CN111094309A

CN111094309A - Preparation method of high-purity riboflavin sodium phosphate

Info

Publication number: CN111094309A
Application number: CN201880037970.5A
Authority: CN
Inventors: 郭涛; 张琦; 张冬民; 程双
Original assignee: Bontac Invitrolife Bio Technology Shenzhen Co ltd; Jiangxi Bonzymes Biotechnology Co ltd; Bontac Bio-Engineering (shenzhen) Co ltd
Current assignee: BONTAC BIO-ENGINEERING (SHENZHEN) Co.,Ltd.; BONTAC INVITROLIFE BIO-TECHNOLOGY (SHENZHEN) Co.,Ltd.; Zhongshan Bangtai Hesheng Biotechnology Co.,Ltd.
Priority date: 2018-09-21
Filing date: 2018-09-21
Publication date: 2020-05-01
Also published as: WO2020056714A1

Abstract

A preparation method of high-purity riboflavin sodium phosphate relates to the technical field of medicine monomer preparation, in particular to a method for preparing high-purity riboflavin sodium phosphate by adopting a separation and purification technology, and aims to solve the technical problems of complex operation, long time consumption, high cost and great pollution existing in the existing method. The method comprises the following steps: adjusting the pH value to 5.0-7.0; controlling the temperature of the solution to be 20-40 ℃, and adding an ethanol water solution with the concentration of 95% (ml/ml); cooling to 3-5 ℃, and waiting for solid precipitation; filtering, and drying the filter cake to obtain the pure riboflavin sodium phosphate. The method is suitable for large-scale industrialized separation and purification of riboflavin sodium phosphate products with the purity of more than 99 percent from biological enzyme reaction liquid and biological fermentation liquid.

Description

Preparation method of high-purity riboflavin sodium phosphate

[0001] The invention relates to the technical field of preparation of medical monomers, in particular to a method for preparing high-purity riboflavin sodium phosphate by adopting a separation and purification technology.

Background

[0002] Riboflavin Sodium Phosphate (RSP) is a Phosphate form of Riboflavin (vitamin B2), is also known as vitamin B2 Sodium Phosphate, flavin Sodium mononucleotide, Riboflavin-5' - (dihydrogenphosphate) monosodium salt and the like, has a CAS number of 130-40-5, and has a structural formula shown in the following figure.

[0004] Riboflavin sodium phosphate is currently widely used as a riboflavin supplement for treating diseases such as angular stomatitis, cheilitis, glossitis, conjunctivitis, scrotitis and the like caused by riboflavin deficiency with arch I, and clinically common dosage forms are injections and oral tablets.

[0005] At present, the method for preparing riboflavin sodium phosphate in China is mainly a chemical synthesis method, which generally takes high-purity riboflavin as an initial raw material and phosphorus oxychloride as a phosphorylation reagent to carry out chemical reaction in the environment of organic solvents such as tetrahydrofuran, acetonitrile, pyridine and the like to generate flavin mononucleotide, and then the flavin mononucleotide is further converted into riboflavin sodium phosphate. However, the chemical synthesis method has serious environmental pollution, and with the gradual emphasis of the country on environmental protection, a substituted biocatalysis method is gradually started, wherein a biological enzyme is used for catalyzing a substrate to be converted into flavin mononucleotide, and the flavin mononucleotide is further converted into riboflavin sodium phosphate.

[0006] Whether the method is a chemical synthesis method or a biological catalysis method, the purity of the riboflavin sodium phosphate product prepared by the method is often not high enough, and further separation and purification treatment is needed. For example, chinese patent documents CN106674225A and CN103396452B both disclose a method for preparing high-purity riboflavin sodium phosphate by crystallization process, but the preparation process thereof needs to use a large amount of toxic and harmful organic solvents, for example, CN106674225A needs to use methyl acetate, acetone, cyclohexanol and methanol, and CN103396452B needs to use acetonitrile, methanol and chloroform, which is not in accordance with the environmental protection concept of sustainable development.

Summary of The Invention

Technical problem

Solution to the problem

Technical solution

[0007] The invention aims to provide a novel method for preparing high-purity riboflavin sodium phosphate, which is energy-saving, environment-friendly and low in cost.

