High performance liquid chromatography method for determining ribavirin injection related substances

Abstract

The invention relates to the technical field of ribavirin injection related substance detection, and discloses a high performance liquid chromatography method for determining ribavirin injection related substances, which comprises the following steps: taking three batches of ribavirin injections, preparing into a related substance detection test solution according to a method under the item of solution preparation, then carrying out sample introduction and determination according to corresponding chromatographic conditions under the item of chromatographic conditions, and finally determining the ribavirin injection related substances according to the determined ribavirin injection related substances. And recording the peak area and calculating the known impurity content by using a reference substance external standard method (other impurities are calculated by using a ribavirin reference substance). The high performance liquid chromatography method for determining the related substances of the ribavirin injection can be used for determining a plurality of ribavirin impurities at one time, and is used for detecting the related substances of the ribavirin injection.

Description

Technical Field

[0001] The present invention relates to the technical field of detection of related substances in ribavirin injection, and specifically to a high performance liquid chromatography method for determining the related substances in ribavirin injection. Background Art

[0002] Ribavirin, also known as ribavirin, has the chemical name of 1-β-D-ribofuranosyl-1H-1,2,4-triazole-3-carboxamide. It is an artificially synthesized broad-spectrum anti-non-retroviral drug that can inhibit a variety of DNA and RNA viruses. Ribavirin preparations include oral solution, granules, tablets (including dispersible tablets and lozenges), capsules, injections, etc.; among them, ribavirin injection is widely used clinically for the treatment of infections caused by respiratory syncytial virus, influenza virus, adenovirus, etc. Due to its wide clinical application, compared with other preparations, its incidence of adverse reactions is high, and the safety problem is becoming increasingly prominent. It is necessary to strictly control its quality to ensure the safety of drug use.

[0003] After consulting relevant domestic and foreign standards, the known impurities controlled in ribavirin preparations are as follows (the names of impurities are shown in the following table: Summary Table of Known Impurities in Ribavirin): For ribavirin granules (YBH06072017), impurities A and D are determined; for EP ribavirin raw materials, impurities A, B, C, D, F, and G are determined; for JP ribavirin raw materials, impurity A is determined; for USP ribavirin tablets, method 1 determines impurities A, C, D, E, F, G, and H, method 2 determines impurities A, D, uridine, and uracil, and for ribavirin capsules, impurity A is determined. It can be seen from each standard that ribavirin has many impurities, and different methods are used for determination respectively, and the methods are relatively cumbersome. This article will refer to relevant literature to establish an HPLC gradient elution method for determining the related substances in ribavirin injection; the established method has good specificity and separation effect, and all the above impurities can be determined by one method. The method is simple and convenient and can be used for the quality control of ribavirin injection.

[0004]

[0005]

[0006] Summary Table of Known Impurities in Ribavirin

[0007] There are the following problems in the inspection of ribavirin raw materials and injections according to the current quality standards: The retention time of the main component ribavirin on the chromatographic column is too short (flow rate 0.4 mL·min-1, Rt is only 4 min), and the front and rear impurities cannot reach baseline separation from the main peak; the current chromatographic conditions only record up to 2 times the retention time of the main peak, and the impurities in the sample cannot be completely eluted; the responses of some impurities are inconsistent with ribavirin, resulting in inaccurate impurity content when calculated by the self-control method; the impurity response is not sensitive, often resulting in non-detection; therefore, the current standard fails to effectively control the quality of the product. Summary of the Invention

[0008] (1) Technical Problem to be Solved

[0009] In view of the deficiencies of the prior art, the present invention provides a high performance liquid chromatography method for determining the related substances of ribavirin injection, which has the advantages of effectively improving the quality of ribavirin injection and solving the problem of inaccurate determination of impurities in ribavirin injection.

