Preparation method of vincristine sulfate impurity

Abstract

The invention discloses a preparation method of vincristine sulfate impurities, which comprises the following steps: under the protection of nitrogen, mixing vincristine sulfate with an organic solvent, regulating the pH value by alkali, and reacting with a hydrogen peroxide aqueous solution at low temperature; adjusting the pH value of the reaction liquid with acid, concentrating under reduced pressure, adjusting the pH value of the concentrate with alkali, and extracting with dichloromethane to obtain a crude product; and purifying the crude product through silica gel column chromatography to obtain the target impurity. Through detection by a Chinese pharmacopoeia method, the relative retention time of the impurity and vincristine sulfate is 0.47, and the purity is greater than or equal to 98%. The method is easy and convenient to operate, complete in reaction and high in product purity, the reference substance meeting the vincristine sulfate quality analysis requirement can be prepared, and technical support is provided for production process optimization and quality standard improvement of vincristine sulfate.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical chemistry technology, specifically relating to a method for preparing an impurity in vincristine sulfate. This impurity can be used as a reference substance in the quality analysis of vincristine sulfate raw materials and preparations for qualitative identification and quantitative detection, thereby supporting the optimization of vincristine sulfate production processes and the improvement of quality standards, ultimately ensuring the safety and efficacy of clinical medication. Background Technology

[0002] Vincristine sulfate, a classic antitumor alkaloid, is mainly used in clinical practice to treat acute lymphoblastic leukemia, Hodgkin's lymphoma, and non-Hodgkin's lymphoma. It is also suitable for the treatment of malignant tumors such as breast cancer, bronchogenic carcinoma, soft tissue sarcoma, and neuroblastoma, and is an indispensable drug in tumor chemotherapy regimens.

[0003] In the pharmaceutical field, drug impurity research is a core component of drug quality control, directly impacting drug safety and efficacy. Impurities in drugs can trigger adverse reactions (such as allergic reactions), and some impurities even possess genotoxic properties, posing potential risks to patient health. Therefore, systematically analyzing the impurity profile of drugs, clarifying impurity structures, and preparing high-purity impurity reference standards are crucial prerequisites for establishing scientifically sound drug quality standards and achieving quality control throughout the production process.

[0004] In the production process of vincristine sulfate, impurities arise from multiple factors: First, the raw material "vincristine" is extracted from plants, and the raw material itself contains many types of impurities with complex structures, making it difficult to completely remove them through pretreatment. Second, during the synthesis of vincristine from vincristine through oxidation, column chromatography, purification, and salt formation, side reactions easily occur, generating various byproducts. These byproducts have similar physicochemical properties to the target product, making them difficult to remove completely using conventional separation methods. Third, the functional groups (such as ester groups and double bonds) in the vincristine molecule have poor stability and are prone to degradation during purification, salt formation, and storage, generating new degradation impurities.

[0005] Currently, research on impurities in vincristine sulfate remains insufficient, particularly regarding the specific impurity with a relative retention time of 0.47 seconds (according to the Chinese Pharmacopoeia method), for which a clear and reproducible high-purity preparation method is lacking. The absence of this impurity hinders accurate qualitative and quantitative analysis of vincristine sulfate, consequently affecting the determination of production process optimization and the refinement of quality standards. Therefore, developing a controllable and high-purity method for preparing this specific impurity in vincristine sulfate has become a pressing technical problem to be solved in this field. Summary of the Invention

[0006] To overcome the shortcomings of existing technologies, the present invention aims to provide a method for preparing impurities in vincristine sulfate. This method can prepare target impurities with a purity ≥98%, which can be used as a reference standard for the quality analysis of vincristine sulfate (qualitative identification and quantitative detection). This provides technical support for optimizing the production process and improving the quality standards of vincristine sulfate, ultimately ensuring the safety of clinical medication.

