A kind of teniposide degradation impurity and its detection and separation method

Abstract

The present invention discloses a teniposide degradation impurity and a detection and separation method thereof. It relates to the field of medical technology. A teniposide degradation impurity has the following structural formula: #imgabs0# wherein R1, R2, R3, R4 and R6 are independently selected from ‑OH or a halogen group; R5 and R7 are independently selected from methoxy, ethoxy, propoxy or phenoxy. The content of the impurity must be strictly controlled in each link of the production of raw materials and preparations to ensure that the teniposide injection can still meet the quality standards at the end of the effective period. The presence of impurities may cause adverse reactions of the drug, especially in the long-term treatment process, the accumulation of impurities will increase the risk of adverse reactions such as allergic reactions and toxic reactions in patients.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a teniposide degradation impurity and a detection and separation method thereof. Background Art

[0002] As one of the major lethal diseases worldwide, cancer has become a major challenge that needs to be solved in the field of modern medicine. With the continuous progress of scientific research and clinical treatment, the treatment methods of cancer have gradually diversified, but at the same time, the development of new anticancer drugs has become increasingly difficult. Especially in the optimization of existing drugs and the research and development of new drugs, there are increasingly severe challenges. Therefore, how to improve the quality and efficacy of existing anticancer drugs has become one of the problems that pharmaceutical researchers need to solve urgently.

[0003] Teniposide (VM-26) is an anti-tumor drug widely used in clinical practice and is highly valued for its excellent therapeutic effects. However, during its production and use, the control of formulation impurities has become a key issue in ensuring its safety and effectiveness.

[0004] Teniposide is a glycoside compound extracted from podophyllotoxin. It is a non-intercalating anti-tumor drug that acts on DNA topoisomerase II. It binds to DNA topoisomerase II, blocks the activity of the enzyme, causes DNA chain breaks, and thus affects DNA replication and transcription. Teniposide mainly acts in the late S and G2 phases of the cell cycle. It prevents cell mitosis by causing single-strand and double-strand breaks in the DNA chain, thereby inhibiting the proliferation of tumor cells. It has a wide spectrum of anti-tumor effects and can effectively treat various types of malignant tumors, including malignant lymphoma, Hodgkin's disease, acute lymphocytic leukemia, glioblastoma, cerebrovascular disease, astrocytoma, bladder cancer, neuroblastoma, and other solid tumors in children. In addition, because teniposide has strong lipophilicity and can cross the blood-brain barrier, it has become one of the first choice drugs for the clinical treatment of brain tumors. Teniposide has relatively low toxicity and can significantly improve the efficacy when used in combination with other anticancer drugs.

[0005] However, despite the significant clinical efficacy of teniposide, its production process still faces many challenges, and there are degradation impurities in teniposide preparations. Degradation impurities refer to other impurity compounds produced by degradation reactions of the original drug molecules caused by the external environment or chemical reactions during the storage, processing, transportation or use of drugs or preparations. These impurities will not only affect the quality of the preparation, but may also lead to reduced drug efficacy or increased adverse reactions. The degradation pathway of teniposide is complex and may be affected by many factors, such as acid-base environment, temperature, oxidation, etc. These impurities already exist in the raw materials of teniposide, and during the production of preparations, due to changes in processing conditions, the content of impurities tends to increase continuously, especially in high temperature, acidic or alkaline environments. More seriously, in the stability study of teniposide preparations, the level of impurities may continue to grow over time, and there is even a risk of approaching the limit at the end of the shelf life.

[0006] In order to ensure the quality and safety of teniposide preparations, impurities must be strictly controlled and managed from the production of raw materials to the production of final preparations. This is not only to reduce the incidence of allergic reactions, but also to ensure that the efficacy of the drug is not affected and that it can continue to exert the expected effect during the treatment process. Impurity control measures include optimizing the synthesis process, improving the quality control of raw materials, and monitoring the links that may introduce impurities during the preparation production process. In addition, long-term stability studies are also needed to predict and control the growth trend of impurities during the use of drugs to ensure that the impurity content is always within a safe level during the shelf life of the drug.

[0007] The removal of impurities in teniposide is not only to improve the quality and stability of the drug, but also related to the safety of patients. In the drug production process, the presence of impurities may lead to adverse drug reactions, especially in long-term treatment, the accumulation of impurities will increase the risk of adverse reactions such as allergic reactions and toxic reactions in patients.

[0008] Therefore, ensuring the rationality of the impurity content in teniposide preparations is crucial to improving the therapeutic effect of teniposide preparations. Summary of the invention

[0009] The object of the present invention is to provide a novel teniposide degradation impurity and a method for detecting, separating and removing the impurity, so as to monitor and ensure the purity, safety, stability and reliability of the production process of the drug. By detecting the impurity, potential quality problems can be discovered in time, and separation and removal methods can be taken to improve the teniposide preparation and / or teniposide raw material.

