Synthetic process and application of luminol monosodium salt precursor for chemiluminescent substrate liquid

Abstract

The invention discloses a synthetic process and application of a luminol monosodium salt precursor for a chemiluminescent substrate solution, and belongs to the technical field of physical and chemical detection. The method comprises the following steps: by taking dimethyl 3-aminophthalate as an initial raw material, crystallizing and purifying, reacting with acetyl chloride to generate dimethyl 3-acetamido phthalate, and further purifying through column chromatography separation and recrystallization to obtain high-purity dimethyl 3-acetamido phthalate. The product reacts with hydrazine hydrate to generate N-acylated luminol, and the compound has long-term storage stability as a precursor of luminol monosodium salt. According to the present invention, the precursor is hydrolyzed under the alkaline condition, the pH value is adjusted, the crystallization is performed in ethanol, the high-purity luminol monosodium salt or the aqueous solution thereof is finally prepared, and the chemiluminescence immunoassay results of the obtained product are superior to the national standard.

Description

Technical Field

[0001] The present invention belongs to the technical field of clinical detection. Specifically, it relates to a synthesis process and application of luminol monosodium salt precursor for chemiluminescent substrate solution. Background Art

[0002] Luminol, chemically named 3-aminophthalhydrazide, is an organic compound with unique chemiluminescent properties. Since its discovery, luminol and its derivatives have been widely used in multiple fields, especially in criminal investigation, environmental monitoring, and biomedical detection. When luminol reacts with an oxidant (such as hydrogen peroxide) under alkaline conditions, it emits blue light, a phenomenon known as "chemiluminescence". This luminescence phenomenon can be used not only for visual detection but also for quantitative analysis by instruments. Therefore, luminol and its salts have important application values in modern science and technology. The sodium salt form of luminol, especially luminol monosodium salt, has become the main form in practical applications due to its good water solubility and ease of operation. Luminol monosodium salt is widely used in the following fields: Criminal investigation: Luminol monosodium salt is often used for the detection of bloodstains at the crime scene. Since iron ions in blood can catalyze the luminescence reaction of luminol, even bloodstains that are difficult to observe with the naked eye after cleaning or over time can be revealed by spraying luminol reagent. This technology has become an indispensable and important means in modern criminal investigation. Biomedical detection: In biomedical fields such as immunoassay and DNA detection, luminol monosodium salt, as a highly sensitive luminescent label, can bind to biomolecules such as antibodies and antigens, and achieve quantitative detection of target molecules through luminescence signals. This luminol-based immunoassay technology has the advantages of high sensitivity, high specificity, and rapid response, and has been widely used in clinical diagnosis and scientific research experiments.

[0003] Although luminol monosodium salt has wide applications in multiple fields, there are still some problems in its preparation process:

[0004] Purity problem: Industrially, luminol monosodium salt usually starts from 3-nitrophthalic acid (or anhydride, dimethyl ester), and the finished product contains nitro unreduced products that are difficult to completely remove. Even low levels of these impurities have a significant impact on the luminescence performance of the product. For high-precision application scenarios such as biomedical detection, the purity of the reagent directly affects the accuracy and sensitivity of the detection results.

[0005] Stability problem: The luminol molecule contains an aniline structure, which is easily oxidized in air, resulting in an increase in errors in biochemical analysis instruments.

[0006] To overcome the above problems, in recent years, researchers have proposed a new approach, which is to design and synthesize a luminol monosodium salt precursor to extend the storage period of the product and improve the product quality. A precursor refers to a relatively stable and easy-to-operate intermediate that can be converted into the target product (i.e., luminol monosodium salt) under appropriate conditions. This method has the following advantages:

[0007] Improve purity: The newly designed synthesis route controls impurities at the raw material end. The raw materials of this route are much more soluble in common organic solvents than luminol and its sodium salt, facilitating large-scale crystallization or column chromatography for impurity removal.