[0007] The objective of this invention is achieved through the following technical solution: The first aspect of this invention provides a method for preparing vincristine sulfate impurities, comprising the following steps: S10. Reaction steps: Under nitrogen protection, vincristine sulfate is mixed with an organic solvent. After adjusting the pH of the system with alkali, the mixture is cooled to the preset temperature and stirred to dissolve the vincristine sulfate. Then, hydrogen peroxide is slowly added dropwise, and the reaction temperature is controlled and the reaction is carried out for at least 3 hours. The reaction solution is monitored by high performance liquid chromatography until the content of vincristine sulfate in the reaction solution is ≤1.0%. S20. Post-processing steps: Add acid dropwise to the reaction solution obtained in step S10 to adjust the system to acidity, then concentrate under reduced pressure to remove organic solvent and obtain a concentrate; adjust the pH of the concentrate to alkaline with saturated sodium bicarbonate aqueous solution, then extract the pH-adjusted concentrate twice with dichloromethane, wash the extract with saturated brine, dry it with anhydrous sodium sulfate, filter it, and concentrate the filtrate under reduced pressure to dryness to obtain crude vincristine impurity. S30. Purification step: Dissolve the crude vincristine impurity in an organic solvent, load the dissolved solution onto a silica gel column, monitor the elution process by thin-layer chromatography, collect the fraction containing the target vincristine impurity, concentrate the fraction under reduced pressure to obtain the vincristine sulfate impurity; wherein, when detected using the Chinese Pharmacopoeia method, the relative retention time of the vincristine sulfate impurity to vincristine sulfate is 0.47, and the structure of the vincristine sulfate impurity is confirmed by nuclear magnetic resonance and mass spectrometry.

[0008] In a first aspect of the invention, as an optional embodiment, the organic solvent in step S10 is selected from one or more of methanol, ethanol, and acetonitrile, and the mass ratio of vincristine sulfate to the organic solvent is 1:5 to 1:10.

[0009] In a first aspect of the invention, as an optional embodiment, the alkali in step S10 is selected from ammonia, diethylamine, or triethylamine, and the pH of the adjusted system is 6-10.

[0010] In a first aspect of the present invention, as an optional embodiment, the step S10 of "cooling down to a preset temperature" specifically means cooling down to -30°C to -20°C, and the reaction temperature is controlled to be -30°C to 0°C.

[0011] In a first aspect of the invention, as an optional embodiment, the concentration of hydrogen peroxide in step S10 is 6%-20%, and the amount of hydrogen peroxide used is 20-30 times the mass of vincristine sulfate; if high performance liquid chromatography shows that the content of vincristine sulfate in the reaction solution is ≥1.0%, then 10 times the mass of vincristine sulfate with a concentration of 10% hydrogen peroxide is added, and the reaction continues for 1 hour until the content of vincristine sulfate in the reaction solution is ≤1.0%.

[0012] In a first aspect of the invention, as an optional embodiment, the acid in step S20 is selected from sulfuric acid, hydrochloric acid, or acetic acid, and the pH value of the adjusted system is 3-4.

[0013] In a first aspect of the invention, as an optional embodiment, step S20, "adjusting the pH of the concentrate to alkaline with a saturated sodium bicarbonate aqueous solution," specifically means adjusting the pH of the concentrate to 7-8.

[0014] In a first aspect of the invention, as an optional embodiment, the organic solvent used to dissolve the crude vincristine impurity in step S30 is selected from dichloromethane or ethyl acetate, and the amount of said organic solvent is 5-10 times the amount of the crude vincristine impurity.

[0015] In a first aspect of the invention, as an optional embodiment, the silica gel column in step S30 has a silica gel size of 200-300 mesh, and the mobile phase used for elution is selected from a dichloromethane-methanol system or an ethyl acetate-methanol system.

[0016] In a first aspect of the present invention, as an optional embodiment, after the reaction in step S10 is completed, the purity of vincristine impurities in the reaction solution is ≥94%; after the concentration of the fraction in step S30, the purity of vincristine sulfuric acid impurities is ≥98%.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention provides the first complete synthetic scheme for this specific impurity, solving the problem in existing technologies where the absence of this impurity leads to inaccurate qualitative / quantitative analysis of vincristine sulfate. It provides a core material basis for subsequent quality research, ensuring the effectiveness and reliability of the product: The structure is confirmed by NMR and mass spectrometry, ensuring that the synthesized impurity has the same structure as the impurity actually generated during vincristine sulfate production; the relative retention time of 0.47 matches the detection method in the Chinese Pharmacopoeia, directly meeting the requirement of "comparing the retention time of the reference standard and the sample impurity" in vincristine sulfate quality analysis, avoiding analytical errors caused by inconsistent impurity structures. This method can prepare the target impurity with a purity ≥98%, which can be used as a reference standard for the quality analysis of vincristine sulfate (qualitative identification and quantitative detection), providing technical support for optimizing the vincristine sulfate production process and improving quality standards, ultimately ensuring clinical drug safety.