[0010] A first aspect of the present invention is:

[0011] A teniposide degradation impurity is provided.

[0012] The second aspect of the present invention is:

[0013] Provided is a method for detecting teniposide degradation impurities. Impurities are degradation byproducts of drugs, and their existence and concentration changes can provide important information for drug quality control.

[0014] The third aspect of the present invention is:

[0015] A method for separating impurities from a teniposide preparation is provided, thereby obtaining a pure impurity.

[0016] Another method of the present invention provides a method for removing impurities from teniposide raw material medicine, thereby reducing the impurity content in the teniposide raw material medicine and improving the purity of the raw material medicine.

[0017] Specifically, the technical solution adopted according to the first aspect of the present invention is:

[0018] A teniposide degradation impurity having the following structural formula:

[0019]

[0020] Wherein, R1, R2, R3, R4 and R6 are independently selected from -OH or a halogen group;

[0021] R5 and R7 are independently selected from methoxy, ethoxy, propoxy or phenoxy.

[0022] According to the embodiments of the present invention, one of the technical solutions has at least one of the following advantages or beneficial effects:

[0023] The present invention finds a type of degradation impurity in teniposide preparations, which has multiple degradation pathways. Teniposide preparations may produce such impurities under acidic, alkaline, high temperature and oxidative conditions, and already exist in the raw material medicine. Such impurities may further increase during the preparation production process and continue to grow during the validity period. Therefore, the content of such impurities must be strictly controlled in each link of the production of the raw material medicine and the preparation to ensure that the teniposide injection can still meet the quality standards at the end of the validity period.

[0024] In addition, the presence of this impurity may cause adverse drug reactions, especially during long-term treatment, the accumulation of impurities will increase the risk of patients experiencing adverse reactions such as allergic reactions and toxic reactions.

[0025] According to an embodiment of the present invention, the teniposide degradation impurity has the following structural formula:

[0026]

[0027] The molecular formula of the impurity is: C 32 H 34 O 14 S, molecular weight: 674.67, named: S2302-M674.

[0028] According to a second aspect of the present invention, a method for detecting teniposide degradation impurities is provided, and the technical solution adopted is:

[0029] A method for detecting teniposide degradation impurities comprises the following steps:

[0030] Teniposide degradation impurities are detected by liquid chromatography, and the chromatographic conditions of the liquid chromatography include:

[0031] The mobile phase was acetonitrile-sodium acetate buffer in a volume ratio of 35-37:60-62;

[0032] The filling material of the chromatographic column is octadecylsilane bonded silica gel.

[0033] According to the embodiments of the present invention, the technical solution has at least the following advantages or beneficial effects:

[0034] By adopting the chromatographic conditions of the present invention, the teniposide degradation impurities can be detected.

[0035] After using teniposide preparations, some people will have allergic reactions, mainly manifested as chills, fever, tachycardia, bronchospasm, dyspnea, hypotension, flushing, sweating or edema, which may be related to teniposide degradation impurities in teniposide preparations or raw materials. The present invention provides a method for detecting teniposide degradation impurities, which can quickly and stably detect impurity S2302-M674, and provide a guarantee for the quality management of teniposide preparations or raw materials.

[0036] According to one embodiment of the present invention, the method for detecting teniposide degradation impurities further includes the following chromatographic conditions: the elution procedure is isocratic elution.

[0037] According to one embodiment of the present invention, the volume ratio of the acetonitrile-sodium acetate buffer is 35:60.

[0038] According to one embodiment of the present invention, the pH of the acetonitrile-sodium acetate buffer is 4.0-4.2.

[0039] According to one embodiment of the present invention, the pH of the acetonitrile-sodium acetate buffer is 4.0.

[0040] According to one embodiment of the present invention, the method for preparing the sodium acetate buffer comprises the following steps: taking 5.44 g of anhydrous sodium acetate, dissolving it in water and diluting it to 2000 mL, and adjusting the pH to 4.0 with glacial acetic acid to obtain a sodium acetate buffer.

[0041] According to one embodiment of the present invention, the column temperature is 30-32°C.

[0042] According to one embodiment of the present invention, the column temperature is 30°C.

[0043] According to one embodiment of the present invention, the chromatographic conditions of the liquid chromatography further include: a flow rate of 1.0-1.2 mL / min. Preferably, the flow rate is 1.0 mL / min.

[0044] According to one embodiment of the present invention, the chromatographic conditions of the liquid chromatography also include: a detection wavelength of 280 nm.

