Method for detecting impurities in moxifloxacin hydrochloride bulk drug

Abstract

The invention relates to the technical field of drug analysis and detection, and provides a detection method of moxifloxacin hydrochloride related substances. The related substances of moxifloxacin hydrochloride are detected by adopting a high performance liquid chromatography method, and the detection conditions are as follows: octadecylsilane chemically bonded silica is taken as a filling agent of a chromatographic column; the wavelength of a UV detector is 296 nm; the column temperature is 35-45 DEG C; the flow velocity of the mobile phase is 1.6-1.8 ml / min; the sample injection volume is 10-50 [mu] l; the mobile phase A is phosphate buffer solution-acetonitrile-methanol; the mobile phase B is phosphate buffer solution-acetonitrile-methanol; the elution mode is gradient elution; the diluent is a mobile phase A; according to the method provided by the invention, effective separation between moxifloxacin hydrochloride and various impurities can be realized, the sensitivity is high, the accuracy is good, the reproducibility is relatively good, a good linear relation is shown in a certain range, and qualitative and quantitative determination can be carried out on various impurities in moxifloxacin hydrochloride bulk drugs.

Description

Field of the Invention

[0001] The invention relates to the technical field of drug analysis and detection, and in particular to a method for determining impurities in a moxifloxacin hydrochloride raw material drug. Background Art

[0002] Moxifloxacin is a fourth-generation broad-spectrum quinolone antibiotic with broad-spectrum antibacterial activity against Gram-positive and Gram-negative bacteria, anaerobic bacteria, acid-fast bacteria, and atypical microorganisms (such as mycoplasma, chlamydia, and Legionella). In clinical applications, moxifloxacin has shown good safety and oral absorption, with an average absolute bioavailability of up to 90%. However, a variety of impurities will be generated during the production, preparation, and storage of moxifloxacin hydrochloride injection; these impurities may not only affect the efficacy of the drug, but also increase the risk of adverse reactions. Therefore, it is necessary to separate and test moxifloxacin hydrochloride and its impurities. By testing the impurity content, it is possible to evaluate whether the quality of the drug meets the standard, thereby avoiding the use of unqualified products; secondly, impurities may have adverse reactions to the human body. Through separation and detection, these impurities can be discovered and controlled in time to ensure the safety of patients' medication; at the same time, through separation and detection, the source and conditions of impurity generation can be understood, which helps to optimize the production process, reduce the generation of impurities, and improve the purity and stability of the product.

[0003] High performance liquid chromatography (HPLC) is the main method for detecting and separating moxifloxacin and its impurities, but in practical applications, there are still problems such as poor specificity, poor separation effect and poor sensitivity. For some impurities with similar structures and similar properties, their molecular structures contain multiple functional groups, such as fluorine, hydroxyl, methoxy, carboxyl and complex heterocyclic structures. The interaction between these functional groups may lead to the formation of hydrogen bonds and π-π stacking between molecules. The similarity of these structures will also cause their physical and chemical properties to be similar. When HPLC is used for detection and separation, the separation efficiency is not high enough, resulting in the impurity peak overlapping with the main peak or being difficult to distinguish, which will affect the accuracy and reliability of the detection; and for impurities with extremely low content, the detection sensitivity of HPLC may be insufficient, and it is difficult to accurately determine their content, which may lead to missed detection or misjudgment.

[0004] According to the analysis of the synthetic process route of moxifloxacin hydrochloride in the literature, methyl ester impurities, impurity F, impurity E, hydroxyquinoline impurities, methoxyquinoline impurities, and quinoline ethyl ester impurities are process impurities that are very likely to be introduced or produced by degradation, and are impurities that need to be monitored. Therefore, a detection method that can monitor the above impurities in moxifloxacin hydrochloride raw materials is needed.

[0005] A variety of determination methods for moxifloxacin hydrochloride and its impurities are disclosed in the related art. However, based on the synthesis process and stability research data of moxifloxacin hydrochloride and the degradation impurities that may appear during the preparation and storage process, the impurities in moxifloxacin hydrochloride are not completely consistent with the disclosed technology, and some determination methods have too long detection time (more than 60 minutes), which cannot meet the actual needs.

[0006] Chinese patent CN118937545A discloses a method for separating moxifloxacin and its impurities; the high performance liquid chromatography detection conditions include: the chromatographic column is a chromatographic column with phenylsilane bonded silica gel as filler; the mobile phase includes mobile phase A and mobile phase B, mobile phase A is a buffer solution, and mobile phase B is methanol; the pH of the buffer solution is 2-3; the column temperature is 40-45°C; the mobile phase flow rate is 1.0-1.8mL / min; the injection volume is 5-15μl; the elution method is gradient elution; the detection wavelength is 277nm. The impurities are impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity ACB-8, impurity ACB-9, impurity ACB-10, impurity I, impurity J, impurity K and impurity L.

