Method for separating and detecting rifapentine intermediate Z1 and impurities thereof

Abstract

The invention belongs to the technical field of pharmaceutical analysis, and particularly relates to a method for separating and detecting a rifapentine intermediate Z1 and impurities thereof. The impurities comprise one or more of an impurity SM1d, an impurity Z1b, an impurity Z1c, an impurity SM1b and an impurity SM1. The method comprises the following steps: a chromatographic column adopts octadecylsilane chemically bonded silica as a filler, a mobile phase A and a mobile phase B are adopted for linear gradient elution and enter a detector for detection, and the mobile phase A consists of sodium dihydrogen phosphate and acetonitrile according to a volume ratio of (55-65): (45-35); the mobile phase B is composed of sodium dihydrogen phosphate and acetonitrile according to the volume ratio of (65-75): (35-25); and finally, according to the chromatogram, calculating the content of each impurity by adopting a correction factor-added principal component self-contrast method and / or an area normalization method. The method disclosed by the invention has the characteristics of good separation degree, good durability, high sensitivity and good reproducibility.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and particularly relates to a method for separating and detecting a rifapentine intermediate Z1 and its impurities. Background Art

[0002] Rifapentine is a macrolide antibiotic developed by Aventis Pharma AG. Its chemical name is 3-[N-(4-cyclopentyl-1-piperazinyl)iminomethyl]rifamycin and its molecular formula is C 47 H 64 N4O 12 , the structural formula is shown in Formula VII. As a semi-synthetic rifamycin, rifapentine has antibacterial effects on Mycobacterium tuberculosis, Mycobacterium leprae, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus, Neisseria meningitidis, and Haemophilus influenzae.

[0003]

[0004] Rifapentine intermediate Z1 is a key intermediate in the synthesis of rifapentine. Studies have found that rifapentine intermediate Z1 is prone to produce five process impurities during the preparation process: impurity SM 1d 、Impurity Z 1b 、Impurity Z 1c 、Impurity SM 1b And impurity SM1. These impurities need to be strictly controlled whether in raw materials or preparations.

[0005] However, there is currently a lack of methods that can quickly and simultaneously separate the above five impurities. In the prior art, patent CN112730657A discloses a method for analyzing and detecting the content of rifapentine, which is determined by high performance liquid chromatography, and the chromatographic column is YMC-Pack C8. The mobile phase consists of an aqueous phase and an organic phase, and isocratic elution is set according to the volume fraction; wherein the organic solvent is selected from methanol, ethanol, acetonitrile or a mixture thereof, and the aqueous phase includes water, buffer salt, sodium perchlorate and acid-base reagents; finally, the external standard method is used for quantitative analysis. This method cannot separate the rifapentine intermediate Z1 and the impurity SM 1d 、Impurity Z 1b 、Impurity Z 1c 、Impurity SM 1b and impurity SM1.

[0006] Therefore, developing an analytical method that can quickly and effectively separate and determine the rifapentine intermediate Z1 and five impurities is of great significance for drug quality control and degradation pathway research. Summary of the invention

[0007] In view of this, one of the objects of the present invention is to provide a method for separating rifapentine intermediate Z1 and its impurities based on high performance liquid chromatography, which method can complete the separation of multiple substances in a relatively short time.

[0008] To achieve the above object, the technical solution of the present invention is:

[0009] A method for separating a rifapentine intermediate Z1 and its impurities based on high performance liquid chromatography, wherein the rifapentine intermediate Z1 and the impurities together constitute a composition, and the impurities include impurity SM 1d 、Impurity Z 1b 、Impurity Z 1c 、Impurity SM 1b and any one or more of impurities SM1;

[0010] In the high performance liquid chromatography method, the chromatographic column uses octadecylsilane bonded silica gel as a filler, and uses mobile phase A and mobile phase B for linear gradient elution; the mobile phase A and mobile phase B are both mixed solutions of sodium dihydrogen phosphate and acetonitrile; in the mobile phase A, the volume ratio of sodium dihydrogen phosphate to acetonitrile is 55-65:45-35; in the mobile phase B, the volume ratio of sodium dihydrogen phosphate to acetonitrile is 65-75:35-25;

[0011] The structural formula of the rifapentine intermediate Z1 and its impurities are as follows:

[0012]

[0013] The separated substances can be used in the next production step.

[0014] The above impurities can be arranged and combined in various ways.

[0015] For example, combination 1: impurity SM 1d 、Impurity Z 1b and impurity Z 1c .

[0016] For example, combination 2: impurity SM 1d 、Impurity SM 1b .

[0017] For example, combination 3: impurity SM 1d 、Impurity Z 1b 、Impurity Z 1c 、Impurity SM 1b and impurity SM1.

[0018] Theoretically, when the upper limit of the substances to be separated, identified and / or detected by the method is n (number), 1-n substances can be detected naturally.

