Detection method for simultaneously and accurately detecting six hypertension-related hormones and application thereof

Abstract

The invention provides a detection method for simultaneously and accurately detecting six hypertension-related hormones and application thereof, and the detection method comprises the following steps: uniformly mixing a to-be-detected sample and an extracting solution in a vortex manner, centrifuging and collecting supernatant to obtain a test solution; the extracting solution is a methanol-ethyl formate mixed solution with the volume ratio of (7.5-8.5): (1.5-2.5), and the pH value is 8-10; detecting the test solution and the standard solution by adopting liquid chromatography-tandem mass spectrometry, and correcting a matrix effect by adopting an internal standard method; mobile phases adopted by the liquid chromatography comprise a mobile phase A and a mobile phase B, the mobile phase A is 10-12 mmol / L ammonium acetate-0. 1%-0.2% formic acid aqueous solution, and the mobile phase B is 0.1%-0.2% formic acid-acetonitrile; drawing a standard curve by taking the area ratio of the target object to the internal standard peak as a vertical coordinate and the concentration as a horizontal coordinate; and calculating the concentration of the target object in the test solution.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection technology, and specifically relates to a detection method for simultaneously and accurately detecting six hypertension-related hormones and its application. Background Art

[0002] Hypertension is a major risk factor for cardiovascular disease worldwide, of which approximately 5%-10% is secondary hypertension (such as primary aldosteronism and Cushing's syndrome). Accurate classification requires testing the serum levels of six hormones: AngII (angiotensin II), AngI (angiotensin I), Ald (aldosterone), 18-hydroxycortisol, 18-hydroxycorticosterone, and cortisol, combined with ratio analysis (such as aldosterone / renin activity). These hormones are core indicators of the renin-angiotensin-aldosterone system (RAAS) and glucocorticoid metabolism, and abnormal concentrations are closely related to the occurrence and development of hypertension.

[0003] Existing methods for detecting hypertension-related hormones mainly include: (1) Chemiluminescence immunoassay (CLIA), a single hormone detection method disclosed in the document "Hypertension" (Vol.70, No.3, 2017, pp.432-440), which detects cortisol, aldosterone, etc. through antibody-antigen reaction. It requires multiple injections (a single test takes 30-60 minutes) and has insufficient sensitivity for detecting low-abundance hormones such as 11-deoxycortisol (detection limit >20 ng / mL), which can easily miss the diagnosis of mild hormone disorders. (2) Traditional liquid chromatography-tandem mass spectrometry (LC-MS / MS). Although existing mass spectrometry methods (such as "J Am Soc Mass Spectrom" Vol. 28, No. 5, 2017, pp. 985-994) can detect a variety of hormones, the pretreatment uses liquid-liquid extraction (LLE) or solid-phase extraction (SPE), which takes 40-60 minutes, and the mobile phase system (such as methanol-water) has poor separation effect on polar hormones such as aldosterone (retention time > 20 minutes), resulting in low detection efficiency.

[0004] Problems with existing technologies include: (1) Low detection efficiency. CLIA requires separate testing for each hormone, and it takes 3-4 hours to complete the six indicators. The traditional mass spectrometry pre-processing steps are cumbersome and cannot meet the rapid detection needs of emergency or large-scale screening. (2) Insufficient separation and sensitivity. The polarity difference between aldosterone (strong polarity, logP = -0.5) and cortisol (weak polarity, logP = 1.8) is large, and traditional C18 columns are difficult to achieve efficient separation at the same time, and peak tailing or overlap often occurs. The serum concentrations of hormones such as 18-hydroxycortisol and 18-hydroxycorticosterone are low (normal range 5-50 ng / mL). The recovery rate of existing methods is only 70%-80%, which is prone to false negative results. (3) Clinical typing relies on experience. There is a lack of a combined detection scheme for the six hormones. Clinicians need to manually integrate different test results and calculate ratios (such as aldosterone / cortisol), which increases human errors and diagnostic time.

[0005] Therefore, there is an urgent need to develop a method that can simultaneously, quickly and accurately detect six hypertension-related hormones. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a method and application for the simultaneous and precise detection of six hypertension-related hormones. This method addresses the existing challenges of low detection efficiency, poor separation, and complex pre-processing, providing a reliable basis for the early diagnosis and classification of secondary hypertension (such as primary aldosteronism and congenital adrenal hyperplasia).

[0007] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a method for simultaneously and accurately detecting six hypertension-related hormones, the method comprising:

[0009] (S1) Vortex mixing the sample to be tested and the extract, centrifuging to collect the supernatant, filtering the membrane to obtain a test solution; the extract is a methanol-ethyl formate mixed solution with a volume ratio of (7.5-8.5):(1.5-2.5), and a pH value of 8-10; the volume ratio can be, for example, 7.5:2.5, 8:2, or 7.5:1.5, and the pH value can be, for example, 8, 9, or 10;

[0010] (S2) using liquid chromatography tandem mass spectrometry to detect the test solution and the standard solution, and using an internal standard method to correct for matrix effects; the mobile phase used in the liquid chromatography includes a mobile phase A and a mobile phase B, the mobile phase A is 10-12 mmol / L (for example, 10 mmol / L, 11 mmol / L, or 12 mmol / L, etc.) ammonium acetate-0.1-0.2% (for example, 0.1%, 0.15%, or 0.2%, etc.) formic acid aqueous solution, and the mobile phase B is 0.1-0.2% (for example, 0.1%, 0.15%, or 0.2%, etc.) formic acid-acetonitrile;

[0011] (S3) Draw a standard curve with the peak area ratio of the target substance to the internal standard as the ordinate and the concentration as the abscissa; calculate the concentration of the target substance in the test solution.

[0012] In the present invention, methanol-ethyl formate extraction was used to rapidly precipitate proteins and extract polar and fat-soluble hormones. Combined with gradient elution, aldosterone (strong polarity) and other glucocorticoids (weak polarity) with significant differences in polarity were separated. Tandem mass spectrometry was used in MRM mode to perform qualitative and quantitative analysis of the six hormones.

[0013] In the present invention, methanol-ethyl formate (7.5-8.5):(1.5-2.5) is used as the extraction solution. The polar group (ester group) of ethyl formate can specifically enhance the solubility of highly polar hormones such as aldosterone, thereby increasing the aldosterone recovery rate from 80% in traditional methanol extraction to more than 95%, while reducing the interference of fat-soluble impurities.

[0014] The aldosterone / renin activity ratio (ARR) is a core indicator for screening and diagnosing primary aldosteronism (PA, an important type of secondary hypertension); if the ARR exceeds the laboratory threshold and the aldosterone level is >15ng / dL, the possibility of PA should be considered. Clinically, hypertension is classified into the following types in combination with other diagnoses: aldosteroneoma (APA); idiopathic aldosteronism (IHA); high-renin hypertension; low-renin essential hypertension. The detection method of the present invention has high sensitivity and high accuracy, and can simultaneously detect the content of 6 hormones in the sample to be tested. Accurate typing can be performed when combined with aldosterone / renin activity ratio analysis.

[0015] In the present invention, in step (S1), the vortex mixing time is 1 to 2 minutes, for example, 1 minute, 1.5 minutes or 2 minutes.

[0016] In the present invention, in step (S1), the centrifugation condition is 12000-13000 rpm (for example, 12000 rpm, 12500 rpm or 13000 rpm, etc.) and centrifugation for 8-10 min (for example, 8 min, 9 min or 10 min, etc.).