[0007] Chinese patent CN118584016A discloses a method for determining impurities in moxifloxacin hydrochloride raw materials. The determination method is as follows: chromatographic column: phenylsilane bonded silica gel as filler; flow rate: 1.2-1.4ml / min; detection wavelength: 280-300nm; injection volume: 10-50μl; column temperature: 40-50℃; mobile phase: potassium dihydrogen phosphate-tetrabutylammonium hydrogen sulfate solution as mobile phase A, methanol as mobile phase B. The impurities are impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, impurity I, impurity J, impurity SM1-1, and impurity SM2.

[0008] Chinese patent 113588837A provides a method for detecting related substances of moxifloxacin hydrochloride. The present invention uses high performance liquid chromatography to detect related substances of moxifloxacin hydrochloride, and the detection conditions include: the chromatographic column is octadecylsilane bonded silica gel as a filler; the UV detector wavelength is 291nm-319nm; the column temperature is 25℃-40℃; the mobile phase flow rate is 0.8-1.3mL / min; the injection volume is 10-50μL; the mobile phase A is a phosphate buffer-acetonitrile-methanol mixed solution; the mobile phase B is a phosphate buffer-acetonitrile-methanol mixed solution; the elution method is gradient elution; the diluent is a phosphate buffer-sulfate mixed solution. The impurities are impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity H, impurity J, impurity K, impurity L, impurity M, impurity P, impurity R, and impurity V. Summary of the invention

[0009] The present application provides a method for detecting impurities in a moxifloxacin hydrochloride raw material drug.

[0010] According to the synthetic process and stability research data of moxifloxacin hydrochloride, this application analyzes the process impurities of moxifloxacin hydrochloride and the impurities that may be degraded during the preparation and storage process, and formulates a corresponding impurity control strategy. The method for determining impurities in the moxifloxacin hydrochloride raw material provided in this application can improve the quality of moxifloxacin hydrochloride.

[0011] The present application provides a method for determining impurities in moxifloxacin hydrochloride raw materials, using the following technical solution:

[0012] A method for determining impurities in a moxifloxacin hydrochloride bulk drug, the method being specifically as follows:

[0013] The chromatographic column is a chromatographic column using octadecylsilane bonded silica gel as filler;

[0014] The mobile phase includes mobile phase A and mobile phase B, mobile phase A is buffer solution-acetonitrile-methanol, and mobile phase B is buffer solution-acetonitrile-methanol; the pH of the buffer solution is 2-3; the column temperature is 35-45°C; the flow rate of the mobile phase is 1.5-1.9 mL / min; the injection volume is 10-30 μL; the elution method is gradient elution; the detection wavelength is 286-306 nm;

[0015] Alternatively, a gradient elution program is as follows

[0016] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 100 0 20 100 0 29 94 6 43 0 100 49 0 100 52 100 0 60 100 0

[0017] Preferably, the pH of the buffer solution is 2.45-2.55; the column temperature is 40° C.; the flow rate of the mobile phase is 1.7 ml / min; and the injection volume is 20 μL.

[0018] Preferably, the buffer solution contains potassium dihydrogen phosphate, phosphoric acid, triethylamine and water.

[0019] More preferably, the preparation method of the buffer solution comprises the following steps: dissolving potassium dihydrogen phosphate in water, adding triethylamine, and then adjusting the pH with phosphoric acid.

[0020] More preferably, the solid-liquid ratio of potassium dihydrogen phosphate, triethylamine and water is 3.4g:0.5ml:1L.

[0021] Preferably, the chromatographic column model is YMC-Pack ODS-A, with dimensions of 250 mm×4.6 mm and 5 μm.

[0022] Optionally, the impurity limits are: methyl ester impurities, impurity F, impurity E, hydroxyquinoline impurities, methoxyquinoline impurities and quinoline ethyl ester impurities shall not exceed 0.10%; other individual impurities shall not exceed 0.10%; the total amount of impurities shall not exceed 0.30%.

[0023] Optionally, the diluent used in the determination method is mobile phase A.

[0024] Preferably, the pH of the buffer solution is 2.45 to 2.55; the column temperature is 40°C;

[0025] The method has good specificity, accuracy and precision, high sensitivity, good linear relationship within the impurity limit concentration range of 0.015% to 0.6%, and can achieve single detection of moxifloxacin and its impurities with low cost and high efficiency.

