A method for removing vitamin d from horse serum

Abstract

The present application relates to the biomedical field, disclose a kind of method for removing vitamin D in horse serum, comprising: material screening;25(OH)D in horse serum is physically adsorbed using selected material;After physical adsorption, horse serum is centrifuged;After centrifugation, horse serum is filtered using filter membrane;Selected material is dextran treatment modified activated carbon, dextran treatment modified activated carbon is repeatedly obtained by immersing treatment of modified activated carbon in saturated dextran solution;The mass ratio of dextran and modified activated carbon is 2-10:1;Modified activated carbon is obtained by bonding hydrophobic activated carbon using nitric acid pretreatment activated carbon and diphenyl dimethoxysilane, then using phytic acid to treat hydrophobic activated carbon and calcining to make, the method is efficient, low cost, can remove 25(OH)D in horse serum in scale, it also has high specificity, only remove 25(OH)D, retain total protein in horse serum;Simple operation, without complex equipment.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a method for removing vitamin D from horse serum. Background Technology

[0002] 25-Hydroxyvitamin D (abbreviated as 25(OH)D) is the main form of vitamin D in horse serum. In the production and research of certain in vitro diagnostic projects, in order to ensure the accuracy of experiments and the stability of production, and to reduce interference factors, it is necessary to remove 25(OH)D from horse serum to obtain horse serum that contains no 25(OH)D or contains very low levels of 25(OH)D. This is suitable for clinical test kits, pretreatment of horse serum matrix materials for reference materials, and biological sample analysis, and can significantly improve the detection accuracy of diagnostic reagents.

[0003] Currently, the removal of 25(OH)D from serum, the raw material for in vitro diagnostic reagents, mainly relies on the following methods: solid-phase extraction (SPE) using C... 18 Hydrophobic materials can adsorb lipid-soluble substances, but their selectivity is poor; organic solvent precipitation methods use solvents such as acetonitrile and methanol for extraction, and organic solvents or harsh conditions can easily denature serum proteins, affecting reagent performance; immunoaffinity chromatography uses 25(OH)D antibody immobilization packing material, but high-purity antibody packing material is expensive and the operation is complicated, making it difficult to apply on a large scale. Summary of the Invention

[0004] To address the shortcomings mentioned in the background section, the present invention aims to provide a method for removing vitamin D from horse serum, thereby overcoming the defects of existing methods for removing 25(OH)D from horse serum, such as poor selectivity, low specificity, high cost, and complex operation.

[0005] The objective of this invention can be achieved through the following technical solutions:

[0006] A method for removing vitamin D from horse serum includes the following steps: (1) material screening; (2) physical adsorption of 25(OH)D from horse serum using the selected material; (3) centrifugation of the physically adsorbed horse serum; and (4) filtration of the centrifuged horse serum using a filter membrane.

[0007] The selected material is dextran-treated modified activated carbon, which is obtained by repeatedly immersing modified activated carbon in a saturated dextran solution.

[0008] The mass ratio of the dextran to the modified activated carbon is 2~10:1;

[0009] The modified activated carbon is prepared by pretreating activated carbon with nitric acid, then bonding the pretreated activated carbon with diphenyldimethoxysilane to obtain hydrophobic activated carbon, and then treating the hydrophobic activated carbon with phytic acid and calcining it at high temperature.

[0010] Preferably, the method for preparing the modified activated carbon includes the following steps:

[0011] (1) Take activated carbon and wash it 3 to 5 times with nitric acid and deionized water with a concentration of 0.5 to 1 mol / L, and then dry it at 110 to 125℃ for 3 to 5 hours to prepare pretreated activated carbon;

[0012] (2) Take the pretreated activated carbon and add anhydrous ethanol to disperse it. Then add diphenyldimethoxysilane, deionized water and glacial acetic acid. After ultrasonic mixing, reflux at 65~70℃ for 4~4.5h. After the reaction is completed, filter, wash and dry to prepare hydrophobic activated carbon.