[0010] (2) Technical Solution

[0011] To achieve the above object, the present invention provides the following technical solution: a high performance liquid chromatography method for determining the related substances of ribavirin injection, comprising the following steps:

[0012] Apparatus

[0013] LC-20AT high performance liquid chromatograph; XS205 type electronic analytical balance; KQ-300V type ultrasonic cleaner;

[0014] Reagents

[0015] Reference substances are as follows: ribavirin, uridine, uracil; impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H; ribavirin injection (1 batch each from 3 manufacturers, specification 100mg·mL-1, manufacturer A, batch number Z23001, manufacturer B, batch number B230419, manufacturer C, batch number 2304335); acetonitrile is chromatographically pure, water is purified water, and other reagents are all analytically pure;

[0016] Chromatographic Conditions

[0017] Chromatographic column: Horizone-C18 (250mm×4.6mm, 5μm), Ghost-CleanerI (50mm×4.6mm) ghost peak trap column; water (adjusted to pH 2.8 with 20% phosphoric acid solution) is used as mobile phase A; acetonitrile is used as mobile phase B; gradient elution: 0 - 15 min, A 100%, 15 - 20 min, A 100% → 93%, 20 - 35 min, A 93% → 60%, 35 - 36 min, A 60% → 20%, 36 - 40 min, A 20%, 40 - 41 min, A 20% → 100%, 41 - 60 min, A 100%; flow rate 1.0 mL·min-1, column temperature 20°C, detection wavelength 220nm, injection volume 20 μL;

[0018] Preparation of Solutions

[0019] Reference solution: Weigh accurately about 10 mg each of ribavirin and impurity reference substances, place them in the same 100 mL volumetric flask, add an appropriate amount of water, sonicate to dissolve, take out, cool to room temperature, dilute to the mark with water, and shake well to obtain a mixed reference stock solution; accurately measure 0.5 mL of the mixed reference stock solution and place it in a 20 mL volumetric flask, dilute to the mark with water, and shake well.

[0020] System suitability solution: Weigh accurately about 10 mg of ribavirin reference substance, place it in a 10 mL volumetric flask, accurately add 1 mL of the mixed reference stock solution under the item of "Reference solution", then dilute to the mark with water, and shake well.

[0021] Test solution: Accurately measure 1 mL of the injection, place it in a 100 mL volumetric flask, dilute to the mark with water, and shake well.

[0022] Interference of solvent and blank excipients

[0023] Take the solvent (water), blank excipient solution (prepare a solution with the corresponding concentration by taking the excipient sodium chloride according to the method under the item of "Test solution"), system suitability solution, reference solution, and test solution, and inject and determine according to the corresponding chromatographic conditions under the item of "Chromatographic conditions". The results show that the solvent and blank excipient solution have no interference on the determination, and the resolution between each substance in the system suitability solution is greater than 2.7, meeting the requirements of system suitability.

[0024] Test

[0025] The test includes destructive tests, calculation of the correction factors of each impurity, accuracy and repeatability tests, solution stability tests, precision tests, determination of detection limit and quantification limit, and the test results of related substances.

[0026] Preferably, for the destructive test: Take about 10 mL each of the samples (from Factory B, batch number B230419), and conduct high-temperature destruction (heat in a water bath at 100 °C for 4 h), sunlight irradiation destruction (place under (5000 ± 500) lx for 168 h), ultraviolet light irradiation destruction (irradiate simultaneously at 365 and 254 nm under (3000 ± 500) lx for 168 h), acid destruction (take 1 mL of the sample, add 1 mL of 1 mol·L-1 hydrochloric acid solution, place at room temperature for 2 h, and add 1 mL of 1 mol·L-1 sodium hydroxide solution to make the pH value neutral), alkali destruction (take 1 mL of the sample, add 1 mL of 1 mol·L-1 sodium hydroxide solution, place for 2 h, and add 1 mL of 1 mol·L-1 hydrochloric acid solution to make the pH value neutral), and oxidation destruction (take 1 mL of the sample, add 1 mL of 10% hydrogen peroxide solution, and place at room temperature for 2 h). After preparing the test solution according to the method under the item of "Preparation of solution", inject and determine according to the chromatographic conditions for related substances under the item of "Chromatographic conditions", record the peak areas, and compare with the non-destructed test solution.

[0027] The results showed that a large amount of impurity A was generated in the alkaline-damaged solution, and the impurity D in the solution was significantly increased under high-temperature damage. There was basically no change under sunlight, acid, and ultraviolet damage, and the impurity D increased slightly under oxidative damage. This indicated that ribavirin had poor stability in high-temperature and alkaline environments, was relatively stable under other conditions, and the degraded impurities and the main peak could be well separated.

