Liquid chromatography-tandem mass spectrometry method for synchronously detecting related indexes of secondary hypertension in human plasma and application of liquid chromatography-tandem mass spectrometry method

Abstract

The invention relates to the technical field of medical detection, in particular to a liquid chromatography-tandem mass spectrometry method for synchronously detecting related indexes of secondary hypertension in human plasma and application of the liquid chromatography-tandem mass spectrometry method. The method combines the advantages of sample solid-phase extraction pretreatment, chromatographic separation and mass spectrum high resolution, and the sensitivity and specificity are greatly improved. For a target compound with relatively high content, the signal intensity of the compound in mass spectrometric detection is reduced by reducing collision energy, and combined detection of 10 compounds with large level difference is realized. The clinical detection time is shortened while the clinical sample consumption is reduced, and the personnel cost of a laboratory is reduced.

Description

Technical Field

[0001] The present invention relates to the field of medical detection technology, and in particular to a liquid chromatography-tandem mass spectrometry method and application thereof for synchronous detection of secondary hypertension-related indicators in human plasma. Background Art

[0002] Hypertension is a cardiovascular syndrome characterized by elevated blood pressure. Accurate diagnosis is particularly important for the accurate treatment of hypertension. Hypertension, as a collection of diseases, is divided into primary and secondary. The proportion of secondary hypertension is often underestimated in clinical diagnosis and treatment. Secondary hypertension usually has a clear cause. When the cause is found and effectively removed or controlled, secondary hypertension can be cured or significantly relieved. Therefore, screening and identification of secondary hypertension are important contents of hypertension diagnosis, evaluation and treatment. Hypertension secondary to hypercortisolism, primary aldosteronism, and apparent mineralocorticoid excess syndrome are common types of secondary hypertension. Accurate and rapid detection of indicators related to secondary hypertension is of great significance for the diagnosis and treatment monitoring of hypertension.

[0003] Angiotensin I (AngⅠ) is converted by renin acting on angiotensinogen, so it can reflect renin activity. The renin-angiotensin-aldosterone system plays an important role in maintaining blood pressure, water and electrolyte balance, and internal environment stability. Because AngⅠ detection is affected by many factors, there are currently few items that can be tested together with AngⅠ. At present, there is no report on the simultaneous detection of angiotensin I (AngⅠ), angiotensin II (AngⅡ), aldosterone (Aldo), 18-hydroxycortisol (18OHF), 18-oxycortisol (18oxoF), cortisol (Cortisol), corticosterone (Corticosterone), 11-deoxycortisol (11deoxy), 21-deoxycortisol (21deoxy), and cortisone (Cortisone) by liquid chromatography-tandem mass spectrometry.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a liquid chromatography-tandem mass spectrometry method and application for synchronous detection of secondary hypertension-related indicators in human plasma.

[0006] Specifically, the technical solution of the present invention is as follows: In the first aspect, the present invention provides a liquid chromatography-tandem mass spectrometry method for the simultaneous detection of secondary hypertension-related indicators in human plasma, wherein the secondary hypertension-related indicators in human plasma include AngⅠ, AngⅡ, Aldo, 18OHF, 18oxoF, Cortisol, Corticosterone, 11deoxy, 21deoxy and Cortisone; before performing liquid chromatography-tandem mass spectrometry, the method first performs solid phase extraction pre-treatment on the human plasma sample; the solid phase extraction uses a mixed strong anion exchange MAX SPE plate; the eluent for the solid phase extraction is 10 vol%±1 vol% methanol and 1 vol%±0.1 vol% ammonia water; the eluent for the solid phase extraction is 50 vol%±5 vol% acetonitrile and 1 vol%±0.1 vol% formic acid water.

[0007] Preferably, the liquid chromatography uses 0.01 vol%±0.001 vol% formic acid aqueous solution as mobile phase A, and methanol as mobile phase B.

[0008] Preferably, the elution gradient is: 0-0.5min, 30% B, 6.5min, 60% B, 7.2min, 90% B, 8.0min, 30% B.

[0009] Preferably, the chromatographic column is ACQUITY UPLC BEH C8.