[0026] The technical solution adopted to achieve the above-mentioned purpose is to provide a method for separating and detecting moxifloxacin and its impurities, comprising the following steps:

[0027] Passing the sample to be tested into high performance liquid chromatography to separate and detect moxifloxacin and its impurities;

[0028] The high performance liquid chromatography detection conditions include: the chromatographic column is a chromatographic column filled with octadecylsilane bonded silica gel; the mobile phase includes mobile phase A and mobile phase B, mobile phase A is buffer solution-acetonitrile-methanol, and mobile phase B is buffer solution-acetonitrile-methanol; the pH of the buffer solution is 2-3; the column temperature is 35-45°C; the mobile phase flow rate is 1.5-1.9 mL / min; the injection volume is 10-30 μL; the elution method is gradient elution; the detection wavelength is 296 nm; the impurities are methyl ester impurities, impurity F, impurity E, hydroxyquinoline impurities, methoxyquinoline impurities, and quinoline ethyl ester impurities, and the specific structural formulas are shown in Table 1.

[0029] Table 1 Impurity information

[0030]

[0031]

[0032] Based on the above, it is necessary to develop a new method for determining impurities in moxifloxacin hydrochloride raw materials to meet the requirements for determining impurities in moxifloxacin hydrochloride raw materials and to improve the quality of moxifloxacin hydrochloride.

[0033] The gradient elution program was

[0034]

[0035] Preferably, the pH of the buffer solution is 2.45-2.55; the column temperature is 40° C.; the flow rate of the mobile phase is 1.7 ml / min; and the injection volume is 20 μL.

[0036] Preferably, the buffer solution contains potassium dihydrogen phosphate, phosphoric acid, triethylamine and water.

[0037] More preferably, the method for preparing the buffer solution comprises the following steps: dissolving potassium dihydrogen phosphate in water, adding triethylamine, and then adjusting the pH with phosphoric acid.

[0038] More preferably, the solid-liquid ratio of potassium dihydrogen phosphate, triethylamine and water is 3.4g:0.5ml:1L.

[0039] Preferably, the chromatographic column model is YMC-Pack ODS-A, with dimensions of 250 mm×4.6 mm and 5 μm.

[0040] It is easy for those skilled in the art to understand that some of the operating steps of the analytical method described in this application can be replaced without affecting the results. For example, the preparation steps of the test solution, the reference solution, and the system suitability solution can be replaced at will.

[0041] In this application, related substances are also expressed as impurities.

[0042] In the present application, an appropriate amount means that the amount of each compound is within the detection line or quantitative limit of its high performance liquid chromatography according to the experimental purpose.

[0043] In the present application, suitable concentration means that the concentration of each compound is within the detection limit or quantitative limit of its high performance liquid chromatography according to the experimental purpose.

[0044] Unless otherwise specified, the abbreviations in this application have the following meanings:

[0045] min: minute;

[0046] ml: milliliter;

[0047] μl: microliter;

[0048] LOQ: limit of quantitation;

[0049] The beneficial effects of the present invention are:

[0050] (1) The method for detecting impurities in the moxifloxacin hydrochloride raw material provided by the present invention can effectively separate moxifloxacin hydrochloride, methyl ester impurities, impurity F, impurity E, hydroxyquinoline impurities, methoxyquinoline impurities and quinoline ethyl ester impurities by selecting a suitable mobile phase and controlling the concentration and flow rate of the mobile phase, the chromatographic column, the column temperature and other conditions, which can help product quality research, provide an effective means for product development and detection and release, more comprehensively guarantee the quality control of moxifloxacin hydrochloride, and further improve the safety of drug use.

[0051] (2) The method for detecting impurities in the moxifloxacin hydrochloride raw material provided by the present invention has a short gradient elution time, improves the analysis efficiency, saves the amount of organic solvent in the mobile phase, reduces the detection cost, and reduces environmental pollution. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 The HPLC separation chromatogram of the embodiment; DETAILED DESCRIPTION

[0053] Before describing the embodiments of the present application in detail, it should be understood that the terms used herein are only used for the purpose of describing specific embodiments. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by ordinary technicians in the field to which the terms belong.

[0054] The endpoints and any values ​​of the ranges disclosed in this application are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0055] In the present application, the terms "comprise" or "include" are open expressions, that is, including the contents specified in the present application but not excluding other contents.

[0056] The impurity limits for raw materials in the ICHQ3A guidelines are shown in Table 2.

[0057] Table 2 ICHQ3A Guidelines China's regulations on impurity limits for raw materials

[0058] Maximum daily dose Reporting Limits Identification Limit Quality control limit ≤2g 0.05％ 0.10% or 1.0mg (the minimum value) 0.15% or 1.0mg (the minimum value) ＞2g 0.03％ 0.05％ 0.05％

[0059] According to the instructions for use of moxifloxacin hydrochloride, the maximum daily dose of moxifloxacin hydrochloride is 400 mg. Combined with Table 2, the reporting limit of impurities in the moxifloxacin hydrochloride raw material is 0.05%, the identification limit is 0.10%, and the quality control limit is 0.15%.

[0060] The present application provides a method for determining impurities in a moxifloxacin hydrochloride raw material.