[0028] Preferably, accurately measure 0.75 mL and 1.0 mL of the mixed reference stock solution under the "reference solution" item and place them in different 100 mL volumetric flasks respectively, and 0.25 mL, 0.5 mL, 0.75 mL, and 1.0 mL in different 10 mL volumetric flasks respectively. Dilute to the mark with water, shake well, and use as the linear reference solution. Inject and measure according to the corresponding chromatographic conditions under the "chromatographic conditions" item, record the peak area, use the concentration as the abscissa and the peak area as the ordinate for linear regression, and calculate the correction factors of each impurity;

[0029] The results showed that each compound had a good linear relationship with the corresponding peak area within a certain concentration range, and the responses of some impurities were inconsistent with that of ribavirin.

[0030] Preferably, for the accuracy and repeatability tests: accurately measure 1 mL each of 1 batch of ribavirin injection samples (from Factory B, batch number B230419) and take 9 portions, place them in 100 mL volumetric flasks respectively, accurately add 1 mL, 2.5 mL, and 5 mL of the mixed reference stock solution under the "reference solution" item (3 portions are added for each volume), dilute to the mark with water, shake well, and use as the test solution for recovery determination. Inject and measure according to the corresponding chromatographic conditions under the "chromatographic conditions" item, record the peak area and calculate the recovery rate. The results showed that the method had good accuracy and repeatability.

[0031] Preferably, for the solution stability test: take the related substances test solution of the sample under the "preparation of solution" item (from Factory B, batch number B230419), inject and measure according to the corresponding chromatographic conditions under the "chromatographic conditions" item at certain time intervals, and record the peak area;

[0032] The results showed that each impurity and ribavirin were stable (RSD of peak area was 0.84% - 6.75%), no new impurities were generated in the test solution, and the test solution was stable within 28 hours.

[0033] Preferably, for the precision test: take the mixed reference solution under the "reference solution" item, inject and measure continuously 6 times according to the corresponding chromatographic conditions under the "chromatographic conditions" item, record the peak area, indicating good precision.

[0034] Preferably, for the determination of the detection limit and quantification limit: precisely take 0.2 mL and 0.5 mL of the mixed reference stock solution under the item of "reference solution", place them in different 100-mL volumetric flasks respectively, dilute to the mark with water, shake well, and use them as solution 1 - 2 for the determination of the detection limit and quantification limit. Determine according to the chromatographic conditions under the item of "chromatographic conditions". Calculate the detection limit with a signal-to-noise ratio of 3 and the quantification limit with a signal-to-noise ratio of 10.

[0035] Preferably, for the detection result of related substances: take 3 batches of ribavirin injection, prepare the test solution for the detection of related substances according to the method under the item of "preparation of solution", and then inject and determine according to the corresponding chromatographic conditions under the item of "chromatographic conditions". Record the peak areas and calculate the impurity contents using the external standard method of reference substances (calculate other impurities using ribavirin reference substance); at the same time, determine according to the current quality standard.

[0036] Compared with the prior art, the present invention provides a high performance liquid chromatography method for the determination of related substances in ribavirin injection, which has the following beneficial effects:

[0037] 1. For the high performance liquid chromatography method for the determination of related substances in ribavirin injection, compared with the EP method, the method established herein can effectively separate multiple impurities; compared with the USP method, all impurities that need to be determined by different methods in USP can be determined by one method; compared with the CHP method, it has good resolution, high sensitivity, all impurities can be eluted, and the external standard method of reference substances is used for calculation, and the impurity contents are more accurate. The method established in the present invention is simple, sensitive, efficient, and accurate, and can be used for the quality control of ribavirin injection.

[0038] 2. For the high performance liquid chromatography method for the determination of related substances in ribavirin injection, multiple ribavirin impurities can be determined at one time. The method is accurate, simple, sensitive, and reliable, and can be used for the detection of related substances in ribavirin injection. Description of the Drawings

[0039] Figure 1 It is the solvent spectrum of the present invention;

[0040] Figure 2 It is the spectrum of the blank excipient solution of the present invention;

[0041] Figure 3 It is the spectrum of the system suitability solution of the present invention;

[0042] Figure 4 It is the spectrum of the reference solution of the present invention;

[0043] Figure 5 It is the typical spectrum of the test solution of the present invention (Factory A, batch number Z23001);

