Method for the preparation of a clinical sample for detecting a target analyte in a sample under test

Abstract

The application relates to the field of biological detection, in particular to a preparation method of a clinical sample for detecting target analytes in a to-be-detected sample. The preparation method of the clinical sample comprises the following steps: adsorbing target analytes in the to-be-detected sample by using magnetic beads; the surface of the magnetic beads is modified with C18 hydrophobic functional groups, and the target analytes include angiotensin I, angiotensin II, aldosterone, cortisol, 18-oxygen cortisol, 18-hydroxycorticosterone and 18-hydroxycortisol; and impurities adsorbed on the magnetic beads are removed, and the target analytes adsorbed on the magnetic beads are step-by-step eluted, and the elution liquid obtained in each elution step is used as the clinical sample for subsequent detection of the target analytes. By using the preparation method, the target analytes can be fully extracted for subsequent detection in one operation, and detection basis is provided for screening, diagnosis and subtype diagnosis of primary aldosteronism.

Description

Technical Field

[0001] This application relates to the field of biological detection, and in particular to a method for preparing a clinical sample for detecting a target analyte in a test sample, and a method for detecting a target analyte in a test sample. Background Technology

[0002] Hypertension can be divided into primary hypertension and secondary hypertension. Endocrine hypertension is the most common type of secondary hypertension, accounting for 15% to 20% of all hypertension cases. Primary aldosteronism (PA) is the most common type of endocrine hypertension. It is caused by adrenal cortex lesions leading to excessive autonomous aldosterone secretion, resulting in sodium retention and potassium excretion, increased blood volume, and inhibition of the renin-angiotensin system. Clinically, it mainly manifests as hypertension and hypokalemia. Studies have found that excessive aldosterone is a significant risk factor for myocardial hypertrophy, heart failure, and kidney damage. Compared to patients with primary hypertension, patients with primary aldosteronism experience more severe damage to target organs such as the heart and kidneys. Therefore, early diagnosis and treatment are crucial.

[0003] Diagnosing primary aldosteronism is a lengthy process, involving screening, confirmation, and subtype diagnosis. For screening, the non-supine ARR (aldosterone-to-renin activity ratio) is the most commonly used indicator. Patients with a positive screening result can be confirmed through a high-sodium diet, fludrocortisone test (FST), saline test (SIT), or captopril test (CCT). Common subtypes of primary aldosteronism, such as unilateral aldosteronoma and idiopathic aldosteronism, require differential diagnosis. Adrenal venous stenosis (AVS) is the gold standard for subtype diagnosis, but it is very expensive and can easily cause adrenal hemorrhage. Simpler, non-invasive, and more economical subtype diagnostic methods are important for clinical testing.

[0004] Patent literature reports a liquid chromatography-tandem mass spectrometry (LC-TMS) method for the simultaneous determination of primary aldosteronism (PAH) genotypic markers in plasma. This method uses HLB solid-phase extraction (SPE) to extract analytes 18-hydroxycorticosterone (18-OHB), 18-hydroxycorticosteroids (18-OHF), and 18-oxocorticosteroids (18-OXOF). Its disadvantages include a complex and time-consuming procedure involving activation, sample loading, rinsing, and elution, with a single batch of samples taking approximately two hours to process. Furthermore, due to the variability of biological sample matrices, extraction efficiencies vary significantly between wells, requiring calibration with isotopic internal standards for all indicators. Additionally, due to the specificity of biological sample matrices, some samples (especially those with hemolysis or lipemia) may cause SPE column blockage, leading to reduced pretreatment efficiency for the entire batch and the need for retesting of blocked samples. The addition of zinc sulfate and phosphoric acid during sample pretreatment, both non-volatile solvents, can easily contaminate the mass spectrometer. Furthermore, this invention can only detect the subtype indicators of primary aldosteronism.

[0005] There is also a patent document reporting a screening kit for primary aldosteronism and a diagnostic system for diagnosis and subtyping. The proposed method only detects aldosterone, angiotensin I, angiotensin II, cortisol, and 18-hydroxycorticosterone, and the subtyping indicators are not comprehensive.

[0006] Therefore, this application is hereby submitted. Summary of the Invention

[0007] Based on this, one or more embodiments of this application provide a method for preparing a clinical sample for detecting a target analyte in a test sample, and a method for detecting the target analyte in a test sample. This includes the following technical solutions:

[0008] One or more embodiments of this application provide a method for preparing a clinical sample for detecting a target analyte in a test sample, the preparation method comprising the following steps:

[0009] The target analytes in the sample are adsorbed using magnetic beads; the surface of the magnetic beads is modified with C18 hydrophobic functional groups, and the target analytes include angiotensin I, angiotensin II, aldosterone, cortisol, 18-oxocortisol, 18-hydroxycorticosterone, and 18-oxocortisol; and,

[0010] Impurities adsorbed on the magnetic beads are removed, and the target analyte adsorbed on the magnetic beads is eluted stepwise. The eluent obtained from each elution step is used as a clinical sample for subsequent detection of the target analyte.

[0011] In some embodiments of this application, the eluent includes a first eluent and a second eluent;

[0012] The first eluent includes aldosterone, cortisol, 18-oxocortisol, 18-hydroxycorticosterone, and 18-oxocortisol;

[0013] The second eluent includes angiotensin I and angiotensin II.

[0014] In some embodiments of this application, stepwise elution includes:

[0015] The target analyte adsorbed on the magnetic beads was eluted using elution buffer I and elution buffer II, respectively, and the first elution buffer and the second elution buffer were collected accordingly.

[0016] The eluent I is methanol or a mixture of methanol and water, wherein the volume percentage of methanol in the mixture is not less than 50%, or / and; the eluent II is a mixture of methanol and ammonia, or a mixture of methanol, ammonia and water, wherein the volume percentage of methanol in the mixture is not less than 50%, and the pH is above 9.0.

[0017] In some embodiments of this application, the method of removing impurities adsorbed on the magnetic beads includes rinsing with a rinsing solution;

[0018] The rinsing solution includes rinsing solution I and rinsing solution II;

[0019] The rinsing solution I comprises an aqueous methanol solution with a volume percentage of 0.8% to 1.2%;

[0020] The rinsing solution II comprises an aqueous methanol solution with a volume percentage of 8% to 12% methanol.

