CN110627849A - Preparation method of N-acetylneuraminic acid standard substance - Google Patents
Preparation method of N-acetylneuraminic acid standard substance Download PDFInfo
- Publication number
- CN110627849A CN110627849A CN201910978700.0A CN201910978700A CN110627849A CN 110627849 A CN110627849 A CN 110627849A CN 201910978700 A CN201910978700 A CN 201910978700A CN 110627849 A CN110627849 A CN 110627849A
- Authority
- CN
- China
- Prior art keywords
- acetylneuraminic acid
- solution
- crystal
- water
- mixed solvent
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H7/00—Compounds containing non-saccharide radicals linked to saccharide radicals by a carbon-to-carbon bond
- C07H7/02—Acyclic radicals
- C07H7/033—Uronic acids
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Saccharide Compounds (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention provides a preparation method of an N-acetylneuraminic acid standard substance, which comprises the processes of dissolving an N-acetylneuraminic acid raw material, adjusting pH, cooling, crystallizing, filtering, washing, drying, redissolving, cooling again, recrystallizing, refiltering, rewashing and redrying, the N-acetylneuraminic acid with the purity of more than 99% can be obtained through the processes, and the N-acetylneuraminic acid standard substance can be obtained and can meet the application requirements in the fields of food, health care, medicine, cosmetics and the like. The preparation method is simple, quick and easy to operate, and the obtained product has high purity and has strong application prospect in the field of preparing standard substances of N-acetylneuraminic acid.
Description
Technical Field
The invention relates to the technical field of standard substances, in particular to a preparation method of an N-acetylneuraminic acid standard substance.
Background
N-acetylneuraminic Acid (N-acetylneuraminic Acid) is the most common one of sialic Acid families, is called SA, NeuAc or NANA for short, is commonly called sialic Acid or cubilose Acid, is a 9-carbon monosaccharide with pyranose structure, and has a molecular formula of C11H19NO9The full name is 5-amino-3, 5-dideoxy-D-glycerol-D-galactononulose. The molecular weight of NeuAc is 309.3, the molecule contains a carboxyl group, the NeuAc is an acidic amino sugar, is very soluble in water and not easily soluble in organic solvents such as ethanol, and is acidic in aqueous solution, and the pH value of 2% aqueous solution is about 1.8-2.3.
NeuAc is an important monosaccharide in human body, mainly exists on cell surface, especially exists in the form of cell membrane glycoprotein and glycolipid, is usually combined with the terminal of sugar chain on the cell membrane surface, has various biological functions, such as neurotransmission, leucocyte secretion, virus or bacterial infection, cell adhesion caused by carbohydrate-protein recognition and the like, and has the effects of resisting oxidation and inhibiting melanin generation.
N-acetylneuraminic acid is a characteristic substance in N-acetylneuraminic acid health-care products or infant milk powder, and is an important index in detection and analysis. There is no standard sample of the compound in China, and the quality of the currently provided reagent or reference substance cannot be guaranteed and is inconsistent in standard. In view of the important role of the standard substance in the food field, the development and development of the related standard substance have very important practical significance.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a preparation method of an N-acetylneuraminic acid standard substance. The preparation method is simple, fast and easy to operate, and the purity of the prepared product can reach more than 99%, so that the application requirements in the fields of food, health care, medicine, cosmetics and the like are met.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides a preparation method of an N-acetylneuraminic acid standard substance, which comprises the following steps:
(1) dissolving N-acetylneuraminic acid raw material in water to obtain a first solution, adjusting the pH value to 0.5-3.5, crystallizing, and filtering to obtain a first crystal;
(2) washing the first crystal obtained in the step (1) by using a mixed solvent of an organic solvent and water, and drying to obtain a second crystal;
(3) dissolving the second crystal obtained in the step (2) in water to obtain a second solution, cooling, crystallizing and filtering to obtain a third crystal;
(4) and (3) washing the third crystal obtained in the step (3) by using a mixed solvent of an organic solvent and water, and drying to obtain the N-acetylneuraminic acid standard substance.
The preparation method comprises the steps of dissolving the raw material of the N-acetylneuraminic acid, adjusting pH, cooling, crystallizing, filtering, washing, drying, redissolving, cooling again, recrystallizing, refiltering, rewashing and redrying, the N-acetylneuraminic acid with the purity of more than 99 percent can be obtained through the processes, and the standard substance of the N-acetylneuraminic acid can be obtained and can meet the application requirements in the fields of food, health care, medicine, cosmetics and the like.
In the step (1) of the preparation method of the present invention, the pH of the first solution is preferably adjusted to 0.5 to 3.5, because when the pH is higher than 3.5, the N-acetylneuraminic acid is insufficiently crystallized, and the 5 th carbon atom of the N-acetylneuraminic acid molecule contains 1 acetylamino group, and when the pH is lower than 0.5, the structure of the N-acetylneuraminic acid is unstable, so that the N-acetylneuraminic acid is easily decomposed, and the purity of the finally obtained product is reduced, and the purity standard of the standard substance cannot be reached.
The N-acetylneuraminic acid standard substance refers to a substance with the purity of an N-acetylneuraminic acid monomer of more than 99%.
Preferably, the content of the N-acetylneuraminic acid monomer in the N-acetylneuraminic acid raw material is more than or equal to 80 percent.
