An analytical packing material for separating glycated hemoglobin and its preparation method

Abstract

This application relates to the technical field of filler preparation, and discloses an analytical filler for separating glycated hemoglobin and a preparation method thereof. A 5-μm non-porous Polymer microsphere is synthesized by dispersion polymerization. The surface of the Polymer microsphere is hydrophilized to obtain a Polymer-based sphere. Finally, an ion exchange group is introduced onto the Polymer-based sphere to obtain an analytical filler for separating glycated hemoglobin. The hydrophilic treatment enhances the hydrophilicity of the matrix surface, reduces non-specific adsorption, and at the same time does not affect the swelling property of the filler in water or the mechanical strength. Through the preparation method of the analytical filler provided by this application, a glycated hemoglobin analytical filler with uniform particle size, good separation effect and long service life can be obtained.

Description

Technical Field

[0001] The present invention relates to the technical field of filler preparation, and particularly to an analytical filler for separating glycated hemoglobin and a preparation method thereof. Background Art

[0002] Glycated hemoglobin (A1c) is a type of hemoglobin that can reflect the average sugar concentration in the blood over the past 1 to 2 months. Therefore, it can be used as a screening test for diabetes and is widely used as a detection item for evaluating the blood glucose control status of diabetic patients. The HPLC method is widely used as a method for measuring hemoglobin A1c because of its good accuracy and the ability to measure in a short time. In the detection, it is required that the filler matrix has a certain hydrophobicity to reduce swelling in water, but too high hydrophobicity will lead to poor dispersibility of the filler and cause problems of non-specific adsorption. Generally, the method for improving the hydrophilicity of the filler is to introduce a large amount of hydrophilic monomers in the synthesis of the base beads, but this will lead to a decrease in the mechanical strength of the filler and swelling in water, resulting in a decrease in accuracy.

[0003] In addition, the fillers for measuring hemoglobin A1c in the past were mostly synthesized by suspension polymerization, and there was a problem of uneven particle size distribution. In order to make the particle size distribution uniform, methods such as classification operations were generally used. However, the classification operation is complex, the product loss is large, and the current classification operation has limitations in adjusting the particle size distribution width and cannot fully suppress the problem of peak broadening caused by sample diffusion due to uneven particle size distribution.

[0004] Therefore, the present application provides a preparation method of an analytical filler for separating glycated hemoglobin to overcome the above problems. Summary of the Invention

[0005] The present application provides an analytical filler for separating glycated hemoglobin and a preparation method thereof. The analytical filler has high durability, can accurately measure glycated hemoglobin, and can achieve good separation of hemoglobin.

[0006] In order to solve the above problems, the technical solutions adopted in the present application are as follows:

[0007] On the one hand, the present application provides a preparation method of an analytical filler for separating glycated hemoglobin, including the following steps:

[0008] (1) Mix and emulsify a monomer, a crosslinking agent, an initiator, and an aqueous solution of polyvinyl alcohol, heat and stir, soak in 95% ethanol and shake to remove the pore-forming agent, and obtain non-porous Polymer seed beads with a particle size of 5 μm;

[0009] (2) React the Polymer seed beads obtained in step (1) with a strong acidic solution, a strong basic solution, or a chloromethylation reagent under heating conditions to obtain Polymer base beads;

[0010] (3) Mix and stir the Polymer-based spheres, allyl glycidyl ether, and Na2SO4 solution obtained in step (2), and then add a strong alkaline solution to react to obtain Polymer-vinyl microspheres.

[0011] (4) Heat and react the Polymer-vinyl microspheres obtained in step (3) with a ligand monomer under oxygen conditions to obtain Polymer-COOH or Polymer-SO3 microspheres, which are 5-μm cation exchange fillers.

[0012] Further, in step (1), the monomer is one or more of methyl methacrylate monomers, ethyl methacrylate, cyclohexyl methacrylate, vinyl glycidyl ether, glycidyl methacrylate monomers, and styrene monomers. The monomer is a hydrophobic monomer, and its function is to prevent the filler from swelling or hardly swelling in an aqueous medium. The monomer can synthesize a hydrophobic cross-linked polymer.

[0013] Further, in step (1), the cross-linking agent is one or more of divinylbenzene, ethylene glycol dimethacrylate, trimethylolpropane trimethacrylate, triethylene glycol dimethyl phthalate, and pentaerythritol tetramethacrylate. Further still, the cross-linking agent is divinylbenzene.

