Primary aldosteronism marker substance spectrum detection kit as well as preparation method and application thereof

Abstract

The invention relates to a primary aldosteronism marker substance spectrum detection kit as well as a preparation method and application thereof. The kit comprises the following components: a calibration product and a quality control product: aldosterone and angiotensin I are added into a stabilizing solution to prepare freeze-dried powder with different concentrations, and the freeze-dried powder is obtained by freeze-drying; the inhibitor A is a solution obtained by dissolving a trypsin inhibitor in water, the inhibitor B is a solution obtained by dissolving benzenesulfonyl fluoride in methanol, the inhibitor C is a solution obtained by dissolving trihydroxymethyl aminomethane and ethylenediaminetetraacetic acid in water, and the internal standard solution is obtained by dissolving aldosterone d8 and angiotensin I-13C6 and 15N4 in a stabilizing solution. Compared with the prior art, a Tris base buffer solution and acetic acid are used for adjusting the pH value to serve as a stabilizing solution, meanwhile, the calibrator and the quality control product are prepared through a freeze-drying technology, the problem that the angiotensin I is poor in stability is solved, production and storage are easy, and the validity period of the kit is remarkably prolonged. The kit is low in detection lower limit, and the method is high in accuracy and precision.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and in particular relates to a primary aldosteronism marker mass spectrometry detection kit and a preparation method and application thereof. Background Art

[0002] Primary aldosteronism is caused by adrenal cortical lesions (adenomas or hyperplasia). The secretion of aldosterone is not dependent on the renin-angiotensin system and is not inhibited by salt load. It is a syndrome caused by excessive autonomous secretion of aldosterone. In the basal state (i.e., supine condition), the aldosterone level of patients with aldosteronism is high and the plasma renin activity is low. Therefore, the plasma aldosterone to renin activity ratio (ARR) is currently widely used as the preferred screening indicator. The calculation formula of the plasma aldosterone to renin activity ratio (ARR) is: ARR = plasma aldosterone concentration (PAC) ÷ renin activity (PRA), and renin activity is expressed as the rate of change of angiotensin I. Traditional plasma renin activity determination and aldosterone detection mostly use immunological methods. In addition, the content of aldosterone in the human body is very low, which will inevitably be interfered by immune cross-reactions, especially for female patients. If they are in the luteal phase, the renin concentration will decrease. Compared with the renin concentration level of men, it is more susceptible to interference from immune cross-byproducts, causing renin concentration fluctuations, which makes cross-reactions more likely to occur. The concentration level of aldosterone in this physiological stage will increase, resulting in an increase in false positive rate, poor accuracy, and inability to detect angiotensin I (AngI) and aldosterone (aldosterone, Ald) at the same time. The test results between different laboratories are also quite different. Therefore, the existing immunological method cannot achieve high accuracy and simultaneous detection of AngI and ALD.

[0003] Liquid chromatography-tandem mass spectrometry has become a powerful means for quantitative detection of trace target analytes in biological samples due to its many advantages such as high sensitivity and strong specificity. However, due to its complex pre-treatment process and high requirements for experimental personnel, it still faces many technical challenges in practical application. In addition, the laboratory's self-built detection methods cannot guarantee accuracy and data reproducibility due to the different solution preparation habits and operation differences of each experimenter, which has become a key bottleneck restricting the development of existing analytical methods.

[0004] The kit based on high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) can minimize the impact of experimental errors by fixing experimental protocols and standardized operating procedures.

[0005] Angiotensin I is a polypeptide composed of 10 amino acids. It is very easy to form a tailing peak in chromatographic separation and also very easy to remain on the chromatographic column, affecting the accuracy of the quantitative results. Acidic additives are usually used to improve the chromatographic peak and residue. However, when aldosterone is detected in the negative ion mode of electrospray mass spectrometry, its response intensity is severely inhibited by acid additives. When screening for primary aldosteronism, the sensitivity of aldosterone is extremely high, usually required to reach 0.02ng / mL, so it is difficult to detect both at the same time. In addition, because angiotensin I is a polypeptide, it is very easy to degrade and has poor stability, which seriously affects the validity period of the kit. Therefore, the preparation of a mass spectrometry kit for simultaneous detection of aldosterone and angiotensin I with high sensitivity, high precision, high accuracy and high stability is particularly important.

[0006] Chinese patent CN 111965369 A discloses a kit and method for detecting angiotensin I in plasma, Chinese patent CN 118501313 A discloses a kit and method for preparing angiotensin I, and Chinese patent CN 114563504 A discloses a method and kit for determining the content of free aldosterone in plasma. The above three patent technologies disclosed can only detect a single angiotensin I or aldosterone, and cannot detect angiotensin I and aldosterone, two primary aldosteronism markers, at the same time. When the disclosed technology is used to detect two markers at the same time, the use of amino acids to add trifluoroacetic acid as a stabilizer can ensure the stability of angiotensin I, but it will also seriously affect the sensitivity of aldosterone, and it is also impossible to detect aldosterone and angiotensin I at the same time.