The content of the N-acetylneuraminic acid monomer in the N-acetylneuraminic acid raw material is preferably more than or equal to 80 percent, so that the step of hydrolyzing and purifying the N-acetylneuraminic acid raw material can be omitted; that is, if the content of the N-acetylneuraminic acid monomer in the N-acetylneuraminic acid raw material used in the present application is less than 80%, the raw material can be used after being subjected to hydrolysis purification.
Preferably, the temperature of the first solution is 45-80 ℃, for example, 45 ℃, 48 ℃, 50 ℃, 54 ℃, 60 ℃, 65 ℃, 70 ℃,73 ℃, 78 ℃ or 80 ℃.
Preferably, the content of N-acetylneuraminic acid in the first solution is 250-450g/L, for example, 250g/L, 260g/L, 280g/L, 300g/L, 320g/L, 350g/L, 400g/L, 420g/L or 450 g/L.
Preferably, step (1) further comprises: the first solution is coarsely filtered using a 0.1-5 μm filter.
The 0.1 to 5 μm filter may be, for example: 0.1 μm, 0.2 μm, 0.5 μm, 0.9 μm,1 μm, 1.5 μm, 2 μm, 2.5 μm, 3 μm, 3.5 μm, 4 μm, 4.5 μm, or 5 μm, etc.
Preferably, the crystallization temperature in step (1) is 4-25 ℃, for example, 4 ℃,6 ℃,8 ℃, 10 ℃, 14 ℃, 15 ℃, 20 ℃, 22 ℃, 24 ℃ or 25 ℃.
Preferably, the crystallization time in step (1) and step (3) is 4-36h, for example, 4h, 6h, 8h, 10h, 14h, 15h, 20h, 24h, 28h, 30h, 34h or 36h, etc.
Preferably, the filtration in step (1) and step (3) is performed by 0.22 μm membrane filtration, microfiltration membrane filtration or centrifugation.
Preferably, the organic solvent in step (2) and step (4) is an N-acetylneuraminic acid-insoluble organic solvent.
Preferably, the organic solvents in step (2) and step (4) are each independently selected from any one of ethanol, isopropanol or acetone or a combination of at least two thereof.
Preferably, the mass percentage of the organic solvent in the mixed solvent in the step (2) and the step (4) is 70-80%, for example, 70%, 71%, 73%, 74%, 75%, 77%, 78%, 79%, 80%, etc.
The mass ratio of the organic solvent to the water in the washing process in the step (2) and the step (4) is preferably selected in the invention, so that the comprehensive removal effect of the water-soluble impurities and the oil-soluble impurities in the N-acetylneuraminic acid raw material is considered. When the content of the organic solvent in the mixed solvent exceeds 80%, the water content is accordingly too low, which results in poor removal of water-soluble impurities from the N-acetylneuraminic acid raw material, and when the content of the organic solvent in the mixed solvent is less than 70%, the water content is too high, which results in poor removal of water-soluble impurities from the N-acetylneuraminic acid raw material.
Preferably, the drying manner in step (2) and step (4) includes any one or a combination of at least two of hot air circulation, vacuum heating or radiation heating.
Preferably, the drying temperature in step (2) and step (4) is 25-80 deg.C, such as 25 deg.C, 28 deg.C, 30 deg.C, 35 deg.C, 40 deg.C, 43 deg.C, 45 deg.C, 50 deg.C, 55 deg.C, 60 deg.C, 65 deg.C, 70 deg.C, 75 deg.C or 80 deg.C etc.
Preferably, the drying time in step (2) and step (4) is 3-36h, such as 3h, 5h, 8h, 10h, 13h, 15h, 20h, 25h, 28h, 30h, 32h, 35h or 36 h.
Preferably, the temperature of the second solution in step (3) is 45-80 deg.C, such as 45 deg.C, 48 deg.C, 50 deg.C, 55 deg.C, 60 deg.C, 65 deg.C, 70 deg.C, 75 deg.C or 80 deg.C.
Preferably, the temperature reduction in step (3) is to reduce the temperature of the second solution to 4-20 deg.C, such as 4 deg.C, 5 deg.C, 6 deg.C, 7 deg.C, 8 deg.C, 9 deg.C, 10 deg.C, 11 deg.C, 12 deg.C, 13 deg.C, 14 deg.C, 15 deg.C, 16 deg.C, 17 deg.C, 18 deg.C, 19 deg.C or 20 deg.C.
Preferably, the preparation method comprises the following steps:
(1) dissolving N-acetylneuraminic acid raw material in water to obtain a first solution, adjusting the pH value to 0.5-3.5, placing at 4-25 ℃ for crystallization for 4-36h, and filtering by adopting a 0.22 mu m membrane to obtain a first crystal, wherein the concentration of the first solution is 250-450 g/L;
(2) washing the first crystal obtained in the step (1) by using a mixed solvent of an organic solvent and water, and drying for 3-36h at 25-80 ℃ to obtain a second crystal, wherein the mass percentage of the organic solvent in the mixed solvent is 70-80%;
(3) dissolving the second crystal obtained in the step (2) in water to obtain a second solution, cooling to 4-20 ℃, crystallizing for 4-36h, centrifuging, and filtering to obtain a third crystal, wherein the temperature of the second solution is 45-80 ℃;
(4) and (3) washing and drying the third crystal obtained in the step (3) by using a mixed solvent of an organic solvent and water to obtain the N-acetylneuraminic acid standard substance, wherein the mass percentage of the organic solvent in the mixed solvent is 70-80%.
