Method for separating and detecting rifabutin and its impurities

Abstract

This invention belongs to the field of pharmaceutical analysis technology, specifically relating to a method for separating and detecting rifabutin and its impurities. The impurities include impurity B, impurity D, impurity E, impurity F, impurity G, impurity I, impurity J, impurity K, and impurity Z. 3b Impurity Z 3c Any one or more of the following. This method is a reversed-phase high-performance liquid chromatography (RP-HPLC) method. The chromatographic column uses octadecyl-bonded silica gel as the packing material, and a linear gradient elution is performed using mobile phases A and B. The chromatogram is obtained by detection using a detector. Mobile phase A is a mixture of phosphate buffer solution and organic solvent, and mobile phase B is a mixture of acid aqueous solution and organic solvent. This method can completely separate and detect rifabutin and its 10 impurities within 60 minutes, and features good resolution, robustness, high sensitivity, and good reproducibility.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical analysis technology, specifically relating to a method for separating and detecting rifabutin and its impurities. Background Technology

[0002] Rifapentine is a semi-synthetic rifamycin derivative with good lipid solubility, allowing it to be widely distributed in tissues and cells. It effectively inhibits bacterial DNA-dependent RNA polymerase, thereby inhibiting and killing bacteria. Current research indicates that rifapentine, a spiroperidol derivative of rifamycin, has approximately four times stronger inhibitory effects against Mycobacterium tuberculosis than rifampin. In clinical trials for the treatment of pulmonary tuberculosis, the combination of levofloxacin and rifapentine showed significantly better efficacy compared to levofloxacin alone, promoting the elimination of Mycobacterium tuberculosis, enhancing the patient's immune function, and exhibiting high safety without causing liver damage. Furthermore, Talicia (omeprazole magnesium / amoxicillin / rifapentine) was approved by the FDA in early November 2019 for the treatment of Helicobacter pylori (H. pylori) infection in adults. This drug is the first FDA-approved three-in-one oral capsule based on the antibiotic rifapentine. Therefore, rifapentine plays an important role in the anti-infective treatment of bacteria such as Mycobacterium tuberculosis and Helicobacter pylori.

[0003] The chemical name of rifabutin is (9S,12E,14S,15R,16S,17R,18R,19R,20S,21S,22E,24Z)-6,16,18,20-tetrahydroxy-1′-isobutyl-14-methoxy-7,9,15,17,19,21,25-heptamethylspiro[9,4-(oxy-bridged pentadecane[1,11,13]trienineimine)-2H-furano[2′,3′:7,8]naphtho[1,2-d]imidazol-2,4′-piperidine]-5,10,26-(3H,9H)-trione,16-acetate, with the molecular formula C 43 H 63 N4O 11 The structural formula is shown in Equation I below.

[0004]

[0005] The raw materials for the synthesis of rifabutin are 3-amino-1,4-dideoxy-1,4-dihydro-4-imino-1-oxorifamycin (referred to as impurity D) and N-isobutylpiperidinone. Studies have found that the following impurities are easily generated during its synthesis: Impurity B (EP impurity), Impurity D (EP impurity), Impurity E (EP impurity / USP impurity), Impurity F (EP impurity / USP impurity), Impurity G (USP impurity), Impurity I (EP impurity / USP impurity), Impurity J, Impurity K, and Impurity Z. 3b Impurity Z3c Etc. To ensure the quality of rifabutin, the content of these impurities needs to be strictly controlled during the drug synthesis process.

[0006] Currently, rifabutin active pharmaceutical ingredient is listed in the European Pharmacopoeia EP11.0 and the United States Pharmacopoeia USPNF 2023, but not in the Chinese Pharmacopoeia. However, the EP11.0 method can only separate five impurities (B, D, E, F, and I), while the USPNF 2023 method can only separate four (E, F, G, and I). Impurities J, K, and Z are also listed. 3b and impurity Z 3c It is a relatively large process impurity that is easily generated during the synthesis of rifabutin active pharmaceutical ingredient, and it is necessary to establish analytical methods to control it.

[0007] Existing literature and patent reports cannot effectively control the aforementioned 10 impurities in rifabutin. Method sensitivity, analytical cost, analytical complexity, and impurity separation are key challenges and areas of focus in the quality control of rifabutin synthesis. Patent CN112946135A discloses a method for separating and detecting the intermediate 3-amino-4-iminorifamycin S and related substances in the rifabutin active pharmaceutical ingredient. This method employs high-performance liquid chromatography (HPLC) using a ZORBAX Eclipse XDB C18 column to detect the content of rifabutin intermediates and impurities. However, this method has limited ability to separate the number of impurities and is only applicable to the separation and detection of intermediates and their impurities.

[0008] Therefore, there is an urgent need to establish a simple and efficient analytical method to separate and detect 10 related impurities in rifabutin, which has important social significance and economic benefits for the quality control of the synthesis process of rifabutin. Summary of the Invention

[0009] In view of this, one of the objectives of the present invention is to provide a method for separating rifabutin and its impurities based on high performance liquid chromatography, which can complete the separation of multiple substances in a short time.

[0010] To achieve the above objectives, the technical solution of the present invention is as follows:

[0011] A method for separating rifabutin and its impurities based on high-performance liquid chromatography, wherein the rifabutin and the impurities together constitute a composition, and the impurities include impurity B, impurity D, impurity E, impurity F, impurity G, impurity I, impurity J, impurity K, and impurity Z. 3b Impurity Z 3c Any one or more of the following; the structural formula of each component of the composition is as follows:

[0012]

[0013] In the high-performance liquid chromatography (HPLC) method, the stationary phase is an octadecyl-bonded silica column; the mobile phases are: mobile phase A is a mixture of phosphate buffer solution and organic solvent, and mobile phase B is a mixture of acid aqueous solution and organic solvent; in mobile phase A, the volume ratio of phosphate buffer solution to organic solvent is 35-50:65-50; in mobile phase B, the volume ratio of organic solvent to acid aqueous solution is 70-90:30-10; rifabutin and its impurities are separated by linear gradient elution.

[0014] After separation and testing, it is used for the next step of production.

[0015] The aforementioned impurities can be arranged and combined in various ways. For example, combination 1: impurity J, impurity K, and impurity Z. 3b For example, combination 2: impurity B, impurity D, impurity E, impurity K. Another example is combination 3: impurity E, impurity K.