[0008] In order to achieve the purpose, the invention provides a preparation method of high-purity riboflavin sodium phosphate, which comprises the following steps: 1) Adjusting the pH value of the crude riboflavin sodium phosphate and/or flavin mononucleotide solution to 5.0-7.0; 2) Controlling the temperature of the solution to be 20-40 ℃, and adding 95% (ml/ml) ethanol water solution which accounts for 0.5-1.5 times of the volume of the crude product solution; 3) Cooling to 3-5 ℃, and waiting for solid precipitation; 4) Filtering, removing the filtrate, and drying the filter cake to obtain the pure riboflavin sodium phosphate.

[0009] Flavin Mononucleotide (Flavin mononucletide, abbreviated as FMN in English), also called riboflavin-5-phosphate, is a prosthetic group of flavoprotein, plays an important role in electron transfer in biological oxidation processes such as respiration, namely, as a prosthetic group of flavoenzyme group, participates in electron transfer from substrate to electron acceptor in a binding state with enzyme protein (apo-enzyme) and plays an important role in basal metabolism, and the CAS number is 146-17-8, and the structural formula is shown in the following figure.

[] [0010] in the method of the present invention, the crude solution of riboflavin sodium phosphate and/or flavin mononucleotide of step 1) means a solution containing a large amount of impurities in addition to riboflavin sodium phosphate and/or flavin mononucleotide, and includes: the method comprises the steps of dissolving riboflavin sodium phosphate and/or flavin mononucleotide which are purchased from the market and have low purity in water to obtain a solution, and preparing a crude product solution obtained by the flavin mononucleotide through a biological catalysis method.

[0011] Preferably, the concentration of the crude riboflavin sodium phosphate and/or flavin mononucleotide solution is controlled to be more than 7.5g/L in terms of riboflavin sodium phosphate so as to ensure that enough solid can be separated out in the later period, thereby ensuring high enough yield.

[0012] Biocatalysis refers to a process of substance conversion (respectively referred to as enzyme catalysis and biofermentation) using a biological enzyme or a biological organism (cell, organelle, tissue, etc.) as a catalyst, and this reaction process is also referred to as biotransformation (biotransformation). The biological organisms commonly used in biocatalysis are mainly microorganisms, and the essence of the biological organisms is to utilize enzymes in microbial cells for catalysis to promote the process of biotransformation. The biological catalysis method has mild action condition (basically completed in the environment of normal temperature, neutrality, water and the like)

Unique and high-efficiency substrate selectivity (because the enzyme in the catalytic process has the characteristic of specificity, namely, one enzyme can only catalyze one specific substrate to react, but one substrate can be catalyzed by a plurality of enzymes); has the advantage of being unique to the synthesis of chiral active pharmaceutical ingredients.

[0013] The crude product solution obtained by preparing the flavin mononucleotide by the biocatalysis method is an enzyme reaction solution obtained by converting a substrate into the flavin mononucleotide by using a biological enzyme, or a biological fermentation liquid containing the flavin mononucleotide and obtained by removing a biological organism after the biological organism containing the biological enzyme is subjected to fermentation culture and induction expression. The term "biological enzyme" refers to an enzyme that specifically catalyzes the conversion of a substrate into flavin mononucleotide, and the term "substrate" refers to a precursor substance that can be converted into flavin mononucleotide. For example, the substrate may be riboflavin and ATP, or a precursor substance capable of converting riboflavin and/or ATP, and the biological enzyme may be riboflavin kinase (EC 2.7.1.26), or a combination of riboflavin kinase (EC 2.7.1.26) and one or more other enzymes. As another example, the substrate may be riboflavin and D-glucose-1-phosphate, or a precursor substance capable of converting riboflavin and/or D-glucose-1-phosphate, and the biological enzyme corresponding thereto may be riboflavin phosphotransferase (EC2.7.1.42), or a combination of riboflavin phosphotransferase (EC2.7.1.42) and one or more other enzymes.