Preferably, the preparation method comprises the following steps:
(1) dissolving N-acetylneuraminic acid raw material in water to obtain a first solution, adjusting the pH value to 1-3, crystallizing at 4-20 ℃ for 4-30h, and filtering by adopting a 0.22 mu m membrane to obtain a first crystal, wherein the concentration of the first solution is 300-400 g/L;
(2) washing the first crystal obtained in the step (1) by using a mixed solvent of an ethanol solvent and water, and drying at 30-75 ℃ for 5-34h to obtain a second crystal, wherein the mass percentage of ethanol in the mixed solvent is 75-80%;
(3) dissolving the second crystal obtained in the step (2) in water to obtain a second solution, cooling to 4-10 ℃, crystallizing for 5-25h, centrifuging, and filtering to obtain a third crystal, wherein the temperature of the second solution is 50-80 ℃;
(4) and (3) washing the third crystal obtained in the step (3) by using a mixed solvent containing isopropanol and water, and drying to obtain the N-acetylneuraminic acid standard substance, wherein the mass percentage of the isopropanol in the mixed solvent is 73-80%.
Preferably, the preparation method comprises the following steps:
(1) dissolving an N-acetylneuraminic acid raw material in water to obtain a first solution, adjusting the pH to 2, crystallizing at 5 ℃ for 6 hours, and filtering by adopting a 0.22-micron membrane to obtain a first crystal, wherein the concentration of the first solution is 350 g/L;
(2) washing the first crystal obtained in the step (1) by using a mixed solvent of an ethanol solvent and water, and drying at 60 ℃ for 26h to obtain a second crystal, wherein the mass percentage of ethanol in the mixed solvent is 75%;
(3) dissolving the second crystal obtained in the step (2) in water to obtain a second solution, cooling to 4 ℃, crystallizing for 20 hours, centrifuging, and filtering to obtain a third crystal, wherein the temperature of the second solution is 70 ℃;
(4) and (3) washing the third crystal obtained in the step (3) by using a mixed solvent containing isopropanol and water, and drying to obtain the N-acetylneuraminic acid standard substance, wherein the mass percentage of the isopropanol in the mixed solvent is 73%.
The N-acetylneuraminic acid standard substance obtained by the preparation method preferably has the following preservation method: the prepared N-acetylneuraminic acid solid is subpackaged in a relatively independent and clean glove box by adopting a 2mL brown sample bottle, sealed by a black bottle cap with a polytetrafluoroethylene lining and independently vacuum-plastically packaged by adopting a plastic bag.
Compared with the prior art, the invention has the following beneficial effects:
(1) the preparation method comprises the steps of dissolving the raw material of the N-acetylneuraminic acid, adjusting the pH value, cooling, crystallizing, filtering, washing, drying, redissolving, cooling again, recrystallizing, refiltering, rewashing and redrying, the N-acetylneuraminic acid with the purity of more than 99 percent can be obtained through the processes, and the standard substance of the N-acetylneuraminic acid can be obtained and can meet the application requirements of the N-acetylneuraminic acid in the fields of food, health care, medicine, cosmetics and the like;
(2) the preparation method of the N-acetylneuraminic acid standard substance is simple, quick and easy to operate.
Drawings
FIG. 1 is a Fourier transform infrared spectrum of the product prepared in example 1 of the present invention.
FIG. 2(a) is a NMR chart of a product obtained in example 1 of the present invention.
FIG. 2(b) is a NMR spectrum of the product obtained in example 1 of the present invention.
FIG. 3 is a mass spectrum of the product prepared in example 1 of the present invention.
FIG. 4 is a high performance liquid chromatography detection spectrum of the product prepared in example 1 of the present invention.
Detailed Description
The present invention is described in further detail below. The following examples are merely illustrative of the present invention and do not represent or limit the scope of the claims, which are defined by the claims.
Example 1
This example provides a method for preparing N-acetylneuraminic acid standard substance, which comprises the following steps:
(1) dissolving an N-acetylneuraminic acid raw material in water to obtain a first solution, adjusting the pH to 2, crystallizing at 4 ℃ for 6 hours, and filtering by adopting a 0.22-micron membrane to obtain a first crystal, wherein the concentration of the first solution is 350g/L, and the content of N-acetylneuraminic acid in the N-acetylneuraminic acid raw material is 95%;
(2) washing the first crystal obtained in the step (1) by using a mixed solvent of ethanol and water, and drying at 60 ℃ for 26h to obtain a second crystal, wherein the mass percentage of the ethanol in the mixed solvent is 75%;
(3) dissolving the second crystal obtained in the step (2) in water to obtain a second solution, cooling to 15 ℃, crystallizing for 20 hours, centrifuging, and filtering to obtain a third crystal, wherein the temperature of the second solution is 70 ℃;
(4) and (3) washing the third crystal obtained in the step (3) by using a mixed solvent containing isopropanol and water, and drying to obtain the N-acetylneuraminic acid standard substance, wherein the mass percentage of the isopropanol in the mixed solvent is 73%.
Example 2
This example provides a method for preparing N-acetylneuraminic acid standard substance, which comprises the following steps:
(1) dissolving an N-acetylneuraminic acid raw material in water to obtain a first solution, adjusting the pH to 0.5, crystallizing at 25 ℃ for 36 hours, and filtering by adopting a 0.22-micron membrane to obtain a first crystal, wherein the concentration of the first solution is 450g/L, and the content of N-acetylneuraminic acid in the N-acetylneuraminic acid raw material is 90%;
(2) washing the first crystal obtained in the step (1) by using a mixed solvent of ethanol and water, and drying at 80 ℃ for 10 hours to obtain a second crystal, wherein the mass percentage of the ethanol in the mixed solvent is 70%;
(3) dissolving the second crystal obtained in the step (2) in water to obtain a second solution, cooling to 20 ℃, crystallizing for 36 hours, centrifuging, and filtering to obtain a third crystal, wherein the temperature of the second solution is 70 ℃;
(4) and (3) washing the third crystal obtained in the step (3) by using a mixed solvent containing acetone and water, and drying to obtain the N-acetylneuraminic acid standard substance, wherein the mass percentage of the acetone in the mixed solvent is 80%.
