Preparation, separation and detection method and application of sodium citicoline impurity with structure of formula I

Abstract

The present application relates to a kind of preparation, separation detection method and application of the impurity of sodium cytidine phosphate with structure I, comprising the following steps: 5'-cytidylic acid, choline phosphate and sodium tert-butanolate aqueous solution are mixed to be prepared into reaction solution, then catalyst is added to carry out reaction, the solution after reaction is collected, chromatographic column chromatography separation, crystallization drying, the impurity of sodium cytidine phosphate with structure I is obtained.The raw material of the preparation method of the present application is environmental protection, process is simple, yield is high, fills the technical blank of the preparation of the impurity of sodium cytidine phosphate with structure I, and the research on the preparation method is conducive to guiding the optimization of cytidine phosphate sodium synthesis process route and ensuring cytidine phosphate sodium safe drug use.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to a preparation, separation and detection method and application of a citicoline sodium impurity with structure I. BACKGROUND

[0002] Citicoline sodium (English name: Citicoline sodium), chemical name: choline cytosine nucleoside diphosphate monosodium salt, is the most common pharmaceutical active form of citicoline in clinical application. Citicoline is an intermediate of endogenous synthesis of phosphatidylcholine and an important component of biological membrane construction. Citicoline can reduce cerebral vascular resistance, increase cerebral blood flow to promote brain metabolism, improve cerebral circulation, thereby promoting the recovery of brain function and consciousness awakening, and is widely used for acute craniocerebral trauma and postoperative consciousness disturbance. At present, the citicoline sodium preparations approved for marketing in China include injections, tablets and capsules.

[0003] Citicoline sodium is usually prepared by a synthetic method, using 5'-cytidylic acid and choline phosphate as starting materials, and the synthetic process route is as follows:

[0004]

[0005] In the process of commercial batch production of citicoline sodium, various impurities are produced. Some of the impurities are collected by the existing analysis method, but there are still some unknown impurities that are difficult to effectively separate and determine by the existing analysis method. For example, Chinese patent CN110174482A discloses a UPLC analysis method for simultaneously determining citicoline sodium and nine related substances, but the method only considers the compounds that may be produced in the reaction process, and does not consider the possible polymerization reaction of citicoline sodium molecules in the system in the later reaction stage. These possible polymers are difficult to effectively control by the existing analysis method for citicoline sodium due to their similar chemical properties to citicoline sodium, and may be mixed into the subsequent preparation as impurities. In order to improve the quality of citicoline sodium, it is necessary to fully study the impurities produced in the production of citicoline sodium, establish a rapid and accurate analysis method, and control them within the safety limit range to ensure the effectiveness and safety of citicoline and its preparations. SUMMARY

[0006] The technical problem to be solved by the present application is that the present application provides a preparation, separation and detection method and application of a citicoline sodium impurity with structure I, which can effectively prepare and detect the citicoline sodium impurity with structure I, and can be used for quality control of citicoline sodium and its preparation products, thereby laying a good foundation for the research of unknown impurities of citicoline sodium.

[0007] In order to solve the above technical problems, the technical scheme adopted by the present application is:

[0008] In a first aspect, the present application provides a preparation method of a sodium cytidine 5'-phosphocholine impurity with structure of formula I, comprising the following steps: mixing 5'-cytidylic acid, choline phosphate and sodium tert-butoxide in an aqueous solution to prepare a reaction solution, then adding a catalyst to react, collecting the solution after reaction, and separating and drying by column chromatography to obtain the sodium cytidine 5'-phosphocholine impurity with structure of formula I.

[0009] The structure of formula I is as follows:

[0010]

[0011] Formula I.

[0012] The preparation method of the sodium cytidine 5'-phosphocholine impurity with structure of formula I provided by the present application has the advantages of environmentally friendly raw materials, simple process, high yield, filling of the technical blank of preparation of the sodium cytidine 5'-phosphocholine impurity with structure of formula I, and the like. The research on the preparation method is conducive to guiding the optimization of the synthesis process route of the sodium cytidine 5'-phosphocholine and ensuring the safe use of the sodium cytidine 5'-phosphocholine. In combination with the synthesis route of the sodium cytidine 5'-phosphocholine, the generation of the impurity may be a condensation reaction between sodium cytidine 5'-phosphocholine molecules in the later stage of the reaction to form a dimer. After a large number of rigorous chemical researches, it is finally confirmed that the chemical structure of the impurity is as shown in formula I.

