Extraction rod, preparation method thereof and method for detecting 25-hydroxyvitamin D

Abstract

The invention provides an extraction rod, a preparation method thereof and a method for detecting 25-hydroxyvitamin D, and belongs to the technical field of analysis and detection. The preparation method comprises the following steps: sequentially carrying out etching treatment, silanization treatment, heat treatment and activation treatment on a glass rod to obtain an activated glass rod; the activated glass rod is placed in a 25-hydroxyvitamin D antibody solution for coupling treatment, and a coupled antibody glass rod is obtained; and sequentially carrying out deactivation treatment and covalent bond fixation on the coupled antibody glass rod to obtain the extraction rod. The extraction rod is prepared on the basis of the glass rod by using an antibody coupling technology, and the 25-hydroxyvitamin D is extracted and then detected by using an antibody immune enrichment technology on the extraction rod, so that the substrate interference is small, the specificity is strong, and the accuracy is good; and the extraction rod is simple in use method, easy to realize automatic operation, and especially suitable for clinical detection of large-batch samples.

Description

Technical Field

[0001] The invention relates to the technical field of analysis and detection, in particular to an extraction rod and a preparation method thereof, and a method for detecting 25-hydroxyvitamin D. Background Art

[0002] At present, the most commonly used method for clinical 25-hydroxyvitamin D detection is liquid chromatography-tandem mass spectrometry, which can distinguish 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 with excellent sensitivity and accuracy. When using liquid chromatography-tandem mass spectrometry to detect 25-hydroxyvitamin D, the sample pretreatment methods mainly include protein precipitation, liquid-liquid extraction, and solid phase extraction (common fillers include C18, polystyrene-divinylbenzene, etc.). However, these pretreatment methods will extract more unnecessary non-target substances, resulting in a strong matrix effect, affecting the accuracy of the test results. Summary of the invention

[0003] The purpose of the present invention is to provide an extraction rod and a preparation method thereof and a method for detecting 25-hydroxyvitamin D. The extraction rod provided by the present invention is used to detect 25-hydroxyvitamin D, with small matrix interference, strong specificity and accurate detection results.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: The present invention provides a method for preparing an extraction rod, comprising the following steps: The glass rod is sequentially subjected to etching treatment, silanization treatment, heat treatment and activation treatment to obtain an activated glass rod; placing the activated glass rod in a 25-hydroxyvitamin D antibody solution for coupling treatment to obtain an antibody-coupled glass rod; The antibody-coupled glass rod is sequentially subjected to deactivation treatment and covalent bond fixation to obtain the extraction rod.

[0005] Preferably, the concentration of 25-hydroxyvitamin D antibody in the 25-hydroxyvitamin D antibody solution is 0.1-0.6 mg / mL; the 25-hydroxyvitamin D antibody is a 25-hydroxyvitamin D monoclonal antibody or a 25-hydroxyvitamin D polyclonal antibody.

[0006] Preferably, the coupling treatment is carried out at a temperature of 30-40° C. and for a time of 1-4 h.

[0007] The present invention provides an extraction rod prepared by the preparation method described in the above technical solution.

[0008] The present invention provides a method for detecting 25-hydroxyvitamin D for non-disease diagnosis purposes, comprising the following steps: The biological sample to be tested is mixed with a dissociation agent, and a dissociation treatment is performed to obtain a dissociation solution; the dissociation agent contains 8-anilino-1-naphthalenesulfonic acid and perfluorooctanoic acid; Placing the extraction rod described in the above technical solution in the dissociation liquid to perform extraction to obtain an enriched extraction rod; The enrichment extraction rod is sequentially cleaned and eluted to obtain a test solution; The liquid to be tested is subjected to liquid chromatography tandem mass spectrometry detection to obtain a chromatogram of the liquid to be tested; According to the standard curve of 25-hydroxyvitamin D and the chromatogram of the test solution, the content of 25-hydroxyvitamin D in the biological sample to be tested is obtained.

[0009] Preferably, the 25-hydroxyvitamin D includes 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3.

[0010] Preferably, the concentration of 8-anilino-1-naphthalenesulfonic acid in the dissociator is 0.1-0.5wt%, and the concentration of perfluorooctanoic acid is 1-5wt%; the pH value of the dissociator is 7.0-7.5; the temperature of the dissociation treatment is 30-40°C, and the time is 10-30min.

[0011] Preferably, the extraction temperature is 30-40°C and the extraction time is 5-30 min; The eluent used for elution includes methanol, acetonitrile or a derivatizing agent solution, and the derivatizing agent includes 4-phenyl-1,2,4-triazine-3,5-dione, 4-[2-(6,7-dimethoxy-4-methyl-3,4-dihydroquinolinyl)ethyl]-1,2,4-triazine-3,5-dione or 4-(4'-dimethylaminophenyl)-1,2,4-triazine-3,5-dione; the elution temperature is 30-40°C and the elution time is 10-30 minutes.

[0012] Preferably, the liquid phase conditions for the liquid chromatography tandem mass spectrometry detection include: The mobile phase includes mobile phase A and mobile phase B, wherein the mobile phase A is an aqueous solution of ammonium acetate with a concentration of 2 mmol / L, and the mobile phase B is a methanol solution of ammonium acetate with a concentration of 2 mmol / L; Gradient elution program: 0~1min, the volume fraction of mobile phase B is 10%; 1~3.5min, the volume fraction of mobile phase B increases from 10% to 100%; 3.5~4.5min, the volume fraction of mobile phase B remains at 100%; 4.5~4.6min, the volume fraction of mobile phase B decreases from 100% to 10%; 4.6~5min, the volume fraction of mobile phase B remains at 10%.