[0044] Figure 6 It is the spectrum of the intact test solution of the present invention;

[0045] Figure 7 The solution spectrum of the test sample damaged by acid for the present invention;

[0046] Figure 8 The solution spectrum of the test sample damaged by alkali for the present invention;

[0047] Figure 9 The blank solution spectrum of the test sample damaged by acid and alkali for the present invention;

[0048] Figure 10 The solution spectrum of the test sample damaged by oxidation for the present invention;

[0049] Figure 11 The blank solution spectrum of the test sample damaged by oxidation for the present invention;

[0050] Figure 12 The solution spectrum of the test sample damaged by high temperature for the present invention;

[0051] Figure 13 The solution spectrum of the test sample damaged by sunlight for the present invention;

[0052] Figure 14 The solution spectrum of the test sample damaged by ultraviolet for the present invention. Detailed implementation manners

[0053] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0054] Please refer to Figure 1-14 , a high performance liquid chromatography method for determining the related substances of ribavirin injection, comprising the following steps:

[0055] 1. Instruments

[0056] LC-20AT high performance liquid chromatograph; XS205 type electronic analytical balance; KQ-300V type ultrasonic cleaner;

[0057] 2. Test drugs

[0058] Reference substances are as follows: ribavirin, uridine, uracil; impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H; ribavirin injection (1 batch from each of 3 manufacturers, specification 100 mg·mL-1, manufacturer A, batch number Z23001, manufacturer B, batch number B230419, manufacturer C, batch number 2304335); acetonitrile is chromatographically pure, water is purified water, and other reagents are all analytically pure;

[0059] 3. Chromatographic conditions

[0060] Chromatographic column: Horizone-C18 (250 mm × 4.6 mm, 5 μm), Ghost-Cleaner I (50 mm × 4.6 mm) ghost peak trapping column. Water (adjusted to pH 2.8 with 20% phosphoric acid solution) was used as mobile phase A; acetonitrile was used as mobile phase B; gradient elution: 0 - 15 min, A 100%, 15 - 20 min, A 100% → 93%, 20 - 35 min, A 93% → 60%, 35 - 36 min, A 60% → 20%, 36 - 40 min, A 20%, 40 - 41 min, A 20% → 100%, 41 - 60 min, A 100%; flow rate 1.0 mL·min-1, column temperature 20 °C, detection wavelength 220 nm, injection volume 20 μL;

[0061] Selection of chromatographic conditions:

[0062] Since ribavirin and its impurities are all soluble in water, a chromatographic column resistant to pure water is required.

[0063] Six impurities (impurities A, B, C, D, F, G) were determined according to the EP method. Chromatographic column: Waters X- HSS T3 (50 mm × 4.6 mm, 5 μm), mobile phase A: 5.0 g of anhydrous sodium sulfate was dissolved in 950 mL of water, 2.0 mL of 5% phosphoric acid solution was added, the pH was adjusted to 2.8 with 5% phosphoric acid solution, and the volume was made up to 1000 mL with water; mobile phase B: acetonitrile - mobile phase A (5:95); gradient elution was carried out. As a result, the resolution between ribavirin and impurity D was greater than 4.0, and the separation of each substance was good. However, when a new impurity to be determined was added on the basis of the determination of the six impurities, the peak of ribavirin broadened, the resolution between ribavirin and impurity D decreased, and uracil and impurity A could not be separated. Attempts were made to replace the new chromatographic column, instrument, and adjust the ion concentration, etc., but none of them could solve the problem, and the requirement of determining multiple impurities by one method could not be met.

[0064] According to the structures of the components, attempts were made to elute using a 0.1% phosphoric acid (pH about 2.3)-acetonitrile system. Impurities A and B overlapped and could not be separated. An attempt was made to elute using a 0.1% formic acid (pH about 2.8)-acetonitrile system, and impurities A and B could be separated. However, after adding impurity G, the baseline and resolution were not good. Under the 0.1% formic acid-acetonitrile system, the isocratic elution time of pure water phase was extended to make the impurities elute before and after the gradient slope, and the column temperature was reduced from 30 °C to 20 °C, and the resolution of the impurities was improved. Under the 0.1% formic acid-acetonitrile system, the pure water-resistant chromatographic columns Horizon C18 / AR, Artchrom SSAQ, Horizon C18 / PFP, and Horizon AQUA C18 chromatographic columns (all with specifications of 250 mm × 4.6 mm, 5 μm) were screened under the optimal conditions. As a result, the resolution of Horizon C18 / AR was the best, and this column was selected for further optimization. Since the cut-off absorption wavelength of formic acid was near the detection wavelength and the gradient peak was obvious, an attempt was made to use phosphoric acid at the same pH value and it was found that the resolution of the two aqueous phases was relatively close. After optimizing the phosphoric acid system gradient, the resolution of each impurity met the requirements, and the chromatographic conditions described in this article were established.