[0013] (3) Take hydrophobic activated carbon, add anhydrous ethanol to disperse it, add 50% phytic acid aqueous solution to impregnate it, sonicate it for 0.5-1h, then impregnate it at room temperature for 20-24h, and then calcine it at 430-460℃ for 1-1.5h. After calcination, the product is washed with deionized water and ethanol until neutral to prepare modified activated carbon.

[0014] Preferably, the addition ratio of the pretreated activated carbon to diphenyldimethoxysilane is 1g:0.5~1mL; the addition ratio of the hydrophobic activated carbon to phytic acid aqueous solution is 1g:3.5~7mL.

[0015] Preferably, the molecular weight of the dextran is 10~30kDa.

[0016] Preferably, the activated carbon has a particle size of 0.5~0.8 mm.

[0017] Preferably, the TP value of the horse serum is 6.5~7.5 g / dL.

[0018] Preferably, the amount of dextran-treated modified activated carbon added is 40~100g / L.

[0019] Preferably, the physical adsorption conditions are room temperature and a treatment time of 10-20 hours.

[0020] Preferably, the centrifugation conditions are 3000~6000 r / min and centrifugation at 4℃ for 20~50 min.

[0021] Preferably, the filter membrane is a PES filter element micromembrane with an asymmetric structure.

[0022] The beneficial effects of this invention are:

[0023] This invention utilizes nitric acid pretreatment of activated carbon to increase the number of oxygen-containing functional groups on the activated carbon surface. Then, the pretreated activated carbon is bonded to diphenyldimethoxysilane, thereby generating hydrophobic functional groups on the activated carbon surface, thus preparing hydrophobic activated carbon to enhance the specific adsorption of 25(OH)D. Phytic acid is then used to treat the hydrophobic activated carbon, with phosphate groups in the phytic acid attaching to the surface of the hydrophobic activated carbon. Furthermore, the dehydration condensation of phytic acid at high temperatures and the catalytic effect of hydrogen protons at high temperatures result in a structure rich in pores in the hydrophobic activated carbon, providing numerous adsorption sites for 25(OH)D. Additionally, dextran treatment is used to modify the activated carbon, forming a hydrophobic core and a hydrophilic shell structure, enhancing the specific adsorption of 25(OH)D and retaining key components such as total protein in horse serum.

[0024] The reagents used in this invention are readily available, low in cost, and easy to operate; this invention does not require complex equipment and is suitable for in vitro diagnostic reagent production processes; this invention can effectively remove 25(OH)D from horse serum, and the key components such as total protein in the obtained horse serum are minimally affected, with a low loss rate and high retention, making it suitable for specific in vitro diagnostic research and production projects without 25(OH)D interference. Detailed Implementation

[0025] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0026] Example 1: A method for preparing modified activated carbon includes the following steps:

[0027] (1) Take activated carbon with a particle size of 0.6 mm and wash it three times with nitric acid and deionized water with a concentration of 0.5 mol / L, and then dry it at 120℃ for 4 h to prepare pretreated activated carbon;

[0028] (2) Take 1g of pretreated activated carbon and add 30mL of anhydrous ethanol to disperse it. Then add 0.6mL of diphenyldimethoxysilane, 0.25mL of deionized water and 3 drops of glacial acetic acid. After ultrasonic mixing, place it under reflux at 70℃ for 4h. After the reaction is completed, filter, wash and dry to prepare hydrophobic activated carbon.

[0029] (3) Take 1g of hydrophobic activated carbon, add 30mL of anhydrous ethanol to disperse it, add 5.5mL of 50% phytic acid aqueous solution to impregnate it, sonicate it for 1h, then impregnate it at room temperature for 24h, and then calcine it at 450℃ for 1h. After calcination, the product is washed with deionized water and ethanol until neutral to prepare modified activated carbon.

[0030] Example 2 A method for removing vitamin D from horse serum, comprising the following steps:

[0031] (1) The modified activated carbon prepared in Example 1 was immersed in a saturated dextran solution three times to obtain dextran-treated modified activated carbon, wherein the molecular weight of dextran was 10 kDa and the mass ratio of dextran to modified activated carbon was 2.5:1;

[0032] (2) Dextran-treated modified activated carbon was used to physically adsorb 25(OH)D in horse serum. The amount of dextran-treated modified activated carbon added was 60 g / L, and the physical adsorption conditions were room temperature and treatment time was 10 h.

