A method for detecting related substances of doxofylline injection

Abstract

The invention mainly relates to a method for determining related substances of doxofylline injection, using high performance liquid chromatography, using octadecylsilane bonded silica gel as a filler; mobile phase A is a phosphoric acid aqueous solution with a pH of 1.5-3.5, and mobile phase B is acetonitrile, and gradient elution is performed. The detection method can completely separate impurities 1-9 in doxofylline injection, with a separation degree greater than 1.5, good system applicability, and the method has high specificity, accuracy and reproducibility, etc., is easy to operate, has no damage to the chromatographic column, and can be used for analysis, detection and quality control of doxofylline injection in industrial production.

Description

Technical Field

[0001] The invention belongs to the field of determination of drug-related substances, and mainly relates to a method for determining related substances of doxofylline injection. Background Art

[0002] Doxofylline is a bronchodilator that can directly act on the bronchi and relax bronchial smooth muscles. It relaxes smooth muscles by inhibiting phosphodiesterase in smooth muscle cells, thereby achieving the effect of inhibiting asthma. Doxofylline is mostly administered by intravenous push or intravenous drip. Doxofylline injection is used to treat bronchial asthma, asthmatic chronic bronchitis and other dyspnea caused by bronchospasm. Doxofylline raw materials can be directly formulated into liquid preparations. The aqueous solution has good heat-pressing stability and can be terminally sterilized. It is one of the safer and more effective drugs to replace theophylline and aminophylline. The safety of injections has always been a concern. As a drug for the treatment of bronchospasm, doxofylline injection, its quality inspection and control are particularly important. Standardized research on related substances and controlling them within a safe and reasonable limit will be directly related to its quality and safety. Doxofylline is prone to produce impurities during the preparation process. Therefore, the study of doxofylline-related substances is of great significance for effectively controlling the quality of doxofylline preparations.

[0003] The existing related substance detection method of doxofylline is as follows:

[0004] Doxofylline Injection (Chinese Pharmacopoeia 2015) adopts high performance liquid chromatography to detect related substances, using a C18 column and acetonitrile-phosphate buffer (pH=5.8) 12:88 as the mobile phase for detection. The disadvantage is that the detected impurities are relatively few and the generated impurities cannot be completely separated.

[0005] Doxofylline injection (import registration standard JX20170001) was detected by high performance liquid chromatography using a C8 column and water-acetonitrile 85:15 as the mobile phase, but this method was unable to separate all the impurities produced.

[0006] The prior art CN111060623A uses octadecyl bonded silica gel or octadecyl bonded silica gel as a reverse phase chromatography column filler; uses acetonitrile and phosphate buffer (pH 4.6-5.0) as mobile phases for gradient elution. This method cannot separate all the impurities generated.

[0007] In addition, the prior art CN101569603A uses a reverse phase chromatography column with octadecyl bonded silica gel or octadecyl bonded silica gel as a filler and acetonitrile-phosphate buffer (pH 5.8) (12:88) as a mobile phase to detect related substances. The above methods are unable to separate all the impurities generated. Summary of the invention

[0008] On the basis of the prior art, the present invention follows the relevant guidelines such as the "Technical Guidelines for Validation of Chemical Drug Quality Control Analytical Methods", "Technical Guidelines for the Standardized Process of Establishing Chemical Drug Quality Standards", "Technical Guidelines for Research on Impurities in Chemical Drugs", ICH and ChP 2015 Appendix, and combines the preparation process of doxofylline injection, the results of process validation such as multi-batch pilot studies, and finally develops a new method for detecting related substances of doxofylline injection, which strictly controls the product quality. The specific detection method is as follows:

[0009] A method for detecting related substances of doxofylline injection is disclosed. The method is a high performance liquid chromatography method. The chromatographic conditions are as follows: using octadecylsilane bonded silica gel as a filler; mobile phase A is a phosphoric acid aqueous solution with a pH of 1.5-3.5, and mobile phase B is acetonitrile. Gradient elution is performed according to the following table:

[0010]

[0011] The detection wavelength is 268nm-278nm or 235nm-245nm; the column temperature is 30℃-45℃; the flow rate is 0.9ml / min -1.2ml / min.