[0065] Selection of detection wavelength

[0066] The chromatograms of the reference solution and the test solution for the detection of related substances were recorded using a diode array detector, and the ultraviolet spectra of each peak were extracted. As a result, uridine had a large absorption at 261 nm and uracil had a large absorption at 258 nm, and the remaining substances all had end absorption. Considering the ultraviolet absorption of each peak, in order to eliminate the interference of end absorption, 220 nm was used as the determination wavelength.

[0067] 4. Preparation of solutions

[0068] Reference solution: Weigh about 10 mg of ribavirin and each impurity reference substance accurately, place them in the same 100 mL volumetric flask, add an appropriate amount of water, sonicate to dissolve, take out, cool to room temperature, and dilute to the scale with water, and shake well to obtain a mixed reference stock solution; accurately measure 0.5 mL of the mixed reference stock solution and place it in a 20 mL volumetric flask, dilute to the scale with water, and shake well.

[0069] System suitability solution: Weigh about 10 mg of ribavirin reference substance accurately, place it in a 10 mL volumetric flask, accurately add 1 mL of the mixed reference stock solution under the item of "reference solution", and then dilute to the scale with water, and shake well.

[0070] Test solution: Accurately measure 1 mL of the injection solution, place it in a 100 mL volumetric flask, dilute to the scale with water, and shake well.

[0071] 5. Interference of solvents and blank excipients

[0072] Take the solvent (water), blank excipient solution (prepare the excipient sodium chloride into a solution with the corresponding concentration according to the item of "test solution"), system suitability solution, reference solution, and test solution, and inject and determine according to the corresponding chromatographic conditions under the item of "chromatographic conditions". The results show that the solvent and the blank excipient solution have no interference with the determination. The resolution between each substance in the system suitability solution is greater than 2.7, meeting the requirements of system suitability. The results are shown in Figures 1 to 5 .

[0073] 6. Tests

[0074] The tests include destructive tests, calculation of the correction factors of each impurity, accuracy and repeatability tests, solution stability tests, precision tests, determination of detection limit and quantification limit, and the test results of related substances.

[0075] I. Destructive tests:

[0076] Take about 10 mL of each sample (from Factory B, batch number B230419), and conduct high-temperature destruction (heat in a water bath at 100 °C for 4 h), sunlight irradiation destruction (place under (5000 ± 500) lx for 168 h), ultraviolet light irradiation destruction (irradiate simultaneously at 365 and 254 nm and place under (3000 ± 500) lx for 168 h), acid destruction (take 1 mL of the sample, add 1 mL of 1 mol·L-1 hydrochloric acid solution, place at room temperature for 2 h, and add 1 mL of 1 mol·L-1 sodium hydroxide solution to make the pH value neutral), alkali destruction (take 1 mL of the sample, add 1 mL of 1 mol·L-1 sodium hydroxide solution, place for 2 h, and add 1 mL of 1 mol·L-1 hydrochloric acid solution to make the pH value neutral), and oxidation destruction (take 1 mL of the sample, add 1 mL of 10% hydrogen peroxide solution, and place at room temperature for 2 h). After preparing the test solution according to the method under the item of "solution preparation", inject and determine according to the chromatographic conditions for related substances under the item of "chromatographic conditions", record the peak areas, and compare with the non-destructed test solution;

[0077] The results show that a large amount of impurity A is generated in the alkali-destroyed solution, and impurity D in the high-temperature-destroyed solution increases significantly. There is basically no change in sunlight, acid, and ultraviolet destruction, and impurity D in the oxidation-destroyed solution increases slightly. This indicates that ribavirin has poor stability in high-temperature and alkaline environments, and is relatively stable under other conditions. The degraded impurities and the main peak can be well separated. The results are shown in Figures 6 to 14 .