[0017] In the present invention, in step (S1), the pore size of the filter membrane is 0.22 to 0.45 μm, for example, 0.22 μm or 0.45 μm.

[0018] Preferably, in step (S2), the chromatographic column used in the liquid chromatography is a C18 column bonded toluene phase, 2.1×100 mm, 1.8 μm, pore size

[0019] The present invention adopts a C18 column bonded with a toluene phase, combined with a specific elution procedure, to achieve the purpose of separating various hormones in a short time.

[0020] Preferably, in step (S2), the elution method adopted by the liquid chromatography is gradient elution, and the procedure of the gradient elution is:

[0021] From minute 0 to minute 2, the volume fraction of mobile phase B changes uniformly from 10-11% to 15-16%, and the rest is mobile phase A;

[0022] From minute 2 to minute 2.5, the volume fraction of mobile phase B changes uniformly from 15-16% to 20-21%, with the remainder being mobile phase A;

[0023] From 2.5 to 3.0 minutes, the volume fraction of mobile phase B changes uniformly from 20 to 21% to 30 to 31%, and the rest is mobile phase A;

[0024] From 3.0 to 3.5 minutes, the volume fraction of mobile phase B changes uniformly from 30-31% to 50-51%, and the rest is mobile phase A;

[0025] From 3.5 to 4.0 minutes, the volume fraction of mobile phase B changes uniformly from 50 to 51% to 70 to 71%, with the remainder being mobile phase A;

[0026] At 4.0-4.5 minutes, the volume fraction of mobile phase B changes uniformly from 70-71% to 90-91%, with the remainder being mobile phase A;

[0027] From 4.5 to 4.7 minutes, the volume fraction of mobile phase B changes uniformly from 90 to 91% to 10 to 11%, with the remainder being mobile phase A;

[0028] From minute 4.7 to minute 5, the volume fraction of mobile phase B is 10-11%, and the rest is mobile phase A.

[0029] The above-mentioned volume fractions of 10-11% can be, for example, 10%, 10.5% or 11%, the volume fractions of 15-16% can be, for example, 15%, 15.5% or 16%, the volume fractions of 20-21% can be, for example, 20%, 20.5% or 21%, the volume fractions of 30-31% can be, for example, 30%, 30.5% or 31%, the volume fractions of 50-51% can be, for example, 50%, 50.5% or 51%, the volume fractions of 70-71% can be, for example, 70%, 70.5% or 71%, the volume fractions of 90-91% can be, for example, 90%, 90.5% or 91%, etc.

[0030] In the present invention, a dual-gradient program of "strong polarity priority elution + weak polarity gradient enrichment" was designed to target the polarity differences between aldosterone (retention time 3.25 min) and cortisol (retention time 3.4 min), achieving baseline separation of all components within 5 minutes, saving 95% of detection time compared to traditional methods (more than 30 min).

[0031] The mobile phase system was optimized: (1) Mobile phase composition: A combination of mobile phase A (10-12 mmol / L ammonium acetate-0.1-0.2% formic acid aqueous solution) and mobile phase B (0.1-0.2% formic acid-acetonitrile) was used. Ammonium acetate can adjust the ionic strength, formic acid improves the peak shape, and acetonitrile, as a strong eluent, can accurately control the retention time of hormones of different polarity. (2) Polarity matching strategy: In view of the polarity difference between aldosterone (strong polarity, logP = -0.5) and cortisol (weak polarity, logP = 1.8), the mobile phase polarity gradient was changed to achieve preferential elution of highly polar substances and gradient enrichment of weakly polar substances.

[0032] Refined design of gradient elution program: (1) Segmented gradient elution: The elution process is divided into 8 time periods. The volume fraction of mobile phase B is precisely controlled in each time period, such as increasing from 10% to 15% from 0 to 2 minutes, gradually increasing the proportion of organic phase to ensure that hormones of different polarity are eluted in sequence. (2) Optimization of elution gradient slope: In the elution intervals of aldosterone (retention time 3.25min) and cortisol (retention time 3.4min), a "slow rise-fast rise-slow fall" gradient change is adopted to avoid peak overlap. For example, in 2.5 to 3.0 minutes, mobile phase B is increased from 20% to 30%, which accelerates the elution of weak polar hormones and shortens the overall detection time.

[0033] Collaborative optimization of chromatographic column and elution conditions: (1) Column selection: A C18 column (2.1×100 mm, 1.8 μm) with a bonded toluene phase was used. Its hydrophobicity and π-π interaction can enhance the retention of weakly polar hormones. Combined with gradient elution, it can improve the separation of hormones with significant polarity differences. (2) Flow rate and column temperature control: The flow rate was set at 0.3-0.4 mL / min and the column temperature was set at 40-42°C to optimize mass transfer efficiency, reduce peak broadening, and ensure that the baseline separation of the six hormones was completed within 5 minutes.

[0034] The detection method of this invention significantly improves detection efficiency: The traditional method requires more than 30 minutes to separate six hormones, but the optimized method only takes 4-5 minutes, increasing detection efficiency by 95%, making it suitable for clinical high-throughput screening and emergency testing. Single-injection multi-component analysis avoids the tedious multiple injections required by traditional CLIA methods (a single test takes 30-60 minutes), reducing sample consumption and human error.

[0035] The detection method of the present invention significantly improves resolution and peak shape: Baseline separation: The six hormones are completely separated in the MRM chromatogram, with clear retention time intervals (e.g., 3.16 minutes for aldosterone and 3.41 minutes for cortisol), symmetrical peak shapes with no tailing, and improved quantitative accuracy. Efficient separation of substances with different polarity: Hormones such as highly polar aldosterone and less polar cortisol are separated within 5 minutes, resolving the peak overlap problem caused by the large polarity differences in traditional C18 columns.

[0036] The detection method of the present invention improves sensitivity and precision: Sensitivity is improved: the minimum detection limit (LOD) for aldosterone reaches 5 pg / mL, which is better than the 20 pg / mL of traditional CLIA. It can detect mild hormone disorders (such as patients with 18-hydroxycortisol <50 ng / mL), and the detection rate is 40% higher than CLIA. The recovery rate and precision are excellent: the serum spike recovery rate is 92%-105% (95% ± 2% for aldosterone), the intra-assay coefficient of variation (CV) is <3.5%, and the inter-assay CV is <5%, meeting clinical diagnostic requirements.

[0037] The detection method of the present invention has important application value in clinical classification: Ratio analysis accuracy: It can directly calculate the aldosterone / cortisol (A / C) ratio. In patients with primary aldosteronism, this ratio is >30 (healthy individuals <15), with a diagnostic sensitivity of 93%, a 17% increase over the CLIA standard of 76%, reducing missed diagnoses and misdiagnoses. Multi-indicator combined diagnosis: The simultaneous detection of six hormones provides more classification parameters (such as the 11-deoxycortisol / 21-deoxycortisol ratio) for the identification of secondary hypertension such as primary aldosteronism and Cushing's syndrome.

[0038] In the present invention, the elution condition parameters were investigated, and the results showed that the detection effect deteriorated after the elution conditions of the mobile phase were changed.

[0039] 1. Impact of mobile phase ratio adjustment

[0040] The initial proportion of mobile phase B is too high: For example, starting with 20% mobile phase B in the 0-2 minute range will shorten the retention time of highly polar hormones (aldosterone, angiotensin), lead to co-elution with matrix impurities, tailing of the peak, and increase in quantitative error.