[0016] All possible permutations and combinations will not be listed here. Theoretically, when the upper limit of the substances that this method can separate, identify and / or detect is n (where n is the number of substances), it can naturally detect 1 to n substances.

[0017] As a preferred embodiment, the impurities are impurity J, impurity K, and impurity Z. 3b Impurity Z 3c Any one or more of the following; or the impurities are impurity J, impurity K, and impurity Z. 3b Impurity Z 3c It consists of any one or more of the following impurities, along with other impurities, including any one or more of impurity B, impurity D, impurity E, impurity F, impurity G, and impurity I.

[0018] As a preferred embodiment, the linear gradient elution procedure is as follows:

[0019] Time - minutes Mobile phase A - (volume parts) Mobile phase B - (volume parts) 0 90-100 0-10 10±1 90-100 0-10 15±1 20-40 80-60 50±1 20-40 80-60 51±1 90-100 0-10 60±1 90-100 0-10 .

[0020] More preferably, the linear gradient elution procedure is as follows:

[0021] Time - minutes Mobile phase A - (volume parts) Mobile phase B - (volume parts) 0 100 0 10 100 0 15 30 70 50 30 70 51 100 0 60 100 0 .

[0022] Furthermore, in the mobile phase, the organic solvent is any one or more of acetonitrile, methanol, ethanol, tetrahydrofuran, and isopropanol, preferably acetonitrile.

[0023] Furthermore, the phosphate buffer solution is any one or more of potassium dihydrogen phosphate solution, dipotassium hydrogen phosphate solution, potassium hexafluorophosphate solution, sodium dihydrogen phosphate solution, disodium hydrogen phosphate solution, ammonium dihydrogen phosphate solution, and diammonium hydrogen phosphate solution, preferably potassium dihydrogen phosphate solution.

[0024] Furthermore, the concentration of the phosphate buffer solution is 0.01 mol / L to 0.05 mol / L, preferably 0.025-0.035 mol / L, and most preferably 0.03 mol / L; the pH value is 7 to 8, preferably 7.5 ± 0.1, and most preferably 7.5 ± 0.05.

[0025] Preferably, the acid used to adjust the pH of the phosphate buffer solution is one or more of phosphoric acid, hydrochloric acid, sulfuric acid, acetic acid, formic acid, and trifluoroacetic acid; the alkaline solution used to adjust the pH of the phosphate buffer solution is one or more of sodium hydroxide, potassium hydroxide, triethylamine, and ammonia.

[0026] As the optimal choice, the pH of the phosphate buffer solution is adjusted using a 2 mol / L potassium hydroxide solution.

[0027] Furthermore, the acid in the acidic aqueous solution is any one or more of phosphoric acid, hydrochloric acid, sulfuric acid, acetic acid, formic acid, and trifluoroacetic acid, preferably phosphoric acid.

[0028] Furthermore, the concentration of the acidic aqueous solution is 0.05% to 0.3%, preferably 0.1%.

[0029] Preferably, in the mobile phase A, the volume ratio of phosphate buffer solution to organic solvent is 41-45:55-59, the phosphate buffer solution is a potassium dihydrogen phosphate solution with a concentration of 0.025-0.035 mol / L and a pH value of 7.5±0.1, and the organic solvent is acetonitrile.

[0030] Preferably, in the mobile phase B, the volume ratio of organic solvent to acid aqueous solution is 78-82:22-18, wherein the organic solvent is acetonitrile and the acid aqueous solution is a 0.1% phosphoric acid aqueous solution.

[0031] As the most preferred embodiment, in the mobile phase A, the volume ratio of phosphate buffer solution to organic solvent is 43:57, the phosphate buffer solution is a potassium dihydrogen phosphate solution with a concentration of 0.03 mol / L and a pH value of 7.5 ± 0.05, and the organic solvent is acetonitrile.

[0032] As the most preferred embodiment, in the mobile phase B, the volume ratio of organic solvent to acid aqueous solution is 80:20, wherein the organic solvent is acetonitrile, and the acid aqueous solution is a 0.1% phosphoric acid aqueous solution.

[0033] Furthermore, the flow rate was 0.7-1.3 mL / min; the column temperature was 30-50℃.

[0034] Preferably, the flow rate is 0.9-1.1 mL / min and the column temperature is 38-42℃.

[0035] The optimal flow rate is 1.0 mL / min, and the column temperature is 40 °C.

[0036] Preferably, the chromatographic column has a size of 250×4.6mm and a diameter of 5μm.

[0037] Preferably, the chromatographic column is a Shim-pack scepter HD-C18 or a Shim-pack scepter C18, with dimensions of 250×4.6mm and a diameter of 5μm.

[0038] As the optimal choice, the chromatographic column selected was the Shim-pack scepter HD-C18, 250×4.6mm, 5μm.

[0039] As a preferred option, the injection volume is 10 μl.

[0040] As a preferred option, the running time is 60 minutes.

[0041] As a preferred embodiment, in the high-performance liquid chromatography (HPLC) method, the chromatographic column uses octadecylsilane-bonded silica gel as the packing material, with dimensions of 250 × 4.6 mm and 5 μm; in mobile phase A, the volume ratio of phosphate buffer solution to organic solvent is 41-45:55-59, the phosphate buffer solution is a potassium dihydrogen phosphate solution with a concentration of 0.025-0.035 mol / L and a pH of 7.5 ± 0.1, and the organic solvent is acetonitrile; in mobile phase B, the volume ratio of organic solvent to aqueous acid solution is 78-82:22-18, the organic solvent is acetonitrile, and the aqueous acid solution is a 0.1% aqueous phosphoric acid solution; the flow rate is 0.9-1.1 mL / min; the column temperature is 38-42℃; gradient elution is performed under the following chromatographic conditions:

[0042] Time - minutes Mobile phase A - (volume parts) Mobile phase B - (volume parts) 0 100 0 10 100 0 15 30 70 50 30 70 51 100 0 60 100 0 .

[0043] The second objective of this invention is to provide a method for identifying rifabutin and its impurities, which can complete the identification of multiple substances in a short time.