[0045] According to one embodiment of the present invention, the chromatographic conditions of the liquid chromatography also include: an injection volume of 20 μL.

[0046] Specifically, the technical solution adopted according to the third aspect of the present invention is:

[0047] A method for separating teniposide degradation impurities from a teniposide preparation comprises the following steps:

[0048] Separating the teniposide degradation impurities in the teniposide preparation by high performance liquid chromatography;

[0049] The chromatographic conditions of the high performance liquid chromatography include:

[0050] The mobile phase is 0.1-0.2% volume concentration of phosphoric acid solution and acetonitrile;

[0051] The filling material of the chromatographic column is octadecylsilane bonded silica gel;

[0052] The elution procedure is:

[0053]

[0054]

[0055] Since the impurities have many degradation channels, the content of the impurities will double during the preparation production process. The impurity separation method of the present invention can be used to prepare pure impurities. The pure impurities can be used to prepare impurity localization solutions, etc., and used in the method of the present invention for detecting teniposide degradation impurities.

[0056] According to one embodiment of the present invention, the chromatographic column is selected from Eclipse XDB C18. The size of the Eclipse XDB C18 filler particles is 5 μm, the inner diameter is 4.6 mm, and the length is 150 mm. The Eclipse XDB C18 chromatographic column is selected to separate the teniposide degradation impurities from the teniposide preparation, so as to achieve more efficient separation.

[0057] According to one embodiment of the present invention, in the method for separating the teniposide degradation impurities from the teniposide preparation, the mobile phase is a 0.1% volume concentration phosphoric acid solution and acetonitrile.

[0058] According to one embodiment of the present invention, in the method for separating the teniposide degradation impurities from the teniposide preparation, the chromatographic conditions of the high performance liquid chromatography further include: an injection volume of 10-12 μL.

[0059] According to one embodiment of the present invention, in the method for separating the teniposide degradation impurities from the teniposide preparation, during the injection, 1 drop of the mixture is taken, DMF (N,N-dimethylformamide), acetonitrile and water are added to dilute to 1.5 mL, and then the injection is performed.

[0060] According to one embodiment of the present invention, the method for separating the teniposide degradation impurities from the teniposide preparation further comprises subjecting the teniposide preparation to at least one treatment, including acid treatment, alkali treatment, high temperature treatment or oxidation treatment, before separating the teniposide preparation by high performance liquid chromatography.

[0061] By subjecting the teniposide preparation to at least one of acid treatment, alkali treatment, high temperature treatment or oxidation treatment, the content of impurity S2302-M674 in the teniposide preparation can reach more than 60%, and then the method for separating the teniposide degradation impurities from the teniposide preparation is used to separate the impurities, so as to obtain pure impurities, and the pure impurities can be used to prepare impurity location solutions, reference solution, test sample impurity spiked solution, etc.

[0062] According to one embodiment of the present invention, the method for separating the teniposide degradation impurities further comprises the following steps: after the teniposide preparation is subjected to at least one of acid treatment, alkali treatment, high temperature treatment or oxidation treatment, and chromatographic separation, a purification operation is performed to obtain pure impurities.

[0063] According to one embodiment of the present invention, the purification operation comprises the following steps: adding ethanol to the sample solution obtained after chromatographic separation, rotary evaporation to obtain a white solid-liquid mixture, filtering, adding water, ethanol and dichloromethane to the filtrate, rotary evaporation, heating in a 50°C water bath, and completely removing the solvent to obtain a solid, washing with water, and filtering to obtain a pure impurity.

[0064] According to one embodiment of the present invention, the volume ratio of water, ethanol and dichloromethane is 80:10:10.

[0065] According to one embodiment of the present invention, the acid treatment comprises the following steps: adding 0.01-0.5 mol / L hydrochloric acid to the teniposide preparation.

[0066] According to one embodiment of the present invention, the acid treatment comprises the following steps: adding 0.01-0.02 mol / L hydrochloric acid to the teniposide preparation.

[0067] According to one embodiment of the present invention, the acid treatment comprises the following steps: adding 0.05-0.06 mol / L hydrochloric acid to the teniposide preparation.

[0068] According to one embodiment of the present invention, the acid treatment comprises the following steps: adding 0.1-0.12 mol / L hydrochloric acid to the teniposide preparation.

[0069] According to one embodiment of the present invention, the acid treatment comprises the following steps: adding 0.2-0.22 mol / L hydrochloric acid to the teniposide preparation.

[0070] According to one embodiment of the present invention, the acid treatment comprises the following steps: adding 0.5-0.52 mol / L hydrochloric acid to the teniposide preparation.