[0014] Further, in step (1), the initiator is one or more of azobisisobutyronitrile, dimethyl azobisisobutyrate, benzoyl peroxide, and diacyl peroxide. Further still, the initiator is azobisisobutyronitrile.

[0015] Further, in step (1), the stabilizer is selected from one or more of polyvinyl alcohol, polyoxyethylene, polyethylene glycol, polyvinylpyrrolidone, hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxymethylpropyl cellulose, and sodium polyaspartate. Further still, the stabilizer is polyvinyl alcohol.

[0016] Further, in step (1), the temperature of the heating and stirring is 70-90 °C, and the time is 10-13 h. Further still, the time is 12 h.

[0017] Further, in step (1), the shaking time is 6 h.

[0018] Further, in step (1), the Polymer seed spheres are Polymer seed spheres with a particle size of 5 μm and no pores on the surface.

[0019] Further, in step (2), the strong acid solution is selected from one or more of sulfuric acid, hydrochloric acid, and nitric acid. Further still, in step (2), the strong acid solution is sulfuric acid.

[0020] Further, in step (2), the strong base solution is selected from one or more of NaOH, KOH, or LiOH. Further still, in step (2), the strong base solution is NaOH.

[0021] Further, in step (2), the chloromethylation reagent is selected from one or more of chloromethyl methyl ether or chloromethyl ethyl ether.

[0022] Further, in step (2), the heating temperature is 25 - 70 °C and the time is 20 - 24 h.

[0023] Further, in step (3), the strong base solution is NaOH.

[0024] Further, in step (3), the stirring conditions are: the rotation speed is 160 rpm and the time is 2 h.

[0025] Further, in step (3), the reaction conditions are: the rotation speed is 160 rpm, the temperature is 25 - 30 °C, and the time is 14 - 16 h.

[0026] Further, in step (4), the ligand monomer is selected from one or more of mercaptosuccinic acid, mercaptoglutaric acid, acrylic acid, crotonic acid, fumaric acid, glutaconic acid, NaHSO3, and Na2SO3. Further still, the ligand monomer is selected from one or more of mercaptosuccinic acid and NaHSO3.

[0027] Further, in step (4), the heating temperature is 40 - 70 °C and the time is 10 - 12 h.

[0028] The mechanism of action of this application is as follows:

[0029] 1. Synthesis of 5 - μm non - porous Polymer - based spheres by dispersion polymerization of hydrophobic monomers:

[0030] To prevent the filler from swelling or hardly swelling in the aqueous medium, hydrophobic monomers are selected to synthesize hydrophobic cross - linked polymers as the base spheres. The specific method is as follows:

[0031] Using hydrophobic monomers such as methyl methacrylate (MMA), glycidyl methacrylate - type monomers (GMA), styrene - type monomers (St), etc., 5 - μm non - porous PMMA microspheres, PGMA microspheres, and PSDVB microspheres with uniform particle size are synthesized by dispersion polymerization, collectively referred to as Polymer seed spheres;

[0032] PMMA microspheres are hydrolyzed under alkaline conditions through ester bonds to form PMMA-COOH microspheres, or PGMA microspheres are hydrolyzed under acidic conditions through epoxy groups to form PGMA-OH microspheres, or PSDVB is synthesized into PSDVB-Cl microspheres by chloromethylation to replace the H on the benzene ring. The above three microspheres are collectively referred to as Polymer-based spheres, that is, hydrophobic cross-linked polymer-based spheres are obtained.

[0033] 2. Hydrophilize the Polymer-based spheres:

[0034] Hydrophilize the surface of the hydrophobic cross-linked polymer-based spheres. After functionalizing the base spheres, hydrophilic blocks are introduced for hydrophilization treatment. For example, the hydrophobic cross-linked polymer-based spheres are carboxylated, hydroxylated, chloromethylated, etc. Select compounds such as allyl glycidyl ether with dual reactive functional groups of allyl and epoxy groups, and react with the functional groups through the ring-opening reaction of the epoxy group for the hydrophilization treatment of the base sphere surface.

[0035] By using allyl glycidyl ether, the functional groups of the Polymer-based spheres react with the epoxy groups through a ring-opening reaction to obtain PMMA-vinyl or PGMA-vinyl or PSDVB-vinyl microspheres with hydrophilic groups on the surface, which are collectively referred to as Polymer-vinyl microspheres.

