Screening, typing and diagnosing marker for automatically and quantitatively determining primary aldosteronism in blood plasma through paramagnetic particle method

Abstract

The invention relates to a screening and typing diagnosis marker for automatically and quantitatively determining primary aldosteronism in plasma through a paramagnetic particle method, and belongs to the technical field of substance detection. The porous magnetic microsphere provided by the invention is applied to adsorption of steroid hormones and polypeptide hormones with molecular weight less than 5000Da, the porous magnetic microsphere has a porous structure, and epoxypropyl methacrylate and pyrrolidone groups are chemically modified, grafted and / or bonded on the surface of the porous structure. The steroid hormone and the polypeptide hormone of which the molecular weight is less than 5000Da can be captured, the steroid hormone and the polypeptide hormone have higher selectivity, the adsorption effect on interfering substances is weak, the interference of antibody cross reaction can be reduced, and the steroid hormone and the polypeptide hormone can be further separated from a blood sample.

Description

Technical Field

[0001] This application relates to the field of substance detection, and particularly to a magnetic bead method for automated quantitative determination of screening and typing diagnostic markers for primary aldosteronism in plasma. Background Art

[0002] The clinical detection of steroid hormones and polypeptide hormones mainly relies on immunological methods. Although immunological methods have the advantages of high automation, fast detection speed, low cost, etc. and have become a common means in clinical experiments, their disadvantages are also very obvious. This technology usually requires a large sample size, is time-consuming and laborious, and the detection results are easily affected by other interfering substances, such as the interference of antibody cross-reaction, reducing the reliability of the results. Therefore, immunological methods are usually only applicable to the analysis of single or a few substances, and are particularly susceptible to insufficient antibody specificity in the detection of polypeptide hormones, resulting in decreased sensitivity and accuracy. Summary of the Invention

[0003] The purpose of this application is to provide a porous magnetic microsphere and its preparation method, a detection kit and a detection method, so as to solve the problem that when detecting steroid hormones and polypeptide hormones in existing blood samples by immunological methods, they are easily affected by other interfering substances, resulting in decreased sensitivity and accuracy.

[0004] In the first aspect, this application provides a porous magnetic microsphere, which is applied to the adsorption of steroid hormones and / or polypeptide hormones with a molecular weight less than 5000 Da. The porous magnetic microsphere includes a polystyrene-divinylbenzene backbone, and glycidyl methacrylate and pyrrolidone groups are grafted and / or bonded to the polystyrene-divinylbenzene backbone.

[0005] The porous magnetic microsphere of this application has a porous structure, and through chemical modification on the surface of the porous structure, glycidyl methacrylate and pyrrolidone groups are grafted and / or bonded, which can capture steroid hormones and polypeptide hormones with a molecular weight less than 5000 Da, and has high selectivity for steroid hormones and polypeptide hormones, while having a weak adsorption effect on other interfering substances, which can reduce the interference of antibody cross-reaction, and thus can separate steroid hormones and polypeptide hormones from blood samples.

[0006] In the second aspect, this application provides a preparation method of a porous magnetic microsphere, including the following steps:

[0007] Carry out a cross-linking polymerization reaction on styrene and divinylbenzene in a reaction system containing an initiator, a pore-forming agent and magnetic particles modified with polyethylene glycol to obtain a polystyrene-divinylbenzene backbone;

[0008] The polystyrene-divinylbenzene framework was modified with glycidyl methacrylate and pyrrolidone groups to obtain porous magnetic microspheres.

[0009] The preparation method of the porous magnetic microspheres in this application can prepare porous structures, which can be applied to the adsorption of steroid hormones and polypeptide hormones with a molecular weight less than 5000 Da. In the cross-linking polymerization reaction process of this application, magnetic particles are introduced and bonded to the polystyrene-divinylbenzene matrix to obtain polymer magnetic microspheres, which have magnetism under a magnetic field and at the same time have the adsorption performance of polystyrene-divinylbenzene, and can be applied to the extraction and purification of acidic, alkaline and / or neutral compounds.

[0010] In the third aspect, this application provides a detection kit for polypeptide hormones, including the porous magnetic microspheres described above or the porous magnetic microspheres prepared by the preparation method described above.

[0011] Precisely because of the porous magnetic microspheres described above or the porous magnetic microspheres prepared by the preparation method described above, the steroid hormones and polypeptide hormones, which are the markers for the screening, diagnosis and typing of primary aldosteronism, can be detected simultaneously to achieve more accurate disease diagnosis and typing. At the same time, the magnetic solid-phase extraction method can be used to quickly and effectively pretreat plasma samples.

[0012] In the fourth aspect, this application provides a detection method for a detection kit, including the following steps:

[0013] After the porous magnetic microspheres are sequentially subjected to activation treatment and equilibration treatment, they are added to the sample to be tested for extraction treatment, wherein the sample to be tested contains steroid hormones and polypeptide hormones;

[0014] After the extraction treatment, the porous magnetic microspheres are rinsed with a rinsing solution and then eluted with an eluent to obtain a test solution;

[0015] The test solution is subjected to liquid chromatography-tandem mass spectrometry analysis to obtain the concentration information of the polypeptide hormones in the sample to be tested.

[0016] The present application uses porous magnetic microspheres as porous magnetic microspheres, and through chemical modification on the surface of the porous structure, grafting and / or bonding with methacrylate and pyrrolidone groups, polypeptide hormones with a molecular weight of less than 5000Da can be captured, and the magnetic field force is used to separate steroid hormones and polypeptide hormones from the sample matrix, and the impurity interference substances with properties close to those of steroid hormones are removed in conjunction with the elution and elution steps, and the desorption of steroid hormones and polypeptide hormones is completed to achieve the removal of impurities and purification of polypeptide hormones. The porous magnetic microspheres of the present application have strong binding performance with polypeptide hormones and high extraction efficiency. The purity of steroid hormones and polypeptide hormones in the obtained test solution is high, and there are fewer impurity interference substances, which is conducive to improving the ionization efficiency and detection sensitivity of mass spectrometry detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 A standard curve of angiotensin I in a standard product obtained by the detection method of the present application;

[0018] Figure 2 A standard curve of angiotensin II in a standard product obtained by the detection method of the present application;

[0019] Figure 3 A standard curve of aldosterone in a standard substance obtained by the detection method of the present application;

[0020] Figure 4 A standard curve of 18-hydroxycorticosterone in a standard substance obtained by the detection method of the present application;

[0021] Figure 5 A standard curve of 18-hydroxycortisol in a standard substance obtained by the detection method of the present application;

[0022] Figure 6 A standard curve of 18-oxycortisol in a standard substance obtained by the detection method of the present application;

[0023] Figure 7 The detection spectrum of angiotensin I in the standard product obtained by the detection method of the present application;