[0013] Optionally, the molar concentration of the 5'-cytidylic acid in the reaction solution is 50 mmol / L, the molar concentration of the choline phosphate is 400 mmol / L, the molar concentration of the sodium tert-butoxide is 20-50 mmol / L, the pH of the reaction solution is 6.5-8.0, the reaction time is 24-48 h, and the reaction temperature is 50°C.

[0014] Optionally, the catalyst is immobilized cytidine phosphate transferase, and the mass-volume ratio of the catalyst to the reaction solution is 50 g / L.

[0015] In a second aspect, the present application provides a separation and detection method of the sodium cytidine 5'-phosphocholine impurity with structure of formula I, comprising separating and detecting the sodium cytidine 5'-phosphocholine impurity with structure of formula I obtained by the above preparation method by using a high-performance liquid chromatography gradient method. The separation and detection conditions are as follows: a reverse-phase chromatographic column is used for chromatographic analysis, the stationary phase of the reverse-phase chromatographic column is hydrophilic octadecylsilane-bonded silica gel as a filler, the mobile phase is composed of mobile phase A and B, gradient elution is used, the mobile phase A is a phosphate buffer, the mobile phase B is a methanol solution, the column temperature is 30°C, the detection wavelength is 276 nm, the flow rate is 1 mL / min, and the injection amount is 10 μL.

[0016] The gradient elution conditions are as follows: 0 min, 96% of mobile phase A and 4% of mobile phase B, 5.5 min, 95% of mobile phase A and 5% of mobile phase B, 8 min, 96% of mobile phase A and 4% of mobile phase B.

[0017] The method for separating and detecting the impurity of sodium citicoline with the structure of formula I can effectively detect the impurity of sodium citicoline with the structure of formula I, and separate the impurity from the characteristic peak of sodium citicoline, which is beneficial to the quality control of sodium citicoline and further lays a foundation for the optimization of the production process of sodium citicoline.

[0018] Optionally, the phosphate buffer is a mixture of a potassium dihydrogen phosphate aqueous solution and a tetrabutylammonium aqueous solution in a volume ratio of 50:50, wherein the concentration of the potassium dihydrogen phosphate aqueous solution is 0.1 mol / L, the concentration of the tetrabutylammonium aqueous solution is 0.01 mol / L, and the pH of the phosphate buffer is 4.5.

[0019] In a third aspect, the application provides an application of the preparation method in the optimization of a synthesis method of sodium citicoline.

[0020] The application provides an application of the preparation method of the impurity of sodium citicoline with the structure of formula I in the optimization of a synthesis method of sodium citicoline, and the content of the impurity of sodium citicoline with the structure of formula I in a final product can be effectively reduced by controlling reaction conditions, so that the expected efficacy of sodium citicoline and a preparation thereof can be ensured.

[0021] In a fourth aspect, the application provides an application of the impurity of sodium citicoline with the structure of formula I obtained by the preparation method or the separating and detecting method in the quality control of sodium citicoline and a preparation thereof. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 A chromatogram of the impurity A of sodium citicoline obtained in Example 3;

[0023] Figure 2 A chromatogram of sodium citicoline obtained in Example 3;

[0024] Figure 3 A chromatogram of sodium citicoline obtained in Example 4;

[0025] Figure 4 A graph of weight change rate in a rat toxicity test obtained in Example 5. DETAILED DESCRIPTION

[0026] For better understanding of the above technical solutions, the above content of the present application is further explained in detail through specific embodiments, but this should not be understood as any limitation on the protection subject of the present application. Any technical solution realized based on the above content of the present application belongs to the scope of the present application.

[0027] The test methods in the following examples are all conventional methods, and the raw materials, reagents and materials used in the following examples are all commercially available products, unless otherwise specified.

[0028] The present application provides a preparation method of a cytidine sodium impurity with structure I, comprising the following steps: mixing 5'-cytidylic acid, choline phosphate and an aqueous solution of sodium tert-butoxide to prepare a reaction solution, then adding a catalyst for reaction, collecting the solution after reaction, and separating by chromatography column, crystallizing and drying to obtain the cytidine sodium impurity with structure I.

[0029] The structure I is as follows:

[0030]

[0031] Structure I.