Example 3
This example provides a method for preparing N-acetylneuraminic acid standard substance, which comprises the following steps:
(1) dissolving an N-acetylneuraminic acid raw material in water to obtain a first solution, adjusting the pH to 3.5, crystallizing at 20 ℃ for 34 hours, and filtering by adopting a 0.22-micron membrane to obtain a first crystal, wherein the concentration of the first solution is 250g/L, and the content of N-acetylneuraminic acid in the N-acetylneuraminic acid raw material is 93%;
(2) washing the first crystal obtained in the step (1) by using a mixed solvent of ethanol and water, and drying at 40 ℃ for 32h to obtain a second crystal, wherein the mass percentage of acetone in the mixed solvent is 80%;
(3) dissolving the second crystal obtained in the step (2) in water to obtain a second solution, cooling to 10 ℃, crystallizing for 22 hours, centrifuging, and filtering to obtain a third crystal, wherein the temperature of the second solution is 60 ℃;
(4) and (3) washing the third crystal obtained in the step (3) by using a mixed solvent containing isopropanol and water, and drying to obtain the N-acetylneuraminic acid standard substance, wherein the mass percentage of the ethanol in the mixed solvent is 75%.
Example 4
This example provides a method for preparing N-acetylneuraminic acid standard substance, which comprises the following steps:
(1) dissolving an N-acetylneuraminic acid raw material in water to obtain a first solution, adjusting the pH to 3, crystallizing at 22 ℃ for 35 hours, and filtering by adopting a 0.22-micron membrane to obtain a first crystal, wherein the concentration of the first solution is 300g/L, and the content of N-acetylneuraminic acid in the N-acetylneuraminic acid raw material is 85%;
(2) washing the first crystal obtained in the step (1) by using a mixed solvent of ethanol and water, and drying at 25 ℃ for 36h to obtain a second crystal, wherein the mass percentage of acetone in the mixed solvent is 70%;
(3) dissolving the second crystal obtained in the step (2) in water to obtain a second solution, cooling to 4 ℃, crystallizing for 4 hours, centrifuging, and filtering to obtain a third crystal, wherein the temperature of the second solution is 55 ℃;
(4) and (3) washing the third crystal obtained in the step (3) by using a mixed solvent containing isopropanol and water, and drying to obtain the N-acetylneuraminic acid standard substance, wherein the mass percentage of the ethanol in the mixed solvent is 80%.
Comparative example 1
This comparative example provides a method for preparing N-acetylneuraminic acid comprising the steps of:
(1) dissolving an N-acetylneuraminic acid raw material in water to obtain a solution, adjusting the pH to 2, crystallizing at 4 ℃ for 6 hours, and filtering by adopting a 0.22-micron membrane to obtain a crystal, wherein the concentration of the solution is 350g/L, and the content of N-acetylneuraminic acid in the N-acetylneuraminic acid raw material is 95%;
(2) and (2) washing the crystal obtained in the step (1) by using a mixed solvent of ethanol and water, and drying at 60 ℃ for 26h to obtain the N-acetylneuraminic acid, wherein the mass percentage of the ethanol in the mixed solvent is 75%.
Comparative example 2
This comparative example provides a method for preparing N-acetylneuraminic acid comprising the steps of:
(1) dissolving an N-acetylneuraminic acid raw material in water to obtain a first solution, adjusting the pH to 2, crystallizing at 4 ℃ for 6 hours, and filtering by adopting a 0.22-micron membrane to obtain a first crystal, wherein the concentration of the first solution is 350g/L, and the content of N-acetylneuraminic acid in the N-acetylneuraminic acid raw material is 95%;
(2) dissolving the first crystal obtained in the step (1) in water to obtain a second solution, cooling to 15 ℃, crystallizing for 20 hours, centrifuging, and filtering to obtain a second crystal, wherein the temperature of the second solution is 70 ℃;
(3) and (3) washing and drying the second crystal obtained in the step (2) by using a mixed solvent containing isopropanol and water to obtain the N-acetylneuraminic acid, wherein the mass percentage of the isopropanol in the mixed solvent is 73%.
Comparative example 3
This comparative example provides a method for preparing N-acetylneuraminic acid comprising the steps of:
(1) dissolving an N-acetylneuraminic acid raw material in water to obtain a first solution, adjusting the pH to 2, crystallizing at 4 ℃ for 6 hours, and filtering by adopting a 0.22-micron membrane to obtain a first crystal, wherein the concentration of the first solution is 350g/L, and the content of N-acetylneuraminic acid in the N-acetylneuraminic acid raw material is 95%;
(2) washing the first crystal obtained in the step (1) by using a mixed solvent of ethanol and water, and drying at 60 ℃ for 36 hours to obtain a second crystal, wherein the mass percentage of the ethanol in the mixed solvent is 75%;
(3) and (3) dissolving the second crystal obtained in the step (2) in water to obtain a second solution, cooling to the temperature, crystallizing for 20 hours, centrifuging, and filtering to obtain the N-acetylneuraminic acid.
