Vitamin D mass spectrometry detection method based on antibody immune enrichment technology

Abstract

According to the vitamin D mass spectrometric detection method based on the antibody immune enrichment technology, 25-hydroxyvitamin D antibodies are coated on a 96-well plate to obtain an extraction plate, then 25-hydroxyvitamin D in a sample is extracted through the antibody immune enrichment effect on the extraction plate and then detected, the method is easy to operate, automation is easy to achieve, the specificity is high, and the detection accuracy is high. No obvious matrix effect exists, and the accuracy is good.

Description

Technical Field

[0001] The present invention belongs to the technical field of analysis and detection, and in particular relates to a vitamin D mass spectrometry detection method based on antibody immune enrichment technology. Background Art

[0002] Vitamin D is an essential fat-soluble vitamin for the human body, which plays an important role in regulating human metabolic function and maintaining physical health. 25-hydroxyvitamin D is the main form of vitamin D in the blood circulation, and the concentration of 25-hydroxyvitamin D in the body can be used to evaluate the nutritional status of vitamin D in the human body.

[0003] At present, the main clinical vitamin D detection methods are immunoassay and liquid chromatography-tandem mass spectrometry. Immunoassay is a reaction technology based on antigen-antibody specific immune binding, mainly including radioimmunoassay, enzyme-linked immunosorbent assay, chemiluminescence immunoassay, electrochemiluminescence immunoassay, etc. Immunoassay for vitamin D detection has the advantages of simple operation and easy automation, but the immunoassay can only detect total vitamin D and cannot distinguish 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 and other vitamin D metabolites. It has been gradually replaced by liquid chromatography-tandem mass spectrometry.

[0004] Liquid chromatography-tandem mass spectrometry has excellent sensitivity, specificity and accuracy, and can distinguish multiple vitamin D metabolites such as 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3. It is currently the internationally recognized gold standard for vitamin D detection. However, the sample pretreatment methods for liquid chromatography-tandem mass spectrometry detection of 25-hydroxyvitamin D mainly include liquid-liquid extraction and solid-phase extraction, which are relatively cumbersome to operate, and these pretreatment methods are non-specific enrichment methods, which will extract more unnecessary non-target substances at the same time. These non-target substances will interfere with the ionization of the target substance in the ESI source (electrospray ionization source) of the mass spectrometer, thereby causing a strong matrix effect and affecting the accuracy of the test results. Summary of the invention

[0005] The purpose of the present invention is to address the deficiencies of the prior art and provide a vitamin D mass spectrometry detection method based on antibody immune enrichment technology.

[0006] The object of the present invention is achieved through the following technical scheme: a vitamin D mass spectrometry detection method based on antibody immune enrichment technology, comprising the following sub-steps:

[0007] (1) Adding 10 to 50 μL of a 25-hydroxyvitamin D antibody solution having a concentration of 0.1 to 1 mg / mL to each well of a 96-well plate, followed by antibody coating treatment to obtain an antibody-coated extraction plate;

[0008] (2) subsequently performing a blocking treatment on the antibody-coated extraction plate to obtain a blocked extraction plate; then washing the blocked extraction plate to obtain an extraction plate coated with 25-hydroxyvitamin D;

[0009] (3) adding the sample to be tested to the extraction plate coated with 25-hydroxyvitamin D and performing enrichment extraction to obtain an extraction plate enriched with the sample to be tested; and then sequentially performing a cleaning treatment and an elution derivatization treatment on the extraction plate enriched with the sample to be tested to obtain a test solution;

[0010] (4) Then, the test liquid is subjected to liquid chromatography-tandem mass spectrometry detection to obtain a chromatogram of the test liquid; and the content of 25-hydroxyvitamin D in the test sample is obtained based on a standard curve of 25-hydroxyvitamin D and the chromatogram of the test liquid.

[0011] Furthermore, the 25-hydroxyvitamin D antibody is a 25-hydroxyvitamin D monoclonal antibody or a 25-hydroxyvitamin D polyclonal antibody.

[0012] Furthermore, in step (1), the antibody coating treatment is specifically: incubating with shaking at 30-40° C. and 100-300 rpm / min for 1-2 hours, and then aspirating the remaining solution in the well.