[0019] Further, in the mobile phase A, the volume ratio of sodium dihydrogen phosphate to acetonitrile is 58-62:42-38; in the mobile phase B, the volume ratio of sodium dihydrogen phosphate to acetonitrile is 68-72:32-28; the concentration of sodium dihydrogen phosphate is 0.01-0.03 mol / L, and the pH is 6.0±0.5. For example, the volume ratio of sodium dihydrogen phosphate to acetonitrile in the mobile phase A is 62:38 ( Fig. 9 ); or the volume ratio of sodium dihydrogen phosphate and acetonitrile in the mobile phase A is 58:42 ( Fig.10 ).

[0020] Preferably, in the mobile phase A, the volume ratio of sodium dihydrogen phosphate to acetonitrile is 60:40; and in the mobile phase B, the volume ratio of sodium dihydrogen phosphate to acetonitrile is 70:30.

[0021] Preferably, the concentration of the sodium dihydrogen phosphate is 0.015 to 0.025 mol / L. For example, the concentration of the sodium dihydrogen phosphate is 0.015 mol / L ( Fig.15 ); or the concentration of sodium dihydrogen phosphate is 0.025 mol / L ( Fig.16 ).

[0022] Preferably, the pH of the sodium dihydrogen phosphate is 5.8 to 6.2. For example, the pH of the sodium dihydrogen phosphate is 5.8 ( Fig.13 ); or sodium dihydrogen phosphate pH 6.2 ( Fig.14 ).

[0023] Most preferably, the concentration of the sodium dihydrogen phosphate is 0.02 mol / L.

[0024] Most preferably, the pH of the sodium dihydrogen phosphate is 6.0±0.1.

[0025] Furthermore, the pH value of sodium dihydrogen phosphate was adjusted using sodium hydroxide having a concentration of 2 mol / L.

[0026] Further, the procedure of the linear gradient elution is as follows:

[0027] At 0 minutes, the volume ratio of the mobile phase A to the mobile phase B was set to 90-100:10-0;

[0028] At 20 minutes, the volume ratio of the mobile phase A to the mobile phase B was set to 40-60:60-40;

[0029] At 30 minutes, the volume ratio of the mobile phase A to the mobile phase B was set to 0-10:100-90;

[0030] At 40 minutes, the volume ratio of the mobile phase A to the mobile phase B was set to 0-10:100-90;

[0031] At 40.1 minutes, the volume ratio of the mobile phase A to the mobile phase B was set to 90-100:10-0;

[0032] At 50 minutes, the volume ratio of the mobile phase A to the mobile phase B was set to 90-100:10-0.

[0033] Preferably, the procedure of the linear gradient elution is as follows:

[0034] At 0 minutes, the volume ratio of the mobile phase A to the mobile phase B was set to 100:0;

[0035] At 20 minutes, the volume ratio of the mobile phase A to the mobile phase B was set to 50:50;

[0036] At 30 minutes, the volume ratio of the mobile phase A to the mobile phase B was set to 0:100;

[0037] At 40 minutes, the volume ratio of the mobile phase A to the mobile phase B was set to 0:100;

[0038] At 40.1 minutes, the volume ratio of the mobile phase A to the mobile phase B was set to 100:0;

[0039] At 50 minutes, the volume ratio of the mobile phase A to the mobile phase B was set to 100:0.

[0040] Furthermore, the flow rate is 0.8-1.2 mL / min; and the column temperature is 20-30°C.

[0041] Preferably, the flow rate is 0.9-1.1 mL / min; the column temperature is 23-27°C. For example, the flow rate is 0.9 mL / min ( Figure 7 ); or flow rate of 1.1 mL / min ( Figure 8 ). For example, the column temperature is 23°C ( Fig.11 ); or column temperature is 27°C ( Fig.12 ).

[0042] Most preferably, the flow rate is 1.0 mL / min and the column temperature is 25°C.

[0043] Preferably, the specifications of the chromatographic column are 250×4.6 mm, 5 μm.

[0044] Most preferably, the chromatographic column is Hypersil GOLD C18 (250×4.6 mm, 5 μm).

[0045] Preferably, the injection volume is 5 μl.

[0046] Preferably, the running time is 50 minutes.

[0047] The second object of the present invention is to provide a method for identifying rifapentine intermediate Z1 and its impurities, which method can complete the identification of multiple substances in a relatively short time.

[0048] To achieve the above object, the technical solution of the present invention is:

[0049] The method for identifying the rifapentine intermediate Z1 and its impurities comprises separating the composition by the aforementioned separation method, and entering the composition into a detector for detection to obtain a chromatogram; and determining whether the test sample contains the rifapentine intermediate Z1 and its impurities by comparing the chromatogram characteristics of the test sample with those of the reference sample.

