Preparation method, product and application of high-purity reduced coenzyme Q10

Abstract

The invention discloses a preparation method, a product and application of high-purity reduced coenzyme Q10, and belongs to the technical field of medicine preparation. The technical problem to be solved is to prepare a reduced coenzyme Q10 product which has the purity of more than or equal to 99.5% and the reduced coenzyme Q9 of less than or equal to 0.3% and does not contain reduced coenzyme Q11. According to the technical scheme, the method is characterized by comprising the following steps: S1, reacting coenzyme Q10 with sodium dithionite, and removing a solvent to obtain a concentrated solution of reduced coenzyme Q10; s2, dissolving the concentrated solution obtained in the step S1 by using a mobile phase, purifying by using a C18 preparative column, eluting by using the mobile phase, and collecting an elution component; s3, removing a solvent from the elution component obtained in the step S2, adding a co-crystallization solvent, and crystallizing to obtain crystallized wet powder; and S4, drying the crystal wet powder obtained in the step S3 to obtain the high-purity reduced coenzyme Q10. According to the method, the product purity of the reduced coenzyme Q10 can be further improved, and the safety risk caused by impurities is reduced.

Description

Technical Field

[0001] This invention relates to a method for preparing high-purity reduced coenzyme Q10, the product, and its application, belonging to the field of pharmaceutical preparation technology. Background Technology

[0002] Coenzyme Q10 is a vitamin-like nutrient found in almost all cells, playing a crucial role in energy production in the mitochondrial respiratory chain. Coenzyme Q10 exists in two forms in the human body: reduced coenzyme Q10 and oxidized coenzyme Q10 (collectively referred to as coenzyme Q10).

[0003] Reduced coenzyme Q10 is a white or off-white powder, insoluble in water, highly lipid-soluble, photosensitive, and unstable in air, readily oxidized to its oxidized form. The chemical structural formula of reduced coenzyme Q10 is shown below:

[0004] .

[0005] The chemical structural formula of coenzyme Q10 is shown below: .

[0006] Among them, the configuration of isopentenyl, a coenzyme Q series substance present in organisms, is all trans.

[0007] Both oxidized and reduced coenzyme Q10 have tails composed of decapentene repeating units.

[0008] Currently, coenzyme Q10 production methods include extraction from animal and plant tissues, animal and plant tissue culture, microbial fermentation, and chemical synthesis. Both extraction and culture methods require extracting coenzyme Q10 from animal and plant tissues. However, because the concentration of coenzyme Q10 in animal and plant tissues is low, extraction requires large amounts of tissue, resulting in low atom economy.

[0009] The chemical synthesis of coenzyme Q10 includes total chemical synthesis and semi-chemical synthesis. Total chemical synthesis uses inexpensive and readily available raw materials, but the synthetic route for the 10-isoprenyl tail is lengthy and involves numerous steps, making intermediate purification difficult and resulting in poor stereoselectivity. Semi-chemical synthesis utilizes solanesol extracted from tobacco leaves as a raw material. Solanesol contains nine isopentenyl groups, and synthesizing coenzyme Q10 based on this significantly reduces the number of synthetic steps. However, since the isopentenyl group closest to the ring needs to be artificially synthesized and directly attached to the ring, the stereoselectivity at this site is poor. Furthermore, the presence of oligomers in the synthetic system results in relatively high viscosity, unfavorable reaction kinetics, and may require the addition of viscosity reducers or other auxiliaries. The addition of reaction auxiliaries and / or catalysts increases the risk of containing harmful biological impurities in the product.

[0010] Currently, the main method for synthesizing commercial coenzyme Q10 is through fermentation engineering using genetically modified or screened microorganisms to express coenzyme Q10 in large quantities. During fermentation, the polyisoprene tail is synthesized by elongating it sequentially. For example, Meganathan R. et al., in their article "Ubiquinone biosynthesis in microorganisms" (FEMS Microbiology Letters, Volume 203, Issue 2, September 2001, Pages 131-139), provided the synthetic pathway of coenzyme Q10 side chains in E. coli and S. cerevisiae. The specific formula is shown below: .

[0011] It is evident that during biosynthesis, due to the simultaneous presence of side chains with 1-10 isopentenyl repeating groups and the identical tail-end active functional groups, biosynthesized CoQ10 inevitably contains CoQ series compounds with varying degrees of isopentenyl polymerization, such as CoQ8, CoQ9, CoQ10, and CoQ11. This results in most commercially available CoQ10 products unavoidably containing these compounds.

[0012] It is generally believed that reduced coenzyme Q10 has antioxidant properties, can scavenge free radicals, and prevent lipid and protein peroxidation. Therefore, it is widely used in the clinical treatment of cardiovascular and cerebrovascular diseases, nervous system diseases, hypertension, hyperlipidemia, diabetes and other diseases.

[0013] Relevant patent documents retrieved: This document, published in China (CN104892370A) on September 9, 2015, discloses a method for preparing reduced coenzyme Q10 from oxidized coenzyme Q10. Existing processes for producing reduced coenzyme Q10 mostly use iron powder, zinc powder, or sodium hydrosulfite, all of which are solids, and the reduction reaction is heterogeneous, making workshop operation inconvenient and the reaction time long. This invention uses oxidized coenzyme Q10 as a raw material and stannous chloride as a reducing agent. A small amount of acid is added dropwise to control the pH of the reaction system to be weakly acidic. The reaction is carried out in a solvent at 50-70°C with continuous stirring and nitrogen protection. After the reaction is complete, the product is purified and crystallized to obtain reduced coenzyme Q10 with a purity ≥95%. The stannous chloride is electrolyzed and recovered after the reaction, and the obtained stannous chloride is used to further reduce coenzyme Q10, allowing for repeated use and continuous reaction. The invention yields highly pure reduced coenzyme Q10, ensuring product safety and reliability. The reducing agent is regenerable and reusable, making it both environmentally friendly and safe—a green method for synthesizing coenzyme Q10. However, the coenzyme Q10 content needs further improvement, and the coenzyme Q9 / Q11 content is not yet controlled.

[0014] Relevant non-patent literature retrieved: This master's thesis from Hebei University of Science and Technology, titled "Preparation and Crystallization Process Study of Reduced Coenzyme Q10," authored by Huang Liqiang and published on June 1, 2024, discloses a process for preparing reduced coenzyme Q10 using oxidized coenzyme Q10 as a raw material and sodium dithionite as a reducing agent. The thesis also discloses a solution-cooling coupled crystallization process for preparing reduced coenzyme Q10, investigating the effects of initial solution concentration, solvent dosage, stirring rate, cooling rate, and crystal growth time on the crystallization process using single-factor experiments. However, the method provided in this thesis only increases the purity of the reduced coenzyme Q10 product to 97.1%, and does not record the content of reduced coenzyme Q9 and reduced coenzyme Q11 in the product.

[0015] The prior art represented by the aforementioned documents has at least the following unresolved technical problems or defects: Currently, the main production process for coenzyme Q10 uses silica gel chromatography to remove 5-demethoxycoenzyme Q10 impurities, but this method is not very effective at removing impurities such as Q9 and Q11. This results in higher levels of these impurities in commercially available coenzyme Q10 products. Q11 impurity, in particular, is difficult to remove using conventional column chromatography or crystallization methods and requires high-pressure preparative chromatography separation.

[0016] For example, Chinese invention patent application CN108084007A discloses a method for separating coenzyme Q10 and coenzyme Q11 by simulated moving bed chromatography. The method includes the following steps: (1) dissolving a mixture of coenzyme Q10 and coenzyme Q11 in an organic solvent to prepare a feed solution; (2) continuously passing the feed solution and eluent into a simulated moving bed chromatography system, continuously collecting the Q11-rich extract from the extraction port of the simulated moving bed chromatography system, and continuously collecting the coenzyme Q10-rich raffinate from the raffinate port; (3) obtaining coenzyme Q10 monomer after post-treatment of the raffinate; and obtaining coenzyme Q11 monomer after post-treatment of the extract.

[0017] For example, Chinese invention patent application CN112920035A discloses a method for preparing a coenzyme Q10 product by removing Q11 impurities via chromatography. This method includes the following steps: dissolving a coenzyme Q10 sample, applying chromatographic separation mode, eluting with one or more organic solvents as the mobile phase, collecting the eluent corresponding to the coenzyme Q10 absorption peaks, and obtaining a coenzyme Q10 product free of Q11 impurities. The packing material used in the chromatographic separation mode is silica gel modified with polar or non-polar groups, wherein the polar groups are: C3-C... 30 A positive-chain alkyl group is attached to silica gel with one or more of the following polar groups: chlorine atom, bromine atom, cyano group, sulfonic acid group, hydroxyl group, glycol group, carboxyl group, amino group, diamino group, amide group, and phenyl group, each C1-C... 30 The number of polar groups on the positive-chain alkyl linker is one or more, and the bonding amount of the polar groups is 0.5-8.0 μmol / m. 2 The nonpolar groups are: C1-C 30 One or more of the following: n-chain alkanes and aromatics, with a nonpolar group bonding amount of 0.5-8.0 μmol / m 2 The types and number of bonding groups can all be controlled.

[0018] However, the aforementioned technologies generally utilize expensive preparative chromatography systems or packing materials. Currently, the market price of Coenzyme Q10 is relatively low, and the high cost of preparative chromatography systems and packing materials limits the application of these technologies.

[0019] Currently, the main industrial method for preparing reduced coenzyme Q10 is to use high-purity coenzyme Q10 as raw material, reducing it with a reducing agent and then crystallizing it to obtain the finished product. However, due to the introduction of impurities in the coenzyme Q10 raw material, the purity of the product is generally not high (approximately 98%) even with simple solvent crystallization methods. It is difficult to remove structurally similar impurities such as coenzyme Q9 and Q11, thus failing to meet the demand for ultra-high purity coenzyme Q10.

[0020] Other content useful for understanding, searching, and examining this invention: According to the latest requirements for Coenzyme Q10 in the European Pharmacopoeia (EP 11.0), Coenzyme Q9 ≤ 0.3% and Coenzyme Q11 (unknown single impurity) ≤ 0.1%. With the increasing quality standards for Coenzyme Q10, the limits for related impurities in its derivative, reduced Coenzyme Q10, will also increase accordingly. Therefore, there is an urgent need to develop a high-purity production process for reduced Coenzyme Q10. Summary of the Invention

[0021] The purpose of this invention is to provide: A method for preparing high-purity reduced coenzyme Q10, and related technologies, to solve technical problems such as increasing the purity of reduced coenzyme Q10 to ≥99.5%, reducing the weight content of reduced coenzyme Q9 relative to reduced coenzyme Q10 to ≤0.3%, and removing reduced coenzyme Q11 contained therein, or combinations thereof.

[0022] Terminology Explanation: Unless otherwise defined, all technical terms in this document have the same meanings as commonly understood by one of ordinary skill in the art to which the subject matter of the claims pertains. Unless otherwise stated, all patents, patent inventions, and publications cited in this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms in this document, the definitions in this chapter shall prevail.

[0023] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0024] Definitions of standard chemical terms can be found in the reference "Lange's Handbook of Chemistry" (published by McGraw-Hill, authors N.A. Lange, J.A. Dean, James G. Speight, translator Shang Jiufang et al.).

[0025] Unless otherwise stated, conventional methods within the scope of the art, such as dissolution and mixing, shall be used.

[0026] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0027] The term "inert gas" as used in this article refers to a gas that does not react with reduced coenzyme Q10 at room temperature (0-45℃) and normal pressure (1±0.05 atmospheres) in the absence of a catalyst, especially a gas that does not oxidize reduced coenzyme Q10 at room temperature (0-45℃) and normal pressure (1±0.05 atmospheres); specifically, it does not include oxidizing gases such as oxygen, ozone, and / or chlorine. Common inert gases under the above definition include helium, neon, argon, krypton, xenon, nitrogen, carbon dioxide, hydrogen, etc., or mixtures of the above gases in any proportion.

[0028] Unless otherwise specified, the terms "content" and "percentage content" in this article refer to "weight content" and "weight percentage content" respectively.

[0029] In a first aspect, the present invention provides a method for preparing high-purity reduced coenzyme Q10, comprising the following steps: S1. Coenzyme Q10 is reacted with sodium dithionite, the solvent is removed, and a concentrated solution of reduced coenzyme Q10 is obtained. S2. Mix the concentrated solution of reduced coenzyme Q10 obtained in step S1 with the mobile phase, purify it by C18 column chromatography, and elute with the mobile phase to obtain the eluent fraction. S3. Remove the solvent from the eluted component obtained in step S2 to obtain a solid, add a co-crystallization solvent, and crystallize to obtain a crystalline wet powder. S4. Dry the crystalline wet powder obtained in step S3 to obtain high-purity reduced coenzyme Q10. The mobile phase is a mixture of methanol and ethyl acetate or a mixture of ethanol and ethyl acetate; When the mobile phase is a mixture of methanol and ethyl acetate, the volume ratio of methanol to ethyl acetate is 75-85:15-25. When the mobile phase is a mixture of ethanol and ethyl acetate, the volume ratio of ethanol to ethyl acetate is 85-91:9-15.

[0030] Further, in step S1, the reaction of coenzyme Q10 with sodium dithionite includes the following steps: Coenzyme Q10, ethyl acetate, and an aqueous solution of sodium dithionite with a weight concentration of 10%-30% were mixed, reacted under inert gas protection, and the organic phase was separated and concentrated to obtain a concentrated solution of reduced coenzyme Q10.

[0031] The weight concentration of the sodium dithionite aqueous solution is selected from any value within the range of 10%-30%, for example, any of the following values ​​or any range between two: 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%; In a preferred embodiment of the present invention, the aqueous solution of sodium dithionite has a weight concentration of 20%.

