Detection kit and detection method for sex hormone

Abstract

The invention relates to a sex hormone detection kit and a detection method, and belongs to the technical field of substance detection. The sex hormone detection kit comprises a magnetic solid-phase extraction filler, an activating solution, an equilibrium solution, an eluent and an eluent, the magnetic solid-phase extraction filler comprises hydrophilic and lipophilic magnetic microspheres; the hydrophilic and lipophilic magnetic microsphere comprises a polystyrene-divinyl benzene magnetic microsphere skeleton and a pyrrolidone group, and the pyrrolidone group is grafted or bonded on the polystyrene-divinyl benzene magnetic microsphere skeleton. The detection kit adopts hydrophilic and lipophilic magnetic microspheres as a magnetic solid phase extraction filler, the hydrophilic and lipophilic magnetic microspheres promote the combination with sex hormones through hydrophilic groups and lipophilic groups, and purification and impurity removal are carried out on the sex hormones in cooperation with leacheate and eluent, so that the accuracy and reliability of subsequent determination are ensured. The pretreatment process for detection is simple in process and less in time consumption, and is beneficial to rapid and accurate detection of sex hormones.

Description

Technical Field

[0001] The present application relates to a detection kit and a detection method for sex hormones, belonging to the technical field of substance detection. Background Art

[0002] Sex hormones (with a chemical nature of lipids) refer to steroid hormones synthesized by the gonads of animals, as well as tissues such as the placenta and the reticular zone of the adrenal cortex, and have the functions of promoting the maturation of sex organs, the development of secondary sexual characteristics, and maintaining sexual function. The ovaries of female animals mainly secrete two sex hormones - estrogen and progesterone, and the testes of male animals mainly secrete androgens mainly composed of testosterone.

[0003] Sex hormones are closely related to female health. Disorders in sex hormone secretion may lead to various endocrine and metabolic diseases in women. For example, an important reproductive endocrine disease: polycystic ovary syndrome (abbreviated as PCOS), with an incidence rate of 5 - 10% in women of childbearing age. Its clinical manifestations are often oligomenorrhea, anovulation, high androgen levels, insulin resistance, polycystic ovaries, obesity, and / or hirsutism, etc. Among them, high androgen levels can be an important indicator for the diagnosis of polycystic ovary syndrome. Therefore, the detection of female sex hormone levels is helpful for the diagnosis of endocrine and metabolic diseases and has important clinical and research value.

[0004] Currently, the following methods are available for the detection of sex hormones:

[0005] (1) Immunological method: Based on the binding reaction between an antibody and an antigen, the detection is carried out by detecting the recognition of sex hormone molecules by the antibody. The immunological method has become a commonly used detection method in current clinical experiments due to its advantages of automated operation, rapid detection, and low cost. However, it is prone to problems such as cross-reaction and false positives, and only one sex hormone can be detected in one experiment. In addition, the immunological method is very unstable for the detection of sex hormones at low levels. For example, when performing immunological detection of female testosterone, the concentration is often overestimated by 2 - 5 times.

[0006] (2) Gas chromatography - mass spectrometry (GC - MS) can also be used for the quantitative detection of sex hormones. It has good specificity and can achieve the simultaneous detection of multiple sex hormones. However, the requirements for the pretreatment process of samples are relatively harsh. For example, steps such as extraction, purification, and derivatization make the entire pretreatment process very cumbersome, and it is difficult to carry out in clinical practice.

[0007] (3) The detection of sex hormones by liquid chromatography-tandem mass spectrometry (LC-MS / MS) has gradually become an important choice for current medical testing laboratories. It has the advantages of high sensitivity, less impurity interference, direct detection, and high specificity. Moreover, the mass spectrometry method can detect multiple sex hormones simultaneously in a short time. Compared with the traditional immunological method that can only detect one sex hormone at a time, it has higher clinical application value in disease diagnosis. However, the liquid chromatography-tandem mass spectrometry technology is still restricted in clinical applications. The most prominent problem is the complex pretreatment process, which often requires a multi-step solid-phase extraction (SPE) process. The prior art has proposed using an automated enzyme-linked immunosorbent assay for sample pretreatment to achieve a one-stop SPE sampling and sample transfer process through the transfer of a robotic arm. However, this process is slow and requires multiple centrifugation, positive pressure and other steps to adsorb or desorb the sample, with a low degree of automation. Manually completing the SPE sampling and sampling steps requires high skills from clinical operators and is prone to human errors.

[0008] In summary, the existing methods for detecting sex hormones have a complex and cumbersome pretreatment process, long time consumption, and are prone to errors, resulting in poor accuracy of test results. Moreover, the types and secretion levels of female sex hormones are different from those of males. Therefore, a method for detecting sex hormones is needed to accurately detect the levels of female sex hormones to promote the diagnosis and research of female sex hormone-related diseases. Summary of the Invention

[0009] The purpose of this application is to provide a test kit and a detection method for sex hormones. The magnetic solid-phase extraction packing is used to bind with the sex hormones in the sample to be tested, and the enrichment and purification of sex hormones are realized through magnetic adsorption transfer, greatly simplifying the operation process of pretreatment, with simple and fast operation, high extraction efficiency, and the ability to perform high-throughput sample detection on multiple sex hormones in the sample to be tested simultaneously.

[0010] In a first aspect, the present application provides a detection kit for sex hormones. The detection kit includes a magnetic solid-phase extraction packing material, an activation solution, a balancing solution, a washing solution, and an elution solution; the magnetic solid-phase extraction packing material includes hydrophilic-lipophilic magnetic microspheres; the hydrophilic-lipophilic magnetic microspheres include a polystyrene-divinylbenzene magnetic microsphere skeleton and a pyrrolidone group represented by Formula I, and the pyrrolidone group is grafted or bonded to the polystyrene-divinylbenzene magnetic microsphere skeleton. The detection kit of the present application uses hydrophilic-lipophilic magnetic microspheres as the magnetic solid-phase extraction packing material. The hydrophilic-lipophilic magnetic microspheres promote the binding with sex hormones through hydrophilic groups (pyrrolidone groups) and lipophilic groups (carbon chains formed by the bonding of phenyl groups and vinyl groups contained in the polystyrene-divinylbenzene magnetic microspheres). The sex hormones can be bound by the hydrophilic-lipophilic balanced magnetic microspheres, and magnetic adsorption technology can be used to extract and enrich the sex hormones. After extracting the sex hormones, the washing solution and the elution solution can be used to purify and remove impurities, ensuring the accuracy and reliability of subsequent determinations. The pre-treatment process of the detection is simple and time-consuming, and it can be combined with an automated magnetic solid-phase extraction system, which is conducive to the rapid and accurate detection of sex hormones, improving the detection efficiency of sex hormones and the accuracy and stability of the detection results, and is conducive to applications such as the diagnosis and prognosis evaluation of related diseases with sex hormones as markers.

[0011] In the present application, the pyrrolidone group is as shown in Formula I: It is obtained by removing the hydrogen atom on the N atom of the α-pyrrolidone molecule.

[0012] The pyrrolidone group can be grafted or bonded to the polystyrene-divinylbenzene magnetic microsphere skeleton. After grafting or bonding, the pyrrolidone group is combined with the polystyrene-divinylbenzene magnetic microsphere skeleton in the form of a functional group. In the present application, grafting refers to the combination of the pyrrolidone group and the polystyrene-divinylbenzene magnetic microsphere skeleton through a chemical reaction after the pyrrolidone group is activated after the polystyrene-divinylbenzene magnetic microsphere skeleton is prepared; bonding refers to adding the pyrrolidone group to the reaction system containing polymerization monomers during the preparation process of the polystyrene-divinylbenzene magnetic microsphere skeleton to react together to form hydrophilic-lipophilic magnetic microspheres.