[0024] Figure 8 The detection spectrum of angiotensin II in the standard product obtained by the detection method of the present application;

[0025] Figure 9 The detection spectrum of aldosterone in the standard product obtained by the detection method of the present application;

[0026] Figure 10 This is the detection spectrum of 18-hydroxycorticosterone in the standard product obtained by the detection method of the present application;

[0027] Figure 11The detection spectrum of 18-hydroxycortisol in the standard product obtained by the detection method of the present application;

[0028] Figure 12 The detection method of the present application is used to determine the 18-oxycortisol in the standard product. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical solution and advantages of the present application more clear, the present application is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0030] For simplicity, this application only explicitly discloses some numerical ranges. However, any lower limit can be combined with any upper limit to form an unclearly recorded range; and any lower limit can be combined with other lower limits to form an unclearly recorded range, and any upper limit can be combined with any other upper limit to form an unclearly recorded range. In addition, although not clearly recorded, each point or single value between the range endpoints is included in the range. Thus, each point or single value can be combined with any other point or single value as its own lower limit or upper limit or with other lower limits or upper limits to form an unclearly recorded range.

[0031] Hormones can be divided into three main types based on their chemical structure: steroids, polypeptides, and amines. Steroid hormones, also known as steroid hormones, are biologically active compounds secreted by the adrenal cortex, gonads, and placenta. These hormones can be divided into mineralocorticoids, glucocorticoids, and sex hormones based on the type of receptors they bind to. They play a vital role in regulating growth, development, reproduction, and maintaining a stable internal environment. Changes in steroid hormone levels are closely related to the diagnosis of a variety of diseases, including endocrine disorders and certain psychiatric disorders. Peptide hormones such as angiotensin I, which is an important component of the renin-angiotensin system (RAAS), are 10 peptides produced by the action of renin on angiotensinogen. Angiotensin I is converted into angiotensin II by the action of convertase in the lungs. The latter is a potent vasoconstrictor that can increase blood pressure and stimulate the adrenal cortex to secrete aldosterone.

[0032] Primary aldosteronism (PA) is a disorder in which the adrenal cortex produces excessive amounts of aldosterone, leading to elevated blood pressure and hypokalemia.

[0033] Angiotensin I and II regulate blood pressure and electrolyte balance under normal circumstances, but in PA, the secretion of aldosterone is detached from the regulation of the RAAS. 18-hydroxycorticosterone (18-OHB), 18-hydroxycortisol (18-OHF), and 18-oxocortisol (18-OXOF), as metabolites of steroid hormones, play important biomarker roles in the diagnosis of PA subtypes.

[0034] The inventors found that with the progress of instrument technology, chromatographic-mass spectrometric techniques (such as gas chromatography-tandem mass spectrometry GC-MS / MS and liquid chromatography-tandem mass spectrometry LC-MS / MS) have shown obvious advantages in detection sensitivity and accuracy. Especially LC-MS / MS, which can be combined with high-throughput analysis and become an ideal tool for the routine detection of steroid hormones. More and more studies have shown that in disease diagnosis, comprehensive analysis of multiple steroid hormones is more diagnostically valuable than a single hormone index. Mass spectrometry technology can quickly detect multiple steroid hormones and polypeptide hormones, and this advantage has gradually made it occupy an important position in clinical detection.

[0035] However, LC-MS / MS still faces challenges in clinical applications, especially the complex sample pretreatment process, which usually requires multiple steps of solid-phase extraction (SPE). Although existing technologies have tried to simplify this process through automated enzyme-linked immunosorbent assays and use robotic arms to achieve sample loading and sample transfer for SPE, the processing speed is slow, and multiple centrifugation and positive pressure steps are still required, with limited automation. In addition, manual operation of SPE requires high skills from technicians and is prone to human errors, further affecting the detection efficiency and accuracy. For polypeptide hormones, the challenge of mass spectrometry detection methods lies in their large molecular weight and complex structure, which make them more vulnerable to interference during pretreatment and detection, further increasing the difficulty of detection.

[0036] Although gas chromatography-mass spectrometry (GC-MS) has high specificity, is suitable for the quantitative analysis of steroid hormones and can analyze multiple hormones simultaneously, its sample pretreatment (such as extraction, purification, derivatization) is complex, increasing the operation difficulty and limiting its wide application in clinical practice. Similarly, in the detection of polypeptide hormones, the GC-MS method has high requirements for the stability of hormone molecules during sample processing, further increasing the complexity and time cost.

[0037] Therefore, the existing technologies face problems of insufficient sensitivity and specificity in the detection of steroid hormones and polypeptide hormones. At the same time, the complex pretreatment process of mass spectrometry detection methods is time-consuming and prone to introducing errors, affecting the accuracy of detection results. In addition, there are differences in hormone secretion levels among different types of primary aldosteronism (PA) patients, which further affect diagnosis and treatment. Therefore, there is an urgent need to develop a technology that can simultaneously detect steroid hormones and polypeptide hormones to achieve more accurate disease diagnosis and classification.

[0038] In view of this, a first aspect of an embodiment of the present application provides a porous magnetic microsphere, which uses the porous magnetic microsphere as an extraction filler, and by chemically modifying the surface of the porous structure, grafting and / or bonding methacrylate and pyrrolidone groups, steroid hormones and polypeptide hormones with a molecular weight of less than 5000Da can be captured simultaneously, and the microsphere has high selectivity for steroid hormones and polypeptide hormones, can remove impurities and interferents as much as possible, and can separate steroid hormones and polypeptide hormones from blood samples.

[0039] In some embodiments, polypeptide hormones with a molecular weight of less than 5000Da can be captured to solve the problem that the polypeptide hormones in blood samples have a large molecular weight and a complex structure, which leads to interference during mass spectrometry detection and increases the difficulty of detection. Specifically, polypeptide hormones include multiple components, among which angiotensin I and angiotensin II have a large molecular weight, and are rich in amino and carboxyl groups, which are easy to bind to proteins in the blood and difficult to extract. The extraction filler of this scheme is a main body of polystyrene divinylbenzene, and at the same time has a porous structure, which can allow angiotensin I and angiotensin II to enter the porous structure of the porous magnetic microspheres, and its benzene ring framework and the methacrylate glycidyl and pyrrolidone groups grafted or / and bonded on the surface of the porous structure can enhance the interaction with angiotensin I and angiotensin II, and perform tight binding, thereby reducing the binding of polypeptide hormones to proteins, which is conducive to reducing adsorption competition, and thus can effectively improve the extraction of polypeptide hormones in blood samples.