[0012] Furthermore, the above detection method also includes:

[0013] 1) Preparation of test solution: Take doxofylline injection and dilute with water to prepare a solution containing 1.0-1.5 mg of doxofylline per 1 ml as the test solution;

[0014] 2) Preparation of control solution: Accurately measure an appropriate amount of the test solution in 1), dilute with water to make a solution containing 5-7.5 μg of doxofylline per 1 ml as the control solution.

[0015] Furthermore, the pH of the mobile phase A is 2.3-2.7, preferably 2.5.

[0016] Furthermore, the gradient elution conditions of the detection method are:

[0017]

[0018] Furthermore, the gradient elution conditions of the detection method are:

[0019]

[0020]

[0021] Furthermore, the detection wavelength of the above detection method is 273nm or 240nm.

[0022] Furthermore, the related substances include at least impurities 1-5, wherein impurity 1 is 1-(2'-methyl-1',3'-dioxolanyl)-4-N-methylamino-5'-N-formamidoimidazole, impurity 2 is theophylline-7-acetaldehyde, impurity 3 is theophylline, impurity 4 is 2-(9-theophylline-methyl)-1,3-dioxolane, and impurity 5 is ethophylline.

[0023] Furthermore, the related substances also contain one or more impurities 6-9, wherein impurity 6 is 7-(2,2-dimethoxyethyl)-1,3-dimethyl-1H-purine-2,6(3H,7H)-dione, impurity 7 is 1,3-dimethyl-6,7,9,10-tetrahydro-7,10-epoxy[1,4]oxazino[5,4-f]purine-2,4(1H,3H)-dione, impurity 8 is 1-((1,3-dioxolan-2-yl)methyl)-4-(1,3-dimethylureido)-1H-imidazole-5-carboxylic acid, and impurity 9 is (1-(1,3-dioxolan-2-yl)methyl)-4-(3-methylureido)-1H-imidazole-5-carboxylic acid.

[0024] The structure of the related substances is as follows:

[0025]

[0026]

[0027] The detection method can be used for the analysis, detection and quality control of doxofylline injection.

[0028] The detection method provided by the invention can completely separate impurities 1-9 in the doxofylline injection, the separation degree is greater than 1.5, the system applicability is good, and the method has high specificity, accuracy and reproducibility, etc., is easy to operate, does not damage the chromatographic column, and can be used for analysis, detection and quality control of the doxofylline injection in industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The detection results when the detection wavelength is 273nm in Example 1;

[0030] Figure 2 The detection results when the detection wavelength is 240nm in Example 1;

[0031] Figure 3 The detection results when the detection wavelength is 268nm in Example 2;

[0032] Figure 4 The detection results when the detection wavelength is 235nm in Example 2;

[0033] Figure 5Comparative Example 1 test results; DETAILED DESCRIPTION

[0034] Example 1

[0035] Chromatographic conditions: octadecylsilane bonded silica gel as filler; column temperature: 40°C; flow rate 1.0 ml / min; detection wavelength 273 nm or 240 nm; phosphoric acid aqueous solution (pH = 2.5) as mobile phase A, acetonitrile as mobile phase B, gradient elution according to the following table:

[0036]

[0037] Detection method:

[0038] Blank solution: water.

[0039] System suitability solution: Accurately weigh appropriate amounts of theophylline reference substance and impurity 1-9 reference substances, add water and ultrasonically dissolve and dilute to make a solution containing approximately 1 mg of doxofylline and 1 μg of each impurity per 1 ml, shake well, and obtain.

[0040] Blank solution and system suitability solution were injected, and the results were displayed (see Figure 1 , Figure 2 ): System suitability: Impurities 1, 8, 3, 2, 9, 5, 4, 7 and 6 in the solution are eluted in sequence, and the minimum separation between each impurity and between the impurity and the main peak is 3.44 (273nm) and 3.49 (240nm), respectively, both greater than 1.5, and the chromatographic conditions are well suited. The details are as follows:

[0041]

[0042] Example 2

[0043] Chromatographic conditions: octadecylsilane bonded silica gel as filler; column temperature: 40°C; flow rate 1.0 ml / min; detection wavelength 273 nm, 240 nm; phosphoric acid aqueous solution (pH = 2.5) as mobile phase A, acetonitrile as mobile phase B, gradient elution according to the following table:

[0044]

[0045] Detection method:

[0046] The blank solution and system suitability solution were the same as in Example 1. The blank solution and system suitability solution were injected once each. The results showed (see Figure 3 , Figure 4): System suitability: Impurities 1, 8, 3, 2, 9, 5, 4, 7 and 6 in the solution are peaked in sequence, and the minimum separation between each impurity and between the impurity and the main peak are 2.70 (wavelength 273nm) and 2.75 (wavelength 240nm), respectively, both greater than 1.5, and the chromatographic conditions are well suited. The details are as follows:

[0047]

[0048] Example 3

[0049] Chromatographic conditions: octadecylsilane bonded silica gel as filler; column temperature: 40°C; flow rate 1.0 ml / min; detection wavelength 273 nm, 240 nm; phosphoric acid aqueous solution (pH = 2.5) as mobile phase A, acetonitrile as mobile phase B, gradient elution according to the following table:

[0050]

[0051] Detection method:

[0052] Preparation of test solution: Accurately measure an appropriate amount of three batches (batch 18120611, batch 18120612, batch 18120711) of doxofylline injection, place in a suitable volumetric flask, add water to dilute to make a solution containing 1.0 mg of doxofylline per 1 ml, as the doxofylline test solution.

[0053] Preparation of control solution: Accurately measure an appropriate amount of the test solution and dilute it with water to make a solution containing 5 μg of doxofylline per 1 ml as the control solution.

[0054] Take 10μl of the control solution and inject it into the liquid chromatograph, adjust the detection sensitivity so that the peak height of the main component chromatographic peak is 20% to 25% of the full scale; take 10μl of the test solution and inject it into the liquid chromatograph. The results show that there are no impurities mentioned above in the test sample, as shown in the following table:

[0055]

[0056] Example 4 Methodology Verification

[0057] Blank solution: water.

[0058] System suitability solution: Accurately weigh appropriate amounts of theophylline reference substance and impurity 1-9 reference substances, add water and ultrasonically dissolve and dilute to make a solution containing approximately 1 mg of doxofylline and 1 μg of each impurity per 1 ml, shake well, and obtain.

[0059] Impurity reference substance stock solution 1: Accurately weigh appropriate amounts of impurity 1-5 and impurity 8-9 reference substances, add water to dissolve them by ultrasonication and dilute them to make solutions containing approximately 100 μg of the corresponding impurities per 1 ml, as impurity reference substance stock solution 1.

[0060] Impurity reference substance stock solution 2: Accurately weigh appropriate amounts of impurity 6-7 reference substances, add appropriate amounts of dimethyl sulfoxide (DMSO) to dissolve them by ultrasonication, and dilute with water to make a solution containing approximately 100 μg of the corresponding impurity per 1 ml, as impurity reference substance stock solution 2.

[0061] Doxofylline reference solution: Accurately weigh an appropriate amount of theophylline reference substance, add water to dissolve it by ultrasonication and dilute it to make a solution containing about 1.0 mg of doxofylline per 1 ml, which is used as the doxofylline reference solution.

[0062] Doxofylline test solution: Accurately measure an appropriate amount of theophylline injection solution, place it in a suitable volumetric flask, and dilute it with water to make a solution containing approximately 1.0 mg of doxofylline per 1 ml, which is used as the doxofylline test solution.

[0063] Detection limit solutions for each impurity and main component: dilute the "Impurity Reference Stock Solution 1", "Impurity Reference Stock Solution 2" and "Doxofylline Reference Solution" step by step to appropriate multiples, and inject the samples. When the signal-to-noise ratio (S / N) is about 3:1, it is the detection limit solution for the impurity and main component.

[0064] Quantitative limit solutions of each impurity and main component: dilute the "Impurity Reference Stock Solution 1", "Impurity Reference Stock Solution 2" and "Doxofylline Reference Solution" step by step to appropriate multiples, and inject the samples. When the signal-to-noise ratio (S / N) is about 10:1, it is the quantitative limit solution of the impurity and main component.

[0065] 1) Exclusivity

[0066] Chromatographic conditions: as in Example 1.