[0044] To achieve the above objectives, the technical solution of the present invention is as follows:

[0045] A method for identifying rifabutin and its impurities involves separating the composition using the aforementioned separation method and detecting it in a detector with a detection wavelength of 254±10 nm to obtain a chromatogram; by comparing the chromatographic characteristics of the test sample and the reference sample, it is determined whether the test sample contains rifabutin and its impurities.

[0046] The ±10 nm setting range is based on a comprehensive consideration of factors such as error tolerance, methodological superiority, and practical application requirements. This setting range helps ensure the reliability of detection results, improve measurement repeatability and flexibility, and meet the requirements of specific experiments.

[0047] The optimal detection wavelength is 254nm.

[0048] Furthermore, the components in the composition can be identified according to the order of retention time. The components of the composition, in ascending order, are: impurity G, impurity Z, etc. 3b Impurity B, Impurity J, Impurity D, Impurity Z 3c Impurity E, Impurity K, Impurity F, Rifabutin, Impurity I.

[0049] As a preferred method, a retention time of 4.5 ± 0.5 min is identified as impurity G; a retention time of 7.7 ± 0.5 min is identified as impurity Z. 3b The retention time was 11.6 ± 0.5 min, identified as impurity B; the retention time was 12.9 ± 0.5 min, identified as impurity J; the retention time was 17.5 ± 0.5 min, identified as impurity D; and the retention time was 19.2 ± 0.5 min, identified as impurity Z. 3c The retention time was 20.8±0.5 min, which was identified as impurity E; the retention time was 22.2±0.5 min, which was identified as impurity K; the retention time was 24.4±0.5 min, which was identified as impurity F; the retention time was 26.3±0.5 min, which was identified as rifabutin; and the retention time was 36.9±0.5 min, which was identified as impurity I.

[0050] Retention time can be used to identify components. Retention time refers to the time required for a sample to travel from the point of entry into the chromatographic column to detection by the detector. This time is calculated based on the migration rate of the components on the column, i.e., the time interval from the start of injection to the chromatographic peak (maximum concentration) of a particular component. It is primarily used to determine the elution order and position of each component in a sample and is one of the fundamental data points in chromatographic analysis. In quality control, changes in retention time can reflect factors such as the state of the chromatographic column, the stability of the mobile phase, and the performance of the instrument.

[0051] The third objective of this invention is to provide a method for determining the impurity content in rifabutin, which can quantify multiple impurities in a rifabutin sample in a relatively short time.

[0052] To achieve the above objectives, the technical solution of the present invention is as follows:

[0053] The method for determining the impurity content in rifabutin includes the following steps:

[0054] (1) Rifabutin and its impurities were separated and identified using the aforementioned method, and chromatograms were obtained;

[0055] (2) Based on the chromatogram obtained in step (1), calculate the content of each impurity by using the external standard method, the self-comparison method with correction factor and / or the self-comparison method by peak area.

[0056] The content determination can further be used to determine whether the impurity content in rifabutin is within acceptable limits. If the test solution contains impurities B, D, E, F, G, I, J, K, and Z... 3b Impurity Z 3c If the peak area of ​​any one or more impurities in the test solution is greater than the peak area of ​​the corresponding impurity in the control solution, it indicates that the impurity content is unqualified; conversely, if the peak area of ​​impurities B, D, E, F, G, I, J, K, and Z in the test solution is greater than the peak area of ​​the corresponding impurity in the control solution, it indicates that the impurity content is unqualified. 3b Impurity Z 3c If the peak area of ​​any one or more impurities in the solution is not greater than the peak area of ​​the corresponding impurity in the control solution, it indicates that the impurity content is qualified.

[0057] The aforementioned judgment method can serve as a drug quality assessment model and further as an indispensable key module in intelligent production processes. This model, through precise control of parameters such as mobile phase composition, flow rate, and column temperature, achieves accurate separation and quantitative analysis of active ingredients, impurities, and degradation products in drugs, providing a scientific basis for comprehensive drug quality assessment. In intelligent production systems, this model is seamlessly integrated, capable of receiving raw data from the production line in real time, automatically executing analysis tasks, and rapidly providing judgment results based on preset quality standards.

[0058] Furthermore, the solvent used to prepare the sample was acetonitrile.

[0059] As a preferred method, the sample solution is prepared as follows: accurately weigh approximately 25 mg of rifabutin, place it in a 25 ml volumetric flask, add diluent to dissolve and dilute to the mark, shake well, and the solution is ready.

[0060] As a preferred method, the preparation of the reference solution is as follows: Take impurity B, impurity D, impurity E, impurity F, impurity G, impurity I, impurity J, impurity K, and impurity Z. 3bImpurity Z 3c Accurately weigh appropriate amounts of each ingredient, dissolve them in acetonitrile, and quantitatively dilute to prepare a solution containing approximately Z impurities per 1 ml. 3c Approximately 25 μg, impurities B, D, J, and K each approximately 37.5 μg, and impurity Z... 3b Prepare a mixed stock solution containing approximately 125 μg each of impurities E, G, and I, and approximately 187.5 μg of impurity F; accurately measure 4.0 ml of this stock solution and 1.0 ml of the test solution, place them in the same 100 ml volumetric flask, dilute to the mark with diluent, and shake well. Alternatively, prepare a control (standard) solution according to the method in Example 1.

[0061] The beneficial effects of this invention are as follows:

[0062] (1) This invention utilizes a novel approach to adjust the pH of the mobile phase, its ratio, and the mixing of acid and base components during the analytical process. A high-carbon content chromatographic column is selected to establish a reversed-phase high-performance liquid chromatography (RP-HPLC) method for the separation and detection of rifabutin and its impurities. This method can effectively separate and determine impurities B, D, E, F, G, I, J, K, and Z in rifabutin. 3b Impurity Z 3c A total of 10 impurities were identified, and the separation results were superior to conventional methods. This invention provides a new approach for the separation and detection of rifabutin and impurities generated during its synthesis.

[0063] (2) The present invention uses reversed-phase high performance liquid chromatography to separate and detect rifabutin and its 10 impurities. Octadecyl bonded silica gel is selected as the packing material. This packing material is durable, environmentally friendly and has low toxicity. It can completely separate and detect rifabutin and its 10 impurities within 60 minutes and has excellent separation performance and durability.

