CN117129605B - Method for detecting 11 antihypertensive drugs and 3 metabolites by liquid chromatography-tandem mass spectrometry - Google Patents

Abstract

The invention relates to the technical field of medicine detection, in particular to a method for detecting 11 antihypertensive medicines and 3 metabolites by using a liquid chromatography-tandem mass spectrometry method. 11 antihypertensive agents and 3 metabolites include: captopril, enalapril, benazepril, benazeprilat, fosinopril, fosinoprilat, ramipril, quinapril, zofenopril, perindopril, imidapril, cilazapril, and lisinopril, the method comprising: and preparing a standard curve equation, preprocessing a sample to be detected and detecting on-machine. The invention can detect 11 antihemorrhagic drugs and 3 active metabolites at one time, has short pretreatment time and detection time, and remarkably improves detection efficiency. The detection method has the advantages of good precision, good repeatability, high sample recovery rate, wide linear range and remarkable improvement of the sensitivity and accuracy of the detection result.

Description

Method for detecting 11 antihypertensive drugs and 3 metabolites by liquid chromatography-tandem mass spectrometry

Technical Field

The invention relates to the technical field of medicine detection, in particular to a method for detecting 11 antihypertensive medicines and 3 metabolites by using a liquid chromatography-tandem mass spectrometry method.

Background

The Angiotensin Converting Enzyme Inhibitor (ACEI) has the effects of lowering blood pressure, delaying and reversing ventricular remodeling, preventing further development of cardiac hypertrophy, improving vascular endothelial function and cardiac function, reducing arrhythmia, improving survival rate, and improving prognosis. Clinically commonly used ACEI are cilazapril, captopril, enalapril (enalapril), benazepril (benazeprilat), fosinopril (fosinoprilat), ramipril, quinapril, zofenopril, perindopril, imidapril, lisinopril.

Cilazapril inhibits the renin-angiotensin-aldosterone system, thereby inhibiting the conversion of angiotensin i to angiotensin ii having a strong vasoconstrictor effect, causing a decrease in peripheral vascular resistance and a decrease in blood pressure. The etanercept is used for primary hypertension and renal hypertension, has obvious curative effect on patients with renal diseases and hypertension, has a certain protection effect on kidneys, can be used for various heart failure, and can improve heart function and delay myocardial remodeling. Quinapril is a long-acting non-mercaptoangiotensin converting enzyme inhibitor useful in the treatment of hypertension and congestive heart failure.

Hypertension can cause cerebral vascular atherosclerosis, cerebral ischemia, cerebral hemorrhage, cerebral infarction and other diseases, and controlling hypertension can effectively reduce the death rate of cardiovascular diseases, so that the blood concentration of the hypertension drugs of angiotensin converting enzyme inhibitors is necessary to be detected.

Disclosure of Invention

In order to solve the technical problems, the invention provides a method for simultaneously detecting 11 antihypertensive drugs and 3 metabolites by using a liquid chromatography-tandem mass spectrometry method.

The invention provides a method for detecting 11 antihypertensive drugs and 3 metabolites in blood by liquid chromatography tandem mass spectrometry, wherein the 11 antihypertensive drugs and the 3 metabolites comprise the following components: captopril, enalapril, benazepril, benazeprilat, fosinopril, fosinoprilat, ramipril, quinapril, zofenopril, perindopril, imidapril, cilazapril, and lisinopril, the method comprising at least the steps of:

s1, preparing a standard curve equation, which comprises the following steps:

preparing an internal standard working solution and a standard working solution, preparing a sample solution for standard yeast, and detecting the sample solution for standard yeast by using a high performance liquid chromatography-mass spectrometer to obtain a standard curve equation for calculating the content of 11 antihypertensive drugs and metabolites;

S2, preprocessing a sample to be tested, including:

adding formic acid and 2-10 mol/L dithiothreitol solution into plasma to be measured to obtain a sample to be measured, uniformly mixing an internal standard working solution and the sample to be measured, adding a protein precipitant, uniformly mixing, centrifuging to obtain a supernatant, adding a diluent into the supernatant, and uniformly mixing to obtain a sample injection sample;

s3, detecting a sample injection sample, which comprises the following steps:

and detecting the sample by using a high performance liquid chromatography-mass spectrometer, and substituting the detection result of the sample into a standard curve equation to obtain the contents of 11 antihypertensive drugs and 3 metabolites in the sample to be detected.

Optionally, S1 includes:

s11, respectively preparing an internal standard working solution and a standard working solution with gradient concentration;

s12, respectively taking an internal standard working solution and a standard working solution, adding water, uniformly mixing to obtain a standard solution with gradient concentration, adding a protein precipitant, uniformly mixing to obtain a mixed solution, adding a diluent into the mixed solution, and uniformly mixing to obtain a sample for standard yeast;

s13, detecting the sample loading solution for the standard curve by using a high performance liquid chromatography mass spectrometer to obtain a standard curve equation.

Optionally, the sample to be tested is a plasma sample or a serum sample, and the volume is 50-100 mu L; in S12, the sum of the volumes of the standard working fluid and the water is equal to the volume of the sample to be measured.

Optionally, the internal standard working fluid contains isotope internal standards of 10 antihypertensive drugs and 3 active metabolites.

Alternatively, the conditions of the high performance liquid phase when detected by using the high performance liquid chromatography mass spectrometer are as follows: the chromatographic column adopts a T3 chromatographic column; mobile phase: the phase A is an aqueous solution containing 0.05-0.2% formic acid and 1-10 mmol/L ammonium acetate by volume percent, and the phase B is acetonitrile; flow rate: 0.4-0.5 mL/min; column temperature: 35-45 ℃; sample injection amount: 5-20 mu L; analysis time: and 6 min.

Compared with the prior art, the technical scheme provided by the embodiment of the invention has the following advantages:

the invention can detect 11 antihemorrhagic drugs and 3 active metabolites at one time, has short pretreatment time, can finish pretreatment within 12-22 min, has short detection time, and can obtain detection results within 6 min after being on machine. The instrument can analyze 10 groups of samples within 1 hour, and the detection efficiency is remarkably improved.

The pretreatment method is simple in operation, and aiming at the stability of captopril, fosinopril and fosinopril, formic acid and dithiothreitol solution are added into blood plasma to serve as stabilizers, then protein is precipitated, and the stabilizers are added into the precipitant to ensure accurate detection of the protein.