[0040] In some embodiments, the pore size of the porous magnetic microsphere is B, The pore size of porous magnetic microspheres can reach Furthermore, the porous magnetic microspheres have a large enough space to accommodate steroid hormones and polypeptide hormones with a molecular weight of less than 5000Da. In addition, the molecular weights of angiotensin I and angiotensin II contained in the polypeptide hormones are both less than 1500Da, so they can smoothly enter the porous structure. Moreover, since glycidyl methacrylate and pyrrolidone groups are grafted and / or bonded to the polystyrene-divinylbenzene skeleton, the adsorption of angiotensin I and angiotensin II is strong, and thus angiotensin I and angiotensin II can be easily captured. Furthermore, by controlling the porosity of the porous magnetic microspheres to 60% to 80%, the adsorption efficiency of steroid hormones and polypeptide hormones can be improved.

[0041] In some embodiments, the molar ratio of the polystyrene-divinylbenzene backbone, glycidyl methacrylate, and pyrrolidone groups is (5 to 15):(1 to 5):(4 to 20). By controlling the appropriate ratio of glycidyl methacrylate and pyrrolidone groups in the porous magnetic microspheres, their polar and non-polar groups are coordinated with those of steroid hormones and polypeptide hormones, thereby enabling stronger binding ability to steroid hormones and polypeptide hormones, which is beneficial to improving the adsorption effect of the porous magnetic microspheres on steroid hormones and polypeptide hormones.

[0042] Furthermore, in the porous magnetic microspheres, the molar ratio of the polystyrene-divinylbenzene backbone, glycidyl methacrylate, and pyrrolidone groups is (5 to 15):(1 to 5):(4 to 20). Further controlling the ratio of glycidyl methacrylate and pyrrolidone groups can enable the porous magnetic microspheres to exhibit excellent binding effects with both polypeptide hormones and steroid hormones in a blood sample, and the binding specificity is higher, reducing the adsorption of non-target impurities.

[0043] The second aspect of the embodiments of the present application provides a method for preparing porous magnetic microspheres, including the following steps:

[0044] Step S10: Styrene and divinylbenzene are subjected to a cross-linking polymerization reaction in a reaction system containing an initiator, a pore-forming agent, and magnetic particles modified with polyethylene glycol to obtain a polystyrene-divinylbenzene backbone;

[0045] Step S20: The porous surface of the polystyrene-divinylbenzene backbone is modified with glycidyl methacrylate and pyrrolidone groups to obtain porous magnetic microspheres.

[0046] In the embodiments of the present application, magnetic particles are introduced during the cross-linking polymerization reaction and bonded to the polystyrene-divinylbenzene matrix to obtain polymer magnetic microspheres, which have magnetism under a magnetic field and at the same time have the adsorption properties of polystyrene-divinylbenzene, and can be applied to the extraction and purification of acidic, basic, and / or neutral compounds.

[0047] In some embodiments, the initiator includes at least one of azobisisobutyronitrile, benzoyl peroxide, and sodium metabisulfite.

[0048] In some embodiments, the pore-forming agent can be selected from at least one of toluene, liquid paraffin, and dibutyl phthalate; the reaction system for the cross-linking polymerization reaction in the present application is an ethanol aqueous solution system. By adjusting the types and amounts of different pore-forming agents, the pore size and porosity can be finely adjusted, which is beneficial to balancing the adsorption capacity and the number of adsorption sites of the porous magnetic beads for steroid hormones and / or polypeptide hormones, and further improving the adsorption efficiency of the porous magnetic beads for steroid hormones and / or polypeptide hormones.

[0049] In some embodiments, the magnetic particles include at least one of iron particles, cobalt particles, nickel particles, and magnetite particles.

[0050] In some embodiments, the magnetite particles modified with polyethylene glycol in the present application are obtained by a preparation method including the following steps: after mixing a ferrous salt, an iron salt, a reducing agent, and polyethylene glycol, a precipitating agent is added for coprecipitation treatment to obtain the magnetite particles modified with polyethylene glycol.

[0051] In some embodiments, when carrying out the crosslinking polymerization reaction, the molar ratio of styrene to divinylbenzene is (1-6):(1-9). Specifically, the conditions of the crosslinking polymerization reaction include: the temperature of the crosslinking polymerization reaction is 50-80 °C, and the time of the crosslinking polymerization reaction is 10-24 h. By controlling the ratio of the styrene and divinylbenzene reactants and the reaction conditions in the present application, the number of benzene rings and crosslinked carbon chains in the polystyrene-divinylbenzene magnetic microspheres is controlled, and further the polarity of the porous magnetic microspheres is controlled, so that they can better bind steroid hormones and polypeptide hormones, reduce the binding with non-target substances, and improve the extraction effect of magnetic solid-phase extraction.

[0052] To further improve the binding effect of the porous magnetic microspheres with steroid hormones and polypeptide hormones, adjust the average pore size and porosity of the porous magnetic microspheres, and enable the polystyrene-divinylbenzene skeleton to better cooperate with hydrophilic groups. When carrying out the crosslinking polymerization reaction, the molar ratio of styrene to divinylbenzene is (0.5-5):(6-10).

[0053] In some embodiments, the porous magnetic microspheres are obtained by a preparation method including the following steps: styrene and divinylbenzene are used as the basic skeleton of the magnetic microspheres and can be obtained by a free radical polymerization reaction with an initiator. The glycidyl methacrylate and pyrrolidone functional groups can be imparted to the microsphere skeleton of polystyrene divinylbenzene by grafting and / or bonding on the basis of free radical polymerization to obtain the hydrophilic and lipophilic magnetic microspheres.

[0054] The third aspect of the embodiments of the present application provides a detection kit for polypeptide hormones, including the porous magnetic microspheres described above or the porous magnetic microspheres prepared by the preparation method of the porous magnetic microspheres.

[0055] The embodiments of the present application provide a detection kit for sex hormones. The porous magnetic microspheres have a porous structure. The porous magnetic microspheres include a polystyrene-divinylbenzene backbone, and glycidyl methacrylate and pyrrolidone groups are grafted and / or bonded to the polystyrene-divinylbenzene backbone. The detection kit of the embodiments of the present application uses the porous magnetic microspheres as extraction fillers. Through the porous structure of the porous magnetic microspheres, and the chemical modification of the porous surface by glycidyl methacrylate and pyrrolidone groups through grafting or bonding means, the adsorption of steroid hormones and polypeptide hormones is promoted. In addition, magnetic adsorption technology can be used to extract and enrich steroid hormones and polypeptide hormones.