[0010] Preferably, the column temperature is 50±2°C.

[0011] Preferably, the mobile phase flow rate is 0.3±0.1 mL / min.

[0012] Preferably, the mass spectrometer adopts electrospray ESI mode, positive and negative switching segmented scanning; automatic sampler temperature: 10±1°C, capillary voltage: 2.5±0.5 kv, nebulizer gas: 7.0±0.5 Bar, ion source temperature: 150±5°C, desolvation temperature: 550±10°C, cone gas flow rate: 150±5 L / h, desolvation gas flow rate: 1000±20 L / h, collision gas flow rate: 0.15±0.05 mL / min.

[0013] Preferably, in mass spectrometry analysis: AngⅠ quantitative ion pair 433.0>269.2, qualitative ion pair 433.0>255.0; and / or, AngⅡ quantitative ion pair 523.97>263.30, qualitative ion pair 523.97>784.5; and / or, Aldo quantitative ion pair 359.33>189.16, qualitative ion pair 359.33>331.16; and / or, 18OHF quantitative ion pair 379.37>267.19, qualitative ion pair 379.37>285.20; and / or, 18oxoF quantitative ion pair 377.37>313.17, qualitative ion pair 377.37>341.19; and / or, Cortisol quantitative ion pair 363.30>121.08, qualitative ion pair 363.30>327.19; and / or, corticosterone quantitative ion pair 347.30>121.08, qualitative ion pair 347.30>91.05; and / or, 11deoxy quantitative ion pair 347.30>97.03, qualitative ion pair 347.30>109.06; and / or, 21deoxy quantitative ion pair 347.30>311.23, qualitative ion pair 347.30>121.08; And / or, the quantitative ion pair of Cortisone is 361.23>163.10, and the qualitative ion pair is 361.23>121.05.

[0014] Preferably, linear regression analysis is performed with the mass concentration of the standard solution of each index compound as the X-axis and the ratio of the standard peak area to the internal standard peak area as the Y-axis to obtain a standard curve regression equation; then the peak area of ​​the component to be measured in the sample is substituted into the standard curve equation to calculate the concentration of each component to be measured in the plasma sample.

[0015] In a second aspect, the present invention provides an application of the liquid chromatography-tandem mass spectrometry method in the synchronous detection of secondary hypertension-related indicators in human plasma for non-disease diagnosis and treatment purposes.

[0016] Beneficial effects: The present invention provides a liquid chromatography-tandem mass spectrometry method and its application for simultaneous detection of secondary hypertension-related indicators in human plasma. The method combines the advantages of sample solid phase extraction pretreatment, chromatographic separation and high-resolution mass spectrometry, and the sensitivity and specificity are greatly improved. For target compounds with high content, the signal intensity of the compound in mass spectrometry detection is reduced by reducing the collision energy, thereby achieving the joint detection of 10 compounds with large level differences. While reducing the amount of clinical samples, the time of clinical testing is also shortened, and the personnel cost of the laboratory is reduced. In addition, the content of aldosterone in the human body is low, and the plasma aldosterone content of healthy adults is usually below 200pg / mL. Acid can inhibit the response of aldosterone, but in the past, when detecting AngⅠ and AngⅡ, 0.1% formic acid is usually added to the mobile phase to increase the chromatographic separation effect and mass spectrometry response of AngⅠ and AngⅡ. The present invention attempts to reduce the addition ratio of formic acid in the mobile phase to 0.01%, while improving the response of aldosterone, it can still meet the separation efficiency and sensitivity of other compounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be described below.

[0018] Figure 1 This is the TIC diagram of the standard solution in Example 1 of the present invention.

[0019] Figure 2 This is the TIC diagram of the quality control sample in Example 1 of the present invention. DETAILED DESCRIPTION

[0020] The present invention aims to establish a method for the combined detection of plasma AngⅠ, AngⅡ, Aldo, 18OHF, 18oxoF, Cortisol, Corticosterone, 11deoxy, 21deoxy, and Cortisone based on a liquid chromatography-tandem mass spectrometry platform. Combining the high sensitivity of mass spectrometry technology and the high separation of chromatography technology, the laboratory gold standard ARR ratio (aldosterone / renin activity) and its typing diagnostic indicators (18OHF, 18oxoF) for the diagnosis of PA in mineralocorticoid hypertension and the information of early screening indicators for glucocorticoid hypertension (Cortisol, Corticosterone, 11deoxy, 21deoxy) can be obtained simultaneously, providing a rapid, efficient, and accurate clinical basis for the early diagnosis, typing diagnosis, and differential diagnosis of hypertension.