[0041] The gradient rise rate is too slow: for example, the time from 30% to 50% of mobile phase B in 3.0-3.5 minutes is extended to 5 minutes, the elution time of weak polar hormones (cortisol, 18-hydroxycortisol) is prolonged, the overall detection cycle is increased, and a long period of low organic phase may lead to column contamination.

[0042] 2. Impact of column parameter replacement

[0043] Replacing with a standard C18 column: C18 columns bonded with a toluene phase have a stronger retention capacity for weakly polar hormones. However, replacing with a standard C18 column shortens the retention time of substances like cortisol, causing peak overlap with aldosterone, resulting in reduced resolution. Changing column length or particle size: Using a 150 mm column or 5 μm particle size packing increases column pressure, prolonging elution time to over 10 minutes, reducing column efficiency, and increasing the limit of detection (e.g., the LOD for aldosterone increases to 15 pg / mL).

[0044] 3. Effect of flow rate and column temperature adjustment

[0045] The flow rate is too high (>0.4mL / min): the column pressure exceeds the tolerance range of the instrument (such as more than 400bar), and the residence time of the mobile phase in the column is shortened, the separation is reduced, and the peaks of aldosterone and angiotensin I overlap (retention time difference <0.1min).

[0046] Column temperature is too low (<40°C): The viscosity of the mobile phase increases, the mass transfer efficiency decreases, the peak broadening is obvious (for example, the peak width of cortisol increases from 0.2min to 0.5min), and the quantitative repeatability deteriorates (CV>10%).

[0047] 4. Impact of changes in gradient elution program

[0048] Omitting the gradient recovery step (e.g., not decreasing to 10% at 4.5-4.7 minutes): A high proportion of acetonitrile remains in mobile phase B, and the retention time of highly polar hormones shifts during the next injection (e.g., the retention time of aldosterone is extended from 3.16 min to 3.5 min), affecting batch-to-batch reproducibility.

[0049] Combined elution stage: For example, combining the 3.0-4.5 minute gradient into a single step up to 90% will cause the co-elution of the medium polar hormone (18-hydroxycorticosterone) and the weak polar hormone, with a peak overlap rate >50%, making accurate quantification impossible.

[0050] Preferably, in step (S2), the standard solution is a mixed solution of 6 hormone standards, including angiotensin II, angiotensin I, aldosterone, 18-hydroxycortisol, 18-hydroxycorticosterone and cortisol.

[0051] Preferably, the preparation method of the standard solution is: dissolving the 6 hormone standards in methanol to prepare single-standard stock solutions, mixing the single-standard stock solutions, and diluting them with 50-55% methanol aqueous solution to obtain a mixed solution of the 6 hormone standards.

[0052] Preferably, the concentration of angiotensin II in the mixed solution is 20-300 pg / mL, the concentration of angiotensin I is 20-300 pg / mL, the concentration of aldosterone is 10-1000 pg / mL, the concentration of 18-hydroxycortisol is 30-300 ng / mL, the concentration of 18-hydroxycorticosterone is 10-100 ng / mL, and the concentration of cortisol is 50-500 ng / mL.

[0053] In the present invention, the concentration of angiotensin II is 20 to 300 pg / mL, for example, 20 pg / mL, 40 pg / mL, 80 pg / mL, 160 pg / mL, or 300 pg / mL.

[0054] The concentration of angiotensin I is 20 to 300 pg / mL, for example, 20 pg / mL, 40 pg / mL, 80 pg / mL, 160 pg / mL, or 300 pg / mL.

[0055] The concentration of aldosterone is 10 to 1000 pg / mL, for example, 10 pg / mL, 20 pg / mL, 40 pg / mL, 160 pg / mL, 320 pg / mL, 640 pg / mL, or 1000 pg / mL.

[0056] The concentration of 18-hydroxycortisol is 30 to 300 ng / mL, for example, 30 ng / mL, 60 ng / mL, 120 ng / mL, 240 ng / mL, or 300 ng / mL.

[0057] The concentration of 18-hydroxycorticosterone is 10 to 100 ng / mL, for example, 10 ng / mL, 20 ng / mL, 40 ng / mL, 80 ng / mL, or 100 ng / mL.

[0058] The concentration of cortisol is 50 to 500 ng / mL, for example, 50 ng / mL, 100 ng / mL, 200 ng / mL, 400 ng / mL, or 500 ng / mL.

[0059] Preferably, in step (S2), the internal standard is a deuterated cortisol solution of 500-550 ng / mL (for example, 500 ng / mL, 510 ng / mL, 520 ng / mL, 530 ng / mL, 540 ng / mL or 550 ng / mL, etc.), and the solvent is methanol.

[0060] In the present invention, the internal standard method is used to correct the matrix effect to ensure the detection accuracy in a complex serum matrix.

[0061] Preferably, in step (S2), the flow rate of the liquid chromatography is 0.3 to 0.4 mL / min, for example, 0.3 mL / min, 0.35 mL / min or 0.4 mL / min.

[0062] Preferably, in step (S2), the column temperature of the liquid chromatography is 40-42°C, for example, 40°C, 41°C or 42°C.

[0063] Preferably, in step (S2), the injection volume of the liquid chromatography is 5 to 6 μL, for example, 5 μL, 5.5 μL or 6 μL.

[0064] Preferably, in step (S2), the mass spectrometry parameters include: ion source: electrospray positive ion mode ESI+; capillary voltage: 3.5~4.1KV (for example, it can be 3.5KV, 3.8KV or 4.1KV, etc.); cone voltage: 25~45V (for example, it can be 25V, 35V or 45V, etc.); collision energy: 12~25eV (for example, it can be 12eV, 15eV, 20eV or 25eV, etc.); detection method: multiple reaction monitoring.

[0065] In the present invention, the electrospray positive ionization mode (ESI+) is used, which is suitable for the positive ionization detection of 6 hormones.

[0066] In the present invention, the capillary voltage is set to 3.5-4.1 kV, which optimizes the ionization efficiency of polar hormones such as aldosterone, and the ion strength is increased by 50% compared with the traditional method.

[0067] In the present invention, the cone voltage is set to 25-45V to enhance the polar hormone desolvation effect (aldosterone desolvation efficiency 95%), while controlling the ion fragmentation rate within 12%.

[0068] In the present invention, the collision energy is set to 12-25 eV, which is optimized according to the specificity of each component, such as the collision energy of aldosterone is 18 eV, and the collision energy of cortisol is 15 eV.

[0069] In the present invention, the monitoring ion pairs of each component are shown in Table 1 below:

[0070] Table 1

[0071] Hormone name Precursor ion (m / z) Product ion (m / z) Aldosterone 361.3 199.2 corticosterone 347.3 121.1 cortisol 363.3 121.1 cortisone 361.3 121.1 11-Deoxycortisol 347.3 121.1 21-Deoxycortisol 345.3 109.1

[0072] The present invention establishes a combined detection method for six hormones. By optimizing mass spectrometry parameters (such as the aldosterone-specific ion pair m / z 361.3, 199.2), key typing parameters such as the aldosterone / cortisol ratio and the 11-deoxycortisol / 21-deoxycortisol ratio are simultaneously obtained. This method has application prospects in the differentiation of primary aldosteronism and Cushing's syndrome.

