Detection method of related substances of fosfomycin tromethamine

Abstract

The present invention provides a method for detecting impurity D in fosfomycin trometamol or its preparation, which adopts high performance liquid chromatography. The present invention uses specific chromatographic columns, column temperatures, test solution concentrations, injection volumes and other conditions to effectively separate impurity D, and has good durability for detecting impurity D and stable chromatographic column efficiency.

Description

Technical Field

[0001] The present invention relates to a drug detection method. Background Art

[0002] Fosfomycin tromethamine is a mixture of fosfomycin and 2-amino-2-hydroxymethyl-1,3-propanediol salt in a ratio of 1:1, with fosfomycin as its active ingredient. Fosfomycin tromethamine overcomes some of the defects of fosfomycin metal salts, and has the advantages of mild efficacy, high oral absorption concentration, and long duration of action. It is a milder and more effective antibacterial drug that can directly block the action of pyruvate transferase necessary for bacterial cell wall synthesis. Its antibacterial spectrum includes Escherichia coli, Shigella dysenteriae, Citrobacter citrate, Proteus, Serratia marcescens, Staphylococcus aureus and Pseudomonas aeruginosa, etc. It is mainly used for lower urinary tract infections caused by pathogenic bacteria that are sensitive to this product, such as cystitis and urethritis.

[0003] The study of impurities is an important part of drug development, which is directly related to the quality and safety of marketed drugs. According to their physical and chemical properties, impurities are generally divided into three categories: organic impurities, inorganic impurities and residual solvents. Organic impurities include impurities and degradation products introduced in the process, which may be known or unknown, volatile or non-volatile. Since the chemical structure of such impurities is generally similar to or related to the active ingredients, they are usually called related substances. The detection methods of related substances include chemical methods, spectroscopy, chromatography, etc. At present, the most widely used methods are high performance liquid chromatography (HPLC), gas chromatography (GC) and capillary electrophoresis.

[0004] Impurity D is included in the relevant substances item of the quality standards of fosfomycin tromethamine raw material in the 43rd edition of the United States Pharmacopoeia, the 10.0 edition of the European Pharmacopoeia, and the 2020 edition of the British Pharmacopoeia. Impurity D is not included in the relevant substance methods in the quality standards of the 2020 edition of the Chinese Pharmacopoeia.

[0005] Fosfomycin tromethamine granules are one of the solid preparations of fosfomycin tromethamine. In view of the fact that the known impurity D is controlled under the related substance detection items of fosfomycin tromethamine in the European Pharmacopoeia 10.0 and the British Pharmacopoeia 2020, the actual situation is that all current pharmacopoeia methods cannot effectively detect impurity D. Therefore, it is of great significance to develop a highly sensitive detection method for impurity D of fosfomycin tromethamine and its preparations for quality control. Summary of the invention

[0006] The present invention aims to provide a method for detecting related substance D in fosfomycin tromethamine, and further provide a quality control method for related preparations.

[0007] In the early stage of the present invention, the method for related substances of fosfomycin tromethamine raw materials recorded in the 43rd edition of the United States Pharmacopoeia was used to detect related substances in fosfomycin tromethamine granules. It can be seen from the results of the early tests that the impurity D peak of this method is immediately after the main peak of fosfomycin ( Figure 1 ), the separation is poor, and the chromatographic column (Zorbax NH24.6 mm×250 mm, 5 μm) filled with aminopropylsilane bonded silica gel has a rapid decrease in column efficiency. Based on the above shortcomings, it is necessary to develop a method for effectively detecting and controlling impurity D in fosfomycin tromethamine particles.

[0008] Specifically, the present invention provides a method for detecting impurity D in fosfomycin tromethamine or its preparation, which adopts high performance liquid chromatography and includes the following contents:

[0009] Chromatographic column: Sepax ME-SAX (Ⅱ), 4.6mm×250mm, 5μm or other chromatographic column with equivalent performance

[0010] Mobile phase: potassium dihydrogen phosphate solution was used as the mobile phase;

[0011] Flow rate: less than 1.0 ml per minute;

[0012] Determined by differential refractometer. Differential detector is one of the commonly used detectors in HPLC. It is a detector that continuously detects the difference in refractive index of liquid between the sample flow path and the reference flow path. When the sample to be measured flows through the measuring cell, the refractive index changes, causing the light beam shining on the dual photoresistor to deflect, causing the resistance of the dual photoresistor to change. At this time, the signal output by the bridge reflects the change in sample concentration.