[0013] Preferably, in the hydrophilic-lipophilic magnetic microspheres, the molar ratio of the pyrrolidone group to the polystyrene-divinylbenzene magnetic microsphere skeleton is (9-15):(8-12). By controlling the appropriate ratio of hydrophilic groups and lipophilic groups in the hydrophilic-lipophilic magnetic microspheres, it can cooperate with the polar and non-polar groups of various sex hormones, so as to have a stronger binding ability to sex hormones, which is beneficial to improving the extraction effect of magnetic microspheres on sex hormones.

[0014] Furthermore, in the hydrophilic-lipophilic magnetic microspheres, the molar ratio of the pyrrolidone group to the polystyrene-divinylbenzene magnetic microsphere skeleton is (13-15):(8-10). By further controlling the ratio of the hydrophilic group to the lipophilic group, the hydrophilic-lipophilic magnetic microspheres can exhibit excellent binding effects with all seven sex hormones in the blood sample, and the binding specificity is higher, reducing the extraction of non-target impurities, improving the extraction efficiency and accuracy, and meeting the clinical requirements for the detection of seven sex hormones.

[0015] Preferably, the polystyrene-divinylbenzene magnetic microsphere skeleton is obtained by a preparation method including the following steps: Styrene and divinylbenzene are subjected to a cross-linking polymerization reaction in a reaction system containing an initiator, a porogen, and magnetic particles modified with polyethylene glycol, and the polystyrene-divinylbenzene magnetic microsphere skeleton is obtained. In the present application, magnetic particles are introduced during the cross-linking polymerization reaction, 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, alkaline, and / or neutral compounds.

[0016] Preferably, the initiator includes azobisisobutyronitrile and / or benzoyl peroxide; the porogen 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.

[0017] Preferably, the magnetic particles include at least one of iron particles, cobalt particles, nickel particles, and magnetite particles.

[0018] 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, and the magnetite particles modified with polyethylene glycol are obtained.

[0019] Preferably, when carrying out the cross-linking polymerization reaction, the molar ratio of styrene to divinylbenzene is (0.5-9):(1-10). Specifically, the conditions for the cross-linking polymerization reaction include: the temperature of the cross-linking polymerization reaction is 40-80°C; the time of the cross-linking polymerization reaction is 6-24 h. In the present application, by controlling the ratio of the styrene and divinylbenzene reactants and the reaction conditions, the number of benzene rings and cross-linked carbon chains in the polystyrene-divinylbenzene magnetic microspheres is controlled, and then the polarity of the hydrophilic-lipophilic magnetic microspheres is controlled, so that they can better bind sex hormones, reduce the binding with non-target substances, and improve the extraction effect of magnetic solid-phase extraction.

[0020] To further improve the binding effect between the hydrophilic-lipophilic magnetic microspheres and sex hormones, and enable the magnetic microsphere matrix to better cooperate with hydrophilic groups, during the cross-linking polymerization reaction, the molar ratio of styrene to divinylbenzene is (0.5 - 5):(6 - 10).

[0021] Optionally, the hydrophilic-lipophilic magnetic microspheres are obtained through a preparation method including the following steps: subjecting the polystyrene-divinylbenzene magnetic microsphere skeleton to chloromethylation treatment, and then adding pyrrolidone for modification to obtain the hydrophilic-lipophilic magnetic microspheres. The hydrophilic-lipophilic magnetic microspheres include the polystyrene-divinylbenzene magnetic microsphere skeleton and the pyrrolidone groups grafted on the skeleton.

[0022] Preferably, the average particle size of the hydrophilic-lipophilic magnetic microspheres is 1 - 50 μm. The average particle size of the hydrophilic-lipophilic magnetic microspheres in this application is in the range of 1 - 50 μm. Using small particle hydrophilic-lipophilic magnetic microspheres with an average particle size of 1 - 5 μm and large particle hydrophilic-lipophilic magnetic microspheres with an average particle size of 30 - 50 μm can both achieve good binding with sex hormones.

[0023] Preferably, the eluent includes a first eluent and a second eluent; the first eluent includes a nitrile compound and water, and the volume ratio of the nitrile compound to water in the first eluent is (5 - 15):(60 - 90); and / or, the second eluent includes a nitrile compound and an aliphatic hydrocarbon compound, and the volume ratio of the nitrile compound to the aliphatic hydrocarbon compound in the second eluent is (10 - 50):(30 - 90). In this application, the first eluent is used to remove a part of the impurities with relatively stronger polarity than sex hormones co-extracted by the magnetic solid phase extraction packing during the sex hormone extraction step; the second eluent is used to remove a part of the impurities with relatively stronger non-polarity than sex hormones co-extracted by the magnetic solid phase extraction packing during the sex hormone extraction step. Through the combined two-step elution for purification and impurity removal, the co-extracted impurities can be removed to the greatest extent, making the final sex hormone sample to be measured cleaner, reducing the interference caused by impurities in subsequent detections, and improving the accuracy of the detection results.

[0024] Preferably, the nitrile compound is selected from at least one of acetonitrile, propionitrile, acrylonitrile; and / or, the aliphatic hydrocarbon compound is selected from at least one of C 5～15 aliphatic hydrocarbon compounds, and the aliphatic hydrocarbon compounds in this application include alkanes and / or alkenes.

[0025] Furthermore, the aliphatic hydrocarbon compound is selected from at least one of C 6～10 alkane compounds, such as n-hexane, n-heptane, etc.

[0026] Preferably, the activation liquid comprises a nitrile compound and water, and the volume ratio of the nitrile compound to water in the activation liquid is (40 - 90):(10 - 50). Before extracting sex hormones from a sample to be tested, the present application uses the activation liquid to activate the magnetic solid-phase extraction packing material. The mixture of the nitrile compound and water can infiltrate the magnetic solid-phase extraction packing material itself, clean trace impurities that may exist in the packing material, and at the same time can also make the magnetic solid-phase extraction packing material have a better adsorption effect.

[0027] Preferably, the equilibration liquid comprises a nitrile compound and water, and the volume ratio of the nitrile compound to water in the equilibration liquid is (5 - 30):(60 - 90). The present application stores the magnetic solid-phase extraction packing material in the equilibration liquid, which not only plays a role in storage, but also, before extracting sex hormones from a sample to be tested, plays a role in equilibrating the solvent system of the magnetic solid-phase extraction packing material itself, and is more suitable for achieving a high extraction efficiency in the next extraction step.

[0028] Preferably, the elution liquid comprises a nitrile compound and water, and the volume ratio of the nitrile compound to water in the elution liquid is (50 - 100):(3 - 25). After the magnetic solid-phase extraction packing material undergoes a rinsing step, the present application extracts and purifies sex hormones through the hydrophilic-lipophilic magnetic microspheres in the magnetic solid-phase extraction packing material, and then realizes the desorption of sex hormones through the elution liquid. While the sex hormones are released, the elution liquid converts the sex hormones into a solution form, which is conducive to subsequent direct analysis and testing by liquid chromatography-tandem mass spectrometry.