[0036] 3. Introduce ion exchange groups into the Polymer-vinyl microspheres:

[0037] Introduce ion exchange groups such as sulfonic acid groups and carboxyl groups onto the hydrophilized polymer-based spheres, which can be introduced through chemical reactions between functional groups, such as the reaction between double bonds and mercapto groups, or the oxidation reaction of double bonds, etc. Specifically:

[0038] React the Polymer-vinyl microspheres with a ligand monomer containing a mercapto group, such as mercaptosuccinic acid or mercaptoglutaric acid, through the reaction between the double bond on the microsphere surface and the mercapto group to form Polymer-COOH microspheres;

[0039] Or oxidize the double bond on the microsphere surface with NaHSO3 to form Polymer-SO3 microspheres.

[0040] On the other hand, the present application also provides an analytical packing material for separating glycated hemoglobin, and the analytical packing material is prepared by the above preparation method.

[0041] Advantages of the present application:

[0042] An analytical packing material for separating glycated hemoglobin provided by the present application uses a hydrophobic monomer to synthesize a hydrophobic cross-linked polymer, i.e., 5-μm non-porous microspheres as the base spheres through dispersion polymerization. At the same time, the hydrophilicity of the matrix surface is enhanced through hydrophilic treatment, reducing non-specific adsorption, without affecting the swelling property of the packing material in water, nor the mechanical strength. The polymer after hydrophilic treatment can improve the salt tolerance and reduce non-specific adsorption. Non-specific adsorption is actually an adsorption effect caused by non-covalent bond forces, which may be triggered by the hydrophobic interaction between the compound to be measured in the solution and the solid surface. In the application of separation packing materials, this non-specific adsorption may lead to non-specific binding between the target molecule and the packing material, thus affecting the separation efficiency and purity. In contrast, specific adsorption is a specific binding between the packing material and the target molecule, based on specific intermolecular interactions. The analytical packing material prepared by the preparation method of the present application has uniform particle size, good separation effect and long service life. Description of the Drawings

[0043] Figure 1 SEM images of PMMA microspheres prepared in Examples 1-2.

[0044] Figure 2 SEM images of PGMA microspheres prepared in Examples 3-4.

[0045] Figure 3 SEM images of PSDVB microspheres prepared in Examples 5-6.

[0046] Figure 4 HPLC linear elution diagram of the packing material prepared in Example 1 injected into a GlyHb-HNmicro 4.6*20mm metal pre-packed column. Detailed Embodiments

[0047] The present invention will be further described below with reference to the drawings. The following examples are only used to illustrate the technical solution of the present invention more clearly, and cannot be used to limit the protection scope of the present invention.

[0048] Before introducing the embodiments of the present application, the following interpretations are made for the relevant terms involved in the embodiments of the present application:

[0049] Hemoglobin: The product of the combination of hemoglobin in red blood cells and glucose in serum. This combination is formed through a non-enzymatic glycosylation reaction, and the main carbohydrate is glucose. Hemoglobin glycosylation is a slow and relatively irreversible process, usually lasting more than three months. The formation of glycated hemoglobin is related to the blood glucose concentration and the contact time between blood glucose and hemoglobin, and is independent of factors such as the blood sampling time, whether the patient is fasting or using insulin.

[0050] Strong cation exchange packing: Strong cation exchange packing is a type of material with high hydrophilicity, usually based on substrates such as glycidyl methacrylate microspheres, and its performance is enhanced through surface modification. This type of packing is mainly used for protein analysis, rapid and efficient separation, analysis, and purification of biological macromolecules.

[0051] SEM test: That is, Scanning Electron Microscope test, which is a microscopic analysis technique that uses a high-energy electron beam to scan the surface of a sample and capture signals to generate high-resolution images. In SEM, a high-energy electron beam is emitted onto the surface of the sample, and these electrons interact with the sample to generate signals such as secondary electrons and backscattered electrons. These electron signals are captured by the detector and converted into electrical signals, and then an image is formed to display the microscopic structure of the sample surface.

[0052] O2 bubbling: It is a technique that injects oxygen into a liquid in the form of bubbles to increase the solubility and utilization rate of oxygen.

[0053] Chromatographic column: A chromatographic column is a key component used in high-performance liquid chromatography (HPLC) and other chromatographic techniques, and its main function is to separate different components in a mixture.