[0014] The reason why the pH value is adjusted to 5.0 to 7.0 in step 1) of the method of the present invention is that riboflavin monosodium phosphate can be formed in an oriented manner in this pH range. When the regulating object is a crude product solution of flavin mononucleotide, adding sodium hydroxide for regulating; and when the adjusting object is a crude riboflavin sodium phosphate solution, adding sodium hydroxide or hydrochloric acid according to different pH values of the solution for adjusting. In order to improve the yield of the target product and obtain the riboflavin sodium phosphate product with better appearance, the pH value of the solution is preferably adjusted to be 5.5-6.5, and the pH value of the solution is more preferably adjusted to be 6.0-6.3.

[0015] In step 2) of the method, 95% ethanol is added as a poor solvent, and simultaneously the polarity of the aqueous solution is reduced, so that the solubility of riboflavin sodium phosphate in the solution is reduced, and the riboflavin sodium phosphate is precipitated and precipitated. Preferably, ethanol water solution with the concentration of 95% (ml/ml) accounting for 0.5-0.9 time of the volume of the solution is added, so that the cost can be saved, and the uniformity of products in different batches can be improved.

[0016] In the step 3) of the method, when the temperature is reduced to 3-5 ℃, a riboflavin sodium phosphate product with high yield and content can be obtained, wherein the yield is low when the temperature is higher than the range, and the content is reduced when the temperature is lower than the range.

[0017] The inventor researches and discovers that the precipitation of the solid is greatly influenced by the temperature, if the temperature reduction speed is too high, the solid is explosively precipitated in a short time, so that the particle size is different, the shape of the product is influenced, even the solid is agglomerated and precipitated, a large amount of impurities are embedded, and the content of the product is not high. In order to obtain a product with higher content and better form, preferably, the temperature reduction process of the step 3) is completed within l-2 hours.

[0018] More preferably, the cooling process of step 3) is operated as follows: the temperature is firstly reduced to 12-15 ℃, and then is reduced to 3-5 ℃ after the solid precipitation rate in the solution reaches more than 40%.

[0019] In order to further increase the amount of the precipitated solid in the step 3), preferably, after the precipitation rate of the solid in the solution reaches more than 40%, adding 95% (ml/ml) ethanol aqueous solution which accounts for 0.3-0.5 times of the volume of the crude product solution.

[0020] The filtration in step 4) of the method is a process of separating solid and liquid in the solution by adopting a physical method, and common filtration methods are all suitable for the method, and include normal pressure filtration, reduced pressure filtration, centrifugal filtration and the like.

[0021] In step 4) of the process of the invention, the filter cake obtained is preferably washed with an aqueous solution of ethanol having a concentration of 75% (ml/ml) before the drying treatment.

[0022] More preferably, after washing with 75% (ml/ml) ethanol aqueous solution, washing with 95% (ml/ml) ethanol aqueous solution is performed to ensure that impurities attached to the surface of the product are washed clean to the maximum extent

[0023] In order to further reduce the impurity residue and increase the product content, preferably, the obtained filter cake is pulped by 50% ethanol water solution before being washed, and then the filter cake is collected by filtration. The beating is a process of adding a solvent having a low solubility for the target substance and a high solubility for other related substances to the solid substance, and stirring to dissolve the other unnecessary related substances in the solvent, thereby suspending the target substance and the solvent.

[0024] Preferably, in step 4) of the method of the present invention, the drying process is performed by vacuum drying at 30 ℃ for 5 to 10 hours.

[0025] In order to further improve the purity of the pure riboflavin sodium phosphate, the filter cake obtained in the step 4) or the pure riboflavin sodium phosphate is preferably dissolved in purified water, and then the steps 1) to 4) of the method are repeated

[0026] When the crude solution of riboflavin sodium phosphate and/or flavin mononucleotide in step 1) of the method of the present invention is a crude product solution obtained by preparing flavin mononucleotide through a biological catalysis method, the solution will contain a part of residual biological enzyme, and in order to prevent the target product from being degraded by the residual enzyme in the separation and purification process of riboflavin sodium phosphate, the crude solution of riboflavin sodium phosphate and/or flavin mononucleotide is preferably subjected to the following pre-treatment before step 1): a) Adjusting the pH value of the crude riboflavin sodium phosphate and/or flavin mononucleotide solution to 11.0-12.0; b) Heating the solution to 40 +/-1 ℃, and keeping the temperature at 40 +/-1 ℃ for 2 hours, and C) removing insoluble matters.

