阅读说明：本技术 一种地夸磷索四钠的含量及有关物质检测方法 (Method for detecting content of diquafosol tetrasodium and related substances ) 是由 刘小斌 胡俊 翟洪 于 2020-05-25 设计创作，主要内容包括：本发明公开了一种地夸磷索四钠的含量测定及有关物质检测方法,该方法主要采用高效液相色谱法对地夸磷索四钠原料药的含量,以及其有关物质进行了测定和检查。该方法省时省力,精密度高,含量测定结果准确,重复性和回收率良好,且该方法通过验证,可以用于地夸磷索四钠原料药和制剂样品的常规分析和质量控制。(The invention discloses a method for measuring the content of diquafosol tetrasodium and detecting related substances. The method is time-saving and labor-saving, has high precision, accurate content measurement result and good repeatability and recovery rate, and can be used for conventional analysis and quality control of the diquafosfomycin tetrasodium raw material medicine and preparation samples through verification.)

1. A method for measuring the content of diquafosol tetrasodium is characterized by comprising the following steps: adopts a reversed-phase high performance liquid chromatography method,

the mobile phase is acetonitrile; 70-90mmol/L potassium dihydrogen phosphate solution =40:60 (V/V) and pH adjusted with phosphoric acid = 6-7.

2. The method for determining the content of quartos phosphate tetrasodium according to claim 1, wherein: the mobile phase is acetonitrile: 80mmol/L potassium dihydrogen phosphate solution =40:60 (V/V) and pH adjusted with phosphoric acid = 6.60.

3. The method for determining the content of quartos tetrasodium according to claim 1 or 2, wherein: column chromatography was C8, 5 μm, 4.6 × 250mm (i.d.); the flow rate is 1.0 ml/min; the column temperature was 40 ℃ and the detection wavelength was 266 nm.

4. A method for measuring related substances of diquafosol tetrasodium is characterized by comprising the following steps: performing gradient elution by reversed-phase high performance liquid chromatography under the following mobile phase conditions: the mobile phase A is 70-90mmol/L potassium dihydrogen phosphate solution, and the pH is adjusted to 6-7 by phosphoric acid; the mobile phase B is acetonitrile or methanol; the gradient of the mobile phase was set as follows:

the method for determining diquafosol tetrasodium related substance according to claim 4, wherein: mobile phase a is 80mmol/L potassium dihydrogen phosphate solution, and pH =6.60 adjusted with phosphoric acid; mobile phase B was acetonitrile.

5. The method for determining diquafosol tetrasodium-related substance according to claim 4 or 5, wherein: column chromatography was C8, 5 μm, 4.6 × 250mm (i.d.); the flow rate is 1.0 ml/min; the column temperature was 40 ℃ and the detection wavelength was 266 nm.

Technical Field

The invention belongs to the technical field of drug analysis, and particularly relates to a quality control method of diquafosol tetrasodium bulk drug, in particular to a content determination method of quafosol tetrasodium and a related substance detection method.

Background

Diquafosol Tetrasodium, british name Diquafosol Tetrasodium, chemical name [ [ (2R,3R,4R,5R) -5- (2, 4-dioxomimetic-1-yl) -3,4-dihydroxy-oxolan-2-yl ] methoxy-hydroxy-phosphatyl ] oxy- [ [ [ (2R,3R,4R,5R) -5- (2, 4-dioxomimetic-1-yl) -3,4-dihydroxy-oxolan-2-yl ] methoxy-phosphatyl ] oxy-phosphatic acid. The 3% diquafosol tetrasodium eye drops are products (trade name: squas) developed by ginseng and instire pharmaceutical companies in combination for the treatment of dry eye. Imported and marketed in 2017 in 10 months.

Dry eye, also known as keratoconjunctival xerosis (KCS) or xerophthalmia, is a disease in which the quality and quantity of tears are abnormal, resulting in instability of tear film and damage to the ocular surface, which leads to ocular discomfort. The common symptoms of dry eye are dry eyes, burning pain and much eye droppings; soreness of the eyes, itching of the eyes, photophobia and impaired vision. Other symptoms include headache, restlessness, fatigue, difficulty in concentrating, keratomalacia and perforation in severe cases, and conjunctival congestion can be seen during examination. The following large populations are susceptible to dry eye:

1. dry eye is present in 75% of people over the age of 65. Since tear production in elderly people decreases with age. Decreased tear secretion leaves the eye vulnerable to irritation and sometimes causes a severe lacrimation reaction, i.e., epiphora.

2. A computer user. Staring at the fluorescent screen for a long time, there is a lack of normal blinking, which can cause tears to better wet the eye. More than 90% of people who work more than 3 hours before the computer each day have problems with their eyes.

