A method for detecting the content of diquafosol tetrasodium and related substances

Abstract

The invention discloses a method for determining the content of diquafosol tetrasodium and detecting related substances, wherein the method mainly adopts high performance liquid chromatography to determine and inspect the content of diquafosol tetrasodium raw material medicine and its related substances. The method is time-saving and labor-saving, has high precision, accurate content determination results, good repeatability and recovery rate, and the method is verified and can be used for routine analysis and quality control of diquafosol tetrasodium raw material medicine and preparation samples.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and specifically relates to a quality control method for a diquafosol tetrasodium raw material drug, and in particular to a content determination method of diquafosol tetrasodium and a related substance detection method. Background Art

[0002] Diquafosol Tetrasodium, English name Diquafosol Tetrasodium, chemical name [[(2R,3R,4R,5R)-5-(2,4-dioxopyrimidin-1-yl)-3,4-dihydroxy-oxolan-2-yl]methoxy-hy droxy-phosphoryl]oxy-[[[(2R,3R,4R,5R)-5-(2,4-dioxopyrimidin-1-yl)-3,4-dihydroxy-oxolan-2-yl]methoxy-hydroxy-phosphoryl]oxy-hydroxy-phosphoryl]oxy-phosphinicacid. 3% diquafosol tetrasodium eye drops are a product jointly developed by Santen and Inspire Pharmaceuticals (trade name: Diquas) for the treatment of dry eye disease. It was imported and launched in China in October 2017.

[0003] Dry eye, also known as keratoconjunctivitis sicca (KCS) or dry eye, refers to a type of disease caused by abnormalities in the quality and quantity of tears, which leads to unstable tear film and damage to the surface of the eye, thus causing eye discomfort. The general symptoms of dry eye are dry and burning eyes, a lot of eye mucus, sore eyes, itchy eyes, fear of light, and decreased vision. Other symptoms include headaches, irritability, fatigue, difficulty concentrating, and in severe cases, corneal softening and perforation may occur. Conjunctival congestion may be seen during examination. The following groups of people are prone to dry eye:

[0004] 1. 75% of people over the age of 65 suffer from dry eyes. This is because the production of tears in the elderly decreases with age. Reduced tear secretion makes the eyes susceptible to irritation, sometimes causing a violent tearing reaction, i.e., epiphora.

[0005] 2. Computer users. Staring at the screen for a long time, they lack normal blinking, which can make tears moisten the eyes better. Among those who work in front of the computer for more than 3 hours a day, more than 90% have eye problems.

[0006] 3. Certain disease factors, such as rheumatoid arthritis, diabetes, thyroid abnormalities, asthma, cataracts, glaucoma and lupus erythematosus.

[0007] 4. Drug factors. Certain drugs can reduce tear secretion and lead to dry eye symptoms, such as antidepressants, antihistamines, diuretics, antihypertensive drugs, anesthetics, anti-ulcer drugs, etc.

[0008] 5. People who wear contact lenses. Contact lenses float on tears, and wearing them will accelerate the evaporation of tears. The discomfort and poor tolerance of wearing contact lenses are mainly caused by dry eyes, especially soft contact lenses, which can quickly evaporate tears, produce a foreign body sensation, and cause protein to precipitate on the lenses. On the other hand, dry eyes can reduce the damage resistance of the cornea of ​​people who wear contact lenses, making contact lenses more likely to scratch the cornea.

[0009] 6. Environmental factors: Smoke, ultraviolet rays, air pollution, high temperature, air conditioning and dry climate can increase the evaporation of tears and cause dry eyes.

[0010] 7. Women in menopause, pregnancy, lactation and taking oral contraceptives. Changes in hormone levels may cause dry eye symptoms.

[0011] 8. Long-term use of certain eye drops, such as vasoconstrictive eye drops, can also easily lead to dry eyes.

[0012] 9. Meibomian gland dysfunction and lacrimal puncta obstruction reduce tear secretion and discharge, leading to dry eyes.

[0013] Diquafosol tetrasodium is the first P2Y2 receptor agonist eye drop approved for marketing worldwide. It treats dry eye disease with a new mechanism of action: improving dry eye disease symptoms by promoting water and mucin secretion, making the tear film closer to a normal state. In clinical studies in Japan, no serious ocular and systemic adverse reactions were found. Diquafosol tetrasodium is well tolerated and can be used for long-term treatment of dry eye disease symptoms.