[0013] Wherein, the potassium dihydrogen phosphate solution contains 10.89 g of potassium dihydrogen phosphate per 1000 ml of water.

[0014] The present invention has found that the combination of a specific type of chromatographic column and a lower flow rate significantly improves the separation and detection results of impurity D. In the present invention, 0.6 to 0.9 ml per minute can be selected, such as 0.6, 0.7, 0.8, 0.9 ml / min.

[0015] Among them, the detector temperature is 35℃±2℃.

[0016] Wherein, the column temperature is 25-35°C, further selected from 30°C±2°C.

[0017] In the present invention, the injection volume of the test solution also has a certain influence on the detection result. The present invention has found that only when the concentration of the test solution is reduced and the injection volume is increased, a better detection result of impurity D can be obtained. Based on the above findings, in the detection method of the present invention, the content of fosfomycin tromethamine is 50-70 mg / ml, further selected from 60 mg / ml; the injection volume is selected from 20 μl.

[0018] Wherein, the preparation is selected from solid preparations, including powders, granules, capsules and the like.

[0019] The preparation may contain saccharin sodium. Saccharin sodium is dissolved in the mobile phase of the present invention, which may affect the detection of the product and related substances. Experiments have shown that the detection conditions of the present invention are also suitable for preparations containing saccharin sodium.

[0020] In the present invention, the preparation of the test solution, the reference solution, etc. can be carried out according to conventional operations. The solvent can refer to the detection method for related substances of fosfomycin tromethamine in the standards of the 43rd edition of the United States Pharmacopoeia or the 10.0 edition of the European Pharmacopoeia.

[0021] The solvent for preparing the test solution, reference solution, etc. in the present invention is the mobile phase.

[0022] The present invention has found that only by using specific chromatographic columns, column temperatures, test solution concentrations and injection volumes and other conditions can the impurity D be effectively separated, and the detection durability of the impurity D is good and the chromatographic column efficiency is stable.

[0023] The impurity content of the present invention can be calculated by various conventional methods such as internal standard method, external standard method, self-comparison method, area normalization method, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Chromatogram of the prior art

[0025] Figure 2 Example 1 Chromatogram

[0026] Figure 3 Chromatogram at a mobile phase flow rate of 0.6 ml / min

[0027] Figure 4 Chromatogram at a mobile phase flow rate of 0.8 ml / min

[0028] Figure 5 Chromatogram at a mobile phase flow rate of 1.0 ml / min

[0029] Figure 6 Impurity D Linearity Graph DETAILED DESCRIPTION

[0030] Example 1

[0031] Use a cryogenic sample injector (temperature 8°C). Take an appropriate amount of this product, weigh it accurately, add the mobile phase to dissolve and dilute it to make a solution containing about 60 mg of fosfomycin tromethamine per 1 ml as the test solution.

[0032] Accurately measure an appropriate amount of the test solution, dilute it with the mobile phase to make a solution containing about 0.6 mg of fosfomycin tromethamine per 1 ml, shake well, and use it as the control solution;

[0033] Accurately measure an appropriate amount of the test solution and dilute it with the mobile phase to make a solution containing approximately 30 μg of fosfomycin tromethamine per 1 ml, which serves as the sensitivity solution.

[0034] Take about 0.3 g of fosfomycin tromethamine raw material, moisten it with 60 μl of water, heat it in an oven at 60° C. for 24 hours, add mobile phase to dissolve and dilute to 20 ml, shake well, and use this as solution (1);

[0035] Take about 0.3 g of fosfomycin tromethamine raw material, add 2.5 ml of solution (1), dissolve it with mobile phase and dilute to 5 ml, as the system suitability solution (the solution contains impurities A, B, C, and D).