[0013] Furthermore, in step (2), the blocking treatment is specifically as follows: adding 10 to 50 μL of a 0.1 to 2 wt% bovine serum albumin solution to each well of the antibody-coated extraction plate, blocking at 30 to 40° C. for 1 to 2 hours, and then aspirating the remaining solution in the well;

[0014] The washing operation is specifically as follows: adding 200 μL of PBS buffer to each well of the blocked extraction plate, aspirating the remaining waste liquid after shaking, and patting dry with a paper towel, and repeating twice.

[0015] Furthermore, the sample to be tested is water, milk or other biological samples.

[0016] Furthermore, in step (3), the enrichment extraction is specifically: incubating with shaking at 30-40° C. and 100-300 rpm / min for 10-30 min, and then aspirating the remaining solution in the well;

[0017] The cleaning process is specifically as follows: adding 100-200 μL of 5-10% methanol aqueous solution to each well of the extraction plate enriched with the sample to be tested, aspirating the remaining waste liquid after shaking, and patting dry with a paper towel, repeating twice;

[0018] The elution derivatization treatment is specifically as follows: 50 to 200 μL of an eluent containing a derivatization agent at a concentration of 0.1 to 1 mg / mL is added to each well of the extraction plate enriched with the sample to be tested after the cleaning treatment, and the elution and derivatization reaction is performed at 30 to 40° C. and 100 to 300 rpm / min for 10 to 30 minutes.

[0019] Further, the derivatizing agent is 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;

[0020] The eluent is methanol or acetonitrile.

[0021] Furthermore, the liquid phase conditions for the liquid chromatography tandem mass spectrometry detection include:

[0022] 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;

[0023] 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%;

[0024] Mobile phase flow rate: 0.2 mL / min; column temperature: 40°C; injection volume: 5 μL.

[0025] Furthermore, the mass spectrometry conditions of the liquid chromatography tandem mass spectrometry detection include:

[0026] 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.

[0027] Furthermore, the 25-hydroxyvitamin D includes 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3.

[0028] The invention has the following beneficial effects: the antibody is spotted on an ELISA plate, and the ELISA plate adsorbs the antibody on its surface through various forces such as van der Waals force, electrostatic attraction, hydrophobic effect, π-π effect, etc., to prepare an extraction plate coated with the antibody; 25-hydroxyvitamin D and other vitamin D metabolites in the sample can be specifically extracted through the immune enrichment effect of the antibody on the extraction plate, and then detected by liquid chromatography-tandem mass spectrometry, with little matrix interference, good specificity and accuracy; at the same time, the sample pretreatment process of the invention is simple, no nitrogen blowing centrifugation and other operations are required, the organic reagent consumption is small, and automatic operation is easy to realize, which is very suitable for the detection needs of clinical samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a chromatogram of the 25-hydroxyvitamin D2 standard solution with a concentration of 1 ng / mL in Example 1 after sample enrichment and target elution;

[0030] Figure 2 This is a chromatogram of the 25-hydroxyvitamin D3 standard solution with a concentration of 1 ng / mL in Example 1 after sample enrichment and target elution;

[0031] Figure 3 It is the standard curve diagram of 25-hydroxyvitamin D2;

[0032] Figure 4 This is the standard curve of 25-hydroxyvitamin D3. DETAILED DESCRIPTION

[0033] In order to make the purpose, technical scheme and advantages of the present invention more clear, the present invention is further described in detail in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0034] The present invention provides a vitamin D mass spectrometry detection method based on antibody immune enrichment technology, comprising the following sub-steps:

[0035] (1) Add 10 to 50 μL of a 0.1 to 1 mg / mL 25-hydroxyvitamin D antibody solution to each well of a 96-well plate, followed by antibody coating to obtain an antibody-coated extraction plate.

[0036] The 25-hydroxyvitamin D antibody is a 25-hydroxyvitamin D monoclonal antibody or a 25-hydroxyvitamin D polyclonal antibody.

[0037] The antibody coating treatment is specifically as follows: incubating at 30-40° C. and 100-300 rpm / min for 1-2 hours with shaking, and then aspirating the remaining solution in the wells.