[0018] 2. In the reaction process of this invention, the pH value is controlled at 8-9, resulting in a complete reaction and high conversion rate. The reaction is incomplete under acidic conditions, and excessively high pH values ​​can easily generate other byproducts. A reaction temperature of -20℃ to -10℃ results in a high product conversion rate; if the temperature increases, the product will be further oxidized into other impurities. A hydrogen peroxide concentration of 6%-10% ensures a complete reaction with a shorter reaction time; too low a concentration significantly increases the reaction time or even leads to incomplete reaction; too high a concentration easily leads to further oxidation of the product. Before concentration, the reaction solution must be adjusted to acidity to prevent product degradation due to increased temperature during concentration; before extraction, the concentrate must be adjusted to alkalinity to improve the yield. After column purification, vincristine is obtained with a purity greater than 98%, indicating high purity. Attached Figure Description

[0019] Figure 1 This is a schematic flowchart of the preparation method of the present invention.

[0020] Figure 2 This is a chemical structure diagram of the vincristine sulfate impurity in Example 1 of the present invention.

[0021] Figure 3 This is a high-performance liquid chromatogram of the vincristine sulfate impurity in Example 1 of the present invention.

[0022] Figure 4 This is a high-performance liquid chromatogram of vincristine sulfate from Example 1 of the present invention.

[0023] Figure 5 This is a high-performance liquid chromatogram of vincristine sulfate and vincristine impurities in Example 1 of the present invention.

[0024] Figure 6 The image shows the 1H NMR spectrum of the vincristine sulfate impurity in Example 1 of this invention.

[0025] Figure 7 This is a mass spectrum of the vincristine sulfate impurity in Example 1 of the present invention. Detailed Implementation

[0026] The present invention will now be further described with reference to the accompanying drawings and specific embodiments. It should be noted that, without conflict, the various embodiments or technical features described below can be arbitrarily combined to form new embodiments. Unless otherwise specified, the materials and equipment used in this embodiment are all commercially available.

[0027] Please refer to Figure 1 The first aspect of this invention provides a method for preparing vincristine sulfate impurities, comprising the following steps: S10. Reaction steps: Under nitrogen protection, vincristine sulfate is mixed with an organic solvent. After adjusting the pH of the system with alkali, the mixture is cooled to the preset temperature and stirred to dissolve the vincristine sulfate. Then, hydrogen peroxide is slowly added dropwise, and the reaction temperature is controlled and the reaction is carried out for at least 3 hours. The reaction solution is monitored by high performance liquid chromatography until the content of vincristine sulfate in the reaction solution is ≤1.0%. S20. Post-processing steps: Add acid dropwise to the reaction solution obtained in step S10 to adjust the system to acidity, then concentrate under reduced pressure to remove organic solvent and obtain a concentrate; adjust the pH of the concentrate to alkaline with saturated sodium bicarbonate aqueous solution, then extract the pH-adjusted concentrate twice with dichloromethane, wash the extract with saturated brine, dry it with anhydrous sodium sulfate, filter it, and concentrate the filtrate under reduced pressure to dryness to obtain crude vincristine impurity. S30. Purification step: Dissolve the crude vincristine impurity in an organic solvent, load the dissolved solution onto a silica gel column, monitor the elution process by thin-layer chromatography, collect the fraction containing the target vincristine impurity, concentrate the fraction under reduced pressure to obtain the vincristine sulfate impurity; wherein, when detected using the Chinese Pharmacopoeia method, the relative retention time of the vincristine sulfate impurity to vincristine sulfate is 0.47, and the structure of the vincristine sulfate impurity is confirmed by nuclear magnetic resonance and mass spectrometry.

[0028] In a preferred embodiment, the organic solvent in step S10 is selected from one or more of methanol, ethanol, and acetonitrile, and the mass ratio of vincristine sulfate to the organic solvent is 1:5-1:10.

[0029] Methanol, ethanol, and acetonitrile are all polar organic solvents that can form a good miscible system with vincristine sulfate and do not undergo side reactions with hydrogen peroxide or alkali. A mass ratio of 1:5 to 1:10 can ensure that vincristine sulfate is completely dissolved, avoiding incomplete reaction due to solid raw material residue, and will not increase the energy consumption and time of subsequent vacuum concentration due to excessive solvent.

[0030] In a preferred embodiment, the alkali in step S10 is selected from ammonia, diethylamine or triethylamine, and the pH value of the adjusted system is 6-10; preferably, the pH value of the adjusted system is 8-9.

[0031] If pH < 6 (strong acidity): the oxidizing power of hydrogen peroxide is weakened, the oxidation reaction rate of vincristine sulfate decreases, and the raw material residue can be > 5% after 3 hours of reaction, and hydroxylation byproducts are easily generated. If pH > 10 (strongly alkaline): hydrogen peroxide is easily decomposed into oxygen, its oxidizing power drops sharply, and the ester group in vincristine molecules is easily hydrolyzed, generating carboxylic acid impurities.