[0056] In some embodiments, the detection kit further includes an activation solution, which can act synergistically with the porous magnetic microspheres to remove interfering substances in the blood sample, thereby improving the accuracy and reliability of the detection. In addition, the activation solution includes a first alcohol compound and a first ketone compound with a volume ratio of (50-100):(0-20). The first alcohol compound is selected from at least one of methanol, ethanol, and isopropanol. The first ketone compound is selected from at least one of acetone, methyl butanone, and methyl isobutyl ketone. By adjusting the types and ratios of the activation solution, the adsorption effect on steroid hormones and polypeptide hormones can be further improved.

[0057] In some embodiments, the detection kit further includes a balance solution, which can act synergistically with the porous magnetic microspheres. The balance solution can provide a stable chemical environment for the blood sample, thereby improving the accuracy and reliability of the detection result. The balance solution includes a second alcohol compound, a second ketone compound, and water with a volume ratio of (5-30):(0-10):(50-90). The second alcohol compound is selected from at least one of methanol, ethanol, and isopropanol. The second ketone compound is selected from at least one of acetone, methyl butanone, and methyl isobutyl ketone. By adjusting the types and ratios of the activation solution, the adsorption effect on steroid hormones and polypeptide hormones can be further improved.

[0058] In some embodiments, the detection kit further includes an elution solution, which can effectively desorb steroid hormones and polypeptide hormones from the porous magnetic microspheres. The elution solution includes a third alcohol compound, a first nitrile compound, and water with a volume ratio of (6-100):(0-60):(0-40). Further, the third alcohol compound is selected from at least one of methanol, ethanol, and isopropanol. The first nitrile compound and the second nitrile compound are selected from at least one of acetonitrile, propionitrile, and acrylonitrile. By adjusting the types and ratios of the activation solution, the accuracy and reliability of the detection can be further improved.

[0059] In some embodiments, the detection kit further includes a first eluent and a second eluent, which can wash away some impurities in the porous magnetic microspheres. The first eluent includes a fourth alcohol compound, a third ketone compound and water, forming a weak elution, mainly washing away impurities with stronger polarity. The second eluent includes a fifth alcohol compound, a second nitrile compound and water, forming a strong elution, mainly washing away impurities with weaker polarity. The volume ratio of the fourth alcohol compound, the third ketone compound and water is (1-10):(0-3):(50-95).

[0060] The volume ratio of the fourth alcohol compound, the second nitrile compound and water is (6-40):(0-10):(60-90). Further, the fourth alcohol compound is selected from at least one of methanol, ethanol, and isopropanol. The fifth alcohol compounds are each independently selected from at least one of methanol, ethanol, and isopropanol. The third ketone compound is selected from at least one of acetone, methyl butanone, and methyl isobutyl ketone. The second nitrile compound is selected from at least one of acetonitrile, propionitrile, and acrylonitrile. By adjusting the types and ratios of the first eluent and the second eluent, the accuracy and reliability of the detection can be further improved.

[0061] In the fourth aspect of the embodiments of the present application, a detection method for a polypeptide hormone detection kit is provided, including the following steps:

[0062] Step S30: After the porous magnetic microspheres are sequentially subjected to activation treatment and equilibration treatment, they are added to a test sample for extraction treatment, wherein the test sample contains steroid hormones and / or polypeptide hormones;

[0063] Step S40: After the extraction treatment, the porous magnetic microspheres are washed with an eluent and then eluted with an eluent to obtain a test solution;

[0064] Step S50: Perform liquid chromatography-tandem mass spectrometry analysis on the test solution to obtain the concentration information of the polypeptide hormones in the test sample.

[0065] In the embodiments of the present application, the hydrophilic-lipophilic magnetic microspheres are tightly combined with steroid hormones and polypeptide hormones. The magnetic solid-phase extraction technology can quickly and effectively extract steroid hormones and polypeptide hormones in blood samples. The transfer of steroid hormones and polypeptide hormones between the eluent and the eluent is realized under the action of a magnetic field, and the steroid hormones and polypeptide hormones are purified and decontaminated. The steps are simple, and it can also be combined with an automated magnetic solid-phase extraction processing system to further simplify manual operations and reduce human errors, improving the accuracy of the detection of steroid hormones and polypeptide hormones.

[0066] In some embodiments, six key substances such as aldosterone, angiotensin I, angiotensin II, 18-hydroxycorticosterone, 18-hydroxycortisol, and 18-oxocortisol in a blood sample can be detected simultaneously, and qualitative and quantitative analysis can be accurately performed.

[0067] Compared with the protein precipitation pretreatment method in the embodiments of the present application, the porous magnetic microspheres in the embodiments of the present application can be directly added to the protein precipitation system for extraction, so that the two pretreatment steps of protein precipitation and extraction purification are combined into one step. On the other hand, due to the limited diffusion and mass transfer rates in the traditional solid-phase extraction process, the equilibrium time of the extraction process is usually long. The porous magnetic microspheres of the present application are completely exposed to the system to be tested and are in full contact with steroid hormones and / or polypeptide hormones. Therefore, steroid hormones and / or polypeptide hormones can be adsorbed and extracted from a large-volume system to be tested in a short time, and then separated from the sample to be tested by using an external magnetic field, avoiding cumbersome processing procedures, thereby absolutely shortening the enrichment and purification processes. The operation time required for the traditional protein precipitation and solid-phase extraction processes is 2-3 hours. The detection method in the embodiments of the present application optimizes the conditions for impurity removal and separation for plasma samples; combined with the magnetic solid-phase extraction technology, the magnetic solid-phase extraction packing can be directly added to the system after impurity removal and separation, and the enrichment of steroid hormones can be completed within 180 seconds, shortening the total operation time of the pretreatment to within 20 minutes, greatly improving the detection efficiency of steroid hormones, and having the advantages of high accuracy and precision. In addition, it can meet the inter-batch precision <15%, the intra-batch precision <15%, and the accuracy of the concentration detection value is between 89.10% and 107.54%, which is beneficial to the diagnosis and prognosis evaluation of primary aldosteronism with steroid hormones and polypeptide hormones as markers.

[0068] In the embodiments of the present application, the porous magnetic microspheres can be used as a magnetic solid-phase extraction packing. Magnetic-solid phase extraction (abbreviated as M-SPE) is a dispersive solid-phase extraction technology using magnetic or magnetizable materials as the adsorbent matrix. On the basis of the solid-phase extraction technology, the packing is given magnetism, and it can be aggregated and transferred under the action of a magnetic field. The target substance is extracted by using the specific adsorption ability of the magnetic solid-phase extraction packing, and the purification and desorption processes are completed by transferring between different functional liquids, and the separation and purification in a complex sample matrix can be realized.