[0013] Preferably, the mass spectrometry conditions of the liquid chromatography tandem mass spectrometry detection include: Acquisition ion mode: ESI positive ion mode; capillary voltage: 3.6 kV; desolvation gas temperature: 500°C; desolvation gas flow rate: 1000 L / h; cone gas: 150 L / h; scanning mode: multiple reaction monitoring mode.

[0014] Beneficial effects: The present invention sequentially performs etching treatment, silanization treatment, heat treatment and activation treatment on the glass rod to obtain an activated glass rod; the activated glass rod is placed in a 25-hydroxyvitamin D antibody solution for coupling treatment to obtain a coupled antibody glass rod; the coupled antibody glass rod is sequentially deactivated and covalently fixed to obtain an extraction rod. The present invention uses antibody immune enrichment technology on the basis of the glass rod to prepare an extraction rod (i.e., vitamin D antibody immunoaffinity extraction rod), and uses the extraction rod to extract 25-hydroxyvitamin D and then detect it, with small matrix interference, strong specificity and good accuracy; and the extraction rod is simple to use and easy to realize automated operation, which is particularly suitable for the detection of large batches of clinical samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 is the standard curve of 25-hydroxyvitamin D2; Figure 2 is the standard curve of 25-hydroxyvitamin D3; Figure 3 This is the chromatogram obtained by LC-MS / MS detection of 25-hydroxyvitamin D2 in spiked serum samples after extraction with an extraction stick; Figure 4 This is the chromatogram obtained by LC-MS / MS detection after 25-hydroxyvitamin D3 in the spiked serum sample was extracted with an extraction stick. DETAILED DESCRIPTION

[0016] The present invention provides a method for preparing an extraction rod, comprising the following steps: The glass rod is sequentially subjected to etching treatment, silanization treatment, heat treatment and activation treatment to obtain an activated glass rod; placing the activated glass rod in a 25-hydroxyvitamin D antibody solution for coupling treatment to obtain an antibody-coupled glass rod; The antibody-coupled glass rod is sequentially subjected to deactivation treatment and covalent bond fixation to obtain the extraction rod.

[0017] The present invention prepares an extraction rod (i.e., a vitamin D antibody immunoaffinity extraction rod) based on antibody immune enrichment technology, and uses the extraction rod to extract 25-hydroxyvitamin D and then detect it. The matrix interference is small, the specificity is strong, and the accuracy is good. The extraction rod is simple to use and easy to realize automated operation, and is particularly suitable for the detection of large batches of clinical samples. The preparation method of the extraction rod of the present invention is described in detail below.

[0018] In the present invention, unless otherwise specified, the raw materials used are commercially available products well known to those skilled in the art or are prepared by methods well known to those skilled in the art.

[0019] The present invention etches a glass rod to obtain an etched glass rod. As an embodiment of the present invention, the glass rod may be a quartz glass rod or a high borosilicate glass rod; the diameter of the glass rod may be 3 mm and the length may be 60 mm; the present invention uses a glass rod to prepare an extraction rod, which is low in cost and easy to obtain compared to other materials such as stainless steel. As an embodiment of the present invention, the glass rod is placed in a piranha solution for etching. For example, one end of the glass rod (referred to as the treatment end) may be placed in a piranha solution for etching. The immersion depth of the treatment end in the piranha solution may be ≥0.5 cm, specifically 1 to 1.5 cm; the piranha solution may be prepared from a hydrogen peroxide aqueous solution and concentrated sulfuric acid; the concentration of the hydrogen peroxide aqueous solution may be 30 wt%, and the concentration of the concentrated sulfuric acid may be 86 wt%; the volume ratio of the hydrogen peroxide aqueous solution to the concentrated sulfuric acid may be 3:7. As an embodiment of the present invention, the etching treatment can be specifically performed at room temperature (25°C); the time can be 0.5~1.5h, specifically 1h; the organic matter on the surface of the glass rod can be removed by the etching treatment, and active hydroxyl groups can be introduced on the glass rod; after the etching treatment, the obtained glass rod is preferably rinsed thoroughly with pure water, anhydrous ethanol and pure water in sequence.

[0020] After obtaining the etched glass rod, the present invention performs a silanization treatment on the etched glass rod to obtain a silanized glass rod. As an embodiment of the present invention, the etched glass rod is placed in a silane modification liquid for silanization treatment. For example, the treated end of the etched glass rod can be placed in the silane modification liquid for silanization treatment. The immersion depth of the treated end in the silane modification liquid can be ≥0.5 cm, specifically 1 to 1.5 cm; the silane modification liquid can be prepared from a silane reagent, water and ethanol; the volume ratio of the silane reagent, water and ethanol can be 5:5:90. As an embodiment of the present invention, the silane reagent can be 3-aminopropyltriethoxysilane (APTES), which contains an amino functional group and three ethoxy functional groups in its chemical structure. These functional groups can react with the active hydroxyl groups on the surface of the etched glass rod, thereby introducing amino groups on the glass rod; the silane reagent can also be dihydro-3-[3-(triethoxysilyl)propyl]furan-2,5-dione (TESPSA), which can carboxylate the surface of the glass rod; the silane reagent can also be 3-(2,3-epoxypropyloxy)propyltrimethoxysilane (GPTMS), which can hydroxylate the surface of the glass rod. As an embodiment of the present invention, the temperature of the silanization treatment can be 20~30℃, specifically room temperature; the time can be 20~30h, specifically 24h; after the silanization treatment, it is preferred to further include thoroughly rinsing the obtained glass rod with pure water and anhydrous ethanol in sequence.