[0007] Chinese patent CN 114895043 A discloses a primary aldosteronism screening kit and a diagnosis and typing system. The patent proposes to use magnetic beads with hydrophilic-lipophilic balanced polymers bonded on the surface for sample pretreatment, optimize the process conditions, and then use liquid chromatography-tandem mass spectrometry to simultaneously and accurately detect five markers of aldosterone, angiotensin I, angiotensin II, cortisol, and 18-hydroxycorticosterone in the sample. The calibration matrix is ​​a PBS buffer solution containing BSA. Chinese patent CN 116106466 A discloses a kit for simultaneously determining multiple causal markers of endocrine hypertension. It proposes to use a solid phase extraction method, and then use LC-MS to simultaneously detect 10 markers of aldosterone, angiotensin I, angiotensin II, metanephrine, epinephrine, dopamine, norepinephrine, normetanephrine, 3-methoxytyramine and cortisol. The calibration matrix is ​​a PBS buffer solution containing BSA. Although the above two patents use more universal stabilizing liquids to prepare calibration products and quality control products in order to detect multiple markers at the same time, neither of them mentions how to prevent the degradation of angiotensin I; at the same time, the processes disclosed in the above two patents do not guarantee the simultaneous detection of multiple indicators. The mobile phase system and elution gradient used affect the sensitivity of aldosterone or the chromatographic peak of angiotensin I to a certain extent, affecting the detection accuracy. Summary of the invention

[0008] Based on the lack of a method for simultaneous detection of aldosterone and angiotensin I with high sensitivity, high precision, high accuracy and high stability in the prior art, the present invention provides a primary aldosteronism marker mass spectrometry detection kit and its preparation method and application. The primary aldosteronism marker mass spectrometry detection kit provided by the present invention can solve the problems of poor stability of existing aldosterone and angiotensin I mass spectrometry simultaneous detection kits and the inability to take into account both aldosterone sensitivity and angiotensin I chromatographic peak type.

[0009] The purpose of the present invention can be achieved by the following technical solutions:

[0010] The present invention first provides a primary aldosteronism marker mass spectrometry detection kit, wherein the markers are aldosterone and angiotensin I, and the kit comprises the following components:

[0011] Calibrators: aldosterone and angiotensin I are added to the stabilizing solution, prepared into different concentrations, and freeze-dried to obtain freeze-dried powder;

[0012] Quality control: aldosterone and angiotensin I are added to the stabilizing solution, prepared into different concentrations, and freeze-dried to obtain freeze-dried powder;

[0013] Inhibitors: including inhibitor A, inhibitor B, inhibitor C,

[0014] Wherein, inhibitor A: a solution of trypsin inhibitor dissolved in water, the concentration of trypsin inhibitor is 3-300 mg / mL; inhibitor B: a solution of phenylmethylsulfonyl fluoride dissolved in methanol, the concentration of phenylmethylsulfonyl fluoride is 50-500 mmol / L; inhibitor C: a solution of tris(hydroxymethylaminomethane) and ethylenediaminetetraacetic acid dissolved in water, the concentration of tris(hydroxymethylaminomethane) is 0.5-2 mol / L, the concentration of ethylenediaminetetraacetic acid is 0.1-1 mol / L, and the pH of inhibitor C is 5.45-5.50;

[0015] Internal standard solution: aldosterone-d8 and angiotensin I- 13 C6, 15 Obtained by dissolving N4 in a stabilizing solution.

[0016] In one embodiment of the present invention, before the calibrator is freeze-dried, aldosterone and angiotensin I are prepared into a mixed series of solutions S1 to S5 using a stabilizing solution, wherein the concentrations of aldosterone are 0.02, 0.08, 0.4, 2, and 6 ng / mL, respectively, and the concentrations of angiotensin I are 0.3, 1.2, 6, 60, and 90 ng / mL, respectively.

[0017] In one embodiment of the present invention, before freeze-drying the quality control product, aldosterone and angiotensin I are prepared into mixed series solutions P1-P2 with stabilizing solutions, wherein the concentrations of aldosterone are 0.2 and 4 ng / mL and the concentrations of angiotensin I are 3 and 60 ng / mL, respectively.

[0018] In one embodiment of the present invention, the stabilizing liquid is a solution obtained by mixing tris(hydroxymethyl)aminomethane, bovine serum albumin and water, wherein the concentration of tris(hydroxymethyl)aminomethane is 0.1-1 mol / L, the concentration of bovine serum albumin is 10-100 mg / mL, and the pH is 5.0-7.5.

[0019] In one embodiment of the present invention, the trypsin inhibitor is selected from trypsin inhibitors derived from soybean.

[0020] In one embodiment of the present invention, in the internal standard solution, the concentration of aldosterone-d8 is 5 to 50 ng / mL, and the concentration of angiotensin I- 13 C6, 15 The concentration of N4 is 10 to 800 ng / mL. Preferably, the concentration of aldosterone-d8 is 20 ng / mL, and the concentration of angiotensin I- 13 C6, 15 The concentration of N4 is 100 ng / mL.

[0021] In one embodiment of the present invention, in the internal standard solution, aldosterone-d8 is a deuterated form of aldosterone, CAS No. 1261254-31-2, and the molecular formula is C21 H 20 D8O5, molecular weight is 368.49.

[0022] In one embodiment of the present invention, in the internal standard solution, angiotensin I- 13 C6, 15 N4 refers to angiotensin I, in which the six carbon atoms on arginine are replaced by 13 C, 4 nitrogen atoms replaced by 15 N, the structural formula is shown below.