[0021] In a more preferred and specific implementation, the solution provided by the present invention mainly includes the following steps: (1) Collect fresh plasma samples and add inhibitors.

[0022] (2) Incubate the samples with inhibitors in a 37°C water bath for 3 hours.

[0023] (3) Add the mixed isotope internal standards to the incubated samples, mix well and centrifuge.

[0024] (4) Perform solid phase extraction on the incubated samples.

[0025] (5) The extracted samples were subjected to liquid chromatography-tandem mass spectrometry analysis to detect AngⅠ, AngⅡ, Aldo, 18OHF, 18oxoF, Cortisol, Corticosterone, 11deoxy, 21deoxy, and Cortisone in plasma.

[0026] Preferably, the mixed stable isotope internal standard used in step (3) includes AngⅠ, AngⅡ, Aldo, 18OHF, Cortisol, Corticosterone, 11deoxy, 21deoxy and Cortisone (since 18oxoF has no isotope internal standard, 18OHB-d4 is used as the internal standard). Internal standard calibration can be used to correct changes in pretreatment, chromatographic separation and mass spectrometry detection.

[0027] Preferably, the present invention uses a mixed mode strong anion exchange (MAX) SPE plate.

[0028] Preferably, the analytical instrument of the present invention uses a Waters UPLC I-Class / Xevo TQ-S system, the chromatographic column is ACQUITY UPLC C8 (2.1 mm×100 mm, 1.7 µm), and the mobile phase is 0.01% v / v formic acid aqueous solution and methanol.

[0029] More preferably, the present invention uses 70%-10% 0.01% v / v formic acid aqueous solution (mobile phase A) and 30%-90% methanol (mobile phase B) for gradient elution, with a flow rate of 0.3 mL / min. The mass spectrometer selects multiple reaction monitoring mode, positive and negative ion mode combined measurement, electrospray ionization, and simultaneously monitors 10 compounds. The specific mass spectrometry conditions are as follows: Capillary voltage: 2.5 KV; Atomizing gas: 7.0 Bar; Ion source temperature: 150 °C; Desolvation temperature: 550 °C; Desolvation gas flow rate: 1100 L / h; Cone hole gas flow: 150 L / h.

[0030] The qualitative and quantitative ion pairs of each compound are as follows: The quantitative ion pair of angiotensin I is 433.0>269.2, and the qualitative ion pair is 433.0>255.0.

[0031] The quantitative ion pair of angiotensin II is 523.97>263.30, and the qualitative ion pair is 523.97>784.5.

[0032] The quantitative ion pair of aldosterone is 359.33>189.16, and the qualitative ion pair is 359.33>331.16.

[0033] The quantitative ion pair of 18-hydroxycortisol is 379.37>267.19, and the qualitative ion pair is 379.37>285.20.

[0034] The quantitative ion pair of 18-oxycortisol is 377.37>313.17, and the qualitative ion pair is 377.37>341.19.

[0035] The quantitative ion pair of cortisol is 363.30>121.08, and the qualitative ion pair is 363.30>327.19.

[0036] The quantitative ion pair of corticosterone was 347.30>121.08, and the qualitative ion pair was 347.30>91.05.

[0037] The quantitative ion pair of 11-deoxycortisol is 347.30>97.03, and the qualitative ion pair is 347.30>109.06.

[0038] The quantitative ion pair of 21-deoxycortisol is 347.30>311.23, and the qualitative ion pair is 347.30>121.08.

[0039] The quantitative ion pair of cortisone is 361.23>163.10, and the qualitative ion pair is 361.23>121.05.