[0021] After obtaining the silanized glass rod, the present invention heat treats the silanized glass rod to obtain a heat-treated glass rod. As an embodiment of the present invention, the temperature of the heat treatment can be 75-85°C, specifically 80°C; the time can be 10-15h, specifically 12h; the heat treatment is preferably carried out in a protective atmosphere, and the protective atmosphere can be nitrogen; taking APTES as a silane reagent as an example, amino group fixation can be achieved through heat treatment, that is, the amino group introduced on the glass rod is more stable. In an embodiment of the present invention, the heat treatment can be specifically carried out in a tubular furnace; after the heat treatment, it is preferred that the obtained glass rod is taken out for cooling.

[0022] After obtaining the heat-treated glass rod, the present invention performs an activation treatment on the heat-treated glass rod to obtain an activated glass rod. As an embodiment of the present invention, the heat-treated glass rod is placed in an activator for activation treatment. For example, the treated end of the heat-treated glass rod can be placed in an activator for activation treatment. The immersion depth of the treated end in the activator can be ≥0.5cm, specifically 1~1.5cm; the activator can be prepared from glutaraldehyde and PBS solution, and the concentration of glutaraldehyde in the activator can be 2~3wt%, specifically 2.5wt%; the pH value of the PBS solution can be 7.0~7.4, specifically 7.0 in the embodiment. As an embodiment of the present invention, the temperature of the activation treatment can be 20~30℃, specifically room temperature; the time can be 4~6h, specifically 5h; after the activation treatment, it is preferably further included to thoroughly rinse the obtained glass rod with pure water. In the embodiment of the present invention, taking the preparation of the activator with glutaraldehyde as an example, the amino group on the heat-treated glass rod can react with the aldehyde group on the glutaraldehyde, thereby introducing the aldehyde group on the glass rod.

[0023] After obtaining the activated glass rod, the present invention places the activated glass rod in a 25-hydroxyvitamin D antibody solution for coupling treatment to obtain a coupled antibody glass rod. As an embodiment of the present invention, the treated end of the activated glass rod can be placed in a 25-hydroxyvitamin D antibody solution for coupling treatment, and the immersion depth of the treated end in the 25-hydroxyvitamin D antibody solution can be 0.5 to 1 cm, specifically not exceeding the immersion depth in other treatment steps. As an embodiment of the present invention, the concentration of 25-hydroxyvitamin D antibody in the 25-hydroxyvitamin D antibody solution can be 0.1~0.6mg / mL, specifically 0.1mg / mL, 0.2mg / mL, 0.3mg / mL, 0.4mg / mL, 0.5mg / mL or 0.6mg / mL; the 25-hydroxyvitamin D antibody can be a 25-hydroxyvitamin D monoclonal antibody or a 25-hydroxyvitamin D polyclonal antibody, preferably a 25-hydroxyvitamin D monoclonal antibody; the 25-hydroxyvitamin D antibody solution can be prepared by 25-hydroxyvitamin D antibody and PBS buffer, the PBS buffer is specifically 1×PBS, pH=7.4; the 25-hydroxyvitamin D antibody is specifically purchased from Oukai Biotechnology Co., Ltd., item number R484j4, purity>95%. As an embodiment of the present invention, the temperature of the coupling treatment can be 30~40°C, specifically 37°C; the time can be 1~4h, specifically 2h; the coupling treatment is preferably carried out under shaking conditions. In the embodiment of the present invention, the aldehyde group on the activated glass rod can undergo a condensation reaction with the amino group and the carboxyl group on the 25-hydroxyvitamin D antibody, thereby coupling the 25-hydroxyvitamin D antibody to the glass rod.

[0024] In the embodiment of the present invention, each 25-hydroxyvitamin D antibody molecule has two antigen binding sites, and the molar ratio of antibody binding to antigen in the saturated state is 1:2. Therefore, during the antibody coupling process, a sufficient amount of antibody can be coupled to the surface of the glass rod according to specific detection requirements to ensure that all target substances can be extracted, and sufficient glass surface area needs to be provided for coupling the antibody; considering the production cost of the extraction rod, the amount of antibody should be as small as possible. The inventors have calculated theoretically and verified experimentally that the μg-level (<10 μg) antibody coupled to each glass rod can fully meet the detection requirements of actual biological samples (such as serum samples), and the cost of this dose of antibody can also meet the actual detection requirements.

[0025] After obtaining the coupled antibody glass rod, the present invention performs a deactivation treatment on the coupled antibody glass rod to obtain a deactivated glass rod. As one embodiment of the present invention, the coupled antibody glass rod is placed in a deactivation reagent for deactivation treatment. For example, the treated end of the coupled antibody glass rod can be placed in a deactivation reagent for deactivation treatment. The immersion depth of the treated end in the deactivation agent can be ≥0.5 cm, specifically 1~1.5 cm; the deactivation reagent can be an ethanolamine aqueous solution; the concentration of ethanolamine in the ethanolamine aqueous solution can be 0.25~0.35 mol / L, specifically 0.3 mol / L; the pH value of the ethanolamine aqueous solution can be 7~7.5, and the pH value of the ethanolamine aqueous solution can be adjusted by concentrated hydrochloric acid. As one embodiment of the present invention, the temperature of the deactivation treatment can be 20~30°C, specifically room temperature; the time can be 1.5~2.5h, specifically 2h; the aldehyde groups remaining on the surface of the glass rod can be removed by deactivation treatment.