[0061] In this determination method, the impurity limits are: methyl ester impurity, impurity F, impurity E, hydroxyquinoline impurity, methoxyquinoline impurity and quinoline ethyl ester impurity shall not exceed 0.10%; other individual impurities shall not exceed 0.10%; the total amount of impurities shall not exceed 0.30%.

[0062] The determination method specifically comprises the following steps:

[0063] Chromatographic column: octadecylsilane bonded silica gel as filler (YMC-Pack ODS-A) (4.6mm×250mm, 5μm); flow rate: 1.6~1.8ml / min; detection wavelength 286~306nm, injection volume: 10~50μl; column temperature: 35~45℃; mobile phase: phosphate buffer-acetonitrile-methanol as mobile phase A, phosphate buffer-acetonitrile-methanol as mobile phase B.

[0064] The elution program is shown in Table 3.

[0065] Table 3 Elution program

[0066] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 100 0 20 100 0 29 94 6 43 0 100 49 0 100 52 100 0 60 100 0

[0067] In a specific embodiment, the determination method specifically comprises the following steps:

[0068] Chromatographic column: octadecylsilane bonded silica gel as filler (YMC-Pack ODS-A) (4.6 mm × 250 mm, 5 μm); flow rate: 1.7 ml / min; detection wavelength 296 nm, injection volume: 20 μl; column temperature: 40 °C;

[0069] Mobile phase: phosphate buffer-acetonitrile-methanol as mobile phase A, phosphate buffer-acetonitrile-methanol as mobile phase B.

[0070] The elution program is shown in Table 4.

[0071] Diluent: mobile phase A;

[0072] System suitability: Take 20 μl of system suitability solution, inject it into liquid chromatograph, record the chromatogram, the order of peaks is methyl ester impurity, impurity F, moxifloxacin hydrochloride, impurity E, hydroxyquinoline impurity, methoxyquinoline impurity and quinoline ethyl ester impurity; the separation degree between moxifloxacin peak and impurity F peak should be greater than 1.5.

[0073] In this application, the solution is prepared as follows:

[0074] System suitability solution: Weigh appropriate amounts of moxifloxacin hydrochloride reference substance and its impurities (the names, structural formulas and sources of the impurities are shown in Table 1; the information of moxifloxacin hydrochloride and each impurity reference substance is shown in Table 2), dissolve in a diluent and dilute to prepare a solution containing approximately 1.0 mg of moxifloxacin and 1.0 μg of each impurity per 1 ml;

[0075] Reference solution: Take an appropriate amount of moxifloxacin hydrochloride reference substance, weigh accurately, add diluent to dissolve and quantitatively dilute to make a solution containing 1.0 μg of moxifloxacin per 1 ml;

[0076] Test solution: Take an appropriate amount of moxifloxacin hydrochloride bulk drug, weigh accurately, add diluent to dissolve and quantitatively dilute to make a solution containing 1.0 mg of moxifloxacin per 1 ml;

[0077] If no specific technology or conditions are specified in this application, the technology or conditions described in the literature in this field or the product instructions shall be followed. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0078] In order to make the purpose, technical solutions and advantages of this application clearer, the technical solutions in the embodiments of this application are clearly and completely described below in conjunction with the accompanying drawings. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without making creative work are within the scope of protection of this application.

[0079] The present application is further described in detail below in conjunction with the embodiments, drawings and test results.

[0080] Example This example provides a method for detecting impurities in moxifloxacin hydrochloride raw material. The test solution is tested using the test method of the present application.

[0081] The determination method specifically comprises the following steps:

[0082] Chromatographic column: octadecylsilane bonded silica gel as filler (YMC-Pack ODS-A) (4.6 mm × 250 mm, 5 μm); flow rate: 1.7 ml / min; detection wavelength 296 nm, injection volume: 20 μl; column temperature: 40 °C;

[0083] Mobile phase: phosphate buffer-acetonitrile-methanol (80:12:8) was used as mobile phase A, and phosphate buffer-acetonitrile-methanol (20:48:32) was used as mobile phase B.

[0084] Phosphate buffer: Take 3.4 g of potassium dihydrogen phosphate (KH2PO4), add 800 ml of water to dissolve, add 0.5 ml of triethylamine, adjust the pH value to 2.5±0.05 with phosphoric acid, and dilute to 1000 ml with water.

[0085] The elution program is shown in Table 4.

[0086] The detection test refers to the "Technical Guidelines for Validation of Chemical Drug Quality Control Analysis Methods" and the "Technical Guidelines for Research on Impurities in Chemical Drugs", and the determination method provided in Example 1 is used for system validation. The validation items and validation results are shown in Table 5.