[0023]

[0024] In one embodiment of the present invention, the kit further comprises instructions for use.

[0025] In one embodiment of the present invention, the kit further comprises a washing solution, a mobile phase stock solution, an eluent and a stop solution.

[0026] In one embodiment of the present invention, the eluent is methanol aqueous solution and n-hexane.

[0027] In one embodiment of the present invention, the mobile phase stock solution is aqueous acetic acid and methanol.

[0028] In one embodiment of the present invention, the eluent is methanol.

[0029] In one embodiment of the present invention, the stop solution is formic acid.

[0030] In one embodiment of the present invention, the kit further comprises deionized water.

[0031] The present invention further provides a method for preparing a mass spectrometry detection kit for primary aldosteronism markers, comprising the following steps:

[0032] (1) Preparation of calibrators and quality control products:

[0033] Dissolve tris(hydroxymethyl)aminomethane in water, adjust the pH with acetic acid to prepare a buffer solution, add bovine serum albumin, and sonicate until it is completely dissolved, and the resulting solution is a stable solution;

[0034] Take aldosterone (Ald) and angiotensin I (Ang I) and add them to the stabilizing solution, stir until completely dissolved, prepare different concentrations, pack them in brown vials, freeze-dry and seal them to obtain calibration products and quality control products;

[0035] (2) Preparation of inhibitors:

[0036] Trypsin inhibitor was dissolved in water to prepare inhibitor A, phenylmethylsulfonyl fluoride was dissolved in methanol to prepare inhibitor B, tris(hydroxymethyl)aminomethane and ethylenediaminetetraacetic acid were added into water, dissolved, and pH was adjusted to between 5.45 and 5.50 with acetic acid, and the obtained solution was inhibitor C;

[0037] (3) Preparation of internal standard solution:

[0038] Take aldosterone-d8 and angiotensin I- 13 C6, 15 N4 is dissolved in the stabilizing solution described in step (1) to prepare a mixed internal standard solution.

[0039] In one embodiment of the present invention, in step (1), the concentration of tris(hydroxymethyl)aminomethane in the buffer solution is 0.1 to 1 mol / L, and the pH is 5.0 to 7.5.

[0040] In one embodiment of the present invention, in step (1), the concentration of bovine serum albumin in the stabilizing solution is 10 to 100 mg / mL.

[0041] In one embodiment of the present invention, in step (1), when packaging into brown vials, the packaging specification is 0.5 to 5 mL per bottle.

[0042] In one embodiment of the present invention, in step (2), the concentration of the inhibitor A is 3 to 300 mg / mL.

[0043] In one embodiment of the present invention, in step (2), the concentration of the inhibitor B is 50 to 500 mmol / L.

[0044] In one embodiment of the present invention, in step (2), when preparing inhibitor C, dissolution is performed by ultrasound, and the ultrasound time is 20 to 60 minutes.

[0045] In one embodiment of the present invention, in step (2), the concentration of tris(hydroxymethyl)aminomethane in the inhibitor C is 0.5-2 mol / L.

[0046] In one embodiment of the present invention, in step (2), the concentration of EDTA in the inhibitor C is 0.1-1 mol / L.

[0047] The present invention further provides the use of a primary aldosteronism marker mass spectrometry detection kit in the preparation of a product for detecting primary aldosteronism.

[0048] The present invention further provides a method for mass spectrometry detection of primary aldosteronism markers for non-disease diagnosis and treatment purposes, which is based on the primary aldosteronism marker mass spectrometry detection kit described above and comprises the following steps:

[0049] S1. Plasma samples were incubated with inhibitors, formic acid was added to the incubated samples to terminate the reaction, internal standard solution was added, water was added to dilute and centrifuged; calibrators were incubated with inhibitors, formic acid was added to the incubated samples to terminate the reaction, internal standard solution was added, water was added to dilute and centrifuged;

[0050] S2, respectively taking the supernatant of the sample and the calibrator after centrifugation for solid phase extraction;

[0051] S3. Analyze the samples and calibration materials after solid phase extraction using LC-MS / MS to obtain the angiotensin I and angiotensin I- 13 C6, 15 peak areas of N4, aldosterone, and aldosterone-d8;

[0052] S4. Calculation of test results: Angiotensin I and angiotensin I- 13 C6, 15 The peak area ratios of N4, aldosterone and aldosterone-d8 were used as the ordinate, and the concentrations of angiotensin I and aldosterone were used as the abscissa. The linear equations were fitted to obtain the standard curves of angiotensin I and aldosterone, respectively. The angiotensin I and angiotensin I- 13 C6, 15 The peak area ratio of N4 was substituted into the standard curve of angiotensin I to calculate the concentration of angiotensin I in the sample, and the peak area ratio of aldosterone and aldosterone-d8 in the sample was substituted into the standard curve of aldosterone to calculate the concentration of aldosterone in the sample. The renin activity of the sample was calculated by the concentration of angiotensin I in the sample to be tested after incubation ÷ incubation time.

[0053] In one embodiment of the present invention, in step S1, the inhibitor is an inhibitor prepared according to a volume ratio of inhibitor A: inhibitor B: inhibitor C = 1:1:1000.