[0026] After obtaining the deactivated glass rod, the present invention fixes the deactivated glass rod with a covalent bond to obtain the extraction rod. As an embodiment of the present invention, the deactivated glass rod is placed in a reducing agent for covalent bond fixation. For example, the treated end of the deactivated glass rod can be placed in a reducing agent for covalent bond fixation. The immersion depth of the treated end in the reducing agent can be ≥0.5 cm, specifically 1~1.5 cm; the reducing agent can be prepared from sodium cyanoborohydride and PBS buffer solution, and the concentration of sodium cyanoborohydride in the reducing agent can be 0.15~0.25 mg / mL, specifically 2 mg / mL; the pH value of the PBS buffer solution can be 7.0~7.4, specifically 7.0 in the embodiment. As an embodiment of the present invention, the temperature of the covalent bond fixation can be 3~5°C, specifically 4°C; the time can be 40~60h, specifically 48h; the imine bond formed by the condensation reaction can be reduced to an amine bond by sodium cyanoborohydride, thereby stabilizing the covalent bond. As an embodiment of the present invention, the extraction rod can be stored in a PBS buffer (pH value is 7.0-7.4) containing 0.02 wt % sodium azide and stored at 4°C.

[0027] The present invention provides an extraction rod prepared by the preparation method described in the above technical solution.

[0028] The present invention provides a method for detecting 25-hydroxyvitamin D for non-disease diagnosis purposes, comprising the following steps: The biological sample to be tested is mixed with a dissociation agent, and a dissociation treatment is performed to obtain a dissociation solution; the dissociation agent contains 8-anilino-1-naphthalenesulfonic acid and perfluorooctanoic acid; Placing the extraction rod described in the above technical solution in the dissociation liquid to perform extraction to obtain an enriched extraction rod; The enrichment extraction rod is sequentially cleaned and eluted to obtain a test solution; The liquid to be tested is subjected to liquid chromatography tandem mass spectrometry detection to obtain a chromatogram of the liquid to be tested; According to the standard curve of 25-hydroxyvitamin D and the chromatogram of the test solution, the content of 25-hydroxyvitamin D in the biological sample to be tested is obtained.

[0029] The present invention combines the advantages of both the immune method and the mass spectrometry method to prepare an extraction rod based on antibody immune enrichment technology. The extraction rod is used in conjunction with the liquid chromatography tandem mass spectrometry (LC-MS / MS) method to determine 25-hydroxyvitamin D (specifically including 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3), which can effectively reduce matrix interference and improve the specificity and accuracy of detection; and the pretreatment process is simple, no nitrogen blowing centrifugation and other operations are required, the reagent consumption is small, and it is easy to realize automated operation, which is suitable for rapid processing and detection of large batches of samples, and is particularly suitable for clinical promotion and popularization. The method of the present invention is described in detail below.

[0030] The present invention mixes the biological sample to be tested with a dissociation agent, performs a dissociation treatment, and obtains a dissociation liquid. As an embodiment of the present invention, the biological sample to be tested may include serum, plasma, urine or tissue fluid. In the embodiment of the present invention, serum is specifically used as an example to verify the detection effect of the scheme of the present invention. It does not need any other treatment. The serum sample to be tested can be directly mixed with the dissociation agent for dissociation treatment. In the present invention, the dissociator contains 8-anilino-1-naphthalenesulfonic acid and perfluorooctanoic acid; as an embodiment of the present invention, the concentration of 8-anilino-1-naphthalenesulfonic acid in the dissociator can be 0.1-0.5wt%, specifically 0.1wt%, 0.15wt%, 0.18wt%, 0.2wt%, 0.3wt%, 0.4wt% or 0.5wt%; the concentration of perfluorooctanoic acid can be 1-5%, specifically 1wt%, 1.5wt%, 1.81wt%, 2wt%, 3wt%, 4wt% or 5wt%; the dissociator can be specifically prepared from 8-anilino-1-naphthalenesulfonic acid, perfluorooctanoic acid and a buffered saline solution, and the buffered saline solution can be made of di(2 -hydroxyethyl)amino(trihydroxymethyl)methane (BIS-TRIS), sodium chloride, N,N-dimethylformamide, dimethyl sulfoxide and water are prepared, the concentration of BIS-TRIS in the buffered salt solution can be 2~3wt%, specifically 2.5wt%; the concentration of sodium chloride can be 0.5~1.0wt%, specifically 0.8wt%; the concentration of N,N-dimethylformamide can be 8.0~8.5wt%, specifically 8.33wt%; the concentration of dimethyl sulfoxide can be 1.4~1.8wt%, specifically 1.6wt%; the pH value of the dissociator can be 7.0~7.5, and hydrochloric acid can be used to adjust the pH value of the dissociator. As an embodiment of the present invention, taking the biological sample to be tested as a serum sample as an example, the volume ratio of the serum sample to the dissociator can be 1:0.8~1.2, specifically 1:1. As an embodiment of the present invention, the temperature of the dissociation treatment can be 30-40°C, specifically 37°C; the time can be 10-30min, specifically 10-15min. In the present invention, the 25-hydroxyvitamin D in the biological sample to be tested, such as the serum sample, will be combined with the vitamin D binding protein or other proteins to form a bound state, and the antibody on the extraction rod cannot bind to the bound antigen. The role of the dissociator in the present invention is to dissociate 25-hydroxyvitamin D from the vitamin D binding protein, from the bound state to the free state, which is conducive to the antibody to capture the antigen. Therefore, the selection of a suitable dissociator is crucial to the detection sensitivity of the biological sample to be tested. The present invention uses 8-anilino-1-naphthalenesulfonic acid and perfluorooctanoic acid to prepare a neutral dissociator, which has a high dissociation efficiency and does not affect the activity of the antibody.

[0031] After obtaining the dissociation liquid, the present invention places the extraction rod described in the above technical solution in the dissociation liquid to perform extraction to obtain an enriched extraction rod. As an embodiment of the present invention, the extraction temperature can be 30-40°C, specifically 37°C; the extraction time is 5-30 minutes, specifically 20 minutes.