[0027] In the previous treatment, the pH value of the solution is adjusted to 11.0-12.0, and then the solution is heated at 40 +/-1 ℃ to inactivate the residual biological enzyme in the solution. Preferably, the flavin mononucleotide can be converted into riboflavin sodium phosphate by adjusting the pH value with sodium hydroxide; particularly, the pH value is preferably in the range of 11.0 to 11.5, and the stability of the target product is the best within the range. [0028] In step c) of the preceding treatment, the insoluble matter is preferably removed by: centrifuging the solution heated in the step b), and collecting supernatant.

[0029] When the crude riboflavin sodium phosphate and/or flavin mononucleotide solution obtained in step 1) of the method is a crude product solution obtained by preparing flavin mononucleotide by a biological catalysis method, the subsequent filtering operation in step 4) is difficult, a filtering membrane is easy to block, and in order to make the filtering operation easier, the previous treatment preferably further comprises a step of pretreating the solution obtained in step c) after removing insoluble substances so as to remove impurities remained in the crude product solution; the pretreatment is to purify the solution from which the insoluble matter is removed by using an ion exchange resin, or to sequentially subject the solution from which the insoluble matter is removed to microfiltration and ultrafiltration.

[0030] Microfiltration, also known as microfiltration, uses a microfiltration membrane as the filtering medium, to trap between 0.1-1 microns of particles and bacteria, propelled by the pressure of 0.1-0.3 MPa, but to allow passage of macromolecular organic matter and inorganic salts. The microfiltration membrane used in the process of the invention is preferably a hollow fibre membrane with a pore size of 0.1 pm.

[0031] Ultrafiltration is also called ultrafiltration, and its principle is the same as microfiltration, but the difference is that the pore size of ultrafiltration membrane used in ultrafiltration is smaller than that of microfiltration membrane, generally below 0.01 micron.

[0032] Preferably, the ion exchange resin is an anion exchange resin.

[0033] More preferably, the anion exchange resin is a weakly basic anion exchange resin or a macroporous adsorption resin.

[0034] Preferably, 0.1-0.4 mol/L sodium chloride aqueous solution is used as eluent of the ion exchange resin; in order to increase the product yield, the concentration of the sodium chloride aqueous solution is more preferably 0.3 to 0.35 mol/L.

[0035] Preferably, the method for regenerating the ion exchange resin comprises the steps of: a. Leaching the resin by using a mixed solution of 2mol/L sodium chloride solution and 0.5mol/L hydrochloric acid solution until no impurity remains in an effluent liquid; b. Eluting the eluent by using purified water until the pH value of the eluent is 5.00-7.00, and eluting the eluent by using 0.5mol/L sodium hydroxide solution until the eluent is strong alkaline; d. And eluting the eluent by using purified water until the pH value of the eluent is 9.00-10.00.

[0036] In order to improve the efficiency of the subsequent operation, reduce the amount of solvent used and improve the yield of the target product, it is preferable that the pretreated solution is concentrated to remove a large amount of water before the step 1)

[0037] The concentration treatment in the method of the present invention is a process of increasing the concentration of a solution by reducing the amount of a solvent by a physical method, and includes a reduced pressure distillation method, an ultrafiltration method, a dialysis method, an adsorption method, a freeze-drying method, and the like.

[0038] Preferably, the concentration treatment mode of the method is nanofiltration concentration. Nanofiltration is a pressure-driven membrane separation process between reverse osmosis and ultrafiltration, and takes a nanofiltration membrane as a filter medium, wherein the pore diameter range of the nanofiltration membrane is about a few nanometers, and solvent molecules or certain solutes with smaller relative molecular mass or low-valent ions are allowed to permeate through the nanofiltration membrane, so that the separation and concentration effects are achieved. Preferably, the nanofiltration membrane selected by the method has the molecular weight cut-off of 300 +/-50 daltons.

[0039] In order to obtain higher product yield, the pretreated solution is preferably concentrated until the concentration of riboflavin sodium phosphate in the solution is more than 7.5 g/L.