[0073] The advantages of the method described in the present invention include: 1. After collision energy optimization, the signal-to-noise ratio of the exclusive ion pair of aldosterone (m / z 361.3→199.2) increased by 108%, indicating that the sensitivity and specificity of mass spectrometry detection were significantly improved, verifying the effectiveness of "mass spectrometry parameter optimization." 2. By jointly detecting six hormones and calculating the ratio, the method of the present invention increased the sensitivity by 17% compared with CLIA. 3. The linear correlation coefficient R of the standard curve of the six hormones was 2 ≥0.998 (such as aldosterone R 2 =0.999378), demonstrating that the detection method can accurately quantify and provide a reliable data basis for ratio calculation. 4. The recovery rate of aldosterone was 93%-98%, and the recovery rate of other hormones was 90%-99%, meeting clinical testing requirements. The intra-batch coefficient of variation (CV) was <3.5%, and the inter-batch CV was <5%, ensuring the repeatability of typing parameter calculation. In summary, the above data have been improved in terms of the optimization effect of mass spectrometry parameters, the clinical diagnostic sensitivity of typing parameters, and the accuracy of the detection method. The detection method of the present invention has application prospects in the differentiation of primary aldosteronism and Cushing's syndrome.

[0074] In a second aspect, the present invention provides an extract for extracting six hypertension-related hormones in liquid chromatography tandem mass spectrometry, wherein the extract is a methanol-ethyl formate mixed solution with a volume ratio of (7.5-8.5): (1.5-2.5) and a pH value of 8-10.

[0075] The volume ratio of methanol to ethyl formate in the extract can be, for example, 7.5:2.5, 8:2 or 7.5:1.5, and the pH value can be, for example, 8, 9 or 10.

[0076] In a third aspect, the present invention provides a six-hormone detection kit for hypertension typing diagnosis, the kit comprising: the extract for extracting six hypertension-related hormones by liquid chromatography tandem mass spectrometry as described in the second aspect, as well as the mobile phase, calibration solution and internal standard solution for liquid chromatography tandem mass spectrometry detection.

[0077] Preferably, the mobile phase includes mobile phase A and mobile phase B, the mobile phase A is 10-12 mmol / L (for example, 10 mmol / L, 11 mmol / L or 12 mmol / L, etc.) ammonium acetate-0.1-0.2% (for example, 0.1%, 0.15% or 0.2%, etc.) formic acid aqueous solution, and the mobile phase B is 0.1-0.2% (for example, 0.1%, 0.15% or 0.2%, etc.) formic acid-acetonitrile.

[0078] Preferably, the standard solution is a mixed solution of six hormone standards, including angiotensin II, angiotensin I, aldosterone, 18-hydroxycortisol, 18-hydroxycorticosterone, and cortisol, and the solvent is 50-55% methanol aqueous solution.

[0079] Preferably, the concentration of angiotensin II in the mixed solution is 20-300 pg / mL, the concentration of angiotensin I is 20-300 pg / mL, the concentration of aldosterone is 10-1000 pg / mL, the concentration of 18-hydroxycortisol is 30-300 ng / mL, the concentration of 18-hydroxycorticosterone is 10-100 ng / mL, and the concentration of cortisol is 50-500 ng / mL.

[0080] Preferably, the internal standard is a deuterated cortisol solution of 500-550 ng / mL (eg, 500 ng / mL, 510 ng / mL, 520 ng / mL, 530 ng / mL, 540 ng / mL, or 550 ng / mL), and the solvent is methanol.

[0081] In a fourth aspect, the present invention provides the application of the detection method for simultaneously and accurately detecting six hypertension-related hormones described in the first aspect in non-disease diagnosis or treatment.

[0082] In the fifth aspect, the present invention provides the use of the extract for extracting 6 hypertension-related hormones by liquid chromatography tandem mass spectrometry as described in the second aspect or the 6-hormone detection kit for hypertension typing diagnosis as described in the third aspect 9 in the preparation of early diagnosis and typing products for secondary hypertension.

[0083] In a sixth aspect, the present invention provides a device for simultaneously and accurately detecting six hypertension-related hormones, the device comprising:

[0084] (1) a sample preparation unit, for vortex mixing the sample to be tested and the extract, collecting the supernatant by centrifugation, filtering the membrane, and obtaining a test solution; the extract is a methanol-ethyl formate mixed solution with a volume ratio of (7.5-8.5):(1.5-2.5), and a pH value of 8-10; the volume ratio can be, for example, 7.5:2.5, 8:2, or 7.5:1.5, and the pH value can be, for example, 8, 9, or 10;

[0085] (2) a detection unit for performing liquid chromatography tandem mass spectrometry detection, wherein the test solution and the standard solution are detected by liquid chromatography tandem mass spectrometry analysis, and the matrix effect is corrected by an internal standard method; the mobile phase used in the liquid chromatography includes a mobile phase A and a mobile phase B, wherein the mobile phase A is 10 to 12 mmol / L (for example, 10 mmol / L, 11 mmol / L, or 12 mmol / L, etc.) ammonium acetate-0.1 to 0.2% (for example, 0.1%, 0.15%, or 0.2%, etc.) formic acid aqueous solution, and the mobile phase B is 0.1 to 0.2% (for example, 0.1%, 0.15%, or 0.2%, etc.) formic acid-acetonitrile;

[0086] (3) Data analysis unit, used for analysis and calculation of test results, drawing a standard curve with the peak area ratio of the target substance to the internal standard as the ordinate and the concentration as the abscissa; calculating the concentration of the target substance in the test solution.

[0087] Preferably, in the detection unit, the chromatographic column used in the liquid chromatography is a C18 column bonded toluene phase, 2.1×100 mm, 1.8 μm, pore size

[0088] Preferably, in the detection unit, the elution mode adopted by the liquid chromatography is gradient elution, and the procedure of the gradient elution is:

[0089] From minute 0 to minute 2, the volume fraction of mobile phase B changes uniformly from 10-11% to 15-16%, and the rest is mobile phase A;

[0090] From minute 2 to minute 2.5, the volume fraction of mobile phase B changes uniformly from 15-16% to 20-21%, with the remainder being mobile phase A;

[0091] From 2.5 to 3.0 minutes, the volume fraction of mobile phase B changes uniformly from 20 to 21% to 30 to 31%, and the rest is mobile phase A;

[0092] From 3.0 to 3.5 minutes, the volume fraction of mobile phase B changes uniformly from 30-31% to 50-51%, and the rest is mobile phase A;

[0093] From 3.5 to 4.0 minutes, the volume fraction of mobile phase B changes uniformly from 50 to 51% to 70 to 71%, with the remainder being mobile phase A;

[0094] At 4.0-4.5 minutes, the volume fraction of mobile phase B changes uniformly from 70-71% to 90-91%, with the remainder being mobile phase A;

[0095] From 4.5 to 4.7 minutes, the volume fraction of mobile phase B changes uniformly from 90 to 91% to 10 to 11%, with the remainder being mobile phase A;

[0096] From minute 4.7 to minute 5, the volume fraction of mobile phase B is 10-11%, and the rest is mobile phase A.

[0097] Preferably, in the detection unit, the standard solution is a mixed solution of 6 hormone standards, including angiotensin II, angiotensin I, aldosterone, 18-hydroxycortisol, 18-hydroxycorticosterone and cortisol.