[0071] According to one embodiment of the present invention, the alkali treatment comprises the following steps: adding 2-2.2 mL of 0.1-0.12 mol / L sodium hydroxide solution to 2-2.2 mL of teniposide preparation, leaving it for 4-5 hours, adding 2-2.2 mL of 0.1-0.12 mol / L hydrochloric acid solution, and a flocculent precipitate is precipitated, which cannot be dissolved by adding mobile phase, and N,N-dimethylformamide is added until it can be dissolved to the scale, thereby completing the alkali treatment.

[0072] According to one embodiment of the present invention, the alkaline treatment comprises the following steps: adding 2-2.2 mL of 0.1-0.12 mol / L sodium hydroxide solution to 2-2.2 mL of teniposide preparation, leaving it for 1-2 hours, adding 2-2.22 mL of 0.1-0.12 mol / L hydrochloric acid solution, adding 5-5.2 mL of N,N-dimethylformamide to dissolve it, and diluting the mobile phase to the scale.

[0073] According to one embodiment of the present invention, the high temperature treatment comprises the following steps: placing 2-2.2 mL of the teniposide preparation in a volumetric flask, placing it in an oven at 105-110°C for 5-6 hours, bringing it to room temperature, diluting the mobile phase to the scale, and completing the high temperature treatment.

[0074] According to one embodiment of the present invention, the oxidation treatment comprises the following steps: placing 2-2.2 mL of teniposide preparation in a volumetric flask, adding 2-2.2 mL of 30-32% hydrogen peroxide solution, leaving for 4-5 hours, diluting the mobile phase to the scale, and completing the oxidation treatment.

[0075] According to one embodiment of the present invention, the teniposide preparation comprises at least one of a teniposide preparation injection, a teniposide dry suspension, a teniposide capsule, a lyophilized teniposide for injection, and a teniposide tablet.

[0076] A method for removing teniposide degradation impurities from teniposide bulk drug comprises the following steps:

[0077] The teniposide raw material is dissolved and filtered to obtain a filtrate, the filtrate is mixed with an organic solvent, and the mixture is subjected to dissolution and crystallization, filtration and vacuum drying to obtain a refined teniposide product.

[0078] According to one embodiment of the present invention, at least one of N,N-dimethylacetamide, N,N-dimethylformamide and dimethyl sulfoxide is used to dissolve the teniposide raw material.

[0079] According to one embodiment of the present invention, the organic solvent includes at least one of ethanol, isopropanol, acetone and butanol.

[0080] According to one embodiment of the present invention, the weight ratio of the organic solvent to the teniposide bulk drug is 10-12:1-1.2.

[0081] According to one embodiment of the present invention, the vacuum drying conditions include: a temperature of 95-100° C., a pressure greater than 0.08 MPa, and a drying time of 6-7 hours.

[0082] According to one embodiment of the present invention, a method for removing teniposide degradation impurities from teniposide bulk drug comprises the following steps:

[0083] A1: dissolving and filtering the teniposide raw material to obtain a filtrate, mixing the filtrate with an organic solvent, performing dissolution and crystallization, filtering and vacuum drying to obtain a primary refined teniposide product;

[0084] A2 repeats step A1 to obtain a secondary refined product of teniposide;

[0085] A3: dissolving and filtering the teniposide secondary refined product to obtain a filtrate, adding water to the filtrate, and performing crystallization, filtering and vacuum drying to obtain a teniposide tertiary refined product.

[0086] By removing the teniposide degradation impurities in the teniposide raw material, the impurities in the raw material can be controlled below 0.07%, the quality of the teniposide drug can be controlled from the source, and the allergic risk of the teniposide drug can be reduced.

[0087] After the teniposide degradation impurities are removed from the teniposide raw material, the raw material is used to prepare a teniposide preparation, such as a teniposide injection, in which there is no or only a very small amount of the teniposide degradation impurities, so that the impurities in the finished preparation are controlled below 0.2%, and a high-quality teniposide preparation is prepared.

[0088] According to one embodiment of the present invention, the yield of the secondary refined teniposide is 30-80%.

[0089] A high-purity teniposide bulk drug comprises a bulk drug obtained by removing teniposide degradation impurities from a teniposide bulk drug through the method for removing teniposide degradation impurities from the bulk drug, wherein the mass percentage of impurities S2302-M674 in the bulk drug is less than 0.07%.

[0090] The words "preferred", "more preferred" and the like in the present invention refer to embodiments of the present invention that can provide certain beneficial effects in certain circumstances. However, other embodiments may also be preferred under the same circumstances or other circumstances. In addition, the description of one or more preferred embodiments does not imply that other embodiments are not applicable, nor is it intended to exclude other embodiments from the scope of the present invention.