[0036] Accurately weigh about 250 mg of the blank auxiliary material of this product into a 10 ml volumetric flask, add the mobile phase to dissolve and dilute to the scale, shake well, and filter to obtain the blank auxiliary material solution.

[0037] According to the high performance liquid chromatography method (Chinese Pharmacopoeia 2020 Edition Part IV General Rules 0512), a chromatographic column (Sepax ME-SAX (Ⅱ), 4.6 mm × 250 mm, 5 μm or a chromatographic column with equivalent performance) was prepared using an anion exchange filled column, and potassium dihydrogen phosphate solution (10.89 g of potassium dihydrogen phosphate was dissolved in 1000 ml of water) was used as the mobile phase at a flow rate of 0.8 ml per minute; the measurement was performed using a differential refractometer, the temperature was 35°C, and the column temperature was 30°C.

[0038] Take 20 μl of the system suitability solution and inject it into the liquid chromatograph. The separation degree between the impurity D peak and the adjacent impurities should be no less than 1.5.

[0039] Take 20 μl of the sensitivity solution and inject it into the liquid chromatograph. The signal-to-noise ratio of the fosfomycin peak height should be greater than 10. Accurately measure 20 μl of the blank excipient solution, control solution and test solution, respectively, and inject them into the liquid chromatograph, and record the chromatogram to 1.5 times the fosfomycin peak time.

[0040] If there is an impurity D peak in the chromatogram of the test solution, the impurity D peak area shall not be greater than the main peak area of ​​the control solution fosfomycin (1.0%).

[0041] See the detection spectrum Figure 2 .

[0042] It can be seen from Example 1 that the system applicability and specificity of this method meet the requirements.

[0043] Blank solution: There is no interference at the main peak of fosfomycin and the peak position of impurity D.

[0044] Blank excipient solution: There is no interference at the main peak of fosfomycin and the peak position of impurity D.

[0045] System suitability solution: The resolution between impurity D peak and adjacent impurities is greater than 1.5.

[0046] Impurity A, impurity B, impurity C and tromethamine reference solution: no interference at the peak positions of impurity D and fosfomycin.

[0047] Sensitivity solution: The signal-to-noise ratio of the main peak of fosfomycin is greater than 10.

[0048] Test solution: The ratio between the impurity D peak and its adjacent peaks before and after is greater than 1.5.

[0049] Control solution: There is no impurity interfering with the elution of the main peak of fosfomycin. The number of theoretical plates based on the main peak of fosfomycin is greater than 5000.

[0050] Example 2

[0051] The solution of Example 1 was used, and the flow rates of 0.6 ml / min, 0.8 ml / min, and 1.0 ml / min were selected for detection. The detection results are shown in Figure 3-5 .

[0052] Example 3 Degradation Specificity

[0053] Referring to conventional methods, experiments such as acid destruction, alkali destruction, oxidation destruction, high temperature destruction, high humidity destruction, and light destruction were carried out to examine the degradation specificity of the detection method.

[0054] Acid destruction: Accurately weigh the sample to be tested, place it in a 10ml volumetric flask, add 1ml of 0.1mol / L hydrochloric acid solution, shake well, destroy it at room temperature for 4h, then add 1ml of 0.1mol / L sodium hydroxide solution, and then add blank solution to dilute to the scale, shake well, and obtain.

[0055] Alkali destruction: Accurately weigh the sample to be tested, put it into a 10ml volumetric flask, add 1ml of 0.1mol / L sodium hydroxide solution, destroy it at room temperature for 4h, then add 1ml of 0.1mol / L hydrochloric acid solution, and then add blank solution to dilute to the scale, shake well, and obtain.

[0056] Oxidative destruction: Add 1 ml of 0.03% H2O2 to a 10 ml volumetric flask, destroy at room temperature for 4 hours, then add blank solution to dilute to the scale, shake well, and the product is obtained.

[0057] High temperature destruction: Accurately weigh the sample to be tested and 250.3 mg of blank auxiliary material, place them in a 10 ml volumetric flask, place them in a 60°C oven for 48 hours, take them out and let them cool, add blank solution to dissolve and dilute to the scale, shake well, and obtain.