[0014] At present, the marketed diquafosol tetrasodium dosage form is eye drops, but due to data protection and other reasons, there is no literature report on the content of the raw material and the analysis method of related substances. In order to ensure the quality of the research and development and production of diquafosol tetrasodium preparations, it is necessary to control the content of the raw material and related substances. Therefore, it is particularly urgent for pharmaceutical manufacturers to study and obtain a method for determining the content of diquafosol tetrasodium and detecting related substances. Summary of the invention

[0015] In view of the deficiencies in the prior art, the inventors have explored the quality control method of diquafosol tetrasodium raw materials and preparations through a large number of experiments, and strictly verified the method to ensure the scientific rigor of the method and meet the needs of research and development and production. Therefore, the object of the present invention is to provide a method for determining the content of diquafosol tetrasodium and its related substance detection method.

[0016] The object of the present invention is achieved in that:

[0017] A method for determining the content of diquafosol tetrasodium is characterized by adopting reverse phase high performance liquid chromatography, wherein the mobile phase is acetonitrile: 70-90 mmol / L potassium dihydrogen phosphate solution = 40:60 (V / V), and phosphoric acid is used to adjust the pH to 6-7.

[0018] In the above method for determining the content of tetrasodium quafosol, the preferred mobile phase is acetonitrile: 80 mmol / L potassium dihydrogen phosphate solution = 40:60 (V / V), and the pH is adjusted to 6.60 with phosphoric acid.

[0019] The above method for determining the content of tetrasodium quafosol is preferably a chromatographic column of C8, 5 μm, 4.6×250 mm (ID); a flow rate of 1.0 ml / min; a column temperature of 40° C., and a detection wavelength of 266 nm.

[0020] A method for determining related substances of diquafosol tetrasodium, using reverse phase high performance liquid chromatography, gradient elution according to the following mobile phase conditions: mobile phase A is 70-90mmol / L potassium dihydrogen phosphate solution, and the pH is adjusted to 6-7 with phosphoric acid; mobile phase B is acetonitrile or methanol; the gradient of the mobile phase is set as follows:

[0021] 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

[0022] In the above method for determining the related substances of diquafosol tetrasodium, it is preferred that the mobile phase A is 80 mmol / L potassium dihydrogen phosphate solution, and the pH is adjusted to 6.60 with phosphoric acid; and the mobile phase B is acetonitrile.

[0023] The above method for detecting diquafosol tetrasodium related substances is as follows: the chromatographic column is C8, 5μm, 4.6×250mm (ID); the flow rate is 1.0ml / min; the column temperature is 40°C, and the detection wavelength is 266nm.

[0024] The diquafosol tetrasodium raw material plays an important role in the treatment of dry eye, but there is no literature report on the content of the raw material and the related substance analysis method. The content determination of diquafosol tetrasodium and the related substance detection method of the present invention save time and effort, have high precision, accurate content determination results, good repeatability and recovery, and the method can be used for the conventional analysis and quality control of diquafosol tetrasodium raw material and preparation samples through verification. In addition, through strict method verification, the needs of research and development and production can be met, and necessary technical support is provided for the early listing of diquafosol tetrasodium medicine in China. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Chromatogram of system suitability study of the method for determining the content of diquafosol tetrasodium in Example 1. DETAILED DESCRIPTION

[0026] The following is a further detailed description of the determination of diquafosol tetrasodium content and the related substance detection method involved in the present invention in the form of embodiments, but this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples, and all technologies realized based on the above content of the present invention belong to the scope of the present invention.

[0027] Example 1 Determination of the content of diquafosol tetrasodium by reversed phase high performance liquid chromatography

[0028] Chromatographic conditions and system suitability: Octyl silica gel as filler (AgilentSB-C8, 5μm, 4.6×250mm), detection wavelength at 266nm, flow rate at 1.0ml / min, column temperature at 40°C. Mobile phase acetonitrile: 80mmol / L potassium dihydrogen phosphate solution = 40:60 (V / V), and adjusted to pH = 6.60 with phosphoric acid, the theoretical plate number should be not less than 3000.