[0032] In a preferred embodiment, "cooling down to a preset temperature" in step S10 specifically means cooling down to -30°C to -20°C, and the reaction temperature is controlled to be -30°C to 0°C; preferably, the reaction temperature is controlled to be -20°C to -10°C.

[0033] Vincristine impurity molecules still contain unsaturated bonds. If the reaction temperature is above 0°C, hydrogen peroxide will further oxidize these unsaturated bonds, generating dihydroxylated byproducts. Temperatures between -30°C and 0°C significantly reduce the oxidizing activity of hydrogen peroxide, completing only the one-step oxidation of vincristine sulfate to the target impurity, thus avoiding over-reaction. The optimal temperature range is -20°C to -10°C: this ensures complete dissolution of vincristine sulfate within 15 minutes and allows the reaction to achieve a raw material residue of ≤1.0% within 3 hours, balancing efficiency and product purity.

[0034] In a preferred embodiment, the concentration of hydrogen peroxide in step S10 is 6%-20%, and the amount of hydrogen peroxide used is 20-30 times the mass of vincristine sulfate. If high-performance liquid chromatography shows that the content of vincristine sulfate in the reaction solution is ≥1.0%, then 10 times the mass of vincristine sulfate with a concentration of 10% hydrogen peroxide is added, and the reaction continues for 1 hour until the content of vincristine sulfate in the reaction solution is ≤1.0%. Preferably, the concentration of hydrogen peroxide is 6%-10%.

[0035] If the concentration is <6% and the effective oxidizing component is insufficient, the raw material residue will be >8% after 3 hours of reaction. Additional hydrogen peroxide needs to be added, which will prolong the process time and increase the cost of subsequent acid neutralization. If the concentration is >20%, the oxidizing power is too strong. In addition to oxidizing vincristine, it will also oxidize the solvent (such as methanol to formaldehyde) and introduce new organic impurities. When the concentration is 6%-10%, the oxidation intensity is just right to meet the requirements of one-step oxidation, the raw material residue is ≤1.0% within 3 hours, no need for multiple replenishments, and there are no solvent oxidation byproducts.

[0036] In a preferred embodiment, the acid in step S20 is selected from sulfuric acid, hydrochloric acid or acetic acid, and the pH value of the adjusted system is 3-4.

[0037] If the residual hydrogen peroxide in the system after the reaction is not treated, it will decompose due to temperature rise during vacuum concentration, causing the system to boil violently. pH 3-4 is the optimal acidic condition for hydrogen peroxide decomposition, allowing for complete removal of hydrogen peroxide before concentration and eliminating safety hazards. Furthermore, vincristine impurities readily undergo indole ring-opening reactions under strongly acidic conditions (pH < 2) and are easily oxidized by oxygen in the air under neutral conditions (pH 5-7). pH 3-4, being a weakly acidic environment, can both prevent ring-opening degradation and inhibit oxidation, ensuring no loss of impurity purity during post-processing.

[0038] In a preferred embodiment, step S20, "adjusting the pH of the concentrate to alkaline with a saturated sodium bicarbonate aqueous solution", specifically means adjusting the pH of the concentrate to 7-8.

[0039] In a preferred embodiment, the organic solvent used to dissolve the crude vincristine impurity in step S30 is selected from dichloromethane or ethyl acetate, and the amount of the organic solvent is 5-10 times the amount of the crude vincristine impurity.

[0040] In a preferred embodiment, the silica gel column in step S30 uses silica gel with a mesh size of 200-300, and the mobile phase used for elution is selected from a dichloromethane-methanol system or an ethyl acetate-methanol system. The particle size of 200-300 mesh silica gel is moderate, providing sufficient adsorption sites without causing the elution rate to be too slow due to excessively fine particles.

[0041] In a preferred embodiment, after the reaction in step S10, the purity of vincristine impurities in the reaction solution is ≥94%; after the concentration of the fraction in step S30, the purity of vincristine sulfate impurities obtained is ≥98%. This ensures that the synthesized impurities can be directly used as reference standards for "external standard quantification" or "qualitative identification" without further purification.

[0042] The following are some embodiments listed in this application, which further illustrate this application.

[0043] Example 1 A method for preparing vincristine sulfate impurity includes the following steps: S10. Reaction Procedure: In a 50 mL three-necked flask under nitrogen protection, 1.0 g of vincristine sulfate was mixed with 8.0 mL of methanol. Subsequently, ammonia was slowly added dropwise to the system to adjust the pH to 8.0. The system was cooled to -25 °C and magnetically stirred at 300 rpm to completely dissolve the vincristine sulfate. After dissolution, 25.0 mL of 6% hydrogen peroxide aqueous solution was slowly added dropwise to the system at a dropping rate of 1 mL / min. After the addition was completed, the reaction temperature was controlled at -20 °C and the reaction was continued for 3 hours. HPLC monitoring of the reaction solution showed that the residual amount of vincristine sulfate was 0.5%, and the purity of the target impurity was 94.8%, at which point the reaction was terminated.