[0064] (3) The method of the present invention has good separation, strong specificity, high sensitivity and simple operation. It has the advantages of being simple and fast, which can ensure the quality control of rifabutin and its preparations and ultimately determine the safety and efficacy of the product. It is of great significance for the quality control of rifabutin.

[0065] (4) The analytical method of the present invention has low limits of quantitation and detection, wherein the limit of quantitation concentration of impurity G is 0.1010 μg / ml and the limit of detection concentration is 0.0202 μg / ml; impurity Z 3bThe limit of quantitation (LOQ) for impurity Z was 0.1009 μg / ml, and the limit of detection (LOD) was 0.0202 μg / ml; the LQ for impurity B was 0.0996 μg / ml, and the LOD was 0.0199 μg / ml; the LQ for impurity J was 0.0939 μg / ml, and the LOD was 0.0188 μg / ml; the LQ for impurity D was 0.0925 μg / ml, and the LOD was 0.0186 μg / ml; the LQ for impurity Z was 0.0939 μg / ml, and the LOD was 0.0188 μg / ml; the LQ for impurity D was 0.0925 μg / ml, and the LOD was 0.0186 μg / ml; the LOD for impurity Z was 0.0939 μg / ml, and the LOD was 0.0188 μg / ml. 3c The limit of quantitation (LOQ) for impurity E was 0.0901 μg / ml, and the limit of detection (LOD) was 0.0300 μg / ml; the LQ for impurity K was 0.0998 μg / ml, and the LOD was 0.0200 μg / ml; the LQ for impurity K was 0.1062 μg / ml, and the LOD was 0.0212 μg / ml; the LQ for impurity F was 0.1023 μg / ml, and the LOD was 0.0205 μg / ml; the LQ for rifabutin was 0.1161 μg / ml, and the LOD was 0.0232 μg / ml; and the LQ for impurity I was 0.1026 μg / ml, and the LOD was 0.0205 μg / ml. Attached Figure Description

[0066] Figure 1 The chromatogram is for the blank solvent;

[0067] Figure 2 The chromatogram of the test solution;

[0068] Figure 3 This is the chromatogram of the reference solution;

[0069] Figure 4 For Z 3c Chromatogram of the reference solution;

[0070] Figure 5 The chromatogram of mixed solution 1 is shown below.

[0071] Figure 6 The chromatogram of mixed solution 2 is shown below.

[0072] Figure 7 Impurity Z 3c Chromatogram of a solution at the limit of quantitation;

[0073] Figure 8 The chromatogram is for the solution at the limit of quantitation.

[0074] Figure 9 Impurity Z 3c Chromatogram of the solution at the detection limit;

[0075] Figure 10 Chromatogram of the solution at the detection limit;

[0076] Figure 11 Chromatogram of robustness-mixed solution 2 under normal conditions;

[0077] Figure 12 Chromatogram for robustness-mixed solution 2 at a flow rate of 0.9 ml / min;

[0078] Figure 13 Chromatogram for robustness-mixed solution 2 under flow rate of 1.1 ml / min conditions;

[0079] Figure 14 Chromatogram for robustness-mixed solution 2-column temperature 38°C;

[0080] Figure 15 Chromatogram of robustness-mixed solution 2-column temperature 42℃;

[0081] Figure 16 Chromatograms for robustness-mixed solution 2-mobile phase ratio (acetonitrile: A 59%, B 82%).

[0082] Figure 17 Chromatograms for robustness-mixed solution 2-mobile phase ratio (acetonitrile: A 55%, B 78%).

[0083] Figure 18 Chromatogram for robustness-mixed solution 2-salt concentration 0.025 mol / L;

[0084] Figure 19 Chromatogram of the robustness-mixed solution 2-salt concentration at 0.035 mol / L.

[0085] Figure 20 Chromatogram of the robustness-mixed solution 2-mobile phase at pH 7.4;

[0086] Figure 21 Chromatogram of the robustness-mixed solution 2-mobile phase at pH 7.6.

[0087] Figure 22 The chromatogram of the mixed solution in Comparative Example 1 is shown below.

[0088] Figure 23 The chromatogram of the mixed solution in Comparative Example 2 is shown below.

[0089] Figure 24 The chromatogram is of the mixed solution in Comparative Example 3. Detailed Implementation

[0090] The technical solution of the present invention will be described more clearly and completely below with reference to specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention.

[0091] Supplementary tables to the accompanying drawings in the specification. Included in this patent. Figures 1-11 Visual aids are provided for understanding and interpretation. In case of any ambiguity, users should refer to the corresponding numbered tables (Tables 1-11) for more detailed information. Conversely, if any information that may cause misunderstanding or ambiguity is found during the review of Tables 1-11, the content of the corresponding numbered figures should be taken as the standard. The above guidelines aim to ensure the correct interpretation of this document and the consistency of its information. Although some text overlaps in the spectra of this application, it is still clearly legible, and the specification details the integration results of each figure. Furthermore, the numbers in the spectra do not affect the scope of protection of the claims or the full disclosure of the technical solutions in the specification.

[0092] Table 1

[0093]

[0094] Table 2

[0095]

[0096] Table 3

[0097]

[0098] Table 4

[0099]

[0100] Table 5

[0101]

[0102] Table 6

[0103]

[0104] Table 7

[0105]

[0106] Table 8

[0107]

[0108] Table 9

[0109]

[0110] Table 10

[0111]

[0112] Table 11

[0113]

[0114] Table 12

[0115]

[0116] Table 13

[0117]

[0118] Table 14

[0119]

[0120] Table 15

[0121]

[0122] Table 16

[0123]

[0124] Table 17

[0125]

[0126] Table 18

[0127]

[0128] Table 19

[0129]

[0130] Table 20

[0131]

[0132] Table 21

[0133]

[0134] Table 22

[0135]

[0136] Table 23

[0137]

[0138] Table 24

[0139]

[0140] To enhance understanding of the present invention, certain key technologies and scientific terms will be clearly defined below. Unless specifically defined herein, all other technical and scientific terms shall follow their generally accepted and understood meanings within the art to which this invention pertains. It should be emphasized that the scope of the present invention is not limited to the specific methods, reagents, compounds, compositions, reference standards, and test items described, but allows for reasonable variations and adjustments in these aspects. Furthermore, please understand that the terminology used herein is intended to illustrate specific embodiments and not to impose a limiting interpretation.