Advantageous effects of the invention

Advantageous effects

[0040] Compared with the existing purification method, the preparation method of the high-purity riboflavin sodium phosphate provided by the invention has the following advantages:

[0041]1, no toxic or harmful solvent is needed in the whole process, the environment is protected, no pollution is caused, and health damage to production operators is avoided;

[0042]2, the process is simple and easy to operate, and no special skill requirement and safety protection requirement exist;

[0043]3, the process steps are fewer, the efficiency and the yield are high, the cost is low, and the economic benefit is remarkable;

[0044]4, preparing the riboflavin sodium phosphate product with the purity of more than 99 percent.

Examples of the invention

Modes for carrying out the invention

[0045] The present invention will be described in further detail with reference to specific examples, which are illustrative of the present invention and are not to be construed as being limited thereto.

[0046] The raw materials and reagents used in the following examples were all purchased from the market, except for those specifically mentioned.

[0047] In the following examples, the purification target in example 1 was a crude flavin mononucleotide product purchased from the market, and the purification targets in examples 2 to 5 were crude flavin mononucleotide solutions prepared by the bioenzyme catalysis method (Shenzhen) Limited, using riboflavin and ATP as substrates and riboflavin kinase as catalysts, from Bangtai bioengineering (Shenzhen).

[0048] Example 1

[0049] The purity of the crude flavin mononucleotide product to be purified is 30.20% and the content is 40.14% through detection.

[0050] The purification process comprises the following steps: dissolving 30.01g of crude flavin mononucleotide to be purified in 1.0L of purified water, adjusting the pH value of the solution to 6.0 by using sodium hydroxide after the crude flavin mononucleotide is fully dissolved, heating to 30 ℃, adding 0.5L of ethanol water solution with the concentration of 95% (ml/ml), and uniformly mixing; slowly cooling to 13 ℃ for waiting for solid precipitation, carrying out on-line detection by HPLC, slowly cooling to 4 ℃ after the precipitation rate of the solid in the solution reaches more than 40%, adding 0.4L ethanol aqueous solution with the concentration of 95% (ml/ml), uniformly mixing, and continuously waiting for solid precipitation; performing on-line detection by HPLC (high performance liquid chromatography), after the precipitation rate of solids in the solution reaches over 90%, filtering, collecting a filter cake, washing the filter cake with an ethanol water solution with the concentration of 75% (ml/ml), and finally, placing the washed filter cake at the temperature of 30 ℃ for vacuum drying for 5 hours to obtain orange-yellow riboflavin sodium phosphate solid powder 10.72 g, wherein the weight loss on drying is 4.48%, the sodium ion content is 4.50%, the specific rotation is + 38.37%, the purity is 99.33%, the content (by taking riboflavin as an external standard and by taking the dry product) is 77.28% (the pharmacopoeia requires the range of 74.0% -79.0%), and the total yield is 80.97% by taking the flavin mononucleotide.

[0051] Example 2

[0052] The solution of the crude product to be purified was found to have a pH of 6.60, a volume of 27L and a content of the xanthosine mononucleotide of 186.02 g.

[0053] The purification process comprises the following steps: adjusting the pH value of the crude product solution to 11.50 by using sodium hydroxide, heating to 40 ℃, centrifuging after heating for 2h, pumping the supernatant into an anion resin column with the model of LXQ510 by a peristaltic pump, eluting impurities by 0.3mol/L sodium chloride aqueous solution 120L and eluting products by 0.35mol/L sodium chloride aqueous solution 120L to obtain product eluent 122L with the product content of 175.04g and the yield of 94.10%, nano-filtering and concentrating the product eluent, allowing small molecules with the molecular weight of less than 300 to pass through the nano-filtering membrane, wherein the volume capacity of the nano-filtering membrane is 6L, the volume of the eluent is finally concentrated to 19.5L, the product content in the concentrated solution is 172.74g based on flavin mononucleotide and the mass concentration is 8.85g/L, adjusting the pH value of the concentrated solution to 6.25 by using 6mol/L hydrochloric acid solution, transferring the mixture into a 50L glass reaction kettle, slowly adding 25.6L (1.3 BV) of 95% (ml/ml) ethanol water solution, controlling the temperature at 25-30 ℃, slowly cooling to 13 ℃ after the addition is finished, carrying out online detection through HPLC (high performance liquid chromatography), continuously cooling to 4 ℃ after the solid precipitation rate in the solution reaches more than 40%, maintaining the stirring at 4 ℃ for 4 hours, filtering, washing a filter cake with a small amount of 75% (ml/ml) ethanol water solution, carrying out vacuum drying at 30 ℃ for 10 hours to obtain 166.59g of orange yellow sodium phosphate solid powder, wherein the dry weight loss is 4.34%, the sodium ion content is 4.72%, the specific rotation is + 38.68%, the purity is 99.28%, and the content (calculated by taking riboflavin as an external standard and by dry product) is 77.86% (the pharmacopoeia requires range is 74.0% -79.0%), the total yield was 81.44% based on flavin mononucleotide.