[0054] In one embodiment of the present invention, in step S1, a total of 5 calibrators are provided, wherein the concentrations of aldosterone are 0.02, 0.08, 0.4, 2, and 6 ng / mL, respectively, and the concentrations of angiotensin I are 0.3, 1.2, 6, 60, and 90 ng / mL, respectively.

[0055] In one embodiment of the present invention, a quality control product is also provided, and the concentrations of angiotensin I and aldosterone in the quality control product are known. In step S1, the quality control product is incubated after adding an inhibitor, formic acid is added to the incubated sample to terminate the reaction, an internal standard solution is added, water is added to dilute and the sample is centrifuged; in step S4, angiotensin I and angiotensin I- in the quality control product are diluted to a concentration of 1:1. 13 C6, 15 The peak area ratio of N4 is substituted into the standard curve of angiotensin I to calculate the concentration of angiotensin I in the quality control product. The peak area ratio of aldosterone and aldosterone-d8 in the quality control product is substituted into the standard curve of aldosterone to calculate the concentration of aldosterone in the quality control product. If the calculated concentrations of angiotensin I and aldosterone in the quality control product are within the acceptable standard range (±15%) compared with the actual concentrations of angiotensin I and aldosterone in the quality control product, it indicates that the detection system can accurately detect the target substance in the sample. If the result is not within the acceptable standard range, the operating conditions of steps S1, S2, and S3 are adjusted to make the calculated concentrations of angiotensin I and aldosterone in the quality control product within the acceptable standard range compared with the actual concentrations of angiotensin I and aldosterone in the quality control product.

[0056] In one embodiment of the present invention, in step S4, the standard curve of angiotensin I is y=0.20646x+0.02536, wherein y is the standard curve of angiotensin I and angiotensin I- 13 C6, 15 The standard curve of aldosterone is y=2.59783+0.01955, wherein y is the peak area ratio of aldosterone and aldosterone-d8, and x is the concentration value of aldosterone.

[0057] The present invention is based on liquid chromatography-tandem mass spectrometry technology because of its high sensitivity, strong specificity and the like, and uses angiotensin I and aldosterone as detection markers to develop a mass spectrometry detection kit that can simultaneously detect two markers.

[0058] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0059] (1) The problem of poor stability of angiotensin I is solved by using a Tris base buffer solution supplemented with acetic acid to adjust the pH as a stabilizing solution and preparing the calibrator and quality control product using a freeze-drying process. This makes the product easy to produce and store, and significantly improves the shelf life of the kit.

[0060] (2) The detection limit of the kit of the present invention is low, and the method has high accuracy and precision. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1Detection spectra of angiotensin I and aldosterone under different mobile phase systems. DETAILED DESCRIPTION

[0062] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0063] This embodiment is implemented based on the technical solution of the present invention, and provides a detailed implementation method and a specific operation process, but the protection scope of the present invention is not limited to the following embodiments.

[0064] The experimental methods described in the examples are conventional methods unless otherwise specified; the percentages involved are volume percentages unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0065] Embodiment 1:

[0066] Kit preparation:

[0067] (1) Preparation of calibrators and quality control materials;

[0068] Take 12.11g of tris(hydroxymethyl)aminomethane and dissolve it in 1000mL of water, adjust the pH to 6.0 with acetic acid to prepare a 0.1mol / L Tris base buffer solution, then add 10g of bovine serum albumin and sonicate until it is completely dissolved. The resulting solution is a stable solution.

[0069] Aldosterone and angiotensin I were prepared into mixed series solutions S1-S5 with stabilizing solution, wherein the concentrations of aldosterone were 0.02, 0.08, 0.4, 2, and 6 ng / mL, and the concentrations of angiotensin I were 0.3, 1.2, 6, 60, and 90 ng / mL, respectively;

[0070] Aldosterone and angiotensin I were prepared into mixed series solutions P1-P2 with stabilizing solution, wherein the concentrations of aldosterone were 0.2 and 4 ng / mL and the concentrations of angiotensin I were 3 and 60 ng / mL respectively;

[0071] Dispense S1-S5 and P1-P2 into brown vials, 2 mL per bottle, freeze-dry and seal. The lyophilized powder obtained from S1-S5 is calibrators 1-5, and the lyophilized powder obtained from P1-P2 is quality control products 1-2;

[0072] (2) Preparation of inhibitors:

[0073] 0.3 g of trypsin inhibitor (a trypsin inhibitor derived from soybean, CAS number: 9035-81-8) was dissolved in 10 mL of water to prepare a solution with a concentration of 30 mg / mL, and the resulting solution was inhibitor A. 0.174 g of phenylmethylsulfonyl fluoride was dissolved in 10 mL of methanol to prepare a solution with a concentration of 100 mmol / L, and the resulting solution was inhibitor B. 121.1 g of tris(hydroxymethyl)aminomethane) and 74 g of ethylenediaminetetraacetic acid were added to 1000 mL of water, and ultrasonicated for 30 min until dissolved. The pH was adjusted to 5.45 with acetic acid, and the resulting solution was inhibitor C.

[0074] (3) Preparation of internal standard solution:

[0075] Take aldosterone-d8 and angiotensin I- 13 C6, 15 N4 was dissolved in the stabilizing solution described in step (1) to prepare a mixed internal standard solution, wherein the concentration of aldosterone-d8 was 20 ng / mL, and the concentration of angiotensin I- 13 C6, 15 The concentration of N4 was 100 ng / mL.