[0054] Example 1

[0055] This example provides a preparation method for an analytical packing for separating glycated hemoglobin, including the following steps:

[0056] (1) Mix 10 g of methyl methacrylate (MMA), 3 g of divinylbenzene (DVB), 0.2 g of azobisisobutyronitrile (AIBN), and an aqueous solution of 5% polyvinyl alcohol, emulsify at 1000 rpm for 5 min, with an average droplet size of 6 μm, and raise the temperature to 70 °C and stir for 12 h. Wash with ethanol and water repeatedly 3 times to remove unreacted monomers and oligomers, obtain PMMA microspheres with a particle size of 5 μm and a non-porous surface, dry at 80 °C and store for later use. Its SEM test image is as Figure 1 shown. Through SEM observation, the surface morphology and particle size of the microspheres can be intuitively seen. It can be seen from Figure 1 that the PMMA microspheres are evenly distributed, with an average particle size of 5 μm.

[0057] (2) Take 10 g of PMMA microsphere powder, add 20 g of 0.8 M NaOH aqueous solution, raise the temperature to 70 °C, react under the condition of 180 rpm for 24 h, add 40 g of 0.8 M HCl, wash with ethanol and water repeatedly 3 times to obtain PMMA-COOH microspheres, that is, Polymer base spheres, wash with ethanol and water repeatedly 3 times, dry at 80 °C and store for later use.

[0058] (3) Take 10 g of Polymer-based spheres, 5 g of allyl glycidyl ether, 0.2 g of Na2SO4, and 100 g of a 50% dimethyl sulfoxide aqueous solution. Mix the solutions and stir at 160 rpm for 2 h. Add NaOH solution and react at 30 °C for 16 h to obtain Polymer-vinyl microspheres. Wash them repeatedly with ethanol and water three times and dry them at 80 °C for storage for later use.

[0059] (4) Take 5 g of Polymer-vinyl microspheres, 2.5 g of mercaptosuccinic acid, 100 g of water, and 0.1 g of azobisisobutyronitrile. React at 180 rpm and 70 °C for 12 h to obtain Polymer-COOH microspheres. Wash them repeatedly with ethanol and water three times and dry them in a blast drying oven at 90 °C to obtain a weakly cation-exchange packing with an average particle size of 5 μm.

[0060] Example 2

[0061] This example provides a method for preparing an analytical packing for separating glycated hemoglobin, which includes the following steps:

[0062] (1) Mix 10 g of methyl methacrylate (MMA), 3 g of divinylbenzene (DVB), 0.2 g of azobisisobutyronitrile (AIBN), and a 5% polyvinyl alcohol aqueous solution. Emulsify at 1000 rpm for 5 min with an average droplet size of 6 μm. Heat up to 70 °C and stir and react for 12 h. Wash repeatedly with ethanol and water three times to remove unreacted monomers and oligomers to obtain PMMA microspheres with a particle size of 5 μm and a non-porous surface. Dry them at 80 °C for storage for later use. Its SEM test image is as Figure 1 shown. Through SEM observation, the surface morphology and particle size of the microspheres can be intuitively seen. From Figure 1 it can be seen that the PMMA microspheres are evenly distributed with an average particle size of 5 μm.

[0063] (2) Take 10 g of PMMA microsphere powder, add 20 g of 0.8 M NaOH aqueous solution, heat up to 70 °C, and react at 180 rpm for 24 h. Add 40 g of 0.8 M HCl and wash repeatedly with ethanol and water three times to obtain PMMA-COOH microspheres, i.e., Polymer-based spheres. Wash repeatedly with ethanol and water three times and dry them at 80 °C for storage for later use.

[0064] (3) Take 10 g of Polymer-based spheres, 5 g of allyl glycidyl ether, 0.2 g of Na2SO4, and 100 g of a 50% dimethyl sulfoxide aqueous solution. Mix the solutions and stir at 160 rpm for 2 h. Add NaOH solution and react at 30 °C for 16 h to obtain Polymer-vinyl microspheres. Wash them repeatedly with ethanol and water three times and dry them at 80 °C for storage for later use.

[0065] (4) Take 5 g of Polymer-vinyl microspheres, 3.2 g of NaHSO3, 100 g of water, bubble O2 at 2 bubbles / s, 180 rpm, react at 40 °C for 12 h to obtain Polymer-SO3 microspheres, wash them repeatedly with ethanol and water 3 times, and dry them in a forced-air drying oven at 90 °C to obtain a strong cation exchange packing with an average particle size of 5 μm.