[0032] The molar concentration of the 5'-cytidylic acid in the reaction solution is 50 mmol / L, the molar concentration of the choline phosphate is 400 mmol / L, the molar concentration of the sodium tert-butoxide is 20-50 mmol / L, the pH of the reaction solution is 6.5-8.0, the reaction time is 24-48 h, and the reaction temperature is 50℃.

[0033] The catalyst is immobilized cytidine phosphate transferase, and the mass-volume ratio of the catalyst to the reaction solution is 50 g / L.

[0034] In some preferred embodiments of the present application, the chromatography separation is specifically as follows: the solution after reaction is subjected to macroporous ion exchange chromatography column, then washed with pure water, eluted with 1% NaCl solution, and the eluate is collected.

[0035] The crystallization and drying are specifically as follows: the eluate collected after chromatography separation is vaporized and concentrated, the temperature is adjusted to 50℃, the impurities are removed by ultrafiltration with a molecular weight of 5000 kDa, the concentrated solution is collected, ethanol is added at room temperature and stirred for 3 h, until white crystals are precipitated, then the solution is left to stand for 8 h until the crystals are completely precipitated, the supernatant is removed by centrifugation, and the precipitate is vacuum dried to obtain the dry product of the cytidine sodium impurity with structure I.

[0036] The impurity of sodium citicoline with structure of formula I obtained by the preparation method is separated and detected by a high performance liquid chromatography gradient method, and the separation and detection conditions are as follows: a reversed phase chromatographic column is used for chromatographic analysis, the stationary phase of the reversed phase chromatographic column is hydrophilic octadecylsilane bonded silica gel as the filler, the mobile phase is composed of mobile phase A and B, gradient elution, wherein the mobile phase A is a phosphate buffer solution, the mobile phase B is a methanol solution, the column temperature is 30°C, the detection wavelength is 276 nm, the flow rate is 1 mL / min, and the injection amount is 10 μL.

[0037] The gradient elution conditions are as follows: at 0 min, the mobile phase A is 96% and the mobile phase B is 4%, at 5.5 min, the mobile phase A is 95% and the mobile phase B is 5%, and at 8 min, the mobile phase A is 96% and the mobile phase B is 4%.

[0038] The phosphate buffer solution is a mixture of potassium dihydrogen phosphate aqueous solution and tetrabutylammonium aqueous solution in a volume ratio of 50:50, wherein the concentration of the potassium dihydrogen phosphate aqueous solution is 0.1 mol / L, the concentration of the tetrabutylammonium aqueous solution is 0.01 mol / L, and the pH of the phosphate buffer solution is 4.5.

[0039] In some preferred embodiments, the separation and detection method further comprises a sample solution preparation method.

[0040] The embodiment of the present application also provides application of the above preparation method in optimization of a citicoline sodium synthesis method.

[0041] In some preferred embodiments, the optimized citicoline sodium synthesis method comprises the following steps:

[0042] (1) A reaction solution is prepared by mixing 5'-cytidylic acid 40 mmol / L, choline phosphate 300 mmol / L and magnesium acetate 50 mmol / L in water, the pH of the reaction solution is adjusted to 6.5-8.0, and then immobilized cytidylic acid phosphotransferase with a mass / volume ratio of 50 g / L is added, and the reaction is carried out at a reaction temperature of 50°C for 6-8 h;

[0043] (2) Sodium bicarbonate with a mass / volume ratio of 2 g / L is added 1 h before the end of the reaction, and the stirring is continued until the end of the reaction;

[0044] (3) The solution after the reaction is collected, and chromatographic column separation, crystallization and drying are performed to obtain citicoline sodium dry product.

[0045] In step (3), the difference between the chromatographic separation and the preparation method of the impurity of sodium citicoline with structure of formula I is that ethanol-NaOH is used instead of NaCl for elution.

[0046] The crystallization drying is different from the preparation method of the sodium citicoline impurity with the structure of formula I in that it further comprises secondary crystallization drying, that is, after the sodium citicoline dry product is obtained by preliminary crystallization separation, water is added for dissolution and the pH of the solution is adjusted to 6.5, and the above-mentioned vaporization concentration, impurity removal by filtration and crystallization drying steps are repeated, so that the sodium citicoline finished product is obtained.

[0047] The application also provides application of the sodium citicoline impurity with the structure of formula I obtained by the preparation method or the separation and detection method in quality control of sodium citicoline and a preparation thereof.