3. Certain disease factors. Such as rheumatoid arthritis, diabetes, thyroid abnormality, asthma, cataract, glaucoma and lupus erythematosus.

4. A pharmaceutical factor. Certain drugs may reduce tear secretion, resulting in dry eye symptoms, such as antidepressants, antihistamines, diuretics, hypotensives, anesthetics, antiulcers, and the like.

5. A person wearing contact lenses. Contact lenses float on tears and wearing contact lenses accelerates tear evaporation. The discomfort and poor tolerance of contact lenses, mainly due to dry eye, especially soft contact lenses, can quickly evaporate tears, creating a foreign body sensation that causes protein to precipitate on the lens. On the other hand, dry eye can reduce the anti-injury ability of the cornea of a person wearing the contact lens, so that the contact lens can scratch the cornea more easily.

6. Environmental factors. Smoke, ultraviolet light, air pollution, high temperature, air conditioning and climatic drying all can increase tear evaporation, leading to dry eye.

7. Women in menopause, pregnancy, lactation and oral contraceptives. Dry eye symptoms may result from altered hormone levels.

8. Using certain eye drops for a long time. Such as vasoconstrictive eye drops, also can easily develop dry eye.

9. Meibomian gland dysfunction, punctal obstruction. Resulting in decreased tear secretion and drainage, leading to dry eye.

Diquafossol tetrasodium is the first P2Y2 receptor agonist eye drop approved on the market worldwide for the treatment of dry eye with a new mechanism of action: dry eye symptoms are ameliorated by promoting secretion of water and mucin, bringing the tear film closer to normal. In clinical studies in japan, no serious ocular and systemic adverse reactions were found. The diquafosol tetrasodium has good tolerance when being used, so the diquafosol tetrasodium can be used for treating dry eye symptoms for a long time.

At present, the diquafosol tetrasodium dosage form on the market is eye drops, but the content of the raw material medicine and the related substance analysis method are not reported in documents due to data protection and the like. In order to ensure the development and production quality of the diquafosol tetrasodium preparation, the content of a raw material medicament and related substances need to be controlled. Therefore, the research for obtaining the content determination of diquafosol tetrasodium and the detection method of related substances is very urgent for pharmaceutical manufacturers.

Disclosure of Invention

In view of the defects of the prior art, the inventor groves a quality control method of the diquafosol tetrasodium raw material and the preparation through a large amount of experiments, strictly performs method verification, ensures the scientific and rigorous method, and meets the requirements of research and development and production. Therefore, the invention aims to provide a method for measuring the content of diquafosol tetrasodium and detecting related substances thereof.

The purpose of the invention is realized as follows:

a method for measuring the content of diquafosol tetrasodium is characterized by comprising the following steps: adopting reversed-phase high performance liquid chromatography with acetonitrile as mobile phase; 70-90mmol/L potassium dihydrogen phosphate solution is 40:60(V/V), and pH is adjusted to 6-7 with phosphoric acid.

In the method for measuring the content of the quartos phosphate tetrasodium, the mobile phase is preferably acetonitrile: 80mmol/L potassium dihydrogen phosphate solution is 40:60(V/V) and pH is adjusted to 6.60 with phosphoric acid.

In the method for measuring the content of the tetraphosphato tetrasodium, a preferable chromatographic column is C8, 5 mu m, 4.6X 250mm (I.D.); the flow rate is 1.0 ml/min; the column temperature was 40 ℃ and the detection wavelength was 266 nm.

A method for measuring related substances of diquafosol tetrasodium adopts reversed-phase high performance liquid chromatography and carries out gradient elution according to the following mobile phase conditions: the mobile phase A is 70-90mmol/L potassium dihydrogen phosphate solution, and the pH value is adjusted to 6-7 by phosphoric acid; the mobile phase B is acetonitrile or methanol; the gradient of the mobile phase was set as follows:

time (min)	Mobile phase A	Mobile phase B
			0	80	20
15	80	20
			25	30	70
30	30	70
			45	80	20
55	80	20

In the method for measuring the diquafosol tetrasodium related substance, the mobile phase A is preferably 80mmol/L potassium dihydrogen phosphate solution, and the pH value is adjusted to 6.60 by phosphoric acid; mobile phase B was acetonitrile.

The method for detecting the relevant substances of the diquafosol tetrasodium comprises the following steps of carrying out chromatographic column chromatography on C8, 5 mu m and 4.6 multiplied by 250mm (I.D.); the flow rate is 1.0 ml/min; the column temperature was 40 ℃ and the detection wavelength was 266 nm.