[0021] In some embodiments of this application, the magnetic beads have a particle size of 8µm to 50µm and a specific surface area of ​​approximately 400m². 2 / g~450m 2 / g, with a pore size of 100Å~130Å.

[0022] In some embodiments of this application, the magnetic beads have a particle size of 8µm to 50µm and a specific surface area of ​​400m². 2 / g~450m 2 / g, pore size 100Å~130Å, carbon content 6wt%~8wt%.

[0023] In some embodiments of this application, the use of magnetic beads to adsorb target analytes in the sample to be tested includes the following steps:

[0024] The test sample and the sample incubation solution are mixed and incubated. The reaction is terminated by adding an internal standard to stop the working solution, and the incubation product is prepared; and,

[0025] The incubation product, magnetic beads activated by the activation solution, and internal standard solution are mixed and combined, and then the solvent is removed.

[0026] In some embodiments of this application, the preparation method satisfies one or more of the following conditions:

[0027] (1) The incubation conditions include: temperature of 36℃~38℃ and time of 1h~3h;

[0028] (2) The ratio of the sample to be tested to the magnetic beads is 350µL~450µL:2mg~5mg;

[0029] (3) The sample to be tested includes plasma samples.

[0030] One or more embodiments of this application provide a method for detecting a target analyte in a sample to be tested, the method comprising:

[0031] Using the aforementioned preparation method, clinical samples are prepared from the sample to be tested; and,

[0032] The clinical samples were tested.

[0033] In some embodiments of this application, detection is performed using liquid chromatography-tandem mass spectrometry.

[0034] Compared with traditional technologies, this application has the following advantages:

[0035] This application selects suitable magnetic beads to adsorb target analytes in the sample to be tested, namely angiotensin I, angiotensin II, aldosterone, cortisol, 18-oxocortisol, 18-hydroxycorticosterone, and 18-oxocortisol, and elutes the target analytes stepwise, thereby forming a specific clinical sample preparation process. Using this process, the target analytes can be fully extracted in a single operation for subsequent detection, providing a detection basis for the screening, diagnosis, and subtype diagnosis of primary aldosteronism. Attached Figure Description

[0036] To more clearly illustrate the technical solutions in the embodiments of this application and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0037] Figures 1 to 7 The chromatograms are, in order, those of angiotensin I, angiotensin II, aldosterone, cortisol, 18-hydroxycorticosterone, 18-hydroxycorticosteroid, and 18-oxocorticosteroid.

[0038] Figure 8a This is the chromatogram of the first injection of angiotensin I in Scheme 2.2 of Example 2.

[0039] Figure 8b This is the chromatogram of the first injection of angiotensin II in Scheme 2.2 of Example 2.

[0040] Figure 9a This is the chromatogram of angiotensin I from the 50th injection in Scheme 2.2 of Example 2.

[0041] Figure 9b This is the chromatogram of angiotensin II from the 50th injection in Scheme 2.2 of Example 2.

[0042] Figure 10a This is the chromatogram of angiotensin I from the 100th injection in Scheme 2.2 of Example 2.

[0043] Figure 10b This is the chromatogram of angiotensin II from the 100th injection in Scheme 2.2 of Example 2.

[0044] Figure 11a This is the chromatogram of the first injection of angiotensin I in Scheme 2.1 of Example 2.

[0045] Figure 11b This is the chromatogram of the first injection of angiotensin II in Scheme 2.1 of Example 2.

[0046] Figure 12a This is the chromatogram of angiotensin I from the 50th injection in Scheme 2.1 of Example 2.

[0047] Figure 12b This is the chromatogram of angiotensin II from the 50th injection in Scheme 2.1 of Example 2.

[0048] Figure 13a This is the chromatogram of angiotensin I from the 100th injection in Scheme 2.1 of Example 2.

[0049] Figure 13b This is the chromatogram of angiotensin II from the 100th injection in Scheme 2.1 of Example 2.

[0050] Figures 14a to 14g The test results are obtained using scheme 4.1 in Example 4.

[0051] Figures 15a to 15g The results are obtained using scheme 4.2 in Example 4.

[0052] Figures 16a to 16g The test results are obtained using scheme 4.3 in Example 4. Detailed Implementation

[0053] The present application will be further described in detail below with reference to the accompanying drawings, embodiments, and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the present application. The purpose of providing these embodiments and examples is to enable a more thorough and comprehensive understanding of the disclosure of the present application. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. Furthermore, numerous specific details are set forth in the following description to provide a fuller understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0054] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the specification of this application is for descriptive purposes only and is not intended to be limiting of the application.

[0055] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0056] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0057] In this application, the terms "multiple", "various", "multiple times", "multi-dimensional", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0058] The terms “combinations of,” “any combination of,” and “any combination of” used in this article include all suitable combinations of any two or more of the listed items.

[0059] In this document, the term "suitable" as used in phrases such as "suitable combination," "suitable method," and "any suitable method" refers to the ability to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.

[0060] In this document, terms such as “preferred,” “better,” “more suitable,” and “ideal” are merely used to describe implementation methods or examples that achieve better results, and should be understood not to limit the scope of protection of this application.

[0061] In this application, terms such as "further," "even more," and "particularly" are used for descriptive purposes and to indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0062] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "optional" entries in a technical solution, unless otherwise specified, and there are no contradictions or mutual constraints, each "optional" entry shall be independent.

[0063] In this application, the terms "first aspect," "second aspect," "third aspect," "fourth aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," "fourth," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0064] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0065] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, the selected numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. In this document, this is equivalent to directly listing every integer. For example, if t is an integer selected from 1 to 10, it means that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0066] Unless otherwise specified, the temperature parameters in this application are permitted to be either constant-temperature treatment or variations within a certain temperature range. It should be understood that the constant-temperature treatment allows temperature fluctuations within the precision range of the instrument control, such as ±5℃, ±4℃, ±3℃, ±2℃, or ±1℃.

[0067] In this application, %(w / w) and wt% both represent weight percentage, %(v / v) refers to volume percentage, and %(w / v) refers to mass-volume percentage.

[0068] All references to this application are incorporated herein by reference as if each document were individually incorporated herein by reference. Unless they conflict with the purpose and / or technical solution of this application, all cited references are incorporated herein by reference in their entirety and for all purposes. When references are cited in this application, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. Examples and preferred embodiments of the cited technical features may also be incorporated herein by reference, but only to the extent that they enable the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or modifications shall be made adaptably to the description in this application.

