CN110627849B - Preparation method of N-acetylneuraminic acid standard substance - Google Patents
Preparation method of N-acetylneuraminic acid standard substance Download PDFInfo
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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, re-cooling, re-crystallizing, re-filtering, re-washing and re-drying, the N-acetylneuraminic acid with the purity of more than 99 percent 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, medicines, 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 C 11 H 19 NO 9 The 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 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 (4) 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.
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 crystallization of N-acetylneuraminic acid is insufficient, 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 N-acetylneuraminic acid is unstable, so that the decomposition easily occurs, 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%.
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 hydrolyzed and purified before use.
Preferably, the temperature of the first solution is 45-80 ℃, for example can be 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 450g/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 deg.C, such as 4 deg.C, 6 deg.C, 8 deg.C, 10 deg.C, 14 deg.C, 15 deg.C, 20 deg.C, 22 deg.C, 24 deg.C or 25 deg.C.
Preferably, the crystallization time in step (1) and step (3) is 4-36h, such as 4h, 6h, 8h, 10h, 14h, 15h, 20h, 24h, 28h, 30h, 34h or 36h.
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 invention preferably selects the mass ratio of the organic solvent to the water in the washing processes in the step (2) and the step (4) so as to give consideration to the comprehensive removal effect of water-soluble impurities and oil-soluble impurities in the N-acetylneuraminic acid raw material. 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 36h.
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 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 250-450g/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 (4) 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 be 1-3, placing at 4-20 ℃ for crystallization 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 300-400g/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 the 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 (4) washing and drying the third crystal obtained in the step (3) by using a mixed solvent containing isopropanol and water 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 N-acetylneuraminic acid raw material in water to obtain a first solution, adjusting the pH to 2, placing at 5 ℃ for crystallization for 6 hours, and filtering by adopting a 0.22 mu m membrane to obtain a first crystal, wherein the concentration of the first solution is 350g/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 chart 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 using 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 32 hours 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 (4) washing and drying the third crystal obtained in the step (3) by using a mixed solvent containing isopropanol and water 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 (4) washing and drying the third crystal obtained in the step (3) by using a mixed solvent containing isopropanol and water to obtain the N-acetylneuraminic acid standard substance, wherein the mass percentage of the ethanol in the mixed solvent is 80%.
Comparative example 1
The present comparative example provides a method for preparing N-acetylneuraminic acid, which comprises the following steps:
(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 crystals, 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) 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 6 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 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 at 60 ℃ for 26h 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 and drying the third crystal obtained in the step (3) 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 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 process 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 0.6ml/min; the detection wavelength is 210nm.
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 example 1, comparative example 1 and comparative example 4 omit the process of secondary washing and recrystallization, the purity of the obtained product is obviously 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.
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 example 1, comparative example 5 has exchanged the conditions of two crystallization, the first crystallization adopts the way of lowering the temperature, the second crystallization adopts the way of adjusting the pH value, but the purity of the obtained product is also lower than that of the N-acetylneuraminic acid obtained in example 1, the purity is lower than 99%, and the product can not be used as a standard substance. This indicates that standard substances with a purity of more than 99% can only be obtained in strict accordance with 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 5 th carbon atom 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 can result in 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 -1 3340cm as free hydroxyl group vibration absorption peak -1 2979cm as a stretching vibration absorption peak of carboxyl -1 Stretching vibration absorption peak of methyl group, 2933cm -1 The 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 -1 And 1263cm -1 There 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 (mJ =12.8,4.9Hz, 1H), 1.92-1.82 (s, 3H), 1.67 (m J =12, 1H).
The above data indicate 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 states that the present invention is described by the above embodiments to explain the detailed structural features of the present invention, but the present invention is not limited to the above detailed structural features, that is, it is not meant to imply that the present invention must be implemented by relying 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 (17)
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 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) 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;
the content of the N-acetylneuraminic acid monomer in the N-acetylneuraminic acid raw material in the step (1) is more than or equal to 80 percent;
the mass percentage of the organic solvent in the mixed solvent in the step (2) and the step (4) is respectively 70-80%.
2. The method according to claim 1, wherein the temperature of the first solution is 45 to 80 ℃.
3. The method according to claim 1, wherein the content of N-acetylneuraminic acid in the first solution is 250 to 450g/L.
4. The method of claim 1, wherein step (1) further comprises: the first solution is coarsely filtered using a 0.1-5 μm filter.
5. The method according to claim 1, wherein the temperature of the crystallization in the step (1) is 4 to 25 ℃.
6. The method according to claim 1, wherein the crystallization time in step (1) and step (3) is 4 to 36 hours.
7. The method according to claim 1, wherein the filtration in the steps (1) and (3) is performed by 0.22 μm membrane filtration, microfiltration membrane filtration or centrifugation.
8. The process according to claim 1, wherein the organic solvent in the step (2) and the step (4) is an N-acetylneuraminic acid-insoluble organic solvent.
9. The method according to claim 1, wherein the organic solvent in step (2) and step (4) is independently selected from any one of ethanol, isopropanol, and acetone, or a combination of at least two thereof.
10. The method according to claim 1, wherein the drying in steps (2) and (4) is performed by any one or a combination of at least two of hot air circulation, vacuum heating, and radiation heating.
11. The method according to claim 1, wherein the drying temperature in the steps (2) and (4) is 25 to 80 ℃.
12. The method according to claim 1, wherein the drying time in the steps (2) and (4) is 3 to 36 hours.
13. The method according to claim 1, wherein the temperature of the second solution in the step (3) is 45 to 80 ℃.
14. The method according to claim 1, wherein the temperature reduction in the step (3) is to reduce the temperature of the second solution to 4 to 20 ℃.
15. The method of claim 1, comprising the steps of:
(1) Dissolving N-acetylneuraminic acid raw material in water to obtain a first solution, adjusting the pH 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 250-450g/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%.
16. The method of claim 1, 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 300-400g/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 the 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%.
17. The method of claim 1, comprising the steps of:
(1) Dissolving 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 mu m membrane to obtain a first crystal, wherein the concentration of the first solution is 350g/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%.
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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 |
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| 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 |
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