[0029] Preferably, the sex hormones include at least one of testosterone (abbreviation: T), androstenedione (abbreviation: A4), dehydroepiandrosterone (abbreviation: DHEA), dehydroepiandrosterone sulfate (abbreviation: DHEA-S), 17α-hydroxyprogesterone (abbreviation: 17α-OHP), progesterone (abbreviation: P), and dihydrotestosterone (abbreviation: DHT). The above seven sex hormones can be used as diagnostic markers for sex hormone-related diseases, and there are significant content differences in blood samples. Especially for female blood samples, for example, the contents of testosterone and dihydrotestosterone in female blood samples are extremely low, only 0.05 - 6 ng / ml, while the content of dehydroepiandrosterone sulfate can reach 50 - 6000 ng / ml. Clinically, immunoluminescence detection reagents can only detect one of the sex hormones in one experiment, and there is also a problem of poor detection accuracy for sex hormones with low contents. The detection kit of the present application can achieve rapid extraction and purification of the above seven sex hormones, and can achieve accurate detection in combination with liquid chromatography-tandem mass spectrometry.

[0030] Furthermore, the sex hormones include testosterone, androstenedione, dehydroepiandrosterone, dehydroepiandrosterone sulfate, 17α-hydroxyprogesterone, progesterone, and dihydrotestosterone. The detection kit for sex hormones of the present application can simultaneously detect seven sex hormones in a test sample, and has the advantages of high detection efficiency and high accuracy.

[0031] In a second aspect, the present application provides a detection method using a detection kit for any one of the above sex hormones, comprising the following steps: S100. After the magnetic solid-phase extraction packing is sequentially subjected to activation treatment and equilibration treatment, it is added to a sample to be tested for extraction treatment, wherein the sample to be tested contains sex hormones; S200. After the extraction treatment, the magnetic solid-phase extraction packing is rinsed with a rinsing solution and then eluted with an elution solution to obtain a solution to be tested; S300. The solution to be tested is subjected to liquid chromatography-tandem mass spectrometry analysis to obtain the concentration information of the sex hormones in the sample to be tested. The present application uses the magnetic solid-phase extraction packing to bind to the sex hormones in the sample to be tested, separates the sex hormones from the sample to be tested by applying an external magnetic field, and cooperates with the rinsing and elution steps to remove impurities, obtaining a solution to be tested containing sex hormones, thereby realizing the pretreatment process of liquid chromatography-tandem mass spectrometry. The experimental process required by the method is simple and efficient. The hydrophilic and lipophilic magnetic microspheres in the magnetic solid-phase extraction packing of the present application can highly enrich trace sex hormones and ensure the complete separation of the sex hormones from the matrix of the sample to be tested, improving the accuracy and reliability of the detection.

[0032] The detection method of the present application can also be combined with an automated magnetic solid-phase extraction system. Each component of the detection kit is placed in different containers (such as a 96-well plate), and the magnetic solid-phase extraction packing is transferred between different liquid systems by applying an external magnetic field, automatically realizing the above activation, equilibration, extraction, rinsing, and elution processes, liberating human labor and also helping to reduce the errors of manual operation.

[0033] In the detection method of the present application, the rinsing can also include a first rinsing and a second rinsing. The time for activation, equilibration, the first rinsing, the second rinsing, and elution in the present application can be 20 to 60 s. Preferably, the time for activation, equilibration, the first rinsing, and the second rinsing is 20 to 30 s. The time for the extraction treatment in the present application can be 60 to 90 s. After each step of activation, equilibration, the first rinsing, the second rinsing, and elution, the time for the magnetic transfer step of transferring the magnetic solid-phase extraction packing by applying an external magnetic field can be 20 to 30 s.

[0034] Preferably, step S100 further includes: before the extraction treatment, an alcohol compound is added to the sample to be tested for impurity removal and separation. Some sex hormones in the blood sample will bind to globulin and / or albumin, which will hinder the binding of sex hormones to the magnetic solid-phase extraction packing material, thus affecting the accuracy of sex hormone detection. In this application, aiming at the interfering impurities affecting sex hormone detection in female blood samples, an alcohol compound is used to destroy the protein structure in the blood sample, so that sex hormones are released from the binding with proteins. In addition, the alcohol compound can also react with macromolecules such as proteins and lipids in the blood sample for impurity removal, reducing or avoiding the influence of macromolecular impurities on the magnetic solid-phase extraction process and improving the magnetic solid-phase extraction effect. After the magnetic solid-phase extraction packing material of this application is activated and balanced, it can be directly added to the liquid system after impurity removal and separation for magnetic solid-phase extraction treatment, which greatly simplifies the operation process compared with traditional solid-phase extraction and is conducive to improving the extraction efficiency.

[0035] To further improve the effect of impurity removal and separation, adding an alcohol compound to the sample to be tested for impurity removal and separation includes: the alcohol compound is selected from at least one of the alcohol compounds of C 1～6 For example, methanol, ethanol, isopropanol, ethylene glycol, etc. In this application, C 1～6 The alcohol compound can specifically remove macromolecular impurities in female blood samples and separate sex hormones from protein macromolecules, so that sex hormones are fully released, which is conducive to the subsequent separation of hydrophilic-lipophilic magnetic microspheres, thereby improving the efficiency and accuracy of sex hormone detection in female blood samples.

[0036] The alcohol compound for impurity removal and separation in this application can be used as an impurity remover and is included in the sex hormone detection kit.

[0037] Preferably, in the impurity removal and separation treatment, the volume ratio of the sample to be tested to the alcohol compound is 1:(1.5 - 3).

[0038] Preferably, the time for impurity removal and separation treatment is 30 - 90 s.

[0039] The conditions for liquid chromatography-tandem mass spectrometry analysis in this application include:

[0040] The liquid chromatography conditions include: the chromatographic column is a C18 chromatographic column; the mobile phase A is an aqueous deionized solution of NH4F, and the mobile phase B is a methanol solution of NH4F; the flow rate is 0.4 mL / min; the column temperature is 50 °C; the injection volume is 10 μL;

[0041] Mass spectrometry conditions included an electrospray ionization source, simultaneous positive and negative ion monitoring mode, and multiple reaction monitoring (MRM). Ion source temperature was 150°C, capillary voltage was 2 kV, desolvation temperature was 500°C, desolvation rate was 1000 L / Hr, and cone gas was 150 L / Hr. Multiple reaction monitoring (MRM) was used for scanning.

[0042] The sex hormone detection kit and detection method provided in this application have the following beneficial effects:

[0043] 1) This application uses hydrophilic and lipophilic magnetic microspheres to tightly bind to sex hormones, and utilizes magnetic solid-phase extraction technology to quickly and effectively extract sex hormones from blood samples. The transfer of sex hormones between the eluent and the eluent is achieved under the action of a magnetic field, thereby purifying and removing impurities from the sex hormones. The steps are simple and can be combined with a magnetic solid-phase extraction automated processing system to further simplify manual operations and reduce human errors, thereby improving the accuracy of sex hormone detection.

[0044] 2) The two-step elution step in the pretreatment process can remove impurities and interferents as much as possible, reducing the impact of impurities and interferents on sex hormone analysis and testing. The elution step can also completely separate sex hormones from the magnetic solid phase extraction filler, thereby improving the accuracy, reliability and test sensitivity of the test results.