[0069] Patent literature reports a screening kit for primary aldosteronism and a diagnostic system for diagnosis and subtyping, involving the following:

[0070] The kit includes an activator, an eluent, and an elution buffer; the activator is a solution containing magnetic beads, the surfaces of which are bonded with a hydrophilic-lipophilic balanced polymer; the magnetic beads are used to adsorb markers in the sample, the markers being aldosterone, angiotensin I, angiotensin II, cortisol, and 18-hydroxycorticosterone. The elution buffer is an aqueous solution containing 50% methanol.

[0071] The kit also includes an equilibration solution and a liquid chromatography mobile phase. The equilibration solution is a 1% formic acid aqueous solution. The liquid chromatography mobile phase includes mobile phase A and mobile phase B. Mobile phase A is an aqueous solution containing a mobile phase additive, and mobile phase B is a methanol solution (containing 5% isopropanol) containing a mobile phase additive. The mobile phase additive is 1mM ammonium fluoride.

[0072] Additionally, a method for detecting biomarkers in blood is provided, comprising the following steps:

[0073] (1) The sample was treated with magnetic beads of a surface-bonded hydrophilic and lipophilic balanced polymer to adsorb the analyte;

[0074] (2) The analyte was eluted with an elution buffer, and the content of the marker in the analyte was analyzed by liquid chromatography-tandem mass spectrometry; the marker was aldosterone, angiotensin I, angiotensin II, cortisol and 18-hydroxycorticosterone.

[0075] Step (1) involves incubating the sample in an acidic environment with a generation buffer containing angiotensin-converting enzyme inhibitor (PMSF). After incubation, a reaction termination solution is added to terminate the incubation. Then, magnetic beads with a surface-bonded hydrophilic-lipophilic balanced polymer are used to treat the incubated sample. The pH value of the generation buffer is 5-6.

[0076] Step (1) involves treating the magnetic beads with an activator and an equilibration solution sequentially to adsorb the sample, followed by rinsing with an eluent. The activator is a 50% ethanol-water solution containing magnetic beads, and the magnetic beads are magnetic core-shell solid-phase extraction particles with a particle size of 30-50 μm and a specific surface area of ​​approximately 600 m². 2 / g, with a pore size of approximately 80 Å; the equilibrium solution is a 1% formic acid aqueous solution; the eluent includes eluent 1 and eluent 2, eluent 1 being a 10% methanol aqueous solution and eluent 2 being isooctane.

[0077] In step (2), the eluent is an aqueous solution containing 50% methanol; the mobile phase of the liquid chromatography includes mobile phase A and mobile phase B, mobile phase A is an aqueous solution containing mobile phase additives, mobile phase B is a methanol solution containing mobile phase additives (containing 5% isopropanol), and the mobile phase additive is 1mM ammonium fluoride.

[0078] The applicant discovered the following technical problems with the solution reported in the patent document:

[0079] (1) Magnetic beads with surface-bonded hydrophilic and lipophilic balanced polymers are used. These magnetic beads can adsorb highly polar compounds and effectively extract and adsorb less polar compounds. At the same time, elution will also elute both less polar and highly polar compounds, resulting in a relatively large number of impurities in the eluent.

[0080] (2) The mobile phase additive is 1mM ammonium fluoride. With this mobile phase additive, the sensitivity of aldosterone can be improved. However, in the detection of large batches of samples, since the chromatographic column is a C8 column with silica matrix, under high pH conditions, the exposed silanol groups on the silica surface will dissociate some hydrogen ions. These hydrogen ions will interact with angiotensin I and angiotensin II, resulting in peak tailing or even difficulty in elution. Therefore, angiotensin I and angiotensin II cannot be stably eluted in the detection of large batches of samples, and can only be stably eluted in small batches of samples.

[0081] In response, this application: (1) utilizes C18 magnetic beads on a silica matrix to extract aldosterone, angiotensin I, angiotensin II, cortisol, 18-hydroxycorticosterone, 18-hydroxycorticosteroid (18-OHF), and 18-oxocorticosteroid (18-OXOF); (2) after extraction, the analytes are eluted stepwise and then analyzed, solving the problem that the methods in the aforementioned patent literature cannot stably elute angiotensin I and angiotensin II in large-scale sample detection; (3) adopts stepwise elution and stepwise analysis to solve the current problem that angiotensin I and angiotensin II cannot be stably retained in large-scale sample detection. The technical solution of this application is as follows:

[0082] In a first aspect, this embodiment provides a method for preparing a clinical sample for detecting a target analyte in a test sample, the preparation method comprising the following steps:

[0083] The target analytes in the sample are adsorbed using magnetic beads; the surface of the magnetic beads is modified with C18 hydrophobic functional groups, and the target analytes include angiotensin I, angiotensin II, aldosterone, cortisol, 18-oxocortisol, 18-hydroxycorticosterone, and 18-oxocortisol; and,

[0084] Impurities adsorbed on the magnetic beads are removed, and the target analyte adsorbed on the magnetic beads is eluted stepwise. The eluent obtained from each elution step is used as a clinical sample for subsequent detection of the target analyte.

[0085] In some embodiments of this application, the eluent includes a first eluent and a second eluent;

[0086] The first eluent includes aldosterone, cortisol, 18-oxocortisol, 18-hydroxycorticosterone, and 18-oxocortisol;

[0087] The second eluent includes angiotensin I and angiotensin II.

[0088] In some embodiments of this application, stepwise elution includes:

[0089] The target analyte adsorbed on the magnetic beads was eluted using elution buffer I and elution buffer II, respectively, and the first elution buffer and the second elution buffer were collected accordingly.

[0090] The eluent I is methanol, or a mixture of methanol and water, wherein the volume percentage of methanol in the mixture is not less than 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%), and / or the eluent II is a mixture of methanol and ammonia, or a mixture of methanol, ammonia, and water, wherein the volume percentage of methanol in the mixture is not less than 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%), and the pH is above 9.0 (e.g., 9, 9.2, 9.4, 9.6, 9.8, 10, 10.2, 10.4, 10.6, 10.8, 11). Ammonia is mainly used to adjust the pH of eluent II. This application does not impose a specific limit on the amount of ammonia added to eluent II; it can be added in appropriate amounts according to the concentration of the selected ammonia to achieve the preset pH.