[0078] II. Linear experiment: Accurately measure 0.75 ml and 1.0 ml of the mixed reference stock solution under the item of "reference solution", respectively, and place them in different 100-ml volumetric flasks; accurately measure 0.25 ml, 0.5 ml, 0.75 ml, and 1.0 ml, respectively, and place them in different 10-ml volumetric flasks. Dilute to the mark with water, shake well, and use them as linear reference solutions. Inject and measure according to the corresponding chromatographic conditions under the item of "chromatographic conditions", record the peak areas, take the concentration as the abscissa and the peak area as the ordinate, perform linear regression, and calculate the correction factors of each impurity;

[0079] As a result, all compounds showed good linear relationships with their corresponding peak areas within a certain concentration range. The responses of some impurities were inconsistent with ribavirin. The results are shown in Table 1:

[0080] Determination results of standard curves

[0081]

[0082] Table 1

[0083] III. Accuracy and repeatability tests: Accurately measure 1 ml each of 1 batch of ribavirin injection samples (from Factory B, batch number B230419), and place them in 100-ml volumetric flasks. Accurately add 1 ml, 2.5 ml, and 5 ml of the mixed reference stock solution under the item of "reference solution" (3 portions for each volume), dilute to the mark with water, shake well, and use them as test solutions for recovery determination. Inject and measure according to the corresponding chromatographic conditions under the item of "chromatographic conditions", record the peak areas and calculate the recoveries. The results show that the method has good accuracy and repeatability. The results are shown in Table 2.

[0084] Determination results of recoveries

[0085]

[0086]

[0087]

[0088] Table 2

[0089] IV. Solution stability test: Take the related substances test solution of the sample (from Factory B, batch number B230419) under the item of "solution preparation", inject and measure according to the corresponding chromatographic conditions under the item of "chromatographic conditions" at certain time intervals, and record the peak areas. As a result, all impurities and ribavirin were stable (RSD of peak areas was 0.84% - 6.75%), no new impurities were generated in the test solution, and the test solution was stable within 28 hours. The results are shown in Table 3.

[0090] Determination results of solution stability

[0091]

[0092]

[0093] Table 3

[0094] V. Precision test: Take the mixed reference solution under the item of "Reference Solution", and inject samples continuously for 6 times according to the corresponding chromatographic conditions under the item of "Chromatographic Conditions", record the peak areas, and the results are shown in Table 4, indicating good precision.

[0095] Results of precision determination

[0096]

[0097] Table 4

[0098] VI. Determination of detection limit and quantitation limit: Accurately take 0.2 mL and 0.5 mL of the mixed reference stock solution under the item of "Reference Solution" respectively, place them in different 100-mL volumetric flasks, dilute to the mark with water, shake well, and use them as solutions 1 - 2 for determination of detection limit and quantitation limit. Determine according to the chromatographic conditions under the item of "Chromatographic Conditions", calculate the detection limit with a signal-to-noise ratio of 3, and calculate the quantitation limit with a signal-to-noise ratio of 10. The results are shown in Table 5.

[0099] Results of determination of detection limit and quantitation limit

[0100]

[0101] Table 5

[0102] VII. Results of related substances detection: Take 3 batches of ribavirin injection, prepare the test solution for related substances detection according to the method under the item of "Preparation of Solution", and then inject samples according to the corresponding chromatographic conditions under the item of "Chromatographic Conditions", record the peak areas and calculate the impurity contents using the external standard method of reference substance (calculate other impurities using ribavirin reference substance); determine according to the current quality standard, and the results are shown in Table 6.

[0103] Results of sample determination

[0104]

[0105]

[0106] Table 6

[0107] " / " in the figure indicates not detected. The final results in the sample determination results table are in percentages. CHP2020 stipulates that the content of a single impurity shall not exceed 0.25%, and the total content of all impurities shall not exceed 1.0%.