The diquafosol tetrasodium bulk drug has an important role in the treatment of the xerophthalmia, but the content of the bulk drug and a related substance analysis method are not reported in documents. The method for measuring the content of the quarto fosfomycin tetrasodium and detecting related substances, disclosed by the invention, is time-saving and labor-saving, high in precision, accurate in content measurement result, good in repeatability and recovery rate, and can be used for conventional analysis and quality control of crude drugs and preparation samples of the quarto fosfomycin tetrasodium through verification. In addition, strict method verification proves that the requirements of research and development and production can be met, and necessary technical support is provided for the early marketing of the diquafosol tetrasodium medicament in China.

Drawings

FIG. 1 is a chromatogram for the system adaptability study of the method for determining the content of quarto fosfomycin tetrasodium according to example 1.

Detailed Description

Hereinafter, the method for measuring the content of diquafosol tetrasodium and the method for detecting a related substance according to the present invention will be described in further detail by way of examples, but it should be understood that the scope of the above subject matter of the present invention is not limited to the following examples, and any technique realized based on the above contents of the present invention falls within the scope of the present invention.

Example 1 reverse phase high performance liquid chromatography determination of Diquafosol tetrasodium content

Chromatographic conditions and system suitability: flow rate of 1.0ml/min at 266nm wavelength detected with octyl silica gel as filler (AgilentSB-C8, 5 μm, 4.6X 250 mm); the column temperature was 40 ℃. Mobile phase acetonitrile: 80mmol/L potassium dihydrogen phosphate solution is 40:60(V/V), and the pH is adjusted to 6.60 by phosphoric acid, and the number of theoretical plates should not be less than 3000.

Taking about 30mg of diquafosol tetrasodium sample, precisely weighing, placing in a 50ml volumetric flask, adding water to dissolve and diluting to a scale, and taking the solution as a test solution; taking a proper amount of diquafosol tetrasodium reference substance, preparing the diquafosol tetrasodium with the same method to obtain the diquafosol tetrasodium with the concentration of about 0.6mg/ml, taking the diquafosol tetrasodium as reference liquid, respectively measuring 20 mu L of each of the test sample and the reference liquid, injecting into a liquid chromatograph, recording a chromatogram, calculating by peak area according to an external standard method, and drying to obtain the product.

The following items of the content measurement method were verified:

1. system adaptability

And analyzing whether the chromatographic condition meets the requirement by using quartfossol tetrasodium. As can be seen from FIG. 1, under the condition, the separation degree of each impurity and the main peak meets the requirement, and the peak purity and the single-point threshold both meet the requirement.

2. Standard curve

Preparing the test samples with each concentration gradient, precisely measuring 100 mu l of the test samples, injecting the test samples into a chromatograph, and recording the chromatogram. The concentration (mg/ml) was plotted on the abscissa and the peak area (A) was plotted on the ordinate, and the regression coefficient was calculated. As can be seen from the results, the content of the product is determined that the diquafosol tetrasodium concentration has good linearity within the concentration range of 10.0-90.0 mg/ml.

3. Repeatability of

6 parts of samples are taken and precisely measured respectively, the content is measured according to a high performance liquid chromatography content measuring method, and the repeatability of the measuring method is inspected. The result shows that the repeatability of the high performance liquid chromatography for measuring the content of the product is good.

4. Recovery rate

Nine parts of 30mg of the product are taken in a 50ml volumetric flask, three concentration gradients of low, medium and high are marked, and three parts of 24mg, 30mg and 36mg of reference substances are respectively added. Measuring by high performance liquid chromatography, and calculating the measured amount and recovery rate. As can be seen from the results, the product has good recovery rate in content determination by high performance liquid chromatography.

5. Stability of solution

Get thisThe stability of the test solution for measuring the content of the product (batch number: DK-1) in the liquid phase method is examined at 0 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours and 24 hours. As can be seen from the results, the HPLC solution for content determination of this product was stable within 24 hours.

Time	Peak area of main peak
		0	11967188
2	11967888
		4	11966422
6	11965311
		8	11961355
12	11967488
		24	11967222
Mean value	11966125
		RSD(％)	0.02

6. Intermediate precision

The reproducibility results were taken as intermediate precision data set 1 and the additional test results were taken as intermediate precision data set 2 and data set 3. Inspecting the content of the product, and measuring the intermediate precision of high performance liquid chromatography. As can be seen from the results, the intermediate precision of the high performance liquid chromatography for measuring the content of the product is good.