The detection method has the advantages of good precision, good repeatability, high sample recovery rate, wide linear range and remarkable improvement of the sensitivity and accuracy of the detection result.

Drawings

FIG. 1 is a chromatogram of each substance in the standard solution in example 1;

FIG. 2 is a chromatogram of each of the substances in the labeled plasma sample of example 1;

FIG. 3 is a chromatogram of comparative example 4 with methanol as the mobile phase;

FIG. 4 is a chromatogram of comparative example 4 with acetonitrile as the mobile phase;

FIG. 5 is a chromatogram of elution mode 1 in comparative example 5;

FIG. 6 is a chromatogram of elution mode 2 in comparative example 5;

FIG. 7 is a chromatogram of elution mode 3 in comparative example 5;

FIG. 8 is a chromatogram of the elution mode of example 1 of the present invention;

FIG. 9 is a chromatogram of mode 1 of comparative example 6;

FIG. 10 is a chromatogram of mode 2 of comparative example 6;

FIG. 11 is a chromatogram of mode 3 of comparative example 6;

FIG. 12 is a chromatogram of mode 4 of comparative example 6;

FIG. 13 is a chromatogram after sample injection of quinapril Li Shan of comparative example 7;

FIG. 14 is a chromatogram of comparative example 7 after sample injection of the imidacloprid Li Shan standard;

fig. 15 is a chromatogram after single sample injection of fosinopril in comparative example 7.

Detailed Description

In order that the above objects, features and advantages of the invention will be more clearly understood, a further description of the invention will be made. It should be noted that, without conflict, the embodiments of the present invention and features in the embodiments may be combined with each other. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced otherwise than as described herein; it will be apparent that the embodiments in the specification are only some, but not all, embodiments of the invention.

The embodiment of the invention provides a method for detecting 11 angiotensin converting enzyme inhibitor antihypertensive drugs and 3 active metabolites by adopting a pretreatment method of a protein precipitation method, wherein the 11 antihypertensive drugs are as follows: quinapril, zofenopril, fosinopril, perindopril, captopril, imidapril, cilazapril, enalapril, benazepril, ramipril, and lisinopril, the 3 active metabolites are: fosinoprilat, benazeprilat, and enalaprilat. Compared with the prior art, the pretreatment method provided by the embodiment of the invention is simpler, faster, easy to operate, economical and applicable.

Captopril, because it has a thiol (-SH), is unstable in plasma and loses the characteristic peak of the substance, derivatization is required in the prior art for detecting captopril. According to the embodiment of the invention, repeated researches show that when formic acid and dithiothreitol are added into blood plasma as stabilizers, methanol is adopted as a protein precipitant, and the accurate detection of captopril, fosinopril and fosinopril can be ensured after the formic acid and the dithiothreitol are added simultaneously.

Specifically, the method for detecting 11 antihypertensive drugs and 3 metabolites in blood by using the liquid chromatography-tandem mass spectrometry provided by the embodiment of the invention at least comprises the following steps:

s1, preparing a standard curve equation, which comprises the following steps:

preparing an internal standard working solution and a standard working solution, preparing a sample solution for standard yeast, and detecting the sample solution for standard yeast by using a high performance liquid chromatography-mass spectrometer to obtain a standard curve equation for calculating the contents of 11 antihypertensive drugs and 3 metabolites;

s2, preprocessing a sample to be tested, including:

adding formic acid and 2-10 mol/L dithiothreitol solution into plasma to be measured to obtain a sample to be measured, uniformly mixing an internal standard working solution and the sample to be measured, adding a protein precipitant, uniformly mixing, centrifuging to obtain a supernatant, adding a diluent into the supernatant, and uniformly mixing to obtain a sample injection sample; wherein the protein precipitant is methanol containing formic acid with the volume percentage concentration of 0.05-0.2% and dithiothreitol with the volume percentage concentration of 2-20 mM; the diluent is an aqueous solution containing 2-20 mM dithiothreitol;

s3, detecting a sample injection sample, which comprises the following steps:

and detecting the sample by using a high performance liquid chromatography-mass spectrometer, and substituting the detection result of the sample into a standard curve equation to obtain the contents of 11 antihypertensive drugs and 3 metabolites in the sample to be detected.

As an improvement of the embodiment of the invention, formic acid and 2-10 mol/L dithiothreitol solution are added into the plasma to be tested as stabilizers. Wherein the concentration of formic acid is 99.6%, and the solvent of dithiothreitol solution is water. Adding a stabilizer to enable the plasma to be tested to contain 0.1-0.2% formic acid and 2-20 mM dithiothreitol by volume percent. Further optimizing to make the volume percentage of formic acid and 5 mM dithiothreitol contained in the plasma to be tested be 0.1 percent.

As an improvement of the embodiment of the invention, the protein precipitant adopts methanol containing formic acid with the volume percentage concentration of 0.05-0.2% and dithiothreitol with the volume percentage concentration of 2-20 mM, and further preferably 0.1% of formic acid and 5 mM of dithiothreitol are added. Under the addition proportion, the detection accuracy of captopril, fosinopril and fosinopril is further improved, and the stability of the captopril, fosinopril and fosinopril is ensured.

As an improvement of the embodiment of the invention, the diluent is an aqueous solution containing 2-20 mM dithiothreitol, and 5 mM dithiothreitol is further preferable and added. Under the addition proportion, the stability of captopril can be ensured, and the consumption of the reducing agent is not excessive, so that the cost is saved.

As an improvement of the embodiment of the invention, the matrix is not added in the preparation process of the standard yeast, and the pretreatment of the matrix is not needed, so that the preparation steps are simplified. Specifically, the standard yeast can be prepared by the following method:

S11, respectively preparing an internal standard working solution and a standard working solution with gradient concentration;

s12, respectively taking an internal standard working solution and a standard working solution, adding water, uniformly mixing to obtain a standard solution with gradient concentration, adding a protein precipitant, uniformly mixing to obtain a mixed solution, adding a diluent into the mixed solution, and uniformly mixing to obtain a sample for standard yeast;

s13, detecting the sample loading solution for the standard curve by using a high performance liquid chromatography mass spectrometer to obtain a standard curve equation.