[0029] Take about 30 mg of diquafosol tetrasodium sample, accurately weigh it, put it in a 50 ml volumetric flask, add water to dissolve and dilute to the scale as the test solution; take an appropriate amount of diquafosol tetrasodium reference substance, and prepare it in the same way to make the concentration of diquafosol tetrasodium about 0.6 mg / ml as the reference solution, measure 20 μL of the test sample and the reference solution respectively, inject them into the liquid chromatograph, record the chromatogram, calculate the peak area according to the external standard method and dry it to obtain the result.

[0030] The following items of the assay method were validated:

[0031] 1. System adaptability

[0032] Diquafosol tetrasodium was used to analyze whether the chromatographic conditions met the requirements. Figure 1 It can be seen that under this condition, the separation degree between each impurity and the main peak meets the requirements, and the peak purity and single point threshold both meet the requirements.

[0033] 2. Standard curve

[0034] Prepare the test sample of each concentration gradient, accurately measure 100μl respectively, inject into the chromatograph, and record the chromatogram. With the concentration (mg / ml) as the horizontal axis and the peak area (A) as the vertical axis, draw a regression curve and calculate the regression coefficient. From the results, it can be seen that the content determination of this product has good linearity in the concentration range of 10.0-90.0mg / ml of diquafosol tetrasodium.

[0035]

[0036] 3. Repeatability

[0037] Six samples were taken and accurately determined respectively, and the repeatability of the determination method was investigated by the HPLC method. The results show that the repeatability of the HPLC method for the determination of the content of this product is good.

[0038]

[0039] 4. Recovery rate

[0040] Take nine portions of 30 mg of this product in a 50 ml volumetric flask, mark them as low, medium and high concentration gradients, add three portions of 24 mg, 30 mg and 36 mg of reference substances respectively. Determine by HPLC for content determination, calculate the measured amount and recovery rate. As can be seen from the results, the recovery rate of this product by HPLC content determination is good.

[0041]

[0042] 5. Solution stability

[0043] The test solution of this product (batch number: DK-1) was taken for content determination, and the stability of the solution of the liquid phase method for content determination of this product was investigated at 0, 2, 4, 6, 8, 12, and 24 hours. The results show that the solution of the HPLC method for content determination of this product is stable within 24 hours.

[0044]

[0045]

[0046] 6. Intermediate precision

[0047] The repeatability results are intermediate precision data set 1, and the other test results are intermediate precision data set 2 and data set 3. The intermediate precision of the high performance liquid chromatography method for the content determination of this product was investigated. From the results, it can be seen that the intermediate precision of the high performance liquid chromatography method for the content determination of this product is good.

[0048]

[0049] Example 2 Reverse phase high performance liquid chromatography determination of related substances of diquafosol tetrasodium Chromatographic conditions and system suitability: Octyl silica gel as filler (AgilentSB-C8, 5μm, 4.6×250mm), detection wavelength of 266nm, flow rate of 1.0ml / min; column temperature of 40°C. Mobile phase A is 80mmol / L potassium dihydrogen phosphate solution, and phosphoric acid is used to adjust pH=6.60; mobile phase B is acetonitrile; the gradient of the mobile phase is set as follows:

[0050] 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

[0051] The following items of the substance detection method were verified:

[0052] 1. System adaptability

[0053] Under the above determined chromatographic conditions, the mixture of diquafosol tetrasodium starting material A (uridine 5'-phosphate sodium salt), diquafosol tetrasodium starting material B (free uridine 5'-phosphate), and intermediate (uridine 5'-phosphate tri-n-butylamine salt) was used to analyze whether the chromatographic conditions met the requirements. Under this condition, the separation degree between each impurity and the main peak and between the impurities met the requirements, and the peak purity and single point threshold value all met the requirements.

[0054] 2. Exclusivity

[0055] Take an appropriate amount of this product and destroy it under various harsh conditions to examine the separation of the destruction product and the main component peak.

[0056] Acid destruction: Take an appropriate amount of this product (containing about 30 mg of diquafosol tetrasodium), place it in a 50 ml volumetric flask, add 2 ml of 4.0 M hydrochloric acid solution, leave it at room temperature for 6 hours, adjust the pH to neutral with 4.0 M sodium hydroxide solution, dilute it to the scale with water, shake well, and you have it.

[0057] Alkali destruction: Take an appropriate amount of this product (containing about 30 mg of diquafosol tetrasodium), place it in a 50 ml volumetric flask, add 2 ml of 4.0 M sodium hydroxide, leave it at room temperature for 6 hours, adjust the pH to neutral with 4.0 M hydrochloric acid solution, dilute it to the scale with water, shake well, and you have it.