[0087] Table 4 Methodological validation items and validation results

[0088]

[0089]

[0090]

[0091] (I) Exclusivity

[0092] (1) Impurity separation test and system suitability test

[0093] Using the chromatographic conditions in the determination method of this application, the elution and separation of each known impurity were investigated, and the suitability of the system was also investigated. Blank solution: mobile phase A.

[0094] The methyl ester impurity localization solution, the impurity F localization solution, the impurity E localization solution, the hydroxyquinoline impurity localization solution, the methoxyquinoline impurity localization solution, the quinoline ethyl ester impurity localization solution and the moxifloxacin hydrochloride localization solution were prepared respectively.

[0095] The method for preparing the localization solution is as follows: take about 2 mg of each impurity reference substance, place it in a 200 ml volumetric bottle, add a diluent to dissolve and dilute to the mark, as the stock solution corresponding to the impurity. Then accurately measure 1 ml and place it in a 10 ml volumetric bottle, add a diluent to dilute to the mark, as the impurity localization solution. Take 10 mg of the moxifloxacin hydrochloride reference substance and place it in a 10 ml volumetric bottle, add a diluent to dissolve and dilute to the mark, as the moxifloxacin localization solution.

[0096] System suitability solution: Take 2 mg of each of methyl ester impurity, N-methyl impurity (impurity F), demethyl impurity (impurity E), hydroxyquinoline impurity, methoxyquinoline impurity and quinoline ethyl ester impurity and place them in different 200 ml volumetric bottles, add mobile phase A to dissolve and dilute to scale. Take 10 mg of moxifloxacin hydrochloride reference substance and place it in a 10 ml volumetric bottle, transfer 1 ml of each impurity solution to the 10 ml volumetric bottle, add mobile phase A to dissolve and dilute to scale, and shake well.

[0097] Accurately measure 20 μl of each of the above solutions, inject into the liquid chromatograph, and record the chromatogram. The results are shown in Table 5.

[0098] Table 5 Summary of specificity results

[0099]

[0100]

[0101] As can be seen from Table 6, the blank solvent does not interfere with the determination, the separation between the impurities and the main peak is greater than 1.5, and the separation between each known impurity is greater than 1.5, indicating that the determination method provided in this application has good specificity.

[0102] (2) Degradation test

[0103] The chromatographic conditions in the determination method of this application were used to investigate the stability of moxifloxacin hydrochloride under acid, alkali, oxidation, high temperature and light conditions. The separation degree between the new impurities and the main peak and the purity of the main peak were investigated under various degradation test conditions. The peak area normalization method was used to calculate the impurity content and evaluate the conservation of materials.

[0104] Blank solution: diluent.

[0105] Undamaged test solution: Take about 10 mg of moxifloxacin hydrochloride bulk drug (batch number: XXXX), accurately weigh, place in a 10 ml volumetric flask, add diluent to dissolve and dilute to the scale, shake well, and obtain.

[0106] 1) Acid degradation test:

[0107] Acid degradation solution: Take about 10 mg of moxifloxacin hydrochloride raw material, place it in a 10 ml volumetric flask, add 1 ml of hydrochloric acid test solution, leave it at room temperature for 3 hours, add 1 ml of sodium hydroxide test solution to neutralize it, add diluent to dilute to the scale, shake well, and obtain.

[0108] 2) Alkali degradation test:

[0109] Alkali degradation solution: Take about 10 mg of moxifloxacin hydrochloride raw material, accurately weigh, place in a 10 ml volumetric flask, add 1 ml of sodium hydroxide test solution, leave at room temperature for 3 hours, add 1 ml of hydrochloric acid test solution to neutralize, add diluent to dilute to the scale, shake well, and obtain.

[0110] 3) Oxidative degradation test

[0111] Oxidative degradation solution: Take about 10 mg of moxifloxacin hydrochloride raw material, accurately weigh it, put it in a 10 ml volumetric bottle, add 3%

[0112] 2 ml of hydrogen peroxide is placed at room temperature for 2 hours, and then mobile phase A is added to dilute to the scale and shake well.

[0113] 4) High temperature degradation test

[0114] High temperature degradation solution: Take an appropriate amount of moxifloxacin hydrochloride raw material, place it in a 105℃ oven for 2 hours, take it out and let it cool, accurately weigh about 10 mg, place it in a 10ml volumetric flask, add mobile phase A to dissolve and dilute to the scale, shake well, and obtain.

[0115] 5) Light degradation test

[0116] Photodegradation solution: Take an appropriate amount of moxifloxacin hydrochloride raw material, place it in a visible light (4500Lux±500Lux) light box for 24 hours, take it out, accurately weigh about 10 mg, place it in a 10ml volumetric flask, add mobile phase A to dilute to the scale, shake well, and obtain.

[0117] Accurately measure 20 μl of the above solutions respectively, inject them into the liquid chromatograph, and record the chromatogram.