[0076] (4) The above calibrators, quality control products, inhibitors and internal standard solutions are placed in the kit and stored at 2-8°C.

[0077] Example 2

[0078] Investigating whether the stabilizing solution in the kit of Example 1 can ensure the stability of angiotensin I

[0079] Angiotensin I is a polypeptide composed of 10 amino acid residues, which is very unstable in solution and easily degraded. In the present application, the Tris base buffer solution of Example 1 (i.e., 12.11 g of tris(hydroxymethyl)aminomethane is dissolved in 1000 mL of water, and the pH is adjusted to 6.0 with acetic acid to prepare a 0.1 mol / L Tris base buffer solution) is used as a stabilizer for angiotensin I.

[0080] Angiotensin I was prepared in a stabilizer solution and stored at 4°C for a long term. The results were shown in Table 1.

[0081] Table 1 Long-term storage stability of angiotensin I stock solution at 4°C

[0082] Month 0 Month 1 Month 3 Month 6 Month 12 average value CV Repeat 1 9031782 9073668 8856866 8888928 8876432 8945535 1.11％ Repeat 2 9000656 9120102 8876127 8951374 8937491 8977150 1.02％ Repeat 3 8995683 9087354 8920630 8946789 8892097 8968511 0.85％ average value 9009374 9093708 8884541 8929030 8902007 8963732 0.97％ CV 0.22％ 0.26％ 0.37％ 0.39％ 0.36％

[0083] In Table 1, the numbers represent the peak area and CV is the coefficient of variation.

[0084] Example 3

[0085] A method for providing a non-disease diagnosis and treatment destination for the mass spectrometry detection of primary aldosteronism markers comprises the following steps:

[0086] S1. Plasma samples were incubated with inhibitors, formic acid was added to the incubated samples to terminate the reaction, internal standard solution was added, water was added to dilute and centrifuged; calibrators were incubated with inhibitors, formic acid was added to the incubated samples to terminate the reaction, internal standard solution was added, water was added to dilute and centrifuged;

[0087] S2, respectively taking the supernatant of the sample and the calibrator after centrifugation for solid phase extraction;

[0088] S3. Analyze the samples and calibration materials after solid phase extraction using LC-MS / MS to obtain the angiotensin I and angiotensin I- 13 C6, 15 peak areas of N4, aldosterone, and aldosterone-d8;

[0089] S4. Calculation of test results: Angiotensin I and angiotensin I- 13 C6, 15 The peak area ratios of N4, aldosterone and aldosterone-d8 were used as the ordinate, and the concentrations of angiotensin I and aldosterone were used as the abscissa. The linear equations were fitted to obtain the standard curves of angiotensin I and aldosterone, respectively. The angiotensin I and angiotensin I- 13 C6, 15 The peak area ratio of N4 was substituted into the standard curve of angiotensin I to calculate the concentration of angiotensin I in the sample, and the peak area ratio of aldosterone and aldosterone-d8 in the sample was substituted into the standard curve of aldosterone to calculate the concentration of aldosterone in the sample. The renin activity of the sample was calculated by the concentration of angiotensin I in the sample to be tested after incubation ÷ incubation time.

[0090] Specifically, the detection method for the sample in this embodiment is described as follows:

[0091] (1) Reagent preparation

[0092] a. Reconstitution of calibrators and quality control products: Since calibrators 1 to 5 and quality control products 1 to 2 are all lyophilized powders, they need to be reconstituted into solutions before use. The method is: let the calibrators and quality control products stand at room temperature for 10 minutes, add 2 mL of water to each bottle, mix for 10 minutes and set aside (since the calibrators and quality control products are in the state of lyophilized powder before reconstitution and are anhydrous, this step of adding water for reconstitution is to replenish the water removed by lyophilization. The purpose of lyophilization is to increase the stability of the calibrators and quality control products, that is, the shelf life.).

[0093] b. Inhibitor: Prepare the required amount according to the number of samples, 65 μL for each sample. Prepare the required amount according to the volume ratio of Inhibitor A: Inhibitor B: Inhibitor C = 1:1:1000, vortex mix for 1 min, and prepare the inhibitor (prepare it before use).

[0094] (2) Pre-treatment:

[0095] Take 400 μL of the calibrator, quality control and plasma sample to be tested, add 65 μL of the inhibitor prepared in step (1) b respectively, mix well, incubate at 37°C for 3 h, then add 14 μL of formic acid to terminate the reaction. Add 20 μL of internal standard solution, add 400 μL of water, mix well, and centrifuge at 15000 rpm for 10 min at room temperature. Take 800 μL of the supernatant and purify by solid phase extraction.

[0096] The conditions for solid phase extraction cleanup were as follows:

[0097] Solid phase extraction (using Cleanert PEP Micro Plate, 2mg / 1ml / well solid phase extraction plate)

[0098] Activation: 500 μL methanol;

[0099] Balance: 500 μL water (containing 1% acetic acid);

[0100] Sample loading: 800 μL of the centrifuged sample was loaded;

[0101] Elution: 650 μL 20% methanol in water and 500 μL n-hexane;

[0102] Elution: Elute with 50 μL methanol into a 96-well plate pre-added with 70 μL water, mix well and then test with LC-MS / MS.