[0038] The present invention uses a 96-well plate as a medium for adsorbing antibodies to prepare an extraction plate. The 96-well plate is a common consumable for sample pretreatment, which is low in cost, easy to obtain, and the related supporting equipment is relatively common. The material of the 96-well plate can be polystyrene, polyvinyl chloride or polycarbonate. As an embodiment of the present invention, the material of the 96-well plate can be polystyrene. Antibodies are proteins and can be adsorbed on materials such as polystyrene through various forces such as van der Waals force, electrostatic attraction, hydrophobic effect, π-π effect, etc.

[0039] In an embodiment of the present invention, the 25-hydroxyvitamin D antibody should be able to cross-react with both 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3. The 25-hydroxyvitamin D antibody solution is prepared by 25-hydroxyvitamin D antibody and PBS buffer, and the antibody concentration can be 0.1-1 mg / mL, specifically 0.5 mg / mL; the PBS buffer is 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 incubation temperature of the antibody coating treatment can be specifically 37°C, the time can be 1-2h, specifically 2h; the incubation treatment is preferably carried out under oscillation conditions, and the oscillation rate can be 100-300rpm / min, specifically 180rpm / min.

[0040] It is worth mentioning that the antigens that antibodies can bind to are limited. Therefore, based on the specific sample testing needs, while ensuring that the surface of the extraction plate is coated with a sufficient amount of antibodies, the amount of antibodies used should be reduced as much as possible to save production costs.

[0041] (2) The antibody-coated extraction plate is then blocked to obtain a blocked extraction plate; and the blocked extraction plate is then washed to obtain an extraction plate coated with 25-hydroxyvitamin D.

[0042] The blocking treatment is specifically as follows: adding 10-50 μL of a 0.1-2 wt% bovine serum albumin solution to each well of the antibody-coated extraction plate, blocking at 30-40° C. for 1-2 hours, and then aspirating the remaining solution in the well.

[0043] The washing operation is specifically as follows: adding 200 μL of PBS buffer to each well of the blocked extraction plate, aspirating the remaining waste liquid after shaking, and patting dry with a paper towel, and repeating twice.

[0044] In an embodiment of the present invention, the bovine serum albumin solution is prepared from bovine serum albumin and PBS buffer, and the concentration of bovine serum albumin can be 0.1-2wt%, specifically 1wt%. The blocking process can be carried out at room temperature, and the blocking incubation time can be 1-2h, specifically 1h. The main purpose of the blocking operation is to reduce the non-specific adsorption of other substances by the 96-well plate; the washing operation can be performed by adding PBS buffer to the wells of the extraction plate, shaking, sucking it out, and then patting it dry, and the number of washing times can be 2 times.

[0045] (3) Subsequently, the sample to be tested is added to the extraction plate coated with 25-hydroxyvitamin D and then enriched and extracted to obtain an extraction plate enriched with the sample to be tested; the extraction plate enriched with the sample to be tested is then sequentially cleaned and eluted and derivatized to obtain a test solution.

[0046] The sample to be tested is water, milk or other biological samples.

[0047] In an embodiment of the present invention, the biological sample may be serum, the incubation temperature of the sample to be tested may be 37° C., preferably incubated under shaking conditions, and the incubation time may be 10 to 30 minutes, specifically 30 minutes. The methanol aqueous solution may be washed twice, and the methanol content in the methanol aqueous solution may be 5 to 10% (v / v), specifically 10% (v / v).

[0048] The enrichment extraction is specifically as follows: after shaking and incubating at 30-40° C. and 100-300 rpm / min for 10-30 min, the remaining solution in the well is sucked out.

[0049] The cleaning treatment is specifically as follows: adding 100-200 μL of 5-10% methanol aqueous solution to each well of the extraction plate enriched with the sample to be tested, aspirating the remaining waste liquid after shaking, and patting dry with a paper towel, repeating twice.

[0050] The elution derivatization treatment is specifically as follows: 50 to 200 μL of an eluent containing a derivatization agent at a concentration of 0.1 to 1 mg / mL is added to each well of the extraction plate enriched with the sample to be tested after the cleaning treatment, and the elution and derivatization reaction is performed at 30 to 40° C. and 100 to 300 rpm / min for 10 to 30 minutes.