[0032] Furthermore, the ratio of the weight of the coenzyme Q10, the volume of the ethyl acetate, and the volume of the aqueous solution of sodium dithionite is 5-15:50-150:50-150, in g:mL:mL.

[0033] Wherein, in the ratio of the weight of coenzyme Q10, the volume of ethyl acetate, and the volume of the aqueous solution of sodium dithionite (in g:mL:mL), the weight of coenzyme Q10 is selected from any value in the range of 5-15, for example, any of the following values ​​or any range between two: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15; the volume of ethyl acetate is selected from any value in the range of 50-150, for example, any of the following values ​​or any range between two: 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150; the volume of the aqueous solution of sodium dithionite is selected from any value in the range of 50-150, for example, any of the following values ​​or any range between two: 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150.

[0034] In a preferred embodiment of the present invention, the ratio of the weight of the coenzyme Q10, the volume of the ethyl acetate, and the volume of the aqueous solution of sodium dithionite is 10:100:100, in g:mL:mL.

[0035] Further, the purity of the coenzyme Q10 is ≥98.5%, the content of impurity coenzyme Q9 in the coenzyme Q10 is ≤0.6%, and the content of impurity coenzyme Q11 in the coenzyme Q10 is ≤0.5%. Further, the purity of the coenzyme Q10 is ≥99%, the content of impurity coenzyme Q9 in the coenzyme Q10 is ≤0.5%, and the content of impurity coenzyme Q11 in the coenzyme Q10 is ≤0.4%.

[0036] Furthermore, the purity of the coenzyme Q10 is ≥99.17%, the content of impurity coenzyme Q9 in the coenzyme Q10 is ≤0.35%, and the content of impurity coenzyme Q11 in the coenzyme Q10 is ≤0.32%.

[0037] Furthermore, the purity of the coenzyme Q10 is ≥99.28%, the content of impurity coenzyme Q9 in the coenzyme Q10 is ≤0.33%, and the content of impurity coenzyme Q11 in the coenzyme Q10 is ≤0.32%.

[0038] Furthermore, the inert gas is one or more of nitrogen, hydrogen, and argon.

[0039] In one specific embodiment of the present invention, the inert gas is nitrogen.

[0040] Furthermore, the reaction temperature is 35-45℃, and the reaction time is 1-3h.

[0041] The temperature of the reaction is selected from any value in the range of 35-45℃, such as the following values ​​or any range between the two: 35℃, 36℃, 37℃, 38℃, 39℃, 40℃, 41℃, 42℃, 43℃, 44℃, 45℃.

[0042] The reaction time is selected from any value within the range of 1-3h, such as the following values ​​or any range between the two: 1h, 1.2h, 1.4h, 1.6h, 1.8h, 2h, 2.2h, 2.4h, 2.6h, 2.8h, 3h.

[0043] In one specific embodiment of the present invention, the reaction temperature is 40°C and the reaction time is 2 hours.

[0044] Furthermore, the concentration is a vacuum concentration to recover the solvent.

[0045] Furthermore, the concentration is to concentrate until no solvent residue remains.

[0046] When the mobile phase is a mixture of methanol and ethyl acetate, the volume ratio of the two is selected from any ratio between 75-85:15-25, such as the following ratios or any range between the two: 75:15, 75:16, 75:17, 75:18, 75:19, 75:20, 75:21, 75:22, 75:23, 75:24, 75:25, 76:15, 76:16, 76:17, 76:18, 76:19, 76:20, 76:21, 76:22, 76:23, 76:24, 76:25, 77:1 5, 77:16, 77:17, 77:18, 77:19, 77:20, 77:21, 77:22, 77:23, 77:24, 77:25, 78:15, 78:16, 78:17, 78:18, 78:19, 78:20, 78:21, 78:22, 78:23, 78:24, 78:25, 79:15, 79:16, 79:17, 79:18, 79:19, 79:20, 79:21, 79:22, 79:23, 79:24, 79:25, 80: 15, 80:16, 80:17, 80:18, 80:19, 80:20, 80:21, 80:22, 80:23, 80:24, 80:25, 81:15, 81:16, 81:17, 81:18, 81:19, 81:20, 81:21, 81:22, 81:23, 81:24, 81:25, 82:15, 82:16, 82:17, 82:18, 82:19, 82:20, 82:21, 82:22, 82:23, 82:24, 82:25, 8 3:15, 83:16, 83:17, 83:18, 83:19, 83:20, 83:21, 83:22, 83:23, 83:24, 83:25, 84:15, 84:16, 84:17, 84:18, 84:19, 84:20, 84:21, 84:22, 84:23, 84:24, 84:25, 85:15, 85:16, 85:17, 85:18, 85:19, 85:20, 85:21, 85:22, 85:23, 85:24, 85:25.

[0047] Furthermore, when the mobile phase is a mixture of methanol and ethyl acetate, the volume ratio of the two is selected from any ratio between 79 and 81:19-21, such as the following ratios or any range between the two: 79:19, 79:20, 79:21, 80:19, 80:20, 80:21, 81:19, 81:20, 81:21.

[0048] Furthermore, and as a specific embodiment of the present invention, when the mobile phase is a mixture of methanol and ethyl acetate, the volume ratio of the two is 80:20.

[0049] When the mobile phase is a mixture of ethanol and ethyl acetate, the volume ratio of the two is selected from any ratio between 85-91:9-15, such as the following ratios or any range between the two: 85:9, 85:10, 85:11, 85:12, 85:13, 85:14, 85:15, 86:9, 86:10, 86:11, 86:12, 86:13, 86:14, 86:15, 87:9, 87:10, 87:11, 87:12, 87:13, 87: 14, 87:15, 88:9, 88:10, 88:11, 88:12, 88:13, 88:14, 88:15, 89:9, 89:10, 89:11, 89:12, 89:13, 89:14, 89:15, 90:9, 90:10, 90:11, 90:12, 90:13, 90:14, 90:15, 91:9, 91:10, 91:11, 91:12, 91:13, 91:14, 91:15.

[0050] Furthermore, when the mobile phase is a mixture of ethanol and ethyl acetate, the volume ratio of the two is selected from any ratio between 87 and 89:11-13, such as the following ratios or any range between the two: 87:11, 87:12, 87:13, 88:11, 88:12, 88:13, 89:11, 89:12, 89:13.

[0051] Furthermore, and as a specific embodiment of the present invention, when the mobile phase is a mixture of ethanol and ethyl acetate, the volume ratio of the two is 88:12.

[0052] Further, in step S2, the weight ratio of the concentrated reduced coenzyme Q10 solution to the volume ratio of the mobile phase is 1:80-100, in g:mL.

[0053] Furthermore, in step S2, prior to purification using C18 column chromatography, the following steps are also included: The mixture is purged with an inert gas and heated in a sealed environment to 40-50°C to dissolve.

[0054] The temperature for closed heating is selected from any value between 40-50℃, such as the following ratios or any range between the two: 40℃, 41℃, 42℃, 43℃, 44℃, 45℃, 46℃, 47℃, 48℃, 49℃, 50℃.

[0055] The inert gas is replaced 1-3 times, preferably 2 times.

[0056] Furthermore, in step S2, the C18 column is a C18 preparation column or a C18 dynamic shaft compression column.

[0057] The C18 preparation column can be selected from the following models: 10mm×150mm, 20mm×250mm, 30mm×250mm, 50mm×250mm, etc.

[0058] The C18 dynamic axis compression column can be selected from the following models: DAC50, DAC100, DAC150, DAC200, DAC300, DAC400, etc.

[0059] In one specific embodiment of the present invention, the C18 preparation column is 30mm × 250mm.

[0060] As a specific embodiment of the present invention, the C18 dynamic shaft compression column is of model DAC50 or DAC300.

[0061] Furthermore, in step S2, the particle size of the packing material in the C18 column is 5-100 μm.

[0062] The filler particle size is any value within the range of 5-100 μm, for example, the following values ​​or any range between two: 5μm, 6μm, 7μm, 8μm, 9μm, 10μm, 11μm, 12μm, 13μm, 14μm, 15μm, 16μm, 17μm, 18μm, 19μm, 20μm, 21μm, 22μm, 23μm, 24μm, 25μm, 26μm, 27μm, 28μm, 29μm, 30μm, 31μm, 32μm, 33μm, 34μm, 35μm, 36μm, 37μm, 38μm, 39μm, 40μm, 41μm, 42μm, 43μm, 44μm, 45μm, 46μm, 47μm, 48μm, 49μm. μm, 50μm, 51μm, 52μm, 53μm, 54μm, 55μm, 56μm, 57μm, 58μm, 59μm, 60μm, 61μm, 6 2μm, 63μm, 64μm, 65μm, 66μm, 67μm, 68μm, 69μm, 70μm, 71μm, 72μm, 73μm, 74μm, 7 5μm, 76μm, 77μm, 78μm, 79μm, 80μm, 81μm, 82μm, 83μm, 84μm, 85μm, 86μm, 87μm, 8 8μm, 89μm, 90μm, 91μm, 92μm, 93μm, 94μm, 95μm, 96μm, 97μm, 98μm, 99μm, 100μm.

[0063] Furthermore, the particle size of the packing material in the C18 column is any value within the range of 10-30 μm, for example, the following values ​​or any range between the two: 10 μm, 11 μm, 12 μm, 13 μm, 14 μm, 15 μm, 16 μm, 17 μm, 18 μm, 19 μm, 20 μm, 21 μm, 22 μm, 23 μm, 24 μm, 25 μm, 26 μm, 27 μm, 28 μm, 29 μm, 30 μm.

[0064] Further, in step S2, the eluted components are analyzed by liquid chromatography.

[0065] Furthermore, in step S2, the chromatographic purity of reduced coenzyme Q10 in the elution fraction is ≥99.27%, the chromatographic purity of reduced coenzyme Q9 is ≤0.12%, and reduced coenzyme Q11 is not detected under liquid chromatography conditions.

[0066] Furthermore, the specific conditions for the liquid chromatography are as follows: mobile phase: acetonitrile and ethanol (50:50, v / v); diluent: methanol and n-hexane (85:15, v / v); detector: UV 290nm; column: 4.6mm × 10cm, 3μm C18 packing; column temperature: 35℃; flow rate: 1.5mL / min; injection volume: 20μL. The retention time of the main peak, reduced coenzyme Q10, is 14-15 min in the liquid chromatography detection. The relative retention time (RRT) of the impurity reduced coenzyme Q9 is 0.64, and the relative retention time (RRT) of the impurity reduced coenzyme Q11 is 1.64. "Q11 impurity not detected" means that no impurity peak appears in the Q11 relative retention time in the liquid chromatography chromatogram.

[0067] Furthermore, in step S2, the specific method for performing liquid chromatography detection and analysis on the eluted components is as follows: collect the eluted components in portions of 80-150 mL, and perform liquid chromatography detection and analysis on each portion of the eluted components.

[0068] As a specific embodiment of the present invention, each portion has a volume of 150 mL.

[0069] Preferably, and as a specific embodiment of the present invention, each portion has a volume of 100 mL.

[0070] Further, in step S3, the co-crystallization solvent is ethanol; the volume ratio of the co-crystallization solvent to the mass ratio of the solid is 10-12:1, in mL:g.

[0071] The volume ratio of the co-crystallization solvent to the mass of the solid is any ratio within the range of 10-12:1, for example, the following ratios or any range between them: 10:1, 10.1:1, 10.2:1, 10.3:1, 10.4:1, 10.5:1, 10.6:1, 10.7:1, 10.8:1, 10.9:1, 11:1, 11.1:1, 11.2:1, 11.3:1, 11.4:1, 11.5:1, 11.6:1, 11.7:1, 11.8:1, 11.9:1, 12:1. The unit is mL:g.

[0072] Further, in step S3, the crystallization process includes the following steps: Replace with an inert gas, heat in a sealed environment to 55-65℃ until completely dissolved, cool to 0-5℃, filter, and obtain crystalline wet powder.

[0073] The temperature of the sealed heating is any value within the range of 55-65℃, such as the following values ​​or any range between two of them: 55℃, 56℃, 57℃, 58℃, 59℃, 60℃, 61℃, 62℃, 63℃, 64℃, 65℃.

[0074] The temperature drop to 0-5℃ can be selected from any value between 0-5℃, such as the following values ​​or any range between the two: 0℃, 1℃, 2℃, 3℃, 4℃, 5℃.

[0075] Furthermore, the cooling rate is 5-8℃ / h.

[0076] The cooling rate is any value between 5 and 8℃ / h, for example, the following values ​​or any range between two: 5℃ / h, 5.1℃ / h, 5.2℃ / h, 5.3℃ / h, 5.4℃ / h, 5.5℃ / h, 5.6℃ / h, 5.7℃ / h, 5.8℃ / h, 5.9℃ / h, 6℃ / h, 6.1℃ / h, 6.2℃ / h, 6.3℃ / h, 6.4℃ / h, 6.5℃ / h, 6.6℃ / h, 6.7℃ / h, 6.8℃ / h, 6.9℃ / h, 7℃ / h, 7.1℃ / h, 7.2℃ / h, 7.3℃ / h, 7.4℃ / h, 7.5℃ / h, 7.6℃ / h, 7.7℃ / h, 7.8℃ / h, 7.9℃ / h, 8℃ / h.

[0077] Furthermore, in step S4, the drying is performed under reduced pressure at 20-40°C.

[0078] The drying temperature is selected from any value between 20 and 40°C, such as the following values ​​or any range between two of them: 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, and 40°C.