[0091] Optionally, the eluent I is a mixture of methanol and water; more preferably, the volume percentage of methanol in the eluent I is 55% to 65%.

[0092] Optionally, the eluent II is a mixture of methanol, ammonia and water, wherein the volume percentage of methanol in the mixture is 55% to 65% and the pH is above 9.0.

[0093] In some embodiments of this application, the method of removing impurities adsorbed on the magnetic beads includes rinsing with a rinsing solution;

[0094] The rinsing solution includes rinsing solution I and rinsing solution II;

[0095] The rinsing solution I comprises an aqueous methanol solution with a methanol volume percentage of 0.8% to 1.2% (e.g., 0.8%, 0.9%, 1%, 1.1%, 1.2%).

[0096] The rinsing solution II comprises an aqueous methanol solution with a methanol volume percentage of 8% to 12% (e.g., 8%, 9%, 10%, 11%, 12%).

[0097] In some embodiments of this application, the magnetic beads have a particle size of 8µm to 50µm (e.g., 8, 10, 15, 20, 25, 30, 35, 40, 45, 50µm) and a specific surface area of ​​approximately 400m². 2 / g~450m 2 / g (e.g., 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450m) 2 / g), with a pore size of 100Å~130Å (e.g., 100, 105, 110, 115, 120, 125, 130Å). Magnetic beads that meet this condition include, but are not limited to, the ME10-C18MC magnetic beads mentioned in the examples.

[0098] In some embodiments of this application, the magnetic beads have a particle size of 8µm to 50µm and a specific surface area of ​​400m². 2 / g~450m 2 / g, pore size of 100Å~130Å, and carbon content of 6wt%~8wt%. Magnetic beads that meet these conditions include, but are not limited to, Navitas C18 LC.

[0099] In some embodiments of this application, the use of magnetic beads to adsorb target analytes in the sample to be tested includes the following steps:

[0100] The test sample and the sample incubation solution are mixed and incubated. The reaction is terminated by adding an internal standard to stop the working solution, and the incubation product is prepared; and,

[0101] The incubation product, magnetic beads activated by the activation solution, and internal standard solution are mixed and combined, and then the solvent is removed.

[0102] In some embodiments of this application, the preparation method satisfies one or more of the following conditions:

[0103] (1) The incubation conditions include: temperature of 36℃~38℃ (e.g., 36, 36.5, 37, 37.5, 38℃) and time of 1h~3h (e.g., 1, 1.5, 2, 2.5, 3h);

[0104] (2) The ratio of the sample to be tested to the magnetic beads is 350µL~450µL:2mg~5mg; for example, 350µL:2mg, 400µL:2mg, 450µL:2mg, 350µL:3mg, 400µL:3mg, 450µL:3mg, 350µL:4mg, 400µL:4mg, 450µL:4mg, 350µL:5mg, 400µL:5mg, 450µL:5mg; (3) The sample to be tested includes plasma samples.

[0105] In some embodiments of this application, the internal standard solution includes 3 μg / mL to 4 μg / mL angiotensin I IS, 0.2 μg / mL to 0.25 μg / mL angiotensin II IS, 0.5 μg / mL to 0.55 μg / mL aldosterone IS, 3.1 μg / mL to 3.2 μg / mL cortisol IS, 0.3 μg / mL to 0.4 μg / mL 18-hydroxycorticosterone IS, and 0.2 μg / mL to 0.3 μg / mL 18-oxocorticol IS. In the internal standard solution of this application, the concentrations of angiotensin I (IS) are, for example, 3, 3.2, 3.4, 3.6, 3.8, and 4 μg / mL; the concentrations of angiotensin II (IS) are, for example, 0.2, 0.21, 0.22, 0.23, 0.24, and 0.25 μg / mL; and the concentrations of aldosterone (IS) are, for example, 0.5, 0.51, 0.52, 0.53, 0.54, and 0.55 μg / mL. The concentrations of cortisol_IS were, for example, 3.1, 3.12, 3.14, 3.16, 3.18, and 3.2 μg / mL; the concentrations of 18-hydroxycorticosterone_IS were, for example, 0.3, 0.32, 0.34, 0.36, 0.38, and 0.4 μg / mL; and the concentrations of 18-oxycorticol_IS were, for example, 0.2, 0.22, 0.24, 0.26, 0.28, and 0.3 μg / mL.

[0106] In some embodiments of this application, the sample incubation solution comprises sample incubation solution A and sample incubation solution B with a volume ratio of 1:(95~105) (e.g., 1:95, 1:96, 1:97, 1:98, 1:99, 1:100, 1:101, 1:102, 1:103, 1:104, 1:105); sample incubation solution A is a mixture of benzyl sulfonyl fluoride and methanol, and the concentration of benzyl sulfonyl fluoride is 95~105 mM (e.g., 95, 95, 97, 98, 99, 100, 101, 102, 103, 104, 105 mM); sample incubation solution B comprises samples with a volume ratio of 110~115 g:1 80~185g:1290~1300mL (e.g., 110g:183g:1295mL, 113g:183g:1295mL, 115g:183g:1295mL, 110g:180g:1300mL, 113g:180g:1300mL, 115g:180g:1300mL, 110g:185g:1290mL, 113g:185g:1290mL, 115g:185g:1290mL) of tris(hydroxymethyl)aminomethane, ethylenediaminetetraacetic acid and water, with a pH of up to 5.6~6 (e.g., 5.6, 5.7, 5.8, 5.9, 6).

[0107] In some embodiments of this application, the internal standard termination working solution includes the internal standard solution, the termination solution, and the internal standard diluent in a volume ratio of (4.5~5.5):(4.5~5.5):(385~395); wherein the internal standard diluent is a methanol solution, and the volume percentage of methanol is 8%~12% (e.g., 8%, 9%, 10%, 11%, 12%), and the termination solution is formic acid.

[0108] In some embodiments of this application, the volume ratio of the test sample, the sample incubation solution, and the internal standard termination working solution is 350μL~450μL:90μL~110μL:190μL~210μL, for example, 350μL:90μL:210μL, 400μL:90μL:210μL, 450μL:90μL:210μL, 350μL:110μL:190μL, 400μL:110μL:190μL, 450μL:100μL:190μL.

[0109] In some embodiments of this application, the activating solution is methanol.