[0008] Enhance stability: Protecting the free aniline structure can effectively inhibit the oxidation process, thereby extending the storage life of the product. In practical applications, this improvement can significantly extend the preservation period of the product.

[0009] In summary, by designing a reasonable luminol monosodium salt precursor and optimizing its application in actual production, many problems in the existing process can be overcome. Summary of the Invention

[0010] 1. Problems to be Solved

[0011] Aiming at the problems existing in the existing synthesis process and storage process of luminol monosodium salt, the present invention provides an improved preparation process of a luminol monosodium salt precursor, and further proposes a new method for preparing luminol monosodium salt using this precursor. By optimizing the reaction steps and conditions, the purity and stability of the product are significantly improved, and many technical problems in the traditional process are solved. The traditional synthesis process of luminol monosodium salt usually uses 3-nitrophthalic acid (or anhydride, dimethyl ester) as the raw material, first reacts with hydrazine hydrate to obtain 3-nitrophthalohydrazide, then catalytically reduces it with sodium sulfide or palladium on carbon to obtain luminol, and then adjusts the pH value and crystallizes in an alcohol solvent to finally obtain luminol monosodium salt.

[0012] However, this method has several significant defects: Purity problem: There are residues of incompletely reduced nitro compounds in the reduction process. Depending on the content, these impurities will cause the aqueous solution to show orange-yellow to brownish-black instead of the ideal colorless and transparent state. Such compounds have an obvious absorption effect on the chemiluminescence of luminol, and even a small amount will have a significant impact on the results in the field of quantitative analysis. Oxidation problem: Luminol and its sodium salt contain an aniline structure and are easily slowly oxidized by oxygen in the air during storage. The oxidation product will also cause the above problems. To overcome the above problems, the present invention proposes a new luminol monosodium salt precursor - N-acyl luminol, and formulates the corresponding preparation process and the process for synthesizing luminol monosodium salt using this precursor.

[0013]

[0014] The process flow proposed by the present invention has the following significant advantages compared with the traditional method:

[0015] High product purity: Through multiple steps of fine purification, the present invention can produce luminol monosodium salt with high purity. After being formulated into an aqueous solution, the solution is colorless and transparent, without impurities that affect the luminescence performance. This makes the product particularly suitable for fields with high requirements for detection sensitivity, such as immunofluorescence analysis, medical detection reagents, etc.

[0016] Strong precursor stability: N-acyl-luminol, as a precursor, has strong antioxidant properties and can be stored for a long time without obvious impurities. This characteristic not only extends the shelf life of the product but also reduces the quality degradation problem caused by oxidation.

[0017] 2. Technical solution

[0018] To solve the above problems, the present invention adopts the following technical solution.

[0019] A synthesis process of luminol monosodium salt precursor for chemiluminescent substrate solution, comprising the following steps:

[0020] Crystallize and purify dimethyl 3-aminophthalate, prepare dimethyl 3-amidophthalate by acylation, obtain high-purity dimethyl 3-amidophthalate through column chromatography separation and recrystallization purification, and react dimethyl 3-amidophthalate with hydrazine hydrate to obtain N-acyl-luminol.

[0021] Preferably, the solvent for crystallization purification is one or more of ethyl acetate, petroleum ether, dichloromethane, methanol, ethanol, DMF, DMSO;

[0022] The crystallization method is one of cooling crystallization, solvent evaporation crystallization, and adding a low-solubility solvent for crystallization.

[0023] Preferably, the acylating reagent is one or more of acetyl chloride, propionyl chloride, n-butyryl chloride, tert-butyryl chloride, n-hexanoyl chloride, benzoyl chloride, cyclohexanecarbonyl chloride;

[0024] Preferably, the chromatography column for column chromatography separation is one of a silica gel column, an alumina column, a C18 column, a macroporous adsorption resin column, and a polyamide adsorption resin column.