[0069] The porous magnetic microspheres of the present application are hydrophilic and lipophilic magnetic microspheres. Through the porous structure and the cooperation of hydrophilic groups and lipophilic groups on the hydrophilic and lipophilic magnetic microspheres, they can achieve rapid and tight binding to steroid hormones and / or polypeptide hormones, so that steroid hormones and / or polypeptide hormones can be adsorbed and extracted from a large-volume sample system to be detected in a short time. The entire extraction process reduces the use of toxic and harmful organic solvents, simplifies the cumbersome sample treatment steps, and can be equipped with a magnetic solid-phase extraction automated processing system to achieve automated processing.

[0070] In some specific embodiments, the present application provides a detection kit. Each component, such as porous magnetic microspheres, activation solution, equilibration solution, washing solution, elution solution, etc., can be pre-loaded into a reaction plate product. For example, in a 96-well plate kit, the 96-well plate kit can be sealed with an aluminum film, etc., which greatly facilitates the use of the product. In actual operation, only need to tear open the sealing film, and then add standards, quality control products, samples to be detected, etc. into the sample wells as required. By applying an external magnetic field to transfer the magnetic solid-phase extraction column packing, the activation, equilibration, sample loading, washing, elution and other procedures involved in the detection method can be realized, and the magnetic solid-phase extraction process of steroid hormones and / or polypeptide hormones can be completed.

[0071] In some specific embodiments, the detection kit of the present application can also be paired with a magnetic solid-phase extraction automated processing system, such as the ZPTQ32 full-automatic sample pretreatment system. The activation, equilibration, sample loading, washing, elution and other procedures involved in the detection method are automatically completed by the program, realizing a full-automatic sample pretreatment solution of porous magnetic microspheres combined with a full-automatic sample pretreatment system, completing automated operation, reducing the error introduced by humans, facilitating the liberation of productivity, and can more quickly and effectively realize the extraction, purification and concentration of steroid hormones and / or polypeptide hormones in blood samples. It not only improves the detection efficiency, but also greatly improves the stability and accuracy of the detection results, and is applicable to the clinical detection of primary aldosteronism screening, with the advantages of convenience and speed.

[0072] The embodiments of the present application provide a detection kit, which is used for analysis and detection in combination with liquid chromatography-tandem mass spectrometry (LC-MS / MS), and can be used for in vitro quantitative testing of substances such as aldosterone, angiotensin I, angiotensin II, 18-hydroxycorticosterone, 18-hydroxycortisol, 18-oxocortisol, etc. in blood samples. These substances are all closely related to primary aldosteronism and are important indicators for clinical evaluation, and are very valuable in clinical and physiological research.

[0073] The detection method of the present application also requires the use of calibration solution and quality control solution.

[0074] The calibration solution may be a mixed solution of aldosterone, angiotensin I, angiotensin II, 18-hydroxycorticosterone, 18-hydroxycortisol, and 18-oxycortisol with known series of concentrations.

[0075] The quality control product can use plasma after removing the hormone background as the matrix, add a mixed solution of aldosterone, angiotensin I, angiotensin II, 18-hydroxycorticosterone, 18-hydroxycortisol, and 18-oxycortisol at specific concentrations, and can be divided into high-concentration quality control products, medium-concentration quality control products, and low-concentration quality control products. The concentrations of steroid hormones and / or polypeptide hormones in high-concentration quality control products, medium-concentration quality control products, and low-concentration quality control products decrease in sequence. Among them, the plasma after removing the hormone background can be obtained from conventional commercial channels.

[0076] In some embodiments, the liquid phase conditions of liquid chromatography tandem mass spectrometry are 35°C, the chromatographic column is an HSS T3 column, and the mobile phases of the liquid chromatography are mobile phase A and mobile phase B, respectively. The mobile phase A is prepared at a volume ratio of formic acid to ultrapure water of 1:1000, the formic acid is LC-MS grade, and the purity is 99.0%. The mobile phase B is prepared at a volume ratio of formic acid to acetonitrile to ultrapure water of 1:1000, the formic acid to acetonitrile is LC-MS grade, and the purity is 99.0%.

[0077] The detection method of the present application also includes: after performing the above-mentioned magnetic solid phase extraction and liquid chromatography tandem mass spectrometry analysis on the calibrator, a standard curve is established based on the analysis results with the concentration of the sex hormone standard in the calibrator as the x-axis and the peak area ratio of the sex hormone standard to the internal standard as the y-axis.

[0078] In some embodiments, after the steroid hormone and / or polypeptide hormone to be tested in the test solution is analyzed by liquid chromatography tandem mass spectrometry, the concentration of the steroid hormone and / or polypeptide hormone to be tested in the test sample is obtained based on the standard curve.

[0079] Example

[0080] The technical solution of the present application is described below in conjunction with specific embodiments. The raw materials used in the following embodiments are all from common commercially available products, and the devices or equipment used are all purchased from conventional market sales channels.

[0081] Example 1-1

[0082] The porous magnetic microspheres of this embodiment have a pore size of The porosity is 60%,

[0083] The porous magnetic microspheres include a polystyrene-divinylbenzene skeleton, and glycidyl methacrylate and pyrrolidone groups are grafted and / or bonded to the polystyrene-divinylbenzene skeleton;

[0084] The molar ratio of the polystyrene-divinylbenzene skeleton, glycidyl methacrylate and pyrrolidone group is (5-15):(1-5):(4-20).

[0085] The porous magnetic microspheres are obtained by a preparation method comprising the following steps:

[0086] Place the three-necked flask on a water bath, the reaction system is an ethanol-water system, turn on the stirring paddle, stir at a constant speed, pass inert gas nitrogen into the three-necked flask to remove oxygen, and then add hydroxymethyl cellulose to the three-necked flask. After stirring and dissolving evenly, add styrene and divinylbenzene, the molar ratio of styrene and divinylbenzene is (0.5-5): (6-10), continue to stir evenly, then add initiators azobisisobutyronitrile and benzoyl peroxide, stir and dissolve, then add the dissolved polyethylene glycol-modified ferrosoferric oxide nanoparticles, and then add toluene as a porogen, continue to stir at a constant speed for 20 minutes, so that the reaction system is mixed evenly.

[0087] Then, the water bath was turned on to heat the reaction system to 70°C, and the temperature was maintained for 16 hours after stabilization for cross-linking polymerization. After the reaction was completed, the reaction system was transferred to a suction filtration bottle for suction filtration, and then washed with water and ethanol in turn to obtain the reaction product: polystyrene-divinylbenzene magnetic microspheres. The washed product was soaked in a hydrochloric acid solution overnight to remove the magnetic substances on the surface of the polystyrene-divinylbenzene magnetic microspheres, and then filtered and washed again through a suction filtration bottle. After washing the reaction product with water until neutral, it was washed three times with ethanol three times the volume of the reaction product, and after drying, it was transferred to a 60°C thermostat for drying overnight to obtain the final polystyrene-divinylbenzene magnetic microsphere skeleton with an average particle size of 40um.