Comparative example 4
This comparative example provides a method for preparing N-acetylneuraminic acid comprising the steps of:
(1) dissolving N-acetylneuraminic acid raw material in water to obtain a solution, cooling to 4 ℃, crystallizing for 20 hours, centrifuging, and filtering to obtain crystals, wherein the temperature of the solution is 70 ℃;
(2) and (2) washing and drying the crystal obtained in the step (1) by using a mixed solvent containing isopropanol and water to obtain the N-acetylneuraminic acid, wherein the isopropanol accounts for 73% of the mixed solvent in percentage by mass.
Comparative example 5
This comparative example provides a method for preparing N-acetylneuraminic acid comprising the steps of:
(1) dissolving an N-acetylneuraminic acid raw material in water to obtain a first solution, cooling to 10 ℃, crystallizing for 20 hours, and filtering by adopting a 0.22-micron membrane to obtain a first crystal, wherein the concentration of the first solution is 350g/L, and the content of N-acetylneuraminic acid in the N-acetylneuraminic acid raw material is 95%;
(2) washing the first crystal obtained in the step (1) by using a mixed solvent of ethanol and water, and drying at 60 ℃ for 36 hours to obtain a second crystal, wherein the mass percentage of the ethanol in the mixed solvent is 75%;
(3) dissolving the second crystal obtained in the step (2) in water to obtain a second solution, adjusting the pH to 2, crystallizing at 4 ℃ for 6h, centrifuging, and filtering to obtain a third crystal, wherein the temperature of the second solution is 70 ℃;
(4) and (3) washing the third crystal obtained in the step (3) by using a mixed solvent containing isopropanol and water, and drying to obtain the N-acetylneuraminic acid, wherein the isopropanol accounts for 73% by mass in the mixed solvent.
Comparative example 6
This comparative example provides a method for preparing N-acetylneuraminic acid comprising the steps of:
(1) dissolving an N-acetylneuraminic acid raw material in water to obtain a first solution, adjusting the pH to 2, crystallizing at 4 ℃ for 6 hours, and filtering by using a 0.22-micron membrane to obtain a first crystal, wherein the concentration of the first solution is 350g/L, and the content of N-acetylneuraminic acid in the N-acetylneuraminic acid raw material is 95%;
(2) washing the first crystal obtained in the step (1) by using ethanol, and drying for 26h at 60 ℃ to obtain a second crystal;
(3) dissolving the second crystal obtained in the step (2) in water to obtain a second solution, cooling to 10 ℃, crystallizing for 20 hours, centrifuging, and filtering to obtain a third crystal, wherein the temperature of the second solution is 70 ℃;
(4) and (4) washing the third crystal obtained in the step (3) by using isopropanol, and drying to obtain the N-acetylneuraminic acid.
Comparative example 7
This comparative example provides a method for preparing N-acetylneuraminic acid comprising the steps of:
(1) dissolving an N-acetylneuraminic acid raw material in water to obtain a first solution, adjusting the pH to 2, crystallizing at 4 ℃ for 6 hours, and filtering by using a 0.22-micron membrane to obtain a first crystal, wherein the concentration of the first solution is 350g/L, and the content of N-acetylneuraminic acid in the N-acetylneuraminic acid raw material is 95%;
(2) washing the first crystal obtained in the step (1) by using a mixed solvent of ethanol and water, and drying at 60 ℃ for 26h to obtain a second crystal, wherein the mass percentage of the ethanol in the mixed solvent is 75%;
(3) dissolving the second crystal obtained in the step (2) in water to obtain a second solution, cooling to 10 ℃, crystallizing for 20 hours, centrifuging, and filtering to obtain a third crystal, wherein the temperature of the second solution is 70 ℃;
(4) and (4) washing the third crystal obtained in the step (3) by using isopropanol, and drying to obtain the N-acetylneuraminic acid.
Comparative example 8
This comparative example provides a method for preparing N-acetylneuraminic acid comprising the steps of:
(1) dissolving an N-acetylneuraminic acid raw material in water to obtain a first solution, adjusting the pH to 2, crystallizing at 4 ℃ for 6 hours, and filtering by using a 0.22-micron membrane to obtain a first crystal, wherein the concentration of the first solution is 350g/L, and the content of N-acetylneuraminic acid in the N-acetylneuraminic acid raw material is 95%;
(2) washing the first crystal obtained in the step (1) by using ethanol, and drying for 26h at 60 ℃ to obtain a second crystal;
(3) dissolving the second crystal obtained in the step (2) in water to obtain a second solution, cooling to 10 ℃, crystallizing for 20 hours, centrifuging, and filtering to obtain a third crystal, wherein the temperature of the second solution is 70 ℃;
(4) and (3) washing the third crystal obtained in the step (3) by using a mixed solvent containing isopropanol and water, and drying to obtain the N-acetylneuraminic acid, wherein the isopropanol accounts for 73% by mass in the mixed solvent.
Comparative example 9
This comparative example provides a method for producing N-acetylneuraminic acid, which is different from example 1 only in that pH is adjusted to 4 in step (1).
Comparative example 10
This comparative example provides a method for producing N-acetylneuraminic acid, which is different from example 1 only in that the pH is adjusted to 0.2 in step (1).