[0054] Example 3

[0055] The solution of the crude product to be purified was found to have a pH of 6.87, a volume of 38L and a content of the xanthosine mononucleotide of 255.29 g. [0056] The purification process comprises the following steps: adjusting the pH value of the crude product solution to 12.00 by using sodium hydroxide, heating to 40 ℃, centrifuging after heating for 2h, and taking supernatant for later use; the anion resin (LXQ510) 90L is regenerated by the method of

Using 2mol/L

Leaching the resin with 90L of sodium chloride +0.5mol/L hydrochloric acid aqueous solution, leaching with purified water until the pH is 5.00-7.00, leaching with 0.5mol/L sodium hydroxide solution 90L until the effluent is strongly alkaline, and finally leaching with purified water until the pH is 9.00-10.00; pumping the above-mentioned supernatant fluid into regenerated anion resin column by means of peristaltic pump, using 180L of 0.3mol/L sodium chloride aqueous solution to elute impurity, using 180L of 0.3mol/L sodium chloride aqueous solution to elute product to obtain 250L of product eluent with its product content being 243.31g and yield being 95.31%, nano-filtering and concentrating the product eluent, using nano-filtering membrane to allow small molecule whose molecular weight is less than 300 to pass through, its volume capacity is 6L, the eluent volume is finally concentrated to 16L, the product content in the concentrated solution is 242.42g as flavin mononucleotide, its mass concentration is 15.15g/L, using 6m ol/L hydrochloric acid solution to regulate pH value of the concentrated solution to 5.86, transferring it into 50L glass reaction still, slowly adding 11.6L (0.73BV) 95% (ml/ml) of ethanol aqueous solution, controlling the temperature to be 25-30 ℃, slowly cooling to 13 ℃ after the addition is finished, carrying out online detection by HPLC, continuously cooling to 4 ℃ after the solid precipitation rate in the solution reaches more than 40%, adding 6.4L (0.4BV) of 95% (ml/ml) ethanol water solution, uniformly mixing, stirring for 4 hours at 4 ℃, filtering, washing a filter cake by using a small amount of 75% (ml/ml) ethanol water solution, and carrying out 30. 233.86g of orange yellow riboflavin sodium phosphate solid powder is obtained after vacuum drying for 10h, the drying weight loss is 4.40%, the sodium ion content is 4.32%, the specific optical rotation is +38.20 degrees, the purity is 99.09%, the content (taking riboflavin as an external standard and calculated by dry products) is 76.04% (the pharmacopoeia requires the range of 74.0-79.0%), and the total yield is 83.62% calculated by flavin mononucleotide.

[0057] Example 4

[0058] The solution of the crude product to be purified was found to have a pH of 6.77, a volume of 35L and a content of xanthosine mononucleotide of 265.81 g.