[0048] In the following examples, the sodium citicoline impurity with the structure of formula I is named as impurity A, and thus the embodiments of the application are described by taking the sodium citicoline impurity A as an example.

[0049] Embodiment 1

[0050] A preparation method of a sodium citicoline impurity A with the structure of formula I comprises the following steps: mixing 5'-cytidylic acid 50 mmol / L, choline phosphate 400 mmol / L and sodium tert-butoxide 50 mmol / L in an aqueous solution to prepare a reaction solution, adjusting the pH of the reaction solution to 6.5, adding a catalyst immobilized cytidine phosphotransferase with a mass / volume ratio of 50 g / L of the reaction solution, reacting at a temperature of 50℃ for 48 h, collecting the solution after reaction, performing chromatographic column separation, and crystallizing and drying, so that the sodium citicoline impurity A is obtained.

[0051] The chromatographic separation is specifically as follows: the solution after reaction is subjected to macroporous ion chromatography exchange column, and then washed by pure water and eluted by 1% NaCl solution, and the eluate is collected.

[0052] The crystallization drying is specifically as follows: the eluate collected by chromatographic separation is vaporized and concentrated, the temperature is adjusted to 50℃, impurities are removed by an ultrafilter with a molecular weight of 5000 kDa, the concentrated solution is collected, ethanol is added at room temperature and stirred for 3 h, until white crystals are precipitated, and then the solution is left to stand for 8 h until the crystallization is completely precipitated, the supernatant is removed by centrifugation, and the precipitate is vacuum dried, so that the dry product of the sodium citicoline impurity A is obtained.

[0053] Results: The yield of the sodium citicoline impurity A obtained in this embodiment is 31.68% calculated based on the input amount of 5'-cytidylic acid.

[0054] The obtained sodium citicoline impurity A is analyzed by nuclear magnetic resonance hydrogen spectrum, and the nuclear magnetic resonance hydrogen spectrum result is as follows:

[0055] 1H NMT: δ = 7.79 (2H), 6.02 (2H), 5.78 (1H), 5.76 (1H), 5.37 (4H), 4.43 (1H), 4.35 (1H), 4.31 (2H), 4.30 (1H), 4.08 (1H), 4.05 (4H), 4.00 (15H), 1.36 (3H).

[0056] Based on the synthesis process, the molecular formula of the impurity A is deduced as C 23 H 36 N7O 18 P3Na2, molecular weight is 837.479, and the chemical name is: P-({1-[(3S,4R)-3,4-dihydroxy-5-[(oxido{[oxido(oxo){[2-(trimethylammonium radical)ethyl]oxy}-lambda 5 -phosphono]oxy}(oxo)-lambda 5 -phosphono)methyl]tetrahydrofuran-2-yl]-2-oximido-pyrimidin-4-yl}amino)-P-({[(3S,4R)-5-(4-amino-2-oximido-pyrimidin-1-yl)-3,4-dihydroxytetrahydrofuran-2-yl]methyl}oxy)phosphinic acid disodium salt, and the chemical structure is shown as formula I:

[0057]

[0058] Formula I.

[0059] Example 2

[0060] The difference between this embodiment and example 1 is that the reaction conditions are different, and the specific adjustment and the yield of the dry product of cytidine sodium impurity A calculated based on the input amount of 5'-cytidine acid are recorded in table 1.

[0061] Table 1 Yield of cytidine sodium impurity A under different reaction conditions

[0062]

[0063] As can be seen from table 1, the pH of the reaction solution and the reaction time are the main factors affecting the yield of cytidine sodium impurity A. The smaller the pH value, the longer the reaction time, and the higher the yield of cytidine sodium impurity A.

[0064] Example 3

[0065] A separation and detection method of cytidine sodium impurity A with formula I structure, which separates and detects the cytidine sodium impurity A prepared in example 1 by high performance liquid chromatography gradient method, specifically:

[0066] (I) High performance liquid chromatography separation and detection conditions:

[0067] The chromatographic analysis is carried out by using a reversed-phase chromatographic column, the stationary phase of which is filled with hydrophilic octadecylsilane-bonded silica gel, the mobile phase is composed of mobile phase A and B, and gradient elution is used, wherein the mobile phase A is phosphate buffer (0.1 mol / L potassium dihydrogen phosphate aqueous solution and tetrabutylammonium aqueous solution (0.01 mol / L tetrabutylammonium hydroxide solution is adjusted to pH 4.5 with phosphoric acid) mixed in equal volumes), the mobile phase B is methanol solution, the column temperature is 30℃, the detection wavelength is 276 nm, the flow rate is 1 mL / min, and the injection amount is 10 μL;

[0068] The conditions of the gradient elution are as follows: at 0 min, the mobile phase A is 96% and the mobile phase B is 4%; at 5.5 min, the mobile phase A is 95% and the mobile phase B is 5%; at 8 min, the mobile phase A is 96% and the mobile phase B is 4%.