[0108] In summary, compared with the EP method, the method established in the present invention can effectively separate multiple impurities; compared with the USP method, all the impurities that need to be determined by different methods in the USP can be determined by one method; compared with the CHP method, it has good resolution, high sensitivity, all impurities can be eluted, and the external standard method with reference substances is used for calculation, and the impurity content is more accurate. Therefore, the method established in the present invention is simple, sensitive, efficient and accurate, and can be used for the quality control of ribavirin injection.

[0109] Although the embodiments of the present invention have been shown and described, those of ordinary skill in the art can understand that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A high performance liquid chromatography method for determining the related substances of ribavirin injection, characterized in that, It includes the following steps: Apparatus LC-20AT high performance liquid chromatograph; XS205 electronic analytical balance; KQ-300V ultrasonic cleaner Reagents Reference substances are as follows: ribavirin, uridine, uracil; impurities A, B, C, D, E, F, G, H; ribavirin injection (1 batch each from 3 manufacturers, specification 100mg·mL-1; Factory A, Kaifeng Kangnuo Pharmaceutical Co., Ltd., batch number Z23001; Factory B, Changzhi Sanbao Biochemical Pharmaceutical Co., Ltd., batch number B230419; Factory C, Guoyao Group Rongsheng Pharmaceutical Co., Ltd., batch number 2304335); acetonitrile is chromatographically pure, water is purified water, and other reagents are all analytically pure Chromatographic conditions Chromatographic column: Horizone-C18 (250mm×4.6mm, 5μm), Ghost-CleanerI (50mm×4.6mm) ghost peak trapping column; water (adjusted to pH 2.8 with 20% phosphoric acid solution) is used as mobile phase A; acetonitrile is used as mobile phase B; gradient elution: 0 - 15 min, A 100%, 15 - 20 min, A 100% → 93%, 20 - 35 min, A 93% → 60%, 35 - 36 min, A 60% → 20%, 36 - 40 min, A 20%, 40 - 41 min, A 20% → 100%, 41 - 60 min, A 100%; flow rate 1.0 mL·min-1, column temperature 20°C, detection wavelength 220 nm, injection volume 20 μL Preparation of solutions Reference substance solution: Take about 10 mg each of ribavirin and reference substances of each impurity, accurately weigh and place them in the same 100 mL volumetric flask, add an appropriate amount of water, ultrasonically dissolve, take out, cool to room temperature, dilute to the scale with water, shake well, and use as the mixed reference substance stock solution; accurately measure 0.5 mL of the mixed reference substance stock solution and place it in a 20 mL volumetric flask, dilute to the scale with water, and shake well System suitability solution: Take about 10 mg of ribavirin reference substance, accurately weigh and place it in a 10 mL volumetric flask, accurately add 1 mL of the mixed reference substance stock solution under the item of "reference substance solution", then dilute to the scale with water, and shake well Test sample solution: Accurately measure 1 mL of the injection and place it in a 100 mL volumetric flask, dilute to the scale with water, and shake well Interference from solvent and blank excipients Take the solvent (water), blank excipient solution (prepare a solution with the corresponding concentration by taking excipient sodium chloride according to the method under the item of "test sample solution"), system suitability solution, reference substance solution, and test sample solution, and inject and measure according to the corresponding chromatographic conditions under the item of "chromatographic conditions". The results show that the solvent and blank excipient solution have no interference on the determination, and the resolution between each substance in the system suitability solution is greater than 2.7, meeting the requirements of system suitability Tests The tests include destructive tests, calculation of the correction factors of each impurity, accuracy and repeatability tests, solution stability tests, precision tests, determination of detection limit and quantification limit, and test results of related substances 2. The high performance liquid chromatography method for determining the related substances of ribavirin injection according to claim 1, wherein Destructive tests: Take about 10 mL of each sample (from Factory B, batch number B230419), and conduct high-temperature destruction (heating in a water bath at 100 °C for 4 h), sunlight irradiation destruction (placing under (5000 ± 500) lx for 168 h), ultraviolet light irradiation destruction (simultaneously irradiating with 365 and 254 nm under (3000 ± 500) lx for 168 h), acid destruction (take 1 mL of the sample, add 1 mL of 1 mol·L-1 hydrochloric acid solution, place at room temperature for 2 h, and then add 1 mL of 1 mol·L-1 sodium hydroxide solution to make the pH value neutral), alkali destruction (take 1 mL of the sample, add 1 mL of 1 mol·L-1 sodium hydroxide solution, place for 2 h, and then add 1 mL of 1 mol·L-1 hydrochloric acid solution to make the pH value neutral), and oxidation destruction (take 1 mL of the sample, add 1 mL of 10% hydrogen peroxide solution, and place at room temperature for 2 h). After preparing the test solution according to the method under "Preparation of Solutions", inject and measure according to the chromatographic conditions for related substances under "Chromatographic Conditions", record the peak areas, and compare with the undamaged test solution; The results showed that a large amount of impurity A was generated in the alkali-damaged solution, the impurity D in the high-temperature-damaged solution increased significantly, there were basically no changes in sunlight, acid, and ultraviolet destruction, and the impurity D increased slightly in oxidation destruction, indicating that ribavirin has poor stability in high-temperature and alkaline environments, is relatively stable under other conditions, and the degraded impurities can be well separated from the main peak.