[0032] After obtaining the enrichment extraction rod, the present invention sequentially washes and elutes the enrichment extraction rod to obtain the test solution. As an embodiment of the present invention, the eluent used for the washing can be water or a methanol aqueous solution, and the concentration of the methanol aqueous solution is ≤10wt%, specifically 3-5wt%; the washing is based on ensuring sufficient washing.

[0033] As an embodiment of the present invention, the eluent used for elution may include methanol, acetonitrile or a derivatizing agent solution, specifically a derivatizing agent solution. In the present invention, the ionization efficiency of 25-hydroxyvitamin D under the ESI source is not high, which affects the detection sensitivity. By using a derivatizing agent solution as an eluent, new derivatives can be generated through a derivatization reaction, thereby improving the ionization efficiency and further improving the sensitivity. As an embodiment of the present invention, the derivatizing agent solution can be prepared by a derivatizing agent and acetonitrile; the derivatizing agent can include 4-phenyl-1,2,4-triazolin-3,5-dione (PTAD), 4-[2-(6,7-dimethoxy-4-methyl-3,4-dihydroquinolinyl)ethyl]-1,2,4-triazolin-3,5-dione (DMEQ-TAD) or 4-(4'-dimethylaminophenyl)-1,2,4-triazolin-3,5-dione (DAPTAD), specifically 4-phenyl-1,2,4-triazolin-3,5-dione (PTAD), and the concentration of the derivatizing agent solution can be 0.1~1 mg / mL, specifically 0.3~0.5 mg / mL; the derivatizing agent can form an adduct with a vitamin D compound having a cis-triene system. As an embodiment of the present invention, taking the biological sample to be tested as a serum sample as an example, the volume ratio of the serum sample to the eluent can be 1:0.8~1.2, specifically 1:1. As an embodiment of the present invention, the elution temperature can be 30~40°C, specifically 37°C; the time can be 10~30min, specifically 25~30min. As an embodiment of the present invention, after the elution is completed, the extraction rod is specifically taken out, and the obtained liquid is mixed with water to obtain the liquid to be tested. As an embodiment of the present invention, taking the biological sample to be tested as a serum sample as an example, the volume ratio of the serum sample to water can be 1:0.4~0.6, specifically 1:0.5; the mixing can be vortex mixing, and the vortex mixing time can be 30s. As an embodiment of the present invention, the extraction rod can also be regenerated after use to restore the antibody activity, and the regeneration step is specifically to place the used extraction rod in methanol for washing twice to remove the target analyte remaining on the extraction rod, and then place it in a PBS solution containing 0.02wt% sodium azide for storage.

[0034] After obtaining the liquid to be tested, the present invention performs liquid chromatography tandem mass spectrometry detection on the liquid to be tested to obtain a chromatogram of the liquid to be tested. As an embodiment of the present invention, the liquid phase conditions for the liquid chromatography tandem mass spectrometry (LC-MS / MS) detection may include: the mobile phase includes mobile phase A and mobile phase B, the mobile phase A is an aqueous solution of ammonium acetate with a concentration of 2 mmol / L, and the mobile phase B is methanol; the gradient elution program: 0~1min, the volume fraction of mobile phase B is 10%; 1~3.5min, the volume fraction of mobile phase B increases from 10% to 100%; 3.5~4.5min, the volume fraction of mobile phase B remains at 100%; 4.5~4.6min, the volume fraction of mobile phase B decreases from 100% to 10%; 4.6~5min, the volume fraction of mobile phase B remains at 10%. As an embodiment of the present invention, the liquid phase conditions of the liquid chromatography tandem mass spectrometry detection may also include: the chromatographic column used is specifically a Waters BEHC18 column (2.1×50 mm, 1.7 μm); the mobile phase flow rate is specifically 0.2 mL / min; the column temperature is specifically 40° C.; and the injection volume is specifically 5 μL.

[0035] As an embodiment of the present invention, the mass spectrometry conditions of the liquid chromatography tandem mass spectrometry detection may include: acquisition ion mode: ESI positive ion mode; capillary voltage: 3.6 kV; desolvation gas temperature: 500 ° C; desolvation gas flow rate: 1000 L / h; cone gas: 150 L / h; scanning mode: multiple reaction monitoring (MRM) mode (MRM parameters are specifically shown in Table 1 in Example 1, and will not be repeated here). As an embodiment of the present invention, the mass spectrometry conditions of the liquid chromatography tandem mass spectrometry detection may also include: the mass spectrometry model is Waters TQ-Absolute.