[0079] Based on further solutions to the technical problems of the present invention, or simultaneous solutions to multiple technical problems, the preferred solution in the technical solution provided in the first aspect of the present invention includes: The first preferred embodiment: A method for preparing high-purity reduced coenzyme Q10, comprising the following steps: S1. Coenzyme Q10 is reacted with sodium dithionite, the solvent is removed, and a concentrated solution of reduced coenzyme Q10 is obtained. S2. Mix the concentrated reduced coenzyme Q10 obtained in step S1 with the mobile phase, purify using a C18 preparative column, and elute with the mobile phase to obtain the eluent fraction. S3. Remove the solvent from the eluted component obtained in step S2 to obtain a solid, add a co-crystallization solvent, and crystallize to obtain a crystalline wet powder. S4. Dry the crystalline wet powder obtained in step S3 to obtain high-purity reduced coenzyme Q10. In step S2, the mobile phase is a mixture of methanol and ethyl acetate or a mixture of ethanol and ethyl acetate; when the mobile phase is a mixture of methanol and ethyl acetate, the volume ratio of methanol to ethyl acetate is 79-81:19-21.

[0080] This technical solution not only solves the technical problem of "increasing the purity of reduced coenzyme Q10 to ≥99.5%, reducing the weight content of reduced coenzyme Q9 relative to reduced coenzyme Q10 to ≤0.3%, and eliminating reduced coenzyme Q11", but also further solves the technical problem of "maintaining the total process yield at 91.1%-92.3%".

[0081] The second preferred embodiment: A method for preparing high-purity reduced coenzyme Q10, comprising the following steps: S1. Coenzyme Q10 is reacted with sodium dithionite, the solvent is removed, and a concentrated solution of reduced coenzyme Q10 is obtained. S2. Mix the concentrated reduced coenzyme Q10 obtained in step S1 with the mobile phase, purify using a C18 preparative column, and elute with the mobile phase to obtain the eluent fraction. S3. Remove the solvent from the eluted component obtained in step S2 to obtain a solid, add a co-crystallization solvent, and crystallize to obtain a crystalline wet powder. S4. Dry the crystalline wet powder obtained in step S3 to obtain high-purity reduced coenzyme Q10. In step S2, the mobile phase is a mixture of methanol and ethyl acetate or a mixture of ethanol and ethyl acetate; when the mobile phase is a mixture of ethanol and ethyl acetate, the volume ratio of ethanol to ethyl acetate is 85:15.

[0082] This technical solution, having already solved the technical problems of "increasing the purity of reduced coenzyme Q10 to ≥99.5%, reducing the weight content of reduced coenzyme Q9 relative to reduced coenzyme Q10 to ≤0.3%, and eliminating reduced coenzyme Q11", further solves the technical problems of "increasing the purity of reduced coenzyme Q10 to 99.73%, reducing the weight content of reduced coenzyme Q9 relative to reduced coenzyme Q10 to 0.08%, and eliminating reduced coenzyme Q11, while achieving a total process yield of 93.1% for reduced coenzyme Q10 and a mass content of 99.48% for reduced coenzyme Q10".

[0083] Secondly, the present invention provides high-purity reduced coenzyme Q10 prepared by the above-mentioned preparation method.

[0084] Furthermore, in the high-purity reduced coenzyme Q10, the purity of reduced coenzyme Q10 is ≥99.5%, the weight content of reduced coenzyme Q9 relative to reduced coenzyme Q10 is ≤0.3%, and it does not contain reduced coenzyme Q11.

[0085] Thirdly, the present invention provides the application of the above-mentioned preparation method in the preparation of high-purity reduced coenzyme Q10.

[0086] Fourthly, the present invention provides the application of the above-described preparation method in the preparation of products containing high-purity reduced coenzyme Q10.

[0087] Fifthly, the present invention provides a product containing the above-mentioned high-purity reduced coenzyme Q10.

[0088] Furthermore, the product is a pharmaceutical product.

[0089] Furthermore, when the product is a pharmaceutical product, the product also contains pharmaceutically acceptable excipients.

[0090] Furthermore, the dosage forms of the product include tablets, pills, powders, granules, lozenges, effervescent tablets, capsules, and gels.

[0091] Regarding the invention involved in this matter: The technical feature “when the mobile phase is a mixture of methanol and ethyl acetate, the volume ratio of methanol to ethyl acetate is 75-85:15-25” is summarized from the corresponding technical features of methanol to ethyl acetate volume ratios of 75:25, 79:21, 80:20, 81:19, 85:15, etc. in the foregoing explanation and / or Examples 1-13, which are summarized by the common feature “the volume ratio of methanol to ethyl acetate is 75-85:15-25”. Therefore, those skilled in the art can reasonably infer that the subordinate concept of the technical feature "when the mobile phase is a mixture of methanol and ethyl acetate, the volume ratio of methanol to ethyl acetate is 75-85:15-25", the essentially equivalent technical means of "when the mobile phase is a mixture of methanol and ethyl acetate, the volume ratio of methanol to ethyl acetate is 75-85:15-25", and the technical means that can replace "when the mobile phase is a mixture of methanol and ethyl acetate, the volume ratio of methanol to ethyl acetate is 75-85:15-25" based on existing technology and conventional technical means and common knowledge should all fall within the protection scope of this invention. For example, replacing "the volume ratio of methanol to ethyl acetate is 75-85:15-25" with "80.1:19.9", "79.9:20.1", etc., while keeping other technical features unchanged, still falls within the protection scope of this invention.

[0092] The technical feature “when the mobile phase is a mixture of ethanol and ethyl acetate, the volume ratio of ethanol to ethyl acetate is 85-91:9-15” is summarized from the corresponding technical features of ethanol to ethyl acetate volume ratios of 85:15, 87:13, 88:12, 89:11, 91:9, etc. in the foregoing explanation and / or Examples 1-13, which are summarized by the common feature “the volume ratio of ethanol to ethyl acetate is 85-91:9-15”. Therefore, those skilled in the art can reasonably infer that the subordinate concept of the technical feature "when the mobile phase is a mixture of ethanol and ethyl acetate, the volume ratio of ethanol to ethyl acetate is 85-91:9-15", the essentially equivalent technical means of "when the mobile phase is a mixture of ethanol and ethyl acetate, the volume ratio of ethanol to ethyl acetate is 85-91:9-15", and the technical means that can replace "when the mobile phase is a mixture of ethanol and ethyl acetate, the volume ratio of ethanol to ethyl acetate is 85-91:9-15" based on existing technology and conventional technical means and common knowledge should all fall within the protection scope of this invention. For example, replacing "the volume ratio of ethanol to ethyl acetate is 85-91:9-15" with "87.9:12.1", "88.1:11.9", etc., while keeping other technical features unchanged, still falls within the protection scope of this invention.

[0093] The technical feature “when the mobile phase is a mixture of ethanol and ethyl acetate, the volume ratio of ethanol to ethyl acetate is 85-91:9-15” is summarized from the corresponding technical features of ethanol to ethyl acetate volume ratios of 85:15, 87:13, 88:12, 89:11, 91:9, etc. in the foregoing explanation and / or Examples 1-13, which are summarized by the common feature “the volume ratio of ethanol to ethyl acetate is 85-91:9-15”. Therefore, those skilled in the art can reasonably infer that the subordinate concept of the technical feature "when the mobile phase is a mixture of ethanol and ethyl acetate, the volume ratio of ethanol to ethyl acetate is 85-91:9-15", the essentially equivalent technical means of "when the mobile phase is a mixture of ethanol and ethyl acetate, the volume ratio of ethanol to ethyl acetate is 85-91:9-15", and the technical means that can replace "when the mobile phase is a mixture of ethanol and ethyl acetate, the volume ratio of ethanol to ethyl acetate is 85-91:9-15" based on existing technology and conventional technical means and common knowledge should all fall within the protection scope of this invention. For example, replacing "the volume ratio of ethanol to ethyl acetate is 85-91:9-15" with "87.9:12.1", "88.1:11.9", etc., while keeping other technical features unchanged, still falls within the protection scope of this invention.

[0094] The technical feature “C18 column is a C18 preparation column or a C18 dynamic shaft compression column” is summarized from the corresponding technical features in the foregoing explanation and / or Examples 1-13, such as “C18 preparation column of model 10mm×150mm”, “C18 preparation column of model 20mm×250mm”, “C18 preparation column of model 30mm×250mm”, “C18 preparation column of model 50mm×250mm”, “C18 dynamic shaft compression column of model DAC50”, “C18 dynamic shaft compression column of model DAC100”, “C18 dynamic shaft compression column of model DAC150”, “C18 dynamic shaft compression column of model DAC200”, “C18 dynamic shaft compression column of model DAC250”, and “C18 dynamic shaft compression column of model DAC300”, through the common feature “C18 column is a C18 preparation column or a C18 dynamic shaft compression column”. Therefore, those skilled in the art can reasonably presume that the subordinate concepts of the technical feature "C18 column is a C18 preparation column or a C18 dynamic shaft compression column", the essentially equivalent technical means of "C18 column is a C18 preparation column or a C18 dynamic shaft compression column", and the technical means that can replace "C18 column is a C18 preparation column or a C18 dynamic shaft compression column" based on the existing technical level and conventional technical means and common knowledge should all fall within the protection scope of this invention. For example, if the C18 column is adjusted to a 2-methyl C18 column, a 2,3-dimethyl C18 column, etc., while other technical features remain unchanged, it still falls within the protection scope of this invention.

[0095] Regarding the invention involved in this matter: The technical feature “aqueous solution of sodium dithionite with a weight concentration of 10%-30%” is summarized from the weight concentrations of sodium dithionite of 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, and 30% in the foregoing explanation and / or Examples 1-13, which are derived from the common feature “weight concentration of 10%-30%”. Therefore, those skilled in the art can reasonably presume that the technical feature "aqueous solution of sodium dithionite with a weight concentration of 10%-30%", the subordinate concept of "aqueous solution of sodium dithionite with a weight concentration of 10%-30%", the technical means that are basically equivalent to "aqueous solution of sodium dithionite with a weight concentration of 10%-30%", and the technical means that can replace "aqueous solution of sodium dithionite with a weight concentration of 10%-30%" based on the existing technical level and common knowledge should all fall within the protection scope of this invention. For example, replacing "aqueous solution of sodium dithionite with a weight concentration of 10%-30%" with "aqueous solution of sodium dithionite with a weight concentration of 14.9%" or "aqueous solution of sodium dithionite with a weight concentration of 15.1%" while keeping other technical features unchanged still falls within the protection scope of this invention.

[0096] Regarding the invention involved in this matter: The technical feature “the ratio of the weight of the coenzyme Q10, the volume of the ethyl acetate, and the volume of the aqueous solution of sodium dithionite is 5-15:50-150:50-150, in g:mL:mL” is derived from the aforementioned explanation and / or the corresponding technical features in Examples 1-13, such as the volume ratio of methanol to ethyl acetate (75:25, 79:21, 80:20, 81:19, 85:15), which are summarized by the common features “ratio of 10:100:100, in g:mL:mL”, “ratio of 5:150:150, in g:mL:mL”, and “ratio of 15:50:50, in g:mL:mL”. Therefore, those skilled in the art can reasonably presume that the technical feature "the ratio of the weight of coenzyme Q10, the volume of ethyl acetate, and the volume of the aqueous solution of sodium dithionite is 5-15:50-150:50-150, in g:mL:mL" and "the ratio of the weight of coenzyme Q10, the volume of ethyl acetate, and the volume of the aqueous solution of sodium dithionite is 5-15:50-150:50-150, in g:mL:mL" are essentially equivalent to the technical means described in "the ratio of the weight of coenzyme Q10, the volume of ethyl acetate, and the volume of the aqueous solution of sodium dithionite is 5-15:50-150:50-150, in g:mL:mL". Furthermore, based on the existing level of technology, and within conventional technical means and common knowledge, "the ratio of the weight of coenzyme Q10, the volume of ethyl acetate, and the volume of the aqueous solution of sodium dithionite is 5-15:50-150:50-150, in g:mL:mL" can be replaced by other technical means. The technical means of "the volume ratio of the sodium sulfite aqueous solution is 5-15:50-150:50-150, in g:mL:mL" should all fall within the protection scope of this invention. For example, if other technical features remain unchanged, replacing "the weight of the coenzyme Q10, the volume of the ethyl acetate, and the volume of the sodium dithionite aqueous solution is 5-15:50-150:50-150, in g:mL:mL" with "the weight of the coenzyme Q10, the volume of the ethyl acetate, and the volume of the sodium dithionite aqueous solution is 9.9:100.1:100.1, in g:mL:mL" or "the weight of the coenzyme Q10, the volume of the ethyl acetate, and the volume of the sodium dithionite aqueous solution is 4.9:150.1:49.9, in g:mL:mL" will still fall within the protection scope of this invention.

[0097] Regarding the invention involved in this matter: The technical feature “the reaction temperature is 35-45℃ and the reaction time is 1-3h” is summarized from the corresponding technical features “reaction temperature is 35℃, 36℃, 37℃, 38℃, 39℃, 40℃, 41℃, 42℃, 43℃, 44℃ or 45℃” and “reaction time is 1h, 1.2h, 1.4h, 1.6h, 1.8h, 2h, 2.2h, 2.4h, 2.6h, 2.8h or 3h” in the foregoing explanation and / or Examples 1-13, etc., through the common feature “reaction temperature is 35-45℃ and reaction time is 1-3h”. Therefore, those skilled in the art can reasonably infer that the subordinate concepts of the technical feature "the temperature of the reaction is 35-45℃ and the reaction time is 1-3h", "the temperature of the reaction is 35-45℃ and the reaction time is 1-3h", the essentially equivalent technical means of "the temperature of the reaction is 35-45℃ and the reaction time is 1-3h", and the technical means that can replace "the temperature of the reaction is 35-45℃ and the reaction time is 1-3h" based on the existing technical level and conventional technical means and common knowledge should all fall within the protection scope of this invention. For example, if other technical features remain unchanged, replacing "the temperature of the reaction is 35-45℃ and the reaction time is 1-3h" with "the temperature of the reaction is 34.9℃ and the reaction time is 3.05h" or "the temperature of the reaction is 45.1℃ and the reaction time is 0.99h" still falls within the protection scope of this invention.