[0045] 3) The traditional protein precipitation and solid-phase extraction process requires an operation time of 2 to 3 hours. The detection method of the present application optimizes the conditions for impurity removal and separation treatment for female blood samples. The use of alcohol compounds can quickly and thoroughly remove large molecular impurities and fully expose the sex hormones to be tested; combined with magnetic solid-phase extraction technology, it can be directly added to the system after impurity removal and separation treatment, and the enrichment of sex hormones can be completed within 90 seconds, shortening the total operation time of the pretreatment to less than 9 minutes, greatly improving the efficiency of sex hormone detection, and having the advantages of high accuracy and precision, and can meet the inter-batch precision of <15%, intra-batch precision of <15%, and accuracy of <15%, which is beneficial to the diagnosis and prognosis evaluation of related diseases with sex hormones as markers. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 This is the detection spectrum of testosterone in the standard product obtained by the detection method of this application;

[0047] Figure 2 The detection spectrum of androstenedione in the standard product obtained by the detection method of the present application;

[0048] Figure 3 This is the detection spectrum of dehydroepiandrosterone in the standard product determined by the detection method of the present application;

[0049] Figure 4 The detection spectrum of dehydroepiandrosterone sulfate in the standard product determined by the detection method of this application;

[0050] Figure 5 The detection spectrum of 17α-hydroxyprogesterone in the standard product determined by the detection method of this application;

[0051] Figure 6 The detection spectrum of progesterone in the standard product determined by the detection method of this application;

[0052] Figure 7 The detection spectrum of dihydrotestosterone in the standard product determined by the detection method of this application;

[0053] Figure 8 The standard curve of testosterone with a concentration of 0.05 - 6 ng / mL established by the detection method of this application;

[0054] Figure 9 The standard curve of androstenedione with a concentration of 0.1 - 12 ng / mL established by the detection method of this application;

[0055] Figure 10 The standard curve of dehydroepiandrosterone with a concentration of 0.5 - 60 ng / mL established by the detection method of this application;

[0056] Figure 11 The standard curve of dehydroepiandrosterone sulfate with a concentration of 50 - 6000 ng / mL established by the detection method of this application;

[0057] Figure 12 The standard curve of 17α-hydroxyprogesterone with a concentration of 0.1 - 12 ng / mL established by the detection method of this application;

[0058] Figure 13 The standard curve of progesterone with a concentration of 0.2 - 24 ng / mL established by the detection method of this application;

[0059] Figure 14 The standard curve of dihydrotestosterone with a concentration of 0.05 - 6 ng / mL established by the detection method of this application. Detailed implementation manners

[0060] In order to make the purpose, technical solutions and advantages of this application clearer, the following further details this application in combination with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain this application and are not used to limit this application.

[0061] For simplicity, only some numerical ranges are explicitly disclosed in this application. However, any lower limit can be combined with any upper limit to form a range not explicitly recited; and any lower limit can be combined with other lower limits to form a range not explicitly recited, and similarly any upper limit can be combined with any other upper limit to form a range not explicitly recited. In addition, although not explicitly recited, each point or single value between the range endpoints is included in the range. Thus, each point or single value can be used as its own lower or upper limit and combined with any other point or single value or with other lower or upper limits to form a range not explicitly recited.

[0062] When detecting sex hormones by liquid chromatography tandem mass spectrometry in the prior art, the protein precipitation method is generally used for pretreatment, and then solid phase extraction is used for secondary enrichment and purification to achieve the pretreatment process of sex hormone detection. This method includes first using a 96-well protein precipitation plate to release the bound part of sex hormones and proteins in the blood sample through the protein precipitation pretreatment method to form a free extract. Then solid phase extraction is carried out through a 96-well SPE solid phase extraction plate to complete the pretreatment work of enrichment, purification and concentration. When the 96-well SPE solid phase extraction plate is applied, it needs to be pre-activated and balanced first, and then the protein precipitation extraction component is pre-diluted. After dilution, it is transferred to the 96-well SPE solid phase extraction plate. The target component is selectively adsorbed by the hydrophilic-lipophilic reversed-phase adsorbent, and then through the elution step, some interfering impurities are washed away on the premise that it does not affect the adsorption of the target component on the adsorbent. Finally, by adding a solvent with a stronger binding ability to the target, the target is eluted from the adsorbent, achieving the purpose of enrichment, purification and concentration. The defect of the protein precipitation method pretreatment is that two main processes of protein precipitation and solid phase extraction are required to complete the experiment, with a large consumption of consumables and reagents. At the same time, the steps are relatively cumbersome, including protein precipitation, filtration, activation, sample loading, elution and other steps, and the processing time is long, which is expected to take 2-3 hours. At the same time, due to the source and specificity of the matrix of blood samples, such as hemolytic or lipemic samples, etc., it may cause blockage of the pretreatment plate, thereby affecting the overall experimental progress. Therefore, a faster and more effective clinical detection method suitable for PCOS symptom screening is very necessary.

[0063] In view of this, the present application provides a detection kit for sex hormones. The hydrophilic-lipophilic magnetic microspheres are used as the magnetic solid-phase extraction packing material. Through the hydrophilic groups and lipophilic groups, they are tightly combined with the sex hormones in the blood sample. By using the magnetic field force, the separation of sex hormones from the sample matrix is realized. Combining with the washing and elution steps, the impurity interfering substances with similar properties to the sex hormones are removed, and the desorption of sex hormones is completed, so as to realize the purification of sex hormones by removing impurities. The magnetic solid-phase extraction packing material of the present application has a strong binding performance with sex hormones, a high extraction efficiency, a high purity of sex hormones in the test solution obtained, and fewer impurity interfering substances, which is beneficial to improving the ionization efficiency and detection sensitivity of mass spectrometry detection. Thanks to the high extraction efficiency of the detection kit of the present application, the amount of blood sample required for detection is less. Only 50-100 μL of blood sample is needed to complete the detection, which is beneficial to reducing the error in the process of blood sample collection, and can also reduce the physical and psychological impact on the examinee.

[0064] Compared with the protein precipitation pretreatment method, the magnetic solid-phase extraction packing material 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 completed in 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 magnetic solid-phase extraction packing material of the present application is completely exposed to the test system and is in full contact with the sex hormones. Therefore, it can adsorb and extract sex hormones from a large volume of the test system in a short time, and then use an external magnetic field to achieve separation from the test sample, avoiding the cumbersome treatment process, so as to absolutely shorten the enrichment and purification process, and shorten the time of the whole pretreatment from 2-3 hours of traditional solid-phase extraction to within 9 minutes, and then it can be more suitable for clinical detection and achieve the purpose of rapid analysis and testing to obtain the detection results.

[0065] In the present application, 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 material 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 material, and the purification and desorption processes are completed by transferring between different functional liquids, so that the separation and purification in a complex sample matrix can be realized.

[0066] Through the cooperation of hydrophilic and lipophilic groups on the hydrophilic-lipophilic magnetic microspheres in this application, rapid and tight binding of sex hormones can be achieved, enabling the adsorption and extraction of sex hormones from a large-volume sample system to be tested in a short time. The entire extraction process reduces the use of toxic and harmful organic solvents, simplifies the cumbersome sample treatment steps, can be automated by loading a magnetic solid-phase extraction automated processing system, and can highly enrich trace sex hormones such as testosterone and dihydrotestosterone in female blood samples to be tested, having extremely high advantages in application.