[0069] (II) Sample solution preparation method:

[0070] System suitability solution: an appropriate amount of 5'-cytidylic acid reference substance is accurately weighed, dissolved and quantitatively diluted with water to prepare a solution containing about 0.25 mg per 1 mL, and an appropriate amount of 5'-cytidylic acid reference substance solution is mixed with an equal amount of colfors sodium reference substance solution, and shaken well.

[0071] Colfors sodium reference substance solution: an appropriate amount of colfors sodium reference substance is accurately weighed, dissolved and quantitatively diluted with water to prepare a solution containing about 0.25 mg per 1 mL.

[0072] Colfors sodium impurity A solution: an appropriate amount of colfors sodium impurity A dry product prepared in Example 1 is accurately weighed, dissolved and quantitatively diluted with water to prepare a solution containing about 0.25 mg per 1 mL.

[0073] Colfors sodium solution: an appropriate amount of colfors sodium dry product prepared in Example 4 is accurately weighed, dissolved and quantitatively diluted with water to prepare a solution containing about 0.25 mg per 1 mL.

[0074] (III) High performance liquid chromatography determination:

[0075] The above solutions are accurately measured and injected into a high performance liquid chromatograph, respectively, and determined according to the separation and detection conditions, and the chromatogram is recorded (to 2.5 times the retention time of the main component peak). Figures 1-2 ) to 2.5 times the retention time of the main component peak.

[0076] (IV) Results:

[0077] 1. The injection detection results of the system suitability solution are shown in Table 2.

[0078] Table 2 Chromatographic results of system suitability solution

[0079]

[0080] 2. The chromatogram of the choline alfoscerate sodium impurity A solution is shown in the following figure: Figure 1

[0081] It can be seen from the above that the high performance liquid chromatography gradient method of the present application can effectively determine the choline alfoscerate sodium impurity A, and the peak time is 6.319 min (Rt=6.319 min). Figure 1 Figure 1

[0082] 3. The chromatogram of the choline alfoscerate sodium solution is shown in the following figure: Figure 2

[0083] It can be seen from the above that the detection method of the present application can effectively separate and determine choline alfoscerate sodium and choline alfoscerate sodium impurity A, and the peak times of choline alfoscerate sodium, choline alfoscerate sodium impurity A and 5'-cytidylic acid are 4.999 min, 6.323 min and 7.913 min (Rt=4.999 min, Rt=6.323 min and Rt=7.913 min) respectively. Figure 2 Figure 2 In addition, the above results further indicate that choline alfoscerate sodium impurity A is indeed produced in the synthesis process of choline alfoscerate sodium.

[0084] Example 4

[0085] The application of a preparation method of choline alfoscerate sodium impurity A with the structure of formula I in the optimization of the synthesis method of choline alfoscerate sodium is as follows:

[0086] (1) A reaction solution was prepared by mixing 5'-cytidylic acid 40 mmol / L, choline phosphate 300 mmol / L and magnesium acetate 50 mmol / L in water, the pH of the reaction solution was adjusted to 8.0, and then immobilized cytidine phosphotransferase with a mass / volume ratio of 50 g / L was added, and the reaction was carried out at a reaction temperature of 50℃ for 6 h;

[0087] (2) At 1 h before the end of the reaction, 2 g / L of stabilizer sodium bicarbonate was added, and the stirring was continued until the end of the reaction;

[0088] (3) The solution after reaction was collected, and chromatography column separation, crystallization and drying were carried out to obtain choline alfoscerate sodium dry product.

[0089] In step (3), the chromatography separation is specifically as follows: the solution after reaction is loaded on a macroporous ion chromatography exchange column, washed with pure water, then eluted with ethanol-NaOH solution, and the eluate is collected.