[0050] As a preferred solution, the detection wavelength of the detector is 254±10nm. The setting range of ±10nm is based on comprehensive consideration of multiple factors such as error considerations, method superiority, and practical application requirements. This setting range helps to ensure the reliability of the detection results, improve the repeatability and flexibility of the measurement, and meet the requirements of specific experiments.

[0051] Most preferably, the detection wavelength of the detector is 254 nm.

[0052] As a preferred solution, the components in the composition can be identified according to the order of relative retention time. The components of the composition are in ascending order: impurity SM 1d 、Impurity Z 1b 、Impurity Z 1c 、Impurity SM 1b , rifapentine intermediate Z1, impurity SM1.

[0053] Generally, relative retention time is used to describe the relative retention of different components in a mixture on a chromatographic column. It refers to the ratio of the retention time of a component to the retention time of a reference component (usually the main peak or a known component). This ratio reflects the relative retention performance of different components on a chromatographic column and is an important parameter for positioning, qualitative and quantitative analysis in chromatographic analysis. The relative retention time is calculated by dividing the retention time of the target component (tR) by the retention time of the reference component (tR0).

[0054] As a preferred method, the rifapentine intermediate Z1 is used as the reference peak; the relative retention time is 0.47, which is determined to be the impurity SM 1d ; The relative retention time is 0.54, which is determined to be impurity Z 1b ; The relative retention time is 0.64, which is determined to be impurity Z 1c ; The relative retention time is 0.86, which is determined to be impurity SM 1b; The relative retention time is 1.14, which is determined to be impurity SM1; the relative retention time of each component fluctuates within 1%.

[0055] In addition to relative retention time, retention time can also be used to determine components. Retention time refers to the time required for a sample to enter the chromatographic column and be detected by the detector. This time is calculated based on the movement speed of the component on the chromatographic column, that is, the time interval from the start of injection to the chromatographic apex (maximum concentration) of a certain component. It is mainly used to determine the peak order and position of each component in the sample and is one of the basic data 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.

[0056] The third object of the present invention is to provide a method for determining the content of rifapentine intermediate Z1 and its impurities, which method can complete the identification and content determination of multiple substances in a relatively short time.

[0057] To achieve the above object, the technical solution of the present invention is:

[0058] The method for determining the content of rifapentine intermediate Z1 and its impurities is to separate and identify rifapentine intermediate Z1 and its impurities by the aforementioned identification method to obtain a chromatogram; based on the obtained chromatogram, the impurity SM is calculated by the main component self-comparison method with a correction factor. 1d 、Impurity Z 1b and impurity Z 1c The content of impurities SM is calculated by area normalization method. 1b , the content of impurity SM1.

[0059] Furthermore, the impurity SM 1d The correction factor is 0.68.

[0060] Furthermore, the calculation formula of the principal component self-control method with correction factor is as follows:

[0061]

[0062] Through the content determination, we can further determine whether the rifapentine intermediate Z1 and its impurity content are qualified. 1d The peak area multiplied by the correction factor is greater than 1.5 times the main peak area of ​​the control solution, indicating that the impurity SM 1d The content is unqualified; on the contrary, if the impurity SM 1d The peak area multiplied by the correction factor is not greater than 1.5 times the main peak area of ​​the control solution, indicating that the impurity SM 1d The content is qualified. If impurity Z 1b The peak area is 1.5 times greater than the main peak area of ​​the control solution, indicating that impurity Z 1bThe content is unqualified; on the contrary, if the impurity Z 1b The peak area is no more than 1.5 times the main peak area of ​​the control solution, indicating that impurity Z 1b The content is qualified. If impurity Z 1c The peak area is 5 times greater than the main peak area of ​​the control solution, indicating that impurity Z 1c The content is unqualified; on the contrary, if the impurity Z 1c The peak area is not more than 5 times the main peak area of ​​the control solution, indicating that impurity Z 1c The content is qualified. If the impurity SM 1b And / or the impurity SM1 peak area is greater than the peak area of ​​the corresponding impurity in the control solution, indicating that the impurity SM 1b And / or impurity SM1 content is unqualified; on the contrary, if impurity SM 1b Impurity SM 1b And / or impurity SM1 The peak area of ​​impurity SM1 is not greater than the peak area of ​​the corresponding impurity in the control solution, indicating that impurity SM 1b And / or the impurity SM1 content is qualified.

[0063] The above determination method can be used as a drug quality determination model, and further as an indispensable key module in the intelligent production process. This model can achieve accurate separation and quantitative analysis of active ingredients, impurities and degradation products in drugs by accurately controlling parameters such as mobile phase composition, flow rate, column temperature, etc., providing a scientific basis for the comprehensive evaluation of drug quality. In the intelligent production system, this model is seamlessly integrated, able to receive raw data from the production line in real time, automatically perform analysis tasks, and quickly feedback the determination results based on preset quality standards.