[0067] Detection method: blank solution, impurity reference stock solution 1, impurity reference stock solution 2, doxofylline reference solution, system suitability solution, and doxofylline test solution were injected once each. The results showed that when the detection wavelengths were 240nm and 273nm, respectively, the blank solution and the impurities did not interfere with the main component detection; the separation between the impurity peaks and between the impurity peaks and the main peak in the test solution and the system suitability solution was not less than 1.5, as shown in the following table:

[0068]

[0069] 2) Limit of detection and limit of quantification

[0070] Chromatographic conditions: as in Example 1.

[0071] Detection method: Inject blank solution, each impurity and main component detection limit solution, each impurity and main component quantitative limit solution in sequence. The results show that the quantitative limits of impurities 1 to 9 and doxofylline are all less than the reporting limit (0.05%), and the detection sensitivity is high, as shown in the following table:

[0072]

[0073] Each impurity and main component quantitative limit solution was injected 6 times in succession. The results showed that the peak area RSD was no more than 10%, and the retention time RSD was no more than 2.0%. The precision was good and the reproducibility was high. The details are as follows:

[0074]

[0075] 3) Linear

[0076] Chromatographic conditions: as in Example 1.

[0077] Detection method: accurately weigh the blank solution and theophylline reference solution, and dilute them with solvent to a series of concentrations of reference solution. Accurately measure 10 μl of the above reference solutions of different concentrations and inject them into the liquid chromatograph for sample injection and determination. The results show that the linear equations of the main components and impurities are shown in the table, the correlation coefficient r is greater than 0.999, the y-axis intercepts are within 25% of the 100% response value, and the RSD of the response factor is less than 15%. The details are as follows:

[0078] Inspection Project Linear equations Correlation coefficient r y-intercept Response Factor RSD% Correction Factor Impurity 1 y=15175x-188.93 0.9998 188.93 4.36 1.3 Impurity 2 y=21582x-1302.7 0.9999 1302.7 10.28 0.9 Impurity 3 y=32359x-273.29 0.9999 273.29 5.06 0.6 Impurity 4 y=17764x-342.5 0.9998 342.5 3.88 1.1 Impurity 5 y=24258x-639.62 0.9998 639.62 7.12 0.8 Doxofylline y=19532x-99.016 1.0000 99.016 6.40 / Impurity 6 y=18845x+203.21 1.000 203.21 0.8 1.0 Impurity 7 y=28629x-1794.3 0.999 1794.3 11.9 0.7 Impurity 8 y=9188.4x-203.4 0.999 203.4 2.7 0.7 Impurity 9 y=16773x+1167.1 1.000 1167.1 8.9 0.4

[0079] 4) Durability

[0080] The parameters of the chromatographic conditions are shown in the table (except for the changed parameters, other parameters are the same as the specified values):

[0081] Chromatographic conditions Specified value Changing parameters Column temperature (℃) 40 35 and 45 Flow rate (ml / min) 1.0 0.9 and 1.2 Wavelength (nm) 273 268 and 278 Initial ratio of mobile phase (mobile phase A: mobile phase B) 94:6 96:4 and 92:8 Chromatographic columns Column A Columns B and C pH value of mobile phase A 2.5 2.3 and 2.7

[0082] Column A: ACE Excel C18-AR (4.6 mm × 250 mm, 5 μm)

[0083] Column B: ACE Excel C18-AR (4.6 mm × 250 mm, 5 μm)

[0084] Column C: Waters Atlantis T3 (4.6 mm × 250 mm, 5 μm)

[0085] Detection method:

[0086] Preparation of system suitability solution 1: Accurately weigh appropriate amount of impurity 1-5 reference substances, add water to dissolve them by ultrasound and dilute them to make solutions containing approximately 100 μg of the corresponding impurity per 1 ml, as the stock solutions of each impurity reference substance; accurately measure appropriate amount of doxofylline injection and each impurity reference substance stock solution, add water to dilute them to make solutions containing approximately 1 mg of doxofylline and 1 μg of each impurity per 1 ml, as system suitability solution 1.

[0087] Under each variable chromatographic condition, the blank solution, system suitability solution 1, theophylline test solution, and theophylline control solution were injected once each. The results showed that under each chromatographic condition, the separation between the impurities and between the main peak and the adjacent impurities in the system suitability solution was greater than 1.5, among which the separation between impurities obtained by the detection method with specified values ​​was the best; no impurities were detected in the test solution.