[0044] S20. Post-processing steps: Slowly add 50% sulfuric acid (by mass) to the above reaction solution to adjust the pH of the system to 4.0; concentrate the system under reduced pressure in a rotary evaporator to remove methanol, obtaining a pale yellow concentrate; add 10 mL of purified water to the concentrate, and then adjust the pH to 7.0 with saturated sodium bicarbonate aqueous solution; add 15 mL of dichloromethane for the first extraction, and collect the organic phase after standing and separating the layers; extract the aqueous phase a second time with 15 mL of dichloromethane, and combine the two organic phases; wash the organic phase once with 10 mL of saturated brine, then add 5.0 g of anhydrous sodium sulfate, and let it stand and dry at room temperature for 2 hours; remove the anhydrous sodium sulfate by suction filtration, and concentrate the filtrate under reduced pressure in a rotary evaporator to dryness, obtaining 0.92 g of crude vincristine impurity.

[0045] S30. Purification Steps: Add 5.0 mL of dichloromethane to 0.92 g of crude vincristine impurity, and stir magnetically to completely dissolve the crude product, obtaining a loading solution. Load the loading solution into a chromatography column packed with 200-300 mesh silica gel (30 cm long, 2 cm inner diameter, 20 g silica gel), and elute with dichloromethane-methanol (95:5 v / v) as the mobile phase (elution rate 1 mL / min). Monitor the elution process by TLC (developing solvent: dichloromethane-methanol = 90:10, colorimetric reagent: potassium bismuth iodide solution), and collect the elution fraction containing the target impurity. Concentrate the collected fraction to dryness under reduced pressure in a rotary evaporator to obtain 0.82 g of vincristine sulfate impurity. HPLC analysis shows that the purity of this impurity is 98.65%.

[0046] Figure 2-7 The synthesized vincristine sulfate impurities were demonstrated to have "clear structures, meet purity standards, be reliably separated, and be compatible with pharmacopoeia" from five dimensions: structural definition, structural confirmation, purity testing, separation verification, and pharmacopoeia compatibility. This fully meets the core requirements for reference standards in the quality analysis of vincristine sulfate. A comprehensive analysis is provided below: I. Structural Validation Closed Loop: Figure 2 , Figure 6 , Figure 7 These three methods work together to verify the accuracy of impurity molecular structures, eliminate the risk of structural misjudgment, and provide a basis for impurity characterization. Figure 2 Establishing the structural foundation: The chemical structure of the impurity was clarified, containing core units such as indole ring, quinoline ring, ester group, and saturated ring, providing a target reference for subsequent spectral analysis. The impurity was defined as a specific oxidation product of vincristine sulfate, with the addition of oxygen atoms, the reduction of hydrogen atoms, and the retention of sulfate and key heterocyclic structures.

[0047] Figure 6 Verification of structural details: Precise matching through chemical shift, peak shape, and integral area ratio. Figure 2 The chemical environment of all hydrogen atoms in the structure: saturated alkyl hydrogens in the high-field region, near-electron-negative alkyl hydrogens in the mid-field region, and aromatic and double-bonded hydrogens in the low-field region correspond to structural units such as indole rings, quinoline rings, and ester groups, respectively. The types, numbers, and proportions of hydrogen atoms are completely consistent, proving that the molecular skeleton and functional group positions are correct.

[0048] Figure 7 Verification of molecular weight and structural fragment: Molecular ion peak m / z 961.3 ([M+Na) + )and Figure 2 The calculated molecular weight (937.0) of the structure is consistent with the theoretical molecular weight, confirming its accuracy. The main fragment ion peaks (m / z 912.7, 841.9, 432.4) correspond to the characteristic cleavage modes of the ester group, indole ring side chain, and quinoline ring, confirming the structure's correct molecular weight. Figure 2 The presence of key functional groups in the structure, together with the hydrogen nuclear magnetic resonance spectrum, forms a closed loop for structural confirmation of the macroscopic framework and microscopic fragments.

[0049] II. Verification of Purity and Separation Effect: Figure 3 , Figure 4 , Figure 5 The three HPLC chromatograms, from two scenarios—individual purity and compatibility with mixed systems—verify that the impurities meet the purity requirements of pharmaceutical reference standards and are suitable for actual detection. Figure 3 Verification of individual purity: Under pharmacopoeia testing conditions, the area of ​​the main impurity peak accounted for 98.65%, and the total impurity peak accounted for <1.35%, directly proving that after purification by silica gel column, the impurity purity was ≥98%, with no obvious by-products or raw material residues, meeting the core indicator of "high purity" of the reference standard.