[0098] Regarding the invention involved in this matter: The technical feature “the weight ratio of the concentrated solution of reduced coenzyme Q10 to the volume ratio of the mobile phase is 1:80-100 (g:mL)” is summarized from the corresponding technical features in the foregoing explanation and / or Examples 1-13, such as the weight ratio of the concentrated solution to the volume ratio of the mobile phase being 1:81, 1:98.6, 1:89.3 (g:mL), etc., which are summarized from the common feature “the weight ratio of the concentrated solution of reduced coenzyme Q10 to the volume ratio of the mobile phase is 1:80-100 (g:mL)”. Therefore, those skilled in the art can reasonably presume that the subordinate concepts of the technical feature "the weight ratio of the concentrated solution of reduced coenzyme Q10 to the volume ratio of the mobile phase is 1:80-100 (g:mL)" and the essentially equivalent technical means of "the weight ratio of the concentrated solution of reduced coenzyme Q10 to the volume ratio of the mobile phase is 1:80-100 (g:mL)", and based on existing technology, can be considered as such. Any technical means that can replace "the weight ratio of the concentrated reduced coenzyme Q10 to the volume ratio of the mobile phase is 1:80-100 (g:mL)" within the scope of conventional technical means and common knowledge should fall within the protection scope of this invention. For example, if "the weight ratio of the concentrated reduced coenzyme Q10 to the volume ratio of the mobile phase is 1:80-100 (g:mL)" is replaced with "1:79.9", "1:100.1", etc., while keeping other technical features unchanged, it should still fall within the protection scope of this invention.

[0099] Regarding the invention involved in this matter: The technical feature "the particle size of the C18 column packing is 5-100μm" refers to the corresponding technical feature in Examples 1-13 of this document, where the particle size of the C18 column packing is 5μm, 6μm, 7μm, 8μm, 9μm, 10μm, 11μm, 12μm, 13μm, 14μm, 15μm, 16μm, 17μm, 18μm, 19μm, 20μm, 21μm. m, 22μm, 23μm, 24μm, 25μm, 26μm, 27μm, 28μm, 29μm, 30μm, 31μm, 32μm, 33μm, 34μm, 35μm , 36μm, 37μm, 38μm, 39μm, 40μm, 41μm, 42μm, 43μm, 44μm, 45μm, 46μm, 47μm, 48μm, 49μm, 5 0μm, 51μm, 52μm, 53μm, 54μm, 55μm, 56μm, 57μm, 58μm, 59μm, 60μm, 61μm, 62μm, 63μm, 64 μm, 65μm, 66μm, 67μm, 68μm, 69μm, 70μm, 71μm, 72μm, 73μm, 74μm, 75μm, 76μm, 77μm, 78μm The particle sizes of the C18 column, such as 79μm, 80μm, 81μm, 82μm, 83μm, 84μm, 85μm, 86μm, 87μm, 88μm, 89μm, 90μm, 91μm, 92μm, 93μm, 94μm, 95μm, 96μm, 97μm, 98μm, 99μm, and 100μm, are summarized by the common feature that "the particle size of the packing material in the C18 column is 5-100μm". Therefore, those skilled in the art can reasonably infer that the subordinate concepts of the technical feature "the particle size of the C18 column is 5-100μm", the essentially equivalent technical means of "the particle size of the C18 column is 5-100μm", and the technical means that can replace "the particle size of the C18 column is 5-100μm" based on the existing technical level and conventional technical means and common knowledge should all fall within the protection scope of this invention. For example, replacing "the particle size of the C18 column is 5-100μm" with "4.9μm" or "100.1μm" while keeping other technical features unchanged still falls within the protection scope of this invention.

[0100] Regarding the invention involved in this matter: The technical feature “the volume ratio of the co-crystallization solvent to the mass of the solid is 10-12:1 (mL:g)” is summarized from the corresponding technical features in the foregoing explanation and / or Examples 1-13, where the volume ratio of the co-crystallization solvent to the mass of the solid is 10:1, 10.1:1, 10.2:1, 10.3:1, 10.4:1, 10.5:1, 10.6:1, 10.7:1, 10.8:1, 10.9:1, 11:1, 11.1:1, 11.2:1, 11.3:1, 11.4:1, 11.5:1, 11.6:1, 11.7:1, 11.8:1, 11.9:1, 12:1 (mL:g), etc., and is derived from the common feature “the volume ratio of the co-crystallization solvent to the mass of the solid is 10-12:1 (mL:g)”. Therefore, those skilled in the art can reasonably infer that the subordinate concepts of the technical feature "the volume ratio of the co-crystallization solvent to the mass of the solid is 10-12:1 (mL:g)", the essentially equivalent technical means of "the volume ratio of the co-crystallization solvent to the mass of the solid is 10-12:1 (mL:g)", and the technical means that can replace "the volume ratio of the co-crystallization solvent to the mass of the solid is 10-12:1 (mL:g)" based on existing technology and common knowledge should all fall within the scope of protection of this invention. For example, if other technical features remain unchanged, replacing "the volume ratio of the co-crystallization solvent to the mass of the solid is 10-12:1 (mL:g)" with "9.9:1", "12.1:1", etc., still falls within the scope of protection of this invention.

[0101] Regarding the invention involved in this matter: The technical feature "sealed heating to 55-65℃" is derived from the aforementioned explanation and / or the corresponding sealed heating temperatures of 55℃, 56℃, 57℃, 58℃, 59℃, 60℃, 61℃, 62℃, 63℃, 64℃, and 65℃ in Examples 1-13, summarized by the common feature "sealed heating to 55-65℃". Therefore, those skilled in the art can reasonably infer that the subordinate concepts of the technical feature "sealed heating to 55-65℃", the essentially equivalent technical means of "sealed heating to 55-65℃", and the technical means that can replace "sealed heating to 55-65℃" based on existing technology and conventional technical means and common knowledge should all fall within the protection scope of this invention. For example, replacing "sealed heating to 55-65℃" with "54.9℃" or "65.1℃" while keeping other technical features unchanged still falls within the protection scope of this invention.

[0102] The technical feature "cooling down to 0-5℃" is derived from the common feature "cooling down to 0-5℃" by the corresponding technical features of cooling down to 0℃, 1℃, 2℃, 3℃, 4℃, and 5℃ in the foregoing explanation and / or Examples 1-13. Therefore, those skilled in the art can reasonably infer that the technical feature "cooling down to 0-5℃", the subordinate concept of "cooling down to 0-5℃", the basically equivalent technical means of "cooling down to 0-5℃", and the technical means that can replace "cooling down to 0-5℃" based on the existing technical level and conventional technical means and common knowledge should all fall within the protection scope of this invention. For example, replacing "cooling down to 0-5℃" with "-0.1℃", "5.1℃", etc., while keeping other technical features unchanged, still falls within the protection scope of this invention.

[0103] Regarding the invention involved in this matter: The technical feature "the cooling rate is 5-8℃ / h" refers to the cooling rates of 5℃ / h, 5.1℃ / h, 5.2℃ / h, 5.3℃ / h, 5.4℃ / h, 5.5℃ / h, 5.6℃ / h, 5.7℃ / h, 5.8℃ / h, 5.9℃ / h, 6℃ / h, 6.1℃ / h, 6.2℃ / h, and 6℃ / h, respectively, as explained above and / or in Examples 1-13. The rates of 3℃ / h, 6.4℃ / h, 6.5℃ / h, 6.6℃ / h, 6.7℃ / h, 6.8℃ / h, 6.9℃ / h, 7℃ / h, 7.1℃ / h, 7.2℃ / h, 7.3℃ / h, 7.4℃ / h, 7.5℃ / h, 7.6℃ / h, 7.7℃ / h, 7.8℃ / h, 7.9℃ / h, and 8℃ / h are summarized by the common feature that "the cooling rate is 5-8℃ / h". Therefore, those skilled in the art can reasonably infer that the technical feature "the cooling rate is 5-8℃ / h", the subordinate concept of "the cooling rate is 5-8℃ / h", the technical means that are basically equivalent to "the cooling rate is 5-8℃ / h", and the technical means that can replace "the cooling rate is 5-8℃ / h" based on the existing technical level and conventional technical means and common knowledge should all fall within the protection scope of this invention. For example, if other technical features remain unchanged, replacing "the cooling rate is 5-8℃ / h" with "4.9℃ / h", "8.1℃ / h", etc., still falls within the protection scope of this invention.

[0104] The present invention has at least the following beneficial effects: Compared with the prior art, the present invention has better technical effects in terms of the purity of reduced coenzyme Q10, the weight content of reduced coenzyme Q9 relative to reduced coenzyme Q10, and the weight content of reduced coenzyme Q11 relative to reduced coenzyme Q10.

[0105] According to experimental tests, this invention increases the purity of reduced coenzyme Q10 from 97.1% in the prior art to ≥99.5%; reduces the weight content of reduced coenzyme Q9 relative to reduced coenzyme Q10 in reduced coenzyme Q10 to 0.3% or less, and eliminates reduced coenzyme Q11 in reduced coenzyme Q10: according to experimental tests, the weight content of reduced coenzyme Q11 relative to reduced coenzyme Q10 in reduced coenzyme Q10 is reduced to undetectable by liquid chromatography.

[0106] Furthermore, according to experimental tests, the method for producing reduced coenzyme Q10 provided by this invention can not only obtain ultra-high purity reduced coenzyme Q10 products with "reduced coenzyme Q10 purity ≥ 99.5%, reduced coenzyme Q9 weight content relative to reduced coenzyme Q10 ≤ 0.3%, and no detected content of reduced coenzyme Q11", but also maintain the total yield of the reduced coenzyme Q10 production process at a level of 88.6%-93.1% and maintain the mass content of reduced coenzyme Q10 at a level of 99.24%-99.48%. Detailed Implementation

[0107] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection of the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, which should also fall within the scope of protection of the present invention.

[0108] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all instruments, devices, equipment, reagents, products, etc., used in the embodiments of the present invention are obtained through conventional commercial means.

[0109] In the following specific embodiments, the detection and analytical conditions for detecting reduced coenzyme Q10, reduced coenzyme Q9, and reduced coenzyme Q11 in the reduced coenzyme Q10 product using liquid chromatography are as follows: Mobile phase: acetonitrile and ethanol (50:50, v / v); diluent: methanol and n-hexane (85:15, v / v); detector: UV 290nm; column: 4.6mm × 10cm, 3μm C18 packing; column temperature: 35℃; flow rate: 1.5mL / min; injection volume: 20μL. The main peak, reduced coenzyme Q10, had a retention time of 14-15 min in the liquid chromatography. The relative retention time (RRT) of the impurity reduced coenzyme Q9 was 0.64, and the relative retention time (RRT) of the impurity reduced coenzyme Q11 was 1.64. "Q11 impurity not detected" means that no impurity peak appeared in the Q11 relative retention time in the liquid chromatography chromatogram.

[0110] In the following specific embodiments, unless otherwise specified, the terms "content" and "percentage content" refer to weight content.

[0111] Example 1 A method for preparing high-purity reduced coenzyme Q10, the specific steps of which are as follows: S1. In a round-bottom flask, add 10g of coenzyme Q10 (purity 99.17%, coenzyme Q9 impurity content 0.35%, coenzyme Q11 impurity content 0.29%), dissolve in 100mL of ethyl acetate, add 100mL of 20% sodium dithionite aqueous solution, purge with nitrogen for protection, and heat to 40℃ for 2h. After the reaction is complete, separate the layers using a separatory funnel, remove the lower aqueous layer, and concentrate the upper ethyl acetate phase under vacuum to remove the solvent, obtaining a concentrated reduced coenzyme Q10 solution.

[0112] S2. Weigh 9.87 g of the concentrated reduced coenzyme Q10 obtained in step S1 and add 800 mL of a methanol:ethyl acetate mixture (80:20 v / v). The weight to volume ratio of reduced coenzyme Q10 to the mobile phase is 1:81. Purge twice with nitrogen, then heat in a sealed container at 45°C and sonicate until completely dissolved. Load the sample solution onto a C18 preparative column (30 mm × 250 mm, 10 μm particle size), and elute with a methanol:ethyl acetate mixture (80:20 v / v). Collect one eluent fraction per 100 mL. Combine the eluent fractions with reduced coenzyme Q9 impurity content ≤0.3% and reduced coenzyme Q11 impurity undetectable. Analyze the combined eluents. The chromatographic purity of reduced coenzyme Q10 is 99.27%, the reduced coenzyme Q9 impurity content is 0.12%, and reduced coenzyme Q11 impurity is undetectable.

[0113] S3. The combined eluent fractions are concentrated under vacuum using a rotary evaporator to remove the solvent. 100 mL of anhydrous ethanol is added, and the concentration flask is purged twice with nitrogen. The mixture is then heated to 60°C under a sealed environment with stirring until completely dissolved. The mixture is then slowly cooled to 0-5°C for crystallization. The slow cooling rate is 5-8°C / h. The mixture is filtered to obtain a wet crystalline powder.

[0114] S4. The crystalline wet powder obtained in step S3 was vacuum dried at 40℃ to obtain 9.13g of white powder, with a total process yield of 91.3%. The finished product was tested and analyzed, and its mass content of reduced coenzyme Q10 was 99.37%, the chromatographic purity of reduced coenzyme Q10 was 99.69%, the weight content of reduced coenzyme Q9 impurity relative to reduced coenzyme Q10 was 0.11%, and it did not contain reduced coenzyme Q11 impurity.