[0025] Preferably, the reaction solvent for reacting with hydrazine hydrate is one or a mixture of several of water, methanol, ethanol, isopropanol, diethylamine, triethylamine, chloroform, 1,2-dichloroethane; the reaction temperature for reacting with hydrazine hydrate is 60 - 120 °C, and the reaction time is 12 - 48 h.

[0026] Application of the luminol monosodium salt precursor prepared by the above synthesis process in the synthesis of luminol monosodium salt. The application method is as follows:

[0027] If the N-acylated luminol has been stored for more than one year, it needs to be identified by TLC. When impurities are generated, recrystallization is carried out to remove impurities; the N-acylated luminol is hydrolyzed in an aqueous solution of sodium hydroxide to obtain a mixture of luminol mono / disodium salts; the pH of the reaction solution is adjusted to obtain a luminol monosodium salt solution; crystallization, filtration, and drying are carried out in an alcohol solvent to obtain a solid powder of luminol monosodium salt.

[0028] Preferably, the reagent for adjusting the pH of the reaction solution is one or a mixture of several of sulfuric acid, hydrochloric acid, nitric acid, hydrobromic acid, sodium bisulfate, sodium dihydrogen phosphate, magnesium chloride, benzoic acid, glacial acetic acid.

[0029] Preferably, the crystallization method is one of dropping the reaction solution into a single alcohol solvent, dropping a single alcohol solvent into the reaction solution, dropping the reaction solution into a mixed alcohol solvent, and dropping the mixed alcohol solvent into the reaction solution.

[0030] Preferably, the alcohol solvent is one or a mixture of several of methanol, ethanol, trifluoroethanol, n-propanol, isopropanol, n-butanol, tert-butanol, cyclohexanol.

[0031] Preferably, the drying method is one or several of drying under normal pressure with heating, vacuum drying at room temperature, freeze drying, and drying in an oven.

[0032] The overall reaction route is as follows:

[0033]

[0034] In addition, the content of the prepared luminol monosodium salt should be not less than 99% (standard curve method), including the following detection steps: Prepare aqueous solutions of 0.2000 g / L, 0.4000 g / L, 0.6000 g / L, 0.8000 g / L, and 1.0000 g / L with a luminol monosodium salt standard product, measure the absorbance at a wavelength of 301 nm with a UV-visible spectrophotometer, plot a standard curve with the concentration as the abscissa and the absorbance as the ordinate, and the linear correlation coefficient > 0.9999. Weigh about 0.5 g of the self-made sample with an analytical balance accurate to 0.0001 g, dissolve it in 1.0 L of deionized water, measure the absorbance at 301 nm, and calculate the content according to the standard curve, which should be not less than 99%.

[0035] The result of quantitative fluorescence analysis of the obtained luminol monosodium salt on a fully automatic chemiluminescence analyzer should be better than the national standard. The verification method is as follows:

[0036] Composition of Substrate Solution 1: Sodium luminol 0.8 g / L, Tris buffer 30.5 g / L, 2-hydroxypyridine 0.3 g / L, 1-ethyl-3-methylimidazolium acetate 1.05 g / L, Brij L4 0.5 g / L.

[0037] Composition of Substrate Solution 2: Hydrogen peroxide 1.2 g / L, p-hydroxybenzoic acid 0.11 g / L, Brij L4 0.1 g / L.

[0038] On the Autolumo A2000Plus fully automatic chemiluminescence analyzer, install Substrate Solution 1 and Substrate Solution 2 according to the instrument requirements. The instrument automatically aspirates serum samples to detect triiodothyronine (T3), thyroxine (T4), thyroid stimulating hormone (TSH), thyroglobulin antibody (TgAb), alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), carbohydrate antigen CA 125, carbohydrate antigen CA19-9, and carbohydrate antigen CA15-3 items. After repeated verification, the precision, accuracy, linear range, and sensitivity indicators of the detected items should be better than the national standards. Specific Embodiment

[0039] The present invention will be further described below in conjunction with specific embodiments.