[0066] Example 3

[0067] This example provides a preparation method of an analytical packing for separating glycated hemoglobin, including the following steps:

[0068] (1) Mix 10 g of glycidyl methacrylate (GMA), 3 g of divinylbenzene (DVB), 0.15 g of azobisisobutyronitrile (AIBN) and 5% polyvinyl alcohol aqueous solution, emulsify at 1200 rpm for 5 min, with an average droplet size of 6 μm, heat up to 70 °C and stir to react for 12 h. Wash repeatedly with ethanol and water 3 times to remove unreacted monomers and oligomers to obtain PGMA microspheres with a particle size of 5 μm and no pores on the surface, dry them at 80 °C for storage and standby. Its SEM test image is as Figure 2 shown. Through SEM observation, the surface morphology and particle size of the microspheres can be visually seen. From Figure 2 it can be seen that the PGMA microspheres are evenly distributed with an average particle size of 5 μm.

[0069] (2) Take 10 g of PGMA sphere powder, add 20 g of 0.8 M H2SO4 aqueous solution, heat up to 70 °C, react under the condition of 180 rpm for 24 h, wash repeatedly with ethanol and water 3 times to obtain PGMA-OH microspheres, that is, Polymer-based spheres, wash repeatedly with ethanol and water 3 times, and dry them at 80 °C for storage and standby.

[0070] (3) Take 10 g of Polymer-based spheres, 5 g of allyl glycidyl ether, 0.2 g of Na2SO4, 100 g of 50% dimethyl sulfoxide aqueous solution, mix the solutions, stir at 160 rpm for 2 h, then add NaOH solution, and react at 30 °C for 16 h to obtain Polymer-vinyl microspheres, wash repeatedly with ethanol and water 3 times, and dry them at 80 °C for storage and standby.

[0071] (4) Take 5 g of Polymer-vinyl microspheres, 2.5 g of mercaptosuccinic acid, 100 g of water, 0.1 g of AIBA, react at 180 rpm and 70 °C for 12 h to obtain Polymer-COOH microspheres, wash repeatedly with ethanol and water 3 times, and dry them in a forced-air drying oven at 90 °C to obtain a weak cation exchange packing with an average particle size of 5 μm.

[0072] Example 4

[0073] This embodiment provides a preparation method of an analytical packing material for separating glycated hemoglobin, which includes the following steps:

[0074] (1) Mix 10 g of glycidyl methacrylate (GMA), 3 g of divinylbenzene (DVB), 0.15 g of azobisisobutyronitrile (AIBN) and 5% polyvinyl alcohol aqueous solution, emulsify at 1200 rpm for 5 min, with an average droplet size of 6 μm, heat up to 70 °C and stir and react for 12 h. Wash repeatedly with ethanol and water for 3 times to remove unreacted monomers and oligomers, obtain PGMA microspheres with a particle size of 5 μm and a pore-free surface, dry at 80 °C and store for later use. Its SEM test diagram is as Figure 2 shown. Through SEM observation, the surface morphology and particle size of the microspheres can be intuitively seen. It can be seen from Figure 2 that the PGMA microspheres are evenly distributed and the average particle size is 5 μm.

[0075] (2) Take 10 g of PGMA microsphere powder and add 20 g of 0.8 M H2SO4 aqueous solution, heat up to 70 °C, and react under the condition of 180 rpm for 24 h. Wash repeatedly with ethanol and water for 3 times to obtain PGMA-OH microspheres, that is, Polymer-based spheres. Wash repeatedly with ethanol and water for 3 times, dry at 80 °C and store for later use.

[0076] (3) Take 10 g of Polymer-based spheres, 5 g of allyl glycidyl ether, 0.2 g of Na2SO4, and 100 g of 50% dimethyl sulfoxide aqueous solution, mix the solutions, stir at 160 rpm for 2 h, then add NaOH solution, and react at 30 °C for 16 h to obtain Polymer-vinyl microspheres. Wash repeatedly with ethanol and water for 3 times, dry at 80 °C and store for later use.

[0077] (4) Take 5 g of Polymer-vinyl microspheres, 3.2 g of NaHSO3, 100 g of water, bubble O2 at 2 bubbles / s, 180 rpm, and react at 40 °C for 12 h to obtain Polymer-SO3 microspheres. Wash repeatedly with ethanol and water for 3 times, and dry in a forced-air drying oven at 90 °C to obtain a strong cation exchange packing material with an average particle size of 5 μm.