[0040] For the determination of the above compounds, calibration curves were prepared with the standard samples of their respective mass concentrations, with the mass concentration of the standard solution as the X-axis and the ratio of the peak area of ​​the standard to the peak area of ​​the internal standard as the Y-axis, and linear regression analysis was performed to obtain the regression equation of the standard curve. The peak area of ​​the component to be tested in the sample was substituted into the standard curve equation to calculate the concentration of each component to be tested in the plasma sample. The ratio of the sample detection signal intensity is proportional to the concentration of the component to be tested in the sample.

[0041] The contents of the 10 compounds in the biological samples vary greatly. The concentrations of cortisol, cortisone, and AngⅠ after incubation in healthy people are at the ng / mL level, and the concentrations of Adlo, AngⅡ, 18OHF, 18oxoF, corticosterone, 11deoxy, and 21deoxy are at the pg / mL level. There are many structural analogs and endogenous interfering substances in biological samples, and corticosterone, 11deoxy, and 21deoxy are isomers among the 10 compounds, with the same molecular weight and similar structure, which makes it difficult to distinguish them in detection. The method provided by the present invention combines the advantages of sample solid phase extraction pretreatment, chromatographic separation, and high-resolution mass spectrometry, and the sensitivity and specificity are greatly improved. For compounds with higher contents, the signal intensity of the compounds in mass spectrometry detection is reduced by reducing the collision energy, thereby achieving the joint detection of 10 compounds with large level differences. While reducing the amount of clinical samples, it also shortens the time of clinical testing and reduces the personnel costs of the laboratory.

[0042] The content of aldosterone in the human body is low, and the plasma aldosterone content of healthy adults is usually below 200pg / mL. Acid can inhibit the response of aldosterone, but previous technologies usually add 0.1% formic acid to the mobile phase to increase the chromatographic separation effect and mass spectrometry response of AngⅠ and AngⅡ. The present invention attempts to reduce the addition ratio of formic acid in the mobile phase to 0.01%, which can improve the response of aldosterone while still meeting the separation efficiency and sensitivity of other compounds.

[0043] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0044] The endpoints and any values ​​of the ranges disclosed in this specification are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0045] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "specific implementations", or "some specific implementations" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the embodiment of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0046] In the examples provided in this specification, if no specific techniques or conditions are specified, the techniques or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased through regular channels.

[0047] Example 1

[0048] This embodiment provides a method for the joint detection of plasma AngⅠ, AngⅡ, Aldo, 18OHF, 18oxoF, Cortisol, Corticosterone, 11deoxy, 21deoxy, and Cortisone based on a liquid chromatography-tandem mass spectrometry platform.

[0049] The method provided in this embodiment is as follows: 1. Preparation of reagents.

[0050] (1) Standard curve working stock solution (Table 1).

[0051] Table 1

[0052] Take 25 uL of mixed stock standard 2 and add 975 uL 1% BSA (Buffer B) to obtain the highest point S10 of the standard curve, and then dilute it in sequence as follows (Table 2) to obtain the standard curve.

[0053] Table 2

[0054] (2) Preparation of internal standard stock solution (Table 3).

[0055] Table 3

[0056] Since there is no isotopic internal standard for 18oxoF, 18OHB-d4 was used as the internal standard.

[0057] (3) Preparation of other reagents (sample incubation buffer solution and stop solution).

[0058] ① Buffer solution: weigh 60.55 g Tris salt, 37 g EDTA, add 400 mL water, dissolve by ultrasonic and make up to 450 mL, adjust pH to 5.45-5.50 with acetic acid, and store in a refrigerator at 4°C.

[0059] ②Soybean trypsin inhibitor (SBTI), diluted with water to 30 mg / mL, stored in aliquots at -20℃.

[0060] ③ Phenylmethylsulfonyl fluoride (PMSF): Accurately weigh 0.175 g PMSF and dissolve it in 10 mL methanol. Prepare a 100 mmol stock solution and store it in a 4°C refrigerator.

[0061] ④ Working buffer solution: Take 100 µL of 30 mg / mL SBTI solution, add 100 µL of 0.1 M PMSF solution, add 10 mL of buffer solution, mix well to get the working buffer solution.