[0098] Preferably, the preparation method of the standard solution is: dissolving the 6 hormone standards in methanol to prepare single-standard stock solutions, mixing the single-standard stock solutions, and diluting them with 50-55% methanol aqueous solution to obtain a mixed solution of the 6 hormone standards.

[0099] Preferably, the concentration of angiotensin II in the mixed solution is 20-300 pg / mL, the concentration of angiotensin I is 20-300 pg / mL, the concentration of aldosterone is 10-1000 pg / mL, the concentration of 18-hydroxycortisol is 30-300 ng / mL, the concentration of 18-hydroxycorticosterone is 10-100 ng / mL, and the concentration of cortisol is 50-500 ng / mL.

[0100] Preferably, in the detection unit, the internal standard is a deuterated cortisol solution of 500-550 ng / mL (for example, 500 ng / mL, 510 ng / mL, 520 ng / mL, 530 ng / mL, 540 ng / mL or 550 ng / mL, etc.), and the solvent is methanol.

[0101] Preferably, in the detection unit, the flow rate of the liquid chromatography is 0.3-0.4 mL / min, for example, 0.3 mL / min, 0.35 mL / min or 0.4 mL / min.

[0102] Preferably, in the detection unit, the column temperature of the liquid chromatography is 40-42°C, for example, 40°C, 41°C or 42°C.

[0103] Preferably, in the detection unit, the injection volume of the liquid chromatography is 5 to 6 μL, for example, 5 μL, 5.5 μL or 6 μL.

[0104] Preferably, in the detection unit, the mass spectrometry parameters include: ion source: electrospray positive ion mode ESI+; capillary voltage: 3.5~4.1KV (for example, it can be 3.5KV, 3.8KV or 4.1KV, etc.); cone voltage: 25~45V (for example, it can be 25V, 35V or 45V, etc.); collision energy: 12~25eV (for example, it can be 12eV, 15eV, 20eV or 25eV, etc.); detection method: multiple reaction monitoring.

[0105] The numerical range described in the present invention includes not only the point values ​​listed above, but also any point values ​​between the above numerical ranges that are not listed. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values ​​included in the range.

[0106] Compared with the prior art, the present invention has the following beneficial effects:

[0107] (1) Detection efficiency is greatly improved: 6 hormone tests can be completed within 4 to 5 minutes with a single injection, which is 80 times more efficient than CLIA (6 tests, 4 hours) and saves 95% of time compared to traditional mass spectrometry (35 minutes), making it suitable for clinical high-throughput screening.

[0108] (2) Breakthrough in sensitivity and accuracy: The minimum detection limit (LOD) is 5 pg / mL for aldosterone and 2 ng / mL for 21-deoxycortisol, both of which are superior to the existing CLIA method (LOD is 20 pg / mL and 10 ng / mL, respectively). Recovery rate: The recovery rate of serum spiked samples is 92%-105% (95%±2% for aldosterone and 93%±3% for 11-deoxycortisol), which is significantly higher than the traditional method (75%-85%).

[0109] (3) Excellent precision: intra-batch coefficient of variation (CV) <3.5%, inter-batch CV <5%, meeting the strict requirements for repeatability in clinical diagnosis (industry standard CV <10%).

[0110] (4) Clinical classification value: The aldosterone / cortisol (A / C) ratio can be directly calculated. In patients with primary aldosterone syndrome, this ratio is greater than 30 (healthy people <15), which increases the accuracy of single indicator detection (75%) to 92%, reducing clinical missed diagnosis and misdiagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0111] Figure 1 The following are the MRM chromatograms of 6 hormone standards.

[0112] Figure 2 is the standard curve of AngII (angiotensin II).

[0113] Figure 3 is the standard curve of AngI (angiotensin I).

[0114] Figure 4 is the Ald (aldosterone) standard curve.

[0115] Figure 5 The standard curve of 18-hydroxycortisol.

[0116] Figure 6 is the standard curve of 18-hydroxycorticosterone.

[0117] Figure 7The standard curve of cortisol. DETAILED DESCRIPTION

[0118] The technical solution of the present invention is further described below by way of specific embodiments. It should be understood by those skilled in the art that the embodiments are merely to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0119] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0120] Example 1

[0121] This embodiment provides an extract for extracting six hypertension-related hormones by liquid chromatography-tandem mass spectrometry. The extract is a methanol-ethyl formate mixed solution with a volume ratio of 8.5:1.5 and a pH value of 8.

[0122] Example 2

[0123] This embodiment provides an extract for extracting six hypertension-related hormones by liquid chromatography-tandem mass spectrometry. The extract is a methanol-ethyl formate mixed solution with a volume ratio of 7.5:2.5 and a pH value of 9.

[0124] Example 3

[0125] This embodiment provides an extract for extracting six hypertension-related hormones by liquid chromatography-tandem mass spectrometry. The extract is a methanol-ethyl formate mixed solution with a volume ratio of 8.0:2.0 and a pH value of 10.

[0126] Comparative Example 1

[0127] This comparative example provides an extract for extracting six hypertension-related hormones by liquid chromatography tandem mass spectrometry. The extract is a methanol-ethyl formate mixed solution with a volume ratio of 6.5:3.5 and a pH value of 10.

[0128] Comparative Example 2

[0129] This comparative example provides an extract for extracting six hypertension-related hormones by liquid chromatography tandem mass spectrometry. The extract is a methanol-ethyl formate mixed solution with a volume ratio of 9:1 and a pH value of 10.

[0130] Comparative Example 3

[0131] This comparative example provides an extracting solution for extracting 6 hypertension-related hormones by liquid chromatography tandem mass spectrometry, wherein the extracting solution is methanol and has a pH value of 10.

[0132] Comparative Example 4

[0133] This comparative example provides an extracting solution for extracting 6 hypertension-related hormones by liquid chromatography tandem mass spectrometry, wherein the extracting solution is n-hexane and has a pH value of 10.

[0134] Example 4

[0135] This example investigates the extraction effects of the extracts in Examples 1-3 and Comparative Examples 1-4.

[0136] 1. Sample pretreatment

[0137] The extracts of Examples 1-3 and Comparative Examples 1-4 were respectively used to extract the serum to be tested, and the extraction steps included:

[0138] Take 50 μL of serum to be tested, add 150 μL of extract, vortex for 1 min, centrifuge at 13000 rpm for 10 min, take the supernatant and pass it through a 0.22 μm filter membrane to obtain the test solution.

[0139] 2. Preparation of standard solution

[0140] The standard solution is a mixed solution of six hormone standards, including angiotensin II, angiotensin I, aldosterone, 18-hydroxycortisol, 18-hydroxycorticosterone, and cortisol, and the solvent is a 50-55% methanol aqueous solution; the concentration of angiotensin II in the mixed solution is 20-300 pg / mL, the concentration of angiotensin I is 20-300 pg / mL, the concentration of aldosterone is 10-1000 pg / mL, the concentration of 18-hydroxycortisol is 30-300 ng / mL, the concentration of 18-hydroxycorticosterone is 10-100 ng / mL, and the concentration of cortisol is 50-500 ng / mL.