[0088] The introduced pyrrolidone and glycidyl methacrylate functional groups can be imparted to the magnetic skeleton of polystyrene divinyl benzene by grafting and / or bonding. The molar ratio of polystyrene divinyl benzene to glycidyl methacrylate and pyrrolidone is (5-15):(1-5):(4-20), and the reaction temperature is 70°C.

[0089] Example 1-2 to Example 1-5

[0090] The differences between Examples 1-2 to 1-5 and Example 1-1 are the pore size, porosity, and molar ratio of the polystyrene-divinylbenzene skeleton, glycidyl methacrylate, and pyrrolidone groups of the porous magnetic microspheres. Other points are the same as those of Example 1-1. The specific contents are shown in Table 1.

[0091] Table 1

[0092]

[0093]

[0094] Example 2-1

[0095] The detection kit of polypeptide hormones in this embodiment includes the porous magnetic microspheres in Example 1-1 and the following other components:

[0096] Activation solution: The volume ratio of ethanol and acetone is (50-100):(0-20).

[0097] Balance solution: The volume ratio of ethanol, acetone and water is (5-30):(0-10):(50-90).

[0098] The volume ratio of the first eluent: ethanol aqueous solution, acetone and water is (1-10):(0-3):(50-95).

[0099] The second eluent: the volume ratio of ethanol, acetonitrile and water is (6-40):(0-10):(60-90).

[0100] Eluent: volume ratio of ethanol, acetonitrile and water (6-100):(0-60):(0-40).

[0101] The detection method of the steroid hormone detection kit of this embodiment adopts the detection kit of this embodiment 1, specifically, comprising the following steps:

[0102] 1. Solution preparation

[0103] 1.1 The preparation scheme of the mixed working solution is shown in Table 2:

[0104] Table 2 Preparation of mixed working solution

[0105]

[0106] As shown in Table 2, firstly, the first-level stock solutions of aldosterone, angiotensin I, angiotensin II, 18-hydroxycorticosterone, 18-hydroxycortisol, and 18-oxycortisol were taken, and the volume was adjusted to 1000uL by adding 50vol% methanol aqueous solution to obtain the second-level stock solution. Then, the corresponding volume of the second-level stock solution was taken, and the volume was adjusted to 5000uL by adding 50vol% methanol aqueous solution to obtain the mixed working solution concentrations corresponding to various steroid hormones and polypeptide hormones as shown in Table 2 above, and used for standby.

[0107] 1.2 Preparation of calibration materials, as shown in Table 3:

[0108] Table 3 Preparation of calibrants

[0109]

[0110] The preparation scheme of the matrix liquid used in the preparation of the calibrator is as follows:

[0111] Blank matrix liquid: Weigh 0.1 g BSA and add it into 10 mL buffer A and mix well.

[0112] Preparation of buffer A: Weigh 1.211 g of tris-base and dissolve it in 100 mL of deionized water. Use acetic acid to adjust the pH to 6 and store it at -20°C.

[0113] As shown in Table 3, take the corresponding volume of mixed working solution, mix it, and add the matrix liquid to prepare calibrators S6 and S7. Then take 45uL of calibrator S7 and add 225uL of matrix liquid to obtain calibrator S5. Calibrators S1 to S4 are prepared according to the formula in Table 3 by using calibrator S5 or S7 and adding the corresponding matrix liquid.

[0114] 1.3 Preparation of quality control products, as shown in Table 4:

[0115] Table 4 Preparation of quality control products

[0116]

[0117] The matrix liquid for the preparation of quality control products is plasma with background removed. First, take 30uL of the mixed working solution and mix it with 1845uL of the quality control product matrix liquid to prepare the high-concentration quality control product HQC. Then take 15uL of the high-concentration quality control product HQC, add 1585uL of the quality control product matrix liquid, and prepare the low-concentration quality control product LQC. According to the same process, take 500uL of the high-concentration quality control product HQC, add 500uL of the quality control product matrix liquid, and prepare the medium-concentration quality control product MQC. After the quality control product is prepared, it should be immediately divided and stored frozen.

[0118] 1.4 Preparation of internal standard working solution, as shown in Table 5:

[0119] Table 5 Preparation of internal standard working solution

[0120] Internal standard name Concentration of internal standard stock solution (ug / mL) Diluent Concentration of internal standard working solution (ng / mL) ALD-IS 10 Methanol 10 AngI-IS 100 Methanol 100 AngII-IS 10 Methanol 10 18-OHB-IS 10 Methanol 10 18-OHF-IS 10 Methanol 10 18-OXOF-IS 10 Methanol 10

[0121] Use methanol as the diluent and dilute the internal standard stock solution to the internal standard working solution of the corresponding concentration according to Table 5.

[0122] 2. Sample pretreatment

[0123] 2.1 Magnetic solid phase extraction pre-treatment and purification process

[0124] 2.11 Prepare the magnetic solid phase extraction 96-well plate format steroid hormone detection kit and tear off the aluminum film covering the plate.

[0125] 2.12 Add calibrators S1 to S7, and quality control products LQC, MQC, and HQC to the corresponding wells according to the sample layout of the magnetic solid phase extraction 96-well plate in Table 6.

[0126] 2.13 Clinical samples: add 250uL of blood sample to be tested and 20uL of internal standard working solution to the remaining wells.

[0127] 2.14 Add 450uL of water to all wells in columns 3 and 9 for impurity removal and separation. The volume ratio of the sample to be tested to ethanol is 1:2, and the time for impurity removal and separation is 30s.

[0128] Table 6 Magnetic solid phase extraction 96-well plate sample layout

[0129]

[0130] Table 6 shows the positional layout of the components of the detection kit of the present application, the samples to be tested, the standard products and the quality control products in the 96-well plate, wherein reagent 1 is an activation solution, reagent 2 is a balance solution, the porous magnetic microspheres are stored in the balance solution, reagent 3 is the first eluent, reagent 4 is the second eluent, and reagent 5 is an eluent. In columns 3 and 9 of the 96-well plate, C1 to C7 are calibration products S1 to S7, LQC, MQC and HQC refer to low-concentration quality control products, medium-concentration quality control products and high-concentration quality control products, respectively, and Sample 1 to 6 refer to the steroid hormones and / or polypeptide hormones in 6 samples to be tested that can be subjected to magnetic solid phase extraction treatment at the same time. After the steroid hormones and / or polypeptide hormones in the calibration products S1 to S7 are subjected to liquid chromatography tandem mass spectrometry analysis to obtain a standard curve of concentration and analysis results, there is no need to test the calibration products in a short time. At this time, a maximum of 16 samples to be tested can be processed in columns 3 and 9 at the same time using a 96-well plate.