[0040] In order to make the technical means, creative characteristics, and achieved purposes of the present invention easy to understand, the technical solutions of the present invention will be further described below through specific embodiments, but the protection scope of the present invention is not limited thereto. For the sake of narrative convenience, the necessary or conventional technical conditions in the following embodiments are omitted, such as the feeding method, weighing of materials, etc. The omitted technical conditions are conventional in the art or easily known to those skilled in the art.

[0041] Example 1

[0042] Preparation method of N-acetylated luminol, comprising the following steps:

[0043] 1. Crystallization purification of dimethyl 3-aminophthalate:

[0044] Add dimethyl 3-aminophthalate (6.30 g, 30 mmol) and 100 mL of ethyl acetate to a round-bottom flask. Place the flask in an oil bath and heat to 80 °C for reflux for 30 minutes. Subsequently, adjust the three-way valve above the condenser to slowly discharge the ethyl acetate vapor until a small amount of turbidity appears in the flask. At this time, close the three-way valve and stop exhausting. Turn off the oil bath heating device and cool and crystallize naturally with magnetic stirring at 100 r / min. After 12 h, filter the obtained crystals, wash them with a small amount of petroleum ether and dry them to obtain 4.40 g of purified dimethyl 3-aminophthalate, with a yield of 69%.

[0045] Identified by TLC plate, using petroleum ether: ethyl acetate (3:1) as the developing agent, the Rf value of the raw material is about 0.4, and there is an orange-yellow impurity band below it. The crystallized product should have no such impurities. If impurities are still present, recrystallization purification needs to be carried out again.

[0046] 2. Preparation of dimethyl 2-acetamido-1,2-benzenedicarboxylate:

[0047] Take the purified dimethyl 3-aminophthalate (2.10 g, 10 mmol) and place it in a 50 mL round-bottom flask, add 10 mL of dichloromethane. Slowly drop acetyl chloride (1.20 g, 15 mmol) under an ice-water bath. After the addition is complete, dissolve 2 mL of triethylamine in 10 mL of dichloromethane and add it dropwise to the round-bottom flask. After reacting for 6 h, monitor the reaction progress by TLC. When the raw material has completely reacted, evaporate the solvent, and use petroleum ether: ethyl acetate (10:1 to 5:1) as the eluent to separate the product on a silica gel column. After the obtained product is evaporated to dryness, use 20 mL of redistilled ethyl acetate as the solvent, heat it to 80 °C and stir until completely dissolved, and then let it cool naturally to crystallize. After 12 h, filter the product, and wash it with a small amount of redistilled petroleum ether. After drying, 1.95 g of dimethyl 2-acetamido-1,2-benzenedicarboxylate is obtained, and the yield is 78%.

[0048] 3. Preparation of N-acetyl-luminol:

[0049] Take dimethyl 2-acetamido-1,2-benzenedicarboxylate (1.25 g, 5 mmol) and place it in a 50 mL round-bottom flask, add 20 mL of methanol and 3 mL of hydrazine hydrate. After purging with nitrogen, heat the mixture to 90 °C and reflux for 12 h. After the reaction is completed, cool the solution to room temperature, filter out the generated solid, and wash the solid with a small amount of methanol. Dry the solid to obtain 740 mg of N-acetyl-luminol product, and the yield is 68%.

[0050] Example 2

[0051] A method for preparing solid powder of luminol monosodium salt from luminol monosodium salt precursor, comprising the following steps:

[0052] 1. Preparation of N-acetyl-luminol:

[0053] Prepare N-acetyl-luminol using the same method as in Example 1.

[0054] 2. Recrystallization of N-acetyl-luminol:

[0055] For samples stored for more than 1 year, TLC identification should be performed before use. If there are impurity spots, recrystallization in methanol is required. Take 5.00 g of N-acetylated luminol, add 30 mL of redistilled methanol, heat to 50 °C and stir for 3 h, then cool to room temperature. Filter out the solid and wash it three times with redistilled methanol, and dry it to obtain the product. Take a sample for TLC identification, there should be no impurity spots, otherwise recrystallize again.