Example 2 reversed-phase high performance liquid chromatography of diquafosol tetrasodium related substance to determine chromatographic conditions and system adaptability: the detection was carried out with octyl silica gel as filler (AgilentSB-C8, 5 μm, 4.6X 250mm)The flow rate is 1.0ml/min when the wavelength is 266 nm; the column temperature was 40 ℃. The mobile phase A is 80mmol/L potassium dihydrogen phosphate solution, and the pH value is adjusted to 6.60 by phosphoric acid; the mobile phase B is acetonitrile; the gradient of the mobile phase was set as follows:

time (min)	Mobile phase A	Mobile phase B
			0	80	20
15	80	20
			25	30	70
30	30	70
			45	80	20
55	80	20

The following items concerning the substance detection method were verified:

1. system adaptability

Under the chromatographic conditions determined above, a mixture of diquafosol tetrasodium starting material A (uridine 5 ' -sodium phosphate salt), diquafosol tetrasodium starting material B (free uridine 5 ' -phosphate), intermediate (uridine 5 ' -tri-n-butylamine phosphate salt), respectively, was then used to analyze whether the chromatographic conditions met the requirements. According to the atlas 2, the separation degree of each impurity from the main peak and impurities under the condition meets the requirement, and the peak purity and the single-point threshold value both meet the requirement.

2. Specificity

Taking a proper amount of the product, respectively destroying under each harsh condition, and inspecting the separation condition of the destroyed product and the main component peak.

Acid destruction: taking a proper amount of the product (about 30mg of diquafosol tetrasodium), putting the product into a 50ml measuring flask, adding 2ml of 4.0M hydrochloric acid solution, standing at room temperature for 6 hours, adjusting the pH to be neutral by using 4.0M sodium hydroxide solution, adding water to dilute to a scale, and shaking up uniformly to obtain the product.

Alkali destruction: taking a proper amount of the product (about 30mg of diquafosol tetrasodium), putting the product into a 50ml measuring flask, adding 2ml of 4.0M sodium hydroxide, standing at room temperature for 6 hours, adjusting the pH to be neutral by using 4.0M hydrochloric acid solution, adding water to dilute to a scale, and shaking up uniformly to obtain the product.

And (3) oxidative destruction: taking a proper amount of the product (about 30mg of diquafosol tetrasodium), putting the product into a 50ml measuring flask, adding 2ml of 3% hydrogen peroxide solution, standing at room temperature for 6 hours, adding water to dilute to scale, and shaking uniformly to obtain the product.

High-temperature destruction: taking a proper amount of the product (about 30mg of diquafosol tetrasodium), placing the product in a crucible, placing the crucible in a 100 ℃ oven for 6 hours, transferring the crucible to a 50ml measuring flask, adding water to dilute the product to a scale, and shaking the product uniformly to obtain the product.

And (3) light damage: taking a proper amount of the product (about 30mg of diquafosol tetrasodium), putting the product into a 50ml measuring flask, adding water to dilute the product to the scale, and shaking the product uniformly to obtain the medicine. The product is placed in an environment with 4500 +/-500 lx light for 2 days.

And (3) sampling samples under each damage condition according to the liquid phase condition of related substances, and recording a chromatogram, wherein the result shows that the product and impurity peaks generated under each damage condition can be well separated from the main component. The impurities generated by the product under various damage conditions have larger absorption near 266 nm.

3. Response values and standard curves of starting materials and intermediates

The initial raw material A, the initial raw material B, the intermediate and the diqua tetrasodium phosphate are taken to be prepared into test solutions (1), (2), (3), (4) and (5) with gradient concentrations respectively. And respectively sucking 20 mu l of each test solution, injecting into a chromatograph, and recording the chromatogram. The concentration (. mu.g/ml) was plotted on the abscissa and the peak area (A) was plotted on the ordinate to calculate the regression coefficient. As can be seen from the results, the product has good linearity with the intermediates of each step in the concentration range of 10.0-90.0. mu.g/ml.

Standard curve for low concentration of starting material A

Standard curve for low concentration of starting material B

Standard curve of low concentration of intermediate

Diqua tetrasodium phosphate low concentration standard curve

4. Stability of solution

Taking the test solution for testing related substances of the product, and examining the solution stability of the test solution for testing related substances of the product at 0, 2,4, 6, 8, 12 and 24 hours respectively. As can be seen from the results, the HPLC solution for content determination of this product was stable within 24 hours.

5. Durability

Since the chromatographic condition of the product is gradient elution and the corresponding type, temperature and wavelength of the chromatographic column are specified, the conditions are correspondingly finely adjusted to investigate the durability of the chromatographic condition.

As can be seen, the model of the chromatographic column is changed, the temperature of the chromatographic column is changed within the range of +/-5 ℃ and +/-2 nm of wavelength, the separation of impurities is better, and the durability is good.

6. Quantitative and detection limits

Taking a proper amount of the product reference substance, precisely weighing, adding water to prepare a diqua tetrasodium phosphate solution, precisely weighing a proper amount of the test solution, gradually diluting, and inspecting by sample injection, wherein the result shows that the quantitative limit of the product is 6.11ng/ml and the detection limit is 3.01 ng/ml.