[0091] When a numerical range is disclosed herein, the above range is considered to be continuous and includes the minimum and maximum values ​​of the range, as well as every value between such minimum and maximum values. Further, when a range refers to an integer, every integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be combined. In other words, unless otherwise indicated, all ranges disclosed herein should be understood to include any and all subranges included therein.

[0092] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0093] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0094] Figure 1 This is the spin-spin coupling diagram of the impurity in Example 1.

[0095] Figure 2 This is a direct correlation experimental diagram of carbon and hydrogen of impurities in Example 1.

[0096] Figure 3 This is the carbon-hydrogen long-range correlation experimental diagram of the impurities in Example 1. DETAILED DESCRIPTION

[0097] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of the present invention.

[0098] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0099] In the embodiment, the preparation method of acetonitrile-sodium acetate buffer is as follows: take 5.44 g of anhydrous sodium acetate, dissolve it in water and dilute it to 2000 mL, adjust the pH to 4.0 with glacial acetic acid, add acetonitrile at a volume ratio of acetonitrile to sodium acetate of 35:60, and obtain acetonitrile-sodium acetate buffer.

[0100] In the embodiment, the preparation method of teniposide bulk drug is as follows: accurately measure 20 mg of teniposide, put it in a 100 mL volumetric flask, slowly add it until the drug solution does not stick to the wall, dilute it to the scale with the mobile phase, shake well, and obtain the teniposide bulk drug.

[0101] In the embodiments, the teniposide bulk drug from which impurities are removed refers to a drug in which the mass percentage of impurities in the bulk drug is controlled to be below 0.07%.

[0102] Example 1

[0103] A method for separating degradation impurities in teniposide injection comprises the following steps:

[0104] S1 Take 2 mL of teniposide injection, add 2 mL of 0.01 mol / L hydrochloric acid, let stand for 6 hours, add 2 mL of 0.01 mol / L sodium hydroxide solution, add mobile phase to dilute to 100 mL, and obtain a mixture;

[0105] S2 separating the teniposide degradation impurities in the mixture by high performance liquid chromatography to obtain teniposide degradation impurities;

[0106] The chromatographic conditions of the high performance liquid chromatography include:

[0107] The mobile phase was 0.1% volume concentration of phosphoric acid solution and acetonitrile;

[0108] The chromatographic column was Eclipse XDB C18, 5 μm, 4.6 × 150 mm;

[0109] The elution procedure is:

[0110]

[0111] The teniposide degradation impurity separated above has the following structural formula:

[0112]

[0113] The H NMR spectrum data of the impurity are shown in Table 1.

[0114] Table 1

[0115]

[0116]

[0117] The carbon NMR spectrum data of the impurity are shown in Table 2.

[0118] Table 2

[0119]

[0120] Spin-spin coupling diagram of the impurity 1 H- 1 H COSY Figure 1 As shown, the carbon-hydrogen direct correlation experimental diagram HSQC of this impurity is as follows Figure 2 As shown, the carbon-hydrogen long-range correlation experimental diagram HMBC of the impurity is as follows Figure 3 shown.

[0121] From Table 1-2, Figure 1-3 The structural characterization can confirm the structural formula of the above impurities.

[0122] Example 2

[0123] The difference between Example 2 and Example 1 is that the concentration of hydrochloric acid is different. In Example 2, 0.05 mol / L hydrochloric acid is added to the teniposide injection.

[0124] After testing, it was found that the method of Example 2 was also capable of separating degradation impurities in teniposide injection.

[0125] Example 3

[0126] The difference between Example 3 and Example 1 is that the concentration of hydrochloric acid is different. In Example 3, 0.1 mol / L hydrochloric acid is added to the teniposide injection.

[0127] After testing, it was found that the method of Example 3 was also capable of separating degradation impurities in teniposide injection.

[0128] Example 4

[0129] The difference between Example 4 and Example 1 is that the concentration of hydrochloric acid is different. In Example 4, 0.2 mol / L hydrochloric acid is added to the teniposide injection.

[0130] After testing, it was found that the method of Example 4 was also capable of separating degradation impurities in teniposide injection.

[0131] Example 5

[0132] The difference between Example 5 and Example 1 is that the concentration of hydrochloric acid is different. In Example 5, 0.5 mol / L hydrochloric acid is added to the teniposide injection.

[0133] After testing, it was found that the method of Example 5 can also separate degradation impurities in teniposide injection.

[0134] Example 6

[0135] The difference between Example 6 and Example 1 is that in Example 6, the teniposide preparation is subjected to alkali treatment and the standing time is 4 hours.