[0078] Example 5

[0079] This embodiment provides a preparation method of an analytical packing material for separating glycated hemoglobin, which includes the following steps:

[0080] (1) 10 g of styrene (St), 3 g of divinylbenzene (DVB), 0.1 g of benzoyl peroxide (BPO), and an aqueous solution of 4% polyvinyl alcohol were mixed and emulsified at 900 rpm for 5 min. The average droplet size was 6 μm. The temperature was raised to 90 °C and the reaction was stirred for 12 h. Ethanol and water were repeatedly washed 3 times to remove unreacted monomers and oligomers, obtaining PSDVB microspheres with an average size of 5 μm and a pore-free surface. They were dried at 80 °C and stored for later use. The SEM test image is as shown in Figure 3 shown. By observing with SEM, the surface morphology and particle size of the microspheres can be visually seen. As shown in Figure 3 it can be seen that the PSDVB microspheres are evenly distributed with an average particle size of 5 μm.

[0081] (2) 10 g of PSDVB microsphere powder was taken, 2.8 g of paraformaldehyde, 90 mL of dichloroethane, and 20 g of concentrated hydrochloric acid were added. The temperature was raised to 25 °C and the reaction was carried out at 180 rpm for 24 h. Ethanol and water were repeatedly washed 3 times to obtain PSDVB-Cl microspheres, i.e., Polymer-based spheres. Ethanol and water were repeatedly washed 3 times and dried at 80 °C for storage. (Among them, dichloroethane is used as a solvent, and paraformaldehyde and concentrated hydrochloric acid are used to chloromethylate the PSDVB microspheres. Through the electrophilic substitution of the benzene ring by paraformaldehyde, formaldehyde reacts with hydrogen chloride to form chloromethyl aromatic compounds with aromatic compounds)

[0082] (3) 10 g of Polymer-based spheres, 2.2 g of ethylene glycol, 5 g of allyl glycidyl ether, 0.2 g of Na2SO4, and 100 g of a 50% aqueous solution of dimethyl sulfoxide were mixed and stirred at 160 rpm for 2 h, then an NaOH solution was added, and the reaction was carried out at 30 °C for 16 h to obtain Polymer-vinyl microspheres. Ethanol and water were repeatedly washed 3 times and dried at 80 °C for storage.

[0083] (4) 5 g of Polymer-vinyl microspheres, 2.5 g of mercaptobutanedioic acid, 100 g of water, 0.1 g of AIBA were taken, and the reaction was carried out at 180 rpm and 70 °C for 12 h to obtain Polymer-COOH microspheres. Ethanol and water were repeatedly washed 3 times and dried in a forced-air drying oven at 90 °C, obtaining a weakly cation-exchange packing with an average particle size of 5 μm.

[0084] Example 6

[0085] This example provides a preparation method of an analytical packing for separating glycated hemoglobin, including the following steps:

[0086] (1) 10 g of styrene (St), 3 g of divinylbenzene (DVB), 0.1 g of benzoyl peroxide (BPO) and an aqueous solution of 4% polyvinyl alcohol were mixed, emulsified at 900 rpm for 5 min, with an average droplet size of 6 μm, heated to 90 °C, and stirred and reacted for 12 h. Ethanol and water were used to wash repeatedly for 3 times to remove unreacted monomers and oligomers, obtaining PSDVB microspheres with an average diameter of 5 μm and a pore-free surface. They were dried at 80 °C and stored for later use. The SEM test image is as shown in Figure 3 shown. Through SEM observation, the surface morphology and particle size of the microspheres can be visually seen. From Figure 3 it can be seen that the PSDVB microspheres are evenly distributed with an average particle size of 5 μm.

[0087] (2) 10 g of PSDVB microsphere powder was taken, 2.8 g of paraformaldehyde, 90 mL of dichloroethane, and 20 g of concentrated hydrochloric acid were added, heated to 25 °C, and reacted at 180 rpm for 24 h. Ethanol and water were used to wash repeatedly for 3 times to obtain PSDVB-Cl microspheres, namely Polymer-based spheres. Ethanol and water were used to wash repeatedly for 3 times, dried at 80 °C and stored for later use. (Among them, dichloroethane is used as a solvent, and paraformaldehyde and concentrated hydrochloric acid are used to carry out chloromethylation modification on PSDVB microspheres. Through electrophilic substitution of the benzene ring by paraformaldehyde, formaldehyde reacts with hydrogen chloride to form chloromethyl aromatic compounds with aromatic compounds)

[0088] (3) 10 g of Polymer-based spheres, 2.2 g of ethylene glycol, 5 g of allyl glycidyl ether, 0.2 g of Na2SO4, and 100 g of a 50% aqueous solution of dimethyl sulfoxide were mixed, stirred at 160 rpm for 2 h, then a NaOH solution was added, and the reaction was carried out at 30 °C for 16 h to obtain Polymer-vinyl microspheres. Ethanol and water were used to wash repeatedly for 3 times, dried at 80 °C and stored for later use.