[0110] One or more embodiments of this application provide a method for detecting a target analyte in a sample to be tested, the method comprising:

[0111] Using the aforementioned preparation method, clinical samples are prepared from the sample to be tested; and,

[0112] The clinical samples were tested.

[0113] In some embodiments of this application, detection is performed using liquid chromatography-tandem mass spectrometry.

[0114] In some embodiments of this application, the liquid chromatography-tandem mass spectrometry method includes:

[0115] The first eluent was detected under the first liquid chromatography conditions; and,

[0116] The second eluent was detected under the second liquid chromatography conditions;

[0117] in,

[0118] The first liquid chromatography conditions include a first mobile phase and a first elution program:

[0119] The first mobile phase includes mobile phase A1 and mobile phase B1; mobile phase A1 is an aqueous solution containing 0.1 mM to 0.3 mM (e.g., 0.1, 0.15, 0.2, 0.25, 0.3 mM) ammonium fluoride, and mobile phase B1 is methanol;

[0120] The first elution procedure is as follows:

[0121] From 0 min to 1.5 min, the volume percentage of the mobile phase A1 decreased from 60% to 20%.

[0122] Within 1.5 to 1.6 minutes, the volume percentage of the mobile phase A1 decreased from 20% to 2%.

[0123] From 1.6 min to 2.6 min, the volume percentage of the mobile phase A1 is maintained at 2%.

[0124] Between 2.6 and 2.7 minutes, the volume percentage of the mobile phase A1 increased from 2% to 60%.

[0125] During the 2.7-3.5 min time, the volume percentage of the mobile phase A1 is maintained at 60%.

[0126] The second liquid chromatography conditions include a second mobile phase and a second elution program:

[0127] The second mobile phase includes mobile phase A2 and mobile phase B2; mobile phase A2 is a mixture of formic acid and water, wherein the volume percentage of formic acid is 0.08%~0.12% (e.g., 0.08%, 0.09%, 0.1%, 0.11%, 0.12%); mobile phase B2 is a mixture of formic acid and methanol, wherein the volume percentage of formic acid is 0.08%~0.12% (e.g., 0.08%, 0.09%, 0.1%, 0.11%, 0.12%).

[0128] The second elution procedure is as follows:

[0129] From 0 min to 0.5 min, the volume percentage of the mobile phase A2 decreased from 60% to 50%.

[0130] Within 0.5 to 2 minutes, the volume percentage of the mobile phase A2 decreased from 50% to 40%.

[0131] Between 2 and 2.1 minutes, the volume percentage of the mobile phase A2 decreased from 40% to 2%.

[0132] From 2.1 min to 3.1 min, the volume percentage of the mobile phase A2 is maintained at 2%.

[0133] Between 3.1 and 3.2 minutes, the volume percentage of the mobile phase A2 increased from 2% to 60%.

[0134] For 3.2 to 4 minutes, the volume percentage of the mobile phase A2 is maintained at 60%.

[0135] In some embodiments of this application, the liquid chromatography-tandem mass spectrometry method further includes one or more of the following conditions:

[0136] (1) A C18 column was used as the stationary phase;

[0137] (2) The flow rate is 0.3 mL / min to 0.45 mL / min (e.g., 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41, 0.42, 0.43, 0.44, 0.45 mL / min); and,

[0138] (3) The column temperature is 35℃~45℃ (e.g., 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45℃).

[0139] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this application, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0140] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0141] Example 1: Accurate detection of each analyte

[0142] I. Preparation of Solution

[0143] 1. Preparation of standard products and quality control products

[0144] The preparation of the calibrator stock solution is shown in Table 1:

[0145] Table 1. Preparation of the calibrator stock solution

[0146]

[0147] The preparation of the mixed mother liquor for quality control products is shown in Table 2:

[0148] Table 2. Preparation of the mixed mother liquor for quality control products

[0149]

[0150] The prepared calibrator stock solution and quality control stock solution were diluted with 5% (w / v) BSA solution (solvent is physiological saline) at a volume ratio of 1:20 to prepare calibrators and quality control products.

[0151] 2. Preparation of internal standard solution

[0152] The internal standard solution is prepared as shown in Table 3:

[0153] Table 3. Preparation of Internal Standard Solution

[0154]

[0155] The prepared internal standard solution was dispensed into 1.5 mL brown tubes at a rate of 120 μL / bottle and stored at -18°C or below.

[0156] 3. Preparation of other solutions

[0157] Magnetic beads: These are surface-bonded C18 polymer silicone beads, specifically ME10-C18MC magnetic beads, manufactured by Navitas, catalog number Q03P13M2; particle size 10µm, specific surface area approximately 400m². 2 / g~450m 2 / g, with a pore size of approximately 100Å~120Å.

[0158] Magnetic bead activating solution: methanol.

[0159] Internal standard diluent: Add 0.3L of methanol to 2.7L of water to prepare the internal standard diluent, and store at 2℃~8℃.

[0160] Eluent I: Add 0.11L of methanol to 10.89L of water to prepare eluent I, and store at 2℃~8℃.

[0161] Eluent II: Add 1.1L of methanol to 9.9L of water to prepare eluent II, and store at 2℃~8℃.

[0162] Eluent I: Add 1.2L of methanol to 0.8L of water to prepare eluent I, and store at 2℃~8℃.

[0163] Eluent II: Prepare eluent II by adding 1.2 L of methanol and 0.04 L of ammonia solution (ammonia mass fraction of 2%) to 0.8 L of water and storing at 2℃~8℃. The pH of eluent II should be above 9.

[0164] Sample incubation solution A: Add 0.261g of PMSF (phenylmethylsulfonyl fluoride) to 15mL of methanol to prepare a 100mM PMSF methanol solution, and store at 2℃~8℃.

[0165] Sample incubation solution B: Add 111g of tris(hydroxymethyl)aminomethane (TRIS) and 182g of ethylenediaminetetraacetic acid (EDTA) to 1298mL of water, then add 33mL of glacial acetic acid to adjust the pH to 5.6~6, and store at 2℃~8℃.

[0166] Termination solution: formic acid.

[0167] Calibration diluent: 5% (w / v) bovine serum albumin (BSA) in physiological saline.

[0168] Prepare a kit using the reagents prepared under the above three conditions.

[0169] II. Sample Extraction Methods

[0170] 1. Preparations before the experiment

[0171] (1) Take the kit out of the refrigerator and place it at 10℃~30℃ for equilibration.