[0058] Oxidative damage: Take an appropriate amount of this product (containing about 30 mg of diquafosol tetrasodium), place it in a 50 ml volumetric flask, add 2 ml of 3% hydrogen peroxide solution, leave it at room temperature for 6 hours, dilute it to the scale with water, shake well, and the product is ready.

[0059] High temperature destruction: Take an appropriate amount of this product (containing about 30 mg of diquafosol tetrasodium), put it in a crucible, place it in a 100°C oven for 6 hours, transfer it to a 50 ml volumetric flask, add water to dilute to the scale, shake well, and obtain.

[0060] Light damage: Take an appropriate amount of this product (containing about 30 mg of diquafosol tetrasodium) and place it in a 50 ml volumetric bottle, add water to dilute to the scale, shake well, and place it in an environment with 4500±500lx light for 2 days.

[0061] Take samples under various destruction conditions, inject them according to the liquid phase conditions of the relevant substances, and record the chromatograms. The results show that the impurity peaks generated by this product under various destruction conditions can be well separated from the main components. The impurities generated by this product under various destruction conditions have a large absorption near 266nm.

[0062] 3. Response values ​​and standard curves of starting materials and intermediates

[0063] Take the starting material A, starting material B, intermediate and tetrasodium diquatphosphate of this product and prepare them into gradient concentration test solutions (1), (2), (3), (4) (5). Take 20μl of each test solution and inject it into the chromatograph to record the chromatogram. Draw a regression curve with concentration (μg / ml) as the horizontal axis and peak area (A) as the vertical axis, and calculate the regression coefficient. It can be seen from the results that this product and each intermediate have good linearity in the concentration range of 10.0-90.0μg / ml.

[0064] Low concentration standard curve of starting material A

[0065]

[0066] Low concentration standard curve of starting material B

[0067]

[0068] Intermediate low concentration standard curve

[0069]

[0070] Low concentration standard curve of tetrasodium diquatphosphate

[0071]

[0072] 4. Solution stability

[0073] The test solution for the determination of related substances of this product was taken and the solution stability of the solution for the determination of related substances of this product was investigated at 0, 2, 4, 6, 8, 12 and 24 hours. The results show that the HPLC solution for the determination of the content of this product is stable within 24 hours.

[0074]

[0075] 5. Durability

[0076] Since the chromatographic conditions of this product are gradient elution, and the corresponding chromatographic column model, column temperature, and wavelength are specified, we made corresponding fine adjustments to these conditions to examine the durability of the chromatographic conditions.

[0077] As can be seen from the figure, when the chromatographic column model is changed, the column temperature changes within ±5℃, the wavelength is within the range of ±2nm, the impurities are well separated, and the durability is good.

[0078] 6. Limit of Quantitation and Limit of Detection

[0079] Take an appropriate amount of the reference substance of this product, weigh it accurately, add water to prepare tetrasodium diquatphosphate solution, then accurately measure an appropriate amount of the test solution, dilute it step by step, and inject it for inspection. The results showed that the quantitative limit of this product was 6.11ng / ml and the detection limit was 3.01ng / ml.

Claims

1. A method for measuring the content of diquafosol tetrasodium, characterized in that: Using reverse phase high performance liquid chromatography, The mobile phase is acetonitrile: 70-90mmol / L potassium dihydrogen phosphate solution = 40:60V / V, and adjust pH to 6-7 with phosphoric acid; chromatographic column is C8, 5μm, 4.6×250mmI.D.; flow rate is 1.0ml / min; column temperature is 40℃, detection wavelength is 266nm; Take about 30 mg of diquafosol tetrasodium sample, accurately weigh it, put it in a 50 ml volumetric flask, add water to dissolve and dilute to the scale as the test solution; take an appropriate amount of diquafosol tetrasodium reference substance, and prepare diquafosol tetrasodium reference solution in the same way, measure 20 μL of the test solution and the reference solution respectively, inject them into the liquid chromatograph, record the chromatogram, and calculate the content of diquafosol tetrasodium in the test solution by the peak area according to the external standard method.

2. The method for measuring the content of diquafosol tetrasodium according to claim 1, characterized in that: The mobile phase was acetonitrile: 80 mmol / L potassium dihydrogen phosphate solution = 40:60 V / V, and the pH was adjusted to 6.60 with phosphoric acid.

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