阅读说明：本技术 一种腺苷晶型、其制备方法和用途 (Adenosine crystal form, preparation method and application thereof ) 是由 陈阳 张磊 付永慧 丁薇 李佳杨 于 2021-08-13 设计创作，主要内容包括：本发明提供了一种腺苷晶型、其制备方法及用途。该晶型使用Cu-Kα辐射、以2θ角度表示的X-射线粉末衍射图谱在5.5°±1、7.1°±1、13.5°±1、14.2°±1、14.8°±1、16.3°±1、18.3°±1和19.6°±1处具有衍射峰。该晶型具有稳定性良好,产品不易降解,能够满足生产、运输、储存的制药要求的优势,并且溶解速度快,显著缩短了制剂工艺时间。(The invention provides an adenosine crystal form, a preparation method and application thereof. The crystal form has diffraction peaks at 5.5 +/-1, 7.1 +/-1, 13.5 +/-1, 14.2 +/-1, 14.8 +/-1, 16.3 +/-1, 18.3 +/-1 and 19.6 +/-1 by using Cu-Kalpha radiation and an X-ray powder diffraction pattern expressed by a2 theta angle. The crystal form has the advantages of good stability, difficult degradation of products, capability of meeting the pharmaceutical requirements of production, transportation and storage, high dissolution speed and remarkable shortening of the preparation process time.)

1. A crystalline form of adenosine, characterized by: which has diffraction peaks at 5.5 ° ± 1, 7.1 ° ± 1, 13.5 ° ± 1, 14.2 ° ± 1, 14.8 ° ± 1, 16.3 ° ± 1, 18.3 ° ± 1 and 19.6 ° ± 1 using a Cu-K α radiation, X-ray powder diffraction pattern expressed in 2 θ angle.

2. The crystalline form of claim 1, characterized in that: an X-ray powder diffraction pattern using Cu-Ka radiation expressed in terms of 2 theta angle having diffraction peaks at 5.5 ° ± 1, 7.1 ° ± 1, 8.2 ° ± 1, 11.4 ° ± 1, 13.5 ° ± 1, 14.2 ° ± 1, 14.8 ° ± 1, 16.3 ° ± 1, 16.5 ° ± 1, 18.3 ° ± 1, 19.6 ° ± 1, 21.4 ° ± 1, 23.2 ° ± 1, 24.7 ° ± 1, 26.4 ° ± 1, 27.9 ° ± 1, 29.8 ° ± 1 and 36.2 ° ± 1;

preferably, it has an X-ray powder diffraction pattern using Cu-Ka radiation expressed in terms of 2 theta angles as shown in FIG. 1.

3. A crystalline form according to claim 1 or 2, characterized by a differential scanning calorimetry trace which has endothermic peaks at 54.2 ± 5 ℃, 110.3 ± 5 ℃, 235.7 ± 5 ℃ and an exothermic peak at 151.7 ± 5 ℃.

4. A crystalline form of adenosine according to any one of claims 1 to 3, wherein the moisture content of the crystalline form of adenosine is 8.0% to 15.0%.

5. A process for preparing a crystalline form according to any one of claims 1 to 4, characterized in that the process comprises: dissolving adenosine in a mixed solution of water and an organic solvent, and standing for crystallization.

6. The preparation method according to claim 5, wherein the volume of the mixed solution to adenosine weight ratio is 10-200 mL:1g, preferably 100-150 mL:1 g;

preferably, the volume ratio of water to the organic solvent in the mixed solution is 1: 0.1-10, preferably 1: 0.25-4.

7. The method according to claim 5 or 6, wherein the dissolving temperature is 30 to 85 ℃.

8. The method according to any one of claims 5 to 7, wherein the organic solvent is one or more selected from tetrahydrofuran, acetonitrile, methanol, ethanol, and acetone.

9. The method according to any one of claims 5 to 8, wherein the crystallization is carried out at a temperature of 0 to 30 ℃, preferably 15 to 30 ℃, under atmospheric pressure or vacuum.

10. The production method according to any one of claims 5 to 9, characterized by further comprising a step of filtering and drying the crystals after the crystallization.

11. A pharmaceutical composition comprising the crystalline form of adenosine according to any one of claims 1 to 4 or prepared by the method of any one of claims 5 to 10, and one or more pharmaceutically acceptable carriers.