[0088]

[0089] 5) Accuracy

[0090] Chromatographic conditions: as in Example 1.

[0091] Detection method:

[0092] (I) Impurities 1 to 5:

[0093] Impurity 1-5 reference substance stock solution 1: Take an appropriate amount of impurities 1-5, weigh accurately, place in the same volumetric flask, add water to dilute to the scale, and prepare a solution containing 50μg / ml of each impurity 1-5 as the mixed impurity reference substance stock solution. Prepare 2 portions in parallel.

[0094] Mixed impurity reference solution 1: Accurately measure appropriate amounts of impurity 1-5 reference stock solution 1, dilute with water to make solutions containing approximately 1 μg of each impurity per 1 ml, and prepare 2 portions of mixed impurity reference solution.

[0095] The recovery test solutions at each concentration level were prepared as shown in the following table. Three solutions were prepared for each concentration level in the same manner.

[0096] Test Level Percentage concentration (%) Mixed control stock solution volume (ml) Volume of doxofylline injection added (ml) Volume of measuring flask (ml) R1 50 1 10 100 R2 100 2 10 100 R3 150 3 10 100

[0097] Control solution of test solution: Accurately measure 1 ml of recovery test solution at each concentration level and place it in a 200 ml volumetric flask, dilute to the scale with water, shake well, and filter.

[0098] (ii) Impurities 6 to 9

[0099] Impurity 6-9 reference substance stock solution 2: Accurately measure appropriate amounts of impurity 6-9 stock solutions respectively, dilute with water to make a solution containing approximately 10 μg of each impurity per 1 ml, as the mixed impurity reference substance stock solution.

[0100] Mixed impurity reference solution 2: Accurately measure appropriate amounts of reference solution 2 of impurities 6-9, dilute with water to make solutions containing about 1 μg of each impurity per 1 ml, as mixed impurity reference solution. Prepare 2 portions in parallel.

[0101] The recovery test solutions at each concentration level are shown in the table. Three copies of each concentration level were prepared in the same way.

[0102]

[0103] Control solution of test solution: Accurately measure 1 ml of recovery test solution at each concentration level and place it in a 200 ml volumetric flask, dilute to the scale with water, shake well, and filter.

[0104] Detection method:

[0105] The blank solution, mixed impurity reference solution 1 and 2, recovery test solutions at each concentration level and their control solutions were injected once each; the mixed impurity reference solution was injected 3 times each. The results showed that the recoveries of each impurity at each concentration level after deducting the known impurities detected in the test sample were between 80% and 120%; the RSDs of the recoveries at the three concentration levels were all less than 10%.

[0106]

[0107] Comparative Example 1

[0108] Stationary phase: Waters T3 (4.6 mm × 250 mm, 5 μm) chromatographic column;

[0109] Mobile phase: phosphate (pH = 5.8): acetonitrile = 85:15;

[0110] Flow rate: 0.8ml / min;

[0111] Injection volume: 10 μl;

[0112] Detection wavelength: 273nm;

[0113] Column temperature: 40°C;

[0114] Detection method: Accurately weigh about 20 mg of theophylline raw material, place in a 20 ml volumetric flask, add 2 ml of 30% hydrogen peroxide, shake, place at room temperature for 24 hours, dilute to the scale with water, shake well; inject the above solution into the liquid chromatograph, the result shows (see Figure 5 ), the impurities in the oxidation-damaged samples with retention times of 8.866min, 9.092min, and 9.092min under isocratic elution conditions were not separated.

[0115] Comparative Example 2

[0116] Stationary phase: Waters T3 (4.6 mm × 250 mm, 5 μm) chromatographic column;

[0117] Flow rate: 1.0ml / min;

[0118] Injection volume: 10 μl;

[0119] Detection wavelength: 273nm;

[0120] Column temperature: 40°C;

[0121] Mobile phase: The aqueous phase is phosphate (pH=5.8), the organic phase is acetonitrile, and the elution conditions are:

[0122]

[0123] Detection method:

[0124] Impurity 1, impurity 3, impurity 4, impurity 5 reference substance stock solutions: accurately weigh about 2 mg of impurity 1, about 4 mg of impurity 3, about 2 mg of impurity 4, and about 1 mg of impurity 5 respectively, place them in a 20 ml volumetric flask, add water and ultrasonically dissolve and dilute to the scale, shake well, and obtain.