[0103] (3) LC-MS / MS test instrument conditions

[0104] Chromatographic column: Kinetex C18 (2.6 μm, 2.1 × 100 mm);

[0105] Mobile phase: A: 0.1% acetic acid in water, B: methanol;

[0106] Flow rate: 0.25 mL / min;

[0107] Column temperature: 50°C;

[0108] Injection volume: 15 μL;

[0109] Table 2 Elution gradient

[0110] Time (min) A(％) B(％) 0 65 35 0.8 65 35 2.5 45 55 2.8 5 95 3.8 5 95 4.0 65 35 5.5 65 35

[0111] Mass spectrometry conditions:

[0112] Ion source: electrospray ionization (ESI);

[0113] Scanning mode: MRM multiple response monitoring;

[0114] Ion source temperature (TEM): 550°C;

[0115] Atomizing gas (Gas1): 45psi;

[0116] Auxiliary heating gas (Gas2): 40psi;

[0117] Gurtain Gas: 30psi;

[0118] Electrospray voltage: 5500 / -4500V;

[0119] The corresponding MRM channels and parameters are shown in Table 3.

[0120] Table 3 Angiotensin I and aldosterone MRM parameters

[0121]

[0122] *Quantitative ion

[0123] In this example, the 0.1% acetic acid water-methanol mobile phase system can effectively improve the chromatographic peak tailing and residual interference of angiotensin I while taking into account the sensitivity of aldosterone detection. The results are shown in Figure 1 . A, C, and E are the MRM channel peaks of angiotensin I (lower limit of quantitative concentration), aldosterone (lower limit of quantitative concentration), and angiotensin I injected with blank solvent immediately after high concentration angiotensin I in a 0.1% formic acid water-methanol mobile phase system; B, D, and F are the MRM channel peaks of angiotensin I (lower limit of quantitative concentration), aldosterone (lower limit of quantitative concentration), and angiotensin I injected with blank solvent immediately after high concentration angiotensin I in a 0.1% acetic acid water-methanol mobile phase system according to the method of the present invention. The results show that compared with A, B has an improved response intensity and a similar signal-to-noise ratio, indicating that the sensitivity of aldosterone has not decreased; compared with D, C has significantly improved the tailing of the chromatographic peak, and the chromatographic peak is sharper and more symmetrical; compared with F, the residual interference of angiotensin I is significantly eliminated.

[0124] The above results indicate that the detection limit is low and the method has high accuracy and precision using the conditions defined in this embodiment.

[0125] Under the detection conditions specified in this embodiment,

[0126] The angiotensin I and angiotensin I-13 C6, 15 The peak area ratios of N4, aldosterone, and aldosterone-d8 were used as the ordinate, and the concentrations of angiotensin I and aldosterone were used as the abscissa. The linear equations were fitted to obtain the standard curves of angiotensin I and aldosterone, respectively;

[0127] Among them, for the standard curve of angiotensin I, angiotensin I and angiotensin I- 13 C6, 15 The peak area ratio of N4 is y, and x is the concentration value of angiotensin I;

[0128] For the standard curve of aldosterone, y is the peak area ratio of aldosterone and aldosterone-d8, and x is the concentration value of aldosterone;

[0129] Weighted linear regression was performed to fit the calibration curve, with a weight of 1 / x (x is the concentration value, unit: ng / mL), and the correlation coefficient r≥0.99 was required.

[0130] The linearity of angiotensin I and aldosterone is shown in Table 4.

[0131] Table 4 Angiotensin I and aldosterone linearity

[0132] Compound Name Linear range (ng / mL) Linear equations Correlation coefficient r Angiotensin I 0.3～90 y=0.20646x+0.02536 0.9999 Aldosterone 0.02～6 y＝2.59783+0.01955 0.9998

[0133] As can be seen from Table 4, the linear correlation coefficients r of angiotensin I and aldosterone are 0.9999 and 0.9998, respectively, which meet the requirements.

[0134] Repeatability and accuracy verification is carried out on the basis of the above

[0135] Repeatability evaluation requires testing at three concentration points: 110% LLMI, 90% ULMI, and 50% (LLMI+ULMI). LLMI is the abbreviation of "Lower Limit of Measurement Interval", which is the lower limit of the measurement interval. It represents the lowest concentration value that the measurement system can reliably measure. ULMI is the abbreviation of "Upper Limit of Measurement Interval", which is the upper limit of the measurement interval. It represents the highest concentration value that the measurement system can accurately measure. Each concentration point is tested 20 times, and the CV is required to be ≤15%. Accuracy verification is evaluated by spiked recovery, and the average recovery of each concentration point is required to be between 85% and 115%.

[0136] The repeatability and accuracy verification results of angiotensin I and aldosterone are shown in Table 5.

[0137] Table 5 Repeatability and spike recovery of angiotensin I and aldosterone

[0138]

[0139] On the basis of the above, the quantitative lower limit

[0140] The lower limit of quantification is an intuitive reflection of the sensitivity of the method. It is the lowest value that the target analyte can be detected under the premise of meeting the accuracy and precision requirements. The lower limit of quantification selects 5 samples with concentrations close to the detection limit, and each concentration is measured 10 times. The total precision (CV) of each concentration sample and the deviation of its concentration measurement mean from the theoretical concentration are evaluated respectively. The measurement mean of the lowest concentration sample with CV < 20% and deviation < 15% is taken as the lower limit of quantification of the method.