[0064] As a preferred solution, the sample preparation solvent is acetonitrile.

[0065] As a preferred solution, the sample concentration is 2 mg / mL.

[0066] As the most preferred solution, the method specifically comprises the following steps:

[0067] S1: Accurately weigh an appropriate amount of rifapentine intermediate Z1, add acetonitrile to dissolve the sample, shake well, and prepare a solution containing 2.0 mg of rifapentine intermediate Z1 per 1 ml as the sample solution.

[0068] S2: Set the chromatographic conditions as follows:

[0069] A chromatographic column with octadecylsilane bonded silica as filler and specifications of 250×4.6mm and 5μm was used; mobile phase A consisted of sodium dihydrogen phosphate with a concentration of 0.015-0.025mol / L and a pH of 5.8-6.2 and acetonitrile in a volume ratio of 58-62:42-38; mobile phase B consisted of sodium dihydrogen phosphate with a concentration of 0.015-0.025mol / L and a pH of 5.8-6.2 and acetonitrile in a volume ratio of 30:70; the mobile phase flow rate was 0.9-1.1ml / min, the detection wavelength was 254nm, and the column box temperature of the chromatographic column was 25°C; the linear gradient elution program was set as follows:

[0070] At 0 minutes, the volume ratio of the mobile phase A to the mobile phase B was set to 100:0;

[0071] At 20 minutes, the volume ratio of the mobile phase A to the mobile phase B was set to 50:50;

[0072] At 30 minutes, the volume ratio of the mobile phase A to the mobile phase B was set to 0:100;

[0073] At 40 minutes, the volume ratio of the mobile phase A to the mobile phase B was set to 0:100;

[0074] At 40.1 minutes, the volume ratio of the mobile phase A to the mobile phase B was set to 100:0;

[0075] At 50 minutes, the volume ratio of the mobile phase A to the mobile phase B was set to 100:0.

[0076] S3: Take the sample solution prepared in S1, such as 5 μl, and inject it into the liquid chromatograph, and detect it according to the chromatographic conditions of S2 to obtain a chromatogram.

[0077] S4: Based on the chromatogram obtained in S3, the impurity SM is calculated using the principal component self-reference method with a correction factor. 1d 、Impurity Z 1b and impurity Z 1c The content of impurities SM is calculated by area normalization method. 1b , the content of impurity SM1.

[0078] The beneficial effects of the present invention are:

[0079] 1. The present invention provides for the first time a high performance liquid chromatography method for separating and detecting rifapentine intermediate Z1 and five process impurities. The method can effectively separate and detect the impurity SM in rifapentine intermediate Z1 within 50 minutes. 1d 、Impurity Z 1b 、Impurity Z 1c 、Impurity SM 1b , impurity SM1, significantly shortening the separation time.

[0080] 2. The method of the present invention is used to separate and detect rifapentine intermediate Z1 and five process impurities without interference from blank solvent, and the main peak and impurity peak are well separated, which has the characteristics of strong specificity.

[0081] 3. The method for separating and detecting rifapentine intermediate Z1 and process impurities provided by the present invention has the characteristics of high sensitivity, wherein the impurity SM 1d The quantitative limit concentration is 0.2598μg / ml, and the detection limit concentration is 0.0779μg / ml; impurity Z 1b The quantitative limit concentration is 0.3012μg / ml, and the detection limit concentration is 0.0904μg / ml; impurity Z 1c The quantification limit concentration of rifapentine intermediate Z1 was 0.2969 μg / ml, and the detection limit concentration was 0.0891 μg / ml.

[0082] 4. The method for separating and detecting rifapentine intermediate Z1 and process impurities provided by the present invention has the characteristics of good durability and good reproducibility. Under this chromatographic system, when the flow rate, column temperature, mobile phase pH, and the volume ratio of sodium dihydrogen phosphate and acetonitrile in mobile phase A fluctuate, the separation degree of rifapentine intermediate Z1 and three process impurities from adjacent peaks all meets the requirement of being greater than 1.5.

[0083] 5. The method of the present invention is simple, feasible, efficient and rapid, and both the tailing factor and the theoretical pedal number can achieve very good results, and can effectively determine the content of each impurity in the rifapentine intermediate Z1. The present invention solves the problem of separation and determination of the rifapentine intermediate Z1 and 5 process impurities, thereby ensuring the quality control of the rifapentine intermediate Z1, and ultimately determining the safety and effectiveness of the product, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0084] Figure 1 is the HPLC chromatogram of blank solvent;

[0085] Figure 2 It is a high performance liquid chromatogram of a mixed solution of rifapentine intermediate Z1 and five process impurities;

[0086] Figure 3 It is the HPLC chromatogram of the test solution of rifapentine intermediate Z1;

[0087] Figure 4 This is the HPLC chromatogram of the quantitative limit determination of rifapentine intermediate Z1 and three process impurities;

[0088] Figure 5This is the HPLC chromatogram of the detection limit of rifapentine intermediate Z1 and three process impurities.