[0033] (3) Centrifuge the physically adsorbed horse serum at 3200 r / min and 4℃ for 45 min.

[0034] (4) The centrifuged horse serum was filtered using a PES filter micromembrane.

[0035] Example 3 A method for removing vitamin D from horse serum, comprising the following steps:

[0036] (1) The modified activated carbon prepared in Example 1 was immersed in a saturated dextran solution three times to obtain dextran-treated modified activated carbon, wherein the molecular weight of dextran was 20 kDa and the mass ratio of dextran to modified activated carbon was 5.5:1;

[0037] (2) Dextran-treated modified activated carbon was used to physically adsorb 25(OH)D in horse serum. The amount of dextran-treated modified activated carbon added was 75 g / L, and the physical adsorption conditions were room temperature and treatment time was 15 h.

[0038] (3) Centrifuge the physically adsorbed horse serum at 4500 r / min and 4℃ for 30 min.

[0039] (4) The centrifuged horse serum was filtered using a PES filter micromembrane.

[0040] Example 4 A method for removing vitamin D from horse serum, comprising the following steps:

[0041] (1) The modified activated carbon prepared in Example 1 was immersed in a saturated dextran solution three times to obtain dextran-treated modified activated carbon, wherein the molecular weight of dextran was 30 kDa and the mass ratio of dextran to modified activated carbon was 9.5:1;

[0042] (2) Dextran-treated modified activated carbon was used to physically adsorb 25(OH)D in horse serum. The amount of dextran-treated modified activated carbon added was 100 g / L, and the physical adsorption conditions were room temperature and treatment time was 20 h.

[0043] (3) Centrifuge the physically adsorbed horse serum at 6000 r / min and 4℃ for 20 min.

[0044] (4) The centrifuged horse serum was filtered using a PES filter micromembrane.

[0045] Example 5 A method for removing vitamin D from horse serum, comprising the following steps:

[0046] (1) The modified activated carbon prepared in Example 1 was immersed in a saturated dextran solution three times to obtain dextran-treated modified activated carbon, wherein the molecular weight of dextran was 30 kDa and the mass ratio of dextran to modified activated carbon was 9.5:1;

[0047] (2) Dextran-treated modified activated carbon was used to physically adsorb 25(OH)D in horse serum. The amount of dextran-treated modified activated carbon added was 100 g / L, and the physical adsorption conditions were room temperature and treatment time was 15 h.

[0048] (3) Centrifuge the physically adsorbed horse serum at 6000 r / min and 4℃ for 20 min.

[0049] (4) The centrifuged horse serum was filtered using a PES filter micromembrane.

[0050] Example 6 A method for removing vitamin D from horse serum, comprising the following steps:

[0051] (1) The modified activated carbon prepared in Example 1 was immersed in a saturated dextran solution three times to obtain dextran-treated modified activated carbon, wherein the molecular weight of dextran was 30 kDa and the mass ratio of dextran to modified activated carbon was 9.5:1;

[0052] (2) Dextran-treated modified activated carbon was used to physically adsorb 25(OH)D in horse serum. The amount of dextran-treated modified activated carbon added was 100 g / L, and the physical adsorption conditions were room temperature and treatment time was 10 h.

[0053] (3) Centrifuge the physically adsorbed horse serum at 6000 r / min and 4℃ for 20 min.

[0054] (4) The centrifuged horse serum was filtered using a PES filter micromembrane.

[0055] Comparative Example 1: A method for removing vitamin D from horse serum, which, compared with Example 2, did not involve impregnating the modified activated carbon in a saturated dextran solution, but the remaining procedures were the same as in Example 2.

[0056] Comparative Example 2: A method for removing vitamin D from horse serum, which, compared with Example 3, did not involve impregnating the modified activated carbon in a saturated dextran solution, but the remaining procedures were the same as in Example 3.

[0057] Comparative Example 3: A method for removing vitamin D from horse serum, which, compared with Example 4, did not involve impregnating the modified activated carbon in a saturated dextran solution, but the remaining procedures were the same as in Example 4.