[0089] (4) 5 g of Polymer-vinyl microspheres, 3.2 g of NaHSO3, 100 g of water, O2 was bubbled at 2 bubbles / s, at 180 rpm, and reacted at 40 °C for 12 h to obtain Polymer-SO3 microspheres. They were washed with water and alcohol, and dried in a hot air drying oven at 90 °C, thus obtaining strong cation exchange packing with an average particle size of 5 μm.

[0090] Performance test:

[0091] 1. Loading capacity test

[0092] The packing prepared in Example 1 was packed into a column (4.6 * 20 mm mL), and the loading capacity test was carried out at a flow rate of 0.5 mL / min.

[0093] The solution preparation method is as follows:

[0094] Equilibrium solution: 10 mM phosphate buffer solution (pH 6.3);

[0095] Sample loading solution: 1 mg / mL lysozyme solution;

[0096] Washing solution: 10 mM phosphate buffer solution (pH 6.3);

[0097] Elution solution: 1 M NaCl 10 mM phosphate buffer solution (pH 7.3).

[0098] The specific experimental method is as follows:

[0099] (1) Chromatographic column installation and equilibration: Correctly install the GlyHb-HNmicro 4.6×20 mm metal pre-packed column into the HPLC system. Flush the chromatographic column with 10 mM phosphate buffer solution (pH 6.3) at an appropriate flow rate of 0.5 - 1.0 mL / min until the baseline is stable to remove possible impurities in the column and reach the equilibrium state.

[0100] (2) Sample preparation: Accurately weigh a certain amount of lysozyme standard, dissolve it with 10 mM phosphate buffer solution (pH 6.3) and dilute it to an appropriate concentration (such as 0.1 - 1.0 mg / mL). Use a microsyringe to inject the sample into the injector of the HPLC system.

[0101] (3) Chromatographic analysis: Start the HPLC system, set appropriate flow rate, column temperature and detection wavelength (usually 280 nm). After injecting the sample, observe and record the chromatogram. Pay attention to the elution time, peak shape and purity of lysozyme.

[0102] During the whole experimental process, it should be ensured that all operations are carried out under sterile conditions to avoid contamination.

[0103] The loading capacity is calculated by the following formula, calculation method:

[0104] Loading capacity = OD 洗脱液 / OD 上样液 ×V 洗脱液 (mg / mL),

[0105] In this Example 1, the loading capacity reaches 11.2 mg / mL, and this packing has excellent adsorption performance.

[0106] 2. Pressure resistance effect test

[0107] The specific experimental method is as follows:

[0108] (1) Pack the filler prepared in Example 1 into a column (4.6 * 20 mm mL), set a series of different flow rate values, usually starting from the minimum flow rate recommended for the chromatographic column, and gradually increase it to the maximum flow rate or close to the system pressure limit. Set the composition and pH value of the mobile phase, and adjust according to the specific requirements of the GlyHb-HNmicro chromatographic column. Set parameters such as column temperature and detection wavelength.

[0109] (2) Run the chromatographic column at each set flow rate in sequence, and record the system pressure at each flow rate. The pressure data can be automatically recorded by the software of the HPLC system or manually recorded. Observe and record the elution curve of the chromatographic column to check for any abnormal phenomena (such as peak shape distortion, tailing, etc.).

[0110] Plot a graph of the relationship between flow rate and pressure, and analyze the change trend of pressure with flow rate. Evaluate the pressure stability of the chromatographic column at different flow rates.

[0111] The test results of the pressure and flow rate of the GlyHb-HNmicro 4.6 * 20 mm metal pre-packed column are shown in Table 1, and the results show that the filler prepared in Example 1 has excellent pressure resistance performance.