[0115] Example 2 A method for preparing high-purity reduced coenzyme Q10, the specific steps of which are as follows: S1. Take 10g of coenzyme Q10 (purity 99.31%, coenzyme Q9 impurity content 0.33%, coenzyme Q11 impurity content 0.27%) and prepare reduced coenzyme Q10 concentrate according to the method in Example 1.

[0116] S2. Weigh 10.14 g of the concentrated solution and add 1000 mL of a mixed solvent of ethanol:ethyl acetate = 88:12 (v / v). The weight ratio of reduced coenzyme Q10 to the volume of the mobile phase is 1:98.6. Purge twice with nitrogen, and then sonicate at 45℃ under sealed conditions until completely dissolved. Load the sample solution onto a C18 preparative column (30 mm × 250 mm, packing particle size 30 μm), and then elute with a mixed solvent of ethanol:ethyl acetate = 88:12 (v / v). Collect one eluent fraction per 100 mL. Combine the eluent fractions with a reduced coenzyme Q9 impurity content ≤0.3% and a reduced coenzyme Q11 impurity not detected. Analyze the combined eluents. The chromatographic purity of reduced coenzyme Q10 is 99.32%, the reduced coenzyme Q9 impurity content is 0.10%, and the reduced coenzyme Q11 impurity is not detected.

[0117] S3. The combined eluent fractions are concentrated under vacuum using a rotary evaporator to remove the solvent. 100 mL of anhydrous ethanol is added, the concentration flask is purged with nitrogen twice, and the mixture is heated to 60°C under a sealed environment to dissolve the solvent. The mixture is then slowly cooled to 0-5°C for crystallization. The slow cooling rate is 5-8°C / h. The mixture is filtered to obtain a wet crystalline powder.

[0118] S4. The crystalline wet powder obtained in step S3 was vacuum dried at 40℃ to obtain 9.08g of white powder, with a total yield of 90.8%. Analysis of the finished product showed that the mass content of reduced coenzyme Q10 was 99.24%, the chromatographic purity of reduced coenzyme Q10 was 99.63%, the weight content of reduced coenzyme Q9 impurity relative to reduced coenzyme Q10 was 0.09%, and it did not contain reduced coenzyme Q11 impurity.

[0119] Example 3 A method for preparing high-purity reduced coenzyme Q10, the specific steps of which are as follows: S1. Take 1200g of coenzyme Q10 (purity 99.28%, coenzyme Q9 impurity content 0.30%, coenzyme Q11 impurity content 0.32%) and prepare reduced coenzyme Q10 concentrate according to the method in Example 1.

[0120] S2. Weigh 1231.52 g of the reduced coenzyme Q10 concentrate obtained in step S1 and add 110 L of a methanol:ethyl acetate = 80:20 (v / v) mixed solvent. The weight ratio of the reduced coenzyme Q10 concentrate to the volume of the mobile phase is 1:89.3. Purge the container twice with nitrogen gas and heat to 50°C with stirring until completely dissolved. Load the eluent onto a C18 dynamic shaft compression column (DAC300, packing particle size 30 μm), and then elute with a methanol:ethyl acetate = 80:20 (v / v) mixed solvent. Collect one eluent fraction every 12 L. Combine the eluent fractions with a reduced coenzyme Q9 impurity content ≤0.3% and a reduced coenzyme Q11 impurity not detected. Analyze the combined eluents. The chromatographic purity of reduced coenzyme Q10 is 99.29%, the reduced coenzyme Q9 impurity content is 0.11%, and the reduced coenzyme Q11 impurity is not detected.

[0121] S3. Transfer the combined eluted fractions to a concentration tank and concentrate under vacuum to remove the solvent. After concentration, turn off the vacuum, introduce nitrogen gas, add 12L of anhydrous ethanol, heat to 60℃ to dissolve, and then slowly cool to 0-5℃ for crystallization. The slow cooling rate is 5-8℃ / h. Filter to obtain crystalline wet powder.

[0122] S4. The crystalline wet powder obtained in step S3 was vacuum dried at 40℃ to obtain 1091.9g of white powder, with a total process yield of 90.9%. Liquid chromatography analysis of the finished product showed that the mass content of reduced coenzyme Q10 was 99.47%, the chromatographic purity of reduced coenzyme Q10 was 99.71%, the weight content of reduced coenzyme Q9 impurity relative to reduced coenzyme Q10 was 0.10%, and there was no reduced coenzyme Q11 impurity.

[0123] Example 4 Compared to Example 1, the only difference is that in step S2, the mobile phase is replaced by a mixture of methanol and ethyl acetate in a volume ratio of 79:21; all other steps are the same. The specific steps are as follows: S1. In a round-bottom flask, add 10g of coenzyme Q10 (purity 99.17%, coenzyme Q9 impurity content 0.35%, coenzyme Q11 impurity content 0.29%), dissolve in 100mL of ethyl acetate, add 100mL of 20% sodium dithionite aqueous solution, purge with nitrogen for protection, and heat to 40℃ for 2h. After the reaction is complete, separate the layers using a separatory funnel, remove the lower aqueous layer, and concentrate the upper ethyl acetate phase under vacuum to remove the solvent, obtaining a concentrated reduced coenzyme Q10 solution.

[0124] S2. Weigh 9.87 g of the concentrated reduced coenzyme Q10 obtained in step S1 and add 800 mL of a methanol:ethyl acetate mixture (79:21 v / v). The weight-to-volume ratio of reduced coenzyme Q10 to the mobile phase is 1:81. Purge twice with nitrogen, then heat in a sealed container at 45°C and sonicate until completely dissolved. Load the sample solution onto a C18 preparative column (30 mm × 250 mm, 10 μm particle size), and elute with a methanol:ethyl acetate mixture (79:21 v / v). Collect one eluent fraction per 100 mL, and combine the eluent fractions with a reduced coenzyme Q9 impurity content ≤0.3% and no detected reduced coenzyme Q11 impurity.

[0125] S3. The combined eluent fractions are concentrated under vacuum using a rotary evaporator to remove the solvent. 100 mL of anhydrous ethanol is added, and the concentration flask is purged twice with nitrogen. The mixture is then heated to 60°C under a sealed environment with stirring until completely dissolved. The mixture is then slowly cooled to 0-5°C for crystallization. The slow cooling rate is 5-8°C / h. The mixture is filtered to obtain a wet crystalline powder.

[0126] S4. The crystalline wet powder obtained in step S3 was vacuum dried at 40℃ to obtain 9.23g of white powder, with a total yield of 92.3%. Analysis of the finished product showed that the mass content of reduced coenzyme Q10 was 99.39%, the chromatographic purity of reduced coenzyme Q10 was 99.61%, the weight content of reduced coenzyme Q9 impurity relative to reduced coenzyme Q10 was 0.12%, and there was no reduced coenzyme Q11 impurity.

[0127] Example 5 Compared to Example 1, the only difference is that in step S2, the mobile phase is replaced with a mixture of methanol and ethyl acetate in a volume ratio of 81:19; all other steps are the same. The specific steps are as follows: S1. In a round-bottom flask, add 10g of coenzyme Q10 (purity 99.17%, coenzyme Q9 impurity content 0.35%, coenzyme Q11 impurity content 0.29%), dissolve in 100mL of ethyl acetate, add 100mL of 20% sodium dithionite aqueous solution, purge with nitrogen for protection, and heat to 40℃ for 2h. After the reaction is complete, separate the layers using a separatory funnel, remove the lower aqueous layer, and concentrate the upper ethyl acetate phase under vacuum to remove the solvent, obtaining a concentrated reduced coenzyme Q10 solution.

[0128] S2. Weigh 9.87 g of the concentrated reduced coenzyme Q10 obtained in step S1 and add 800 mL of a methanol:ethyl acetate mixture of 81:19 (v / v). The weight-to-volume ratio of reduced coenzyme Q10 to the mobile phase is 1:81. Purge twice with nitrogen, then heat in a sealed container at 45°C and sonicate until completely dissolved. Load the sample solution onto a C18 preparative column (30 mm × 250 mm, 10 μm particle size), and elute with a methanol:ethyl acetate mixture of 81:19 (v / v). Collect one eluent fraction per 100 mL, and combine the eluent fractions with a reduced coenzyme Q9 impurity content ≤0.3% and no detected reduced coenzyme Q11 impurity.

[0129] S3. The combined eluent fractions are concentrated under vacuum using a rotary evaporator to remove the solvent. 100 mL of anhydrous ethanol is added, and the concentration flask is purged twice with nitrogen. The mixture is then heated to 60°C under a sealed environment with stirring until completely dissolved. The mixture is then slowly cooled to 0-5°C for crystallization. The slow cooling rate is 5-8°C / h. The mixture is filtered to obtain a wet crystalline powder.

[0130] S4. The crystalline wet powder obtained in step S3 was vacuum dried at 40℃ to obtain 9.11g of white powder, with a total process yield of 91.1%. The finished product was tested and analyzed, and its mass content of reduced coenzyme Q10 was 99.34%, the chromatographic purity of reduced coenzyme Q10 was 99.59%, the weight content of reduced coenzyme Q9 impurity relative to reduced coenzyme Q10 was 0.10%, and it did not contain reduced coenzyme Q11 impurity.

[0131] Example 6 Compared to Example 1, the only difference is that in step S2, the mobile phase is replaced with a mixture of methanol and ethyl acetate in a volume ratio of 75:25; all other steps are the same. The specific steps are as follows: S1. In a round-bottom flask, add 10g of coenzyme Q10 (purity 99.17%, coenzyme Q9 impurity content 0.35%, coenzyme Q11 impurity content 0.29%), dissolve in 100mL of ethyl acetate, add 100mL of 20% sodium dithionite aqueous solution, purge with nitrogen for protection, and heat to 40℃ for 2h. After the reaction is complete, separate the layers using a separatory funnel, remove the lower aqueous layer, and concentrate the upper ethyl acetate phase under vacuum to remove the solvent, obtaining a concentrated reduced coenzyme Q10 solution.

[0132] S2. Weigh 9.87 g of the concentrated reduced coenzyme Q10 obtained in step S1 and add 800 mL of a methanol:ethyl acetate mixture (75:25 v / v). The weight to volume ratio of reduced coenzyme Q10 to the mobile phase is 1:81. Purge twice with nitrogen, then heat in a sealed container at 45°C and sonicate until completely dissolved. Load the sample solution onto a C18 preparative column (30 mm × 250 mm, 10 μm particle size), and elute with a methanol:ethyl acetate mixture (75:25 v / v). Collect one eluent fraction per 100 mL, and combine the eluent fractions with a reduced coenzyme Q9 impurity content ≤0.3% and no detected reduced coenzyme Q11 impurity.

[0133] S3. The combined eluent fractions are concentrated under vacuum using a rotary evaporator to remove the solvent. 100 mL of anhydrous ethanol is added, and the concentration flask is purged twice with nitrogen. The mixture is then heated to 60°C under a sealed environment with stirring until completely dissolved. The mixture is then slowly cooled to 0-5°C for crystallization. The slow cooling rate is 5-8°C / h. The mixture is filtered to obtain a wet crystalline powder.

[0134] S4. The crystalline wet powder obtained in step S3 is vacuum dried at 40℃ to obtain 8.98g of white powder, with a total process yield of 89.8%. The finished product is tested and analyzed. The mass content of reduced coenzyme Q10 is 99.44%, the chromatographic purity of reduced coenzyme Q10 is 99.71%, the weight content of reduced coenzyme Q9 impurity is 0.09% higher than that of reduced coenzyme Q10, and there is no reduced coenzyme Q11 impurity.

[0135] Example 7 Compared to Example 1, the only difference is that in step S2, the mobile phase is replaced by a mixture of methanol and ethyl acetate in a volume ratio of 85:15; all other steps are the same. The specific steps are as follows: S1. In a round-bottom flask, add 10g of coenzyme Q10 (purity 99.17%, coenzyme Q9 impurity content 0.35%, coenzyme Q11 impurity content 0.29%), dissolve in 100mL of ethyl acetate, add 100mL of 20% sodium dithionite aqueous solution, purge with nitrogen for protection, and heat to 40℃ for 2h. After the reaction is complete, separate the layers using a separatory funnel, remove the lower aqueous layer, and concentrate the upper ethyl acetate phase under vacuum to remove the solvent, obtaining a concentrated reduced coenzyme Q10 solution.

[0136] S2. Weigh 9.87 g of the concentrated reduced coenzyme Q10 obtained in step S1 and add 800 mL of a methanol:ethyl acetate mixture (85:15 v / v). The weight-to-volume ratio of reduced coenzyme Q10 to the mobile phase is 1:81. Purge twice with nitrogen, then heat in a sealed container at 45°C and sonicate until completely dissolved. Load the sample solution onto a C18 preparative column (30 mm × 250 mm, 10 μm particle size), and elute with a methanol:ethyl acetate mixture (85:15 v / v). Collect one eluent fraction per 100 mL, and combine the eluent fractions with a reduced coenzyme Q9 impurity content ≤0.3% and no detected reduced coenzyme Q11 impurity.

[0137] S3. The combined eluent fractions are concentrated under vacuum using a rotary evaporator to remove the solvent. 100 mL of anhydrous ethanol is added, and the concentration flask is purged twice with nitrogen. The mixture is then heated to 60°C under a sealed environment with stirring until completely dissolved. The mixture is then slowly cooled to 0-5°C for crystallization. The slow cooling rate is 5-8°C / h. The mixture is filtered to obtain a wet crystalline powder.