[0118] The results are shown in Table 6

[0119] Table 6 Separation degree between peaks and material conservation analysis of relevant substance degradation test

[0120] Degradation test Separation degree between main peak and preceding and following impurity peaks Material balance rate (%) Alkali destruction test solution 1.6 100.2 Acid destruction of test solution 1.9 104.0 Oxidative damage to test solution 1.6 100.0 High temperature destroys the test solution 1.6 99.8 Light destruction of test solution 1.8 99.6

[0121] As can be seen from Table 6, the separation between the impurity peak and the main peak produced after the degradation of moxifloxacin hydrochloride raw material by acid, alkali, oxidation, high temperature and light meets the requirements, the balance index is between 0.996 and 1.040, and the conservation of materials meets the requirements, indicating that the determination method provided in this application has strong specificity and is suitable for the detection of related substances.

[0122] (II) Injection precision

[0123] Reference solution:

[0124] Using the determination method provided in Example 1, accurately measure 20 μl, inject into the liquid chromatograph, inject 6 times continuously, calculate the peak area RSD, the results are shown in Table 7.

[0125] Table 7 Results of instrument injection precision test for related substances

[0126]

[0127] As shown in Table 7, after 6 consecutive injections, the RSD of the main component peak area was less than 2.0%, indicating that the precision of the instrument met the requirements.

[0128] (III) Repeatability

[0129] Using the chromatographic conditions in the determination method of this application, accurately measure 20 μl of each of the system suitability solution, reference solution, and test solution, inject them into the liquid chromatograph, and record the chromatogram. Six replicates were measured in parallel. The results are shown in Table 8.

[0130] Table 8 Reproducibility test results of related substances

[0131]

[0132]

[0133] As shown in Table 8, the test sample was measured 6 times, and there was no significant difference in the content of each impurity, indicating that the determination method of the present application has good repeatability.

[0134] (IV) Intermediate precision

[0135] In order to investigate the influence of random variation factors on precision, different personnel conducted the test at different times using different instruments. The solution preparation was tested by repeatability test. 20 μl of system suitability solution, reference solution and test solution were accurately measured and injected into the liquid chromatograph to record the chromatogram. RSD, i.e. intermediate precision, was calculated together with the repeatability test results. The results are shown in Table 9.

[0136] Table 9 Results of intermediate precision tests on related substances

[0137]

[0138]

[0139] From the results in Table 9, it can be seen that different personnel determined the relevant substances using a repeatable method at different times, and there was no significant change in the impurity content of the 12 measurement results, indicating that the intermediate precision was good.

[0140] (V) Solution stability

[0141] System Suitability Solutions:

[0142] Reference solution:

[0143] Test solution: Take about 10 mg of moxifloxacin hydrochloride raw material, place it in a 10 ml volumetric flask, add diluent to dissolve and dilute to the scale, shake well, and obtain.

[0144] Take 20 μl of system suitability solution, reference solution, and test solution, respectively, and inject them into the liquid chromatograph to record the chromatogram. The stability of the reference solution and the test solution at room temperature was investigated using the change in the peak area of ​​the main component in the reference solution and the change in the impurity content in the test solution as indicators. The results are shown in Tables 10 and 11.

[0145] Table 10 Results of stability test of relevant substance solutions - reference solution

[0146]

[0147] Table 11 Results of stability test of relevant substance solutions - test solution

[0148]

[0149]

[0150] From the results in Table 10 and Table 11, it can be seen that within 24 hours, the ratio of the main component peak area to the 0h peak area of ​​the reference solution is between 99% and 100%, indicating that the reference solution is stable within 24 hours at room temperature. Within 24 hours, the content of each known impurity and unknown impurity in the test solution has no significant change, indicating that the test solution is stable within 24 hours at room temperature. Meet the detection requirements.

[0151] (VI) Limit of Quantitation and Limit of Detection

[0152] Using the assay method provided in Example 1, take an appropriate amount of moxifloxacin hydrochloride reference substance and dilute it step by step until the signal-to-noise ratio (S / N) is about 10, which is the limit of quantification, and the signal-to-noise ratio (S / N) is about 3, which is the limit of detection. The summary results of the limit of quantification and the limit of detection are shown in Tables 12 and 13.

[0153] Table 12 Results of quantitative limit test of related substances

[0154]

[0155]

[0156] Table 13 Results of detection limit test of related substances

[0157]

[0158] The quantitative limit results were obtained by continuous injection of 6 injections. The results are shown in Table 14.

[0159] Table 14 Results of the quantitative limit test of related substances.