[0062] ⑤ Incubation stop solution containing internal standards: dilute the mixed internal standard stock solution (9mix) 1000 times with 2% (v / v) ammonia solution to obtain a stop solution containing 9 mixed internal standards (e.g., 10 µL of mixed internal standard stock solution plus 10 mL of 2% ammonia solution).

[0063] 2. Sample pretreatment.

[0064] (1) Sample preparation.

[0065] No incubation batch: add 200 µL of working buffer to 200 µL of plasma / standard / control, vortex to mix, then add 300 µL of incubation stop solution containing internal standard, vortex to mix again.

[0066] Incubation batch: 200 µL plasma / standard / quality control plus 200 µL working buffer, vortex to mix, and incubate at 37°C for 3 hours. Then add 300 µL incubation stop solution containing internal standard and vortex to mix to terminate the reaction.

[0067] After treatment, the samples were centrifuged at 3000 rpm for 10 min.

[0068] (2) Solid phase extraction.

[0069] Activate the SPE plate: add 200 µL of methanol to the Oasis MAX μElution SPE plate and blow it off; add 200 µL of 1% (v / v) ammonia water to the Oasis MAX μElution SPE plate and blow it off.

[0070] Loading: 600 µL of supernatant was added to the Oasis MAX μElution SPE plate.

[0071] Eluent: 200 µL 10% methanol 1% ammonia.

[0072] Change the receiving plate and pre-add 40 µL of water.

[0073] Elution: 40 µL 50% acetonitrile 1% formic acid in water.

[0074] After elution, the samples were shaken on a 96-well plate thermostat for 1 min before LC-MS / MS analysis.

[0075] 3. Liquid chromatography tandem mass spectrometry conditions.

[0076] Waters Xevo TMTQS MS ACQUITY UPLC-MS / MS was used for detection. The chromatographic column was ACQUITY UPLC BEH C8 column (2.1 mm × 100 mm, 1.7 µm). The mobile phase was 0.01% (v / v) formic acid aqueous solution A and methanol B. The injection of 10 µL was performed for gradient elution as shown in the following gradient (Table 4).

[0077] Table 4

[0078] The mass spectrometer was operated in electrospray ionization (ESI) mode with positive and negative switching segmented scanning.

[0079] Autosampler temperature: 10 °C, capillary voltage: 2.5 kV, ion source temperature: 150 °C, desolvation temperature: 550 °C, cone gas flow rate: 150 L / h, desolvation gas flow rate: 1000 L / h, collision gas flow rate: 0.15 mL / min.

[0080] The qualitative and quantitative ion pairs are shown in Table 5: Table 5

[0081] 4. Test results.

[0082] (1) A linear regression equation was fitted based on the test results, as shown in Table 6 below.

[0083] Table 6

[0084] (2) Prepare quality control products of a certain concentration and test the intra-batch imprecision and total imprecision, as shown in Table 7 below.

[0085] Table 7

[0086] (3) The specificity of the method and the plasma matrix effects from different sources are shown in Table 8.

[0087] Table 8

[0088] In summary, the present invention adopts solid phase extraction for sample pretreatment, without the need for complicated essence purification steps, and combined with LC-MS / MS, it can simultaneously and quantitatively analyze and detect AngⅠ, AngⅡ, Aldo, 18OHF, 18oxoF, Cortisol, Corticosterone, 11deoxy, 21deoxy and Cortisone at one time. The analysis results are accurate and reliable and can serve the detection of clinical samples.

[0089] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A liquid chromatography-tandem mass spectrometry method for simultaneous detection of secondary hypertension-related indicators in human plasma, characterized in that: The secondary hypertension-related indicators in human plasma include AngⅠ, AngⅡ, Aldo, 18OHF, 18oxoF, Cortisol, Corticosterone, 11deoxy, 21deoxy and Cortisone; the method comprises performing solid phase extraction pretreatment on human plasma samples before performing liquid chromatography tandem mass spectrometry; the solid phase extraction uses a mixed strong anion exchange MAX SPE plate; the eluent for the solid phase extraction is 10 vol%±1 vol% methanol and 1 vol%±0.1 vol% ammonia water; the eluent for the solid phase extraction is 50 vol%±5 vol% acetonitrile and 1 vol%±0.1 vol% formic acid water.