[0089] Figure 6 For durability - mixed solution - chromatogram under normal conditions;

[0090] Figure 7 This is the chromatogram under the conditions of durability-mixed solution-flow rate 0.9ml / min;

[0091] Figure 8 This is the chromatogram under the conditions of durability-mixed solution-flow rate 1.1ml / min;

[0092] Fig. 9 Durability - mixed solution - chromatogram under the condition that the volume ratio of sodium dihydrogen phosphate and acetonitrile in mobile phase A is 62:38;

[0093] Fig.10 This is the chromatogram under the condition of durability-mixed solution-the volume ratio of sodium dihydrogen phosphate and acetonitrile in mobile phase A is 58:42;

[0094] Fig.11 This is the chromatogram under the conditions of durability-mixed solution-column temperature 23°C;

[0095] Fig.12 This is the chromatogram under the conditions of durability-mixed solution-column temperature 27°C;

[0096] Fig.13 This is the chromatogram under the conditions of durability-mixed solution-buffer salt pH 5.8;

[0097] Fig.14 This is the chromatogram under the conditions of durability-mixed solution-buffer salt pH 6.2;

[0098] Fig.15 This is the chromatogram under the conditions of durability-mixed solution-buffer salt concentration of 0.015M;

[0099] Fig.16 This is a chromatogram of the durability-mixed solution-buffer salt concentration of 0.025M. DETAILED DESCRIPTION

[0100] The technical solution of the present invention will be further described clearly and completely in conjunction with specific embodiments below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work belong to the protection scope of the present invention.

[0101] Supplementary table to the drawings in the specification. Figure 1-Figure 16 Visual aids are provided for the purpose of understanding and interpretation. In case of any ambiguity, the user should refer to the corresponding numbered table ( Figures 1 to 16 Corresponding to Tables 1-16 respectively), the data is used to obtain more detailed information. On the contrary, if any information that may cause misunderstanding or ambiguity is found in the process of reviewing Tables 1-16, the content in the corresponding numbered figure shall prevail. The above guidelines are intended to ensure the correct interpretation of this document and the consistency of its information. Although some of the characters in the spectrum of this application document overlap, they can still be viewed clearly, and the integration results of each figure are recorded in detail in the specification. In addition, the numbers in the spectrum have no effect on the scope of protection of the claims and the full disclosure of the technical solution in the specification.

[0102] Table 1

[0103]

[0104] Table 2

[0105]

[0106] Table 3

[0107]

[0108] Table 4

[0109]

[0110] Table 5

[0111]

[0112] Table 6

[0113]

[0114] Table 7

[0115]

[0116] Table 8

[0117]

[0118] Table 9

[0119]

[0120] Table 10

[0121]

[0122] Table 11

[0123]

[0124] Table 12

[0125]

[0126] Table 13

[0127]

[0128] Table 14

[0129]

[0130] Table 15

[0131]

[0132] Table 16

[0133]

[0134] In Tables 1 to 16 above, the peaks without names are unknown impurities.

[0135] In order to enhance the understanding of the present invention, some key technologies and scientific terms are clearly defined below. Unless specifically defined herein, all other technical and scientific terms follow the meanings generally accepted and understood in the field to which the present invention belongs. It should be emphasized that the scope of the present invention is not limited to the specific methods, reagents, compounds, compositions, reference substances, and test substances described, but reasonable flexibility and adjustment in these aspects are allowed. At the same time, please understand that the terms used in this article are intended to illustrate specific embodiments rather than to be interpreted restrictively.

[0136] In addition, all literature cited herein, including but not limited to patents, patent applications, academic papers, textbooks and further citations therein, are deemed to be incorporated herein by reference as a whole to the extent not directly cited. If there is any inconsistency or conflict between the contents of these cited documents or similar materials and this application, especially in terms of term definitions, term usage, technical descriptions, etc., the contents of this application shall prevail.

[0137] the term

[0138] The limit of quantitation refers to the lowest amount of the substance to be measured in the sample, and the measurement result should have a certain degree of accuracy and precision. In other words, the limit of quantitation is the lowest level at which the analytical method can accurately and reliably measure the concentration of the substance to be measured in the sample. In HPLC, the determination of the limit of quantitation usually relies on the signal-to-noise ratio (S / N) method, that is, the concentration of the substance to be measured corresponding to a certain level of signal-to-noise ratio is used as the limit of quantitation. The determination of the limit of quantitation is crucial to ensure the accuracy and reliability of the analysis results.