12. Use of the crystalline form of adenosine according to any one of claims 1 to 4 or prepared by the method of any one of claims 5 to 10 in the manufacture of a medicament for the prevention and/or treatment of a cardiac disease; preferably, the heart disease is angina pectoris, cardiac infarction, myocardial ischemia or paroxysmal supraventricular tachycardia.

13. Use of the crystalline form of adenosine as claimed in any one of claims 1 to 4 or prepared by the method of any one of claims 5 to 10 in the manufacture of a medicament for the diagnosis of cardiovascular disease, assessment of sinoatrial node function, assessment of myocardial viability and prognostic assessment of coronary heart disease; preferably, the cardiovascular disease is tachycardia, recessive pre-excitation syndrome, coronary artery disease and Chronic Coronary Syndrome (CCS).

Technical Field

The invention belongs to the field of medicinal chemistry, and particularly relates to an adenosine crystal form, and a preparation method and application thereof.

Background

Adenosine (Ado, adenosine) is known as adenosine and is formed by the combination of adenine and pentose. The CAS number is 58-61-7, the chemical name is 6-amino-9-beta-D-ribofuranosyl-9H-purine, and the chemical structural formula is shown as formula I.

Adenosine, as an endogenous active substance, has wide application in clinical aspects such as coronary heart disease diagnosis and the like. Adenosine is both a precursor of adenine nucleotide and a metabolite thereof, a physiological regulator widely present in organisms. Adenosine can activate adenosine receptors (a receptors) to exert physiological and pharmacological effects. To date, 4 receptors for adenosine have been found, namely the a1, A2, A2b, A3 receptors. Al receptors in the heart are mainly distributed on the surfaces of atrial muscles, ventricular muscles, sinoatrial nodes and atrioventricular cells, adenosine and analogues thereof generate negative time-varying, force-varying and variable-conducting effects by exciting the Al receptors, and generate myocardial protection effects through a signal transduction mechanism behind the receptors. The A2 receptor is classified by its affinity for adenosine into the high affinity A2 receptor and the low affinity A2b receptor. Adenosine binds to the a2 receptor and increases cAMP, thereby dilating vascular smooth muscle. Adenosine binds to a3 receptor, increases the activity of superoxide dismutase (SOD) and glutathione enzymes, initiates the antioxidant system of the cell, and reduces myocardial damage. The activated a3 receptor, in turn, increases the release of mast cell mediators of allergy by promoting mast cell degranulation. The mechanism of protective action of adenosine on the myocardium may be: (1) adenosine promotes maintenance of microvascular blood flow; (2) adenosine inhibits neutrophil function; (3) adenosine can improve myocardial energy supply; (4) adenosine inhibits the production of oxygen radicals; (5) adenosine restores calcium balance; (6) adenosine promotes vascular repair, inhibits ventricular remodeling; (7) adenosine mediated ischemic pretreatment and ischemic posttreatment; (8) adenosine inhibits apoptosis of cardiomyocytes. The cardioprotective effects of adenosine are associated with agonism at the a1, a2 and A3 receptors.

Adenosine can be prepared by various routes, including chemical synthesis, biological fermentation, and isolation and purification from biological tissues. Patent CN1629178 discloses a method for preparing adenosine, which comprises obtaining needle-shaped, column-shaped or flake-shaped crystals by means of crystallization to improve the yield of adenosine preparation, but does not disclose specific crystal form information.

The concentration of the formulation of adenosine injection was 3mg/mL, but the solubility of adenosine in water was 5.1 mg/mL. Therefore, in the preparation concentration process, the adenosine solubility is close to the saturation concentration, so that the adenosine dissolution needs a longer time, and becomes one of the longest working procedures in the preparation process of the adenosine injection. And may leave a small undissolved adenosine product remaining during the dissolution process, thereby risking low levels of adenosine injection or unacceptable insoluble particles.

Therefore, when used as a raw material of an injection, it is necessary to develop an adenosine raw material which has good stability and a high dissolution rate, and can effectively prolong the effective period of the injection as a medicine, thereby shortening the preparation time and improving the safety of the injection.

Disclosure of Invention

The inventor of the invention inspects the crystalline products of adenosine obtained under different crystallization conditions, and carries out X-diffraction, infrared detection, thermogravimetric analysis and DSC detection on the obtained crystalline products, and finds that adenosine crystal form with good stability can be obtained under certain specific crystallization conditions.