[0036] After obtaining the chromatogram of the liquid to be tested, the present invention obtains the content of 25-hydroxyvitamin D in the biological sample to be tested according to the standard curve of 25-hydroxyvitamin D and the chromatogram of the liquid to be tested. As an embodiment of the present invention, specifically according to the standard curve of 25-hydroxyvitamin D2 and the standard curve of 25-hydroxyvitamin D3 and the chromatogram of the liquid to be tested, the contents of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 in the biological sample to be tested are obtained respectively. The present invention does not specifically limit the method for obtaining the standard curve of the 25-hydroxyvitamin D, and a method well known to those skilled in the art can be used. In the embodiment of the present invention, the standard curve of the 25-hydroxyvitamin D is specifically a linear equation of the mass concentration of 25-hydroxyvitamin D and the chromatographic peak area of ​​25-hydroxyvitamin D. Specifically, the standard curve of the 25-hydroxyvitamin D takes the chromatographic peak area of ​​25-hydroxyvitamin D as the ordinate and the mass concentration of 25-hydroxyvitamin D as the abscissa. In the embodiment of the present invention, the 25-hydroxyvitamin D standard solution is specifically subjected to dissociation treatment, extraction, cleaning, elution and liquid chromatography tandem mass spectrometry detection according to the above scheme to obtain the chromatographic peak area of ​​25-hydroxyvitamin D; a standard curve is drawn according to the chromatographic peak area of ​​25-hydroxyvitamin D and the mass concentration of 25-hydroxyvitamin D. As an embodiment of the present invention, the 25-hydroxyvitamin D standard solution can be specifically prepared by 25-hydroxyvitamin D and bovine serum albumin (BSA) solution, and the concentration of the BSA solution can be 4wt%; the concentration of the 25-hydroxyvitamin D standard solution can be specifically 1ng / mL, 2ng / mL, 5ng / mL, 10ng / mL, 20ng / mL, 50ng / mL and 100ng / mL. According to the standard curve of 25-hydroxyvitamin D and the chromatogram of the test liquid, the content of 25-hydroxyvitamin D in the test liquid can be obtained, and then the content of 25-hydroxyvitamin D in the biological sample to be tested can be obtained. The standard curve of 25-hydroxyvitamin D2 and the standard curve of 25-hydroxyvitamin D3 in the embodiment of the present invention can be obtained according to the above method, which will not be repeated here.

[0037] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0038] The high borosilicate glass rods used in the following examples were purchased from Yongsheng Quartz Products Store. The high borosilicate glass rods had a diameter of 3 mm and a length of 60 mm.

[0039] Example 1 Prepare the vitamin D affinity extraction stick as follows: A hydrogen peroxide solution is added to concentrated sulfuric acid in a ratio of 3:7 by volume of a hydrogen peroxide solution (concentration of 30wt%) to concentrated sulfuric acid (concentration of 95-98wt%) to obtain a piranha solution; at room temperature, one end (referred to as the treated end) of a high borosilicate glass rod is placed in the piranha solution (immersion depth of 1cm) for etching for 1h, and then rinsed thoroughly with pure water, anhydrous ethanol and pure water in sequence to obtain an etched glass rod; 3-aminopropyltriethoxysilane, water and anhydrous ethanol are mixed in a volume ratio of 5:5:90 to obtain a silane modification solution; the etched glass rod is placed in the silane modification solution (the treated end is immersed to a depth of 1 cm), and silanized for 24 hours at room temperature, and then thoroughly rinsed with pure water and anhydrous ethanol in turn to obtain a silanized glass rod; The silanized glass rod is placed in a tube furnace, protected by nitrogen, and heat-treated at 80° C. for 12 h, then taken out and cooled to obtain a heat-treated glass rod; The heat-treated glass rod is placed in a PBS solution (pH=7.0) containing 2.5wt% glutaraldehyde (immersion depth of the treated end is 1m), and activated for 5 hours at room temperature, and then thoroughly rinsed with pure water to obtain an activated glass rod; The activated glass rod was placed in a PBS solution (pH=7.0) containing 0.5 mg / mL vitamin D monoclonal antibody (the treated end was immersed to a depth of 0.5 cm), and oscillated for reaction at 37°C for 2 hours; then the obtained glass rod was placed in an ethanolamine solution with a concentration of 0.3 mol / L and a pH value of 7.5 (the pH value was adjusted with concentrated hydrochloric acid) (the treated end was immersed to a depth of 1 cm), and was placed at room temperature for 2 hours to deactivate the remaining aldehyde groups; then the obtained glass rod was placed in a PBS solution (pH=7.0) containing 0.2 mg / mL sodium cyanoborohydride (the treated end was immersed to a depth of 1 cm), and was placed at 4°C for 48 hours to reduce the imine to an amine, thereby obtaining a vitamin D antibody immunoaffinity extraction rod (referred to as the extraction rod); finally, the prepared extraction rod was placed in a PBS solution (pH=7.0) containing 0.02 wt% sodium azide and stored at 4°C.

[0040] Example 2 The extraction rod in Example 1 was used to extract and detect serum 25-hydroxyvitamin D, and the steps were as follows: 1. The sample pretreatment steps are as follows: (1) Take 100 μL of 25-hydroxyvitamin D standard solution or serum sample, add it to a 1.5 mL centrifuge tube, then add 100 μL of dissociation agent, vortex mix, and incubate at 37° C. for 10 min; wherein the dissociation agent is a neutral dissociation agent prepared by 8-anilino-1-naphthalenesulfonic acid, perfluorooctanoic acid and buffer solution (the pH value is adjusted to 7 using hydrochloric acid), the concentration of 8-anilino-1-naphthalenesulfonic acid in the dissociation agent is 0.18wt%, the concentration of perfluorooctanoic acid is 1.81wt%, and the buffer solution is composed of bis(2-hydroxyethyl)amino(trihydroxymethyl)methane (BIS-TRIS), sodium chloride, N,N-dimethylformamide, dimethyl sulfoxide and water, and the contents of each component are 2.5wt% BIS-TRIS, 0.8wt% sodium chloride, 8.33wt% N,N-dimethylformamide, 1.6wt% dimethyl sulfoxide, and the rest is water; (2) placing the extraction rod in a centrifuge tube, incubating with shaking at 37°C for 20 min, then taking out the extraction rod and washing it with a 5 wt% methanol aqueous solution; (3) Place the cleaned extraction stick in the insert tube of the injection bottle containing 100 μL of 0.5 mg / mL 4-phenyl-1,2,4-triazolidine-3,5-dione (PTAD) acetonitrile solution, and perform elution and derivatization reaction at 37°C for 30 min; (4) Add 50 μL of pure water into the insert tube of the injection bottle, vortex mix, and then perform LC-MS / MS detection.