[0056] 3. Preparation of solid powder of luminol monosodium salt:

[0057] Take 3.00 g of N-acetylated luminol, add 20 mL of 3N NaOH solution deoxygenated by passing nitrogen, heat to 90 °C and react for 4 h. After the reaction is completed, cool to room temperature, and adjust the pH to 9.0 with concentrated hydrochloric acid. This solution is slowly added dropwise to 70 mL of redistilled isopropanol with a constant pressure dropping funnel under stirring. After 12 h, filter out the precipitated solid and dry it to constant weight to obtain luminol monosodium salt.

[0058] Example 3

[0059] Preparation of aqueous solution of luminol monosodium salt from luminol monosodium salt precursor and its application method in chemiluminescence immunoassay analyzer, comprising the following steps:

[0060] 1. Preparation of N-acetylated luminol:

[0061] Prepare N-acetylated luminol using the same method as in Example 1.

[0062] 2. Recrystallization of N-acetylated luminol:

[0063] For samples stored for more than 1 year, TLC identification should be performed before use. If there are impurity spots, recrystallization in methanol is required. Take 5.00 g of N-acetylated luminol, add 30 mL of redistilled methanol, heat to 50 °C and stir for 3 h, then cool to room temperature. Filter out the solid and wash it three times with redistilled methanol, and dry it to obtain the product. Take a sample for TLC identification, there should be no impurity spots, otherwise recrystallize again.

[0064] 3. Preparation of aqueous solution of luminol monosodium salt:

[0065] Weigh 220 mg of N-acetylated luminol, add 5 mL of 3N NaOH solution deoxygenated by passing nitrogen, react at 90 °C for 4 h. After the reaction is completed, naturally cool to room temperature, adjust the pH to 9.0 with redistilled glacial acetic acid, let it stand in the dark for 1 h, then adjust the pH to 9.0 again and make up the volume to 100 mL. The resulting solution is a 10 mmol / L luminol monosodium salt solution, which is diluted according to the required concentration of the instrument and equipment before use.

[0066] 4. Application in chemiluminescence immunoassay:

[0067] Composition of Substrate Solution 1: Sodium luminol 0.8 g / L, Tris buffer 30.5 g / L, 2-hydroxypyridine 0.3 g / L, 1-ethyl-3-methylimidazolium acetate 1.05 g / L, Brij L40 0.5 g / L; among which the sodium luminol monosodium salt component can be directly prepared with the solid powder of sodium luminol monosodium salt obtained in Example 3, or can be diluted with the sodium luminol monosodium salt solution in Step 3 of this example.

[0068] Composition of Substrate Solution 2: Hydrogen peroxide 1.2 g / L, p-hydroxybenzoic acid 0.11 g / L, Brij L40 0.1 g / L.

[0069] On the Autolumo A2000Plus fully automatic chemiluminescence analyzer, install Substrate Solution 1 and Substrate Solution 2 according to the instrument requirements. The instrument automatically aspirates serum samples to detect items such as triiodothyronine (T3), thyroxine (T4), thyroid stimulating hormone (TSH), thyroglobulin antibody (TgAb), alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), carbohydrate antigen CA 125, carbohydrate antigen CA19-9, carbohydrate antigen CA15-3, etc. After repeated verification for many times, the precision, accuracy, linear range, and sensitivity indexes of the detected items are all better than the national standards.

[0070] The above content further elaborates on the present invention in combination with specific implementation manners. It cannot be determined that the specific implementation of the present invention is only limited to these descriptions. For those of ordinary skill in the technical field to which the present invention pertains, without departing from the concept of the present invention, several simple deductions or substitutions can still be made, and all should be regarded as belonging to the protection scope determined by the claims submitted for the present invention.