The present invention provides a crystalline form of adenosine which has diffraction peaks at 5.5 ° ± 1, 7.1 ° ± 1, 13.5 ° ± 1, 14.2 ° ± 1, 14.8 ° ± 1, 16.3 ° ± 1, 18.3 ° ± 1 and 19.6 ° ± 1 on an X-ray powder diffraction pattern expressed in 2 θ degrees using Cu-K α radiation.

Preferably, said crystalline form of adenosine has an X-ray powder diffraction pattern, expressed in degrees 2 θ using Cu-Ka radiation, at 5.5 ° ± 1, 7.1 ° ± 1, 8.2 ° ± 1, 11.4 ° ± 1, 13.5 ° ± 1, 14.2 ° ± 1, 14.8 ° ± 1, 16.3 ° ± 1, 16.5 ° ± 1, 18.3 ° ± 1, 19.6 ° ± 1, 21.4 ° ± 1, 23.2 ° ± 1, 24.7 ° ± 1, 26.4 ° ± 1, 27.9 ° ± 1, 29.8 ° ± 1 and 36.2 ° ± 1, with diffraction peaks.

More preferably, the crystalline form of adenosine has an X-ray powder diffraction pattern expressed in degrees 2 θ using Cu-ka radiation as shown in figure 1.

The differential scanning calorimetry analysis curve of the adenosine crystal form has endothermic peaks at 54.2 +/-5 ℃, 110.3 +/-5 ℃, 235.7 +/-5 ℃ and exothermic peaks at 151.7 +/-5 ℃.

Preferably, the differential scanning calorimetry analysis curve of the adenosine crystal form is shown in figure 2.

Preferably, the thermogravimetric analysis curve of the adenosine crystalline form is shown in figure 3.

Preferably, the moisture content of the adenosine crystal form is 8.0-15.0%.

In another aspect, the present invention also provides a preparation method of the above adenosine crystal form, which comprises: dissolving adenosine in a mixed solution of water and an organic solvent, and standing for crystallization.

In the above preparation method, the volume ratio of the mixed solution to the weight of adenosine may be 10 to 200mL:1g, preferably 100 to 150mL:1 g.

In the preparation method, the volume ratio of the water to the organic solvent in the mixed solution may be 1: 0.1-10, preferably 1: 0.25-4.

In the preparation method, the dissolving temperature can be 30-85 ℃.

In the above preparation method, the organic solvent may be selected from one or more of tetrahydrofuran, acetonitrile, methanol, ethanol, or acetone.

In the above preparation method, the crystallization is performed at a temperature of 0 to 30 ℃, preferably 15 to 30 ℃ under atmospheric pressure or vacuum.

Preferably, the method further comprises the step of filtering and drying the crystals after crystallization.

In another aspect, the present invention also provides a pharmaceutical composition comprising the above crystalline form of adenosine or comprising the crystalline form of adenosine prepared by the above preparation method, and one or more pharmaceutically acceptable carriers, diluents or excipients.

On the other hand, the invention also provides application of the adenosine crystal form or the adenosine crystal form prepared by the preparation method in preparing a medicament for preventing and/or treating heart diseases.

Preferably, the heart disease is angina pectoris, cardiac infarction, myocardial ischemia or paroxysmal supraventricular tachycardia.

On the other hand, the invention also provides the application of the adenosine crystal form or the adenosine crystal form prepared by the preparation method in preparing a medicament for diagnosing cardiovascular diseases, evaluating the sinus node function, judging the myocardial activity and evaluating the prognosis of coronary heart diseases; preferably, the cardiovascular disease is tachycardia, recessive pre-excitation syndrome, coronary artery disease and Chronic Coronary Syndrome (CCS).

The invention has the following beneficial effects:

1. the prepared adenosine crystal form has good stability, the product is not easy to degrade, and the pharmaceutical requirements of production, transportation and storage can be met;

2. the product has high dissolution speed, obviously shortens the preparation process time, can avoid the generation of impurities in the preparation process, can reduce the risk of sample pollution and improves the safety of the preparation;

3. the new crystal form has good stability under the conditions of illumination, high temperature, high humidity, heating and the like, and the product is not easy to degrade;

4. the product has the advantages that the product has prolonged effective period as a raw material of the medicine, and can meet the pharmaceutical requirements of production, transportation and storage;

5. the production process is stable, repeatable and controllable, and can meet the requirement of industrial production.

Drawings

Specific embodiments of the present invention are illustrated in conjunction with the following figures:

FIG. 1 is an X-ray powder diffraction pattern of the crystalline form of adenosine prepared in example 1 of the present invention.