The internal standard working solution in the embodiment of the invention contains the isotope internal standard of 10 antihypertensive drugs and 3 metabolites, realizes accurate quantification, effectively avoids matrix effect and has good recovery rate. The specific method can be as follows: the isotope internal standard of captopril is captopril-d ₃ The method comprises the steps of carrying out a first treatment on the surface of the Isotopes of enalaprilThe internal standard is enalapril-d ₅ The method comprises the steps of carrying out a first treatment on the surface of the The isotopic internal standard of enalapril is enalapril-d ₅ The method comprises the steps of carrying out a first treatment on the surface of the Isotopic internal standard of cilazapril and benazepril is benazepril-d ₅ The method comprises the steps of carrying out a first treatment on the surface of the The isotopic internal standard of benazeprilat is benazeprilat-d ₅ The method comprises the steps of carrying out a first treatment on the surface of the The isotope internal standard of fosinopril is fosinopril-d ₅ The method comprises the steps of carrying out a first treatment on the surface of the The isotope internal standard of fosinopril is fosinopril-d ₇ The method comprises the steps of carrying out a first treatment on the surface of the The isotope internal standard of ramipril is ramipril-d ₅ The method comprises the steps of carrying out a first treatment on the surface of the The isotope internal standard of quinapril is quinapril-d ₅ The method comprises the steps of carrying out a first treatment on the surface of the The isotope internal standard of zofenopril is zofenopril-d ₅ The method comprises the steps of carrying out a first treatment on the surface of the The isotopic internal standard of perindopril is perindopril-d ₄ The method comprises the steps of carrying out a first treatment on the surface of the The isotope internal standard of the imidapril is imidapril-d ₅ The method comprises the steps of carrying out a first treatment on the surface of the The isotopic internal standard of lisinopril is lisinopril-d ₅ 。

As an improvement of the embodiment of the invention, the preparation method of the internal standard working solution comprises the following steps:

respectively taking the internal standard substances, preparing standard stock solutions by adopting methanol, then respectively taking 13 standard stock solutions, taking a proper amount of standard stock solutions, mixing, diluting by using 70% methanol aqueous solution to obtain an internal standard working solution, and preserving at the temperature of minus 20 ℃. Further preferably, the concentration of each internal standard substance in the internal standard working fluid is as shown in table 1:

table 1: concentration of each internal standard substance in the internal standard working solution

As an improvement of the embodiment of the invention, the preparation method of the standard working solution with gradient concentration comprises the following steps:

respectively taking the 14 standard substances to be tested, preparing a standard stock solution by adopting methanol, then respectively taking 14 standard stock solutions, mixing a proper amount of standard stock solutions, diluting with 70% methanol aqueous solution to obtain a standard working solution, and preserving at the temperature of minus 20 ℃. Further preferably, the concentration of each standard in the standard working solution with gradient concentration is shown in table 2:

Table 2: concentration of each standard substance in standard working solution with gradient concentration

As an improvement of the embodiment of the invention, in S2, the volume ratio of the plasma to be measured, formic acid and dithiothreitol solution is 1000:0.5 to 1.5:0.5 to 1.5, more preferably 1000:1:1.

as an improvement of the embodiment of the present invention, in S12: the volume ratio of the standard working solution to the internal standard working solution is 1:2.

as an improvement of the embodiment of the present invention, in S12: the volume ratio of the standard working solution to the water is 1:9, a step of performing the process; the water is used for replacing the matrix, and experiments prove that the matrix effect investigation of the matrix-free matrix and the matrix target added with different proportions is qualified. Therefore, water can be used for replacing matrix in the process of preparing standard yeast, so that the pretreatment flow is optimized, and the time is saved.

As an improvement of the embodiment of the present invention, in S12: the volume ratio of the sum of the volumes of the standard working solution and the water to the volume ratio of the protein precipitant is 1: 10-12. At the same time in S2: the volume ratio of the sample to be tested to the protein precipitant is 1: 10-12. The protein precipitant has qualified target recovery rate under the condition of the proportion, can reduce the influence of matrix, and does not oversaturate the instrument response. Too much precipitation ratio can result in partial target object with low response, which cannot meet detection requirements, and too little ratio can saturate signals, which affects quantification.

As an improvement of the embodiment of the present invention, in S12, the volume ratio of the mixed solution to the diluent is 1: 1-3: 2, s2, the volume ratio of supernatant to diluent is 1: 1-3: 2. the embodiment of the invention reduces the solvent effect through dilution, and simultaneously ensures that the response of the target object in the instrument cannot be supersaturated. The reason for this ratio is that each target has a good signal response at this ratio. Too much dilution can result in partial target object with low response, which cannot meet the detection requirement, and too little dilution can saturate signals, which affects quantification.

As an improvement of the embodiment of the present invention, in S12, the volume ratio of the sum of the volumes of the standard working solution and the water to the volume ratio of the internal standard working solution is 5:1, a step of; in S2, the volume ratio of the sample to be detected to the internal standard working solution is 5: and 1, the sum of the volumes of the standard working solution and water is equal to the volume of the sample to be tested.

As an improvement of the embodiment of the invention, the detection method of the embodiment of the invention can detect the plasma sample and the serum sample. Specifically, the volume of the blood sample is 50. Mu.L-100. Mu.L, and 50. Mu.L can be used. According to the embodiment of the invention, the 50 mu L blood sample can be used for simultaneously and accurately quantitatively detecting 14 substances.

As an improvement of the embodiment of the present invention, in S2: the mixing condition of the internal standard working solution and the sample to be measured is 1500-2500 r/min vortex mixing for 30 s-1 min, preferably 2500-r/min vortex mixing for 1 min; s12: the mixing condition of the internal standard working solution, the sample to be tested and the water is 1500-2500 r/min vortex mixing for 30 s-1 min, preferably 2500-r/min vortex mixing for 1 min.

As an improvement of the embodiment of the present invention, in S2 and S12: the conditions for mixing after adding the protein precipitant are as follows: vortex mixing for 5-10 min at 1500-2500 r/min; the centrifugation conditions are 12000-15000 r/min for 5-12 min.

As an improvement of the embodiment of the present invention, in S2 and S12: the conditions for mixing after adding the protein precipitant are as follows: 2500 Vortex mixing for 5 min at r/min; the centrifugation condition is 14000 r/min for 5 min.

As an improvement of the embodiment of the present invention, in S2 and S12: mixing conditions after adding the diluent are 1500-2500 r/min vortex mixing for 30 s-1 min, preferably 2500-r/min vortex mixing for 1 min.