[0131] 2.2 Automated operation process of magnetic solid phase extraction

[0132] The prepared pre-treatment plate is placed in the ZPTQ32 fully automatic sample processing system for automated magnetic solid phase extraction of the sample, specifically including: the porous magnetic microspheres stored in the balancing solution of column 2 are transferred to the activation solution of column 1 for activation treatment by applying an external magnetic field force, and then the porous magnetic microspheres are transferred to column 2 for balancing treatment. After the balancing treatment, the fully automatic sample processing system automatically transfers the porous magnetic microspheres to column 3 for magnetic solid phase extraction treatment. After the set processing time is reached, the porous magnetic microspheres are transferred to columns 4, 5 and 6 in turn for the first elution treatment, the second elution treatment and the elution treatment, respectively. After the elution treatment is completed, the target markers after extraction and purification are left in column 6, and the purified sample is obtained as the test liquid, waiting for subsequent on-machine testing.

[0133] In the 96-well plate, the magnetic solid phase extraction process of columns 7 to 12 is the same as that of columns 1 to 6 and is performed in parallel with columns 1 to 6. The time required for the automated operation steps of magnetic solid phase extraction is shown in Table 7 below.

[0134] Table 7 Pre-treatment steps for automated magnetic solid phase extraction

[0135] Serial number Steps Column number in 96-well plate Mixing duration (S) Magnetic absorption duration (S) Volume of well content (uL) 1 Magnetic transfer 2 30 30 400 2 Activation 1 30 30 400 3 Equilibration 2 30 30 400 4 Sample loading 3 90 60 650 5 Washing 1 4 30 30 200 6 Washing 2 5 30 30 200 7 Elution 6 60 30 100 8 Magnetic discard 2 10 0 400

[0136] 3. Sample testing

[0137] After the sample elution treatment is completed, the purified samples obtained in columns 6 and 12 are transferred to the sample injection plate as the test solution for on-machine analysis by liquid chromatography tandem mass spectrometry.

[0138] 3.1 Chromatographic conditions

[0139] When conducting liquid chromatography tandem mass spectrometry analysis, the liquid chromatography uses gradient elution, and the reverse phase chromatography method establishes the separation conditions of the analyte as follows: the chromatographic column is an HSS T3 chromatographic column with a specification of 2.1×100mm, 1.7μm, a flow rate of 0.4mL / min, and a column temperature of 35°C; wherein, the mobile phase A is prepared according to a volume ratio of formic acid to ultrapure water of 1:1000, the formic acid is LC-MS grade, and the purity is 99.0%, and the mobile phase B is prepared according to a volume ratio of formic acid to acetonitrile to ultrapure water of 1:1000, the formic acid to acetonitrile is LC-MS grade, and the purity is 99.0%, and the injection volume is 10uL. The specific chromatographic conditions and gradient elution program are shown in Table 8 below:

[0140] Table 8 Chromatographic gradient elution conditions

[0141]

[0142]

[0143] 3.2 Mass spectrometry conditions

[0144] Electrospray ionization (ESI) source, ESI+ and ESI- ionization positive and negative simultaneous monitoring mode (Note:

[0145] ALD and 18-OHB were in ESI-mode). The ion source temperature was 120°C, the capillary voltage was 2.00 kV, the cone voltage was 30 V, the desolvation temperature was 600°C, the desolvation gas was 1000 L / Hr, and the cone gas was 150 L / Hr. The multiple reaction monitoring (MRM) method was used for scanning. The mass spectrometry ion pair parameters and other information are shown in Table 9 below:

[0146] Table 9 Ion pair parameters

[0147]

[0148] 3.3 Standard curve establishment

[0149] The standard samples are tested, and the detection spectra of various steroid hormones and / or polypeptide hormones in the standard samples are as follows: Figures 1-6 As shown, Figures 1 to 6 They are respectively aldosterone, angiotensin I, angiotensin II, 18-hydroxycorticosterone, 18-hydroxycortisol, and 18-oxycortisol in the calibrator obtained by the detection method of the present application. The internal standard method is used with the concentration of the standard as the x-axis and the ratio of the peak area of ​​the calibrator to the internal standard as the ordinate to obtain a standard curve. The results are as follows Figures 7-12 The linear relationship verification results are shown in Table 10.

[0150] Table 10 Standard curve

[0151] Analyte Weight Correlation coefficient r Linear range Linear equation 18-oxocortisol / 18-OXOF <![CDATA[1 / X 2 > 0.996484 0.01～4 Y = 1.30615*X + 0.00325699 18-hydroxycortisol / 18-OHF <![CDATA[1 / X 2 > 0.999884 0.02～8 Y = 2.45225*X + -0.00554819 Angiotensin I / AngI <![CDATA[1 / X 2 > 0.999824 0.25～100 Y = 0.521805*X + 0.0165041 Angiotensin II / AngII <![CDATA[1 / X 2 > 0.997322 0.02～8 Y = 1.00355*X + 0.00199214 Aldosterone / ALD <![CDATA[1 / X 2 > 0.999465 0.02～8 Y = 0.967343*X + 0.00349978 18-hydroxycorticosterone / 18-OHB <![CDATA[1 / X 2 > 0.999247 0.02～8 Y = 2.94781*X + -0.0244652

[0152] Depend on Figures 7-12 As shown in Table 10, the linear relationship of each standard curve is good, and the correlation coefficient r≥0.9950. It can be seen that the detection results obtained by the detection method of the present application are well correlated with the concentration, which is beneficial to the accuracy of the detection and can meet the detection requirements.

[0153] <Parallel Experiment>

[0154] The detection method of Example 2-1 was used to conduct three parallel experiments on the quality control product for three consecutive days, and the average value of the three experimental test values ​​was taken as the measured value. The accuracy of the test results was investigated, and the results were as follows:

[0155] Table 11 Accuracy test results

[0156]

[0157] The detection method of Example 2-1 was used to perform five parallel experiments on samples of three concentrations, low, medium and high, for five consecutive days. The average of the five experimental test values ​​was taken as the measured value. The intra-batch and overall precision of the test results were investigated. The results are as follows:

[0158] Table 12 Intra-batch precision test results

[0159]

[0160] In this application, accuracy is calculated by the formula: accuracy = measured value / theoretical value × 100%, where the measured value is the average of three experimental test values. Precision is expressed by the index of variation (CV), which is calculated by the formula: precision = standard deviation / measured value average × 100%. The smaller the CV value, the higher the precision. It can be seen from the quality control product test results in Tables 11 and 12 that the detection method of this application can simultaneously detect 6 steroid hormones such as aldosterone, angiotensin I, angiotensin II, 18-hydroxycorticosterone, 18-hydroxycortisol, and 18-oxycortisol in plasma samples, with inter-batch precision <15%, intra-batch precision <15%, and the accuracy of the concentration detection value is 95.35%-103.74%, with high accuracy and precision. In terms of precision, it can meet the requirements of low-value CV%<20% near the lower limit of quantitative concentration within the batch, and CV of other concentrations<15%, low-value total CV%<20% near the inter-batch concentration, and CV of other concentrations<15%; in terms of accuracy, the detection value is within ±15% of the target value, and the low concentration point is within ±20%. The detection results can meet the standards, which is conducive to the clinical application of steroid hormones and / or polypeptide hormones detection.