[0138] S4. The crystalline wet powder obtained in step S3 is vacuum dried at 40℃ to obtain 9.09g of white powder, with a total process yield of 90.9%. The finished product is tested and analyzed. The mass content of reduced coenzyme Q10 is 99.34%, the chromatographic purity of reduced coenzyme Q10 is 99.61%, the weight content of reduced coenzyme Q9 impurity relative to reduced coenzyme Q10 is 0.13%, and there is no reduced coenzyme Q11 impurity.

[0139] Example 8 Compared to Example 2, the only difference is that in step S2, the mobile phase is replaced with a mixture of ethanol and ethyl acetate in a volume ratio of 87:13 (v / v); all other steps are the same. The specific steps are as follows: S1. Take 10g of coenzyme Q10 (purity 99.31%, coenzyme Q9 impurity content 0.33%, coenzyme Q11 impurity content 0.27%) and prepare reduced coenzyme Q10 concentrate according to the method in Example 1.

[0140] S2. Weigh 10.14 g of the concentrate and add 1000 mL of a mixed solvent of ethanol:ethyl acetate = 87:13 (v / v). The weight ratio of reduced coenzyme Q10 to the volume of the mobile phase is 1:98.6. Purge twice with nitrogen, and then heat in a sealed container at 45°C and sonicate until completely dissolved. Load the sample solution onto a C18 preparative column (30 mm × 250 mm, packing particle size 30 μm), and then elute with a mixed solvent of ethanol:ethyl acetate = 87:13 (v / v). Collect one eluent fraction per 100 mL, and combine the eluent fractions with a reduced coenzyme Q9 impurity content ≤0.3% and a reduced coenzyme Q11 impurity not detected.

[0141] S3. The combined eluent fractions are concentrated under vacuum using a rotary evaporator to remove the solvent. 100 mL of anhydrous ethanol is added, the concentration flask is purged with nitrogen twice, and the mixture is heated to 60°C under a sealed environment to dissolve the solvent. The mixture is then slowly cooled to 0-5°C for crystallization. The slow cooling rate is 5-8°C / h. The mixture is filtered to obtain a wet crystalline powder.

[0142] S4. The crystalline wet powder obtained in step S3 was vacuum dried at 40℃ to obtain 9.16g of white powder, with a total yield of 91.6%. Analysis of the finished product showed that the content of reduced coenzyme Q10 was 99.39%, the chromatographic purity of reduced coenzyme Q10 was 99.69%, the weight content of reduced coenzyme Q9 impurity relative to reduced coenzyme Q10 was 0.10%, and there was no reduced coenzyme Q11 impurity.

[0143] Example 9 Compared to Example 2, the only difference is that in step S2, the mobile phase is replaced with a mixture of ethanol and ethyl acetate in a volume ratio of 89:11; all other steps are the same. The specific steps are as follows: S1. Take 10g of coenzyme Q10 (purity 99.31%, coenzyme Q9 impurity content 0.33%, coenzyme Q11 impurity content 0.27%) and prepare reduced coenzyme Q10 concentrate according to the method in Example 1.

[0144] S2. Weigh 10.14 g of the concentrate and add 1000 mL of a mixed solvent of ethanol:ethyl acetate = 89:11 (v / v). The weight ratio of reduced coenzyme Q10 to the volume of the mobile phase is 1:98.6. Purge twice with nitrogen, and then heat in a sealed container at 45°C and sonicate to dissolve completely. Load the sample solution onto a C18 preparative column (30 mm × 250 mm, 30 μm particle size), and then elute with a mixed solvent of ethanol:ethyl acetate = 89:11 (v / v). Collect one eluent fraction every 100 mL, and combine the eluent fractions with Q9 impurities ≤ 0.3% and Q11 impurities not detected.

[0145] S3. The combined eluent fractions are concentrated under vacuum using a rotary evaporator to remove the solvent. 100 mL of anhydrous ethanol is added, the concentration flask is purged with nitrogen twice, and the mixture is heated to 60°C under a sealed environment to dissolve the solvent. The mixture is then slowly cooled to 0-5°C for crystallization. The slow cooling rate is 5-8°C / h. The mixture is filtered to obtain a wet crystalline powder.

[0146] S4. The crystalline wet powder obtained in step S3 was vacuum dried at 40℃ to obtain 9.09g of white powder. The total yield in the process was 90.9%. The finished product was tested and analyzed. The mass content was 99.47%, the chromatographic purity was 99.71%, the Q9 impurity was 0.10%, and the Q11 impurity was not detected.

[0147] Example 10 Compared to Example 2, the only difference is that in step S2, the mobile phase is replaced with a mixture of ethanol and ethyl acetate in a volume ratio of 85:15; all other steps are the same. The specific steps are as follows: S1. Take 10g of coenzyme Q10 (purity 99.31%, Q9 impurity 0.33%, Q11 impurity 0.27%) and prepare reduced coenzyme Q10 concentrate according to the method in Example 1.

[0148] S2. Weigh 10.14 g of the concentrate and add 1000 mL of a mixed solvent of ethanol:ethyl acetate = 85:15 (v / v). The weight ratio of reduced coenzyme Q10 to the volume of the mobile phase is 1:98.6. Purge twice with nitrogen, and then heat in a sealed container at 45°C and sonicate until completely dissolved. Load the sample solution onto a C18 preparative column (30 mm × 250 mm, packing particle size 30 μm), and then elute with a mixed solvent of ethanol:ethyl acetate = 85:15 (v / v). Collect one eluent fraction per 100 mL, and combine the eluent fractions with a reduced coenzyme Q9 impurity content ≤0.3% and a reduced coenzyme Q11 impurity not detected.

[0149] S3. The combined eluent fractions are concentrated under vacuum using a rotary evaporator to remove the solvent. 100 mL of anhydrous ethanol is added, the concentration flask is purged with nitrogen twice, and the mixture is heated to 60°C under a sealed environment to dissolve the solvent. The mixture is then slowly cooled to 0-5°C for crystallization. The slow cooling rate is 5-8°C / h. The mixture is filtered to obtain a wet crystalline powder.

[0150] S4. The crystalline wet powder obtained in step S3 was vacuum dried at 40℃ to obtain 9.31g of white powder, with a total yield of 93.1%. Analysis of the finished product showed that the mass content of reduced coenzyme Q10 was 99.48%, the chromatographic purity of reduced coenzyme Q10 was 99.73%, the weight content of reduced coenzyme Q9 impurity relative to reduced coenzyme Q10 was 0.08%, and there was no reduced coenzyme Q11 impurity.

[0151] Example 11 Compared to Example 2, the only difference is that in step S2, the mobile phase is replaced with a mixture of ethanol and ethyl acetate in a volume ratio of 91:9; all other steps are the same. The specific steps are as follows: S1. Take 10g of coenzyme Q10 (purity 99.31%, coenzyme Q9 impurity content 0.33%, coenzyme Q11 impurity content 0.27%) and prepare reduced coenzyme Q10 concentrate according to the method in Example 1.

[0152] S2. Weigh 10.14 g of the concentrate and add 1000 mL of a 91:9 (v / v) mixture of ethanol and ethyl acetate, resulting in a weight-to-volume ratio of reduced coenzyme Q10 to the mobile phase of 1:98.6. Purge twice with nitrogen, then heat in a sealed container at 45°C and sonicate until completely dissolved. Load the sample solution onto a C18 preparative column (30 mm × 250 mm, 30 μm particle size), and elute with a 91:9 (v / v) mixture of ethanol and ethyl acetate. Collect one eluent fraction per 100 mL, and combine the eluent fractions with a reduced coenzyme Q9 impurity content ≤0.3% and no detected reduced coenzyme Q11 impurity.

[0153] S3. The combined eluent fractions are concentrated under vacuum using a rotary evaporator to remove the solvent. 100 mL of anhydrous ethanol is added, the concentration flask is purged with nitrogen twice, and the mixture is heated to 60°C under a sealed environment to dissolve the solvent. The mixture is then slowly cooled to 0-5°C for crystallization. The slow cooling rate is 5-8°C / h. The mixture is filtered to obtain a wet crystalline powder.

[0154] S4. The crystalline wet powder obtained in step S3 is dried under vacuum at 40℃ to obtain 9.25g of white powder. The overall yield in the process is 92.5%. Analysis of the finished product shows that the mass content of reduced coenzyme Q10 is 99.31%, the chromatographic purity of reduced coenzyme Q10 is 99.74%, the weight content of reduced coenzyme Q9 impurity relative to reduced coenzyme Q10 is 0.08%, and there is no reduced coenzyme Q11 impurity.

[0155] Example 12 Compared to Example 1, the only difference is that the C18 preparation column is a dynamic axial compression column DAC50; all other steps are the same. The specific steps are as follows: S1. Take 10g of coenzyme Q10 (purity 99.17%, coenzyme Q9 impurity content 0.35%, coenzyme Q11 impurity content 0.29%) and prepare reduced coenzyme Q10 concentrate according to the method in Example 1.

[0156] S2. Weigh 10.21 g of the concentrated reduced coenzyme Q10 obtained in step S1 and add 900 mL of a methanol:ethyl acetate mixture of 80:20 (v / v). The weight to volume ratio of reduced coenzyme Q10 to the mobile phase is 1:88. Purge twice with nitrogen, then heat in a sealed container at 45°C and sonicate until completely dissolved. Load the sample solution onto a C18 preparative column (DAC50, 10 μm particle size), and elute with a methanol:ethyl acetate mixture of 80:20 (v / v). Collect one eluent fraction per 100 mL. Combine the eluent fractions with a reduced coenzyme Q9 impurity content ≤0.3% and no detected reduced coenzyme Q11 impurity. Analyze the combined eluents; the chromatographic purity of reduced coenzyme Q10 is 99.32%, the reduced coenzyme Q9 impurity content is 0.09%, and no detected reduced coenzyme Q11 impurity.

[0157] S3. The combined eluent fractions are concentrated under vacuum using a rotary evaporator to remove the solvent. 100 mL of anhydrous ethanol is added, and the concentration flask is purged twice with nitrogen. The mixture is then heated to 60°C under a sealed environment with stirring until completely dissolved. The mixture is then slowly cooled to 0-5°C for crystallization. The slow cooling rate is 5-8°C / h. The mixture is filtered to obtain a wet crystalline powder.

[0158] S4. The crystalline wet powder obtained in step S3 was vacuum dried at 40℃ to obtain 9.29g of white powder, with a total process yield of 92.9%. The finished product was tested and analyzed, and its mass content of reduced coenzyme Q10 was 99.44%, the chromatographic purity of reduced coenzyme Q10 was 99.79%, the weight content of reduced coenzyme Q9 impurity relative to reduced coenzyme Q10 was 0.08%, and it did not contain reduced coenzyme Q11 impurity.

[0159] Example 13 Compared to Example 1, the only difference is that in step S2, one elution fraction is collected every 150 mL; all other steps are the same. The specific steps are as follows: S1. Take 10g of coenzyme Q10 (purity 99.31%, coenzyme Q9 impurity content 0.33%, coenzyme Q11 impurity content 0.27%) and prepare reduced coenzyme Q10 concentrate according to the method in Example 1.

[0160] S2. Weigh 10.14 g of the concentrate and add 1000 mL of a methanol:ethyl acetate = 80:20 (v / v) mixed solvent. The weight ratio of reduced coenzyme Q10 to the volume of the mobile phase is 1:98.6. Purge twice with nitrogen, and then heat in a sealed container at 45 °C and sonicate to dissolve completely. Load the sample solution onto a C18 preparative column (30 mm × 250 mm, packing particle size 30 μm), and then elute with a methanol:ethyl acetate = 80:20 (v / v) mixed solvent. Collect one eluent fraction every 150 mL, and combine the eluent fractions with a reduced coenzyme Q9 impurity content ≤0.3% and a reduced coenzyme Q11 impurity not detected.

[0161] S3. The combined eluent fractions are concentrated under vacuum using a rotary evaporator to remove the solvent. 100 mL of anhydrous ethanol is added, the concentration flask is purged with nitrogen twice, and the mixture is heated to 60°C under a sealed environment to dissolve the solvent. The mixture is then slowly cooled to 0-5°C for crystallization. The slow cooling rate is 5-8°C / h. The mixture is filtered to obtain a wet crystalline powder.

[0162] S4. The crystalline wet powder obtained in step S3 was vacuum dried at 40℃ to obtain 8.86g of white powder, with a total yield of 88.6%. Analysis of the finished product showed that the mass content of reduced coenzyme Q10 was 99.35%, the chromatographic purity of reduced coenzyme Q10 was 99.66%, the weight content of reduced coenzyme Q9 impurity relative to reduced coenzyme Q10 was 0.11%, and there was no reduced coenzyme Q11 impurity.

[0163] Comparative Example 1 Compared to Example 1, the main difference lies in step S2, where the volume ratio of methanol to ethyl acetate is changed to 70:30. The specific steps are as follows: S1. Take 10g of coenzyme Q10 (purity 99.18%, coenzyme Q9 impurity content 0.36%, coenzyme Q11 impurity content 0.28%) and prepare reduced coenzyme Q10 concentrate according to the method in Example 1.

[0164] S2. Add 9.87 g of the concentrated solution to 1000 mL of a methanol:ethyl acetate mixture (v / v) of 70:30. Purge twice with nitrogen, then heat under sealed conditions at 45°C and sonicate until completely dissolved. Load the sample solution onto a C18 preparative column (200 mL column volume, 30 μm particle size). Elute with a methanol:ethyl acetate mixture (v / v), collecting one eluent fraction per 100 mL. Combine the eluent fractions containing ≤0.3% reduced coenzyme Q9 and undetectable reduced coenzyme Q11. Analysis of the combined eluents showed a chromatographic purity of 99.21% for reduced coenzyme Q10, a reduced coenzyme Q9 impurity content of 0.24%, and undetectable reduced coenzyme Q11.