Purity determination and structural characterization
(1) And (3) purity determination: the purity of the products obtained in the examples and comparative examples was measured by Shimadzu LC-15C High Performance Liquid Chromatography (HPLC) detection method, and the specific results are shown in Table 1. Wherein, the detection Column Bio-Rad AMINEX HPX 87H organic analysis Column (300X 7.8 mm); the column temperature is 60 ℃; the mobile phase is 6mmol sulfuric acid, and the flow rate is 0.6 ml/min; the detection wavelength is 210 nm.
The products prepared in the examples and the comparative examples are detected by high performance liquid chromatography, and the specific results are shown in table 1. Illustratively, only the HPLC analysis pattern of the product prepared in example 1 is shown (the result is shown in FIG. 4), and the purity is 99.0%.
TABLE 1
As is clear from the data in Table 1, the N-acetylneuraminic acid obtained in examples 1 to 4 has a purity of more than 99% and can be used as a standard substance in the fields of foods, health care, medicines, cosmetics and the like.
Compared with the example 1, the comparative example 1 and the comparative example 4 omit the processes of secondary washing and recrystallization, and the purity of the obtained product is obviously lower than that of the N-acetylneuraminic acid obtained in the example 1, the purity is lower than 99 percent, and the product cannot be used as a standard substance.
Compared with the example 1, the comparative example 2 and the comparative example 3 omit the process of secondary washing, and the purity of the obtained product is lower than that of the N-acetylneuraminic acid obtained in the example 1, the purity is lower than 99 percent, and the product cannot be used as a standard substance.
Compared with the example 1, the comparative example 5 replaces the conditions of two times of crystallization, the first time of crystallization adopts a cooling mode, the second time of crystallization adopts pH value adjustment, but the purity of the obtained product is lower than that of the N-acetylneuraminic acid obtained in the example 1, the purity is lower than 99 percent, and the product cannot be used as a standard substance. This indicates that standard substances with a purity of more than 99% can only be obtained by the preparation process described in the present application.
Compared with the example 1, the comparative example 6 only adopts the organic solvent for washing in two times of washing, and the purity of the obtained product is lower than that of the N-acetylneuraminic acid obtained in the example 1, the purity is lower than 99 percent, and the product cannot be used as a standard substance. This is probably due to the presence of water-soluble impurities in the N-acetylneuraminic acid starting material which cannot be completely removed by washing with an organic solvent.
Compared with example 1, comparative example 7 only uses organic solvent in the first washing, and comparative example 8 only uses organic solvent in the second washing, and the purity of the obtained product is lower than that of the N-acetylneuraminic acid obtained in example 1, the purity is lower than 99%, and the product cannot be used as a standard substance.
In comparison with example 1, comparative example 9, in which the pH was adjusted to 4 in step (1), out of the range of 0.5 to 3.5 in step (1) of the production process of the present invention, also obtained a product having a purity lower than that of N-acetylneuraminic acid obtained in example 1, having a purity lower than 99%, and could not be used as a standard substance. This is because N-acetylneuraminic acid is insufficiently crystallized.
In comparison with example 1, comparative example 10, in which the pH was adjusted to 0.2 in step (1), out of the range of 0.5 to 3.5 in step (1) of the production process according to the present invention, also obtained a product having a purity lower than that of N-acetylneuraminic acid obtained in example 1, having a purity lower than 99%, and could not be used as a standard. This is because the carbon atom at position 5 of the N-acetylneuraminic acid molecule contains 1 acetylamino group, and when the pH is less than 0.5, the structure of N-acetylneuraminic acid is unstable and easy to decompose, which leads to the reduction of the purity of the finally obtained product.
(2) Structural characterization: the product obtained in example 1 was tested for fourier transform infrared spectroscopy, ultraviolet spectroscopy, nuclear magnetic resonance spectroscopy and mass spectroscopy, and the spectra obtained were shown in fig. 1, fig. 2, fig. 3 and fig. 4, respectively.
As can be seen from FIG. 1, 3492,3462cm-1Vibrating telescopically for free hydroxylAbsorption Peak, 3340cm-12979cm as a stretching vibration absorption peak of carboxyl-1Stretching vibration absorption peak of methyl group, 2933cm-1The stretching vibration absorption peak of methylene is the stretching vibration absorption peak of carbonyl at 1725cm-1，1655cm-1，1530cm-1,1263cm-1,1473cm-1,1373cm-1,1373cm-1,1124cm-1(ii) a Ether bond at 1373cm-1And 1263cm-1There is an absorption peak. The absorption peak positions show that the compound contains groups such as methyl, methylene, ether bond, amide bond, hydroxyl, carboxyl, carbonyl and the like.
Data from the nuclear magnetic resonance carbon spectrum in fig. 2 (a): 13C NMR (151MHz, dmso):174.90,174.42,97.78,73.45,72.92,72.28,68.83,66.73,56.14,42.95, 25.69.
This indicates that the compound has 11 carbons, 1 primary, 2 secondary, 5 tertiary, and 3 quaternary.
Data from the nuclear magnetic resonance hydrogen spectrum in fig. 2 (b): 1H NMR (600MHz, dmso) 12.88(s,1H),8.05(d, J ═ 8.4Hz,1H),6.15(s,1H),4.77(s,1H),4.51(s,1H),4.32(s,1H),4.21(s,1H),3.80(m,1H),3.71(m, J ═ 10.6Hz,1H),3.57(d, J ═ 10.7Hz,1H),3.45(m, J ═ 18.7,8.9Hz,2H),3.25(m, J ═ 10.9,6.9Hz,1H),3.15(m, J ═ 15.3Hz,1H),1.95(m J ═ 12.8,4.9, 1H),1.92-1.82(s, 1H), 1H (t, 1H), 1.67(t, 1H), 1.7, 1H).