[0050] Figure 5 Verification of the separation effect of the mixed system: A scenario simulating an excess of the main component (vincristine sulfate) and trace amounts of impurities in a real sample was presented. The results showed: The separation degree between impurities and main components reaches 8.7 (far exceeding the pharmacopoeia standard of ≥1.5), with no peak overlap interference, ensuring that even if the impurity content is extremely low (such as 0.1%), it can be accurately identified in actual testing; The relative retention time of impurities remained constant at 0.47, compared to... Figure 3 The results were consistent, proving that the chromatographic behavior of impurities is stable in the presence of the main component, which can be used as the core basis for qualitative identification. The relative retention time (RRT) is calculated as follows: impurity retention time ÷ main component (vincristine sulfate) retention time. Figure 5 Measured value: Retention time of vincristine sulfate (main component) = 16.103 min; Target impurity retention time = 7.525 min; The calculation yields: 7.525 ÷ 16.103 ≈ 0.47.

[0051] The fact that the peak area ratio of the two samples is consistent with the concentration ratio indicates that their response factors are similar, and the external standard method can be used directly for quantification, simplifying the actual detection operation.

[0052] III. Pharmacopoeia compatibility is maintained throughout the entire process: Figure 3-4 The HPLC test conditions were strictly in accordance with the "Related Substances Test" section of the Chinese Pharmacopoeia for vincristine sulfate. Figure 4 The qualitative and quantitative feasibility of impurity identification under these conditions was further verified, forming an application closed loop of method adaptability and reliable results: Qualitative fit: The constant value of relative retention time of 0.47 can be directly used for the qualitative identification of this impurity in vincristine sulfate samples (by comparing the relative retention times of the sample peak and the reference peak). Quantitative adaptation: High purity (≥98%) and stable response enable impurities to be used as external standard references, establishing a concentration-peak area standard curve, accurately calculating the content of the impurity in the sample, and meeting the pharmacopoeia's requirement for "limit control of related substances"; IV. Overall Conclusion: Figure 2-7 A complete chain of evidence was formed, including structural confirmation, purity verification, reliable separation, and pharmacopoeia compatibility. Ultimately, it was proven that the synthesized product is the target vincristine sulfate impurity, with a unique and well-defined structure, eliminating the risk of structural misjudgment; the impurity purity is ≥98%, meeting the purity requirements of pharmaceutical reference standards; the impurity and vincristine sulfate are completely separated under pharmacopoeia detection conditions, exhibiting stable chromatographic behavior, and can be directly used for the qualitative identification and quantitative detection of vincristine sulfate raw materials and preparations.

[0053] Example 2 A method for preparing vincristine sulfate impurity includes the following steps: S10. Reaction Procedure: In a 50 mL three-necked flask under nitrogen protection, 1.0 g of vincristine sulfate was mixed with 10.0 mL of methanol. Subsequently, ammonia was slowly added dropwise to the system to adjust the pH to 7.5. The system was cooled to -30 °C and magnetically stirred (300 rpm) to completely dissolve the vincristine sulfate. After dissolution, 30.0 mL of 10% hydrogen peroxide aqueous solution was slowly added dropwise to the system (dropping rate 1 mL / min). After the addition was complete, the reaction temperature was controlled at -15 °C and the reaction was continued for 4 hours. HPLC monitoring of the reaction solution showed that the residual amount of vincristine sulfate was 0.4%, and the purity of the target impurity was 94.2%, at which point the reaction was terminated.

[0054] S20. Post-processing steps: Slowly add 37% hydrochloric acid dropwise to the above reaction solution to adjust the pH of the system to 3.0; concentrate the system under reduced pressure in a rotary evaporator to remove methanol and obtain a pale yellow concentrate; add 10 mL of purified water to the concentrate, and then adjust the pH to 8.0 with saturated sodium bicarbonate aqueous solution; add 15 mL of dichloromethane for the first extraction, and collect the organic phase after standing and separating the layers; extract the aqueous phase a second time with 15 mL of dichloromethane, and combine the two organic phases; wash the organic phase once with 10 mL of saturated brine, and then add 5.0 g of anhydrous sodium sulfate, and let it stand and dry at room temperature for 2 hours; remove the anhydrous sodium sulfate by suction filtration, and concentrate the filtrate under reduced pressure in a rotary evaporator to dryness to obtain 0.91 g of crude vincristine impurity.