[0059] The purification process comprises the following steps: adjusting the pH value of the crude product solution to 11.00 by using sodium hydroxide, heating to 40 ℃, centrifuging after heating for 2h, and taking supernatant for later use; microfiltering the supernatant with a microfiltration membrane with the aperture of O.lum, ultrafiltering the microfiltered liquid with an ultrafiltration membrane, directly pumping the ultrafiltrate into a nanofiltration machine for nanofiltration concentration, allowing small molecules with the molecular weight of less than 300 to pass through the nanofiltration membrane, adjusting the volume capacity of the nanofiltration membrane to be 12L, finally concentrating the volume of the eluent to 18L, washing the wall of a barrel with 1L of purified water, slowly adding 11.4L (0.6BV) of 95% (ml/ml) ethanol aqueous solution, controlling the temperature to be 25-35 ℃, slowly cooling to 13 ℃ after the addition is finished, wherein the content of a product in the concentrated solution is 255.74g based on flavin mononucleotide, the mass concentration is 14.21g/L, the yield is 96.21 percent, adjusting the pH value of the concentrated solution to be 6.10 with 6mol/L hydrochloric acid, transferring into 50L of glass reaction , performing on-line detection by HPLC, after the solid precipitation rate in the solution reaches more than 40%, continuously cooling to 6 ℃, keeping stirring at 6 ℃ for 4h, filtering, washing a filter cake with 75% (ml/ml) of ethanol aqueous solution, then washing with 95% (ml/ml) of ethanol aqueous solution, and drying for 30 ° (: vacuum drying for 10h to obtain 266.95g of orange yellow riboflavin sodium phosphate solid powder, wherein the drying weight loss is 4.60%, the sodium ion content is 4.54%, the specific rotation is +38.89 °, the purity is 99.43%, the content (by taking riboflavin as an external standard and by taking dry products) is 77.64% (the pharmacopoeia requires range is 74.0% -79.0%), and the total yield is 91.25% by taking flavin mononucleotide.

[0060] Example 5

[0061] The pH of the crude product solution to be purified was found to be 6.65, the volume was found to be 41L, and the content of xanthosine mononucleotide in the solution was found to be 341.94 g.

[0062] The purification process comprises the following steps: adjusting the pH value of the crude product solution to 11.75 by using sodium hydroxide, heating to 40 ℃, centrifuging after heating for 2h, and taking supernatant for later use; filtering the supernatant with a microfiltration membrane with a membrane aperture of O.lum, directly transferring the filtrate after ultrafiltration into a nanofiltration machine for nanofiltration concentration, wherein the ultrafiltration membrane mainly intercepts polypeptide and heteroprotein with a molecular weight of more than 1000, the nanofiltration membrane allows small molecules with a molecular weight of less than 300 to pass through, the volume capacity of the nanofiltration membrane is 12L, the volume of the eluate is finally concentrated to 21L, the product content in the concentrate is 338.24g based on flavin mononucleotide, the mass concentration is 16.11g/L, adjusting the pH value of the concentrate to 5.10 with 6mol/L hydrochloric acid solution, transferring into a 50L glass reaction kettle, slowly adding 11.00L (0.6BV) of 95% (ml/ml) ethanol aqueous solution, controlling the temperature to 25-30 ℃, slowly cooling to 10 ℃ after addition, performing online detection by HPLC, after the precipitation rate of solids in the solution reaches more than 40 percent, continuously cooling to 6 ℃, adding 6.4L (0.4BV) of 95 percent (ml/ml) ethanol aqueous solution, uniformly mixing, keeping stirring at 6 ℃, stirring for 4 hours, filtering, pulping a filter cake with 2L of 50 percent ethanol aqueous solution for 2 hours, controlling the temperature to be 10-15 ℃, filtering, washing the filter cake with a small amount of 75 percent (ml/ml) ethanol aqueous solution, drying in vacuum at 30 ℃ for 10 hours to obtain 339.07g of orange-yellow sodium riboflavin phosphate solid powder, drying and losing weight of 4.86 percent, sodium ion content of 4.45 percent, specific rotation of +38.83 percent, purity of 99.34 percent, content (taking riboflavin as an external standard and calculated by dry products) of 77.30 percent (required by pharmacopoeia range of 74.0-79.0 percent), total yield 89.93 calculated as flavin mononucleotide

%

Claims

Claims

[ claim 1] A process for producing high-purity riboflavin sodium phosphate, characterized by comprising the steps of: i) Adjusting the pH value of a crude riboflavin sodium phosphate and/or flavin mononucleotide solution to 5.07.0, 2) controlling the temperature of the solution to be 20-40 ℃, and adding an ethanol water solution with the concentration of 95% (ml/ml) accounting for 0.5-1.5 times of the volume of the crude product solution; 3) Cooling to 3-5 ℃, and waiting for solid precipitation; 4) Filtering, removing the filtrate, and drying the filter cake to obtain the pure riboflavin sodium phosphate.