[0136] Specific:

[0137] A method for separating degradation impurities in teniposide injection comprises the following steps:

[0138] S1: Take 2 mL of teniposide injection, add 2 mL of 0.1 mol / L sodium hydroxide, let stand for 4 hours, add 2 mL of 0.1 mol / L hydrochloric acid solution, and a flocculent precipitate is precipitated, and dilute to 100 mL with N,N-dimethylformyl to obtain a mixture;

[0139] S2 uses high performance liquid chromatography to separate the teniposide degradation impurities in the mixture; the chromatographic conditions of the high performance liquid chromatography include:

[0140] The mobile phase was 0.1% volume concentration of phosphoric acid solution and acetonitrile;

[0141] The chromatographic column was Eclipse XDB C18, 5 μm, 4.6 × 150 mm;

[0142] The elution procedure is:

[0143]

[0144] Example 7

[0145] The difference between Example 7 and Example 6 is that in Example 7, the placement time is 1 hour.

[0146] The chromatographic separation results of Example 7 are shown in Table 3.

[0147] Table 3

[0148]

[0149]

[0150] After calculation, the retention time ratio of the impurity to the main peak is 0.41. From Table 3, it can be seen that when the relative retention time is 0.41, the retention time is 6.793 min, so the retention time of the impurity is 6.793 min, indicating that alkali treatment can also destroy teniposide injection, causing teniposide to degrade into impurity S2302-M674.

[0151] Example 8

[0152] The difference between Example 8 and Example 6 is that in Example 8, the standing time is 10 minutes.

[0153] The chromatographic separation results of Example 8 are shown in Table 4.

[0154] Table 4

[0155]

[0156]

[0157] After calculation, the retention time ratio of the impurity to the main peak is 0.41. From Table 4, we know that when the relative retention time is 0.41, the retention time is 6.795min. Therefore, the retention time of the impurity is 6.795min, and the relative retention time is 0.41, indicating that 10 minutes of alkali treatment can also destroy the teniposide injection, causing teniposide to degrade into impurity S2302-M674. However, due to the short time and low degradation amount, the area normalized number of the impurity in Table 4 is only 68.54%.

[0158] Example 9

[0159] The difference between Example 9 and Example 1 is that in Example 9, the teniposide preparation is subjected to high temperature treatment.

[0160] A method for separating degradation impurities in teniposide injection comprises the following steps:

[0161] S1: Take 2 mL of teniposide injection, place in an oven at 105°C for 5 hours, return to room temperature, and dilute the mobile phase to 100 mL to obtain a mixture;

[0162] S2 separating the teniposide degradation impurities in the mixture by high performance liquid chromatography;

[0163] The chromatographic conditions of the high performance liquid chromatography include:

[0164] The mobile phase was 0.1% volume concentration of phosphoric acid solution and acetonitrile;

[0165] The chromatographic column was Eclipse XDB C18, 5 μm, 4.6 × 150 mm;

[0166] The elution procedure is:

[0167]

[0168] The chromatographic results of Example 9 are shown in Table 5.

[0169] Table 5

[0170] Retention time min Relative retention time Area normalized % 2.636 0.16 - 2.737 0.17 0.04 3.930 0.24 0.06 5.004 0.30 - 5.618 0.34 0.03 6.271 0.38 0.03 6.803 0.41 0.12 7.844 0.48 0.13 9.682 0.59 - 14.823 0.90 0.05 16.474 1.00 99.36 18.556 1.13 0.18

[0171] It can be seen from Table 5 that the retention time of the impurity is 6.803 min and the relative retention time is 0.41, indicating that high temperature treatment can also destroy the teniposide injection, causing teniposide to degrade into impurity S2302-M674.

[0172] Example 10

[0173] The difference between Example 10 and Example 1 is that in Example 10, the teniposide preparation is subjected to oxidation treatment.

[0174] Specific:

[0175] A method for separating degradation impurities in teniposide injection comprises the following steps:

[0176] S1 Take 2 mL of teniposide injection, add 2 mL of 30% hydrogen peroxide solution, let stand for 4 hours, add mobile phase to dilute to 100 mL, and obtain a mixture;

[0177] S2 separating the teniposide degradation impurities in the mixture by high performance liquid chromatography;

[0178] The chromatographic conditions of the high performance liquid chromatography include:

[0179] The mobile phase was 0.1% volume concentration of phosphoric acid solution and acetonitrile;

[0180] The chromatographic column was Eclipse XDB C18, 5 μm, 4.6 × 150 mm;

[0181] The elution procedure is:

[0182]

[0183] The chromatographic results of Example 10 are shown in Table 6.