Therefore, the pretreatment can be completed in the embodiment of the invention with the main duration of 5-10 min of mixing and 5-12 min of centrifugation, and the total of 12-22 min of two times of vortex mixing time of 30 s-1 min.

The 11 antihypertensive drugs and 3 active metabolites tested in the examples of the present invention have a plurality of structural analogs, for example: enalapril and Enalaprilat have the following structural formulas respectively;

；/>；

benazepril and benazeprilat have the following structural formulas:

；

fosinopril and fosinoprilat, the structural formulas are as follows:

；/>；

The compounds to be tested, except the above drugs and their active metabolites, have similar structures:

；/>；/>；

captopril Li Lainuo Pr Li Leimi Pr

；/>；

Cilazapril perindopril

；/>；/>。

Quinapril Li Mida pralidoxime Li Zuofen pralidoxime

Therefore, in the embodiment of the invention, when 14 target objects are detected, chromatographic separation is needed to be realized on the structural analogues, so that ion crosstalk is avoided, and the detection accuracy is influenced. Therefore, the embodiment of the invention improves the high performance liquid chromatography condition. Specifically, the high performance liquid chromatography column used in the detection by the high performance liquid chromatography mass spectrometer adopts a T3 chromatography column, preferably a Waters CORTES T3 chromatography column, the particle size is 2.7 μm, the diameter is 3.0 mm, and the column length is 100 mm.

As an improvement of the embodiment of the invention, the mobile phase in the high performance liquid phase is detected by a high performance liquid chromatography mass spectrometer: the phase A is an aqueous solution containing 0.05-0.2% formic acid and 1-10 mmol/L ammonium acetate by volume percent, and the phase B is acetonitrile. Further preferred, the mobile phase: phase A is aqueous solution containing 0.1% formic acid and 5 mmol/L ammonium acetate by volume percentage, and phase B is acetonitrile.

Flow rate: 0.4-0.5 mL/min, preferably 0.4 mL/min;

Column temperature: 35-45 ℃, preferably 40 ℃;

sample injection amount: 5-20 mu L, preferably 10 mu L;

analysis time: 6.00 And (5) min.

As an improvement of the embodiment of the invention, the gradient elution condition of the high performance liquid phase during detection by using the high performance liquid chromatography mass spectrometer is as follows:

0-0.20 min, A:90%, B:10%;

0.20-0.30 min, A:90% -60%, B:10% -40%;

0.30-0.80 min, A:60%, B:40%;

0.80 to 0.90 minutes, A:60% -40%, B:40% -60%;

0.90-1.50 minutes, A:40%, B:60 percent;

1.51-2.50 minutes, A:20%, B:80%;

2.51-4.50 minutes, A:2%, B:98 percent;

4.51-4.60 minutes, A:2% -90%, B:98% -10%;

4.60 to 6.00 minutes, A:90%, B:10%.

By adopting the high performance liquid chromatography condition of the embodiment of the invention, 14 target objects can be detected simultaneously within 6 minutes, the analysis time is short, and the method is more beneficial to the detection of samples with large flux.

As an improvement of the embodiment of the invention, the mass spectrum conditions when the high performance liquid chromatography-mass spectrometer is used for detection are as follows:

adopting an electrospray ion source, a positive ion mode and multi-reaction monitoring;

atomizing gas flow rate: 3.0 L/min;

DL temperature: 250 ℃;

Drying gas temperature: 400 ℃;

drive gas flow rate: 10 L/min;

heating air flow rate: 10 L/min;

interface temperature: 300 ℃;

ion pairs expressed as parent/daughter ions are:

quantitative ion pair of quinapril: 439.30/117.15 qualitative ion pair of quinapril: 439.30/234.20, ion pair of quinapril internal standard: 444.30/239.20;

quantitative ion pair of perindopril: 369.30/172.35; qualitative ion pair of perindopril: 369.30/98.20; ion pair of perindopril internal standard: 373.30/176.35;

quantitative ion pair of fosinopril: 436.00/390.05 qualitative ion pair of fosinopril: 564.30/436.20 ion pair of fosinopril internal standard: 441.00/395.05;

quantitative ion pair of captopril: 218.10/116.10, qualitative ion pair of captopril: 218.10/70.15, ion pair of captopril internal standard: 221.10/73.15;

quantitative ion pair of imidapril: 406.20/234.20 qualitative ion pair of imidapril: 406.20/117.10, ion pair of the imidapril internal standard: 411.20/239.20;

quantitative ion pair of zofenopril: 430.10/105.10, qualitative ion pair of zofenopril: 430.10/280.15, ion pair of zofenopril internal standard: 435.10/110.10;

Quantitative ion pair of fosinoprilat: 436.20/109.15 qualitative ion pair of fosinoprilat: 436.20/418.20, ion pair of fosinoprilat internal standard: 443.15/153.15;

quantitative ion pair of cilazapril: 418.35/211.15, qualitative ion pair of cilazapril: 418.35/70.15;

quantitative ion pair of enalapril: 377.30/234.25, qualitative ion pair of enalapril: 377.30/117.20, ion pair of enalapril internal standard: 382.30/239.25;

quantitative ion pair of enalaprilat: 349.10/206.40, qualitative ion pair of enalaprilat: 349.10/117.15, ion pair of enalapril Li Lana standard: 354.20/211.15;

quantitative ion pair of benazepril: 425.30/351.15, qualitative ion pair of benazepril: 425.30/190.10, ion pair of benazepril internal standard: 430.30/356.15;

quantitative ion pair of benazeprilat: 397.30/351.15, qualitative ion pair of benazeprilat: 397.30/118.15, benazepril Li Lana target ion pair: 402.30/356.15;

quantitative ion pair of ramipril: 417.30/234.20, qualitative ion pair of ramipril: 417.30/117.15 ion pairs of ramipril internal standard: 422.30/239.20;

quantitative ion pair of lisinopril: 406.30/84.15 qualitative ion pair of lisinopril: 406.30/246.20, ion pair of lisinopril internal standard: 411.30/84.30.

Fosinopril and fosinoprilat have the same ion pair 436.00/390.05, there is ion cross talk. Thus, fosinoprilat was quantified using 436.20/109.15, 436.20/418.20;

quinapril, imidapril, fosinopril interferes with the captopril 218.1-70.15 ion pair. Thus, captopril was quantified using a 218.1/116.1 ion pair.