[0139] The durability of chromatographic conditions refers to the ability of the chromatographic analysis system to maintain stable analytical performance and not significantly affect the measurement results when there is a slight change in the measurement conditions. This durability is crucial to ensure the reliability, repeatability and stability of the analysis results.

[0140] The limit of detection (LOD) refers to the lowest concentration or amount of the substance in the sample that can be detected. It reflects the sensitivity and noise of the analytical method and instrument, and also indicates the level of the blank (background) value after the sample is processed.

[0141] The correction factor is a coefficient or parameter used to correct the results during the analysis process. It is intended to improve data accuracy and reliability. In HPLC analysis, since the same detector has different response values ​​for different substances, when different substances of the same mass pass through the detector, the peak areas generated are not necessarily equal. In order to make the peak area accurately reflect the content of the component to be measured, standard substances are used for correction, the correction factor is calculated, and it is applied to the measurement results of the sample to be measured.

[0142] The ratio of peak height to noise (S / N, i.e. signal-to-noise ratio) is used in HPLC to evaluate the detection sensitivity and resolution of the instrument and is an important indicator for measuring instrument performance. Among them: peak height refers to the signal value output by the detector when the component to be tested is eluted from the column; and noise refers to the fluctuation of the baseline signal, that is, the signal value measured by the blank sample. The signal-to-noise ratio is the ratio of the signal measured by a sample of known concentration to the signal measured by a blank sample. The larger the signal-to-noise ratio, the more accurately the instrument can separate and identify the target component when detecting the sample, while also reducing the interference of background noise.

[0143] In the examples of the present invention, information on the rifapentine intermediate Z1 and its related impurities is shown in Table A.

[0144] Table A. Compound information table

[0145]

[0146]

[0147] In the examples of the present invention, the test solution was prepared by accurately weighing an appropriate amount of the product (rifapentine intermediate Z1), adding acetonitrile to dissolve the sample, shaking well, and preparing a solution containing 2.0 mg of rifapentine intermediate Z1 per 1 ml.

[0148] In the embodiments of the present invention, the instruments and chromatographic conditions used are as follows:

[0149] High performance liquid chromatograph: Thermo Fisher Vanquish; chromatographic column: Hypersil GOLD C18 (250×4.6mm, 5μm); mobile phase: mobile phase A is 0.02mol / L sodium dihydrogen phosphate (adjust the pH value to 6.0±0.1 with 2mol / L sodium hydroxide)-acetonitrile (60:40); mobile phase B is 0.02mol / L sodium dihydrogen phosphate (adjust the pH value to 6.0±0.1 with 2mol / L sodium hydroxide)-acetonitrile (30:70); mobile phase A and mobile phase B are linearly eluted with gradient according to Table B below; detector detection wavelength: 254nm; mobile phase flow rate: 1.0ml / min; column box temperature: 25℃; injection volume: 5μl; running time: 50min; diluent (solvent for dissolving reference substance and test sample): acetonitrile.

[0150] Table B. Gradient elution schedule

[0151] Time (min) 0 20 30 40 40.1 50 Mobile phase A (%) 100 50 0 0 100 100 Mobile phase B (%) 0 50 100 100 0 0

[0152] In the embodiments of the present invention, if there are any unmentioned conditions in the chromatographic conditions, they can be determined by referring to the high performance liquid chromatography method (Chinese Pharmacopoeia 2020 Edition, Part 4, Part 0512).

[0153] In the embodiment of the present invention, the impurity SM 1d 、Impurity Z 1b and impurity Z 1c The impurity SM is calculated by the principal component self-comparison method with the correction factor added. 1d The peak area multiplied by the correction factor 0.68 shall not be greater than 1.5 times (0.15%) the main peak area of ​​the control solution; impurity Z 1b The peak area shall not be greater than 1.5 times (0.15%) of the main peak area of ​​the control solution; impurity Z 1c The peak area shall not be greater than 5 times (0.5%) the main peak area of ​​the control solution. 1b , impurity SM1 was quantified using the area normalization method.

[0154] The calculation formula of the principal component self-control method with correction factor is as follows:

[0155]

[0156] Example 1. Specificity experiment of rifapentine intermediate Z1 and five process impurities

[0157] (1) Prepare the test solution

[0158] Impurity Z 1b Stock solution: Accurately weigh impurities Z1b Place 12.5 mg in a 25 ml volumetric flask, add acetonitrile to dissolve and dilute to the mark, shake well, and you have it.

[0159] Impurity Z 1c Stock solution: Accurately weigh impurity Z 1c Place 12.5 mg in a 25 ml volumetric flask, add acetonitrile to dissolve and dilute to the mark, shake well, and you have it.

[0160] Impurity SM 1d Stock solution: Accurately weigh the impurity SM 1d Place 12.5 mg in a 25 ml volumetric flask, add acetonitrile to dissolve and dilute to the mark, shake well, and you have it.