The data above shows that this compound contains 19 protons.
The mass spectral data in figure 3 show that the molecular weight of this compound is 309.
It was confirmed from the above-mentioned spectrograms 1 to 4 that N-acetylneuraminic acid was confirmed to be obtained by the preparation method of the present invention.
The applicant declares that the present invention illustrates the detailed structural features of the present invention through the above embodiments, but the present invention is not limited to the above detailed structural features, that is, it does not mean that the present invention must be implemented depending on the above detailed structural features. It should be understood by those skilled in the art that any modifications of the present invention, equivalent substitutions of selected components of the present invention, additions of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
Claims (10)
1. A preparation method of an N-acetylneuraminic acid standard substance is characterized by comprising the following steps:
(1) dissolving N-acetylneuraminic acid raw material in water to obtain a first solution, adjusting the pH value to 0.5-3.5, crystallizing, and filtering to obtain a first crystal;
(2) washing the first crystal obtained in the step (1) by using a mixed solvent of an organic solvent and water, and drying to obtain a second crystal;
(3) dissolving the second crystal obtained in the step (2) in water to obtain a second solution, cooling, crystallizing and filtering to obtain a third crystal;
(4) and (3) washing the third crystal obtained in the step (3) by using a mixed solvent of an organic solvent and water, and drying to obtain the N-acetylneuraminic acid standard substance.
2. The method according to claim 1, wherein the content of N-acetylneuraminic acid monomers in the N-acetylneuraminic acid raw material is not less than 80%.
3. The method of claim 1 or 2, wherein the temperature of the first solution is 45 to 80 ℃;
preferably, the content of the N-acetylneuraminic acid in the first solution is 250-450 g/L;
preferably, step (1) further comprises: the first solution is coarsely filtered using a 0.1-5 μm filter.
4. The production method according to any one of claims 1 to 3, wherein the temperature of the crystallization in the step (1) is 4 to 25 ℃;
preferably, the crystallization time in step (1) and step (3) is 4-36 h;
preferably, the filtration in step (1) and step (3) is performed by 0.22 μm membrane filtration, microfiltration membrane filtration or centrifugation.
5. The production method according to any one of claims 1 to 4, wherein the organic solvent in the step (2) and the step (4) is an N-acetylneuraminic acid-insoluble organic solvent;
preferably, the organic solvents in step (2) and step (4) are each independently selected from any one of ethanol, isopropanol or acetone or a combination of at least two thereof;
preferably, the mass percentage of the organic solvent in the mixed solvent in the step (2) and the step (4) is 70-80% respectively and independently.
6. The production method according to any one of claims 1 to 5, wherein the manner of drying in step (2) and step (4) comprises any one of hot air circulation, vacuum heating, or radiation heating or a combination of at least two thereof;
preferably, the temperature of the drying in the step (2) and the step (4) is 25-80 ℃;
preferably, the drying time in step (2) and step (4) is 3-36 h.
7. The method according to any one of claims 1 to 6, wherein the temperature of the second solution in the step (3) is 45 to 80 ℃;
preferably, the temperature reduction in step (3) is to reduce the temperature of the second solution to 4-20 ℃.
8. The method of any one of claims 1 to 7, comprising the steps of:
(1) dissolving N-acetylneuraminic acid raw material in water to obtain a first solution, adjusting the pH value to 0.5-3.5, placing at 4-25 ℃ for crystallization for 4-36h, and filtering by adopting a 0.22 mu m membrane to obtain a first crystal, wherein the concentration of the first solution is 250-450 g/L;
(2) washing the first crystal obtained in the step (1) by using a mixed solvent of an organic solvent and water, and drying for 3-36h at 25-80 ℃ to obtain a second crystal, wherein the mass percentage of the organic solvent in the mixed solvent is 70-80%;
(3) dissolving the second crystal obtained in the step (2) in water to obtain a second solution, cooling to 4-20 ℃, crystallizing for 4-36h, centrifuging, and filtering to obtain a third crystal, wherein the temperature of the second solution is 45-80 ℃;
(4) and (3) washing and drying the third crystal obtained in the step (3) by using a mixed solvent of an organic solvent and water to obtain the N-acetylneuraminic acid standard substance, wherein the mass percentage of the organic solvent in the mixed solvent is 70-80%.
9. The method of any one of claims 1 to 8, comprising the steps of:
(1) dissolving N-acetylneuraminic acid raw material in water to obtain a first solution, adjusting the pH value to 1-3, crystallizing at 4-20 ℃ for 4-30h, and filtering by adopting a 0.22 mu m membrane to obtain a first crystal, wherein the concentration of the first solution is 300-400 g/L;
(2) washing the first crystal obtained in the step (1) by using a mixed solvent of an ethanol solvent and water, and drying at 30-75 ℃ for 5-34h to obtain a second crystal, wherein the mass percentage of ethanol in the mixed solvent is 75-80%;
(3) dissolving the second crystal obtained in the step (2) in water to obtain a second solution, cooling to 4-10 ℃, crystallizing for 5-25h, centrifuging, and filtering to obtain a third crystal, wherein the temperature of the second solution is 50-80 ℃;
(4) and (3) washing the third crystal obtained in the step (3) by using a mixed solvent containing isopropanol and water, and drying to obtain the N-acetylneuraminic acid standard substance, wherein the mass percentage of the isopropanol in the mixed solvent is 73-80%.