[0067] In some specific embodiments, for the sex hormone detection kit provided in this application, each component such as the magnetic solid-phase extraction packing material, activation solution, equilibration solution, washing solution, and elution solution 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, greatly facilitating the use of the product. In actual operation, only need to tear open the sealing film, and then add standards, quality control products, and samples to be tested into the sample wells as required. By applying an external magnetic field to transfer the magnetic solid-phase extraction column packing material, procedures such as activation, equilibration, sample loading, washing, and elution involved in the detection method can be achieved, completing the magnetic solid-phase extraction process of sex hormones.

[0068] In some specific embodiments, the detection kit of this application can also be paired with a magnetic solid-phase extraction automated processing system, such as the ZPTQ32 fully automatic sample pretreatment system, to automatically complete procedures such as activation, equilibration, sample loading, washing, and elution involved in the detection method through a program, realizing a fully automatic sample pretreatment solution that combines the magnetic solid-phase extraction packing material with the fully automatic sample pretreatment system, completing automated operation, reducing errors introduced by humans, facilitating the liberation of productivity, and enabling more rapid and effective extraction, purification, and concentration of sex hormones in blood samples. It not only improves the detection efficiency but also greatly enhances the stability and accuracy of the detection results, being applicable to the actual clinical detection of PCOS symptom screening and having the advantages of convenience and speed.

[0069] The sex hormone detection kit provided in this application is used for analytical 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 testosterone, androstenedione, dehydroepiandrosterone, dehydroepiandrosterone sulfate, 17α-hydroxyprogesterone, progesterone, and dihydrotestosterone in blood samples. These several sex hormones are important markers for the clinical evaluation of PCOS and other related diseases and are very valuable in clinical and physiological research.

[0070] The detection method of this application also requires the use of calibration solution and quality control solution.

[0071] The calibration solution can be a mixed solution of testosterone, androstenedione, dehydroepiandrosterone, dehydroepiandrosterone sulfate, 17α-hydroxyprogesterone, progesterone, and dihydrotestosterone with known series concentrations.

[0072] The quality control product can use the plasma after removing the hormone background as the matrix, and add a mixed solution of testosterone, androstenedione, dehydroepiandrosterone, dehydroepiandrosterone sulfate, 17α-hydroxyprogesterone, progesterone and dihydrotestosterone at a specific concentration. It can be divided into high-concentration quality control products, medium-concentration quality control products and low-concentration quality control products. The concentrations of sex hormones in the high-concentration quality control product, medium-concentration quality control product and low-concentration quality control product decrease in turn. Among them, the plasma after removing the hormone background can be obtained through conventional commercial channels.

[0073] In some embodiments, for the liquid phase conditions of liquid chromatography-tandem mass spectrometry, the mobile phases of the liquid chromatography are mobile phase A and mobile phase B respectively. Mobile phase A is ultrapure water containing an additive, and mobile phase B is a methanol solution containing an additive. The additive is 0.5 mM ammonium fluoride.

[0074] In this application, 0.5 mM ammonium fluoride is added to the mobile phase, which can simultaneously meet the ionization of both the positive mode and negative mode of the mass spectrometry. At the same time, it can also improve the detection sensitivity of the markers in the sample and improve the ionization efficiency of the markers.

[0075] The detection method of this 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 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 according to the analysis results.

[0076] In some embodiments, after performing liquid chromatography-tandem mass spectrometry analysis on the sex hormones to be detected in the test solution, the concentration of the sex hormones to be detected in the test sample is obtained based on the standard curve.

[0077] Example

[0078] The technical solution of this application will be described below in conjunction with specific embodiments. The raw materials used in the following embodiments are all from ordinary commercially available products, and the devices or equipment used are all purchased from conventional market sales channels.

[0079] Example 1

[0080] The sex hormone detection kit of this embodiment includes:

[0081] Magnetic solid-phase extraction packing: The magnetic solid-phase extraction packing includes hydrophilic-lipophilic magnetic microspheres; the hydrophilic-lipophilic magnetic microspheres include a polystyrene-divinylbenzene magnetic microsphere skeleton and a pyrrolidone group. The molar ratio of the pyrrolidone group to the polystyrene-divinylbenzene magnetic microsphere skeleton is 13:10; the pyrrolidone group is grafted on the polystyrene-divinylbenzene magnetic microsphere skeleton.

[0082] Activating solution: An acetonitrile aqueous solution, and the volume ratio of acetonitrile to water is 60:30.

[0083] Balanced solution: acetonitrile aqueous solution, with the volume ratio of acetonitrile to water being 15:70.

[0084] First eluent: acetonitrile aqueous solution, with the volume ratio of acetonitrile to water being 10:70.

[0085] Second eluent: a mixture of acetonitrile and n-hexane, where the volume ratio of acetonitrile to n-hexane is 30:60.

[0086] Eluent: isopropyl cyanide aqueous solution, with the volume ratio of isopropyl cyanide to water being 70:15.

[0087] The polystyrene-divinylbenzene magnetic microsphere framework is obtained through a preparation method including the following steps: Place a three-necked flask on a water bath. The reaction system is an ethanol-water system. Turn on the stirring paddle and stir evenly. Pass inert gas nitrogen into the three-necked flask to remove oxygen, and then add PEG4000 to the three-necked flask. After stirring evenly, add styrene and divinylbenzene respectively, and the molar ratio of styrene to divinylbenzene is 5:6. After continuing to stir evenly, add initiators azobisisobutyronitrile and benzoyl peroxide, stir until dissolved, then add the dissolved polyethylene glycol-modified iron oxide nanoparticles, and then add toluene as a pore-forming agent, and continuously stir evenly for 30 min to make the reaction system mix evenly.

[0088] After that, turn on the water bath to heat the reaction system to 60 °C, and maintain it for 12 h for cross-linking polymerization reaction after the temperature is stable. After the reaction is completed, transfer the reaction system to a suction flask for suction filtration, and then wash it with water and ethanol in sequence to obtain the reaction product: polystyrene-divinylbenzene magnetic microsphere framework. Soak the washed product in hydrochloric acid solution overnight to remove the magnetic substances on the surface of polystyrene-divinylbenzene, and then filter and wash the filler again through a suction flask. Wash the reaction product with water until it is neutral, and then wash it three times with ethanol with a volume three times that of the reaction product. After drying by suction, transfer it to a 60 °C incubator to dry overnight to obtain the final polystyrene-divinylbenzene magnetic microsphere framework with an average particle size of 2 - 3 μm.

[0089] The hydrophilic-lipophilic magnetic microspheres are obtained through a preparation method including the following steps:

[0090] Chloromethylate the polystyrene-divinylbenzene magnetic microsphere framework, and then add pyrrolidone for modification to obtain the hydrophilic-lipophilic magnetic microspheres.

[0091] The detection method of the sex hormone detection kit in this example uses the detection kit of Example 1. Specifically, it includes the following steps:

[0092] 1. Solution preparation

[0093] 1.1 Preparation scheme of the mixed working solution is shown in Table 1:

[0094] Table 1 Preparation of the mixed working solution

[0095]

[0096] As shown in Table 1, first take the primary stock solutions of testosterone, androstenedione, dehydroepiandrosterone, dehydroepiandrosterone sulfate, 17α-hydroxyprogesterone, progesterone, and dihydrotestosterone with corresponding concentrations and volumes, add a 50 vol% methanol aqueous solution to make up to 1000 μL to obtain the secondary stock solutions. Then take the corresponding volumes of the secondary stock solutions respectively, add a 50 vol% methanol aqueous solution to make up the volume, and obtain the mixed working solution concentrations corresponding to each sex hormone as shown in Table 1 above for standby.