Figure 2 is a DSC profile of a crystalline form of adenosine prepared in example 1 of the present invention.

Figure 3 is a TGA profile of a crystalline form of adenosine prepared by example 1 of the present invention.

Fig. 4 is a DVS profile of a crystalline form of adenosine prepared in example 1 of the present invention.

Fig. 5 is a graph comparing X-ray powder diffraction patterns before and after DVS testing of the adenosine crystalline form prepared in example 1 of the present invention.

FIG. 6 is an X-ray powder diffraction pattern of the adenosine crystal form prepared in example 1 of the present invention after 30 days of examination at 60 ℃.

FIG. 7 is an X-ray powder diffraction pattern of the adenosine crystalline form prepared in example 1 of the present invention after 30 days of illumination.

FIG. 8 is an X-ray powder diffraction pattern of the crystalline form of adenosine prepared in example 1 of the present invention after 6 months of investigation under accelerated conditions (40 ℃, RH 75%).

Figure 9 is a TGA profile of a crystalline form of adenosine prepared by the method disclosed in patent CN 1629178.

Fig. 10 is an HPLC chromatogram of a prepared solution after the adenosine crystal form prepared in example 1 of the present invention was completely dissolved.

FIG. 11 is an HPLC chromatogram of a prepared solution of the crystal form obtained in patent CN1629178A at 60min in the preparation process.

FIG. 12 is an HPLC chromatogram of a prepared solution after all the crystal form obtained in patent CN1629178A is dissolved (120 min).

Detailed Description

The present invention will be further described with reference to the following examples so that those skilled in the art can more fully understand the present invention, but the examples themselves are not intended to limit the present invention in any way.

Powder X-ray diffraction (XRPD) data of the adenosine crystalline forms of the invention were measured by bruker X-ray diffractometer under the following test conditions:

ray: monochromatic Cu — K α rays (λ ═ 1.5406);

the scanning mode is as follows: θ/2 θ, scan range: 5-40 degrees;

voltage: 45KV, current: 40 mA;

DSC and TGA test conditions are as follows:

parameter(s)	TAG	DSC
			Method	Linear temperature rise	Linear temperature rise
Sample plate	Aluminum dish, open mouth	Aluminium pan, gland/non-gland
			Temperature range	Room temperature to set end point temperature	25 ℃ to a set end point temperature
Scanning speed (. degree. C./min)	10	10
			Protective gas	Nitrogen gas	Nitrogen gas

DVS test conditions were:

example 1 preparation of crystalline forms of adenosine

Adenosine 10g was dissolved in 1200mL of an aqueous acetonitrile solution (acetonitrile/water: 1/1) at 85 ℃, and the solution was naturally evaporated under vacuum at 20 ℃ to precipitate crystals, which were then filtered and dried to obtain adenosine 8.9g with a purity of 99.7%. FIG. 1 is an X-ray powder diffraction (XRPD) pattern for the crystalline form having diffraction peaks at 5.5 ° ± 1, 7.1 ° ± 1, 8.2 ° ± 1, 11.4 ° ± 1, 13.5 ° ± 1, 14.2 ° ± 1, 14.8 ° ± 1, 16.3 ° ± 1, 16.5 ° ± 1, 18.3 ° ± 1, 19.6 ° ± 1, 21.4 ° ± 1, 23.2 ° ± 1, 24.7 ° ± 1, 26.4 ° ± 1, 27.9 ° ± 1, 29.8 ° ± 1 and 36.2 ° ± 1. FIG. 2 shows a DSC spectrum of the crystalline form, which has endothermic peaks at 54.2 + -5 deg.C, 110.3 + -5 deg.C, 235.7 + -5 deg.C, and an exothermic peak at 151.7 + -5 deg.C. This crystalline form is defined as a new crystalline form of adenosine. Figure 3 shows a TGA profile of the crystalline form, and it can be seen that the moisture content of the adenosine crystals is 12.1%.

Example 2 preparation of crystalline forms of adenosine

10g of adenosine was dissolved in 2000mL of tetrahydrofuran aqueous solution (tetrahydrofuran/water: 10/1) at 30 ℃, and the solution was naturally volatilized under vacuum at 30 ℃ to precipitate crystals, which were then filtered and dried to obtain 8.8g of adenosine with a purity of 99.8%. The X-ray diffraction pattern of the obtained crystal sample is determined to be consistent with the crystal form obtained in example 1 through research and comparison. The water content of the adenosine crystals was 8.0%.