[0141] The standard solution preparation method includes weighing 10 mg of each of the six hormone standards, dissolving them in methanol, and diluting the volume to 10 mL to prepare a 1 mg / mL single-standard stock solution. An appropriate amount of each stock solution is diluted with methanol-water (1:1) to prepare a mixed intermediate solution with a concentration of 1000 ng / mL (cortisol, etc.) or 1000 pg / mL (aldosterone). This is further diluted to prepare a series of calibrators (0.1-5000 ng / mL or 0.1-5000 pg / mL).

[0142] 3. Preparation of internal standard solution

[0143] The internal standard is a 500-550 ng / mL deuterated cortisol solution, and the solvent is methanol.

[0144] Preparation of internal standard solution: Weigh 10 mg of deuterated cortisol (d4-cortisol) and dilute to 100 mL with methanol to prepare a 100 μg / mL stock solution, which was diluted to 500 ng / mL before use.

[0145] 4. Liquid chromatography tandem mass spectrometry detection

[0146] The test and standard solutions were analyzed by liquid chromatography-tandem mass spectrometry, and matrix effects were corrected using an internal standard method. The internal standard was a 500 ng / mL deuterated cortisol solution in methanol.

[0147] The mobile phases used in the liquid chromatography include mobile phase A and mobile phase B, wherein the mobile phase A is 10-12 mmol / L ammonium acetate-0.1-0.2% formic acid aqueous solution, and the mobile phase B is 0.1% formic acid-acetonitrile; the chromatographic column used in the liquid chromatography is a C18 column bonded toluene phase, 2.1×100 mm, 1.8 μm, pore size

[0148] The flow rate of the liquid chromatography was 0.3 mL / min, the column temperature was 40° C., and the injection volume was 5 μL.

[0149] The elution mode adopted by the liquid chromatography is gradient elution, and the procedure of the gradient elution is:

[0150] From minute 0 to minute 2, the volume fraction of mobile phase B changes uniformly from 10-11% to 15-16%, and the rest is mobile phase A;

[0151] From minute 2 to minute 2.5, the volume fraction of mobile phase B changes uniformly from 15-16% to 20-21%, with the remainder being mobile phase A;

[0152] From 2.5 to 3.0 minutes, the volume fraction of mobile phase B changes uniformly from 20 to 21% to 30 to 31%, and the rest is mobile phase A;

[0153] From 3.0 to 3.5 minutes, the volume fraction of mobile phase B changes uniformly from 30-31% to 50-51%, and the rest is mobile phase A;

[0154] From 3.5 to 4.0 minutes into the 15th minute, the volume fraction of mobile phase B changed from 50 to 51% to 70 to 71% at a constant rate, with the remainder being mobile phase A;

[0155] At 4.0-4.5 minutes, the volume fraction of mobile phase B changes uniformly from 70-71% to 90-91%, with the remainder being mobile phase A;

[0156] At 4.5-4.7 minutes in the 22nd minute, the volume fraction of mobile phase B changed from 90-91% to 10-11% at a constant rate, and the rest was mobile phase A;

[0157] From minute 4.7 to minute 5, the volume fraction of mobile phase B is 10-11%, and the rest is mobile phase A.

[0158] The mass spectrometry parameters include:

[0159] Ion source: electrospray positive ionization mode (ESI+).

[0160] Capillary voltage: 3.8kV.

[0161] Cone voltage: 35V.

[0162] Collision energy: 12-25 eV.

[0163] Detection method: Multiple reaction monitoring (MRM), the monitoring ion pairs of each component are shown in Table 1.

[0164] 5. Standard curve drawing

[0165] Calibrators were prepared using blank serum matrix, and the standard curve was drawn with the peak area ratio of the target substance to the internal standard as the ordinate and the concentration as the abscissa. The linear correlation coefficient R2 was ≥0.998.

[0166] The detection results of the samples extracted with the extract in Example 3 are as follows:

[0167] Figure 1 The following is the MRM chromatogram of six hormone standards. The horizontal axis represents retention time (min), and the vertical axis represents ion intensity (cps). The figure shows that AngII (angiotensin II) (3.07 min), AngI (angiotensin I) (3.22 min), Ald (aldosterone) (3.16 min), 18-hydroxycortisol (3.32 min), 18-hydroxycorticosterone (3.37 min), and cortisol (3.41 min) are completely separated within 4 minutes, with symmetrical peaks and no tailing.

[0168] Figure 2 The standard curve for AngII (angiotensin II) is shown in Figure 1. The horizontal axis represents the concentration of AngII (angiotensin II) (pg / mL), and the vertical axis represents the peak area ratio of the target compound to the internal standard. The linear equation is y = 1.56863x + 0.0131564, R 2 =0.999219, showing a good linear relationship in the range of 20-300 pg / mL.

[0169] Figure 3 The standard curve of AngI (angiotensin I) is plotted on the horizontal axis for the concentration of AngI (pg / mL) and on the vertical axis for the peak area ratio of the target compound to the internal standard. The linear equation is y = 0.0534247x + -0.00401352, R 2=0.999816, showing a good linear relationship in the range of 20-300 pg / mL.

[0170] Figure 4 This is the Ald (aldosterone) standard curve. The abscissa is aldosterone concentration (pg / mL), and the ordinate is the peak area ratio of the target compound to the internal standard. The linear equation is y = 0.511156x + -0.000163064, with R² = 0.999378, indicating good linearity in the range of 10-1000 pg / mL.

[0171] Figure 5 The standard curve for 18-hydroxycortisol is shown in Figure 2. The horizontal axis represents the concentration of 18-hydroxycortisol (pg / mL), and the vertical axis represents the peak area ratio of the target compound to the internal standard. The linear equation is y = 0.288293x + 0.000676816, R 2 =0.999521, showing a good linear relationship in the range of 30-300 ng / mL.

[0172] Figure 6 The standard curve of 18-hydroxycorticosterone is shown in Figure 1. The horizontal axis is the concentration of 18-hydroxycorticosterone (pg / mL), and the vertical axis is the peak area ratio of the target substance to the internal standard. The linear equation is y = 0.350522x + -0.0041892, R 2 =0.998201, showing a good linear relationship in the range of 10-100 ng / mL.

[0173] Figure 7 The standard curve for cortisol is shown in Figure 2. The horizontal axis represents the cortisol concentration (pg / mL) and the vertical axis represents the peak area ratio of the target compound to the internal standard. The linear equation is y = 0.00101435x + -0.000372547, R 2 =0.998709, showing a good linear relationship in the range of 50-500 ng / mL.

[0174] The test results of a certain sample showed: aldosterone 500pg / mL (normal <150pg / mL), cortisol 200ng / mL, A / C ratio 25 (>30 is the critical value for primary aldosteronism).

[0175] The effects of the extracts in Examples 1-3 and Comparative Examples 1-4: The extract in Example 3 is the best result (the mass spectrometry response of the 6 targets is the best, and the interference peak is weak and the background is clean). The effects of Examples 1 and 2 are slightly worse than those of Example 3, and good detection can also be achieved. In Comparative Example 1, the proportion of ethyl formate is high, and 18-hydroxycortisol and 18-hydroxycortisol cannot be completely separated. In Comparative Example 2, the proportion of ethyl formate is low, and 18-hydroxycortisol and 18-hydroxycortisol cannot be completely separated. Comparative Examples 3 and 4 are extracted with methanol and n-hexane, respectively, and 18-hydroxycortisol, 18-hydroxycortisol and cortisol cannot be completely separated.

[0176] Example 5

[0177] Recovery and precision experiments

[0178] 1. The detection method and detection conditions are the same as those in Example 4.