[0112] Table 1 Pressure and Flow Rate Data Table

[0113] Flow rate (mL / min) 1 2 3 4 5 6 7 Pressure (Mpa) 1.96 3.63 5.25 6.75 8.16 9.43 10.80

[0114] 3. Linear Elution Test

[0115] Test solution: Whole blood

[0116] Solution A: Phosphate buffer solution with pH 5.0;

[0117] Solution B: Phosphate buffer solution with pH 7.2;

[0118] Solution C: EDTA hemolytic agent.

[0119] The specific experimental method is as follows:

[0120] Inject the filler prepared in Example 1 into the injector of the HPLC system, and start the system for analysis.

[0121] During the analysis process, the HPLC system will pump the mobile phase into the GlyHb-HNmicro 4.6 * 20 mm metal pre-packed column according to the set parameters. The components in the sample are separated in the column and are detected by the detector in sequence. The software of the PLC system will automatically record the elution time (or elution volume) of each component and the corresponding signal intensity (such as peak height or peak area).

[0122] Export these data and plot them into a linear elution graph. As Figure 4As shown, the abscissa usually represents the elution time, and the ordinate represents the signal intensity. The results show that the packing material has excellent separation performance for a1b1, F, LA1C, and SA1C proteins in whole blood.

[0123] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the technical principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A method for preparing an analytical packing material for separating glycated hemoglobin, characterized in that, It includes the following steps: (1) Mix and emulsify the monomer, crosslinking agent, initiator and stabilizer, heat and stir, soak with ethanol and shake to obtain non-porous Polymer seed balls with a particle size of 5 μm; (2) React the Polymer seed balls obtained in step (1) with a strong acid solution or a strong base solution or a chloromethylating reagent under heating conditions to obtain Polymer base balls; (3) Mix and stir the Polymer base balls obtained in step (2), allyl glycidyl ether and Na2SO4 solution, and then add a strong base solution to react to obtain Polymer-vinyl microspheres; (4) React the Polymer-vinyl microspheres obtained in step (3) with a ligand monomer under heating conditions to obtain Polymer-COOH or Polymer-SO3 microspheres, which are the analytical packing materials for separating glycated hemoglobin; In step (1), the monomer is one or more of methyl methacrylate monomers, ethyl methacrylate, cyclohexyl methacrylate, vinyl glycidyl ether, glycidyl methacrylate monomers, and styrene monomers; In step (2), the strong acid solution is one or more of sulfuric acid, hydrochloric acid, and nitric acid, the strong base solution is one or more of NaOH, KOH, and LiOH, and the chloromethylating reagent is selected from one or more of chloromethyl methyl ether or chloromethyl ethyl ether; In step (4), the ligand monomer is one or more of mercaptosuccinic acid, mercaptoglutaric acid, acrylic acid, crotonic acid, fumaric acid, glutaconic acid, NaHSO3, and Na2SO3.

2. The preparation method of an analytical packing material for separating glycated hemoglobin according to claim 1, wherein, In step (1), the crosslinking agent is one or more of divinylbenzene, ethylene glycol dimethacrylate, trimethylolpropane trimethacrylate, triethylene glycol dimethyl phthalate, and pentaerythritol tetramethacrylate; 3. The preparation method of an analytical packing material for separating glycated hemoglobin according to claim 1, characterized in that, In step (1), the initiator is one or more of azobisisobutyronitrile, dimethyl azobisisobutyrate, benzoyl peroxide, and diacyl peroxide; 4. The preparation method of an analytical packing material for separating glycated hemoglobin according to claim 1, characterized in that, In step (1), the stabilizer is selected from one or more of polyvinyl alcohol, polyoxyethylene, polyethylene glycol, polyvinylpyrrolidone, hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxymethylpropyl cellulose, and sodium polyaspartate; 5. The preparation method of an analytical packing material for separating glycated hemoglobin according to claim 1, characterized in that, In step (1), the temperature of heating and stirring is 70-90 °C, and the time is 10-13 h.

6. The preparation method of an analytical packing material for separating glycated hemoglobin according to claim 1, characterized in that, In step (2), the heating temperature is 25-70 °C, and the time is 20-24 h.

7. The preparation method of an analytical packing material for separating glycated hemoglobin according to claim 1, characterized in that, In step (3), the reaction temperature is 25-30 °C, and the time is 14-16 h.

8. The preparation method of an analytical packing material for separating glycated hemoglobin according to claim 1, characterized in that, In step (4), the heating temperature is 40-70 °C, and the time is 10-12 h.

9. An analytical packing material for separating glycated hemoglobin, characterized in that, The analytical packing material is prepared by the preparation method described in any one of claims 1-8.

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