[0058] High humidity damage test solution: Accurately weigh the sample to be tested, spread it flat in an open weighing bottle with a thickness of no more than 5mm, and place it in a constant humidity chamber at a humidity of 75% for 9 hours. Take it out, add blank solution to dissolve it and transfer it to a 10ml volumetric flask. Dilute it to the scale with mobile phase and shake well.

[0059] Light-induced sample solution (liquid): Accurately weigh the sample to be tested, place it in a 10ml volumetric bottle, dissolve it with mobile phase and dilute it to the scale, shake it well, and place it under light (5000Lx) + UV (90μw / cm 2 ) conditions for 4 hours, take it out, and you have it.

[0060] Results: After the test sample was destroyed by acid, alkali, oxidation, high humidity, high temperature and light conditions, the maximum peak area percentage of impurity D was 0.06% and the minimum was 0.03%, both of which were less than the preparation limit (1.0%). Other impurities except impurity D were degraded under various destruction conditions. The degradation rate of acid destruction was 7.16%, and the degradation rate of high humidity destruction was 3.03%, indicating that the degradation conditions were more severe, but impurity D was not easy to degrade. Under various destruction conditions, the separation between impurity D and its adjacent peaks was greater than 1.5.

[0061] Example 4 Durability Test

[0062] Table 1 System suitability solution durability test results

[0063]

[0064] The chromatographic conditions of this method are durable, and the separation between the impurity D peak and the adjacent peaks is greater than 1.5.

[0065] Example 5 Detection Limit and Quantification Limit

[0066] Table 2 Results of detection limit and quantification limit

[0067]

[0068] The sensitivity of this method is good. The detection limit of impurity D is equivalent to 0.007% of the test sample concentration; the quantification limit of impurity D is equivalent to 0.020% of the test sample concentration.

[0069] Example 6 Linearity of Impurity D

[0070] Table 3 Linearity results of impurity D

[0071]

[0072] This method has good linearity. Impurity D and the main component have good linearity with their peak areas within the verified concentration range, and the correlation coefficient r is 0.9999 (see also Figure 6 ), the Y-intercept corresponds to 0.2% of the 100% limit concentration of the formulation.

[0073] Example 7 Accuracy

[0074] Table 4 Recovery results of impurity D

[0075]

[0076] The method has high accuracy. The recovery rate calculated by the external standard method at the limit concentrations of 50%, 100%, and 150% of the measured concentration is between 90.0% and 108.0%; the RSD of the recovery rate at each concentration is less than 10.0%, and the RSD of the recovery rate at all concentrations is less than 10.0%.

Claims

1. A method for detecting impurity D in fosfomycin tromethamine, characterized in that: It uses high performance liquid chromatography and includes the following: Chromatographic column: Sepax ME-SAX (Ⅱ), 4.6 mm × 250 mm, 5 μm; Mobile phase: potassium dihydrogen phosphate solution was used as the mobile phase; Flow rate: less than 1.0 ml per minute; Measured by differential refractometer; The injection volume was 20 μl; Take about 0.3 g of fosfomycin tromethamine raw material, moisten it with 60 μl of water, heat it in a 60°C oven for 24 hours, add mobile phase to dissolve and dilute to 20 ml, shake well, and use this as solution 1; Take about 0.3 g of fosfomycin tromethamine bulk drug, add solution 1 to 2.5 ml, dissolve it with mobile phase and dilute to 5 ml, as the test solution.

2. The detection method according to claim 1, characterized in that: The potassium dihydrogen phosphate solution contains 10.89 g of potassium dihydrogen phosphate in every 1000 ml of water.

3. The detection method according to claim 1, characterized in that: The flow rate is selected from 0.6 to 0.9 ml per minute.

4. The detection method according to claim 3, characterized in that: The flow rate was selected to be 0.8 ml per minute.

5. The detection method according to claim 1, characterized in that: The detector temperature is 35℃±2℃.

6. The detection method according to claim 1, characterized in that: The column temperature was 25-35°C.

7. The detection method according to claim 1, characterized in that: The column temperature is 30℃±2℃.

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