[0165] S3. The combined eluent fractions are concentrated under vacuum using a rotary evaporator to remove the solvent. 100 mL of anhydrous ethanol is added, the concentration flask is purged with nitrogen twice, and the mixture is heated to 60°C under a sealed environment to dissolve the solvent. The mixture is then slowly cooled to 0-5°C for crystallization. The slow cooling rate is 5-8°C / h. The mixture is filtered to obtain a wet crystalline powder.

[0166] S4. The wet powder was vacuum dried at 40℃ to obtain 5.63g of white powder, with a total yield of 56.3%. Liquid chromatography analysis of the finished product showed that the mass content of reduced coenzyme Q10 was 98.93%, the chromatographic purity of reduced coenzyme Q10 was 99.23%, the weight content of reduced coenzyme Q9 impurity relative to reduced coenzyme Q10 was 0.23%, and there was no reduced coenzyme Q11 impurity.

[0167] In this example, a high proportion of ethyl acetate in the elution mobile phase leads to poorer impurity separation and a significant reduction in process yield.

[0168] Comparative Example 2 Compared to Example 1, the difference lies in omitting the column chromatography separation step. The specific steps are as follows: Take 10g of coenzyme Q10 (purity 99.18%, coenzyme Q9 impurity 0.36%, coenzyme Q11 impurity 0.28%) and prepare a reduced coenzyme Q10 concentrate according to the method in Example 1.

[0169] 9.87 g of the concentrated solution was added to 100 mL of anhydrous ethanol, purged with nitrogen, and heated to 60 °C to dissolve. The solution was then slowly cooled to 0-5 °C to crystallize. After filtration, the wet powder was vacuum dried at 40 °C to obtain 9.32 g of white powder, with a total yield of 93.2%. Liquid chromatography analysis of the finished product showed that the content of reduced coenzyme Q10 was 98.51% by weight, the chromatographic purity of reduced coenzyme Q10 was 98.61%, the weight percentage of reduced coenzyme Q9 impurity relative to reduced coenzyme Q10 was 0.34%, and the weight percentage of reduced coenzyme Q11 impurity relative to reduced coenzyme Q10 was 0.27%.

[0170] In this comparative example, a simple crystallization process showed almost no removal effect on the impurities reduced coenzyme Q9 and reduced coenzyme Q11.

[0171] Comparative Example 3 Reduced coenzyme Q10 prepared according to Example 1 described in Chinese patent application CN104892370A.

[0172] Comparative Example 4 Compared to Example 1, the only difference is that the volume ratio of methanol to ethyl acetate in the mobile phase is changed to 95:5. The specific steps are as follows: S1. In a round-bottom flask, add 10g of coenzyme Q10 (purity 99.17%, coenzyme Q9 impurity 0.35%, coenzyme Q11 impurity 0.29%), dissolve in 100mL of ethyl acetate, add 100mL of 20% sodium dithionite aqueous solution, purge with nitrogen for protection, and heat to 40℃ for 2h. After the reaction is complete, separate the layers using a separatory funnel, remove the lower aqueous layer, and concentrate the upper ethyl acetate phase under vacuum to remove the solvent, obtaining a concentrated reduced coenzyme Q10 solution.

[0173] S2. Weigh 9.87 g of the concentrated reduced coenzyme Q10 obtained in step S1 and add it to 800 mL of a methanol:ethyl acetate mixture (95:5 v / v). The weight-to-volume ratio of reduced coenzyme Q10 to the mobile phase is 1:81. Purge twice with nitrogen, then heat in a sealed container at 45°C and sonicate until completely dissolved. Load the sample solution onto a C18 preparative column (30 mm × 250 mm, 10 μm particle size), and elute with a methanol:ethyl acetate mixture (95:5 v / v), collecting one eluent fraction per 100 mL.

[0174] The results showed that the mobile phase was too polar and could not elute the product, resulting in product residue inside the column.

[0175] Comparative Example 5 Compared to Example 1, the only difference is that ethyl acetate is used instead of ethyl acetate as the mobile phase; all other steps are the same. The specific steps are as follows: S1. In a round-bottom flask, add 10g of coenzyme Q10 (purity 99.17%, coenzyme Q9 impurity 0.35%, coenzyme Q11 impurity 0.29%), dissolve in 100mL of ethyl acetate, add 100mL of 20% sodium dithionite aqueous solution, purge with nitrogen for protection, and heat to 40℃ for 2h. After the reaction is complete, separate the layers using a separatory funnel, remove the lower aqueous layer, and concentrate the upper ethyl acetate phase under vacuum to remove the solvent, obtaining a concentrated reduced coenzyme Q10 solution.

[0176] S2. Weigh 9.87 g of the concentrated reduced coenzyme Q10 obtained in step S1 and add 800 mL of ethyl acetate as the mobile phase. The weight to volume ratio of reduced coenzyme Q10 to the mobile phase is 1:81. The solution is purged twice with nitrogen and then completely dissolved by sonication at 45°C under a sealed environment. The sample solution is loaded onto a C18 preparative column (30 mm × 250 mm, 10 μm particle size), and then eluted with ethyl acetate. One eluent fraction is collected every 100 mL. Analysis revealed that each eluent fraction contained reduced coenzyme Q11 impurities, and the content of reduced coenzyme Q9 impurities was >0.3%.

[0177] In this comparative example, only ethyl acetate was used as the mobile phase. The product had a very short retention time in the preparative column, and the preparative column purification process had no effect on removing the impurities reduced coenzyme Q9 and reduced coenzyme Q11.

[0178] Comparative Example 6 Compared with Example 1, the only difference is that the eluent is a mixture of isopropanol and ethyl acetate in a volume ratio of 80:20, otherwise the same.

[0179] S1. In a round-bottom flask, add 10g of coenzyme Q10 (purity 99.17%, coenzyme Q9 impurity content 0.35%, coenzyme Q11 impurity content 0.29%), dissolve in 100mL of ethyl acetate, add 100mL of 20% sodium dithionite aqueous solution, purge with nitrogen for protection, and heat to 40℃ for 2h. After the reaction is complete, separate the layers using a separatory funnel, remove the lower aqueous layer, and concentrate the upper ethyl acetate phase under vacuum to remove the solvent, obtaining a concentrated reduced coenzyme Q10 solution.

[0180] S2. Weigh 9.87 g of the concentrated reduced coenzyme Q10 obtained in step S1 and add 800 mL of a mixture of isopropanol and ethyl acetate at a volume ratio of 80:20 (v / v) as the mobile phase. The weight to volume ratio of reduced coenzyme Q10 to the mobile phase is 1:81. Purge twice with nitrogen, and then sonicate completely at 45°C under sealed conditions. Load the sample solution onto a C18 preparative column (30 mm × 250 mm, 10 μm particle size), and then elute with a mixture of isopropanol and ethyl acetate at a volume ratio of 80:20 (v / v). Collect one eluent fraction per 100 mL. Since each eluent fraction contains reduced coenzyme Q11 impurity, combine the eluent fractions with a reduced coenzyme Q9 impurity content ≤0.3%.

[0181] S3. The combined eluent fractions are concentrated under vacuum using a rotary evaporator to remove the solvent. 100 mL of anhydrous ethanol is added, and the concentration flask is purged twice with nitrogen. The mixture is then heated to 60°C under a sealed environment with stirring until completely dissolved. The mixture is then slowly cooled to 0-5°C for crystallization. The slow cooling rate is 5-8°C / h. The mixture is filtered to obtain a wet crystalline powder.

[0182] S4. The crystalline wet powder obtained in step S3 was vacuum dried at 40℃ to obtain 9.21g of white powder, with a total process yield of 92.1%. The finished product was analyzed, and its reduced coenzyme Q10 content was 98.53%, its chromatographic purity was 98.93%, the weight content of reduced coenzyme Q9 impurity relative to reduced coenzyme Q10 was 0.21%, and the weight content of reduced coenzyme Q11 impurity relative to reduced coenzyme Q10 was 0.12%.

[0183] In this comparative example, using only isopropanol and ethyl acetate as the mobile phase had almost no effect on removing the impurities reduced coenzyme Q9 and reduced coenzyme Q11.

[0184] Comparative Example 7 Compared with Example 1, the only difference is that the C18 preparation column is replaced with a C8 preparation column (the column size is 30mm × 250mm). The specific steps are as follows: S1. Take 10g of coenzyme Q10 (purity 99.17%, coenzyme Q9 impurity content 0.35%, coenzyme Q11 impurity content 0.29%) and prepare reduced coenzyme Q10 concentrate according to the method in Example 1.

[0185] S2. Weigh 9.87 g of the concentrated reduced coenzyme Q10 obtained in step S1 and add 800 mL of a methanol:ethyl acetate mixture (80:20 v / v). The weight to volume ratio of reduced coenzyme Q10 to the mobile phase is 1:81. Purge twice with nitrogen, then heat in a sealed container at 45°C and sonicate until completely dissolved. Load the sample solution onto a C8 preparative column (30 mm × 250 mm, 10 μm particle size), and elute with a methanol:ethyl acetate mixture (80:20 v / v). Collect one eluent fraction per 100 mL. Combine the eluent fractions containing ≤0.3% reduced coenzyme Q9 impurity and undetectable reduced coenzyme Q11 impurity. Analyze the combined eluent; the chromatographic purity of reduced coenzyme Q10 is 99.13%, the content of reduced coenzyme Q9 impurity is 0.19%, and the reduced coenzyme Q11 impurity is undetectable.

[0186] S3. The combined eluent fractions are concentrated under vacuum using a rotary evaporator to remove the solvent. 100 mL of anhydrous ethanol is added, and the concentration flask is purged twice with nitrogen. The mixture is then heated to 60°C under a sealed environment with stirring until completely dissolved. The mixture is then slowly cooled to 0-5°C for crystallization. The slow cooling rate is 5-8°C / h. The mixture is filtered to obtain a wet crystalline powder.

[0187] S4. The crystalline wet powder obtained in step S3 was vacuum dried at 40℃ to obtain 6.38g of white powder, with a total process yield of 63.8%. The finished product was tested and analyzed, and its mass content of reduced coenzyme Q10 was 99.29%, the chromatographic purity of reduced coenzyme Q10 was 99.55%, the weight content of reduced coenzyme Q9 impurity relative to reduced coenzyme Q10 was 0.16%, and it did not contain reduced coenzyme Q11 impurity.

[0188] In this example, using a C8 preparative column for purification results in poorer impurity separation and a significant reduction in process yield.

[0189] Comparative Example 8 Compared to Example 2, the only difference is that the volume ratio of ethanol to ethyl acetate in the eluent is changed to 80:20; all other steps are the same. The specific steps are as follows: S1. Take 10g of coenzyme Q10 (purity 99.31%, coenzyme Q9 impurity content 0.33%, coenzyme Q11 impurity content 0.27%) and prepare reduced coenzyme Q10 concentrate according to the method in Example 1.

[0190] S2. Weigh 10.14 g of the concentrate and add 1000 mL of a mixed solvent of ethanol:ethyl acetate = 80:20 (v / v). The weight ratio of reduced coenzyme Q10 to the volume of the mobile phase is 1:98.6. Purge twice with nitrogen, and then heat in a sealed container at 45°C and sonicate until completely dissolved. Load the sample solution onto a C18 preparative column (30 mm × 250 mm, 30 μm particle size), and then elute with a mixed solvent of ethanol:ethyl acetate = 80:20 (v / v). Collect one eluent fraction per 100 mL. Combine the eluent fractions with a reduced coenzyme Q9 impurity content ≤0.3% and no detected reduced coenzyme Q11 impurity.

[0191] S3. The combined eluent fractions are concentrated under vacuum using a rotary evaporator to remove the solvent. 100 mL of anhydrous ethanol is added, the concentration flask is purged with nitrogen twice, and the mixture is heated to 60°C under a sealed environment to dissolve the solvent. The mixture is then slowly cooled to 0-5°C for crystallization. The slow cooling rate is 5-8°C / h. The mixture is filtered to obtain a wet crystalline powder.

[0192] S4. The crystalline wet powder obtained in step S3 was vacuum dried at 40℃ to obtain 8.04g of white powder, with a total yield of 80.4%. Analysis of the finished product showed that the mass content of reduced coenzyme Q10 was 99.21%, the chromatographic purity of reduced coenzyme Q10 was 99.33%, the weight content of reduced coenzyme Q9 impurity relative to reduced coenzyme Q10 was 0.20%, and there was no reduced coenzyme Q11 impurity.

[0193] Comparative Example 9 Compared to Example 2, the only difference is that the volume ratio of ethanol to ethyl acetate in the mobile phase is changed to 99:1. The specific steps are as follows: S1. Take 10g of coenzyme Q10 (purity 99.31%, coenzyme Q9 impurity content 0.33%, coenzyme Q11 impurity content 0.27%) and prepare reduced coenzyme Q10 concentrate according to the method in Example 1.

[0194] S2. Weigh 10.14 g of the concentrate and add 1000 mL of a 99:1 (v / v) mixture of ethanol and ethyl acetate, resulting in a weight-to-volume ratio of reduced coenzyme Q10 to the mobile phase of 1:98.6. Purge twice with nitrogen, then heat in a sealed container at 45°C and sonicate until completely dissolved. Load the sample solution onto a C18 preparative column (30 mm × 250 mm, 30 μm particle size), and elute with a 99:1 (v / v) mixture of ethanol and ethyl acetate, collecting one eluent fraction per 100 mL.

[0195] Because the mobile phase is too polar, it cannot elute the product, resulting in product residue inside the column.