[0141] Furthermore, all references cited in this document, including but not limited to patents, patent applications, academic papers, textbooks, and further citations therein, are considered to be incorporated into this document in their entirety through citation, unless directly cited, as a reference. If there are any inconsistencies or conflicts between the content of these cited references or similar materials and this application, particularly regarding terminology definitions, usage, or technical descriptions, the content of this application shall prevail.

[0142] If any chromatographic conditions are not mentioned, refer to the high performance liquid chromatography method (Chinese Pharmacopoeia 2020 Edition, Part IV, 0512) for determination.

[0143] the term

[0144] The limit of quantitation (LOQ) is the lowest amount of an analyte in a sample that can be quantitatively determined, and the measurement result should have a certain degree of accuracy and precision. In other words, the LQ is the lowest level at which an analytical method can accurately and reliably determine the concentration of the analyte in a sample. In HPLC, the determination of the LQ usually relies on the signal-to-noise ratio (S / N) method, that is, the concentration of the analyte corresponding to a certain level of signal-to-noise ratio is taken as the LQ. Determining the LQ is crucial for ensuring the accuracy and reliability of analytical results.

[0145] Chromatographic robustness refers to the ability of a chromatographic analysis system to maintain stable analytical performance and unaffected analytical results when measurement conditions are slightly changed. This robustness is crucial for ensuring the reliability, repeatability, and stability of analytical results.

[0146] The limit of detection (LOD) is the lowest concentration or amount of an analyte in a sample that can be detected. It reflects the sensitivity and noise level of the analytical method and instrument, and also indicates the level of the blank (background) value after sample processing.

[0147] A correction factor is a coefficient or parameter used to correct analytical results. It aims to improve data accuracy and reliability. In HPLC analysis, because the same detector responds differently to different substances, peak areas produced when the same mass of different substances passes through the detector may not be equal. To ensure that the peak area accurately reflects the content of the analyte, standard substances are used for correction, a correction factor is calculated, and this factor is applied to the measurement results of the sample.

[0148] The peak height to noise ratio (S / N, or signal-to-noise ratio) is used in high-performance liquid chromatography (HPLC) to evaluate the detection sensitivity and resolution of an instrument, and is an important indicator of instrument performance. Peak height refers to the signal value output by the detector when the analyte elutes from the column; noise refers to the fluctuation of the baseline signal, i.e., the signal value measured for a blank sample. The signal-to-noise ratio is the ratio of the signal measured for a sample of known concentration to the signal measured for a blank sample. A higher signal-to-noise ratio means that the instrument can more accurately separate and identify the target component when detecting samples, while also reducing interference from background noise.

[0149] In this embodiment of the invention, information on rifabutin and its related impurities is shown in Table A.

[0150] Table A. Compound Information Table

[0151]

[0152]

[0153] Example 1. Method for separating and determining rifabutin and its impurities

[0154] This invention presents a self-developed reversed-phase high-performance liquid chromatography (RP-HPLC) method for the separation and determination of rifabutin and its impurities. These impurities include: impurity B, impurity D, impurity E, impurity F, impurity G, impurity I, impurity J, impurity K, and impurity Z. 3b Impurity Z 3c The operation steps are as follows:

[0155] (1) Preparation of the test solution

[0156] Diluent: Acetonitrile.

[0157] Test solution: Accurately weigh about 25 mg of rifabutin, place it in a 25 ml volumetric flask, add diluent to dissolve and dilute to the mark, shake well, and the solution is ready.

[0158] Reference solution: Take impurity B, impurity D, impurity E, impurity F, impurity G, impurity I, impurity J, impurity K, and impurity Z. 3b Accurately weigh appropriate amounts of each of the following, dissolve them in acetonitrile, and quantitatively dilute to prepare a solution containing approximately 37.5 μg each of impurities B, D, J, and K, and impurity Z per 1 ml.3b Prepare a mixed stock solution containing approximately 125 μg each of impurities E, G, and I, and approximately 187.5 μg of impurity F; accurately measure 4.0 ml of this stock solution and 1.0 ml of the test solution, place them in the same 100 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the final product.

[0159] Z 3c Reference solution: Take impurity Z 3c Weigh an appropriate amount accurately, dissolve in acetonitrile, and dilute quantitatively to prepare a solution containing approximately Z impurities per 1 ml. 3c A solution of approximately 1 μg of each.

[0160] (2) Instruments and chromatographic conditions

[0161] High-performance liquid chromatograph (HPLC): Thermo UltiMate 3000; Column: octadecylsilane-bonded silica gel (Shim-pack scepter HD-C18, 250×4.6mm, 5μm or equivalent); Mobile phase A: 0.03mol / L potassium dihydrogen phosphate solution (pH adjusted to 7.5±0.05 with 2mol / L potassium hydroxide solution) - acetonitrile (43:57); Mobile phase B: acetonitrile - 0.1% phosphoric acid solution (80:20); Detector wavelength: 254nm; Mobile phase flow rate: 1.0ml / min; Column oven temperature: 40℃; Injection volume: 10μl; Gradient elution program is shown in Table B below:

[0162] Table B. Gradient Elution Program Table

[0163]

[0164]

[0165] (3) Measurement

[0166] Take the above-mentioned test solution, reference (standard) solution and Z 3c The reference solutions were injected separately for analysis, and the chromatograms were recorded. Based on the chromatograms, the content of each impurity was calculated using either the external standard method or the self-comparison method with correction factors, or the self-comparison method. The correction factors and quantification methods for each impurity are shown in Table C; the reference (standard) solutions and Z... 3c The chromatograms of the reference solutions are as follows: Figure 3 , Figure 4 As shown.

[0167] Table C. Correction factors and quantification methods for each impurity

[0168]

[0169] Example 2. Specificity determination

[0170] (1) Preparation of the test solution

[0171] Diluent / blank solution: acetonitrile.

[0172] Impurity B stock solution: Accurately weigh about 25 mg of impurity B reference standard, place it in a 50 ml volumetric flask, add diluent to dissolve and dilute to the mark, shake well, and the solution is ready.

[0173] Impurity D stock solution: Accurately weigh about 25 mg of impurity D reference standard, place it in a 50 ml volumetric flask, add diluent to dissolve and dilute to the mark, shake well, and the solution is ready.