2. The liquid chromatography-tandem mass spectrometry method for synchronous detection of secondary hypertension-related indicators in human plasma according to claim 1, characterized in that: For liquid chromatography, 0.01 vol% ± 0.001 vol% formic acid aqueous solution was used as mobile phase A, and methanol was used as mobile phase B.

3. The liquid chromatography-tandem mass spectrometry method for synchronous detection of secondary hypertension-related indicators in human plasma according to claim 2, characterized in that: The elution gradient was: 0-0.5min, 30% B, 6.5min, 60% B, 7.2min, 90% B,8.0min, 30% B.

4. The liquid chromatography-tandem mass spectrometry method for synchronous detection of secondary hypertension-related indicators in human plasma according to any one of claims 1 to 3, characterized in that: The chromatographic column used was ACQUITY UPLC BEH C8.

5. The liquid chromatography-tandem mass spectrometry method for synchronous detection of secondary hypertension-related indicators in human plasma according to any one of claims 1 to 4, characterized in that: The column temperature is 50±2℃.

6. The liquid chromatography-tandem mass spectrometry method for synchronous detection of secondary hypertension-related indicators in human plasma according to any one of claims 1 to 5, characterized in that: The mobile phase flow rate was 0.3 ± 0.1 mL / min.

7. The liquid chromatography-tandem mass spectrometry method for synchronous detection of secondary hypertension-related indicators in human plasma according to any one of claims 1 to 6, characterized in that: The mass spectrometer adopted electrospray ESI mode with positive and negative switching segmented scanning; autosampler temperature: 10±1℃, capillary voltage: 2.5±0.5 kv, nebulizer gas: 7.0±0.5 Bar, ion source temperature: 150±5℃, desolvation temperature: 550±10℃, cone gas flow rate: 150±5 L / h, desolvation gas flow rate: 1000±20 L / h, collision gas flow rate: 0.15±0.05 mL / min.

8. The liquid chromatography-tandem mass spectrometry method for synchronous detection of secondary hypertension-related indicators in human plasma according to any one of claims 1 to 7, characterized in that: In mass spectrometry: AngⅠ quantitative ion pair 433.0>269.2, qualitative ion pair 433.0>255.0; and / or, AngⅡ quantitative ion pair 523.97>263.30, qualitative ion pair 523.97>784.5; and / or, Aldo quantitative ion pair 359.33>189.16, qualitative ion pair 359.33>331.16; and / or, 18OHF quantitative ion pair 379.37>267.19, qualitative ion pair 379.37>285.20; and / or, 18oxoF quantitative ion pair 377.37>313.17, qualitative ion pair 377.37>341.19; and / or, Cortisol quantitative ion pair 363.30>121.08, qualitative ion pair 363.30>327.19; and / or, corticosterone quantitative ion pair 347.30>121.08, qualitative ion pair 347.30>91.05; and / or, 11deoxy quantitative ion pair 347.30>97.03, qualitative ion pair 347.30>109.06; and / or, 21deoxy quantitative ion pair 347.30>311.23, qualitative ion pair 347.30>121.08; And / or, the quantitative ion pair of Cortisone is 361.23>163.10, and the qualitative ion pair is 361.23>121.

05.

9. The liquid chromatography-tandem mass spectrometry method for synchronous detection of secondary hypertension-related indicators in human plasma according to any one of claims 1 to 8, characterized in that: The mass concentration of the standard solution of each index compound is taken as the X-axis, and the ratio of the peak area of ​​the standard to the peak area of ​​the internal standard is taken as the Y-axis, and a linear regression analysis is performed to obtain the regression equation of the standard curve; Then substitute the peak area of ​​the component to be tested in the sample into the standard curve equation to calculate the concentration of each component to be tested in the plasma sample.

10. Use of the liquid chromatography-tandem mass spectrometry method according to any one of claims 1 to 9 in the simultaneous detection of secondary hypertension-related indicators in human plasma for non-disease diagnosis and treatment purposes.

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