[0141] The lower limits of quantification for angiotensin I and aldosterone are shown in Table 6.

[0142] Table 6 Quantitative limits of angiotensin I and aldosterone

[0143]

[0144] The results showed that the lower limits of quantification of angiotensin I and aldosterone were 0.3 ng / mL and 0.02 ng / mL, respectively, indicating that the kit of the present invention can ensure the reliability of the results when detecting low-concentration samples.

[0145] Comparative Example 1

[0146] Since aldosterone has good stability, while angiotensin I has poor stability, in order to compare the stability of angiotensin I in different systems, this comparative example uses 20% methanol aqueous solution to prepare calibrants containing low, medium and high concentrations of angiotensin I, which are stored at 4°C after packaging. The test results at different intervals of days are shown in Table 7 below. It can be seen from Table 7 that within 6 days, the relative standard deviations of the peak areas of low, medium and high concentrations of angiotensin I in methanol aqueous solution are all greater than 10%, which seriously affects the accuracy of the quantitative results.

[0147] Table 7 Stability of angiotensin I at different concentrations in 20% methanol aqueous solution

[0148] time Low concentration Medium concentration High concentration day 0 3533.99 90438.63 1360858.97 day 1 3471.75 89425.54 1259169.94 day 2 3394.23 123960.14 1686676.87 day 3 3296.93 104248.30 1604181.74 day 4 4493.79 123332.80 1658604.19 day 5 3852.84 127194.05 1736037.85 day 6 3296.46 107172.84 1560996.94 ave 3620.00 109396.04 1552360.93 CV 11.87％ 14.51％ 11.42％

[0149] Comparative Example 2:

[0150] The stabilizing solution in Example 1 was replaced with a 10 mg / mL BSA solution, which is often used as a simulated matrix for blood samples in clinical testing. The accuracy of the method was evaluated by spike recovery. At the same time, the pretreatment method was replaced with the magnetic bead method. The specific operation was as follows: weigh an appropriate amount of reversed phase C18 magnetic beads, and use a mixed solution of methanol: water = 1:1 to prepare a magnetic bead solution with a concentration of 20 mg / mL; pipette 200 μL of the magnetic bead solution into a 1.5 mL centrifuge tube, and activate it with 200 μL of methanol and 1% formic acid water in turn; load 800 μL of the supernatant after pretreatment in Example 1, vortex oscillate for 1 min, let stand for 1 min, and discard the supernatant after magnetic separation; use 200 μL 20% methanol water and 200 μL n-hexane to wash the magnetic beads respectively; add 200 μL of methanol to the magnetic bead centrifuge tube, vortex mix for 1 min, let stand for 1 min, take all the supernatant after magnetic separation, blow with nitrogen, and re-dissolve with 100 μL 20% methanol water for testing. The results are shown in Table 8 below. As can be seen from Table 8, the deviation between the theoretical value and the measured value of Ang I at low and high concentrations exceeds the requirement of ±15%, indicating that the accuracy of the Ang I detection result is poor; while the recovery rates of Ald at low and high concentrations are 104.0% and 104.%, respectively, indicating that the accuracy of this method in detecting Ald is high. In summary, this method is not suitable for the simultaneous detection of angiotensin I and aldosterone.

[0151] Table 8 Repeatability and spike recovery of angiotensin I and aldosterone in BSA solution system

[0152]

[0153] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A mass spectrometry detection kit for primary aldosteronism markers, wherein the markers are aldosterone and angiotensin I, characterized in that: The kit includes the following components: Calibrators: aldosterone and angiotensin I are added to the stabilizing solution, prepared into different concentrations, and freeze-dried to obtain freeze-dried powder; Quality control: aldosterone and angiotensin I are added to the stabilizing solution, prepared into different concentrations, and then freeze-dried to obtain freeze-dried powder; Inhibitors: including inhibitor A, inhibitor B, inhibitor C, Wherein, inhibitor A: a solution of trypsin inhibitor dissolved in water, the concentration of trypsin inhibitor is 3-300 mg / mL; inhibitor B: a solution of phenylmethylsulfonyl fluoride dissolved in methanol, the concentration of phenylmethylsulfonyl fluoride is 50-500 mmol / L; inhibitor C: a solution of tris(hydroxymethylaminomethane) and ethylenediaminetetraacetic acid dissolved in water, the concentration of tris(hydroxymethylaminomethane) is 0.5-2 mol / L, the concentration of ethylenediaminetetraacetic acid is 0.1-1 mol / L, and the pH of inhibitor C is 5.45-5.50; Internal standard solution: aldosterone-d8 and angiotensin I- 13 C6, 15 Obtained by dissolving N4 in a stabilizing solution.

2. A primary aldosteronism marker mass spectrometry detection kit according to claim 1, characterized in that: Before freeze-drying the calibrator, aldosterone and angiotensin I were prepared into mixed series solutions S1-S5 with stabilizing solutions, wherein the concentrations of aldosterone were 0.02, 0.08, 0.4, 2, and 6 ng / mL, respectively, and the concentrations of angiotensin I were 0.3, 1.2, 6, 60, and 90 ng / mL, respectively.