[0051] The derivatizing agent is 4-phenyl-1,2,4-triazolin-3,5-dione, 4-[2-(6,7-dimethoxy-4-methyl-3,4-dihydroquinolinyl)ethyl]-1,2,4-triazolin-3,5-dione or 4-(4'-dimethylaminophenyl)-1,2,4-triazolin-3,5-dione. The eluent is methanol or acetonitrile.

[0052] In an embodiment of the present invention, the eluent may be methanol or acetonitrile. As an embodiment of the present invention, the eluent containing a derivatizing agent may be prepared by a derivatizing agent and acetonitrile, and the derivatizing agent may be 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); the concentration of the derivatizing agent may be 0.1 to 1 mg / mL, specifically 0.5 mg / mL; the derivatizing agent may react with 25-hydroxyvitamin D to form a new adduct, thereby improving the ionization efficiency and the detection sensitivity. The temperature of the elution reaction may be 30 to 40°C, specifically 40°C, and the time may be 10 to 30 min, specifically 30 min. As an embodiment of the present invention, an appropriate amount of water is added to the obtained feed liquid after the elution is completed, and the volume ratio of the eluent to water can be 1:0.5-1, specifically 1:0.5, to obtain a test liquid. The purpose of adding water is to reduce the solvent effect. The test liquid is subjected to liquid chromatography tandem mass spectrometry detection to obtain a test result.

[0053] (4) Then the test solution is subjected to liquid chromatography tandem mass spectrometry detection to obtain a chromatogram of the test solution; according to the standard curve of 25-hydroxyvitamin D and the chromatogram of the test solution, the content of 25-hydroxyvitamin D in the test sample is obtained.

[0054] The liquid phase conditions of the liquid chromatography tandem mass spectrometry detection include:

[0055] 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;

[0056] 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%;

[0057] Mobile phase flow rate: The chromatographic column used was specifically a Waters BEH C18 column (2.1×50 mm, 1.7 μm); 0.2 mL / min; column temperature: 40° C.; injection volume: 5 μL.

[0058] The mass spectrometry conditions of the liquid chromatography tandem mass spectrometry detection include:

[0059] 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. The mass spectrometry conditions of the liquid chromatography tandem mass spectrometry detection may also include: the mass spectrometry model is Waters TQ-Absolute.

[0060] The 25-hydroxyvitamin D includes 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3.

[0061] 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. In the embodiment of the present invention, the standard curve of 25-hydroxyvitamin D is specifically a linear equation of 25-hydroxyvitamin D mass concentration and chromatographic peak area. Specifically, the standard curve of 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. As an embodiment of the present invention, the 25-hydroxyvitamin D standard solution can be specifically prepared by 25-hydroxyvitamin D and PBS buffer solution, and 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 liquid to be tested, the content of 25-hydroxyvitamin D in the liquid to be tested 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.

[0062] Example 1

[0063] (1) Preparation of extraction plate:

[0064] The 96-well plate used in the following examples was purchased from Zhenghong Industrial Products Store, and the brand is Corning. 20 μL of 0.5 mg / mL 25-hydroxyvitamin D antibody solution was added to each well of the 96-well plate, and after shaking and incubating for 2 hours at 37°C and 180 rpm / min, the remaining solution in the well was aspirated to obtain an antibody-coated extraction plate.

[0065] Then, 100 μL of a 1wt% bovine serum albumin solution was added to each well of the antibody-coated extraction plate, and after blocking at 37°C for 1 hour, the remaining solution in the well was aspirated to obtain a blocked extraction plate. Then, 200 μL of PBS buffer was added to each well of the blocked extraction plate, and the remaining waste liquid was aspirated after slight shaking, and the solution was patted dry with a paper towel, and the process was repeated twice to obtain an extraction plate coated with 25-hydroxyvitamin D. The 25-hydroxyvitamin D extraction plate was stored in a refrigerator at 4°C.

[0066] (2) Preparation of test samples: Use PBS buffer solution to prepare 25-hydroxyvitamin D2 standard solutions with a concentration of 1 ng / mL, 2 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL or 100 ng / mL and 25-hydroxyvitamin D3 standard solutions with a concentration of 1 ng / mL, 2 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL or 100 ng / mL as test solutions.