[0161] Impurity SM1 positioning solution: Accurately weigh 1.0 ml of impurity SM1 stock solution, place it in a 10 ml volumetric flask, add diluent to dissolve and dilute to the scale, shake well, and obtain.

[0162] Impurity SM 1b Positioning solution: Accurately weigh the impurity SM 1b Place 1.0 ml of stock solution in a 10 ml volumetric flask, add diluent to dissolve and dilute to the scale, shake well, and you have it.

[0163] Impurity mixed solution: Precisely remove impurities SM 1d 、Impurity Z 1b 3.0 ml of stock solution, impurity Z 1c , impurity SM1 and impurity SM 1b Place 10 ml of each stock solution in a 50 ml volumetric flask, add acetonitrile to dilute to the mark, shake well, and you have it.

[0164] Test solution: Accurately weigh 20.24 mg of the product, place in a 10 ml volumetric flask, add diluent to dissolve and dilute to the scale, shake well, and obtain.

[0165] Mixed solution: Accurately weigh 19.89 mg of the test sample and place it in a 10 ml volumetric flask. Accurately transfer 1.0 ml of the impurity mixed solution and place it in the same 10 ml volumetric flask. Add acetonitrile to dissolve and dilute to the scale. Shake well to obtain.

[0166] (2) Detection

[0167] Take the diluent, test solution and mixed solution respectively and inject them according to the above-mentioned chromatographic conditions, and record the chromatogram.

[0168] The results are shown in Table C. Figure 1 to Figure 3 , Table 1 to Table 3. The blank diluent does not interfere with the sample determination; the separation between the main peak and the adjacent impurity peak is greater than 1.5; the separation between each known impurity peak is greater than 1.5; the results show that the main peak and the impurity peak are well separated and have strong specificity.

[0169] Table C. Specificity test results

[0170] name Retention time (min) Relative retention time (RRT) Separation <![CDATA[Impurity SM 1d > 8.309 0.47 4.30 <![CDATA[Impurity Z 1b > 9.476 0.54 6.42 <![CDATA[Impurity Z 1c > 11.267 0.64 6.58 <![CDATA[Impurity SM 1b > 14.984 0.86 3.60 <![CDATA[Rifapentine Intermediate Z1]]> 17.500 - 2.11 <![CDATA[Impurity SM1]]> 19.976 1.14 5.75

[0171] Example 2. Chromatographic system for the rifapentine intermediate Z1 and three process impurities (impurity SM 1d 、Impurity Z 1b 、Impurity Z 1c Basic research on the limit of quantification and detection of

[0172] (1) Prepare the test solution

[0173] Z1 reference substance stock solution: Accurately weigh 12.52 mg of Z1 reference substance, place in a 25 ml volumetric flask, dilute to the scale with diluent, shake well, and obtain.

[0174] Quantitative limit stock solution: Accurately pipette 1.0 ml of Z1 reference stock solution, and accurately pipette impurity SM 1d 、Impurity Z 1b 、Impurity Z 1c Place 1.0 ml of each stock solution in a 100 ml volumetric flask, dilute to the mark with diluent, shake well, and the solution is ready.

[0175] Quantitation limit solution: Accurately pipette 6.0 ml of the quantitation limit stock solution into a 100 ml volumetric flask, dilute to the mark with diluent, shake well, and the solution is ready.

[0176] Detection limit solution: Accurately pipette 3.0 ml of the quantitative limit solution into a 10 ml volumetric flask, add diluent to dilute to the scale, shake well, and obtain.

[0177] (2) Measurement method

[0178] The above quantitative limit solution was sampled continuously for 6 times, and the detection limit solution was sampled continuously for 3 times, the ratio of the main peak height to the noise (signal-to-noise ratio) was calculated, and the chromatogram was recorded.

[0179] The calculation formulas for the limit of quantification and the limit of detection are as follows:

[0180]

[0181] Results: The quantitative limit test results are shown in Table D. Figure 4 and Table 4, and the detection limit results are shown in Tables E and Figure 5 From the data in Table D and Table E, it can be seen that under this chromatographic system, the detection limit and quantification limit of the three process impurities meet the requirement of S / N=3(10):1.

[0182] Table D. Quantitation limit determination results

[0183]

[0184]

[0185] Table E. Detection limit determination results

[0186]

[0187] Example 3. Durability test

[0188] Mixed solution: Accurately weigh the product and each impurity reference substance in appropriate amounts to prepare a solution containing approximately 12.0 mg of rifapentine intermediate Z and 12.0 mg of impurity SM per 1 ml. 1d 3.0μg, impurity Z 1b 3μg, impurity SM 1b , impurity SM1 and impurity Z 1c 10.0 μg of each solution.