[0174] Impurity E stock solution: Accurately weigh about 31 mg of impurity E reference standard, place it in a 25 ml volumetric flask, add diluent to dissolve and dilute to the mark, shake well, and the solution is ready.

[0175] Impurity F stock solution: Accurately weigh about 25 mg of impurity F reference standard, place it in a 25 ml volumetric flask, add diluent to dissolve and dilute to the mark, shake well, and the solution is ready.

[0176] Impurity G stock solution: Accurately weigh about 16 mg of impurity G reference standard, place it in a 25 ml volumetric flask, add 80% acetonitrile to dissolve and dilute to the mark, shake well, and the solution is ready.

[0177] Impurity I stock solution: Accurately weigh about 16 mg of impurity I reference standard, place it in a 25 ml volumetric flask, add diluent to dissolve and dilute to the mark, shake well, and the solution is ready.

[0178] Impurity J stock solution: Accurately weigh about 20 mg of impurity J reference standard, place it in a 50 ml volumetric flask, add diluent to dissolve and dilute to the mark, shake well, and the solution is ready.

[0179] Impurity K stock solution: Accurately weigh about 20 mg of impurity K reference standard, place it in a 50 ml volumetric flask, add diluent to dissolve and dilute to the mark, shake well, and the solution is ready.

[0180] Impurity Z 3b Stock solution: Accurately weigh about 20 mg of impurity Z3b reference standard, place it in a 50 ml volumetric flask, add diluent to dissolve and dilute to the mark, shake well, and the solution is ready.

[0181] Impurity Z 3c Stock solution: Accurately weigh about 25 mg of impurity Z3c reference standard, place it in a 50 ml volumetric flask, add diluent to dissolve and dilute to the mark, shake well, and the solution is ready.

[0182] Impurity B positioning solution: Accurately measure 0.1 ml of impurity B stock solution, place it in a 25 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.

[0183] Impurity D positioning solution: Accurately measure 0.1 ml of impurity D stock solution, place it in a 25 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.

[0184] Impurity E positioning solution: Accurately measure 0.1 ml of impurity E stock solution, place it in a 25 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.

[0185] Impurity F positioning solution: Accurately measure 0.2 ml of impurity F stock solution, place it in a 25 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.

[0186] Impurity G positioning solution: Accurately measure 0.2 ml of impurity G stock solution, place it in a 25 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.

[0187] Impurity H positioning solution: Accurately measure 0.2 ml of impurity H stock solution, place it in a 25 ml volumetric flask, add diluent to dilute to the mark, and shake well to obtain the solution.

[0188] Impurity I positioning solution: Accurately measure 0.2 ml of impurity I stock solution, place it in a 25 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.

[0189] Impurity J positioning solution: Accurately measure 0.1 ml of impurity J stock solution, place it in a 25 ml volumetric flask, add diluent to dilute to the mark, and shake well to obtain the solution.

[0190] Impurity K positioning solution: Accurately measure 0.1 ml of impurity K stock solution, place it in a 25 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.

[0191] Impurity Z 3b Positioning solution: Accurately measure 0.1 ml of impurity Z3b stock solution, place it in a 25 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.

[0192] Impurity Z 3c Positioning solution: Accurately measure 0.1 ml of impurity Z3c stock solution, place it in a 25 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.

[0193] Rifabutin test solution: Accurately weigh about 25 mg of rifabutin, place it in a 25 ml volumetric flask, add diluent to dissolve and dilute to the mark, shake well, and the solution is ready.

[0194] Mixed Solution 1: Accurately weigh approximately 25 mg of rifabutin and place it in a 25 ml volumetric flask. Accurately measure impurities B, D, E, J, K, and Z. 3b Place 0.1 ml of each stock solution and 0.2 ml of each stock solution of impurity F, impurity G and impurity I into the same 25 ml volumetric flask, add diluent to dissolve and dilute to the mark, shake well, and the product is ready.

[0195] Mixed Solution 2: Accurately weigh approximately 25 mg of rifabutin and place it in a 25 ml volumetric flask. Accurately measure 0.1 ml each of the stock solutions of impurities B, D, E, J, K, Z3b, and Z3c, and 0.2 ml each of the stock solutions of impurities F, G, and I. Place these solutions in the same 25 ml volumetric flask, add diluent to dissolve and dilute to the mark, and shake well to obtain the solution.

[0196] (2) Detection

[0197] The blank solution, the localization solutions of each impurity, mixed solution 1 and mixed solution 2 were injected sequentially and detected according to the chromatographic conditions of Example 1, and the chromatograms were recorded.

[0198] The measurement results are shown in Table D. Figures 1-2 , Figures 5-6 The results are shown in Tables 1-2 and 5-6. The data show that the blank diluent does not interfere with the sample determination; the resolution between each impurity and its adjacent peaks, as well as between each impurity and rifabutin, is greater than 1.5, and the resolution between each impurity itself is also greater than 1.5. These results indicate that rifabutin is well separated from all known impurity peaks and has strong specificity.

[0199] Table D. Results of Specificity Test

[0200]

[0201]

[0202] Example 3. Determination of the limit of quantitation

[0203] (1) Preparation of the test solution

[0204] Diluent: Acetonitrile.

[0205] Limit of Quantitation (LOQ) Stock Solution: Accurately measure 0.2 ml of impurity E stock solution, 0.3 ml of impurity F stock solution, 0.5 ml each of impurity B and impurity D stock solutions, 0.6 ml each of rifabutin, impurity G and impurity I stock solutions, and impurity K, impurity J and impurity Z stock solutions. 3b Place 0.7 ml of the stock solution in the same 50 ml volumetric flask, add diluent to dilute to the mark, and shake well to obtain the final product.

[0206] Limit of Quantitation Solution: Accurately measure 3.0 ml of the limit of quantitation stock solution, place it in the same 50 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.

[0207] Impurity Z 3c Limit of Quantity (LOQ) Stock Solution: Accurately measure impurity Z from "Example 2. Specificity". 3c Place 1.0 ml of the stock solution into a 50 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the final product.

[0208] Impurity Z 3c Limit of Quantitation Solution: Accurately measure 0.75 ml of the reference standard stock solution, place it in a 50 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.