[0041] 2. Chromatographic and mass spectrometric detection conditions are as follows: 1. Chromatographic conditions Chromatographic column: Waters BEH C18 column (2.1×50 mm, 1.7 μm).

[0042] Mobile phase A: 2 mM ammonium acetate aqueous solution; mobile phase B: pure methanol.

[0043] Gradient elution program: 0 min, the volume fraction of mobile phase B was 10%; 1 min, the volume fraction of mobile phase B was maintained at 10%; 3.5 min, the volume fraction of mobile phase B was 100%; 4.5 min, the volume fraction of mobile phase B was maintained at 100%; 4.6 min, the volume fraction of mobile phase B was 10%; 5 min, the volume fraction of mobile phase B was maintained at 10%.

[0044] Mobile phase flow rate: 0.2 mL / min; column temperature: 40°C; injection volume: 5 μL.

[0045] 2. Mass spectrometry conditions Mass spectrometer model and acquisition ion mode: Waters TQ-Absolute, ESI positive ion mode.

[0046] Capillary voltage: 3.6 kV; desolvation gas temperature: 500 °C; desolvation gas flow rate: 1000 L / h; cone gas: 150 L / h.

[0047] Scanning mode: multiple reaction monitoring (MRM). The mass spectrometry parameters of each compound are shown in Table 1.

[0048] Table 1 25-Hydroxyvitamin D mass spectrometry MRM parameters

[0049] Example 3 Serum samples and 4 wt% bovine serum albumin (BSA) solution samples were taken, and 25-hydroxyvitamin D standard was added to make the concentrations of 5 ng / mL, 10 ng / mL, 20 ng / mL and 50 ng / mL, respectively. The samples were then treated according to the sample pretreatment steps in Example 2, and LC-MS / MS detection was performed according to the chromatographic and mass spectrometric conditions in Example 2.

[0050] Table 2 shows the peak area of ​​25-hydroxyvitamin D extracted from different samples based on the extraction rod with the addition of a dissociator. The results show that the extraction rod can extract 25-hydroxyvitamin D in both serum samples and 4wt% BSA solution samples, and under each added concentration condition, the response of the serum sample after background deduction is close to that of the 4wt% BSA solution sample, indicating that the dissociator can fully dissociate the 25-hydroxyvitamin D in the serum sample.

[0051] Table 2 Peak areas of 25-hydroxyvitamin D extracted from different samples

[0052] Example 4 A 25-hydroxyvitamin D2 standard solution and a 25-hydroxyvitamin D3 standard solution were prepared using a 4 wt % BSA solution with concentrations of 1 ppb, 2 ppb, 5 ppb, 10 ppb, 20 ppb, 50 ppb, and 100 ppb, respectively; the samples were then treated according to the sample pretreatment steps in Example 2, and LC-MS / MS detection was performed according to the chromatographic and mass spectrometry conditions in Example 2 to obtain the retention time and linear range of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3.

[0053] Table 3 shows the retention time and linear range of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3. Figure 1 is the standard curve of 25-hydroxyvitamin D2, Figure 2is the standard curve of 25-hydroxyvitamin D3; the results show that the linear correlation coefficient R 2 >0.99, indicating that it is acceptable within the linear range and meets the actual experimental requirements.

[0054] Table 3 Retention time and linear range of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3

[0055] Example 5 Low, medium and high concentrations of 25-hydroxyvitamin D standards were added to the serum samples to obtain experimental group samples, which were then processed according to the sample pretreatment steps in Example 2, and LC-MS / MS detection was performed according to the chromatographic and mass spectrometric conditions in Example 2. The basic sample concentration in the serum sample and the sample concentration of each experimental group were calculated based on the standard curve, and the recovery rate = (detection concentration-basic sample concentration) / theoretical added concentration × 100%.

[0056] Figure 3 This is the chromatogram obtained by LC-MS / MS detection of 25-hydroxyvitamin D2 in spiked serum samples after extraction with an extraction stick. Figure 4 The chromatogram obtained by LC-MS / MS detection of 25-hydroxyvitamin D3 in spiked serum samples after extraction with an extraction stick; the spiked concentration is 50 ng / mL. The test results of the spiked serum samples are shown in Table 4. From the results in the table, it can be seen that the spiked recoveries of low, medium and high concentrations of 25-hydroxyvitamin D in serum samples are all between 85 and 115%, which meets the actual experimental requirements.

[0057] Table 4 25-Hydroxyvitamin D spike recovery

[0058] Comparative Example 1 Comparison of the dissociation effects of 4 dissociation agents: Dissociation agent 1: commercially available dissociation agent (purchased from Okey Biotechnology Co., Ltd., product number D10a1); Dissociator 2: prepared from perfluorooctanoic acid and a PBS solution containing ethylene glycol, wherein the concentration of ethylene glycol in the PBS solution containing ethylene glycol is 10wt%, and the concentration of perfluorooctanoic acid in the dissociator 2 is 2wt%; Dissociator 3: Perfluorohexanoic acid is mixed with a 5 wt% methanol aqueous solution, and then the pH value is adjusted to 7.5 with sodium hydroxide. The concentration of perfluorohexanoic acid in the dissociator 3 is 0.75 wt%; Dissociation agent 4: the dissociation agent used in Example 2.

[0059] Four serum samples were taken, and the same amount of 25-hydroxyvitamin D standard was added to each of them. The samples were treated according to the sample pretreatment steps in Example 2, wherein the dissociators used were the above-mentioned dissociator 1, dissociator 2, dissociator 3 and dissociator 4, respectively. Then, LC-MS / MS detection was performed according to the chromatographic and mass spectrometric conditions in Example 2.