[0179] 2. Spike recovery rate

[0180] Low (10% upper limit of normal), medium (50% upper limit of normal), and high (150% upper limit of normal) concentrations of mixed standards were added to blank serum. The results are shown in Table 2 below.

[0181] Table 2

[0182] Hormone name Low concentration recovery rate (%) Recovery rate of medium concentration (%) High concentration recovery rate (%) Aldosterone 93±1 96±2 98±1 AngII (angiotensin II) 91±2 94±1 97±1 AngI (angiotensin I) 92±1 95±2 98±1 18-hydroxycortisol 90±2 93±1 96±2 18-hydroxycorticosterone 89±2 92±2 95±1 cortisol 94±1 97±1 99±1

[0183] From Table 2, we can see that the spiked recovery meets the requirements.

[0184] 3. Precision test

[0185] Intra-batch precision: The same quality control sample was injected 10 times continuously, and the CV was less than 3.5% (e.g., CV of cortisol = 2.8%).

[0186] Inter-batch precision: Three batches of kits were prepared on different days and tested on the same sample, and the CVs were all <5% (e.g., 18-hydroxycortisol CV = 4.2%).

[0187] Example 6

[0188] This embodiment provides a six-hormone detection kit for hypertension typing diagnosis, the kit comprising: an extract for extracting six hypertension-related hormones by liquid chromatography-tandem mass spectrometry, and a mobile phase, a calibrator solution, and an internal standard solution for liquid chromatography-tandem mass spectrometry detection.

[0189] The extract is a methanol-ethyl formate mixed solution with a volume ratio of 8:2 and a pH value of 10.

[0190] The mobile phase comprises mobile phase A and mobile phase B, wherein the mobile phase A is 10-12 mmol / L ammonium acetate-0.1-0.2% formic acid aqueous solution, and the mobile phase B is 0.1-0.2% formic acid-acetonitrile.

[0191] The standard solution is a mixed solution of six hormone standards, including angiotensin II, angiotensin I, aldosterone, 18-hydroxycortisol, 18-hydroxycorticosterone, and cortisol. The concentration of angiotensin II in the mixed solution is 20-300 pg / mL, the concentration of angiotensin I is 20-300 pg / mL, the concentration of aldosterone is 10-1000 pg / mL, the concentration of 18-hydroxycortisol is 30-300 ng / mL, the concentration of 18-hydroxycortisol is 10-100 ng / mL, and the concentration of cortisol is 50-500 ng / mL.

[0192] The internal standard is a 500-550 ng / mL deuterated cortisol solution, and the solvent is methanol.

[0193] Comparative experiment using the kit in this implementation and the chemiluminescent immunoassay (CLIA) method

[0194] The steps of the chemiluminescent immunoassay (CLIA) method include:

[0195] 1. Sample pretreatment

[0196] Collect serum samples from patients and centrifuge them (usually at 3000-4000 rpm for 10-15 minutes) to remove cellular components and impurities. If interfering substances (such as hemolysis or lipemia) are present in the sample, pretreatment (such as dilution or filtration) is required.

[0197] 2. Antibody-antigen reaction preparation

[0198] Specific antibodies (such as monoclonal antibodies against aldosterone and cortisol) are added to the reaction cup. The antibodies have been pre-labeled with a chemiluminescent substrate (such as luminol and acridinium ester).

[0199] When the serum sample to be tested is added, the antibody specifically binds to the target hormone (antigen) in the sample to form an "antibody-antigen" complex.

[0200] 3. Incubation and separation

[0201] Incubate at a specific temperature (such as 37°C) for 30-60 minutes to promote full binding of antibodies and antigens.

[0202] Add a separation agent (such as magnetic beads, solid phase carriers) to separate the "antibody-antigen" complex from the unbound free antigen, and wash to remove the free components.

[0203] 4. Chemiluminescence detection

[0204] Add a luminescent excitation solution (such as hydrogen peroxide, acid / base solution) to the reaction system to trigger the chemiluminescent substrate labeled on the antibody to emit light.

[0205] The luminescence intensity is measured by a luminometer, and is positively correlated with the concentration of the target hormone in the sample.

[0206] 5. Standard curve drawing and quantification

[0207] Use hormone standards of known concentration (e.g., aldosterone, cortisol) to create a standard curve, with luminescence intensity as the ordinate and concentration as the abscissa. Calculate the concentration of the target hormone from the standard curve based on the luminescence intensity of the sample.

[0208] 6. Multi-index step-by-step detection (for multiple hormones)

[0209] If six hormones need to be tested (such as aldosterone, cortisol, etc.), six independent tests are required, each test targeting one hormone.

[0210] Blood samples from 80 hypertensive patients (30 with primary aldosteronism and 50 with essential hypertension) were collected and the hormone levels of six hormones were detected using the kit of this example in combination with the method of Example 4 and CLIA.

[0211] Table 3 shows the comparison of diagnostic sensitivity of primary aldosteronism (n=30).

[0212] Table 3

[0213] Detection method Number of positive tests Sensitivity (%) Method of the present invention 28 / 30 93％ CLIA Methods 23 / 30 76％ difference +5 cases +17％

[0214] As shown in Table 3, among the 30 patients with primary aldosteronism, the sensitivity of the method of the present invention was significantly higher than that of CLIA (93% vs 76%), with an absolute difference of 17%.

[0215] Table 4 shows the comparison of detection rates in patients with mild hormone disorders (18-hydroxycortisol <50 ng / mL).

[0216] There were 25 patients with mild hormone disorders (based on the fact that some patients with primary aldosteronism met the requirements).

[0217] Table 4

[0218] Detection method Number of positive tests Detection rate (%) Method of the present invention 20 / 25 80％ CLIA Methods 10 / 25 40％ difference +10 cases +40％

[0219] As shown in Table 4, in mild patients with 11-deoxycortisol <10 ng / mL, the detection rate of the method of the present invention is 40% higher than that of CLIA (mild patients account for 83% of all primary aldosteronism patients, i.e., 25 / 30 cases).

[0220] Example 7

[0221] This example optimizes the mass spectrometry parameters.

[0222] 1. Specific collision energy (CE) optimization. The optimization results are shown in Table 5.

[0223] Table 5

[0224]

[0225]

[0226] As can be seen from Table 5, the optimized CE has obvious effects, and the S / N ratios are improved to varying degrees.

[0227] 2. Capillary voltage optimization: The capillary voltage value was investigated. The results showed that setting the capillary voltage to 3.5-4.1 kV can optimize the ionization efficiency of polar hormones such as aldosterone, and the ion strength is increased by 50% compared with the traditional method.

[0228] 3. Cone voltage optimization: The cone voltage value was investigated. The results showed that setting the cone voltage to 25-45V can enhance the desolvation effect of polar hormones (95% desolvation efficiency of aldosterone) while controlling the ion fragmentation rate within 12%.

[0229] Example 8

[0230] This example optimizes the elution procedure of liquid chromatography

[0231] The standard solution was separated and detected using the following elution procedures 1-3 and the elution conditions of Example 4, respectively. The remaining chromatographic conditions were the same as those of Example 4.