[0097] 1.2 Preparation of the calibrator is shown in Table 2:

[0098] Table 2 Preparation of the calibrator

[0099]

[0100] The matrix liquid used in the preparation of the calibrator is a PBS (1X) buffer solution containing 5% BSA (bovine serum albumin) and stored at -20 °C. Take the corresponding volumes of the mixed working solution as shown in Table 2 and mix them, then add the matrix liquid. First, calibrators S6 and S7 are prepared. Then take 45 μL of calibrator S7 and add 225 μL of the matrix liquid to obtain calibrator S5. Calibrators S1 - S4 are prepared according to the formula in Table 2 by using calibrator S5 or S7 respectively and adding the corresponding matrix liquid.

[0101] 1.3 Preparation of the quality control sample is shown in Table 3:

[0102] Table 3 Preparation of the quality control sample

[0103]

[0104] The matrix liquid for the preparation of the quality control sample is plasma with the background removed. First, take 30 μL of the mixed working solution and mix it with 720 μL of the quality control sample matrix liquid to prepare the high-concentration quality control sample HQC. Then take 15 μL of the high-concentration quality control sample HQC and add 465 μL of the quality control sample matrix liquid to prepare the low-concentration quality control sample LQC. According to the same procedure, take 250 μL of the high-concentration quality control sample HQC and add 250 μL of the quality control sample matrix liquid to prepare the medium-concentration quality control sample MQC. After the preparation of the quality control sample is completed, it is immediately aliquoted and stored frozen.

[0105] 1.4 Preparation of the internal standard working solution is shown in Table 4:

[0106] Table 4 Preparation of the internal standard working solution

[0107] Internal Standard Name Concentration of Internal Standard Stock Solution (ug / mL) Diluent Concentration of Internal Standard Working Solution (ng / mL) T-IS 10 Methanol 10 A4-IS 10 Methanol 10 DHEA-IS 10 Methanol 100 DHEAS-IS 10 Methanol 100 17α-OHP-IS 10 Methanol 10 P-IS 10 Methanol 10 DHT-IS 10 Methanol 10

[0108] Using methanol as the diluent, dilute the internal standard stock solution to the internal standard working solution with corresponding concentrations according to Table 4.

[0109] 2. Sample pretreatment

[0110] 2.1 Purification operation process of magnetic solid-phase extraction pretreatment

[0111] 2.11 Prepare a test kit for sex hormones with a 96-well plate for magnetic solid-phase extraction, and tear off the aluminum film covering the plate.

[0112] 2.12 Add the calibrators S1 - S7, and the quality control products LQC, MQC, and HQC to the corresponding wells according to the sample layout of the 96-well plate for magnetic solid-phase extraction in Table 5.

[0113] 2.13 For clinical samples, add 100 μL of the blood sample to be tested and 10 μL of the internal standard working solution to the remaining other wells.

[0114] 2.14 Add 200 μL of ethanol 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 30 s.

[0115] Table 5 Sample layout table of 96-well plate for magnetic solid-phase extraction

[0116]

[0117] Table 5 shows the position layout of each component, the sample to be tested, the standard product, and the quality control product of the test kit for sex hormones of this application in the 96-well plate. Among them, reagent 1 is the activation solution, reagent 2 is the equilibration solution, the magnetic solid-phase extraction packing is stored in the equilibration solution, reagent 3 is the first elution solution, reagent 4 is the second elution solution, and reagent 5 is the elution solution. In columns 3 and 9 of the 96-well plate, C1 - C7 are the calibrators S1 - S7, LQC, MQC, and HQC respectively refer to the low-concentration quality control product, medium-concentration quality control product, and high-concentration quality control product, and Sample1 - 6 means that the sex hormones in 6 samples to be tested can be subjected to magnetic solid-phase extraction treatment simultaneously. After obtaining the standard curve of concentration and analysis result by liquid chromatography-tandem mass spectrometry analysis of the sex hormones in the calibrators S1 - S7, the calibrators do not need to be tested for a short time. At this time, with the 96-well plate, up to 16 samples to be tested can be processed simultaneously in columns 3 and 9.

[0118] 2.2 Automatic operation process of magnetic solid-phase extraction

[0119] Place the prepared pretreatment plate in the ZPTQ32 fully automatic sample processing system for automated magnetic solid-phase extraction of the sample, which specifically includes: transferring the magnetic solid-phase extraction packing stored in two columns of equilibration solution to one column of activation solution by applying an external magnetic force for activation treatment, and then transferring the magnetic solid-phase extraction packing to two columns for equilibration treatment. After the equilibration treatment, the fully automatic sample processing system automatically transfers the magnetic solid-phase extraction packing to three columns for magnetic solid-phase extraction treatment. After reaching the set processing time, transfer the magnetic solid-phase extraction packing to columns four, five, and six in sequence for the first rinsing treatment, the second rinsing treatment, and the elution treatment respectively. After the elution treatment, the target markers after extraction and purification remain in column six, and the purified sample is obtained as the test solution to await subsequent machine detection.

[0120] In the 96-well plate, the magnetic solid-phase extraction process for columns 7 - 12 is the same as that for columns 1 - 6 and is carried out in parallel with columns 1 - 6. The time required for the automated operation steps of magnetic solid-phase extraction is shown in Table 6 below.

[0121] Table 6 Pretreatment steps for automated operation of magnetic solid-phase extraction

[0122] Serial Number Steps Column in 96-well Plate Mixing Duration (s) Magnetic Absorption Duration (s) Volume of Solvent in Well (uL) 1 Magnetic Transfer 2 30 30 400 2 Activation 1 20 30 400 3 Equilibration 2 20 30 400 4 Sample Loading 3 90 30 600 5 Washing 1 4 30 30 200 6 Washing 2 5 30 30 200 7 Elution 6 60 30 60 8 Magnetic Discarding 2 10 0 400

[0123] 3. Sample Detection

[0124] After the sample elution treatment is completed, transfer the purified samples obtained from columns 6 and 12 as the test solutions to the sample introduction plate for on-machine analysis by liquid chromatography-tandem mass spectrometry.

[0125] 3.1 Chromatographic Conditions

[0126] When performing liquid chromatography-tandem mass spectrometry analysis, gradient elution is used in liquid chromatography, and the separation conditions for the analytes are established by reversed-phase chromatography as follows: the chromatographic column is a C18 chromatographic column with a specification of 2.1×100 mm, 1.7 μm, the flow rate is 0.4 mL / min, and the column temperature is 50°C; among them, mobile phase A is an aqueous solution of 0.5 mM NH4F deionized water, mobile phase B is a methanol solution of 0.5 mM NH4F, and the injection volume is 10 μL. The specific chromatographic conditions and gradient elution program are shown in Table 7 below:

[0127] Table 7 Chromatographic gradient elution conditions

[0128] Time (min) Flow Rate (mL / min) A(％) B(％) 0 0.5 50 50 0.2 0.5 50 50 3 0.5 20 80 3.5 0.5 5 95 4.5 0.5 5 95 4.51 0.5 50 50 6 0.5 50 50