[0060] Table 5 shows the peak area of ​​25-hydroxyvitamin D extracted from serum samples based on the extraction stick when different dissociators were added. The results show that for the same sample, the response of 25-hydroxyvitamin D detected after dissociation with dissociator 4 was the highest, indicating that dissociator 4 has the best dissociation ability, while other dissociators have poor dissociation efficiency and incomplete dissociation.

[0061] Table 5 Peak areas of 25-hydroxyvitamin D extracted from serum samples with the addition of different dissociation agents

[0062] Comparative Example 2 Serum samples and 4 wt % BSA solution samples were taken, and the same amount of 25-hydroxyvitamin D was added to each sample. Except for not adding the dissociation agent, the rest of the sample pretreatment steps in Example 2 were followed, and LC-MS / MS detection was performed according to the chromatographic and mass spectrometric conditions in Example 2.

[0063] Table 6 shows the peak area of ​​25-hydroxyvitamin D extracted from different samples without adding a dissociating agent. The results show that the extraction rod can extract 25-hydroxyvitamin D in a BSA solution sample with a concentration of 4wt%, but 25-hydroxyvitamin D is not extracted in a serum sample. The reason is that after 25-hydroxyvitamin D is added to the serum sample, it will bind to proteins such as the vitamin D binding protein in the serum to form a bound state and cannot be extracted by the antibody on the extraction rod.

[0064] Table 6 Peak areas of 25-hydroxyvitamin D extracted from different samples without adding dissociation agent

[0065] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing an extraction rod, characterized in that: The following steps are involved: The glass rod is sequentially subjected to etching treatment, silanization treatment, heat treatment and activation treatment to obtain an activated glass rod; placing the activated glass rod in a 25-hydroxyvitamin D antibody solution for coupling treatment to obtain an antibody-coupled glass rod; The antibody-coupled glass rod is sequentially subjected to deactivation treatment and covalent bond fixation to obtain the extraction rod.

2. The preparation method according to claim 1, characterized in that: The concentration of the 25-hydroxyvitamin D antibody in the 25-hydroxyvitamin D antibody solution is 0.1-0.6 mg / mL; the 25-hydroxyvitamin D antibody is a 25-hydroxyvitamin D monoclonal antibody or a 25-hydroxyvitamin D polyclonal antibody.

3. The preparation method according to claim 1 or 2, characterized in that: The coupling treatment is carried out at a temperature of 30-40° C. and for a time of 1-4 hours.

4. An extraction rod prepared by the preparation method according to any one of claims 1 to 3.

5. A method for detecting 25-hydroxyvitamin D for non-disease diagnosis purposes, characterized in that: The following steps are involved: The biological sample to be tested is mixed with a dissociation agent, and a dissociation treatment is performed to obtain a dissociation solution; the dissociation agent contains 8-anilino-1-naphthalenesulfonic acid and perfluorooctanoic acid; placing the extraction rod according to claim 4 in the dissociation solution to perform extraction to obtain an enriched extraction rod; The enrichment extraction rod is sequentially cleaned and eluted to obtain a test solution; The liquid to be tested is subjected to liquid chromatography tandem mass spectrometry detection to obtain a chromatogram of the liquid to be tested; According to the standard curve of 25-hydroxyvitamin D and the chromatogram of the test solution, the content of 25-hydroxyvitamin D in the biological sample to be tested is obtained.

6. The method according to claim 5, characterized in that The 25-hydroxyvitamin D includes 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3.

7. The method according to claim 5, characterized in that The concentration of 8-anilino-1-naphthalenesulfonic acid in the dissociator is 0.1-0.5wt%, and the concentration of perfluorooctanoic acid is 1-5wt%; the pH value of the dissociator is 7.0-7.5; the temperature of the dissociation treatment is 30-40°C, and the time is 10-30min.

8. The method according to any one of claims 5 to 7, characterized in that: The extraction temperature is 30-40°C and the extraction time is 5-30min; The eluent used for elution includes methanol, acetonitrile or a derivatizing agent solution, and the derivatizing agent includes 4-phenyl-1,2,4-triazine-3,5-dione, 4-[2-(6,7-dimethoxy-4-methyl-3,4-dihydroquinolinyl)ethyl]-1,2,4-triazine-3,5-dione or 4-(4'-dimethylaminophenyl)-1,2,4-triazine-3,5-dione; the elution temperature is 30-40°C and the elution time is 10-30 minutes.

9. The method according to any one of claims 5 to 7, characterized in that: The liquid phase conditions of the liquid chromatography tandem mass spectrometry detection include: The mobile phase includes mobile phase A and mobile phase B, wherein the mobile phase A is an aqueous solution of ammonium acetate with a concentration of 2 mmol / L, and the mobile phase B is a methanol solution of ammonium acetate with a concentration of 2 mmol / L; Gradient elution program: 0~1min, the volume fraction of mobile phase B is 10%; 1~3.5min, the volume fraction of mobile phase B increases from 10% to 100%; 3.5~4.5min, the volume fraction of mobile phase B remains at 100%; 4.5~4.6min, the volume fraction of mobile phase B decreases from 100% to 10%; 4.6~5min, the volume fraction of mobile phase B remains at 10%.

10. The method according to claim 9, characterized in that The mass spectrometry conditions of the liquid chromatography tandem mass spectrometry detection include: Acquisition ion mode: ESI positive ion mode; capillary voltage: 3.6 kV; desolvation gas temperature: 500°C; desolvation gas flow rate: 1000 L / h; cone gas: 150 L / h; scanning mode: multiple reaction monitoring mode.

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