[0172] (2) Preparation of magnetic bead working solution: Add magnetic bead activation solution to magnetic bead dry powder and vortex for 2 minutes to obtain magnetic bead working solution. The magnetic bead working solution needs to be activated for at least 2 hours before use. This solution should be sealed and stored at 2℃~8℃ for 14 days.

[0173] (3) Preparation of internal standard termination working solution: Mix the internal standard solution, termination solution and internal standard diluent in a volume ratio of 5:5:390 to obtain the internal standard termination working solution.

[0174] (4) Preparation of incubation solution: Mix sample incubation solution A and sample incubation solution B at a volume ratio of 1:100 to obtain the sample incubation solution. The sample incubation solution should be prepared fresh for each use.

[0175] 2. Sample pretreatment methods

[0176] (1) Calibration and quality control loading

[0177] Transfer 1.5 mL of the calibrator to a 2.0 mL screw cap tube and label it Cal 8. Take 7 clean 2.0 mL screw cap tubes and place them in the calibration position of the instrument in sequence.

[0178] Transfer 500 μL of the low-value / high-value quality control sample to a 2.0 mL screw cap tube and place it in the instrument's quality control position; transfer 4 mL of the calibrator diluent restored to 10℃~30℃ to a 5 mL cryovial and place it in the instrument's calibrator diluent position.

[0179] (2) Reagent loading

[0180] a. Sample incubation solution: Transfer the sample incubation solution to a clean reagent tank and place it at the incubation level of the instrument.

[0181] b. Magnetic bead working solution: Transfer the magnetic bead working solution to a clean reagent tank and place it at the magnetic bead working solution level of the instrument.

[0182] c. Internal standard termination solution: Transfer the internal standard termination solution to a clean reagent tank and place it at the internal standard working level of the instrument.

[0183] d. Eluent: Transfer eluent I and eluent II to clean reagent tanks and place them in the corresponding rinsing positions on the instrument.

[0184] e. Eluent: Transfer eluent I and eluent II to clean reagent tanks and place them in the elution position of the instrument.

[0185] (3) Sample loading

[0186] Place the samples to be tested (ensuring each sample volume is ≥500μL, mixed plasma sample S0) sequentially into the sample position of the instrument, ready for use.

[0187] (4) Standard curve preparation

[0188] The instrument will retrieve the corresponding standard curve preparation program from its program and perform the following operations:

[0189] a. The instrument dispenses the calibrator diluent into seven 2mL screw-cap tubes;

[0190] b. Then transfer Cal 8 to the adjacent 2mL screw cap tube, mix by blowing and stirring to complete the preparation of Cal 7, and so on to complete the preparation of the calibrator standard curve.

[0191] (5) Sample preparation

[0192] Place the 96-well deep-well plate and pipette tip that come with the instrument into the corresponding positions on the instrument. Then, retrieve the preparation program in the instrument software, enter the sample quantity (excluding calibrators and quality control samples), and click the "Run" button. The instrument will then perform the following steps:

[0193] A. Sample incubation

[0194] Transfer 400 μL of the sample to be tested and 100 μL of the sample incubation solution, and then incubate at 37 °C for 2 h. After incubation, add 200 μL of internal standard stop solution to terminate the reaction and prepare the incubated sample.

[0195] B. Sample Extraction

[0196] b1. Activation: Activate the magnetic beads with magnetic bead activation solution. After activation, remove the magnetic bead activation solution.

[0197] b2. Sample loading: Add 700 μL of the incubated sample, 3 mg of magnetic beads and internal standard solution to bind. After binding is complete, remove the solution.

[0198] b3. Rinsing: The magnetic beads are rinsed with rinsing solution I and rinsing solution II respectively to remove impurities attached to the magnetic beads. After each rinsing step is completed, the rinsing solution is removed.

[0199] b4. Elution: Elute the analytes on the magnetic beads with elution buffer I (perform the first elution step) and elution buffer II (perform the second elution step). Collect the first elution buffer (containing aldosterone, cortisol, 18-hydroxycortisol, 18-hydroxycorticosterone and 18-oxocortisol) and the second elution buffer (containing angiotensin I and angiotensin II) in a 96-well deep plate.

[0200] b5. Loading: Remove the loading plate containing the eluent, seal it, and load it for testing.

[0201] III. Sample Testing Methods

[0202] When performing liquid chromatography-tandem mass spectrometry analysis on samples, the liquid chromatography conditions include:

[0203] The chromatographic column was a C18 column, Phenomenex kinetex 2.6μm C18 100×2.1mm;

[0204] The flow rate was 0.4 mL / min;

[0205] The column temperature is 40℃;

[0206] Detector: Mass spectrometer;

[0207] The step-by-step washout process is as follows:

[0208] The first elution procedure uses mobile phases A1 and B1. Mobile phase A1 is an aqueous solution containing 0.2 mM ammonium fluoride, and mobile phase B1 is methanol. The elution gradient is shown in Table 4. These conditions were used to detect aldosterone, cortisol, 18-hydroxycorticosteroids, 18-hydroxycorticosterone, and 18-oxocortisol.

[0209] The second elution procedure consists of mobile phases A2 and B2. Mobile phase A2 is an aqueous solution of formic acid containing 0.1% (v / v) formic acid, and mobile phase B2 is methanol containing 0.1% (v / v) formic acid. The elution gradient is shown in Table 5. These conditions are used to detect angiotensin I and angiotensin II.

[0210] Chromatogram as shown Figures 1 to 7 As shown.

[0211] Table 4. Liquid Chromatography Conditions 1

[0212]

[0213] Table 5. Liquid Chromatography Conditions 2

[0214]

[0215] For mass spectrometry detection, a triple quadrupole mass spectrometer was used for quantification. The instrument model was LMSQ-2000, independently developed by BGI Genomics. Mass spectrometry detection was performed using positive and negative ion modes and multiple reaction monitoring (MRM) mode of electrospray ionization source. The corresponding ion channel parameters are shown in Table 6.

[0216] Table 6. Ion Channel Parameters

[0217]

[0218] Plotting the standard curve: Plot the concentration of each analyte in the calibrator as the abscissa (x) and the ratio of the peak area of ​​each analyte to the peak area of ​​the corresponding internal standard as the ordinate (y). Obtain the linear regression equation y=ax+b and calculate the correlation coefficient (r). The validation results of the standard curve are shown in Table 7.