[0055] S30. Purification Steps: Add 6.0 mL of ethyl acetate to 0.91 g of crude vincristine impurity, and stir magnetically to completely dissolve the crude product, obtaining a loading solution. Load the loading solution into a chromatography column packed with 200-300 mesh silica gel (30 cm long, 2 cm inner diameter, 20 g silica gel), and elute with ethyl acetate-methanol (92:8 v / v) as the mobile phase (elution rate 1 mL / min). Monitor the elution process by TLC (developing solvent: ethyl acetate-methanol = 85:15, colorimetric reagent: potassium bismuth iodide solution), and collect the elution fraction containing the target impurity. Concentrate the collected fraction to dryness under reduced pressure in a rotary evaporator to obtain 0.80 g of vincristine sulfate impurity. HPLC analysis shows that the purity of this impurity is 98.7%.

[0056] Example 3 A method for preparing vincristine sulfate impurity includes the following steps: S10. Reaction Procedure: In a 50 mL three-necked flask under nitrogen protection, 1.0 g of vincristine sulfate was mixed with 6.0 mL of ethanol. Subsequently, ammonia was slowly added dropwise to the system to adjust the pH to 8.5. The system was cooled to -20 °C and magnetically stirred (300 rpm) to completely dissolve the vincristine sulfate. After dissolution, 20.0 mL of 8% hydrogen peroxide aqueous solution was slowly added dropwise to the system (dropping rate 1 mL / min). After the addition was complete, the reaction temperature was controlled at -15 °C and the reaction was continued for 3 hours. HPLC monitoring of the reaction solution showed that the residual amount of vincristine sulfate was 0.5%, and the purity of the target impurity was 95.2%, at which point the reaction was terminated.

[0057] S20. Post-processing steps: Slowly add 50% sulfuric acid (by mass) to the above reaction solution to adjust the pH of the system to 3.0; concentrate the system under reduced pressure in a rotary evaporator to remove ethanol, obtaining a pale yellow concentrate; add 10 mL of purified water to the concentrate, and then adjust the pH to 7.5 with saturated sodium bicarbonate aqueous solution; add 15 mL of dichloromethane for the first extraction, and collect the organic phase after standing and separating the layers; perform a second extraction on the aqueous phase with 15 mL of dichloromethane, and combine the two organic phases; wash the organic phase once with 10 mL of saturated brine, then add 5.0 g of anhydrous sodium sulfate, and let it stand and dry at room temperature for 2 hours; remove the anhydrous sodium sulfate by suction filtration, and concentrate the filtrate to dryness under reduced pressure in a rotary evaporator to obtain 0.94 g of crude vincristine impurity.

[0058] S30. Purification Steps: Add 6.0 mL of dichloromethane to 0.94 g of crude vincristine impurity, and stir magnetically to completely dissolve the crude product, obtaining a loading solution. Load the loading solution into a chromatography column packed with 200-300 mesh silica gel (30 cm long, 2 cm inner diameter, 20 g silica gel), and elute with dichloromethane-methanol (93:7 v / v) as the mobile phase (elution rate 1 mL / min). Monitor the elution process by TLC (developing solvent: dichloromethane-methanol = 90:10, colorimetric reagent: potassium bismuth iodide solution), and collect the elution fraction containing the target impurity. Concentrate the collected fraction to dryness under reduced pressure in a rotary evaporator to obtain 0.83 g of vincristine sulfate impurity. HPLC analysis shows that the purity of this impurity is 98.9%.

[0059] Comparative Example 1 The difference in this comparative example is as follows: S10, Reaction Procedure: In a 50 mL three-necked flask under nitrogen protection, 1.0 g of vincristine sulfate was mixed with 6.0 mL of methanol; subsequently, ammonia was slowly added dropwise to the system to adjust the pH to 8.0; the system was cooled to -20 °C and magnetically stirred (300 rpm) to completely dissolve the vincristine sulfate; after dissolution, 20.0 mL of 30% hydrogen peroxide aqueous solution was slowly added dropwise to the system (dropping rate 1 mL / min); after the addition was complete, the reaction temperature was controlled at -15 °C and the reaction was continued for 3 hours; the purity of the target impurity was 56.7% as monitored by HPLC. When the concentration of hydrogen peroxide solution exceeded 20%, the purity of the target impurity in the reaction solution was very low.

