of the filtrate obtained in Examples 31 - 40 and add it to a colorimetric tube containing 2.0 mL of Griess reagent. Mix well and let it stand for 15 minutes to allow the Griess reagent to develop color.

[0110] (2) After the color development is completed, measure the absorbance at 545 nm using a spectrophotometer and perform quantitative analysis. The results are shown in Table 4.

[0111] The results show that the detection results of the present invention are similar to those of the national standard method, verifying the accuracy of the detection method described in the present invention.

[0112] Table 4 Detection results of Experimental Examples 28 - 37 and Comparative Examples 23 - 32

[0113]

[0114]

Claims

1. Use of 3,3'-dimethoxy-5,5'-dimethylbenzidine in nitrite colorimetric detection, characterized in that, The structural formula of 3,3'-dimethoxy-5,5'-dimethylbenzidine is shown in Formula I: Formula I.

2. Use of 3,3'-dimethoxy-5,5'-dimethylbenzidine according to claim 1 in nitrite colorimetric detection, characterized in that, The 3,3'-dimethoxy-5,5'-dimethylbenzidine is used as a chromogenic substrate for nitrite detection.

3. A color-developing solution for nitrite detection, characterized in that, The substrate in the chromogenic solution is 3,3'-dimethoxy-5,5'-dimethylbenzidine; the pH range of the chromogenic solution is 2.0 - 4.

0.

4. A chromogenic solution for nitrite detection according to claim 3, characterized in that, The pH of the chromogenic solution is 2.6 - 3.

2.

5. A chromogenic solution for nitrite detection according to claim 4, wherein, The pH of the chromogenic solution is 2.8, 3.0, 3.

2.

6. A qualitative detection method for nitrite, characterized in that, The qualitative detection method is as follows: First, extract nitrite from the analyte to obtain an extract, then mix the extract and the chromogenic solution. If the color of the mixed solution changes after standing, it indicates that the analyte contains nitrite, thus realizing the qualitative analysis of nitrite; wherein, the substrate in the chromogenic solution is 3,3'-dimethoxy-5,5'-dimethylbenzidine.

7. The qualitative detection method of nitrite according to claim 6, characterized in that, The chromogenic solution includes a 3,3'-dimethoxy-5,5'-dimethylbenzidine solution and a disodium hydrogen phosphate-citric acid buffer. The concentration of 3,3'-dimethoxy-5,5'-dimethylbenzidine in the chromogenic solution is 0.4 - 0.7 mM; the pH range of the chromogenic solution is 2.0 - 4.0; the volume ratio of the extract to the chromogenic solution is 1:4 - 1:6; the standing time is 10 - 90 minutes.

8. The qualitative detection method of nitrite according to claim 7, characterized in that, The pH of the chromogenic solution is 2.6 - 3.2; the volume ratio of the extract to the chromogenic solution is 1:4; the standing time is 15 - 30 minutes.

9. The qualitative detection method of nitrite according to claim 8, characterized in that, The pH of the chromogenic solution is 2.8, 3.0, 3.

2.

10. A semi - quantitative detection method for nitrite, characterized in that, The semi-quantitative detection method is as follows: First, extract nitrite from the analyte to obtain an extract, then mix the extract and the chromogenic solution. After standing, compare the chromogenic result with the corresponding value on the nitrite concentration colorimetric card to semi-quantitatively analyze nitrite; wherein, the substrate in the chromogenic solution is 3,3'-dimethoxy-5,5'-dimethylbenzidine.

11. The semi - quantitative detection method of nitrite according to claim 10, characterized in that, The chromogenic solution is composed of a 3,3'-dimethoxy-5,5'-dimethylbenzidine solution and a disodium hydrogen phosphate-citric acid buffer. The concentration of 3,3'-dimethoxy-5,5'-dimethylbenzidine in the chromogenic solution is 0.4 - 0.7 mM; the pH range of the chromogenic solution is 2.6 - 3.2; the volume ratio of the extract to the chromogenic solution is 1:4 - 1:6; the standing time is 15 - 30 minutes.

12. The semi - quantitative detection method of nitrite according to claim 11, characterized in that, The concentration of 3,3'-dimethoxy-5,5'-dimethylbenzidine in the chromogenic solution is 0.5 mM; the pH of the chromogenic solution is 3.0; the volume ratio of the extract to the chromogenic solution is 1:4; the standing time is 15 minutes.

13. A quantitative detection method for nitrite, characterized in that, The quantitative detection method is as follows: First, extract nitrite from the analyte to obtain an extract, then mix the extract and the chromogenic solution, and let the mixture develop color after standing. Then calculate the content of nitrite in the analyte through the following formula: ； In the formula, X refers to the content of nitrite in the analyte, calculated as NaNO2, with the unit of mg / kg; V refers to the total volume of the extraction solution, with the unit of mL; c refers to the concentration of nitrite in the mixed solution, with the unit of μM; V1 refers to the volume of the extraction solution taken for mixing with the color-developing solution, with the unit of mL; V2 refers to the volume of the color-developing solution taken, with the unit of mL; m refers to the mass of the analyte, with the unit of g; Among them, the substrate in the color-developing solution is 3,3'-dimethoxy-5,5'-dimethylbenzidine.

14. The quantitative detection method of nitrite according to claim 13, characterized in that, The concentration of nitrite in the mixed solution is calculated using the following formula: A = 0.0146c + 0.0104, where A refers to the maximum absorbance of the mixed solution, measured by a spectrophotometer; The color-developing solution is composed of a 3,3'-dimethoxy-5,5'-dimethylbenzidine solution and a disodium hydrogen phosphate-citric acid buffer solution. The concentration of 3,3'-dimethoxy-5,5'-dimethylbenzidine in the color-developing solution is 0.4 - 0.7 mM; The pH of the color-developing solution is 2.6 - 3.2; The volume ratio of the extraction solution to the color-developing solution is 1:4 - 1:6; The standing time is 15 - 30 minutes.

15. The quantitative detection method of nitrite according to claim 14, characterized in that, The concentration of 3,3'-dimethoxy-5,5'-dimethylbenzidine in the color-developing solution is 0.5 mM; the pH of the color-developing solution is 3.0; the volume ratio of the extraction solution to the color-developing solution is 1:4; the standing time is 15 minutes.

16. A nitrite detection kit, the detection kit includes a color-developing solution and a colorimetric tube, and the substrate in the color-developing solution is 3,3'-dimethoxy-5,5'-dimethylbenzidine.

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