[0209] (2) Measurement method

[0210] Take the above-mentioned limit of quantitation solution and impurity Z 3c The limit of quantitation solution was injected three times consecutively, and the detection was performed under the chromatographic conditions of Example 1. The chromatograms were recorded, and the ratio of the main peak height to the noise (signal-to-noise ratio) was calculated.

[0211]

[0212] The test results are shown in Table E. Figures 7-8 Tables 7 and 8 show the data. The data indicates that the limit of quantitation (LOQ) for impurity G is 0.1010 μg / ml, with an average S / N ratio of 48.6; impurity Z... 3b The limit of quantitation (LOQ) for impurity A was 0.1009 μg / ml, with an average S / N of 42.9; the LQ for impurity B was 0.0996 μg / ml, with an average S / N of 25.3; the LQ for impurity J was 0.0939 μg / ml, with an average S / N of 33.9; the LQ for impurity D was 0.0925 μg / ml, with an average S / N of 51.4; and the LQ for impurity Z was 0.0996 μg / ml, with an average S / N of 25.3; the LQ for impurity J was 0.0939 μg / ml, with an average S / N of 33.9; and the LQ for impurity D was 0.0925 μg / ml, with an average S / N of 51.4. 3c The limit of quantitation (LOQ) for impurity E was 0.0901 μg / ml, with an average S / N of 19.8; the LQ for impurity K was 0.0998 μg / ml, with an average S / N of 33.0; the LQ for impurity K was 0.1062 μg / ml, with an average S / N of 38.2; the LQ for impurity F was 0.1023 μg / ml, with an average S / N of 32.1; the LQ for rifabutin was 0.1161 μg / ml, with an average S / N of 32.4; and the LQ for impurity I was 0.1026 μg / ml, with an average S / N of 29.2. These results indicate that this method has good sensitivity.

[0213] Table E. Results of Limit of Quantitation Experiment

[0214]

[0215]

[0216]

[0217] Example 4. Determination of detection limit

[0218] Diluent: Acetonitrile.

[0219] Limit of Detection Solution: Accurately transfer 1.0 ml of the limit of quantitation stock solution into a 50 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the limit of detection solution.

[0220] Impurity Z 3c Detection limit solution: Accurately transfer Z 3c Place 3.0 ml of the limit of quantitation solution into a 10 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the limit of detection solution.

[0221] Take the above-mentioned detection limit solution and impurity Z 3c The detection limit solution was injected three times consecutively, and the detection was performed under the chromatographic conditions of Example 1. The chromatograms were recorded, and the ratio of the main peak height to the noise (signal-to-noise ratio) was calculated.

[0222]

[0223] The test results are shown in Table F. Figures 9-10 Tables 9 and 10 show that the detection limit for impurity G is 0.0202 μg / ml, with an average S / N ratio of 4.7; impurity Z... 3b The detection limit concentration (LOC) for impurity A was 0.0202 μg / ml, with an average S / N of 6.1; the LOC for impurity B was 0.0199 μg / ml, with an average S / N of 4.4; the LOC for impurity J was 0.0188 μg / ml, with an average S / N of 5.0; the LOC for impurity D was 0.0186 μg / ml, with an average S / N of 6.9; and the LOC for impurity Z was... 3c The detection limit (LOD) for impurity E was 0.0300 μg / ml, with an average S / N of 7.5; the LOD for impurity K was 0.0200 μg / ml, with an average S / N of 4.4; the LOD for impurity K was 0.0212 μg / ml, with an average S / N of 5.8; the LOD for impurity F was 0.0205 μg / ml, with an average S / N of 4.4; the LOD for rifabutin was 0.0232 μg / ml, with an average S / N of 4.7; and the LOD for impurity I was 0.0205 μg / ml, with an average S / N of 4.1. These results indicate that the method has good sensitivity.

[0224] Table F. Results of Detection Limit Experiment

[0225]

[0226]

[0227] Example 5. Durability

[0228] Take the mixed solution 2 from Example 2, and adjust each experimental condition (flow rate ±0.1 ml / min, column temperature ±2℃, mobile phase ratio ±2%, salt concentration ±5 mmol / L, pH value ±0.1). After the instrument system stabilizes, test each condition to examine the resolution between each peak.

[0229] Results: As shown in Table G. Figures 11-21 As shown in Tables 11 to 21, under this chromatographic system, when the flow rate, column temperature, and initial mobile phase ratio fluctuate, the resolution of rifabutin and 10 process impurities from adjacent peaks all meet the requirement of greater than 1.5, proving that this method has good robustness.

[0230] Table G. Results of Durability Test Resolution

[0231]

[0232]

[0233] Comparative Example 1. Comparison with the method of EP11.0

[0234] Impurities B, D, E, F, G, I, J, K, and Z in rifabutin were separated and detected using the EP11.0 related substances method. 3b Impurities Z3c The chromatographic conditions included: High Performance Liquid Chromatography (HPLC): Thermo UltiMate 3000; Column: Whatman Partisphere C8; Mobile phase: Acetonitrile: Potassium dihydrogen phosphate buffer solution (13.6 g / L potassium dihydrogen phosphate aqueous solution, pH adjusted to 6.5 with 2 mol / L sodium hydroxide solution) = 50:50; Detector wavelength: 254 nm; Mobile phase flow rate: 1.0 ml / min; Column oven temperature: 30 °C; Injection volume: 20 μl; Acquisition time: 40 min. All other conditions were the same as in Example 1.

[0235] Take the mixed solution 2 from Example 2, inject it under the chromatographic conditions described above, and record the chromatogram.

[0236] The measurement results are shown in Table H. Figure 22 See Table 22. The data shows that the EP11.0 related substances method can only separate the impurities mentioned in the EP standard, and the peaks are relatively fast, with multiple impurities completely overlapping, indicating poor separation of each impurity.

[0237] Table H. Results of the determination of Comparative Example 1

[0238]

[0239] Comparative Example 2. The mobile phase B was acetonitrile-water.

[0240] The separation and determination of rifabutin and its impurities were investigated when mobile phase B was acetonitrile-water. Specifically, mobile phase B was set as follows: acetonitrile:water = 80:20. Except for the use of mobile phase B, all other conditions were the same as in Example 1.