[0060] Comparative Example 2 The difference in this comparative example is as follows: S10, Reaction Procedure: In a 50 mL three-necked flask under nitrogen protection, 1.0 g of vincristine sulfate was mixed with 8.0 mL of methanol; subsequently, ammonia was slowly added dropwise to the system to adjust the pH to 8.5; the system was cooled to -20 °C and magnetically stirred (300 rpm) to completely dissolve the vincristine sulfate; after dissolution, 25.0 mL of 10% hydrogen peroxide aqueous solution was slowly added dropwise to the system (dropping rate 1 mL / min); after the addition was complete, the reaction temperature was controlled at 20 °C and the reaction was continued for 3 hours; the purity of the target impurity was 34.1% as monitored by HPLC. When the reaction temperature exceeded 0 °C, the purity of the target impurity in the reaction solution was very low.

[0061] The above embodiments are merely preferred embodiments of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-substantial changes and substitutions made by those skilled in the art based on the present invention shall fall within the scope of protection claimed by the present invention.

Claims

1. A method for preparing vinblastine sulfate impurity, comprising the following steps: S10, a reaction step: under the condition of nitrogen protection, vinblastine sulfate is mixed with an organic solvent, and after adding a base to adjust the pH value of the system, the temperature is reduced to a preset temperature and stirring is performed to dissolve the vinblastine sulfate, then hydrogen peroxide is slowly added dropwise, the reaction temperature is controlled and the reaction is carried out for at least 3 hours, the reaction solution is monitored by high performance liquid chromatography until the content of vinblastine sulfate in the reaction solution is ≤1.0%; S20, a post-treatment step: after adding an acid dropwise to the reaction solution obtained in step S10 and adjusting the system to be acidic, the organic solvent is removed by concentration under reduced pressure to obtain a concentrate; the pH value of the concentrate is adjusted to be alkaline by saturated sodium bicarbonate aqueous solution, and the concentrate after pH adjustment is extracted twice with dichloromethane; the extract is washed with saturated brine, dried with anhydrous sodium sulfate, and then filtered; the filtrate is concentrated to dryness under reduced pressure to obtain a crude vinblastine sulfate impurity; S30, a purification step: the crude vinblastine sulfate impurity is dissolved with an organic solvent, and the dissolved solution is loaded onto a silica gel column; the elution process is monitored by thin layer chromatography; the fraction containing the target vinblastine sulfate impurity is collected; and the fraction is concentrated under reduced pressure to obtain the vinblastine sulfate impurity; wherein the relative retention time of the vinblastine sulfate impurity and vinblastine sulfate is 0.47 when detected by the method of Chinese Pharmacopoeia, and the structure of the vinblastine sulfate impurity is confirmed by nuclear magnetic resonance and mass spectrometry. The organic solvent in step S10 is selected from one or more of methanol, ethanol and acetonitrile, and the mass ratio of vinblastine sulfate to the organic solvent is 1:5-1:

10. The base in step S10 is selected from ammonia, diethylamine or triethylamine, and the pH value of the adjusted system is 6-10. In step S10, the temperature is reduced to a preset temperature, specifically to -30℃ to -20℃, and the reaction temperature is controlled to be -30℃ to 0℃.

2. The production method according to claim 1, wherein The concentration of hydrogen peroxide in step S10 is 6%-20%, and the amount of hydrogen peroxide is 20-30 times the mass of vinblastine sulfate; if the high performance liquid chromatography detection shows that the content of vinblastine sulfate in the reaction solution is ≥1.0%, then 10 times the mass of vinblastine sulfate is added with 10% hydrogen peroxide, and the reaction is continued for 1 hour until the content of vinblastine sulfate in the reaction solution is ≤1.0%.

3. The production method according to claim 1, wherein The acid in step S20 is selected from sulfuric acid, hydrochloric acid or acetic acid, and the pH value of the adjusted system is 3-4.

4. The production method according to claim 1, wherein In step S20, "adjusting the pH value of the concentrate to be alkaline with saturated sodium bicarbonate aqueous solution" specifically refers to adjusting the pH value of the concentrate to 7-8.

5. The production method according to claim 1, wherein The organic solvent used to dissolve the crude vinblastine sulfate impurity in step S30 is selected from dichloromethane or ethyl acetate, and the amount of the organic solvent is 5-10 times the mass of the crude vinblastine sulfate impurity.

6. The production method according to claim 1, wherein The silica gel specification of the silica gel column in step S30 is 200-300 mesh, and the mobile phase used for elution is selected from dichloromethane-methanol system or ethyl acetate-methanol system.

7. The production method according to claim 1, wherein After the reaction in step S10 is completed, the purity of vinblastine sulfate impurity in the reaction solution is ≥94%; and after the fraction is concentrated in step S30, the purity of the obtained vinblastine sulfate impurity is ≥98%.

8. The production method according to claim 1, wherein ​ 9. The production method according to claim 1, wherein ​ 10. The production method according to claim 1, wherein ​

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