[0219] Table 7. Validation Results of Standard Curve

[0220]

[0221] After verification of accuracy and precision (Table 8), the linear relationship of the detection was good within the concentration range.

[0222] Table 8. Accuracy and precision of method validation

[0223]

[0224] Example 2: The effect of different magnetic beads on extraction efficiency

[0225] In this embodiment, for the mixed plasma sample (S1), the following two different methods are used to detect each analyte:

[0226] Option 2.1: C18 magnetic beads based on a silica matrix, using the method described in Example 1 above. The silica matrix C18 magnetic beads are: ME10-C18MC magnetic beads, Navi, product number Q03P13M2; particle size 10µm, specific surface area approximately 400~450m². 2 / g, with a pore size of approximately 100~120Å).

[0227] Scheme 2.2: Magnetic beads based on surface-bonded hydrophilic-lipophilic balanced polymers, using the method described in Example 1 of document 202211268276.9. The magnetic beads bonded with the hydrophilic-lipophilic balanced polymer are: HLB magnetic beads, Boyun Biotechnology, catalog number BNMA7300001-0; particle size 31µm, specific surface area approximately 647m². 2 / g, with a pore size of approximately 67Å.

[0228] In scheme 2.1, C18 magnetic beads are used. The compounds eluted in the first step are aldosterone, cortisol, 18-hydroxycortisol, 18-hydroxycorticosterone and 18-oxocortisol. The compounds eluted in the second step are angiotensin I and angiotensin II.

[0229] In scheme 2.2, HLB magnetic beads are used, and all analytes are eluted in the first step: aldosterone, cortisol, 18-hydroxycorticosteroid, 18-hydroxycorticosterone, 18-oxocorticosteroid, angiotensin I, and angiotensin II.

[0230] The detection results of schemes 2.1 and 2.2 were examined, and the peak areas of the seven markers in plasma sample S1 were measured as shown in Table 9.

[0231] Table 9. Effects of different treatment methods on analyte extraction efficiency

[0232]

[0233] As shown in Table 9, when different magnetic beads were used to treat the samples, C18 magnetic beads showed better extraction performance for the seven analytes than HLB magnetic beads. The main reason is that HLB magnetic beads have both hydrophilic and lipophilic bonded phases, resulting in more complex compound extraction and relatively poor specificity; C18 magnetic beads, on the other hand, use polar interactions and have a weaker grasp of hydrophilic compounds, thus exhibiting stronger specificity.

[0234] In Scheme 2.2, HLB magnetic beads are used to elute all analytes in one step. However, in mobile phase A, which is an aqueous solution containing 1 mM ammonium fluoride, and mobile phase B, which is methanol containing 1 mM ammonium fluoride, the pH of the mobile phase system is 5-6. Angiotensin I and angiotensin II are small peptide compounds, which cannot be completely eluted in a C18 column with a silica matrix and a mobile phase pH of 5-6. The accumulation of residual small peptides (angiotensin I and angiotensin II) on the column will lead to increased column pressure and shortened column life.

[0235] In Scheme 2.1, the analyte can only be completely eluted in a C18 column with a silica gel matrix and a mobile phase pH of 2-3 (mobile phase A2 is an aqueous solution containing 0.1% formic acid, and mobile phase B2 is methanol containing 0.1% formic acid).

[0236] During the large-scale plasma sample testing (one injection per plasma sample) using scheme 2.2, the chromatograms of angiotensin I and angiotensin II from the first injection are as follows: Figure 8a and Figure 8b As shown, the chromatograms of angiotensin I and angiotensin II from the 50th injection are as follows. Figure 9a and Figure 9b As shown, the chromatograms of angiotensin I and angiotensin II from the 100th injection are as follows. Figure 10a and Figure 10b As shown in the figure, the results indicate that during large-scale sample processing, scheme 2.2 suffers from angiotensin instability in eluting.

[0237] During the large-scale plasma sample testing (one injection per plasma sample) using scheme 2.1, the chromatograms of angiotensin I and angiotensin II from the first injection are as follows: Figure 11a and Figure 11b As shown, the chromatograms of angiotensin I and angiotensin II from the 50th injection are as follows. Figure 12a and Figure 12b As shown, the chromatograms of angiotensin I and angiotensin II from the 100th injection are as follows. Figure 13a and Figure 13b As shown in the figure, the results indicate that angiotensin II using scheme 2.1 can achieve stable peak elution during large-scale sample processing.

[0238] Example 3: Effect of flow relative on angiotensin I and angiotensin II sensitivity

[0239] This embodiment focuses on plasma sample S2, and the only difference from Example 1 is that three schemes are set up as shown in Table 10 below to detect the second eluent (containing angiotensin I and angiotensin II):

[0240] Option 3.1: For the second eluent, detection was performed using the same chromatographic conditions 2 as in Example 1.

[0241] Scheme 3.2: For the second eluent, detection was performed using the same chromatographic conditions as in Example 1.

[0242] Scheme 3.3: For the second eluent, the following chromatographic conditions are used: The only difference from chromatographic conditions 1 in Example 1 is the concentration of ammonium fluoride in the mobile phase A2.

[0243] The results are shown in Table 10:

[0244] Table 10. Comparison of detection effects of different mobile phase additives

[0245]

[0246] As can be seen from Table 10, angiotensin I and angiotensin II compounds showed better sensitivity in the aqueous solution of A2: 0.1% formic acid and the mobile phase of B2: 0.1% formic acid and methanol.

[0247] Example 4

[0248] (1) Microspheres

[0249] Table 11

[0250]

[0251] (2) The detection method differs from that in Example 1 only in the use of different microspheres and eluents. Specifically:

[0252] Option 4.1: Use methanol as the eluent only.

[0253] Option 4.2: Use only a mixture of formic acid and methanol as the eluent, with formic acid accounting for 2% by volume.

[0254] Option 4.3: Use only a mixture of ammonia and methanol as the eluent, wherein the volume percentage of ammonia (2% by mass of ammonia) is 2%.

[0255] The test results of scheme 4.1 are as follows: Figures 14a to 14g As shown in the figure. The results indicate that, except for Mannhager magnetic beads, the extraction efficiencies of other magnetic beads for hormone compounds (ALD, F, 18-OHB, 18-OHF, 18-OXOF) are basically the same. For ANGII and ANGI, only HLB magnetic beads can elute with pure methanol. Furthermore, HLB magnetic beads are currently based on a polymer matrix, while other magnetic beads are based on a silica gel matrix.