[0058] Comparative Example 4: A method for removing vitamin D from horse serum, comprising the following steps:

[0059] (1) The modified activated carbon prepared in Example 1 was immersed in a saturated dextran solution three times to obtain dextran-treated modified activated carbon, wherein the molecular weight of dextran was 30 kDa and the mass ratio of dextran to modified activated carbon was 9.5:1;

[0060] (2) Dextran-treated modified activated carbon was used to physically adsorb 25(OH)D in horse serum. The amount of dextran-treated modified activated carbon added was 100 g / L, and the physical adsorption conditions were room temperature and treatment time was 5 h.

[0061] (3) Centrifuge the physically adsorbed horse serum at 6000 r / min and 4℃ for 20 min.

[0062] (4) The centrifuged horse serum was filtered using a PES filter micromembrane.

[0063] Comparative Example 5: A method for removing vitamin D from horse serum, comprising the following steps:

[0064] (1) Activated carbon with a particle size of 0.6 mm was immersed in a saturated dextran solution three times to obtain dextran-treated activated carbon, wherein the molecular weight of dextran was 30 kDa and the mass ratio of dextran to activated carbon was 9.5:1;

[0065] (2) Dextran was used to treat activated carbon for physical adsorption of 25(OH)D in horse serum. The amount of activated carbon added for dextran treatment was 100 g / L, and the physical adsorption conditions were room temperature and treatment time was 20 h.

[0066] (3) Centrifuge the physically adsorbed horse serum at 6000 r / min and 4℃ for 20 min.

[0067] (4) The centrifuged horse serum was filtered using a PES filter micromembrane.

[0068] Comparative Example 6: A method for removing vitamin D from horse serum, comprising the following steps:

[0069] (1) Activated carbon with a particle size of 0.6 mm was immersed in a saturated dextran solution three times to obtain dextran-treated activated carbon, wherein the molecular weight of dextran was 30 kDa and the mass ratio of dextran to activated carbon was 9.5:1;

[0070] (2) Dextran was used to treat activated carbon for physical adsorption of 25(OH)D in horse serum. The amount of dextran added to the activated carbon was 100 g / L, and the physical adsorption conditions were room temperature and treatment time was 15 h.

[0071] (3) Centrifuge the physically adsorbed horse serum at 6000 r / min and 4℃ for 20 min.

[0072] (4) The centrifuged horse serum was filtered using a PES filter micromembrane.

[0073] Comparative Example 7: A method for removing vitamin D from horse serum, comprising the following steps:

[0074] (1) Activated carbon with a particle size of 0.6 mm was immersed in a saturated dextran solution three times to obtain dextran-treated activated carbon, wherein the molecular weight of dextran was 30 kDa and the mass ratio of dextran to activated carbon was 9.5:1;

[0075] (2) Dextran was used to treat activated carbon for physical adsorption of 25(OH)D in horse serum. The amount of activated carbon added for dextran treatment was 100 g / L, and the physical adsorption conditions were room temperature and treatment time was 10 h.

[0076] (3) Centrifuge the physically adsorbed horse serum at 6000 r / min and 4℃ for 20 min.

[0077] (4) The centrifuged horse serum was filtered using a PES filter micromembrane.

[0078] Performance testing

[0079] A. The total protein content of the horse serum obtained after processing in Examples 2-4 was detected, and the data results are shown in Table 1.

[0080] Table 1 Total Protein Detection Results

[0081]

[0082] As can be seen from the data in Table 1, the total protein in horse serum obtained in Examples 2-4 of this invention is less affected, has a low loss rate, and a high degree of retention.

[0083] B. The horse serum obtained after treatment in Examples 2-4 and Comparative Examples 1-3 was tested for 25(OH)D content, and the data results are shown in Table 2.

[0084] Table 2. Results of 25(OH)D content detection in Examples 2-4 and Comparative Examples 1-3

[0085]

[0086] As can be seen from the data in Table 2, Examples 2-4 of the present invention can effectively remove 25(OH)D from horse serum. Among them, Comparative Examples 1-3, which did not utilize dextran-treated modified activated carbon, showed higher 25(OH)D content after treatment and filtration compared to Examples 2-4. This indicates that dextran-treated modified activated carbon can more effectively remove 25(OH)D from horse serum, and no residual impurities were found after centrifugation.