[0129] 3.2 Mass Spectrometry Conditions

[0130] Electrospray ionization (ESI) source, ESI+ and ESI- ion ionization positive and negative simultaneous monitoring mode (Note: DHEAS is in ESI- mode). Ion source temperature is 150 °C, capillary voltage Capillary: 2 kV, desolvation gas temperature Desolvation Temp: 500 °C, desolvation gas Desolvation: 1000 L / Hr, cone gas Cone: 150 L / Hr. Multiple reaction monitoring (MRM) is used for scanning. The information such as mass spectrometry ion pair parameters is shown in Table 8 below:

[0131] Table 8 Ion pair parameters

[0132]

[0133] 3.3 Standard curve establishment

[0134] The standard products are detected, and the detection spectra of each sex hormone in the standard products are as Figures 1 - 7 shown Figures 1 - 7 The 7 markers of testosterone, androstenedione, dehydroepiandrosterone, dehydroepiandrosterone sulfate, 17α-hydroxyprogesterone, progesterone, and dihydrotestosterone in the calibration products determined by the detection method of this application are respectively. Using the internal standard method, with the concentration of the standard as the x-axis and the ratio of the peak area of the calibration product to the internal standard as the y-axis, the standard curve is obtained. The results are as Figures 8 - 14 shown, and the linear relationship verification results are shown in Table 9

[0135] Table 9 Standard curve

[0136] Chinese Name English Abbreviation Weight Correlation Coefficient r Linear Range Linear Equation Testosterone T <![CDATA[1 / X 2 > 0.998395 0.05 - 6 ng / mL Y = 0.145887x ± 0.00314246 Androstenedione A4 <![CDATA[1 / X 2 > 0.997983 0.1 - 12 ng / mL Y = 1.82504x ± 0.0696257 Dehydroepiandrosterone DHEA <![CDATA[1 / X 2 > 0.999186 0.5 - 60 ng / mL Y = 0.129826x ± 0.0294188 Dehydroepiandrosterone Sulfate DHEA-S <![CDATA[1 / X 2 > 0.997673 50 - 6000 ng / mL Y = 0.010471x ± 0.309622 17α-Hydroxyprogesterone 17α-OHP <![CDATA[1 / X 2 > 0.998699 0.1 - 12 ng / mL Y = 1.68385x ± 0.0407559 Progesterone P <![CDATA[1 / X 2 > 0.999325 0.2 - 24 ng / mL Y = 2.38739x ± 0.0385032 Dihydrotestosterone DHT <![CDATA[1 / X 2 > 0.999103 0.05 - 6 ng / mL Y = 0.788417x ± 0.023291

[0137] From Figures 8 - 14 and Table 9, it can be seen that the linear relationship in each standard curve is good, and the correlation coefficient r ≥ 0.9970. It can be seen that the detection result has a good correlation with the concentration, which is beneficial to the accuracy of the detection and can meet the detection requirements

[0138] Example 2

[0139] Using the detection method of Example 1, the quality control products are subjected to 3 parallel experiments every day for 3 consecutive days, and the accuracy and within-batch and between-batch precision of the detection results are investigated. The results are as follows:

[0140] Table 10 Testosterone accuracy, within-batch, between-batch precision

[0141]

[0142] In this application, the accuracy is calculated by the formula: Accuracy = 1 - Average of measured values / Theoretical value × 100%, where the average of measured values is the average of three measured values. A positive accuracy value indicates that the analysis result is on the low side, and a negative value indicates that the analysis result is on the high side. The smaller the absolute value of the accuracy, the smaller the deviation from the actual value, and the higher the accuracy. The precision is expressed by the coefficient of variation (CV) and is calculated by the formula: Precision = Standard deviation / Average of measured values × 100%. The smaller the CV value, the higher the precision. From the test results of the quality control products in Table 10, it can be seen that the accuracy of the detection method of this application for testosterone is between 0.53% and 8.18%, the within-batch precision is between 1.28% and 12.29%, and the between-batch precision is between 2.29 and 3.52. It has high accuracy and precision, and the test results can meet the standards and can be applied to the clinical test of testosterone.

[0143] Table 11 Androstenedione accuracy, within-batch, between-batch precision

[0144]

[0145] From the test results of the quality control products in Table 11, it can be seen that the accuracy of the detection method of this application for androstenedione is between 0.74% and 3.47%, the within-batch precision is between 0.31% and 6.34%, and the between-batch precision is between 0.38 and 2.37. It has high accuracy and precision, and the test results can meet the standards and can be applied to the clinical test of androstenedione.

[0146] Table 12 Dehydroepiandrosterone accuracy, within-batch, between-batch precision

[0147]

[0148] From the test results of the quality control products in Table 12, it can be seen that the accuracy of the detection method of this application for dehydroepiandrosterone is between 0.23% and 5.35%, the within-batch precision is between 2.98% and 12.44%, and the between-batch precision is between 1.92 and 4.18. It has high accuracy and precision, and the test results can meet the standards and can be applied to the clinical test of dehydroepiandrosterone.

[0149] Table 13 Dehydroepiandrosterone sulfate accuracy, within-batch, between-batch precision

[0150]

[0151] From the quality control product test results in Table 13, it can be seen that the accuracy of the detection method of this application for dehydroepiandrosterone sulfate is between 0.11% and 7.38%, the within-batch precision is between 3.52% and 8.88%, and the between-batch precision is between 2.07 and 3.23. It has high accuracy and precision, and the test results can meet the standards and can be applied to the clinical test of dehydroepiandrosterone sulfate.

[0152] Table 14 Accuracy, within-batch, and between-batch precision of 17α-hydroxyprogesterone

[0153]

[0154] From the quality control product test results in Table 14, it can be seen that the accuracy of the detection method of this application for 17α-hydroxyprogesterone is between 0.67% and 10.51%, the within-batch precision is between 2.83% and 10.62%, and the between-batch precision is between 2.34 and 9.79. It has high accuracy and precision, and the test results can meet the standards and can be applied to the clinical test of 17α-hydroxyprogesterone.

[0155] Table 15 Accuracy, within-batch, and between-batch precision of progesterone

[0156]

[0157] From the quality control product test results in Table 15, it can be seen that the accuracy of the detection method of this application for progesterone is between 1.02% and 8.61%, the within-batch precision is between 2.39% and 13.50%, and the between-batch precision is between 1.85 and 4.97. It has high accuracy and precision, and the test results can meet the standards and can be applied to the clinical test of progesterone.

[0158] Table 16 Accuracy, within-batch, and between-batch precision of dihydrotestosterone

[0159]

[0160] From the quality control product test results in Table 16, it can be seen that the accuracy of the detection method of this application for dihydrotestosterone is between 0.22% and 7.90%, the within-batch precision is between 1.70% and 12.62%, and the between-batch precision is between 2.25 and 3.61. It has high accuracy and precision, and the test results can meet the standards and can be applied to the clinical test of dihydrotestosterone.

[0161] Based on the above test results, for the detection method of the sex hormone detection kit provided by this application, when simultaneously detecting seven sex hormones, namely testosterone, androstenedione, dehydroepiandrosterone, dehydroepiandrosterone sulfate, 17α-hydroxyprogesterone, progesterone, and dihydrotestosterone, in female blood samples, in terms of precision, it can meet the requirement that the low-value CV% near the lower limit of quantitation within a batch is <20%, and the CV of the remaining concentrations is <15%; the total CV% of the low values near the inter-batch concentrations is <20%, and the CV of the remaining concentrations is <15%; in terms of accuracy, it meets the requirement that the measured value is within ±15% of the target value, and at the low concentration point, it is within ±20%. It shows high accuracy and precision, which is conducive to its application in clinical sex hormone detection.