3. A primary aldosteronism marker mass spectrometry detection kit according to claim 1, characterized in that: Before freeze-drying the quality control product, aldosterone and angiotensin I were prepared into mixed series solutions P1-P2 with stabilizing liquid, wherein the concentrations of aldosterone were 0.2 and 4 ng / mL and the concentrations of angiotensin I were 3 and 60 ng / mL respectively.

4. A primary aldosteronism marker mass spectrometry detection kit according to claim 1, characterized in that: The stabilizing solution is a solution obtained by mixing tris(hydroxymethyl)aminomethane, bovine serum albumin and water, wherein the concentration of tris(hydroxymethyl)aminomethane is 0.1-1 mol / L, the concentration of bovine serum albumin is 10-100 mg / mL, and the pH is 5.0-7.

5.

5. A primary aldosteronism marker mass spectrometry detection kit according to claim 1, characterized in that: In the internal standard solution, the concentration of aldosterone-d8 is 5-50 ng / mL, and the concentration of angiotensin I- 13 C6, 15 The concentration of N4 is 10-800 ng / mL.

6. A primary aldosteronism marker mass spectrometry detection kit according to claim 1, characterized in that: The kit also includes instructions for use; The kit also includes a washing solution, a mobile phase stock solution, an eluent and a stop solution; The eluent is methanol aqueous solution and n-hexane, the mobile phase stock solution is acetic acid aqueous solution and methanol, the eluent is methanol, and the stop solution is formic acid; The kit also includes deionized water.

7. A method for preparing a mass spectrometry detection kit for primary aldosteronism markers according to any one of claims 1 to 6, characterized in that: The steps include: (1) Preparation of calibrators and quality control products: Dissolve tris(hydroxymethyl)aminomethane in water, adjust the pH with acetic acid to prepare a buffer solution, add bovine serum albumin, and sonicate until it is completely dissolved, and the resulting solution is a stable solution; Take aldosterone and angiotensin I and add them to the stabilizing solution, stir until completely dissolved, prepare different concentrations, pack them in brown vials, freeze-dry and seal them to obtain calibration products and quality control products; (2) Preparation of inhibitors: Trypsin inhibitor was dissolved in water to prepare inhibitor A, phenylmethylsulfonyl fluoride was dissolved in methanol to prepare inhibitor B, tris(hydroxymethyl)aminomethane and ethylenediaminetetraacetic acid were added into water, dissolved, and pH was adjusted to between 5.45 and 5.50 with acetic acid, and the obtained solution was inhibitor C; (3) Preparation of internal standard solution: Take aldosterone-d8 and angiotensin I- 13 C6, 15 N4 is dissolved in the stabilizing solution described in step (1) to prepare a mixed internal standard solution.

8. The method for preparing a primary aldosteronism marker mass spectrometry detection kit according to claim 7, characterized in that: In step (1), the concentration of tris(hydroxymethyl)aminomethane in the buffer solution is 0.1 to 1 mol / L, and the pH is 5.0 to 7.5; In step (1), the concentration of bovine serum albumin in the stabilizing solution is 10 to 100 mg / mL. In step (2), the concentration of the inhibitor A is 3 to 300 mg / mL; In step (2), the concentration of the inhibitor B is 50 to 500 mmol / L; In step (2), the concentration of tris(hydroxymethyl)aminomethane in the inhibitor C is 0.5-2 mol / L, and the concentration of ethylenediaminetetraacetic acid in the inhibitor C is 0.1-1 mol / L.

9. Use of a primary aldosteronism marker mass spectrometry detection kit according to any one of claims 1 to 6 in the preparation of a product for detecting primary aldosteronism.

10. A method for mass spectrometry detection of primary aldosteronism markers for non-disease diagnosis and treatment purposes, characterized in that: Based on a primary aldosteronism marker mass spectrometry detection kit according to any one of claims 1 to 6, The following steps are involved: S1. Plasma samples were incubated with inhibitors, formic acid was added to the incubated samples to terminate the reaction, internal standard solution was added, water was added to dilute and centrifuged; calibrators were incubated with inhibitors, formic acid was added to the incubated samples to terminate the reaction, internal standard solution was added, water was added to dilute and centrifuged; S2, respectively taking the supernatant of the sample and the calibrator after centrifugation for solid phase extraction; S3. Analyze the samples and calibration materials after solid phase extraction using LC-MS / MS to obtain the angiotensin I and angiotensin I- 13 C6, 15 peak areas of N4, aldosterone, and aldosterone-d8; S4. Calculation of test results: Angiotensin I and angiotensin I- 13 C6, 15 The peak area ratios of N4, aldosterone and aldosterone-d8 were used as the ordinate, and the concentrations of angiotensin I and aldosterone were used as the abscissa. The linear equations were fitted to obtain the standard curves of angiotensin I and aldosterone, respectively. The angiotensin I and angiotensin I- 13 C6, 15 The peak area ratio of N4 was substituted into the standard curve of angiotensin I to calculate the concentration of angiotensin I in the sample, and the peak area ratio of aldosterone and aldosterone-d8 in the sample was substituted into the standard curve of aldosterone to calculate the concentration of aldosterone in the sample. The renin activity of the sample was calculated by the concentration of angiotensin I in the sample to be tested after incubation ÷ incubation time.

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