[0067] (3) Sample enrichment: 100 μL of the sample to be tested was then added to each well of the extraction plate coated with 25-hydroxyvitamin D, followed by incubation at 37°C and 180 rpm / min for 30 min, and then the remaining solution in the well was aspirated to obtain an extraction plate coated with 25-hydroxyvitamin D.

[0068] (4) Elution of the target: 200 μL of a 10 wt% methanol-water solution was then added to each well of the extraction plate enriched with the sample to be tested, the remaining waste liquid was aspirated after shaking, and the solution was patted dry with a paper towel, and repeated twice; then 100 μL of a 0.5 mg / mL acetonitrile solution containing 4-phenyl-1,2,4-triazolin-3,5-dione was added to each well of the extraction plate enriched with the sample to be tested after cleaning, and the elution and derivatization reaction were carried out at 37° C. for 30 minutes to obtain the remaining solution in the extraction plate as the test solution.

[0069] (5) Mass spectrometry detection: The test liquid is then subjected to liquid chromatography-tandem mass spectrometry detection to obtain a chromatogram of the test liquid; based on the standard curve of 25-hydroxyvitamin D and the chromatogram of the test liquid, the content of 25-hydroxyvitamin D in the test sample is obtained.

[0070] The liquid phase conditions for the liquid chromatography tandem mass spectrometry detection include: chromatographic column: Waters BEH C18 column (2.1×50 mm, 1.7 μm).

[0071] Mobile phase A: 2 mM ammonium acetate in water; mobile phase B: 2 mM ammonium acetate in methanol.

[0072] Gradient elution program: 0 min, the volume fraction of mobile phase B is 10%; 1 min, the volume fraction of mobile phase B is maintained at 10%; 3.5 min, the volume fraction of mobile phase B is 100%; 4.5 min, the volume fraction of mobile phase B is maintained at 100%; 4.6 min, the volume fraction of mobile phase B is 10%; 5 min, the volume fraction of mobile phase B is maintained at 10%.

[0073] Mobile phase flow rate: 0.2 mL / min; column temperature: 40°C; injection volume: 5 μL.

[0074] Mass spectrometry conditions for the liquid chromatography tandem mass spectrometry detection:

[0075] Mass spectrometer model and acquisition ion mode: Waters TQ-Absolute, ESI positive ion mode.

[0076] Capillary voltage: 3.6 kV; desolvation gas temperature: 500 °C; desolvation gas flow rate: 1000 L / h; cone gas: 150 L / h.

[0077] Scanning mode: Multiple reaction monitoring (MRM) The mass spectrometry parameters of each compound are shown in Table 1.

[0078] Table 1: Mass spectrometry parameters of 255-hydroxyvitamin D

[0079] Compound Name Precursor ion Product ions Cone voltage (V) Collision energy (V) 25-Hydroxyvitamin D3-qn 558.4 280.1 20 28 25-Hydroxyvitamin D3-ql 558.4 298.2 20 10 25-Hydroxyvitamin D2-qn 570.4 280.1 20 28 25-Hydroxyvitamin D2-ql 570.4 298.2 20 14

[0080] After performing liquid chromatography tandem mass spectrometry detection on the test samples of different concentrations in Example 1, the retention time and linear range of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 were obtained. Figure 1 This is a chromatogram of the 25-hydroxyvitamin D2 standard solution with a concentration of 1 ng / mL in Example 1 after sample enrichment and target elution; Figure 2 This is a chromatogram of the 25-hydroxyvitamin D3 standard solution with a concentration of 1 ng / mL in Example 1 after sample enrichment and target elution.

[0081] Table 2 shows the retention time and linear range of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3; Figure 3 It is the standard curve diagram of 25-hydroxyvitamin D2; Figure 4 This is the standard curve of 25-hydroxyvitamin D3.