[0090] ​​​​​The crystallization drying is specifically as follows: the eluate collected after chromatographic separation is concentrated by vaporization, the temperature is adjusted to 50 DEG C, impurities are removed by an ultrafilter with a molecular weight of 5000 kDa, and the concentrated solution is collected; ethanol is added to the concentrated solution at room temperature, and stirred for 3 hours until white crystals are precipitated, and then the solution is left to stand for 8 hours until the crystallization is completely precipitated; the supernatant is removed by centrifugation, and the precipitate is dried under vacuum to obtain the crude product of sodium citicoline; water is further added to dissolve the product, and the pH of the solution is adjusted to 6.5, and the above-mentioned steps of vaporization concentration, filtration and crystallization drying are repeated to obtain the dried product of sodium citicoline.

[0091] The dried product of sodium citicoline obtained above is accurately weighed, dissolved in water, and quantitatively diluted to prepare a solution containing about 0.25 mg per 1 mL. The solution is detected by the high performance liquid chromatography gradient method of Example 3, and the results are recorded in Table 3. Figure 3 The content of sodium citicoline impurity A is calculated by the peak area ratio of sodium citicoline and sodium citicoline impurity A: (sodium citicoline impurity A peak area / sodium citicoline peak area)*100%, and the results are shown in Table 3.

[0092] Table 3 Chromatographic results of various components

[0093]

[0094] It is known from Example 2 that the reaction conditions are the main factors affecting the yield of sodium citicoline impurity A, therefore, in this example, by optimizing the control of the pH of the reaction solution to 8.0, the reaction time to 6 hours, and the addition of 2 g / L of stabilizer ammonium bicarbonate to the reaction solution, the content of sodium citicoline impurity A in the dried product of sodium citicoline can be reduced to 0.036%, thereby reducing the potential toxicity of sodium citicoline impurity A, and being beneficial to the quality control and safe use of sodium citicoline.

[0095] Example 5

[0096] Toxicological study of sodium citicoline impurity A prepared in Example 1: observe the toxic effects of sodium citicoline containing different contents of sodium citicoline impurity A on rats for 14 consecutive days, and the specific method is as follows:

[0097] 1. Experimental animals

[0098] Thirty 6-8 week old, 180-220 g SD male rats were selected and randomly divided into 3 groups, 10 rats in each group.

[0099] 2. Experimental grouping

[0100] According to the content of sodium citicoline impurity A, it is divided into low dose (0.05%), medium dose (0.10%) and high dose (0.25%) three groups.

[0101] 3. Administration method

[0102] Dosing method: oral gavage, the volume of administration is 10 mL / kg.

[0103] Dosing frequency: once a day, continuous administration for 14 days.

[0104] The dosing amount is shown in Table 4 below:

[0105] Table 4 Dosing amount design table

[0106]

[0107] Drug solution preparation: the cytidine choline sodium impurity A dry product prepared in Example 1 was precisely weighed, dissolved in water and quantitatively diluted to a solution of about 125 mg per 1 mL. The cytidine choline sodium solution was precisely weighed from commercially available cytidine choline sodium tablets, dissolved in water and quantitatively diluted to a solution of about 250 mg per 1 mL. Cytidine choline sodium solutions with cytidine choline sodium impurity A contents of 0.05%, 0.10% and 0.25% were prepared as sample solution for low, medium and high dose groups, respectively.

[0108] 4. Monitoring index

[0109] During the administration period, general observation, body weight examination and food intake examination were performed on rats in each group. After the end of administration, rats were euthanized for gross autopsy examination.

[0110] 4.1. General observation

[0111] After each administration, all animals in each group were observed for no less than 1 h. The observation contents included appearance signs, behavior activities, respiratory rate, etc.

[0112] 4.2. Body weight examination

[0113] The body weight of each rat in each group was measured before administration, and once every two days during the administration period. With 1 g as the minimum recording unit, the body weight change rate was recorded and calculated, and the body weight change rate = 100% * (body weight on the measurement day - initial test body weight) / initial test body weight.

[0114] 4.3. Food intake examination

[0115] On the feeding day, 300 g of rat feed was weighed for each group, and the remaining feed was weighed at the same time the next day. The average food intake of each rat in 24 h was recorded and calculated, and the average food intake = (feeding weight - remaining weight) / number of animals per group.

[0116] 4.4. Gross autopsy examination

[0117] After the end of administration, the rats in each group were euthanized and subjected to gross necropsy examination. The examination items included: surface and subcutaneous examination, neck examination, chest and thoracic organ examination, pelvic and pelvic organ examination, abdominal and abdominal organ examination, head examination, lymph examination, breast, spinal cord and sciatic nerve examination.