Example 3 preparation of crystalline forms of adenosine

Dissolving adenosine 10g in 100mL aqueous solution of methanol and acetone (methanol/acetone/water: 4/1/50) at 50 deg.C, standing at 15 deg.C for natural volatilization to precipitate crystal, filtering, and drying to obtain adenosine 9.0g with purity of 99.9%. The X-ray diffraction pattern of the obtained crystal sample is determined to be consistent with the crystal form obtained in example 1 through research and comparison. The water content of the adenosine crystals was 15.0%.

Example 4 preparation of crystalline forms of adenosine

Dissolving 10g adenosine in 1000mL ethanol water solution (ethanol/water: 1/4) at 40 deg.C, naturally volatilizing under vacuum condition at 25 deg.C to precipitate crystal, filtering, and drying to obtain 8.5g adenosine with purity of 99.9%. The X-ray diffraction pattern of the obtained crystal sample is determined to be consistent with the crystal form obtained in example 1 through research and comparison. The water content of the adenosine crystals was 10.0%.

Example 5 preparation of crystalline forms of adenosine

Dissolving adenosine 10g in 1500mL aqueous solution of acetone and ethanol (acetone/ethanol/water: 2/2/1) at 30 deg.C, standing at 0 deg.C for natural volatilization to precipitate crystal, filtering, and drying to obtain adenosine 7.8g with purity of 99.8%. The X-ray diffraction pattern of the obtained crystal sample is determined to be consistent with the crystal form obtained in example 1 through research and comparison. The water content of the adenosine crystals was 14.0%.

Example 6 adenosine Crystal TGA experiment

The TGA test conditions were:

parameter(s)	TAG
		Method	Linear temperature rise
Sample plate	Aluminum dish, open mouth
		Temperature range	Room temperature to set end point temperature
Scanning speed (. degree. C./min)	10
		Protective gas	Nitrogen gas

TGA measurements were carried out on the samples of example 1 and on the adenosine powder obtained by the method of patent CN1629178A, respectively. The results are shown in fig. 3 and 9.

The result shows that the sample of example 1 of the present invention is a hydrate, the moisture content is 12.1%, while the sample prepared in patent CN1629178A has no weight loss before 200 ℃, the weight loss begins only around 260 ℃, and the temperature is the degradation temperature of adenosine, which indicates that the weight loss is caused by degradation of adenosine rather than water loss, so the adenosine powder is judged to be anhydrous.

EXAMPLE 7 adenosine Crystal DVS experiment

The sample of example 1 was selected for hygroscopicity assessment and the results are shown in fig. 4, where the weight change of the adenosine crystal form is small between 20% RH and 100% RH, and only under 10% RH, significant weight loss occurs, but with increasing humidity, the lost water molecules can recombine with adenosine, thus maintaining the stability of the crystal form. And as shown in figure 5, the adenosine crystal forms were unchanged by comparing XRPD patterns before and after DVS experiments. The crystal form is a stable crystal form and has better stability.

Example 8 adenosine Crystal Effect experiment

The adenosine crystal form prepared in example 1 was uniformly spread in an open petri dish with a thickness of about 5mm, and placed under high temperature (60 ℃) and illumination (4500lx ± 500lx) conditions, respectively, for sampling detection for 5 days, 10 days and 30 days, and compared with the results of 0 day, and the results are shown in tables 1 and 2. Wherein, the X-ray powder diffraction pattern of the adenosine crystal form at 60 ℃ after 30 days is shown in figure 6, and the X-ray powder diffraction pattern of the adenosine crystal form after 30 days of illumination is shown in figure 7.

TABLE 1 high temperature stability test results for adenosine crystal forms

Time (sky)	Appearance of the product	Purity (%)
			0	White powder	99.7％
5	White powder	99.7％
			10	White powder	99.7％
30	White powder	99.7％

TABLE 2 adenosine Crystal light stability test results

Time (sky)	Appearance of the product	Purity (%)
			0	White powder	99.7％
5	White powder	99.7％
			10	White powder	99.7％
30	White powder	99.7％

And (4) test conclusion: the crystal of the invention has good stability under high temperature and illumination, and can keep stable appearance and purity within 30 days.