[0162] Example 3

[0163] The difference between the sex hormone detection method of this example and that of Example 1 is only that in the hydrophilic-lipophilic magnetic microspheres in the detection kit, the molar ratio of the pyrrolidone group to the polystyrene-divinylbenzene magnetic microsphere skeleton is 12:12.

[0164] Example 4

[0165] The difference between the sex hormone detection kit of this example and that of Example 1 is only that the preparation method of the polystyrene-divinylbenzene magnetic microsphere skeleton includes: when carrying out the cross-linking polymerization reaction, the molar ratio of styrene to divinylbenzene is 9:1.

[0166] Test Example

[0167] The quality control product QCM was pretreated using the sex hormone detection kits of Examples 1, 3, and 4, and detected in combination with liquid chromatography-tandem mass spectrometry. Through operations such as automated extraction treatment using the ZPTQ32 full-automatic sample pretreatment system, where Sample 1-6 in 9 columns are all quality control product QCM, after pretreatment using the sex hormone detection kits of Examples 1, 3, and 4 respectively, the average detection values of liquid chromatography-tandem mass spectrometry and the average accuracy of the seven sex hormones are shown in Table 17 below.

[0168] Table 17 Detection Results of Quality Control Product QCM in Examples 1, 3, and 4

[0169]

[0170] As shown in Table 17, the sex hormone detection kit of the present application combines liquid chromatography tandem mass spectrometry to simultaneously extract and detect seven sex hormones. The absolute values of the detection accuracy of the detection kits in Examples 1, 3, and 4 for the seven sex hormones are between 0.55% and 13.90%, all of which can meet the standard of accuracy < 15%, which is beneficial to the detection accuracy in clinical applications. In addition, the absolute values of the detection accuracy of Example 3 for the seven sex hormones are between 5.05% and 11.65%, and the absolute values of the detection accuracy of Example 4 for the seven sex hormones are between 8.03% and 13.90%. Compared with Example 1, the absolute value of the accuracy reaches 0.55% to 3.94%. It can be seen that by controlling the molar ratio of the reactants styrene and divinylbenzene in the cross-linking polymerization reaction to be (0.5 - 5):(6 - 10), and further controlling the ratio of hydrophilic groups to lipophilic groups to be (13 - 15):(8 - 10), the magnetic solid-phase extraction packing can better bind to sex hormones, thereby improving the extraction efficiency and accuracy, and meeting the high-accuracy detection requirements for seven sex hormones in clinical practice.

[0171] The above are only the preferred embodiments of the present application and are not intended to limit the present application. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A detection kit for sex hormones, characterized in that, The detection kit includes a magnetic solid-phase extraction filler, an activation solution, a equilibration solution, a washing solution, and an elution solution; The magnetic solid-phase extraction filler includes hydrophilic-lipophilic magnetic microspheres; The hydrophilic-lipophilic magnetic microspheres include a polystyrene-divinylbenzene magnetic microsphere framework and pyrrolidone groups; The pyrrolidone groups are grafted or bonded to the polystyrene-divinylbenzene magnetic microsphere framework.

2. The detection kit according to claim 1, wherein In the hydrophilic-lipophilic magnetic microspheres, the molar ratio of the pyrrolidone groups to the polystyrene-divinylbenzene magnetic microsphere framework is (9-15):(8-12).

3. The detection kit according to claim 2, characterized in that In the hydrophilic-lipophilic magnetic microspheres, the molar ratio of the pyrrolidone groups to the polystyrene-divinylbenzene magnetic microsphere framework is (13-15):(8-10).

4. The detection kit according to claim 1, wherein The polystyrene-divinylbenzene magnetic microsphere framework is obtained by a preparation method including the following steps: 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 the polystyrene-divinylbenzene magnetic microsphere framework.

5. The detection kit according to claim 4, wherein The preparation method of the polystyrene-divinylbenzene magnetic microsphere framework includes at least one of the following conditions: Condition 1: The initiator includes azobisisobutyronitrile and / or benzoyl peroxide; Condition 2: The reaction system is an ethanol aqueous solution system; Condition 3: The magnetic particles include at least one of iron particles, cobalt particles, nickel particles, and magnetite particles; Condition 4: The molar ratio of styrene to divinylbenzene is (0.5-9):(1-10); Condition 5: The temperature of the cross-linking polymerization reaction is 40-80°C; the time of the cross-linking polymerization reaction is 6-24 h.

6. The detection kit according to claim 1, characterized in that The washing solution includes a first washing solution and a second washing solution; The first washing solution includes a nitrile compound and water, and the volume ratio of the nitrile compound to water in the first washing solution is (5-15):(60-90); and / or, The second washing solution includes a nitrile compound and an aliphatic hydrocarbon compound, and the volume ratio of the nitrile compound to the aliphatic hydrocarbon compound in the second washing solution is (10-50):(30-90).

7. The detection kit according to claim 6, wherein The nitrile compound is selected from at least one of acetonitrile, propionitrile, and acrylonitrile; and / or, The aliphatic hydrocarbon compound is selected from C 5～15 At least one of the aliphatic hydrocarbon compounds.

8. The detection kit according to claim 1, wherein The detection kit includes at least one of the following conditions: Condition A: The activation solution includes a nitrile compound and water, and the volume ratio of the nitrile compound to water in the activation solution is (40-90):(10-50); Condition B: The equilibration solution includes a nitrile compound and water, and the volume ratio of the nitrile compound to water in the equilibration solution is (5-30):(60-90); Condition C: The elution solution includes a nitrile compound and water, and the volume ratio of the nitrile compound to water in the elution solution is (50-100):(3-25).

9. The detection kit according to claim 1, wherein The sex hormones include at least one of testosterone, androstenedione, dehydroepiandrosterone, dehydroepiandrosterone sulfate, 17α-hydroxyprogesterone, progesterone, and dihydrotestosterone.

10. The detection kit according to claim 1, wherein The sex hormones include testosterone, androstenedione, dehydroepiandrosterone, dehydroepiandrosterone sulfate, 17α-hydroxyprogesterone, progesterone, and dihydrotestosterone.

11. A detection method using a detection kit for sex hormones according to any one of claims 1 to 10, characterized in that, including the following steps: S100. After the magnetic solid-phase extraction packing is sequentially subjected to activation treatment and equilibration treatment, it is added to the sample to be tested for extraction treatment, wherein the sample to be tested contains sex hormones; S200. After the extraction treatment, the magnetic solid-phase extraction packing is rinsed with a rinsing solution and then eluted with an elution solution to obtain a solution to be tested; S300. The solution to be tested is analyzed by liquid chromatography-tandem mass spectrometry to obtain the concentration information of sex hormones in the sample to be tested.

12. The detection method according to claim 11, characterized in that Step S100 further includes: before the extraction treatment, an alcohol compound is added to the sample to be tested for impurity removal and separation treatment.

13. The detection method according to claim 12, wherein The adding of an alcohol compound to the sample to be tested for impurity removal and separation treatment includes at least one of the following conditions: Condition a, the alcohol compound is selected from at least one of the alcohol compounds of C 1～6 ; Condition b. The volume ratio of the sample to be tested to the alcohol compound is 1:(1.5 - 3); Condition c. The time for the impurity removal and separation treatment is 30 - 90 s.

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