[0125] Test solution: Accurately weigh about 25 mg of theophylline raw material, place it in a 25 ml volumetric flask, add water and ultrasonically dissolve it and dilute it to the scale, shake well, and obtain it.

[0126] Impurity mixed solution: Accurately measure 1 ml each of impurity 1, impurity 3, impurity 4, impurity 5 and test sample solution, place in a 50 ml volumetric flask, add water to dilute to the scale, shake well, and obtain.

[0127] Preparation of acid-destroyed samples: Accurately weigh about 20 mg of doxofylline, place in a 20 ml volumetric flask, add 2 ml of 1 mol / L hydrochloric acid, shake, and leave at room temperature for 1 hour; add 2 ml of 1 mol / L sodium hydroxide solution to neutralize, cool, dilute to the scale with water, shake well, and obtain.

[0128] Acid-destroyed impurity mixed sample solution: Accurately measure 1 ml of acid-destroyed sample solution and 1 ml of impurity mixed solution, mix well, and obtain.

[0129] The acid-destroyed impurity mixed sample solution was injected and tested under gradient elution conditions. The results showed that under the test conditions, the separation between the impurities and between the impurities and the main peak in the acid-destroyed impurity mixed sample solution was >1.5. However, the chromatographic column was severely damaged and could not be reused, which was not conducive to subsequent sample testing and greatly increased the cost.

[0130] Comparative Example 3

[0131] Chromatographic conditions:

[0132] Stationary phase: Agilent Eclipse XDB-C18 4.6 mm × 250 mm, 5 μm;

[0133] Detection wavelength: 273nm;

[0134] Flow rate: 1.0ml / min;

[0135] Column temperature: 40°C;

[0136] Test solution: 0.3 mg / ml;

[0137] Injection volume: 20 μl;

[0138] Mobile phase: acetonitrile-water, gradient elution:

[0139]

[0140] Detection method: Accurately weigh appropriate amounts of doxofylline reference substance and impurity 1 to impurity 5 reference substances, add water ultrasonic solution and dilute to make a solution containing about 0.3 mg of doxofylline and 3 μg of each impurity per 1 ml, shake well, and obtain; inject one injection of the solution, the results show that the separation between the impurities and between the impurities and the main peak is unstable (>1.5 and <1.5 appear), and the method has poor reproducibility when using high performance liquid chromatographs from different manufacturers or different high performance liquid chromatographs from the same manufacturer, or when detecting different batches of samples.

Claims

1. A method for detecting related substances of doxofylline injection, the detection method being a high performance liquid chromatography method, characterized in that The chromatographic conditions are as follows: the chromatographic column is filled with octadecylsilane bonded silica gel, with a specification of 4.6 mm × 250 mm, 5 μm; the mobile phase A is a phosphoric acid aqueous solution with a pH of 2.3-2.7, and the mobile phase B is acetonitrile, and the gradient elution is performed as shown in the following table: The detection wavelength is 268nm-278nm or 235nm-245nm; the column temperature is 35℃-45℃; the flow rate is 0.9ml / min-1.2ml / min, Wherein, the related substances include impurities 1-9, and their structures are shown below:

2. The detection method according to claim 1, further comprising: 1) Preparation of test solution: Take doxofylline injection and dilute with water to prepare a solution containing 1.0-1.5 mg of doxofylline per 1 ml as the test solution; 2) Preparation of control solution: Accurately measure an appropriate amount of the test solution and dilute it with water to make a solution containing 5-7.5 μg of doxofylline per 1 ml as the control solution.

3. The detection method as claimed in claim 1, wherein the pH of the mobile phase A is 2.

5.

4. The detection method as claimed in claim 1, wherein the gradient elution conditions are:

5. The detection method according to claim 1, wherein the detection wavelength is 273 nm or 240 nm.

6. The detection method according to any one of claims 1 to 5, which is used for the analysis, detection and quality control of doxofylline injection.

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