10. The method of any one of claims 1 to 9, comprising the steps of:
(1) dissolving an N-acetylneuraminic acid raw material in water to obtain a first solution, adjusting the pH to 2, crystallizing at 4 ℃ for 6 hours, and filtering by adopting a 0.22-micron membrane to obtain a first crystal, wherein the concentration of the first solution is 350 g/L;
(2) washing the first crystal obtained in the step (1) by using a mixed solvent of an ethanol solvent and water, and drying at 60 ℃ for 26h to obtain a second crystal, wherein the mass percentage of ethanol in the mixed solvent is 75%;
(3) dissolving the second crystal obtained in the step (2) in water to obtain a second solution, cooling to 4 ℃, crystallizing for 20 hours, centrifuging, and filtering to obtain a third crystal, wherein the temperature of the second solution is 70 ℃;
(4) and (3) washing the third crystal obtained in the step (3) by using a mixed solvent containing isopropanol and water, and drying to obtain the N-acetylneuraminic acid standard substance, wherein the mass percentage of the isopropanol in the mixed solvent is 73%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910978700.0A CN110627849B (en) | 2019-10-15 | 2019-10-15 | Preparation method of N-acetylneuraminic acid standard substance |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910978700.0A CN110627849B (en) | 2019-10-15 | 2019-10-15 | Preparation method of N-acetylneuraminic acid standard substance |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110627849A true CN110627849A (en) | 2019-12-31 |
| CN110627849B CN110627849B (en) | 2023-02-17 |
Family
ID=68975174
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910978700.0A Active CN110627849B (en) | 2019-10-15 | 2019-10-15 | Preparation method of N-acetylneuraminic acid standard substance |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110627849B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH02235897A (en) * | 1989-03-08 | 1990-09-18 | Mect Corp | N-acetylneuraminic acidsodium-trihydrate |
| CN109180749A (en) * | 2018-10-18 | 2019-01-11 | 中国科学院合肥物质科学研究院 | A method of high purity N-n acetylneuraminic acid n hydrate is prepared using supersaturated crystallisation |
| CN110305177A (en) * | 2019-07-04 | 2019-10-08 | 嘉必优生物技术（武汉）股份有限公司 | A kind of preparation method of N-acetyl-neuraminate |
-
2019
- 2019-10-15 CN CN201910978700.0A patent/CN110627849B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH02235897A (en) * | 1989-03-08 | 1990-09-18 | Mect Corp | N-acetylneuraminic acidsodium-trihydrate |
| CN109180749A (en) * | 2018-10-18 | 2019-01-11 | 中国科学院合肥物质科学研究院 | A method of high purity N-n acetylneuraminic acid n hydrate is prepared using supersaturated crystallisation |
| CN110305177A (en) * | 2019-07-04 | 2019-10-08 | 嘉必优生物技术（武汉）股份有限公司 | A kind of preparation method of N-acetyl-neuraminate |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110627849B (en) | 2023-02-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102659741B (en) | Method for extracting and preparing active ingredients of Ningxia wolfberries | |
| JP6114308B2 (en) | Rutin-enriched extract and method for producing the same | |
| WO2016101432A1 (en) | Crystal form i of canagliflozin and preparation method thereof | |
| CN110105458A (en) | The method that polysaccharide and PEARLITOL 25C are extracted in waste liquid is extracted from mogroside | |
| CN108250320B (en) | Low-ash ganoderma lucidum polysaccharide extract and preparation method thereof | |
| CN109701032A (en) | A kind of supercritical CO2Anti-solvent technology prepares myricetin/HP- beta-CD inclusion ultra-fine grain method | |
| CN109400569A (en) | A kind of method of extraction purification anthocyanidin in butterfly Tofu pudding | |
| CN110627849B (en) | Preparation method of N-acetylneuraminic acid standard substance | |
| CN108822229B (en) | Extraction method of lycium ruthenicum polysaccharide | |
| JP6768970B2 (en) | How to prepare rubusoside | |
| WO2016201905A1 (en) | New industrial crystallisation method for cefuroxime sodium and preparation thereof | |
| CN106496022B (en) | A method of extracting pyruvic acid from microbial fermentation solution or enzymatic conversion liquid | |
| CN108892732B (en) | Preparation method and application of Jinchang jujube polysaccharide with immunoregulation function | |
| CN109181352B (en) | Method for modifying black sesame melanin into water-soluble melanin | |
| CN116332822A (en) | Method for preparing low-chroma 5-hydroxytryptophan from garna seeds | |
| CN101683332B (en) | high purity scutellarin salt bulk drug and preparation method thereof | |
| CN110563584A (en) | method for extracting and purifying chlorogenic acid in chrysanthemum morifolium ramat | |
| CN105837546A (en) | Method for separating and purifying alnustone, pinocembrin, cardamomin and alpinetin from alpinia katsumadai | |
| CN109821027A (en) | A method of preparing hesperetin/HP- beta-CD inclusion | |
| CN104496815B (en) | A kind of preparation method of medicinal chlorogenic acid | |
| CN108440324B (en) | Ornithine aspartate and crystallization method thereof | |
| CN104402678B (en) | A kind of nerol, geraniol isomers inclusion separation method | |
| CN110240544A (en) | A kind of chlorogenic acid method for extraction and purification and application | |
| CN108440635B (en) | Separation method for improving yield of tea saponin | |
| CN106496020B (en) | A method of extracting α-ketoglutaric acid from microbial fermentation solution or enzymatic conversion liquid |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |