Synchronous Detection Method of Medicaments Influencing ARR in the Detection Process of Renin Activity by Liquid Chromatography-Tandem Mass Spectrometry

Abstract

The present invention provides a synchronous detection method of medicaments influencing ARR in the detecting process of renin activity by liquid chromatography-tandem mass spectrometry. The method achieves the qualitative screening of medicaments influencing ARR values while detecting plasma renin activity by liquid chromatography-tandem mass spectrometry; moreover, the method can be used to assist to analyze and judge ARR as negative, positive, false negative or false positive. The detecting method achieves effective extraction of angiotensin I and 43 hypertension therapeutics synchronously through protein precipitation to samples, and enables to perform synchronous detection of a plurality of indices including angiotensin I and 43 hypertension therapeutics on the extracted sample by liquid chromatography-tandem mass spectrometry technology of high throughput, high specificity and high sensitivity. Moreover, the detection method utilizes the MRM mass spectrum parameters for preliminary screening of the medicaments, and rechecks the medicaments according to the retention time thereof. The ARR detection value is combined with the medicament screening result for co-analysis, which effectively distinguishes the false positive or false negative result of the detection while avoiding the patient's withdrawal of medicament for treatment, thereby improving the detection accuracy of the actual primary aldosteronism, and facilitating clinical promotion and application.

Claims

1. A method for synchronous qualitative detection of medicaments influencing ARR in the detection process of renin activity by liquid chromatography-tandem mass spectrometry, comprising: detecting a production rate of angiotensin I and hypertension therapeutics influencing ARR in a plasma sample; combining a concentration of aldosterone to obtain ARR values, thus analyzing and judging ARR as one of a negative, positive, false negative or false positive results.

2. The detection method of claim 1, wherein the hypertension therapeutics influencing ARR, including 9 β-receptor blockers, 5 potassium-removing diuretics, 3 potassium-retaining diuretics, 10 angiotensin converting enzyme inhibitors, 6 angiotensin receptor antagonists, 8 calcium channel blockers, 1 central α2 receptor agonist and 1 non-steroid anti-inflammatory drug.

3. The detection method of claim 2, wherein the ARR has a computing formula as follows:
ARR=concentration of aldosterone/production rate of angiotensin I.

4. The detection method of claim 3, wherein the hypertension therapeutics influencing ARR are: the 9 α-receptor blockers comprising Arotinolol, Atenolol, Bisoprolol, Esmolol, Labetalol, Metoprolol, Nebivolol, Propranolol, and Sotalol; the 5 potassium-removing diuretics comprising Bumetanide, Furosemide, Hydrochlorothiazide, Indapamide, and Torasemidel; the 3 potassium-retaining diuretics comprising Amiloride, Eplerenone, and Spironolactone; the 10 angiotensin converting enzyme inhibitors comprising Benazepril, Captopril, Enalapril, Fosinopril, Imidapril, Lisinopril, Perindopril, Quinapril, Ramipril, and Trandolapril; the 6 angiotensin receptor antagonists comprising Candesartan, Irbesartan, Losartan, Olmesartan, Telmisartan, and Valsartan; the 8 calcium ion antagonists comprising Amlodipine, Benidipine, Felodipine, Flunarizine, Lercanidipine, Nicardipine, Nifedipine, and Nimodipine; the 1 central α2 receptor agonist comprising Methyldopa; and the 1 non-steroid anti-inflammatory drug comprising aspirin.

5. The detection method of claim 4, wherein the β-receptor blockers, the central α2 receptor agonist, and the non-steroid anti-inflammatory drug can increase ARR to show a false positive result; the potassium-removing diuretics, the potassium-retaining diuretics, the angiotensin converting enzyme inhibitors, the angiotensin receptor antagonists, and the calcium channel blockers can decrease ARR to show a false negative result; the procedure for analysis of ARR values are as follows: analyzing and judging ARR as one of a negative, positive, false negative or false positive results by a clinical medication AI guidance system, wherein the clinical medication AI guidance system comprises a patient information module, a database module and a decision tree system.

6. The detection method of claim 5, wherein the information module is used to record a patient's basic information; the database module is used to store detection results of ARR and detection results of hypertension therapeutics; and the decision tree system automatically analyzes and judges an ARR index as one of a negative, positive, false negative or false positive result according to the detection results.

7. The detection method of any one of claim 6, wherein the analysis and judgment method of the decision tree system is as follows: analyzing and judging the ARR indices as one of a negative, positive, false negative or false positive results according to the qualitative detecting results of the medicaments influencing ARR, wherein when the ARR detection result is less than 30, and meanwhile if the patient is detected to contain a medicament capable of decreasing ARR, the ARR indices may be judged as false negative, and an experiment for confirmation is further needed; when the ARR detection result is less than 30, and meanwhile medicament capable of increasing ARR positive, the ARR indices may be judged as false positive, and an experiment for conformation is necessary; and when the ARR detection result is higher than 30, and meanwhile if the patient is detected to contain a medicament capable of decreasing ARR, the ARR indices may be judged as positive.

8. The detecting method of claim 7, wherein the production rate of the angiotensin I is obtained by detecting concentrations of angiotensin I in the sample before and after incubation respectively and calculated according to the following formula: production rate of angiotensin I=(concentration of angiotensin I after incubation−concentration of angiotensin I before incubation)/incubation time.

9. The detection method of claim 8, wherein the detecting procedures are as follows: (1) sample pretreatment: 1) taking two parts of 100 μL parallel samples, adding a generation buffer solution to the samples respectively and mixing evenly, subjecting one part to incubation, and subjecting another part to next step directly without incubation; 2) adding a terminating solution to samples where the generation buffer solution is added, and mixing evenly; 3) adding a protein precipitant with internal standard to the samples where the terminating solution is added in the step 2), and vortex mixing evenly; and 4) mixing the samples where the protein precipitant is added in step 3) and then performing centrifugation, and taking supernatant for detection on an instrument; (2) performing the detection of angiotensin I and screening of 43 hypertension therapeutics influencing ARR with sample before incubation obtained in the above steps with a high performance liquid chromatography-tandem mass spectrometry system; (3) respecting to the hit for medicament screening result, a double-check is necessary with simultaneous reference to the retention time of the standard substance of each hypertension therapeutic in a “mixed medicament-screening working solution”, thus ensuring the accuracy of the hit of the medicament screening from two dimensions of mass spectrum parameters and retention time.

10. The detection method of claim 9, wherein the protein precipitant with internal standard is prepared by: methanol, a zinc sulfate aqueous solution and an angiotensin I isotope internal standard.

11. The detection method of claim 9, wherein the generation buffer solution is: a PMSF-containing Tris and EDTA buffer solution; and the terminating solution is: formic acid or acetic acid.

12. The detection method of claim 9, wherein the mixed medicament-screening working solution is a mixed medicament-screening working solution prepared by standard substances of the 43 hypertension therapeutics influencing ARR.

13. The detection method of claim 9, wherein the incubation refers that a sample is added to a generation buffer solution and mixed evenly, then put to a 37° C. water bath for 3 h; and a non-incubated sample is used for detecting Angiotensin I before incubation and screening the 43 hypertension therapeutics influencing ARR.

14. The detection method of claim 9, wherein mobile phase A of the liquid chromatography is 0.1% formic acid aqueous solution, mobile phase B is methanol solution with 0.1% formic acid; and the gradient is shown in the Table below: TABLE-US-00012 Time Flow rate Mobile phase Mobile phase (min) (mL/min) A (%) B (%) 0 0.6 90 10 1 0.6 90 10 2 0.6 5 95 3.2 0.6 5 95 3.25 0.6 90 10 3.8 0.6 90 10

15. The detection method of claim 9, wherein the retention time of the standard substance of each hypertension therapeutic in the “mixed medicament-screening working solution” is shown in the table below: TABLE-US-00013 Limit of detection (LOD) of English name medicament Retention Type of medicament of medicament (ng/mL) time (min) Potassium-retaining Amiloride 25 1.06 diuretics CCBs Amlodipine 25 2.19 β-receptor blocker Arotinolol 5 1.97 Non-steroid aspirin 250 2.33 anti-inflammatory drug β-receptor blocker Atenolol 25 0.65 ACEI Benazepril 5 2.19 CCBs Benidipine 5 2.16 β-receptor blocker Bisoprolol 5 2.05 Potassium-removing Bumetanide 25 2.29 diuretics ARBs Candesartan 25 2.32 ACEI Captopril 100 1.95 ACEI Enalapril 5 2.1 Potassium-retaining Eplerenone 25 2.16 diuretics β-receptor blocker Esmolol 5 1.98 CCBs Felodipine 250 2.39 CCBs Flunarizine 5 2.25 ACEI Fosinopril 100 2.46 Potassium-removing Furosemide 250 2.61 diuretics Potassium-removing Hydrochlorothia 250 1.97 diuretics zide ACEI Imidapril 5 2.13 Potassium-removing Indapamide 25 2.17 diuretics ARBs Irbesartan 5 2.27 β-receptor blocker Labetolol 25 2.04 CCBs Lercanidipine 5 2.26 ACEI Lisinopril 25 1.77 ARBs Losartan 5 2.26 Centralα2 receptor Methyldopa 250 0.39 agonist β-receptor blocker Metoprolol 25 1.94 β-receptor blocker Nebivolol 25 2.2 CCBs Nicardipine 5 2.15 CCBs Nifedipine 250 2.27 CCBs Nimodipine 25 2.34 ARBs Olmesartan 25 2.12 ACEI Perindopril 5 2.14 β-receptor blocker Propranolol 25 2.08 ACEI Quinapril 5 2.22 ACEI Ramipril 5 2.2 β-receptor blocker Sotalol 25 0.53 Potassium-retaining Spironolactone 250 2.29 diuretics ARBs Telmisartan 25 2.24 Potassium-removing Torasemide 5 2.13 diuretics ACEI Trandolapril 5 2.25 ARBs Valsartan 100 2.34

16. The detection method of claim 9, wherein during the mass spectrometric detection, a triple quadrupole mass spectrometer with a model of SCIEX 4500MD is used; and the mass spectrometric detection is performed by adopting a positive ion mode of an electrospray ionization ion source (ESI+) and a multiple-reaction monitoring MRM mode; and the corresponding mass spectrometric detection method is configured as shown in the following table: TABLE-US-00014 Mass spectrometry conditions Value Curtain gas CUR 30 psi Atomization gas GS1 50 psi Auxiliary heating gas GS2 50 psi Heating temperature of ion 500° C. sources Collision gas CAD 10 psi Spray voltage 5500 V

17. The detection method of claim 16, wherein during the mass spectrometric detection, mass spectrum parameters of each substance to be detected are shown in the table below: TABLE-US-00015 Dwell Compound Q1 Q3 Time DP CE CXP ANG I-1 433.1 647.6 30 74 24 12 ANG I-2 433.1 619.6 5 87 26 12 ANG I-IS 437.3 660.4 30 70 22 12 Metoprolol 268.1 133 4 90 37 9 Esmolol 296.1 145.1 4 78 38 9 Labetolol 329.2 162 4 78 35 9 Bisoprolol 326.5 116.2 4 96 25 9 Flunarizine 405.2 203 4 79 21 9 Ramipril 417.3 234.1 4 78 29 9 Lercanidipine 612.2 280.2 4 94 31 9 Benazepril 425.2 351.1 4 105 30 9 Quinapril 439.1 234 4 90 27 9 Losartan 423.2 207 4 69 33 9 Trandolapril 431.2 234 4 90 31 9 Telmisartan 515.4 276 4 153 59 9 Nifedipine-1 347.1 315.1 4 79 13 9 Nifedipine-2 347.3 195.1 4 65 58 6 Nicardipine 480.2 315.1 4 78 34 9 Benidipine 506.2 174 4 78 37 9 Torasemide 349.1 264.1 4 63 22 9 Nebivolol 406.1 151 4 105 41 9 Indapamide 366 132 4 78 20 9 Imidapril 406.2 234 4 75 27 9 Eplerenone 415.2 162.9 4 107 28 9 Olmesartan 447.1 207 4 83 37 9 Irbesartan 429.4 206.8 4 108 38 9 Nimodipine 419.2 343.1 4 82 13 9 Valsartan-1 436.3 206.9 4 90 38 9 Valsartan-2 436.4 207.2 4 90 38 4 Felodipine 384 338 4 76 13 9 Spironolactone 417.2 341.2 4 90 12 9 Candesartan 441.1 263 4 97 18 9 Atenolol 267.2 145 4 78 35 9 Methyldopa 212 166 4 75 21 9 Amiloride 230.1 170.9 4 69 24 9 Sotalol 273.1 133 4 70 36 9 Arotinolol 372 316.1 4 78 22 9 Lisinopril 406.2 84 4 78 52 9 Enalapril 377.2 234.1 4 70 26 9 Perindopril 369.1 172.1 4 90 31 9 Propranolol 260 116 4 90 24 9 Amlodipine-1 409 237.9 4 69 12 9 Amlodipine-2 409.2 206 4 55 37 10 Fosinopril 564.4 492.2 4 84 11 9 Captopril-1 218.1 116.1 4 73 20 9 Captopril-2 218.2 70 4 70 25 6 Nifedipine Meta-1 331.2 270.2 4 165 47 9 Felodipine Meta 384 320 4 165 47 9 Spironolactone Meta 341.1 107.1 4 165 47 9 Telmisartan Meta 691.4 515.4 4 165 47 9 Aspirin-1 163.2 64.9 4 42 39 10 Aspirin-2 163.2 120.8 4 43 17 7 Bumetanide 365.2 240.1 4 128 24 18 Furosemide 331 80.9 4 131 26 8 Hydrochlorothiazide 297.3 98.9 4 168 40 12

18. The detection method of claim 1, wherein influences of the hypertension medicaments on ARR can be analyzed and predicted by AI software.

19. A kit for synchronous qualitative detection of antihypertensive medicaments influencing ARR in the detection process of renin activity by liquid chromatography-tandem mass spectrometry, the kit comprising a protein precipitant containing an internal standard, a generation buffer solution, a terminating solution, a mixed medicament-screening working solution and liquid mobile phases, wherein the protein precipitant with internal standard is prepared by methanol, a zinc sulfate aqueous solution and an angiotensin I isotope-labeled internal standard; the generation buffer solution is: a PMSF-containing Tris and EDTA buffer solution; and the terminating solution is formic acid or acetic acid; the mixed medicament-screening working solution is a mixed medicament-screening working solution prepared by standard substances of 43 hypertension therapeutics influencing ARR at certain concentration; mobile phase A of the liquid chromatography is a 0.1% formic acid aqueous solution, and mobile phase B is a methanol solution with of 0.1% formic acid.

Description

BRIEF DESCRIPTION OF THE FICIURES

[0062] FIG. 1 is a detection flow of a sample in Example 1;

[0063] FIG. 2 shows a detection chromatogram of aldosterone in Example 1;

[0064] FIG. 3 is a screening report of primary aldosteronism in Example 1.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0065] The present invention will be further described in detail with reference to the drawings and examples. It should be indicated that the examples below aim at facilitating the understanding of the present invention, but not limiting the present invention. Reagents used in the examples are known products, and are commercially available.

EXAMPLE 1: QUALITATIVE DETECTION METHOD OF ARR PROVIDED BY THE PRESENT INVENTION

I. Solution Preparation

[0066] Preparation of a ZnSO.sub.4 solution: 3.56 g ZnSO.sub.4.7H.sub.2O were weighed, and dissolved into 40 mL water to prepare 89 mg/mL ZnSO.sub.4.7H.sub.2O aqueous solution. The volume of the solution could be adjusted according to the ratio as required, and the solution was stored at room temperature.

[0067] Generation buffer solution: 12.11 g trismetyl aminomethane (TRIS) and 7.4 g ethylenediamine tetraacetic acid (EDTA) were added to a 100 mL volumetric flask, and deionized water was added to 90 mL, the remaining solution was dissolved evenly under ultrasonic for 30 min. Deionized water was added to a scale line, and the solution was mixed evenly. Afterwards, the solution was transferred to a reservoir vessel made of polypropylene. pH was regulated within 5.45-5.50 by acetic acid, and the remaining solution was stored at −20° C. The preparation of generating buffer was performed on the same day of detection and analysis, 100 μL 100 mM PMSF solution (0.174 g PMSF was dissolved in 10 mL methanol) was added to a 10 mL generation buffer solution to get generation buffer solution (pH=5.4-5.6).

[0068] Terminating solution: formic acid or acetic acid.

[0069] A protein precipitant containing internal standard: 50 μL 1 μg/mL angiotensin I internal standard stock solution (methanol: water=1:1), and 100 μL 89 mg/mL zinc sulfate (ZnSO4.7H2O) were transferred to a 10 mL centrifugal tube, and 9850 μL methanol was added to the centrifugal tube, and mixed evenly to obtain a protein precipitant containing angiotensin I isotope-labeled internal standard.

[0070] A mixed medicament-screening working solution: standards of the 43 medicaments to be detected and partial metabolite standards were dissolved into DMSO, and prepared into a corresponding stock solution; and the stock solution was prepared into a mixed medicament-screening working solution having a LOD concentration based on the LOD concentration listed in the “Screening List and Limit of Detection of Medicaments Influencing ARR Values”.

II. Sample Detection

[0071] the detection flow diagram was shown in FIG. 1.

[0072] (1) Reagent preparation: 20 μL generation buffer solution was added to two clean 1.2 mL 96-well collection plate first, and used for the following sample pretreatment.

[0073] (2) Sample thawing: a plasma sample to be detected was put to ice water (0° C.) and thawed out.

[0074] (3) Sampling: 2 parts of 100 μL parallel plasma samples were taken and transferred to two plates prepared in the step (1), then non-incubated and incubated samples were pretreated and detected respectively; and the rest samples were immediately frozen at −20° C.; and non-incubated sample was taken for the medicament screening.

[0075] (4) Incubated sample treatment: a batch of samples in step (3) were sealed by a silicone pad, and vortex mixed for a short time, then put to a 37° C. water bath for 3 h; and 12 μL terminating solution (formic acid) was added and vortex mixed, and 200 μL internal standard working solution containing 5 ng/mL angiotensin I isotope-labeled internal standard ANG I—.sup.13C.sup.15N was added and vortex mixed, afterwards, centrifuged for 10 min at 4° C. and 15000 rpm; 100 μL supernatant was transferred to a 96-well sample injecting plate for analysis by liquid chromatography-tandem mass spectrometry.

[0076] (5) Non-incubated sample treatment: another batch of samples remaining in step (3) were sealed by a sealing gasket and vortex mixed evenly. 12 μL terminating solution, formic acid was immediately added and vortex mixed, and 200 μL internal standard working solution containing an angiotensin I isotope internal standard was added, and vortex mixed evenly; afterwards, centrifuged for 10 min at 4° C. and 15000 rpm, 100 μL supernatant was transferred to a 96-well sample injection plate for analysis by liquid chromatography-tandem mass spectrometry (non-incubated samples were taken in the medicament screening, non-incubated samples could be not only used for the calculation of renin activity, but also for screening the medicament disturbing ARR).

[0077] (6) Calculation of renin activity: renin activity=production rate of angiotensin I, where the unit was ng/mL/hr, and calculating formula was: (concentration of angiotensin I after incubation−concentration of angiotensin I before incubation)/incubation time.

[0078] (7) During the liquid chromatography-tandem mass spectrometry, gradient elution was taken in liquid chromatography; and separation conditions of a substance to be detected was established by a reversed phase chromatography were as follows: the chromatographic column was Phenomenex C18 (2.6 μm, 50*2.1 mm), flow rate was 0.6 mL/min; column temperature was 40° C.; where mobile phase A was 0.1% formic acid aqueous solution, mobile phase B was a methanol solution of 0.1% formic acid; and volume ratio of mobile phase A to mobile phase B was 90-5%:10-95%. Gradient elution procedure was shown in Table 1; and the retention time of angiotensin I and its isotope-labeled internal standard was 1.92 min.

TABLE-US-00002 TABLE 1 Gradient elution procedure Time Flow rate Mobile phase Mobile phase (min) (mL/min) A (%) B (%) 0 0.6 90 10 1 0.6 90 10 2 0.6 5 95 3.2 0.6 5 95 3.25 0.6 90 10 3.8 0.6 90 10

[0079] The retention time of the standard substance of each hypertension therapeutic in the “mixed medicament-screening working solution” was shown in Table 2 below:

TABLE-US-00003 TABLE 2 Retention time of the standard substance of each hypertension therapeutic Limit of detection (LOD) of Retention- Type of Medicament medicament time medicament in English (ng/mL) (min) Potassium- Amiloride 25 1.06 retaining diuretics CCBs Amlodipine 25 2.19 β-receptor Arotinolol 5 1.97 blocker Non-steroid anti- aspirin 250 2.33 inflammatory drug β-receptor Atenolol 25 0.65 blocker ACEI Benazepril 5 2.19 CCBs Benidipine 5 2.16 β-receptor Bisoprolol 5 2.05 blocker Potassium- Bumetanide 25 2.29 removing diuretics ARBs Candesartan 25 2.32 ACEI Captopril 100 1.95 ACEI Enalapril 5 2.1 Potassium- Eplerenone 25 2.16 retaining diuretics β-receptor Esmolol 5 1.98 blocker CCBs Felodipine 250 2.39 CCBs Flunarizine 5 2.25 ACEI Fosinopril 100 2.46 Potassium- Furosemide 250 2.61 removing diuretics Potassium- Hydrochlorothiazide 250 1.97 removing diuretics ACEI Imidapril 5 2.13 Potassium- Indapamide 25 2.17 removing diuretics ARBs Irbesartan 5 2.27 β-receptor Labetolol 25 2.04 blocker CCBs Lercanidipine 5 2.26 ACEI Lisinopril 25 1.77 ARBs Losartan 5 2.26 Centralα2 Methyldopa 250 0.39 receptor agonist β-receptor Metoprolol 25 1.94 blocker β-receptor Nebivolol 25 2.2 blocker CCBs Nicardipine 5 2.15 CCBs Nifedipine 250 2.27 CCBs Nimodipine 25 2.34 ARBs Olmesartan 25 2.12 ACEI Perindopril 5 2.14 β-receptor Propranolol 25 2.08 blocker ACEI Quinapril 5 2.22 ACEI Ramipril 5 2.2 β-receptor Sotalol 25 0.53 blocker Potassium- Spironolactone 250 2.29 retaining diuretics ARBs Telmisartan 25 2.24 Potassium- Torasemide 5 2.13 removing diuretics ACEI Trandolapril 5 2.25 ARBs Valsartan 100 2.34

[0080] During the mass spectrometric detection, a triple quadrupole mass spectrometer with a model of SCIEX 4500MD was used for the quantitative detection of angiotensin I and qualitative screening of antihypertensive medicaments before and after incubation; and the mass spectrometric detection was performed by adopting a positive ion mode of an electrospray ionization ion source (ESI+) and a multiple-reaction monitoring MRM mode; and the corresponding mass spectrometric detection method was configured as shown in Table 3 and Table 4:

TABLE-US-00004 TABLE 3 Parameter settings for mass spectrometric detection Mass Parameters Value Curtain gas CUR 30 psi Atomization gas GS1 50 psi Auxiliary heating gas 50 psi GS2 Heating temperature 500° C. of ion sources Collision gas CAD 10 psi Spray voltage 5500 V

TABLE-US-00005 TABLE 4 Mass spectrometry parameters of each substance to be detected Dwell Compound Q1 Q3 Time DP CE CXP ANG I-1 433.1 647.6 30 74 24 12 ANG I-2 433.1 619.6 5 87 26 12 ANG I-IS 437.3 660.4 30 70 22 12 Metoprolol 268.1 133 4 90 37 9 Esmolol 296.1 145.1 4 78 38 9 Labetolol 329.2 162 4 78 35 9 Bisoprolol 326.5 116.2 4 96 25 9 Flunarizine 405.2 203 4 79 21 9 Ramipril 417.3 234.1 4 78 29 9 Lercanidipine 612.2 280.2 4 94 31 9 Benazepril 425.2 351.1 4 105 30 9 Quinapril 439.1 234 4 90 27 9 Losartan 423.2 207 4 69 33 9 Trandolapril 431.2 234 4 90 31 9 Telmisartan 515.4 276 4 153 59 9 Nifedipine-1 347.1 315.1 4 79 13 9 Nifedipine-2 347.3 195.1 4 65 58 6 Nicardipine 480.2 315.1 4 78 34 9 Benidipine 506.2 174 4 78 37 9 Torasemide 349.1 264.1 4 63 22 9 Nebivolol 406.1 151 4 105 41 9 Indapamide 366 132 4 78 20 9 Imidapril 406.2 234 4 75 27 9 Eplerenone 415.2 162.9 4 107 28 9 Olmesartan 447.1 207 4 83 37 9 Irbesartan 429.4 206.8 4 108 38 9 Nimodipine 419.2 343.1 4 82 13 9 Valsartan-1 436.3 206.9 4 90 38 9 Valsartan-2 436.4 207.2 4 90 38 4 Felodipine 384 338 4 76 13 9 Spironolactone 417.2 341.2 4 90 12 9 Candesartan 441.1 263 4 97 18 9 Atenolol 267.2 145 4 8 35 9 Methyldopa 212 166 4 75 21 9 Amiloride 230.1 170.9 4 69 24 9 Sotalol 273.1 133 4 70 36 9 Arotinolol 372 316.1 4 78 22 9 Lisinopril 406.2 84 4 78 52 9 Enalapril 377.2 234.1 4 70 26 9 Perindopril 369.1 172.1 4 90 31 9 Propranolol 260 116 4 90 24 9 Amlodipine-1 409 237.9 4 69 12 9 Amlodipine-2 409.2 206 4 55 37 10 Fosinopril 564.4 492.2 4 84 11 9 Captopril-1 218.1 116.1 4 73 20 9 Captopril-2 218.2 70 4 70 25 6 Nifedipine Meta-1 331.2 270.2 4 165 47 9 Felodipine Meta 384 320 4 165 47 9 Spironolactone Meta 341.1 107.1 4 165 47 9 Telmisartan Meta 691.4 515.4 4 165 47 9 Aspirin-1 163.2 64.9 4 42 39 10 Aspirin-2 163.2 120.8 4 43 17 7 Bumetanide 365.2 240.1 4 128 24 18 Furosemide 331 80.9 4 131 26 8 Hydrochlorothiazide 297.3 98.9 4 168 40 12

[0081] (8) Detection of aldosterone: for sample pretreatment of aldosterone detection in plasma, supportive liquid-liquid extraction (SLE) was used for extraction, and liquid chromatography-tandem mass spectrometry was used for detection; specific steps were as follows: 300-450 μL plasma was transferred and added to 50 μL aldosterone internal standard solution, samples were loaded on an SLE plate, standing for 10 min, and eluted by a 1.5 mL (2*750 ul) mixed solvent of ethyl acetate and n-hexane (1:1). After eluent was dried, the resident was redissolved and detected by liquid chromatography-tandem mass spectrometer; an ESI+detection mode was taken; quantitative MRM ion pair of Aldosterone was 361.3/315.1; MRM ion pair of an Aldosterone internal standard Aldosterone-d8 was 369.4/323.1; and the representative detection chromatogram was shown in FIG. 2. A great number of studies have proved that the 43 medicaments influencing ARR cannot be synchronously screened during the pretreatment and detection of aldosterone; and the reason is probably that the pretreatment process is not suitable for the extraction of all 43 medicaments influencing ARR; and the experimental data is omitted.

[0082] (9) Test report of the medicament screening; in the plasma PA screening report (as shown in FIG. 3), the screening results of the medicament factors leading to false positive or false negative ARR were collected and in clinical combination with ARR values and medicament screening results to effectively eliminate partial false positive or false negative results caused by medicament interference.

EXAMPLE 2: VERIFICATION OF FALSE NEGATIVE CLINICAL SAMPLES

[0083] In this example, clinical samples were detected for PA Screening; and the detection method used in Example 1 was taken for ARR detection and qualitative judgment; and the detection results of the clinical samples were shown in Table 5.

TABLE-US-00006 TABLE 5 Detection results of clinical samples Unit of Item Result measurement Reference interval Aldosterone 222.46 pg/ml ≥18 years old: [ALD] Upright position: 8:00-10:00 A.M. ≤280, 4:00-6:00 P.M. ≤210; Supine position: 8:00-10:00 A.M. 30-160 Aldosterone/renin 28.78 activity ratio [ARR] Angiotensin I 2.52 ng/ml (after incubation) Angiotensin I 0.21 ng/ml (before incubation) Plasma Renin 0.77 ng/ml/h 0.25-5.82 Activity [PRA]

[0084] Based on the above detection results of the sample, ARR was less than the cut-off value (30) of disease diagnosis; therefore, the patient in the sample was judged as negative. But, in combination with the screening results of antihypertensive medicaments of the patient in Table 4, ARB and CCB medicaments were detected as positive, that is, both might lead to a false negative result. Through comprehensive assessment, the ARR result of the patient sample was lower than the cut-off value, the patient also might be judged as positive, and recommended to receive a further experiment for confirming diagnosis.

TABLE-US-00007 TABLE 6 Patient's medicament screening result Influence Screening Name of Influence on on Renin Influence Medicament factors results medicament Aldosterone Activity onARR β-receptor blocker − No ↓ ↓↓ ↑(False positive) Centralα2receptor agonist − No ↓ ↓↓ ↑(False positive) NSAID − No ↓ ↓↓ ↑(False positive) Potassium-removing diuretics − No .fwdarw.↑ ↑↑ ↓(False negative) Potassium-retaining diuretics − No ↑ ↑↑ ↓(False negative) ACEI − No ↓ ↑↑ ↓(False negative) ARBs + Valsartan ↓ ↑↑ ↓(False negative) Dihydropyridine CCBs + Amlodipine .fwdarw.↓ ↑ ↓(False negative)

[0085] The patient was definitely diagnosed with primary aldosteronism via a Captopril experiment for a confirmation diagnosis.

EXAMPLE 3: DETECTION AND CONFIRMATION OF FALSE NEGATIVE CLINICAL SAMPLES

[0086] In this example, clinical samples were detected for PA screening; and the detection method used in Example 1 was taken for ARR detection and qualitative judgment; and the detection results of the clinical samples were shown in Table 7.

TABLE-US-00008 TABLE 7 Detection results of clinical samples Unit of Reference Item Result measurement interval Aldosterone 134.32 pg/ml ≥18 years old: [ALD] Upright position: 8:00-10:00 A.M. ≤280, 4:00-6:00 P.M. ≤210; Supine position: 8:00-10:00 A.M. 30-160 Aldosterone/renin 33.58 activity ratio [ARR] Angiotensin I (after 1.20 ng/ml incubation) Angiotensin I (before <0.2 ng/ml incubation) Plasma Renin 0.40 ng/ml/h 0.25-5.82 Activity [PRA]

[0087] Based on the above detection results of the sample, ARR was higher than the cut-off value (30) of disease diagnosis; therefore, the sample was judged as positive. But, in combination with the screening results of antihypertensive medicaments in Table 8, the β-receptor blocker and central α2 receptor agonist were detected as positive, while both might lead to a false negative result. Through comprehensive assessment, the ARR result of the patient sample was higher than the cut-off value, the patient also might be judged as negative, and recommended to receive a further experiment for a definite diagnosis.

TABLE-US-00009 TABLE 8 Patient's medicament screening result Influence Screening Name of Influence on on renin Influence Medicament factors results medicament aldosterone activity on ARR β-receptor blocker + Metoprolol ↓ ↓↓ ↑(False positive) Centralα2receptor agonist + Methyldopa ↓ ↓↓ ↑(False positive) NSAID − − ↓ ↓↓ ↑(False positive) Potassium-removing diuretics − − .fwdarw.↓ ↑↑ ↓(False negative) Potassium-retaining diuretics − − ↑ ↑↑ ↓(False negative) ACEI − − ↓ ↑↑ ↓(False negative) ARBs − − ↓ ↑↑ ↓(False negative) Dihydropyridine CCBs − − .fwdarw.↓ ↑ ↓(False negative)

[0088] The patient was diagnosed as negative after Captopril confirmation test.

EXAMPLE 4: DETECTION AND CONFIRMATION OF POSITIVE CLINICAL SAMPLES

[0089] In this example, clinical samples were detected for PA screening; and the detection method used in Example 1 was taken for ARR detection and qualitative judgment; and the detecting results of the clinical samples were shown in Table 9.

TABLE-US-00010 TABLE 9 Detection results of the clinical samples Unit of Reference Item Result measurement interval Aldosterone 201.53 pg/ml ≥18 years old: [ALD] Upright position: 8:00-10:00 A.M. ≤280, 4:00-6:00 P.M. ≤210; Supine position: 8:00-10:00 A.M.30-160 Aldosterone/renin 49.15 activity ratio [ARR] Angiotensin I (after 1.58 ng/ml incubation) Angiotensin I (before 0.35 ng/ml incubation) Renin Renin 0.41 ng/ml/h 0.25-5.82 Activity[PRA]

[0090] Based on the above detection results of the sample, ARR was higher than the cut-off value (30); therefore, the patient was judged as positive. Meanwhile, in combination with the screening results of antihypertensive medicaments of the patient in Table 10, ARB and potassium-removing diuretics were detected as positive, that is, both might lead to a false negative result of the detected ARR. Through comprehensive assessment, positive ARB and potassium-removing diuretics might lead to false negative, while the ARR result was still higher than the cut-off value, the patient might be judged as a positive sample, therefore the patient was recommended to receive a further confirmation experiment.

TABLE-US-00011 TABLE 10 Patient's medicament screening result Screening Name of Influence on Influence on Influence Medicament factors results medicament aldosterone renin activity on ARR β-receptor blocker − − ↓ ↓↓ ↑(False positive) Centralα2receptor agonist − − ↓ ↓↓ ↑(False positive) NSAID − − ↓ ↓↓ ↑(False positive) Potassium-removing diuretics + Hydrochlorothiazide .fwdarw.↑ ↑↑ ↓(False negative) Potassium-retaining diuretics − − ↑ ↑↑ ↓(False negative) ACEI − − ↓ ↑↑ ↓(False negative) ARBs + Candesartan ↓ ↑↑ ↓(False negative) Dihydropyridine CCBs − − .fwdarw.↓ ↑ ↓(False negative)

[0091] Even though the present invention is disclosed above, the present invention is not limited thereto. A person skilled in the art can make various alterations and modifications within the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be subjected to the scope defined by the claims.