[0189] Take the mixed solution and adjust the experimental conditions (mobile phase pH ± 0.2, buffer salt concentration ± 0.005M flow rate ± 0.1ml / min, column temperature ± 2°C, volume ratio of sodium dihydrogen phosphate and acetonitrile in mobile phase A ± 2). After the instrument system is stable, test them separately to examine the separation between the peaks.

[0190] Results: Figure 6-Figure 16 As shown in Table F, Table G, and Tables 6 to 16, under the present chromatographic system, when the flow rate, column temperature, mobile phase pH, and the ratio of sodium dihydrogen phosphate and acetonitrile in mobile phase A fluctuate, the separation degrees of rifapentine intermediate Z1 and the three process impurities from adjacent peaks all meet the requirement of being greater than 1.5, demonstrating the good durability of the present method.

[0191] Table F. Durability test results (separation)

[0192]

[0193] Table G. Durability test results (retention time)

[0194]

Claims

1. A method for separating rifapentine intermediate Z1 and its impurities based on high performance liquid chromatography, characterized in that: The rifapentine intermediate Z1 and the impurities together constitute a composition, and the impurities include impurity SM 1d 、Impurity Z 1b 、Impurity Z 1c 、Impurity SM 1b and any one or more of impurities SM1; In the high performance liquid chromatography method, the chromatographic column uses octadecylsilane bonded silica gel as a filler, and uses mobile phase A and mobile phase B for linear gradient elution; the mobile phase A and mobile phase B are both mixed solutions of sodium dihydrogen phosphate and acetonitrile; in the mobile phase A, the volume ratio of sodium dihydrogen phosphate to acetonitrile is 55-65:45-35; in the mobile phase B, the volume ratio of sodium dihydrogen phosphate to acetonitrile is 65-75:35-25; The structural formula of the rifapentine intermediate Z1 and its impurities are as follows:

2. The method according to claim 1, characterized in that In the mobile phase A, the volume ratio of sodium dihydrogen phosphate to acetonitrile is 58-62:42-38; in the mobile phase B, the volume ratio of sodium dihydrogen phosphate to acetonitrile is 68-72:32-28; The concentration of the sodium dihydrogen phosphate is 0.01-0.03 mol / L, and the pH is 6.0±0.

5.

3. The method according to claim 1, characterized in that The procedure of the linear gradient elution is as follows: At 0 minutes, the volume ratio of the mobile phase A to the mobile phase B was set to 90-100:10-0; At 20 minutes, the volume ratio of the mobile phase A to the mobile phase B was set to 40-60:60-40; At 30 minutes, the volume ratio of the mobile phase A to the mobile phase B was set to 0-10:100-90; At 40 minutes, the volume ratio of the mobile phase A to the mobile phase B was set to 0-10:100-90; At 40.1 minutes, the volume ratio of the mobile phase A to the mobile phase B was set to 90-100:10-0; At 50 minutes, the volume ratio of the mobile phase A to the mobile phase B was set to 90-100:10-0.

4. The method according to claim 1, characterized in that The flow rate is 0.8-1.2 mL / min; the column temperature is 20-30°C.

5. A method for identifying rifapentine intermediate Z1 and its impurities, characterized in that: The composition is separated by the method described in any one of claims 1 to 4, and enters a detector for detection to obtain a chromatogram; by comparing the chromatogram characteristics of the test sample and the reference sample, it is determined whether the test sample contains rifapentine intermediate Z1 and its impurities.

6. The method according to claim 5, characterized in that The detection wavelength of the detector is 254±10 nm.

7. The method according to claim 5, characterized in that According to the order of relative retention time, the components in the composition can be identified. The components of the composition are in ascending order: impurity SM 1d 、Impurity Z 1b 、Impurity Z 1c 、Impurity SM 1b , rifapentine intermediate Z1, impurity SM1.

8. The method according to claim 5, characterized in that The rifapentine intermediate Z1 was used as the reference peak; the relative retention time was 0.47, which was determined to be impurity SM 1d ; The relative retention time is 0.54, which is determined to be impurity Z 1b ; The relative retention time is 0.64, which is determined to be impurity Z 1c ; The relative retention time is 0.86, which is determined to be impurity SM 1b ; The relative retention time is 1.14, which is determined to be impurity SM1; the relative retention time of each component fluctuates within 1%.

9. A method for determining the content of rifapentine intermediate Z1 and its impurities, characterized in that: The method according to any one of claims 5 to 8 is used to separate and identify the rifapentine intermediate Z1 and its impurities to obtain a chromatogram; based on the obtained chromatogram, the impurity SM is calculated by the principal component self-reference method with a correction factor. 1d 、Impurity Z 1b and impurity Z 1c The content of impurities SM is calculated by area normalization method. 1b , the content of impurity SM1.

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

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