The detection method provided by the embodiment of the invention has the advantages of good precision, good repeatability, high sample recovery rate, wide linear range, coverage of the reference value range and the warning value, and simultaneously, the sensitivity and the accuracy of the detection result are obviously improved.

Example 1

The embodiment provides a liquid chromatography analysis method for detecting the contents of 11 antihypertensive drugs and 3 active metabolites in blood plasma, which comprises the following steps:

calibration of standard solution

1.1 preparation of standard working solution:

accurately weighing a captopril standard substance 3.631 mg, and dissolving the captopril standard substance 3.631 mg in 1.816 mL methanol solution to obtain a standard stock solution A; accurately weighing quinapril hydrochloride standard substance 6.768 mg, and dissolving in 3.061 mL methanol solution to obtain standard stock solution B; precisely weighing 2.825 mg of fosinopril sodium standard, and dissolving in 1.350 mL methanol solution to obtain a standard stock solution C; accurately weighing a fosinopril standard substance 5.448 mg, and dissolving the fosinopril standard substance 5.448 mg in 5.099 mL methanol solution to obtain a standard stock solution D; accurately weighing a perindopril tert-butylamine standard substance 2.061 mg, and dissolving in 0.854 mL methanol solution to obtain a standard stock solution E; accurately weighing a imidapril hydrochloride standard substance 3.591 mg, and dissolving the imidapril hydrochloride standard substance in a 1.647-mL methanol solution to obtain a standard stock solution F; accurately weighing zofenopril calcium standard 4.742 mg, and dissolving the zofenopril calcium standard 4.742 mg in 2.220 mL methanol solution to obtain a standard stock solution G; precisely weighing a cilazapril standard 2.192 mg, and dissolving in 2.130 mL methanol solution to obtain a standard stock solution H; precisely weighing benazepril hydrochloride 4.456 mg, and dissolving in 4.095 mL methanol solution to obtain a standard stock solution I; precisely weighing benazeprilat hydrochloride standard 7.560 and mg, and dissolving in 7.544 mL methanol solution to obtain standard stock solution J; accurately weighing an enalapril hydrochloride standard substance 5.384 mg, and dissolving the enalapril hydrochloride standard substance 5.384 mg in 4.107 mL methanol solution to obtain a standard stock solution K; accurately weighing enalapril 9.482 mg, and dissolving in 4.177 mL methanol solution to obtain a standard stock solution L; precisely weighing ramipril standard 6.400 mg, and dissolving the ramipril standard 6.400 mg in 3.184 mL methanol solution to obtain standard stock solution M; precisely weighing lisinopril standard 5.963 mg, and dissolving in 5.408 mL methanol solution to obtain standard stock solution N; an appropriate amount of standard stock solution A, B, C, D, E, F, G, H, I, J, K, L, M, N was diluted with 70% aqueous methanol, and the concentrations of the respective standards in the obtained standard working solutions were shown in table 2.

1.2 preparation of internal standard working solution:

captopril-d ₃ Internal standard 5 mg, dissolved in 2.464 mL methanol solution, yielded standard stock solution 1; quinapril-d ₅ Dissolving an internal standard 5 mg in 2.282 mL diluent solution to obtain a standard stock solution 2; fosinopril-d ₅ Internal standard 5 mg, dissolved in 2.347 mL dilution solution, gave standard stock 3; fosinoprilat-d ₇ Internal standard 1 mg is dissolved in 0.98 mL diluent solution to obtain standard stock solution 4; taking perindopril-d ₄ Dissolving an internal standard 5 mg in 2.459 mL methanol solution to obtain a standard stock solution 5; taking Midapril-d ₅ Internal standard 5 mg, dissolved in 2.270 mL methanol solution, yielded standard stock solution 6; taking zofenopril-d ₅ Internal standard 5 mg, dissolved in 2.498 mL methanol solution, gave standard stock 7; taking benazepril-d ₅ Dissolving an internal standard 5 mg in 2.291 mL methanol solution to obtain a standard stock solution 8; taking benazeprilat-d ₅ Internal standard 1 mg, dissolved in 0.986 mL diluent solution, gives standard stock solution 9; taking enalapril-d hydrochloride ₅ Internal standard 5 mg, dissolved in 1.879 mL diluent solution, yielded standard stock solution 10; taking enalaprilat-d ₅ Internal standard 1 mg, dissolved in 0.921 mL dilution solution to give standard stock solution 11; ramipril-d ₅ Internal standard 5 mg, dissolved in 2.492 mL methanol solution, yielded standard stock solution 12; lisinopril-d ₅ Dissolving an internal standard 5 mg in 2.399 mL methanol solution to obtain a standard stock solution 13; appropriate amounts of standard stock solutions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 were diluted with 70% aqueous methanol, and the concentrations of the internal standards in the obtained internal standard working solutions were as shown in table 1. The internal standard working solution is preserved at-20deg.C.

1.3 standard yeast preparation:

firstly, respectively transferring 5 mu L of standard working solution, 10 mu L of internal standard working solution and 45 mu L of standard working solution by a pipette, respectively placing the standard working solution and the 45 mu L of internal standard working solution in a 1.5 mL centrifuge tube, mixing the standard solutions into eight standard solutions with different concentrations by vortex at the rotating speed of 2500 rpm for 1 min, adding 500 mu L of protein precipitant (methanol containing 0.1% formic acid and 5 mM dithiothreitol), mixing the mixed solution by vortex at the rotating speed of 2500 rpm for 5 min, sucking 150 mu L of mixed solution, adding 100 mu L of diluent (water containing 5 mM dithiothreitol) and mixing the mixed solution by vortex for 1 min to obtain a sample for standard curve.

Detecting sample injection samples for standard curve by using a high performance liquid chromatography-mass spectrometer to obtain standard solution chromatograms of 14 targets and internal standards with eight different concentrations, respectively obtaining peak areas of the targets and the internal standards from the standard solution chromatograms of the targets and the internal standards, and respectively taking the ratio of the peak areas of the standard solutions of the 14 targets with eight different concentrations to the peak areas of the internal standards as the ordinate y of a standard curve equation ₁ The ratio of the concentration in the 14 target standard working fluids to the concentration of the internal standard working fluid is taken as the abscissa x of the standard curve equation ₁ Performing linear regression on the eight data with different concentrations obtained by the detection, and fitting to obtain a standard curve equation of y ₁ =a*x ₁ +b, and yields linear equation coefficients a, b.

(II) sample treatment to be measured

And taking 500 mu L of plasma to be tested, and respectively adding 0.5 mu L of formic acid and 5 mol/L of dithiothreitol aqueous solution to obtain a sample to be tested. Transferring 50 mu L of a sample to be detected by a pipetting gun into a 1.5 mL centrifuge tube, adding 10 mu L of an internal standard working solution, mixing for 1 min by vortex at 2500 rpm, adding 500 mu L of a protein precipitant (methanol containing 0.1% formic acid and 5 mM dithiothreitol), mixing for 5 min by vortex at 2500 rpm, centrifuging for 5 min at 14000 r/min, absorbing 150 mu L of supernatant, adding 100 mu L of a diluent (water containing 5 mM dithiothreitol), and mixing for 1 min by vortex to obtain the sample to be detected;

(III) detection of sample to be tested

The step (II) was performed by using a high performance liquid chromatography mass spectrometer) Detecting the sample to be detected to obtain chromatograms of 14 target objects and internal standard in the sample to be detected, and obtaining the ratio y of the 14 target objects to the internal standard peak area from the chromatograms of the 14 target objects and the internal standard ₁ Substituting the standard curve equation y of the step (one) ₁ =a*x ₁ In +b, the relative concentration x of 14 targets and internal standard in the sample to be detected is obtained by calculation ₁ The concentration of the internal standard working fluid is known, and thus the concentration of 14 target substances in the plasma sample to be detected is calculated.

The chromatographic column used for the chromatographic analysis was Waters CORTES T3 (2.7 μm, 3.0X100 mm); mobile phase: phase A is an aqueous solution containing 5 mmol/L ammonium acetate and 0.1% formic acid, and phase B is acetonitrile.

Gradient elution is shown in Table 3, the flow rate is 0.4 mL/min, the column temperature is 40 ℃, and the sample injection amount is 10 mu L; analysis time was 6 min.

Table 3: gradient elution conditions

Fig. 1 is a chromatogram of each substance in a standard solution, and fig. 2 is a chromatogram of each substance in a labeled plasma sample. Quinapril has a retention time of 2.617 min, zofenopril has a retention time of 3.134 min, perindopril has a retention time of 2.318 min, captopril has a retention time of 2.033 min, idapril has a retention time of 2.160 min, cilazapril has a retention time of 2.329 min, enalapril has a retention time of 2.241 min, enalapril has a retention time of 1.864 min, benazepril has a retention time of 2.636 min, benazeprilat has a retention time of 1.960 min, ramipril has a retention time of 2.473 min, and lisinopril has a retention time of 1.827 min.

The mass spectrum detector was a shimadzu MS8050 detector, using electrospray ion source (ESI), positive ion mode, multiple Reaction Monitoring (MRM), nebulizing Gas Flow:3.0 L/min; DL Temperature:250 ℃; heat Block Temperature:400 ℃; driving Gas Flow:10 L/min; heating Gas Flow:10 L/min; interface Temperature:300 ℃. The ion pair parameters are shown in table 4:

table 4: ion pair parameters

Example 2

The technical process in example 1 is demonstrated as follows:

1. linearity of the method

The prepared standard working solutions of 14 targets at the concentration of 5. Mu.L were added with 10. Mu.L of the internal standard working solution and 45. Mu.L of pure water, respectively, and the pretreatment was performed as in example 1, and 10. Mu.L to LC-MS/MS analysis was performed.

Quinapril concentration in the range of 10 ng/mL to 5000 ng/mL, zofenopril concentration in the range of 2 ng/mL to 1000 ng/mL, fosinopril concentration in the range of 20 ng/mL to 10000 ng/mL, fosinopril concentration in the range of 20 ng/mL to 10000 ng/mL, perindopril concentration in the range of 10 ng/mL to 5000 ng/mL, captopril concentration in the range of 10 ng/mL to 5000 ng/mL, imidapril concentration in the range of 1 ng/mL to 500 ng/mL, cilazapril concentration in the range of 0.4 ng/mL to 200 ng/mL, enalapril concentration in the range of 2 ng/mL to 1000 ng/mL, enalapril concentration was in the range of 1 ng/mL to 500 ng/mL, benazepril concentration was in the range of 0.5 ng/mL to 250 ng/mL, benazepril concentration was in the range of 2 ng/mL to 1000 ng/mL, lisinopril concentration was in the range of 2 ng/mL to 1000 ng/mL, ramipril concentration was in the range of 0.4 ng/mL to 200 ng/mL, and the concentration was measured from low to high under the measurement conditions of this example, and the ratio of the chromatographic peak area of 14 targets to the chromatographic peak area of the internal standard was plotted as a ratio of the concentration of-14 targets to the concentration of the internal standard, to obtain a standard curve.

The results show that the linearity of each substance to be measured is good and the correlation coefficient R is good within the linear range shown in Table 5 ² ﹥0.99。

Table 5: linear range

2. Quantitative limit and detection limit of example 1 method

Plasma samples with certain concentrations are prepared from 14 standard working solutions of target substances respectively, and are subjected to quantitative limit and detection limit experiments, and the results are shown in Table 6.

Table 6: quantitative limit and detection limit

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3. The recovery rate and precision of the method

Standard working solutions of all substances are respectively prepared into low, medium and high concentrations for sample addition recovery rate experiments and precision experiments, the average recovery rates and the relative standard deviation results of 14 target substances in the ranges of the low, medium and high addition levels are shown in table 7.

Table 7: yield and precision of the addition mark

In combination with the verification test, the recovery rate and the precision of the embodiment 1 meet the technical indexes, and the method is used for detecting the contents of 14 target substances in the blood plasma, has good reproducibility and high sample adding recovery rate, and improves the accuracy of detection results. The plasma is directly sampled after protein precipitation, the detection process is simple, convenient and rapid, the experimental cost is reduced, the analysis time is short, and the method is more beneficial to the detection of samples with large flux.

Example 3

The present example is used to illustrate the results of investigation of the matrix effect:

matrix effect studies were performed using the formulation methods shown in table 8.

Table 8: design of experiments on matrix effects

The experimental results are shown in tables 9 to 22.

Table 9: cilazapril matrix effect investigation results

Table 10: results of investigation of Enalapril Li Jizhi Effect

Table 11: benazepril Li Jizhi effect investigation results

Table 12: ramipril Li Jizhi effect investigation results

Table 13: lisinopu Li Jizhi effect investigation results

Table 14: enalaprilat matrix effect investigation results

Table 15: benazeprilat matrix effect investigation results

Table 16: quinapril Li Jizhi effect investigation results

Table 17: perindopril Li Jizhi effect investigation results

Table 18: captopril matrix effect investigation results

Table 19: midap Li Jizhi effect investigation results

Table 20: zofenopril Li Jizhi effect investigation results

Table 21: fosinopril matrix effect investigation results

Table 22: fosinoprilla matrix effect investigation results

As shown in the experimental results in tables 9-22, the matrix effect investigation of the matrix-free object and the matrix object added with different proportions is qualified. Therefore, water can be used for replacing matrix in the process of preparing the standard yeast, so that the pretreatment flow is optimized, and the time is saved.

Comparative example 1:

the comparative example examined the effect of the selection of the stabilizer on the detection result:

1. the recovery rate of captopril after a certain period of time after different stabilizers were added to the captopril-labeled plasma is shown in table 23:

table 23: recovery of captopril under different stabilizer conditions

2. The recovery of fosinopril after a certain period of time following the addition of various stabilizers to fosinopril-labeled plasma is shown in table 24:

table 24: recovery of fosinopril under different stabilizer conditions

2. The recovery of fosinoprilat after a certain period of time after addition of various stabilizers to fosinoprilat plus standard plasma is shown in table 25:

table 25: recovery of fosinoprilat under different stabilizer conditions

From the experimental results shown in tables 23 to 25, it is clear that for captopril: the sample to be tested is placed 24 and h unstably, and the sample to be tested can be kept 48 h stably by adding a stabilizer to enable the sample to be tested to contain 0.1% formic acid and 5 mM dithiothreitol.

For fosinopril/fosinoprilat: the sample to be tested is placed in 24 and h unstable, and is stabilized by adding formic acid, and when the sample to be tested is monitored simultaneously with captopril, the sample to be tested can be stabilized by adding a stabilizer to enable the sample to be tested to contain 0.1% formic acid and 5 mM dithiothreitol, and 24 h can be stabilized. Only fosinopril/fosinopril was measured, and the test sample was kept stable at 24 h by adding formic acid to make the test sample contain 0.1% formic acid.

Comparative example 2

The comparative example examined the effect of the selection of precipitants on the detection results: 1. the test was conducted in accordance with the method of example 1 except that methanol containing 0.1% formic acid was used as a precipitant, and recovery rate examination results obtained are shown in Table 26:

table 26: results of investigation on recovery of cilazapril/methanol (0.1% formic acid)

2. The test was performed as in example 1, except that methanol was used as the precipitant, and recovery rates were examined as shown in Table 27:

table 27: cilazapril/methanol recovery investigation results

3. The test was performed as in example 1, except that acetonitrile was used as a precipitant, and recovery rates were examined as shown in Table 28:

table 28: cilazapril/acetonitrile recovery rate investigation results

4. The test was carried out as in example 1, except that methanol (5 mM dithiothreitol) was used as a precipitant, and the recovery rate was examined as shown in Table 29:

table 29: fosinopril/methanol (5 mM dithiothreitol) recovery examination results

5. The test was carried out as in example 1, except that methanol (0.01% formic acid+1 mM dithiothreitol) was used as a precipitant, and the recovery rate was examined as shown in Table 30:

Table 30: results of examination of recovery of captopril/methanol (0.01% formic acid+1 mM dithiothreitol)

6. The test was performed as in example 1, except that methanol (0.3% formic acid+1 mM dithiothreitol) was used as a precipitant, and recovery rates obtained were examined as shown in tables 31 and 32:

table 31: results of examination of recovery of captopril/methanol (0.3% formic acid+1 mM dithiothreitol)

Table 32: results of investigation on recovery of cilazapril-methanol (0.3% formic acid+1 mM dithiothreitol)

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7. The test was carried out as in example 1, except that methanol (0.01% formic acid+30 mM dithiothreitol) was used as a precipitant, and the recovery rate was examined as shown in Table 33:

table 33: results of examination of recovery of captopril/methanol (0.01% formic acid+30 mM dithiothreitol)

8. The test was carried out as in example 1, except that methanol (0.3% formic acid+30 mM dithiothreitol) was used as a precipitant, and the recovery rate was examined as shown in Table 34:

table 34: results of examination of recovery of captopril/methanol (0.3% formic acid+30 mM dithiothreitol)

Fosinopril: from the data in tables 7 and 29, it was found that methanol (0.1% formic acid+5 mM dithiothreitol) was used as a precipitant and the recovery rate was acceptable. Methanol (5 mM dithiothreitol) was used as a precipitant, and the recovery rate was low.

Captopril: from the data in tables 7, 30, 31, 33 and 34, it was found that the recovery rate was acceptable by using methanol (0.1% formic acid+5 mM dithiothreitol) as a precipitant. Methanol (0.01% formic acid+1 mM dithiothreitol) is adopted as a precipitator, and the recovery rate is high. Methanol (0.3% formic acid+1 mM dithiothreitol) is adopted as a precipitator, and the recovery rate is high. Methanol (0.01% formic acid+30 mM dithiothreitol) is adopted as a precipitator, and the recovery rate is high. Methanol (0.3% formic acid+30 mM dithiothreitol) is adopted as a precipitator, and the recovery rate is high.

Cilazapril: from the data in tables 7, 26, 27, 28 and 32, it was found that the recovery rate was acceptable by using methanol (0.1% formic acid+5 mM dithiothreitol) as a precipitant. Methanol (0.1% formic acid) is adopted as a precipitator, and the recovery rate is qualified. Methanol is adopted as a precipitator, and the recovery rate is qualified but slightly higher. Acetonitrile is adopted as a precipitator, the recovery rate is high, and the recovery rate exceeds the acceptance standard. Methanol (0.3% formic acid+1 mM dithiothreitol) is used as a precipitator, and the recovery rate is low and exceeds the acceptance standard.