Example 9 accelerated adenosine stability test

The packaged adenosine crystal form prepared in example 1 was placed in a stability box at 40 ℃ and RH 75% for six months, sampled at the end of 3 and 6 months, and compared with the results of 0 month. The results are shown in Table 3. The X-ray powder diffractogram of the 6 month sample is shown in figure 8.

TABLE 3 accelerated stability test results for adenosine crystals

Time (moon)	Appearance of the product	Purity (%)
			0	White powder	99.7％
3	White powder	99.7％
			6	White powder	99.6％

And (4) test conclusion: the crystal of the invention has good stability under the conditions of 40 ℃ and RH 75%, and can keep stable appearance shape and purity within 6 months.

Example 10 dissolution Rate experiments for adenosine crystalline forms

The adenosine crystal form (prepared in example 1) of the present invention and the adenosine powder obtained by the method of patent CN1629178A were dissolved in normal saline, respectively, the concentration process was simulated, and the dissolution rates were compared, and the results are shown in table 4:

TABLE 4 adenosine Crystal dissolution Rate test results

The experimental results show that: the dissolution speed of the adenosine crystal prepared in the embodiment 1 of the invention is obviously higher than that of the adenosine powder obtained by the method of the patent CN1629178A, thereby obviously shortening the production working hours of the preparation.

Example 11 dissolution rate experiment of adenosine crystal form at different temperatures

The adenosine crystal form of the present invention (prepared by example 1) and the adenosine powder obtained by the method of patent CN1629178A were dissolved in physiological saline at different temperatures, respectively, the concentration process was simulated, and the dissolution rates were compared, and the results are shown in table 5:

TABLE 5 adenosine Crystal dissolution Rate test results

The experimental results show that: the dissolution speed of the adenosine crystal prepared in the embodiment 1 of the invention is obviously higher than that of the adenosine powder obtained by the method of the patent CN1629178A at different temperatures, and particularly, the advantage of the dissolution speed is more obvious compared with that of the adenosine powder obtained by the method of the patent CN1629178A at the temperature of 20 ℃ close to room temperature, which shows that the solubility of the adenosine crystal prepared by the invention is better, so that the production man-hour of a preparation can be obviously shortened, the generation of impurities in the preparation process can be avoided, and the risk of sample pollution can be reduced.

Example 12 stability test of adenosine Crystal form preparation solution

The adenosine crystal form (prepared from example 1) of the present invention and the adenosine powder obtained by the method of patent CN1629178A were dissolved in physiological saline, respectively, to simulate the concentration process. Specific experimental parameters are shown in table 6.

After the adenosine crystal form is completely dissolved (5min), related substances are measured; the adenosine powder obtained by patent CN1629178A is used for measuring related substances at 60min and after complete dissolution (120min) in the preparation process.

The related substance determination method adopts adenosine related substance determination method in Chinese pharmacopoeia 2020 edition. Fig. 10 is an HPLC chromatogram of the prepared solution after the adenosine crystal form of the present invention is completely dissolved, and fig. 11 and 12 are HPLC chromatograms of the prepared solution at 60min and after (120min) the crystal form obtained in patent CN1629178A is completely dissolved in the preparation process, respectively.

TABLE 6 simulation of specific experimental parameters of the process of the concentrated blending

And (4) conclusion: after the adenosine crystal form is completely dissolved in normal saline, related substances in a sample solution are detected, an HPLC chromatogram is shown in figure 10, the content of other impurities except adenosine is extremely low, and the purity of the sample solution is high. Sampling the adenosine powder obtained by the method disclosed in patent CN1629178A at 60min in the preparation process, detecting related substances in the sample solution, and increasing the content of other impurities besides adenosine as shown in an HPLC chromatogram as shown in figure 11; after the adenosine powder is completely dissolved in the physiological saline (120min), relevant substances in the sample solution are detected, an HPLC chromatogram is shown in figure 12, except adenosine, other impurities such as adenine are obviously increased, and the purity of the sample solution is influenced.

From the above, it is known that, in a long-term formulation process, the contact time of the adenosine raw material with the external environment is increased, which may cause degradation of the raw material and increase of the impurity content. Therefore, the preparation time of the adenosine solution is an important factor influencing the quality of subsequent preparations in the test and production processes.

Although the present invention has been described to a certain extent, it is apparent that appropriate changes in the respective conditions may be made without departing from the spirit and scope of the present invention. It is to be understood that the invention is not limited to the described embodiments, but is to be accorded the scope consistent with the claims, including equivalents of each element described.