[ claim 2] the method for producing highly pure riboflavin sodium phosphate according to claim 1, characterized in that

The concentration of the crude riboflavin sodium phosphate and/or flavin mononucleotide solution is more than 7.5g/L calculated by the riboflavin sodium phosphate.

[ claim 3] the method for producing highly pure riboflavin sodium phosphate according to claim 1, characterized in that

In the step 1), the pH value of the solution is adjusted to 5.5-6.5.

[ claim 4] the method for producing highly pure riboflavin sodium phosphate according to claim 1, characterized in that

In the step 2), 95% (ml/ml) ethanol water solution accounting for 0.5-0.9 time of the volume of the solution is added.

[ claim 5] the method for preparing highly pure riboflavin sodium phosphate according to claim 1, wherein the cooling process of the step 3) is performed by the following steps: the temperature is firstly reduced to 12-15 ℃, and then reduced to 3-5 ℃ after the solid precipitation rate in the solution reaches more than 40%.

[ claim 6] the method for producing highly pure riboflavin sodium phosphate according to claim 1, characterized in that

In the step 3), ethanol water solution with the concentration of 95% (ml/ml) accounting for 0.3-0.5 time of the volume of the crude product solution is added after the precipitation rate of the solid in the solution reaches more than 40%.

[ claim 7] the method for producing highly pure riboflavin sodium phosphate according to claim 1, characterized in that

In the step 4), the filter cake is washed with an aqueous ethanol solution having a concentration of 75% (ml/ml) before the drying treatment.

[ claim 8] the method for producing highly pure riboflavin sodium phosphate according to claim 7, characterized in that

The filter cake is pulped with 50% ethanol water solution before being washed, and then filtered to collect the filter cake.

[ claim 9] the method for producing highly pure riboflavin sodium phosphate according to claim 1, characterized in that

In the step 1), the crude product solution of riboflavin sodium phosphate and/or flavin mononucleotide is a crude product solution obtained by preparing flavin mononucleotide by a biological catalysis method.

[ claim 10] the method for producing highly pure riboflavin sodium phosphate according to claim 9, characterized in that, before the step 1), the crude riboflavin sodium phosphate and/or flavin mononucleotide solution is subjected to the following preliminary treatment: a) Adjusting the pH value of a crude riboflavin sodium phosphate and/or flavin mononucleotide solution to 11.0-12.0, b) heating the solution to 40 +/-1 ℃, keeping the temperature at 40 +/-1 ℃ for 2 hours, and C) removing insoluble substances.

[ claim 11] the method for preparing highly pure riboflavin sodium phosphate according to claim 10, wherein said preliminary treatment further comprises a step of pretreating the solution obtained in the step c) after removing the insoluble matter by purifying the solution after removing the insoluble matter with an ion exchange resin or subjecting the solution after removing the insoluble matter to a microfiltration treatment and an ultrafiltration treatment in this order.

[ claim 12] the method for producing highly pure riboflavin sodium phosphate according to claim 11, characterized in that

The ion exchange resin is anion exchange resin.

[ claim 13] the method for producing highly pure riboflavin sodium phosphate according to claim 11, characterized in that

Using 0.l-0.4 mol/L sodium chloride water solution as the eluent of ion exchange resin.

[ claim 14] the method for producing highly pure riboflavin sodium phosphate according to claim 11, characterized in that

Before the step 1), the pretreated solution is concentrated.

[ claim 15] the method for producing highly pure riboflavin sodium phosphate according to claim 11, characterized in that said ion exchange resin is subjected to a regeneration treatment by: a. Leaching the resin by using a mixed solution of 2mol/L sodium chloride solution and 0.5mol/L hydrochloric acid solution until no impurities are left in an effluent liquid; b. Eluting the eluent by using purified water until the pH value of the eluent is 5.00-7.00, and eluting the eluent by using 0.5mol/L sodium hydroxide solution until the eluent is strongly alkaline; d. And eluting the eluent by using purified water until the pH value of the eluent is 9.00-10.00.