[0161] Example 2-2

[0162] The steroid hormone detection method of this embodiment is different from that of Embodiment 2-1 only in that the porous magnetic microspheres are the porous magnetic microspheres of Embodiment 1-2.

[0163] Example 2-3

[0164] The steroid hormone detection method of this embodiment is different from that of Embodiment 2-1 only in that the porous magnetic microspheres are the porous magnetic microspheres of Embodiment 1-3.

[0165] Embodiment 2-4

[0166] The steroid hormone detection method of this embodiment is different from that of Embodiment 2-1 only in that the porous magnetic microspheres are the porous magnetic microspheres of Embodiment 1-4.

[0167] Embodiment 2-5

[0168] The steroid hormone detection method of this embodiment is different from that of Embodiment 2-1 only in that the porous magnetic microspheres are the porous magnetic microspheres of Embodiment 1-5.

[0169] Test example

[0170] The quality control product QCM was pretreated using the detection kits of Examples 2-1 and 2-3, and detected with liquid chromatography tandem mass spectrometry. The ZPTQ32 fully automatic sample pretreatment system was used for automated extraction and other operations, where Samples 1 to 6 in the 9 columns were all quality control products QCM, and the average values ​​of the detection values ​​of liquid chromatography tandem mass spectrometry and the average values ​​of the accuracy of the 6 steroid hormones and / or polypeptide hormones were shown in Table 13 below.

[0171] Table 13 QCM test results of quality control products of Examples 2-1 and 2-3

[0172]

[0173] As shown in Table 13, the detection kit for steroid hormones and / or polypeptide hormones of the present application is combined with liquid chromatography tandem mass spectrometry to extract and detect 6 steroid hormones at the same time. The detection accuracy of the detection kits of Examples 2-1 and 2-3 for 6 steroid hormones is between 91.25% and 102.58%, and the detection value is within ±15% of the target value, which is beneficial to the detection accuracy in clinical applications. In addition, the detection accuracy of Example 2-3 for 6 steroid hormones is between 91.25% and 97.44%, and compared with Example 2-1, the accuracy reaches 94.98% to 102.58%. It can be seen that the present application further controls the pore size of the porous magnetic microspheres to be The porosity is 70%, which enables the porous magnetic microspheres to better combine with steroid hormones and polypeptide hormones, thereby improving the extraction efficiency and detection accuracy, and meeting the clinical needs for high-accuracy detection of 6 types of steroid hormones and polypeptide hormones.

[0174] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present application should be included in the protection scope of the present application.

Claims

1. A porous magnetic microsphere, characterized in that: The porous magnetic microspheres are applied to the adsorption of steroid hormones and / or polypeptide hormones with a molecular weight less than 5000Da. The porous magnetic microspheres include a polystyrene-divinylbenzene skeleton, and glycidyl methacrylate and pyrrolidone groups are grafted and / or bonded to the polystyrene-divinylbenzene skeleton.

2. The porous magnetic microsphere according to claim 1, characterized in that: The pore size of the porous magnetic microsphere is B, and / or, The porosity of the porous magnetic microspheres is 60% to 80%; and / or, The molar ratio of the polystyrene-divinylbenzene skeleton, glycidyl methacrylate and pyrrolidone group is (5-15):(1-5):(4-20).

3. A method for preparing the porous magnetic microspheres according to claim 1 or 2, characterized in that: The steps include: Styrene and divinylbenzene are subjected to cross-linking polymerization in a reaction system containing an initiator, a porogen and magnetic particles modified with polyethylene glycol to obtain a polystyrene-divinylbenzene skeleton; Glycidyl methacrylate and pyrrolidone groups are introduced to graft and / or bond the polystyrene-divinylbenzene to obtain the porous magnetic microspheres.

4. A detection kit for polypeptide hormones, characterized in that: The porous magnetic microspheres include the porous magnetic microspheres according to claim 1 or 2 or the porous magnetic microspheres prepared by the preparation method according to claim 3.

5. The detection kit according to claim 4, characterized in that: The detection kit also includes an activation solution, and the activation solution includes a first alcohol compound and a first ketone compound in a volume ratio of (50-100):(0-20).

6. The detection kit according to claim 4, characterized in that: The detection kit also includes a balancing solution, and the balancing solution includes a second alcohol compound, a second ketone compound and water in a volume ratio of (5-30):(0-10):(50-90).

7. The detection kit according to claim 4, characterized in that: The detection kit also includes an eluent, and the eluent includes a third alcohol compound, a first nitrile compound and water in a volume ratio of (6-100):(0-60):(0-40).

8. The detection kit according to any one of claims 4 to 7, characterized in that: The detection kit further comprises a first eluent and a second eluent, wherein the first eluent comprises a fourth alcohol compound, a third ketone compound and water, and the second eluent comprises a fifth alcohol compound, a second nitrile compound and water.

9. The detection kit according to claim 8, characterized in that: The volume ratio of the fourth alcohol compound, the third ketone compound and water is (1-10):(0-3):(50-95); and / or, The volume ratio of the fourth alcohol compound, the second nitrile compound and water is (6-40):(0-10):(60-90); and / or, The first alcohol compound, the second alcohol compound, the third alcohol compound, the fourth alcohol compound, and the fifth alcohol compound are each independently selected from at least one of methanol, ethanol, and isopropanol; and / or, The first ketone compound, the second ketone compound and the third ketone compound are each independently selected from at least one of acetone, methyl butyl ketone and methyl isobutyl ketone; and / or, The first nitrile compound and the second nitrile compound are each independently selected from at least one of acetonitrile, propionitrile and acrylonitrile.

10. A detection method of the detection kit according to any one of claims 4 to 9, characterized in that: The steps include: The porous magnetic microspheres are sequentially activated and balanced, and then added to a sample to be tested for extraction, wherein the sample to be tested contains steroid hormones and / or polypeptide hormones; After the extraction treatment, the porous magnetic microspheres are eluted with an eluent, and then eluted with an eluent to obtain a test solution; The liquid to be tested is subjected to liquid chromatography tandem mass spectrometry analysis to obtain the concentration information of steroid hormones and / or polypeptide hormones in the sample to be tested.

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