[0160] Moxifloxacin hydrochloride 1 2 3 4 5 6 average value RSD% Retention time 21.293 21.304 21.315 21.301 21.291 21.343 21.308 0.1 Peak area 4532 4446 4441 4632 4239 4526 4469 3.0

[0161] As shown in Tables 12 to 14, the limit of quantification of moxifloxacin hydrochloride is 0.052 μg / ml, equivalent to 0.005% of the test sample concentration, and the limit of detection is 0.021 μg / ml, equivalent to 0.002% of the test sample solution concentration. The impurity detection requirements are met, and the repeatability peak area RSD of the limit of quantification is less than 5%, indicating that the determination method provided in this application has high sensitivity.

[0162] (VII) Linearity and Range Take appropriate amount of each impurity and moxifloxacin hydrochloride reference substance, add solvent to prepare a series of concentration solutions (50%, 75%, 100%,

[0163] 150%, 200%). Accurately measure 20 μl of each of the above solutions, inject into liquid chromatograph, record the chromatogram, and perform linear regression with concentration as the horizontal axis (X) and peak area as the vertical axis (Y). The results are shown in Table 15.

[0164] Table 15 Linearity test results of related substances

[0165]

[0166]

[0167] It can be seen from Table 15 that within the concentration range of 0.0522 μg / ml to 2.088 μg / ml, the concentration and peak area of ​​moxifloxacin hydrochloride present a good linear relationship, and the linear equation is y=71332x+828.27, r=0.9999.

[0168] Correction Factor

[0169] Different personnel, on different dates, used non-instrumentation methods to prepare linear solutions according to the linear solution preparation method under "Linearity and Range" and injected the samples for determination. Linear fitting was performed with concentration as the abscissa and peak area as the ordinate. Impurity correction factor = slope of the linear regression equation of moxifloxacin hydrochloride / slope of the linear regression equation of each impurity. The correction factor results are summarized in Table 16.

[0170] Table 16 Determination results of correction factors for related substances

[0171] name Person 1 Person 2 average Methyl ester impurity 1.091 1.148 1.12 Impurity F 1.135 1.106 1.12 Impurity E 4.098 3.911 4.00 Hydroxyquinoline Impurity 4.946 5.053 5.00 Methoxyquinoline Impurity 4.513 4.591 4.55 Quinoline Ethyl Ester Impurity 3.504 3.399 3.45

[0172] (VIII) Accuracy

[0173] Test solution: Take 10 mg of Moxifloxacin hydrochloride API, place in a 10 ml volumetric flask, add solvent to dissolve and dilute to the mark.

[0174] Shake well and serve.

[0175] Stock solution of reference substance: Take 5 mg of each impurity, place it in a 100 ml volumetric flask, add solvent to dissolve and dilute to the scale.

[0176] Control solution: Take 1 ml of the reference stock solution, place it in a 50 ml volumetric flask, and add solvent to dilute to the scale.

[0177] Low concentration test solution (50%): Take 10 mg of moxifloxacin hydrochloride raw material, put it in a 10 ml volumetric flask, add 1 ml of reference substance stock solution, then add solvent to dilute to the scale, shake well, as the low concentration test solution, prepare three times in parallel.

[0178] Medium concentration test solution (100%): Take 10 mg of moxifloxacin hydrochloride raw material, place it in a 10 ml volumetric flask, add 2 ml of reference substance stock solution, then add solvent to dilute to the scale, shake well, and use it as the low concentration test solution. Prepare three copies in parallel.

[0179] High concentration test solution (150%): Take 10 mg of moxifloxacin hydrochloride raw material, put it in a 10 ml volumetric flask, add 3 ml of reference substance stock solution, then add solvent to dilute to the scale, shake well, and use it as the low concentration test solution. Prepare three copies in parallel.

[0180] Accurately measure 20 μl of each of the above solutions, inject into the liquid chromatograph, record the chromatogram, and calculate the recovery rate of each impurity. The results are shown in Table 17.

[0181] Table 17 Results of the test on the recovery rate of related substances

[0182]

[0183]

[0184] It can be seen from Table 17 that, in the range of 50%-150% equivalent to the limit concentration, the recovery rate of each impurity calculated by the external standard method is between 95% and 105%, indicating that the method is highly accurate and meets the requirements for the determination of relevant substances.

[0185] (IX) Durability

[0186] The flow rate, column temperature, mobile phase ratio, phosphate concentration, and different batches of chromatographic columns in the chromatographic conditions were appropriately changed, and the robustness of the method was investigated using the change in separation between the peaks of each component in the system suitability solution as an indicator. The chromatographic conditions are shown in Table 18.

[0187] Table 18 Chromatographic conditions for durability of related substances detection

[0188]

[0189] Blank solution: solvent (mobile phase A).

[0190] System suitability solution: Prepare the system suitability solution in the same manner as the system suitability solution under the specificity item.

[0191] Accurately measure 20 μl of each of the above solutions and inject them according to the above chromatographic conditions. The results are shown in Table 19.