[0118] 5. Experimental results

[0119] 5.1. The general observation results are shown in Table 5. As shown in Table 5, no obvious abnormalities were observed in the control group and the low-dose group, the activity of the animals in the medium-dose group was reduced, and the activity of the animals in the high-dose group was significantly reduced.

[0120] Table 5: General observation record table of each group on the 14th day of administration

[0121]

[0122] 5.2. The body weight change rate of the rats is shown in Figure 4 . As shown in Figure 4 , the body weight of the control group increased slightly, the body weight of the low-dose group increased slightly, the body weight of the medium-dose group decreased, and the body weight of the high-dose group decreased significantly.

[0123] 5.3. The results of the change in the food intake of the rats are shown in Table 6. As shown in Table 6, the average daily food intake of the control group and the low-dose group showed no significant change and increased slightly with the growth of the rats; the average daily food intake of the medium-dose group decreased slightly; and the average daily food intake of the high-dose group decreased significantly.

[0124] Table 6: Examination table of the average daily food intake of rats in each group (g)

[0125]

[0126] 5.4. The results of the necropsy examination showed that no abnormalities were observed.

[0127] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application, and are not limiting; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that they can still modify the technical solutions described in the foregoing examples, or make equivalent substitutions for part or all of the technical features; and such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A process for the preparation of a sodium cytosine phosphate impurity having the structure of Formula I, ###00001### Formula I characterized in that, The preparation method comprises the following steps: mixing an aqueous solution of 5'-cytidylic acid, choline phosphate and sodium tert-butoxide to prepare a reaction solution, adding a catalyst to the reaction solution, collecting the reaction solution, and separating and drying the reaction solution through a chromatographic column to obtain the cytidine phosphate choline sodium impurity with the structure of formula I. ​ The structure of formula I is shown as follows: Formula I.

2. The production method according to claim 1, wherein The molar concentration of the 5'-cytidylic acid in the reaction solution is 50 mmol / L, the molar concentration of the choline phosphate is 400 mmol / L, the molar concentration of the sodium tert-butoxide is 20-50 mmol / L, the pH of the reaction solution is 6.5-8.0, the reaction time is 24-48 h, and the reaction temperature is 50 DEG C.

3. The production method according to claim 1, wherein The catalyst is immobilized cytidine phosphate transferase, and the mass-volume ratio of the catalyst to the reaction solution is 50 g / L.

4. A method for separating and detecting the impurity of sodium cytidine phosphate having the structure of formula I, characterized in that, The cytidine phosphate choline sodium impurity with the structure of formula I obtained by the preparation method of claim 1 is separated and detected by using a high performance liquid chromatography gradient method, and the separation and detection conditions are as follows: a reversed-phase chromatographic column is used for chromatographic analysis, the stationary phase of the reversed-phase chromatographic column is hydrophilic octadecylsilane bonded silica gel as a filler, the mobile phase is composed of mobile phase A and B, gradient elution is used, the mobile phase A is a phosphate buffer solution, the mobile phase B is a methanol solution, the column temperature is 30 DEG C, the detection wavelength is 276 nm, the flow rate is 1 mL / min, and the injection volume is 10 μL. The gradient elution conditions are as follows: at 0 min, the volume ratio of the mobile phase A to the mobile phase B is 96:4, at 5.5 min, the volume ratio of the mobile phase A to the mobile phase B is 95:5, and at 8 min, the volume ratio of the mobile phase A to the mobile phase B is 96:

4.

5. The separation and detection method according to claim 4, wherein The phosphate buffer solution is a mixture of a potassium dihydrogen phosphate aqueous solution and a tetrabutylammonium aqueous solution in a volume ratio of 50:50, the concentration of the potassium dihydrogen phosphate aqueous solution is 0.1 mol / L, the concentration of the tetrabutylammonium aqueous solution is 0.01 mol / L, and the pH of the phosphate buffer solution is 4.

5.

6. The preparation method of any one of claims 1-3 is used in the optimization of a cytidine phosphate choline sodium synthesis method.

7. The cytidine phosphate choline sodium impurity with the structure of formula I obtained by the preparation method of any one of claims 1-3 or the separation and detection method of any one of claims 4-5 is used in the quality control of cytidine phosphate choline sodium and its preparation.

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