3. The high performance liquid chromatography method for determining the related substances of ribavirin injection according to claim 1, wherein: Precisely measure 0.75 and 1.0 mL of the mixed reference stock solution under "Reference Solution" and place them in different 100 mL volumetric flasks respectively, and 0.25, 0.5, 0.75, and 1.0 mL in different 10 mL volumetric flasks respectively. Dilute to the mark with water, shake well, and use as linear reference solutions. Inject and measure according to the corresponding chromatographic conditions under "Chromatographic Conditions", record the peak areas, use the concentration as the abscissa and the peak area as the ordinate for linear regression, and calculate the correction factors of each impurity; The results showed that each compound had a good linear relationship with the corresponding peak area within a certain concentration range, and the responses of some impurities were inconsistent with that of ribavirin.

4. The high performance liquid chromatography method for determining the related substances of ribavirin injection according to claim 1, wherein Accuracy and repeatability tests: Precisely measure 1 mL of each of 9 samples of a batch of ribavirin injection (from Factory B, batch number B230419) and place them in 100 mL volumetric flasks respectively. Precisely add 1, 2.5, and 5 mL of the mixed reference stock solution under "Reference Solution" (3 portions for each volume), dilute to the mark with water, shake well, and use as the test solution for recovery determination. Inject and measure according to the corresponding chromatographic conditions under "Chromatographic Conditions", record the peak areas and calculate the recoveries. The results showed that the method had good accuracy and repeatability.

5. The high performance liquid chromatography method for determining related substances in ribavirin injection according to claim 1, wherein Solution stability test: Take the related substances test solution (from Factory B, batch number B230419) under "Preparation of Solutions", inject and measure according to the corresponding chromatographic conditions under "Chromatographic Conditions" at certain intervals, and record the peak areas; The results showed that each impurity and ribavirin were stable (RSD of peak areas was 0.84% - 6.75%), no new impurities were generated in the test solution, and the test solution was stable within 28 hours.

6. The high performance liquid chromatography method for determining the related substances of ribavirin injection according to claim 1, characterized in that, Precision test: Take the mixed reference solution under the item of "Reference Solution", and inject samples continuously for 6 times according to the corresponding chromatographic conditions under the item of "Chromatographic Conditions", record the peak areas, indicating good precision.

7. The high performance liquid chromatography method for determining the related substances of ribavirin injection according to claim 1, characterized in that, Determination of detection limit and quantitation limit: Accurately take 0.2 mL and 0.5 mL of the mixed reference stock solution under the item of "Reference Solution" and place them in different 100-mL volumetric flasks respectively, dilute to the mark with water, shake well, and use them as solutions 1 - 2 for the determination of detection limit and quantitation limit. Determine according to the chromatographic conditions under the item of "Chromatographic Conditions", calculate the detection limit with a signal-to-noise ratio of 3, and calculate the quantitation limit with a signal-to-noise ratio of 10.

8. The high performance liquid chromatography method for determining the related substances of ribavirin injection according to claim 1, wherein Results of related substances detection: Take 3 batches of ribavirin injection, prepare the test solution for related substances detection according to the method under the item of "Preparation of Solution", then inject samples and determine according to the corresponding chromatographic conditions under the item of "Chromatographic Conditions", record the peak areas and calculate the impurity contents using the external standard method with reference substances (calculate other impurities using ribavirin reference substance); at the same time, determine according to the current quality standard.

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