[0192] Table 19: Durability test results of related substances - System suitability solutions

[0193]

[0194]

[0195]

[0196]

[0197] As shown in Table 19, when the determination method of the present application is used to determine the related substances, the flow rate, column temperature, the amount of phosphoric acid in the mobile phase buffer salt phase, and the use of a chromatographic column of a different batch number from the same manufacturer have little effect on the content of impurities, the separation between the peaks in the system applicability solution and between the peaks and the main peak meet the requirements, and the number of theoretical plates meets the requirements. That is, a slight change in the flow rate, column temperature, the amount of phosphoric acid in the mobile phase buffer salt phase, and the chromatographic column in the chromatographic conditions does not affect the test results, indicating that the durability is good.

[0198] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit it. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present application.

[0199] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process conversion made using the specification and drawings of the present invention, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A method for detecting impurities in a moxifloxacin hydrochloride bulk drug, characterized in that: The related substances are selected from one or more of the compounds described in Table 1 below, and are qualitatively or quantitatively detected by high performance liquid chromatography. The detection conditions of liquid chromatography include: chromatographic column: a chromatographic column or an equivalent chromatographic column with octadecylsilane bonded silica as filler. The mobile phase includes mobile phase A and mobile phase B: a mixture of phosphate buffer solution-acetonitrile-methanol is used as mobile phase A and mobile phase B, and gradient elution. The pH of the buffer solution is 2-3; the column temperature is 35-45°C; the mobile phase flow rate is 1.5-1.9mL / min; the injection volume is 10-30μL; the elution method is gradient elution; the detection wavelength is 286-306nm; the impurities involved in the present invention and their corresponding structures are shown in Table 1. Table 1: Structure and source list of impurities of moxifloxacin hydrochloride 2. The detection method according to claim 1, wherein the concentration of the phosphate buffer is 0.32% to 0.36%; preferably, the concentration of the phosphate buffer is 0.34%; wherein the phosphate buffer solution needs to be adjusted to a pH value of 2.0 to 3.0 with an acid, preferably to a pH value of 2.5±0.05, and the acid is selected from one of formic acid, acetic acid, trifluoroacetic acid, and phosphoric acid, and preferably, the acid is phosphoric acid.

3. The detection method according to claim 1, wherein the reverse phase chromatography column uses non-polar stationary as filler; preferably, the reverse phase chromatography uses octadecylsilane bonded silica gel as filler; more preferably, the reverse phase chromatography column is YMC-Pack ODS-A, with a size of 250 mm×4.6 mm, 5 μm.

4. The detection method according to claim 1, wherein the column temperature is 35°C to 45°C; preferably, the column temperature is 40°C.

5. The detection method according to claim 1, wherein the flow rate of the eluent is 1.5 to 1.9 ml / min; preferably, the flow rate of the eluent is 1.7 ml / min.

6. The detection method according to claim 1, wherein the detection method is performed on a high performance liquid chromatograph using a diode array detector or an ultraviolet detector; preferably, the analysis method is performed on a high performance liquid chromatograph using an ultraviolet detector, and its detection wavelength is 286nm to 306nm; preferably 296nm.

7. The detection method according to claim 1, characterized in that The following steps are involved: (1) The concentration of phosphate buffer is 0.32% to 0.36%; preferably, the concentration of phosphate buffer is 0.34%; wherein the pH value of the phosphate buffer solution needs to be adjusted to 2.0 to 3.0 with acid, preferably to 2.5±0.

05. (2) Solvent: The solvent is mobile phase A, and the ratio is phosphate-acetonitrile-methanol = (790:125:85) to (810:115:75), preferably 80:12:

8. (3) Preparation of test solution: Take an appropriate amount of moxifloxacin hydrochloride, add a solvent to dissolve and dilute to a solution of appropriate concentration; the concentration of moxifloxacin hydrochloride is 0.1 mg / ml to 2 mg / ml, preferably, the concentration is 1 mg / ml. (4) Preparation of reference solution: Take an appropriate amount of moxifloxacin hydrochloride, add a solvent to dissolve and dilute to a solution of appropriate concentration; the concentration is 0.1 μg / ml to 2 μg / ml, preferably, the concentration of each impurity is 1 μg / ml. (5) Preparation of system suitability solution: Take appropriate amounts of methyl ester impurity, impurity F, moxifloxacin hydrochloride, impurity E, hydroxyquinoline impurity, methoxyquinoline impurity, and quinoline ethyl ester impurity, add solvent to dissolve and dilute to a solution of appropriate concentration; the concentration of moxifloxacin hydrochloride is 0.1 mg / ml to 2 mg / ml, preferably, the concentration is 1 mg / ml. The concentrations of methyl ester impurity, impurity F, impurity E, hydroxyquinoline impurity, methoxyquinoline impurity, and quinoline ethyl ester impurity are 0.1 μg / ml to 2 μg / ml, preferably, the concentration of each impurity is 1 μg / ml.

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