[0184] Table 6

[0185] Retention time min Relative retention time Area normalized % 2.746 0.17 14.98 3.038 0.18 - 5.006 0.30 - 5.290 0.32 1.98 5.645 0.34 1.56 6.731 0.41 24.42 7.840 0.48 0.03 9.477 0.57 2.03 10.131 0.61 0.03 11.773 0.71 0.64 12.610 0.76 0.08 14.866 0.90 0.03 16.503 1.00 54.26

[0186] It can be seen from Table 6 that the retention time of the impurity is 6.732 min and the relative retention time is 0.41, indicating that oxidation treatment can also destroy the teniposide injection, causing teniposide to degrade into impurity S2302-M674.

[0187] Embodiment 11

[0188] A method for removing teniposide degradation impurities in teniposide bulk drug comprises the following steps:

[0189] A1: Dissolve 300g of teniposide API with 1.5 times the mass of N,N-dimethylacetamide in a hot water bath, and filter while hot to obtain a filtrate; add 10 times the mass of the API in an 80% ethanol solution in a 5L conical flask, slowly add the filtrate to the 80% ethanol solution, stir to crystallize, and place in a refrigerator overnight. The next day, filter, wash with 80% ethanol 3 times, using 250mL each time, drain, and vacuum dry at 95-100℃ / 0.08MPa or above for 6 hours to obtain a refined teniposide product.

[0190] A2 repeats step A1 to obtain a secondary refined product of teniposide.

[0191] A3: heat dissolve the teniposide secondary refined product with 1.5 times the mass of N,N-dimethylacetamide, filter to obtain a filtrate; add 10 times the mass of purified water into a 5L conical flask, stir, drop the filtrate to crystallize, the drop addition is completed within 30 minutes, then stir for 1 hour, filter, and wash with 250mL×2 purified water, dry on a plate when it is evacuated to anhydrous, vacuum dry at 50°C / 0.08MPa or above for 2 hours, vacuum dry at 70°C / 0.08MPa or above for 2 hours, and finally vacuum dry at 95-100°C / 0.08MPa or above for about 16 hours to constant weight, pass the dried material through a 60-mesh sieve to obtain a teniposide tertiary refined product, the mass percentage of impurities of which is controlled to be less than 0.07%.

[0192] From Examples 1-10, it is known that there are many degradation pathways of impurity S2302-M674. The present invention can prepare pure impurities by removing impurity S2302-M674 in teniposide preparations. From Example 11, it is known that the present invention can prepare a raw material drug that does not contain or contains only a very small amount of impurity S2302-M674 by removing impurity S2302-M674 in teniposide raw material drugs. The teniposide preparation prepared with the raw material drug is a high-quality teniposide preparation product, which can effectively reduce the incidence of allergic reactions.

[0193] Example 12

[0194] A method for purifying teniposide degradation impurities comprises the following steps:

[0195] Take the mobile phase solution obtained after the separation treatment in Example 1, add ethanol to the solution for rotary evaporation, precipitate a white solid (inorganic salt crystal), add ethanol and filter to obtain a white solid-liquid mixture, take the filtrate and add water, ethanol and dichloromethane in a volume ratio of 80:10:10, after rotary evaporation and heating in a 50°C water bath, completely remove the solvent to obtain a solid (teniposide degradation impurity), wash with water and filter to obtain a pure impurity.

[0196] Example 13

[0197] A method for detecting degradation impurities in teniposide injection is provided, wherein the degradation impurities in teniposide injection are detected by a method comprising the following steps: liquid chromatography is used to detect the degradation impurities in teniposide injection, and the detection results are shown in Table 7. The chromatographic conditions of the liquid chromatography include:

[0198] The mobile phase was acetonitrile-sodium acetate buffer with a pH of 4.0 and a volume ratio of 35:60;

[0199] The filling material of the chromatographic column is octadecylsilane bonded silica gel;

[0200] Isocratic elution;

[0201] The column temperature was 30°C;

[0202] Flow rate: 1.0 mL / min;

[0203] The detection wavelength is 280nm;

[0204] The injection volume was 20 μL.

[0205] Table 7 Chromatographic detection results of degradation impurities in teniposide injection

[0206]

[0207]

[0208] In Table 7, it is known that the retention time of the degradation impurity S2302-M674 is 6.745 min, and the relative retention time is 0.41 min, indicating that the degradation impurity S2302-M674 can be detected from teniposide injection by the method of Example 13.

[0209] Embodiment 14

[0210] Example 14 The teniposide bulk drug was tested using the same chromatographic conditions as in Example 13. The test results are shown in Table 8.