[0241] Take the mixed solution 1 from Example 2, inject it under the chromatographic conditions described above, and record the chromatogram.

[0242] The measurement results are shown in Table I. Figure 23 See Table 23. The data shows that when the mobile phase B is acetonitrile-water, the resolution of impurity E is less than 1.5, and impurities K and F cannot be separated from the main peak, which does not meet the requirements for effective separation and determination of rifabutin and its impurities.

[0243] Table I. Results of the determination of Comparative Example 2

[0244]

[0245] Comparative Example 3. Investigating mobile phase A and chromatographic column

[0246] The separation and determination of rifabutin and its impurities were investigated using mobile phase A with different pH values ​​and chromatographic columns with different packing materials. Chromatographic conditions included: Mobile phase A: 4.08 g of potassium dihydrogen phosphate (0.03 mol / L) was weighed into a 1000 ml volumetric flask, dissolved in water and diluted to the mark, and the pH was adjusted to 6.5 ± 0.1 with sodium hydroxide; Column: Agilent ZORBAX XDB-C18 (250 × 4.6 mm, 5 μm); Except for the mobile phase A and the chromatographic column, all other conditions were the same as in Example 1.

[0247] Take the mixed solution 2 from Example 2, inject it under the chromatographic conditions described above, and record the chromatogram.

[0248] The measurement results are shown in Table J. Figure 24 See Table 24. The data shows that the separation of rifabutin and its impurities was significantly reduced under different conditions of mobile phase pH and chromatographic column.

[0249] Table J. Results of the determination of Comparative Example 3

[0250]

Claims

1. A method for separating rifabutin and its impurities based on high performance liquid chromatography, characterized in that, The rifabutin and the impurities together constitute a composition, wherein the impurities include impurity B, impurity D, impurity E, impurity F, impurity G, impurity I, impurity J, impurity K, and impurity Z. 3b Impurity Z 3c Any one or more of the following; the structural formula of each component of the composition is as follows: In the high-performance liquid chromatography (HPLC) method, the stationary phase is an octadecyl-bonded silica column; the mobile phases are: mobile phase A is a mixture of phosphate buffer solution and organic solvent, and mobile phase B is a mixture of acid aqueous solution and organic solvent; in mobile phase A, the volume ratio of phosphate buffer solution to organic solvent is 35-50:65-50; in mobile phase B, the volume ratio of organic solvent to acid aqueous solution is 70-90:30-10; rifabutin and its impurities are separated by linear gradient elution.

2. The method according to claim 1, characterized in that, The procedure for linear gradient elution is as follows: 。 3. The method according to claim 1, characterized in that, In the mobile phase, the organic solvent is any one or more of acetonitrile, methanol, ethanol, tetrahydrofuran, and isopropanol; the phosphate buffer solution is any one or more of potassium dihydrogen phosphate solution, dipotassium hydrogen phosphate solution, potassium hexafluorophosphate solution, sodium dihydrogen phosphate solution, disodium hydrogen phosphate solution, ammonium dihydrogen phosphate solution, and diammonium hydrogen phosphate solution; the concentration of the phosphate buffer solution is 0.01 mol / L to 0.05 mol / L, and the pH value is 7 to 8; the acid in the acid aqueous solution is any one or more of phosphoric acid, hydrochloric acid, sulfuric acid, acetic acid, formic acid, and trifluoroacetic acid; the concentration of the acid aqueous solution is 0.05% to 0.3%.

4. The method according to claim 1, characterized in that, The flow rate is 0.7-1.3 mL / min; the column temperature is 30-50℃.

5. The method according to claim 1, characterized in that, In the high-performance liquid chromatography (HPLC) method, the chromatographic column uses octadecylsilane-bonded silica gel as the packing material, with dimensions of 250 × 4.6 mm and 5 μm. In mobile phase A, the volume ratio of phosphate buffer solution to organic solvent is 41-45:55-59. The phosphate buffer solution is a potassium dihydrogen phosphate solution with a concentration of 0.025-0.035 mol / L and a pH of 7.5 ± 0.

1. The organic solvent is acetonitrile. In mobile phase B, the volume ratio of organic solvent to aqueous acid solution is 78-82:22-18. The organic solvent is acetonitrile, and the aqueous acid solution is a 0.1% aqueous phosphoric acid solution. The flow rate is 0.9-1.1 mL / min, and the column temperature is 38-42℃. Gradient elution was performed under the following chromatographic conditions: 。 6. A method for identifying rifabutin and its impurities, characterized in that, The composition is separated using the method described in any one of claims 1-5 and detected by a detector with a detection wavelength of 254±10nm to obtain a chromatogram; the presence of rifabutin and its impurities in the test sample is determined by comparing the chromatographic characteristics of the test sample and the reference sample.

7. The method according to claim 6, characterized in that, The components in the composition can be identified according to the order of retention time. The components of the composition, in ascending order, are: impurity G, impurity Z, etc. 3b Impurity B, Impurity J, Impurity D, Impurity Z 3c Impurity E, Impurity K, Impurity F, Rifabutin, Impurity I.

8. The method according to claim 7, characterized in that, A retention time of 4.5 ± 0.5 min is identified as impurity G; a retention time of 7.7 ± 0.5 min is identified as impurity Z. 3b The retention time was 11.6 ± 0.5 min, identified as impurity B; the retention time was 12.9 ± 0.5 min, identified as impurity J; the retention time was 17.5 ± 0.5 min, identified as impurity D; and the retention time was 19.2 ± 0.5 min, identified as impurity Z. 3c The retention time was 20.8±0.5 min, which was identified as impurity E; the retention time was 22.2±0.5 min, which was identified as impurity K; the retention time was 24.4±0.5 min, which was identified as impurity F; the retention time was 26.3±0.5 min, which was identified as rifabutin; and the retention time was 36.9±0.5 min, which was identified as impurity I.

9. A method for determining the impurity content in rifabutin, characterized in that, Includes the following steps: (1) Rifabutin and its impurities are separated and identified by the method described in any one of claims 6-8, and a chromatogram is obtained; (2) Based on the chromatogram obtained in step (1), calculate the content of each impurity by using the external standard method, the self-comparison method with correction factor and / or the self-comparison method by peak area.

10. The method according to claim 9, characterized in that, The solvent used to prepare the sample was acetonitrile.

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