[0256] The test results of scheme 4.2 are as follows Figures 15a to 15g As shown, the detection results of scheme 4.3 are as follows: Figures 16a to 16g As shown, Boyun HLB corresponds to serial number 5 in Table 11. The results show that HLB magnetic beads can still be completely eluted under acidic and alkaline conditions; Agilent PEP magnetic beads, silica-based magnetic beads, can elute ANGII and Angi under acidic conditions; Navitas C18 magnetic beads cannot be eluted under acidic conditions; PEP has poor extraction effect on 18-OXOF; Navitas MC18 and LC18 magnetic beads can elute ANGII and Angi under alkaline conditions, and their sensitivity is equivalent to that of HLB magnetic beads in all aspects.

[0257] The technical features of the above-described embodiments and examples can be combined in any suitable manner. For the sake of brevity, not all possible combinations of the technical features in the above-described embodiments and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0258] The embodiments described above are merely illustrative of several implementation methods of this application, intended to facilitate a detailed understanding of the technical solutions of this application, but should not be construed as limiting the scope of protection of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the scope of protection of this application. Furthermore, it should be understood that after reading the above teachings of this application, those skilled in the art can make various alterations or modifications to this application, and the equivalent forms obtained also fall within the scope of protection of this application. It should also be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided in this application through logical analysis, reasoning, or limited experimentation are all within the scope of protection of the appended claims. Therefore, the scope of protection of this patent application should be determined by the content of the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A method for preparing a clinical sample for detecting a target analyte in a test sample, characterized in that, The preparation method includes the following steps: The target analytes in the sample are adsorbed using magnetic beads; the surface of the magnetic beads is modified with C18 hydrophobic functional groups, and the target analytes include angiotensin I, angiotensin II, aldosterone, cortisol, 18-oxocortisol, 18-hydroxycorticosterone, and 18-hydroxycortisol; and, Impurities adsorbed on the magnetic beads are removed, and the target analytes adsorbed on the magnetic beads are eluted stepwise. The eluents obtained from each elution step are used as clinical samples for subsequent detection of the target analytes. The eluent includes a first eluent and a second eluent; The first eluent includes aldosterone, cortisol, 18-oxocortisol, 18-hydroxycorticosterone, and 18-hydroxycorticosteroid; The second eluent includes angiotensin I and angiotensin II; Stepwise elution includes: The target analyte adsorbed on the magnetic beads was eluted using elution buffer I and elution buffer II, respectively, and the first elution buffer and the second elution buffer were collected accordingly. The eluent I is a mixture of methanol and water, in which methanol accounts for 60% by volume; the eluent II is a mixture of methanol and ammonia, or a mixture of methanol, ammonia and water, in which methanol accounts for 60% by volume and the pH is above 9.

0. Methods for removing impurities adsorbed on magnetic beads include rinsing with a rinsing solution; The rinsing solution includes rinsing solution I and rinsing solution II; The rinsing solution I comprises a methanol aqueous solution with a methanol volume percentage of 1%; The rinsing solution II comprises a methanol aqueous solution with a methanol volume ratio of 10%. The magnetic beads have a particle size of 8µm to 50µm and a specific surface area of ​​400m². 2 / g~450m 2 / g, pore size 100Å~130Å, carbon content 12wt%~14wt%.

2. The method for preparing a clinical sample for detecting a target analyte in a test sample according to claim 1, characterized in that, The process of using magnetic beads to adsorb target analytes from a sample includes the following steps: The test sample and the sample incubation solution are mixed and incubated. The reaction is terminated by adding an internal standard to stop the working solution, and the incubation product is prepared; and, The incubation product, magnetic beads activated by the activation solution, and internal standard solution are mixed and combined, and then the solvent is removed.

3. The method for preparing a clinical sample for detecting a target analyte in a test sample according to claim 2, characterized in that, The preparation method satisfies one or more of the following conditions: (1) The incubation conditions include: temperature of 36℃~38℃ and time of 1h~3h; (2) The ratio of the sample to be tested to the magnetic beads is 350µL~450µL:2mg~5mg; (3) The sample to be tested includes plasma samples.

4. The method for preparing a clinical sample for detecting a target analyte in a test sample according to any one of claims 2 to 3, characterized in that, The sample incubation solution includes sample incubation solution A and sample incubation solution B in a volume ratio of 1:(95~105); The sample incubation solution A is a mixture of benzyl sulfonyl fluoride and methanol, with the concentration of benzyl sulfonyl fluoride being 95~105 mM; The sample incubation solution B comprises tris(hydroxymethyl)aminomethane, ethylenediaminetetraacetic acid, and water in a ratio of 110-115g:180-185g:1290-1300mL, with a pH of 5.6-6.

5. The method for preparing a clinical sample for detecting a target analyte in a test sample according to any one of claims 2 to 3, characterized in that, The internal standard termination working solution comprises an internal standard solution, a termination solution, and an internal standard diluent in a volume ratio of (4.5~5.5):(4.5~5.5):(385~395); wherein the internal standard diluent is a methanol solution with a volume percentage of 8%~12% and the termination solution is formic acid.

6. The method for preparing a clinical sample for detecting a target analyte in a test sample according to any one of claims 2 to 3, characterized in that, The ratio of the volume of the sample to be tested, the sample incubation solution, and the internal standard termination solution is 350μL~450μL:90μL~110μL:190μL~210μL.

7. The method for preparing a clinical sample for detecting a target analyte in a test sample according to any one of claims 2 to 3, characterized in that, The activation solution is methanol.

8. The method for preparing a clinical sample for detecting a target analyte in a test sample according to any one of claims 2 to 3, characterized in that, The internal standard solutions include 3 μg / mL to 4 μg / mL angiotensin I IS, 0.2 μg / mL to 0.25 μg / mL angiotensin II IS, 0.5 μg / mL to 0.55 μg / mL aldosterone IS, 3.1 μg / mL to 3.2 μg / mL cortisol IS, 0.3 μg / mL to 0.4 μg / mL 18-hydroxycorticosterone IS, and 0.2 μg / mL to 0.3 μg / mL 18-oxocorticol IS.

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