[0211] Table 8 Chromatographic detection results of degradation impurities in teniposide bulk drug

[0212] Retention time min Relative retention time Area normalized % 2.677 0.16 0.10 2.754 0.17 0.10 5.622 0.34 0.02 6.282 0.38 0.03 6.733 0.41 0.08 7.838 0.47 0.02 14.887 0.90 0.05 16.517 1.00 99.38 25.043 1.52 0.21

[0213] In Table 8, it is known that the retention time of the degradation impurity S2302-M674 is 6.733, and the relative retention time is 0.41 min, indicating that the degradation impurity S2302-M674 can be detected from the teniposide bulk drug by the method of Example 14.

[0214] Comparative Example 1

[0215] Comparative Example 1 The mobile phase in the method for detecting teniposide degradation impurities was adjusted relative to Example 13, and the mobile phase was adjusted to an acetonitrile-sodium acetate buffer solution with a pH of 3.01. The mobile phase preparation method was as follows: 10.88187 g of anhydrous sodium acetate was taken, 4000 mL of water was added to dissolve it, the pH was adjusted to 3.01 with glacial acetic acid, filtered, 600 ml was accurately measured, 350 ml of acetonitrile was added, mixed, and ultrasonicated to obtain a mobile phase.

[0216] The chromatographic conditions of Comparative Example 1 were unable to detect impurities from the teniposide injection.

[0217] Comparative Example 2

[0218] Comparative Example 2 The mobile phase in the method for detecting teniposide degradation impurities was adjusted relative to Example 13, and the mobile phase was adjusted to an acetonitrile-sodium acetate buffer solution with a pH of 4.98. The preparation method of the mobile phase is as follows: 10.88187 g of anhydrous sodium acetate was taken, 4000 mL of water was added to dissolve it, the pH was adjusted to 4.98 with glacial acetic acid, filtered, 600 ml was accurately measured, 350 ml of acetonitrile was added, mixed, and ultrasonicated to obtain a mobile phase.

[0219] The chromatographic conditions of Comparative Example 2 were unable to detect impurities from the teniposide injection.

[0220] The above are only embodiments of the present invention, and are not intended to limit the patent scope of the present invention. Any equivalent transformations made using the contents of the present invention specification, or directly or indirectly applied in related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A teniposide degradation impurity, characterized in that: Its structural formula is as follows: Wherein, R1, R2, R3, R4 and R6 are independently selected from -OH or a halogen group; R5 and R7 are independently selected from methoxy, ethoxy, propoxy or phenoxy.

2. The teniposide degradation impurity according to claim 1, characterized in that Its structural formula is as follows:

3. A method for detecting teniposide degradation impurities as claimed in claim 1 or 2, characterized in that: The following steps are involved: Teniposide degradation impurities are detected by liquid chromatography, and the chromatographic conditions of the liquid chromatography include: The mobile phase was acetonitrile-sodium acetate buffer in a volume ratio of 35-37:60-62; The filling material of the chromatographic column is octadecylsilane bonded silica gel.

4. The method according to claim 3, characterized in that The pH of the acetonitrile-sodium acetate buffer is 4.0-4.

2.

5. A method for separating the teniposide degradation impurities according to claim 1 or 2 from a teniposide preparation, characterized in that: The following steps are involved: Separating the teniposide degradation impurities in the teniposide preparation by high performance liquid chromatography; The chromatographic conditions of the high performance liquid chromatography include: The mobile phase is 0.1-0.2% volume concentration of phosphoric acid solution and acetonitrile; The filling material of the chromatographic column is octadecylsilane bonded silica gel; The elution procedure is:

6. The method according to claim 5, characterized in that The chromatographic column is selected from Eclipse XDB C18, the size of the filler particles is 5 μm, and the inner diameter×length is 4.6×150 mm.

7. The method for separating teniposide degradation impurities from a teniposide preparation according to claim 5, characterized in that: Before the teniposide preparation is separated by high performance liquid chromatography, the method further comprises subjecting the teniposide preparation to at least one treatment, including acid treatment, alkali treatment, high temperature treatment or oxidation treatment.

8. The method for separating teniposide degradation impurities from a teniposide preparation according to claim 5, characterized in that: The teniposide preparation comprises at least one of teniposide injection, teniposide dry suspension, teniposide capsule and teniposide tablet.

9. A method for removing the teniposide degradation impurities as claimed in claim 1 or 2 from teniposide bulk drug, characterized in that: The following steps are involved: The teniposide raw material is dissolved and filtered to obtain a filtrate, the filtrate is mixed with an organic solvent, and the mixture is subjected to dissolution and crystallization, filtration and vacuum drying to obtain a refined teniposide product.

10. A high-purity teniposide bulk drug, characterized in that: The invention comprises a raw material drug obtained by removing teniposide degradation impurities through the method of claim 9.

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