Claims

1. A process for synthesizing a luminol monosodium salt precursor for a chemiluminescent substrate solution, characterized in that: The following steps are involved: 3-Aminophthalic acid dimethyl ester is crystallized and purified, acylated to prepare 3-amidophthalic acid dimethyl ester, separated by column chromatography and purified by recrystallization to obtain high-purity 3-amidophthalic acid dimethyl ester, and 3-amidophthalic acid dimethyl ester is reacted with hydrazine hydrate to obtain N-acylated luminol.

2. The synthesis process of luminol monosodium salt precursor for chemiluminescent substrate solution according to claim 1, characterized in that: The solvent for crystallization and purification is one or a mixed solution of ethyl acetate, petroleum ether, dichloromethane, methanol, ethanol, DMF, and DMSO; The crystallization method is one of cooling crystallization, evaporating solvent crystallization, and adding low-solubility solvent crystallization.

3. The synthesis process of luminol monosodium salt precursor for chemiluminescent substrate solution according to claim 2, characterized in that: The acylation reagent is one or more of acetyl chloride, propionyl chloride, n-butyryl chloride, tert-butyryl chloride, n-hexanoyl chloride, benzoyl chloride, and cyclohexanecarbonyl chloride.

4. The synthesis process of luminol monosodium salt precursor for chemiluminescent substrate solution according to claim 3, characterized in that: The chromatography column for column chromatography separation is one of a silica gel column, an alumina column, a C18 column, a macroporous adsorption resin column, and a polyamide adsorption resin column.

5. The synthesis process of luminol monosodium salt precursor for chemiluminescent substrate solution according to claim 4, characterized in that: The reaction solvent for the reaction with hydrazine hydrate is one or a mixture of water, methanol, ethanol, isopropanol, diethylamine, triethylamine, chloroform, 1,2-dichloroethane; the reaction temperature for the reaction with hydrazine hydrate is 60-120°C, and the reaction time is 12-48h.

6. Use of a luminol monosodium salt precursor prepared by any one of the synthesis processes of claims 1 to 5 in the synthesis of luminol monosodium salt, characterized in that: The application method is as follows: If the N-acylated luminol is less than one year from the date of production, it is directly hydrolyzed. If the N-acylated luminol has been stored for more than one year, it needs to be identified by TLC, and impurities are removed by recrystallization when they are produced; the acyl group of N-acylated luminol is hydrolyzed in an aqueous solution of sodium hydroxide to obtain a mixture of luminol mono / disodium salts; the pH of the reaction solution is adjusted to obtain a luminol monosodium salt solution; crystallization is performed in an alcohol solvent, filtration, and drying to obtain a luminol monosodium salt solid powder.

7. Use of the luminol monosodium salt precursor prepared by the synthesis process according to claim 6 in the synthesis of luminol monosodium salt, characterized in that: The reagent for adjusting the pH of the reaction solution is one or a mixture of sulfuric acid, hydrochloric acid, nitric acid, hydrobromic acid, sodium hydrogen sulfate, sodium dihydrogen phosphate, magnesium chloride, benzoic acid, and glacial acetic acid.

8. The use of the luminol monosodium salt precursor prepared by the synthesis process according to claim 7 in the synthesis of luminol monosodium salt, characterized in that: The crystallization method is one of adding the reaction solution dropwise into a single alcohol solvent, adding the single alcohol solvent dropwise into the reaction solution, adding the reaction solution dropwise into a mixed alcohol solvent, and adding the mixed alcohol solvent dropwise into the reaction solution.

9. Use of the luminol monosodium salt precursor prepared by the synthesis process according to claim 8 in the synthesis of luminol monosodium salt, characterized in that: The alcohol solvent is one or a mixture of methanol, ethanol, trifluoroethanol, n-propanol, isopropanol, n-butanol, tert-butanol and cyclohexanol.

10. Use of the luminol monosodium salt precursor prepared by the synthesis process according to claim 9 in the synthesis of luminol monosodium salt, characterized in that: The drying method is one or more of normal pressure heating drying, normal temperature vacuum drying, freeze drying, and drying oven drying.

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