[0082] Table 2: Retention time and linear range of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3

[0083]

[0084] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A vitamin D mass spectrometry detection method based on antibody immune enrichment technology, characterized in that: It includes the following sub-steps: (1) Adding 10 to 50 μL of a 25-hydroxyvitamin D antibody solution having a concentration of 0.1 to 1 mg / mL to each well of a 96-well plate, followed by antibody coating treatment to obtain an antibody-coated extraction plate; (2) subsequently performing a blocking treatment on the antibody-coated extraction plate to obtain a blocked extraction plate; then washing the blocked extraction plate to obtain an extraction plate coated with 25-hydroxyvitamin D; (3) adding the sample to be tested to the extraction plate coated with 25-hydroxyvitamin D and performing enrichment extraction to obtain an extraction plate enriched with the sample to be tested; and then sequentially performing a cleaning treatment and an elution derivatization treatment on the extraction plate enriched with the sample to be tested to obtain a test solution; (4) Then, the test liquid is subjected to liquid chromatography-tandem mass spectrometry detection to obtain a chromatogram of the test liquid; and the content of 25-hydroxyvitamin D in the test sample is obtained based on a standard curve of 25-hydroxyvitamin D and the chromatogram of the test liquid.

2. The vitamin D mass spectrometry detection method based on antibody immune enrichment technology according to claim 1, characterized in that: The 25-hydroxyvitamin D antibody is a 25-hydroxyvitamin D monoclonal antibody or a 25-hydroxyvitamin D polyclonal antibody.

3. The vitamin D mass spectrometry detection method based on antibody immune enrichment technology according to claim 1, characterized in that: In step (1), the antibody coating treatment is specifically as follows: incubating with shaking at 30-40° C. and 100-300 rpm / min for 1-2 hours, and then aspirating the remaining solution in the well.

4. The vitamin D mass spectrometry detection method based on antibody immune enrichment technology according to claim 1, characterized in that: In step (2), the blocking treatment is specifically as follows: adding 10 to 50 μL of a 0.1 to 2 wt% bovine serum albumin solution to each well of the antibody-coated extraction plate, blocking at 30 to 40° C. for 1 to 2 h, and then aspirating the remaining solution in the well; The washing operation is specifically as follows: adding 200 μL of PBS buffer to each well of the blocked extraction plate, aspirating the remaining waste liquid after shaking, and patting dry with a paper towel, and repeating twice.

5. The vitamin D mass spectrometry detection method based on antibody immune enrichment technology according to claim 1, characterized in that: The sample to be tested is water, milk or other biological samples.

6. The vitamin D mass spectrometry detection method based on antibody immune enrichment technology according to claim 1, characterized in that: In step (3), the enrichment extraction is specifically as follows: incubating at 30-40° C. and 100-300 rpm / min for 10-30 min with shaking, and then aspirating the remaining solution in the well; The cleaning process is specifically as follows: adding 100-200 μL of 5-10% methanol aqueous solution to each well of the extraction plate enriched with the sample to be tested, aspirating the remaining waste liquid after shaking, and patting dry with a paper towel, repeating twice; The elution derivatization treatment is specifically as follows: 50 to 200 μL of an eluent containing a derivatization agent at a concentration of 0.1 to 1 mg / mL is added to each well of the extraction plate enriched with the sample to be tested after the cleaning treatment, and the elution and derivatization reaction is performed at 30 to 40° C. and 100 to 300 rpm / min for 10 to 30 minutes.

7. The method for mass spectrometry detection of vitamin D based on antibody immune enrichment technology according to claim 6, characterized in that: The derivatizing agent is 4-phenyl-1,2,4-triazolin-3,5-dione, 4-[2-(6,7-dimethoxy-4-methyl-3,4-dihydroquinolinyl)ethyl]-1,2,4-triazolin-3,5-dione or 4-(4'-dimethylaminophenyl)-1,2,4-triazolin-3,5-dione; The eluent is methanol or acetonitrile.

8. The vitamin D mass spectrometry detection method based on antibody immune enrichment technology according to claim 1, 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%; Mobile phase flow rate: 0.2 mL / min; column temperature: 40°C; injection volume: 5 μL.

9. The vitamin D mass spectrometry detection method based on antibody immune enrichment technology according to claim 1, 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.

10. The vitamin D mass spectrometry detection method based on antibody immune enrichment technology according to claim 1, characterized in that: The 25-hydroxyvitamin D includes 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3.

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