[0196] The characterization results of each component of the final product (high-purity reduced coenzyme Q10) obtained in each embodiment and comparative example are summarized in Table 1 below.

[0197] Table 1

[0198] In the table above, " / " indicates that experimental results could not be obtained.

[0199] The calculation method for the overall process yield in the table above is as follows: Overall process yield (%) = (weight of reduced coenzyme Q10 product / weight of coenzyme Q10) × 100%; The method for calculating mass content is as follows: Mass content (%) = (r u / r s ) / (c s / c u ) ×100%; Where r u To test the peak area of ​​the sample; r s c represents the peak area of ​​the reference standard. s Reference concentration (mg / mL); c u The concentration of the test sample is (mg / mL).

[0200] The method for calculating the chromatographic purity of reduced coenzyme Q10 is as follows: Chromatographic purity of Q10 (%) = (peak area of ​​reduced coenzyme Q10 / total peak area) × 100%; The method for calculating the impurity content of reduced coenzyme Q9 in reduced coenzyme Q10 is as follows: Impurity content of Q9 (%) = (peak area of ​​reduced coenzyme Q9 / total peak area) × 100%.

[0201] The method for calculating the impurity content of reduced coenzyme Q11 in reduced coenzyme Q10 is as follows: Impurity content of Q11 (%) = (peak area of ​​reduced coenzyme Q11 / total peak area) × 100%.

[0202] Note: For the impurity content of reduced coenzyme Q9 and reduced coenzyme Q11, the correction factor can be considered to be close to 1. Therefore, the weight content of reduced coenzyme Q9 / Q11 impurities can be regarded as the content of reduced coenzyme Q9 / Q11 impurities.

[0203] As shown in Table 1 above, the preparation methods provided in Examples 1-13 of this invention can simultaneously achieve the following technical effects: the total yield of reduced coenzyme Q10 is 88.6%-93.1%, the mass content of reduced coenzyme Q10 is 99.24%-99.48%, the chromatographic purity of reduced coenzyme Q10 is 99.59%-99.79%, the content of reduced coenzyme Q9 is controlled at 0.08%-0.13%, and it does not contain reduced coenzyme Q11.

[0204] The main difference between Comparative Example 1 and Example 1 is that a volume ratio of methanol to ethyl acetate of 70:30 was used. The overall yield in this comparative example was only 56.3%, far lower than the overall yield of Example 1 of this invention; simultaneously, the content of reduced coenzyme Q9 impurities reached 0.23%, which is below the level achievable by the preparation methods of Examples 1-13.

[0205] The main difference between Comparative Example 2 and Example 1 is that the column chromatography separation step was omitted. In this comparative example, although the overall yield reached 93.2%, the impurity content of reduced coenzyme Q9 was as high as 0.34%, and it also contained reduced coenzyme Q11 (0.27%), which did not reach the level achievable by the preparation methods of Examples 1-13.

[0206] Comparative Example 3 is the reduced coenzyme Q10 prepared in Example 1 as described in prior art CN104892370A. Testing revealed that this reduced coenzyme Q10 contained a high content of reduced coenzyme Q9 (0.33%) and also contained reduced coenzyme Q11 (0.26%), failing to reach the levels achievable by the preparation methods of Examples 1-13. This demonstrates that not all arbitrarily chosen prior art can achieve the technical effects achievable by this invention, particularly the technical effect of "controlling the content of reduced coenzyme Q9 to 0.08%-0.13% and eliminating reduced coenzyme Q11."

[0207] Comparative Example 4 differed from Example 1 only in that the volume ratio of methanol to ethyl acetate in the mobile phase was changed to 95:5. The results showed that the mobile phase was too polar, failing to elute the product and resulting in a large amount of product residue remaining in the column, making the process unsuccessful. This demonstrates that an arbitrary choice of methanol to ethyl acetate volume ratio is not sufficient to achieve the same results as the preparation methods in Examples 1-13, which, while controlling the content of reduced coenzyme Q9 and reduced coenzyme Q11, also maintain a certain overall yield of reduced coenzyme Q10 (88.6%-93.1%).

[0208] Comparative Example 5 differs from Example 1 only in that the mobile phase is changed to ethyl acetate. Comparative Example 6 differs from Example 1 only in that the eluent is changed to a mixture of isopropanol and ethyl acetate in a volume ratio of 80:20. Comparative Example 5 uses only ethyl acetate as the mobile phase, resulting in a very short product retention time in the preparative column. The preparative column purification process has no effect on removing impurities such as reduced coenzyme Q9 and reduced coenzyme Q11. In Comparative Example 6, the content of reduced coenzyme Q9 is as high as 0.21%, and reduced coenzyme Q11 is also detected, with a content as high as 0.12%. Neither Comparative Example 5 nor Comparative Example 6 achieved the effect of "controlled reduced coenzyme Q9 content of 0.08%-0.13% and no reduced coenzyme Q11" achieved by Examples 1-13 of this invention. This shows that the technical effects achieved by the embodiments of this invention cannot be achieved by arbitrarily choosing the type of mobile phase.

[0209] Comparative Example 7 differs from Example 1 only in that the C18 preparative column was replaced with a C8 preparative column of the same size (30 mm × 250 mm). The content of reduced coenzyme Q9 in the reduced coenzyme Q10 of Comparative Example 7 was 0.16%, which does not achieve the effect of "controlled reduced coenzyme Q9 content between 0.08% and 0.13%" achieved in Examples 1-13 of this invention. This demonstrates that not all arbitrarily chosen chromatographic columns can achieve the effects achievable in Examples 1-13 of this invention.

[0210] Compared to Example 2, Comparative Example 8 differs only in that the volume ratio of ethanol to ethyl acetate in the eluent is changed to 80:20. Comparative Example 9 differs only in that the volume ratio of ethanol to ethyl acetate in the mobile phase is changed to 99:1; all other aspects are the same. The reduced coenzyme Q10 in Comparative Example 8 contains 0.20% reduced coenzyme Q9, which does not achieve the effect of "controlled reduced coenzyme Q9 content between 0.08% and 0.13%" achieved in Examples 1-13 of this invention. Furthermore, in Comparative Example 9, due to the excessive polarity of the mobile phase, the product could not be eluted, resulting in a large amount of product residue remaining in the column. This demonstrates that not all arbitrarily chosen ethanol-ethyl acetate mixtures can achieve the effects achieved in Examples 1-13 of this invention, regardless of their volume ratio.

[0211] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution created by the present invention do not depart from the essence and scope of the technical solution created by the present invention.

Claims

1. A method for preparing high-purity reduced coenzyme Q10, characterized in that, Includes the following steps: S1. Coenzyme Q10 is reacted with sodium dithionite, the solvent is removed, and a concentrated solution of reduced coenzyme Q10 is obtained. S2. Mix the concentrated reduced coenzyme Q10 obtained in step S1 with the mobile phase, purify using a C18 column, and elute with the mobile phase to obtain the eluent fraction. S3. Remove the solvent from the eluted component obtained in step S2 to obtain a solid, add a co-crystallization solvent, and crystallize to obtain a crystalline wet powder. S4. Dry the crystalline wet powder obtained in step S3 to obtain high-purity reduced coenzyme Q10. In step S2, the mobile phase is a mixture of methanol and ethyl acetate or a mixture of ethanol and ethyl acetate, and the C18 column is a C18 preparative column or a C18 dynamic axial compression column. When the mobile phase is a mixture of methanol and ethyl acetate, the volume ratio of methanol to ethyl acetate is 75-85:15-25. When the mobile phase is a mixture of ethanol and ethyl acetate, the volume ratio of ethanol to ethyl acetate is 85-91:9-15.

2. The preparation method according to claim 1, characterized in that, In step S1, the reaction of coenzyme Q10 with sodium dithionite includes the following steps: Coenzyme Q10, ethyl acetate, and an aqueous solution of sodium dithionite with a weight concentration of 10%-30% were mixed, reacted under inert gas protection, and the organic phase was separated and concentrated to obtain a concentrated solution of reduced coenzyme Q10.

3. The preparation method according to claim 2, characterized in that, The aqueous solution of sodium dithionite has a weight concentration of 20%.

4. The preparation method according to claim 2, characterized in that, The ratio of the weight of the coenzyme Q10, the volume of the ethyl acetate, and the volume of the aqueous solution of sodium dithionite is 5-15:50-150:50-150, in g:mL:mL.

5. The preparation method according to claim 4, characterized in that, The ratio of the weight of the coenzyme Q10, the volume of the ethyl acetate, and the volume of the aqueous solution of sodium dithionite is 10:100:100, in g:mL:mL.

6. The preparation method according to claim 2, characterized in that, The purity of the coenzyme Q10 is ≥98.5%, the content of impurity coenzyme Q9 in the coenzyme Q10 is ≤0.6%, and the content of impurity coenzyme Q11 in the coenzyme Q10 is ≤0.5%.

7. The preparation method according to claim 6, characterized in that, The purity of the coenzyme Q10 is ≥99.17%, the content of impurity coenzyme Q9 in the coenzyme Q10 is ≤0.35%, and the content of impurity coenzyme Q11 in the coenzyme Q10 is ≤0.32%.

8. The preparation method according to claim 7, characterized in that, The purity of the coenzyme Q10 is ≥99.28%, the content of impurity coenzyme Q9 in the coenzyme Q10 is ≤0.33%, and the content of impurity coenzyme Q11 in the coenzyme Q10 is ≤0.32%.

9. The preparation method according to claim 2, characterized in that, The inert gas is one or more of nitrogen or argon.

10. The preparation method according to claim 9, characterized in that, The inert gas is nitrogen.

11. The preparation method according to claim 2, characterized in that, The reaction temperature is 35-45℃, and the reaction time is 1-3h.

12. The preparation method according to claim 11, characterized in that, The reaction temperature is 40°C, and the reaction time is 2 hours.

13. The preparation method according to claim 2, characterized in that, The concentration is achieved through vacuum concentration to recover the solvent.

14. The preparation method according to claim 13, characterized in that, The concentration is to concentrate until no solvent residue remains.

15. The preparation method according to claim 1, characterized in that, The volume ratio of methanol to ethyl acetate is 79-81:19-21.

16. The preparation method according to claim 15, characterized in that, The volume ratio of methanol to ethyl acetate is 80:

20.

17. The preparation method according to claim 1, characterized in that, The volume ratio of ethanol to ethyl acetate is 87-89:11-13.

18. The preparation method according to claim 17, characterized in that, The volume ratio of ethanol to ethyl acetate is 88:

12.

19. The preparation method according to claim 1, characterized in that, In step S2, the weight ratio of the concentrated reduced coenzyme Q10 solution to the volume ratio of the mobile phase is 1:80-100, in g:mL.

20. The preparation method according to claim 1, characterized in that, In step S2, before purification using C18 column chromatography, Includes the following steps: The mixture is purged with an inert gas and heated in a sealed environment to 40-50°C to dissolve.

21. The preparation method according to claim 1, characterized in that, In step S2, the particle size of the packing material in the C18 column is 5-100 μm.

22. The preparation method according to claim 21, characterized in that, The packing particle size of the C18 column is 10-30 μm.

23. The preparation method according to claim 1, characterized in that, In step S2, the eluted components are analyzed by liquid chromatography.

24. The preparation method according to claim 23, characterized in that, In step S2, the chromatographic purity of reduced coenzyme Q10 in the elution fraction is ≥99.27%, the chromatographic purity of reduced coenzyme Q9 is ≤0.12%, and reduced coenzyme Q11 is not detected under liquid chromatography conditions.

25. The preparation method according to claim 23, characterized in that, The specific liquid chromatography conditions are as follows: mobile phase: a mixture of acetonitrile and ethanol in a volume ratio of 1:1; diluent: a mixture of methanol and n-hexane in a volume ratio of 85:15; detector: UV 290nm; column: 4.6mm × 10cm, 3μm, C18 packing; column temperature: 35℃; flow rate: 1.5mL / min; injection volume: 20μL.

26. The preparation method according to claim 1, characterized in that, In step S3, the co-crystallization solvent is ethanol; the volume ratio of the co-crystallization solvent to the mass ratio of the solid is 10-12:1, in mL:g.

27. The preparation method according to claim 1, characterized in that, In step S3, the crystallization process includes the following steps: Replace with an inert gas, heat in a sealed environment to 55-65℃ until completely dissolved, cool to 0-5℃, filter, and obtain crystalline wet powder.

28. The preparation method according to claim 27, characterized in that, The cooling rate is 5-8℃ / h.

29. The preparation method according to claim 27, characterized in that, The complete dissolution refers to complete dissolution under ultrasound assistance.

30. The preparation method according to claim 1, characterized in that, In step S4, the drying is performed under reduced pressure at 20-40°C.

31. High-purity reduced coenzyme Q10 prepared by the preparation method according to any one of claims 1-30.

32. The high-purity reduced coenzyme Q10 according to claim 31, characterized in that, The purity of reduced coenzyme Q10 is ≥99.5%, the weight content of reduced coenzyme Q9 relative to reduced coenzyme Q10 is ≤0.3%, and it does not contain reduced coenzyme Q11.

33. The application of the preparation method according to any one of claims 1-30 in the preparation of high-purity reduced coenzyme Q10.

34. The application of the preparation method according to any one of claims 1-30 in the preparation of products containing high-purity reduced coenzyme Q10.

35. A drug containing high-purity reduced coenzyme Q10, characterized in that, Contains the high-purity reduced coenzyme Q10 as described in any one of claims 31-32.

36. The pharmaceutical product according to claim 35, characterized in that, The dosage forms of the medicine include tablets, pills, powders, granules, lozenges, effervescent tablets, capsules, and gels.

Images