[0087] C. The 25(OH)D content of horse serum obtained after treatment in Examples 4-6 and Comparative Examples 4-7 was detected, and the data results are shown in Table 3.

[0088] Table 3. Results of 25(OH)D content detection in Examples 4-6 and Comparative Examples 4-7

[0089]

[0090] As can be seen from the data in Table 3, Examples 4-6 of the present invention exhibit good effects in removing 25(OH)D from horse serum under both low temperature and room temperature conditions. The treatment times in Examples 4-6 were 20h, 15h, and 10h, respectively. Comparative Example 4 further reduced the treatment time to 5h. Comparative Examples 5-7 did not involve any modification of the activated carbon, and the treatment times in Comparative Examples 5-7 were 20h, 15h, and 10h, respectively. The measured effects of Comparative Examples 4-7 in removing 25(OH)D from horse serum were significantly lower than those in Examples 4-6.

[0091] In the description of this specification, references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0092] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention.

Claims

1. A method for removing vitamin D from horse serum, characterized by, It comprises the following steps: (1) material screening; (2) using the selected material to physically adsorb 25(OH)D in horse serum; (3) centrifuging the horse serum after physical adsorption; (4) filtering the horse serum after centrifugation by using a filter membrane; The selected material is dextran-treated modified activated carbon, which is obtained by repeatedly immersing modified activated carbon in saturated dextran solution for three times; The mass ratio of the dextran to the modified activated carbon is 2-10:

1. The modified activated carbon is prepared by using nitric acid pretreatment activated carbon, then bonding the pretreated activated carbon with diphenyl dimethoxysilane to prepare hydrophobic activated carbon, and then treating the hydrophobic activated carbon with phytic acid and calcining at high temperature; The preparation method of the modified activated carbon comprises the following steps: (1) taking activated carbon, washing it with 0.5-1 mol / L nitric acid and deionized water for 3-5 times respectively, and then drying it at 110-125 DEG C for 3-5 h to prepare pretreated activated carbon; (2) taking the pretreated activated carbon, dispersing it in anhydrous ethanol, then adding diphenyl dimethoxysilane, deionized water and glacial acetic acid, ultrasonic mixing, and then refluxing at 65-70 DEG C for 4-4.5 h, and then filtering, washing and drying after the reaction to prepare hydrophobic activated carbon; (3) taking the hydrophobic activated carbon, dispersing it in anhydrous ethanol, adding a 50% phytic acid aqueous solution for immersion, ultrasonic treatment for 0.5-1 h, then immersing at room temperature for 20-24 h, and then calcining at 430-460 DEG C for 1-1.5 h, and then washing the product with deionized water and ethanol until it is neutral to prepare modified activated carbon; The addition ratio of the pretreated activated carbon to diphenyl dimethoxysilane is 1g:0.5-1 mL; and the addition ratio of the hydrophobic activated carbon to the phytic acid aqueous solution is 1g:3.5-7 mL.

2. The method of removing vitamin D from horse serum according to claim 1, characterized in that, The molecular weight of the dextran is 10-30 kDa.

3. The method of removing vitamin D from horse serum according to claim 1, characterized in that, The particle size of the activated carbon is 0.5-0.8 mm.

4. The method of removing vitamin D from horse serum according to claim 1, wherein, The TP value of the horse serum is 6.5-7.5 g / dL.

5. The method of removing vitamin D from horse serum according to claim 1, wherein, The addition amount of the dextran-treated modified activated carbon is 40-100 g / L.

6. The method of removing vitamin D from horse serum according to claim 1, wherein, The physical adsorption conditions are room temperature environment and a treatment time of 10-20 h.

7. The method of removing vitamin D from horse serum according to claim 1, wherein, The centrifugation conditions are 3000-6000 r / min, 4 DEG C and centrifuging for 20-50 min.

8. The method of removing vitamin D from horse serum according to claim 1, wherein, The filter membrane is a PES filter core micro membrane with an asymmetric structure.

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