[0232] Elution program 1 (Table 6):

[0233] Time (min) Mobile phase A (%) Mobile phase B (%) 0 90 10 2 90 10 4.7 10 90 5 90 10

[0234] Elution program 2 (Table 7):

[0235]

[0236]

[0237] Elution program 3 (Table 8):

[0238] Time (min) Mobile phase A (%) Mobile phase B (%) 0 95 5 2 95 5 4.7 10 90 4.8 95 5 5 95 5

[0239] The above three elution procedures could not achieve complete separation of the six target compounds within 4 to 5 minutes. The results showed that the elution conditions in Example 4 were the best and could achieve complete separation of the six target compounds in a short time.

[0240] In summary, the present invention provides a method for simultaneously and accurately detecting six hypertension-related hormones. Compared with traditional mass spectrometry (35 minutes), it saves 95% of time and improves efficiency by 80 times, which has important application prospects in hormone detection.

[0241] The applicant declares that the above is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention fall within the scope of protection and disclosure of the present invention.

Claims

1. A method for simultaneously and accurately detecting six hypertension-related hormones, characterized in that: The detection method comprises: (S1) vortexing the sample to be tested and the extract, centrifuging and collecting the supernatant to obtain a test solution; the extract is a methanol-ethyl formate mixed solution with a volume ratio of (7.5-8.5): (1.5-2.5) and a pH value of 8-10; (S2) using liquid chromatography tandem mass spectrometry to detect the test solution and the standard solution, and using an internal standard method to correct for matrix effects; the mobile phase used in the liquid chromatography includes mobile phase A and mobile phase B, the mobile phase A is 10-12 mmol / L ammonium acetate-0.1-0.2% formic acid aqueous solution, and the mobile phase B is 0.1-0.2% formic acid-acetonitrile; (S3) Draw a standard curve with the peak area ratio of the target substance to the internal standard as the ordinate and the concentration as the abscissa; calculate the concentration of the target substance in the test solution.

2. The method for simultaneously and accurately detecting six hypertension-related hormones according to claim 1, characterized in that: In step (S2), the chromatographic column used in the liquid chromatography is a C18 column bonded toluene phase, 2.1×100 mm, 1.8 μm, pore size 3. The method for simultaneously and accurately detecting six hypertension-related hormones according to claim 1 or 2, characterized in that: In step (S2), the elution mode adopted by the liquid chromatography is gradient elution, and the procedure of the gradient elution is: From minute 0 to minute 2, the volume fraction of mobile phase B changes uniformly from 10-11% to 15-16%, and the rest is mobile phase A; From minute 2 to minute 2.5, the volume fraction of mobile phase B changes uniformly from 15-16% to 20-21%, with the remainder being mobile phase A; From 2.5 to 3.0 minutes, the volume fraction of mobile phase B changes uniformly from 20 to 21% to 30 to 31%, and the rest is mobile phase A; From 3.0 to 3.5 minutes, the volume fraction of mobile phase B changes uniformly from 30-31% to 50-51%, and the rest is mobile phase A; From 3.5 to 4.0 minutes, the volume fraction of mobile phase B changes uniformly from 50 to 51% to 70 to 71%, with the remainder being mobile phase A; At 4.0-4.5 minutes, the volume fraction of mobile phase B changes uniformly from 70-71% to 90-91%, with the remainder being mobile phase A; From 4.5 to 4.7 minutes, the volume fraction of mobile phase B changes uniformly from 90 to 91% to 10 to 11%, with the remainder being mobile phase A; From minute 4.7 to minute 5, the volume fraction of mobile phase B is 10-11%, and the rest is mobile phase A.

4. The method for simultaneously and accurately detecting six hypertension-related hormones according to any one of claims 1 to 3, characterized in that: In step (S2), the standard solution is a mixed solution of 6 hormone standards, including angiotensin II, angiotensin I, aldosterone, 18-hydroxycortisol, 18-hydroxycorticosterone and cortisol; Preferably, the preparation method of the standard solution is: dissolving the six hormone standards in methanol to prepare single-standard stock solutions, mixing the single-standard stock solutions, and diluting them with 50-55% methanol aqueous solution to obtain a mixed solution of the six hormone standards; Preferably, the concentration of angiotensin II in the mixed solution is 20-300 pg / mL, the concentration of angiotensin I is 20-300 pg / mL, the concentration of aldosterone is 10-1000 pg / mL, the concentration of 18-hydroxycortisol is 30-300 ng / mL, the concentration of 18-hydroxycorticosterone is 10-100 ng / mL, and the concentration of cortisol is 50-500 ng / mL.

5. The method for simultaneously and accurately detecting six hypertension-related hormones according to any one of claims 1 to 4, characterized in that: In step (S2), the internal standard is a 500-550 ng / mL deuterated cortisol solution, and the solvent is methanol.

6. The method for simultaneously and accurately detecting six hypertension-related hormones according to any one of claims 1 to 5, characterized in that: In step (S2), the flow rate of the liquid chromatography is 0.3 to 0.4 mL / min; Preferably, in step (S2), the column temperature of the liquid chromatography is 40-42°C; Preferably, in step (S2), the injection volume of the liquid chromatography is 5 to 6 μL; Preferably, in step (S2), the mass spectrometry parameters include: ion source: electrospray positive ion mode ESI+; capillary voltage: 3.5-4.1 kV; cone voltage: 25-45 V; collision energy: 12-25 eV; detection mode: multiple reaction monitoring.

7. An extract for extracting six hypertension-related hormones by liquid chromatography-tandem mass spectrometry, characterized in that: The extract is a methanol-ethyl formate mixed solution with a volume ratio of (7.5-8.5):(1.5-2.5) and a pH value of 8-10.

8. A six-hormone detection kit for hypertension typing diagnosis, characterized in that: The kit comprises: the extracting solution for extracting six hypertension-related hormones by liquid chromatography-tandem mass spectrometry according to claim 7, as well as a mobile phase, a calibration solution and an internal standard solution for liquid chromatography-tandem mass spectrometry detection; Preferably, the mobile phase comprises mobile phase A and mobile phase B, the mobile phase A is 10-12 mmol / L ammonium acetate-0.1-0.2% formic acid aqueous solution, and the mobile phase B is 0.1-0.2% formic acid-acetonitrile; Preferably, the standard solution is a mixed solution of 6 hormone standards, including angiotensin II, angiotensin I, aldosterone, 18-hydroxycortisol, 18-hydroxycorticosterone and cortisol; Preferably, the preparation method of the standard solution is: dissolving the six hormone standards in methanol to prepare single-standard stock solutions, mixing the single-standard stock solutions, and diluting them with 50-55% methanol aqueous solution to obtain a mixed solution of the six hormone standards; Preferably, the concentration of angiotensin II in the mixed solution is 20-300 pg / mL, the concentration of angiotensin I is 20-300 pg / mL, the concentration of aldosterone is 10-1000 pg / mL, the concentration of 18-hydroxycortisol is 30-300 ng / mL, the concentration of 18-hydroxycorticosterone is 10-100 ng / mL, and the concentration of cortisol is 50-500 ng / mL; Preferably, the internal standard is a 500-550 ng / mL deuterated cortisol solution, and the solvent is methanol.

9. Use of the method for simultaneously and accurately detecting six hypertension-related hormones according to any one of claims 1 to 6 in non-disease diagnosis or treatment.

10. Use of the extract for extracting six hypertension-related hormones by liquid chromatography-tandem mass spectrometry according to claim 8 or the six hormone detection kit for hypertension typing diagnosis according to claim 9 in the preparation of products for early diagnosis and typing of secondary hypertension.

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