Comparative example 3

The comparative example examined the effect of diluent selection on the detection results:

1. The test was performed as in example 1, except that: the diluent is pure water. The results of examining the recovery rate of captopril are shown in table 35:

table 35: captopril/pure water recovery rate investigation results

2. The test was performed as in example 1, except that: the dilution was 1 mM dithiothreitol in water. The results of the recovery rate investigation of captopril are shown in table 36:

table 36: examination result of recovery rate of captopril/1 mM dithiothreitol aqueous solution

3. The test was performed as in example 1, except that: the dilution was 30 mM dithiothreitol in water. The results of the recovery rate investigation of captopril are shown in table 37:

table 37: examination result of recovery rate of captopril/30 mM dithiothreitol aqueous solution

4. The test was performed as in example 1, except that: the dilution was 0.1% formic acid in water. The recovery rates obtained are shown in Table 38:

table 38: examination result of recovery rate of captopril/0.1% formic acid aqueous solution

When the precipitant is methanol (0.1% formic acid+5 mM dithiothreitol), the diluent adopts 5mM dithiothreitol water and pure water, the recovery rate is qualified, the recovery rate is better under the condition of 5mM dithiothreitol water solution, and meanwhile, the stability of the treated sample is considered, and the 2-20 mM dithiothreitol water solution is adopted as the diluent.

When the diluted solution is 1 mM dithiothreitol aqueous solution, 30 mM dithiothreitol aqueous solution and 0.1% formic acid aqueous solution, the recovery rate is high.

Comparative example 4

This comparative example is used to illustrate the effect comparison of phase B in the mobile phase:

1. the test was performed as in example 1, except that: the phase B adopts methanol. The resulting chromatogram is shown in FIG. 3.

As can be seen from fig. 3, lisinop Li Fengxing is wide and tailing is severe.

2. The test was performed as in example 1, except that: acetonitrile is used for phase B. The resulting chromatogram is shown in FIG. 4.

As can be seen from fig. 4, the peak shape of lisinopril is normal and baseline separation of benazepril from enalapril can be achieved.

Comparative example 5

The comparative example examined the effect of the elution method on the detection result:

1. the assay was performed as in example 1, except that the following elution procedure was used:

table 39: gradient elution condition 1

The resulting chromatogram is shown in FIG. 5.

2. The assay was performed as in example 1, except that the following elution procedure was used:

table 40: gradient elution Condition 2

The resulting chromatogram is shown in FIG. 6.

3. The assay was performed as in example 1, except that the following elution procedure was used:

table 41: gradient elution Condition 3

The resulting chromatogram is shown in FIG. 7.

4. The detection was carried out as in example 1, and the elution method was as shown in Table 3.

The resulting chromatogram is shown in FIG. 8.

The elution times in the above method are shown in table 42.

Table 42: elution time of different elution modes

As can be seen from FIGS. 5-8 and Table 42, the method 1 shows a peak front of captopril Li Sepu, and methods 2 and 3 can lead each target to completely show a peak, but most targets show a peak at the same retention time, and finally partial targets are separated for detection by adopting the elution mode of Table 3.

Comparative example 6

Mode 1: the test was conducted in accordance with the method of example 1, and the amount of the protein precipitant added was 800. Mu.L. The resulting chromatogram is shown in FIG. 9.

As can be seen from fig. 9, the lisinopril instrument has insufficient response.

Mode 2: the test was carried out as in example 1, and the amount of the protein precipitant added was 300. Mu.L. The resulting chromatogram is shown in FIG. 10.

As can be seen from FIG. 10, the perindopril and quinapril instrument responses are saturated.

Mode 3: the test was carried out as in example 1, and the amount of the diluent added was 50. Mu.L. The resulting chromatogram is shown in FIG. 11.

As can be seen from FIG. 11, perindopril and quinapril instrument responses are saturated.

Mode 4: the test was carried out as in example 1, and the amount of the diluent added was 200. Mu.L. The resulting chromatogram is shown in FIG. 12.

As can be seen from fig. 12, the lisinopril instrument response was low, resulting in poor precision.

Comparative example 7

This comparative example is used to illustrate the effect of ion pair selection on detection results:

the procedure of example 1 was followed except that the elution conditions were as shown in Table 43:

table 43: elution conditions:

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fosinopril and fosinopril have the same ion pair 436.00/390.05, there is ion cross talk, solution: fosinoprilat was quantified using 436.20/109.15, and characterized by 436.20/418.20.

Quinapril, imidapril, fosinopril interfering with captopril 218.1-70.15 ion pairs, as shown in figures 13, 14, 15, solution: captopril was ion pair quantified using 218.1-116.1.

Captopril 218.1-70.15 ion pairs were detected after quinapril Li Shan labeling: as shown in fig. 13.

Captopril 218.1-70.15 ion pairs were detected after sample injection of imidacloprid Li Shan: as shown in fig. 14.

The captopril 218.1-70.15 ion pair is detected after single sample injection of fosinopril: as shown in fig. 15.

The foregoing is only a specific embodiment of the invention to enable those skilled in the art to understand or practice the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown and described herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims (9)

1. A method for detecting 11 antihypertensive drugs and 3 metabolites in blood by liquid chromatography tandem mass spectrometry, wherein the 11 antihypertensive drugs and 3 metabolites comprise: a method of captopril, enalapril, benazepril, benazeprilat, fosinopril, fosinoprilat, ramipril, quinapril, zofenopril, perindopril, imidapril, cilazapril, and lisinopril comprising at least the steps of:

s1, preparing a standard curve equation, which comprises the following steps:

preparing an internal standard working solution and a standard working solution, preparing a sample solution for standard yeast, and detecting the sample solution for standard yeast by using a high performance liquid chromatography-mass spectrometer to obtain a standard curve equation for calculating the content of 11 antihypertensive drugs and metabolites;

s2, preprocessing a sample to be tested, including:

adding formic acid and 2-10 mol/L dithiothreitol solution into plasma to be detected to obtain a sample to be detected, uniformly mixing an internal standard working solution and the sample to be detected, adding a protein precipitant, uniformly mixing, and centrifuging to obtain a supernatant, and adding a diluent into the supernatant to be uniformly mixed to obtain a sample injection sample;

the protein precipitant is methanol containing formic acid with the volume percentage concentration of 0.05-0.2% and dithiothreitol with the concentration of 2-20 mM;

The diluent is an aqueous solution containing 2-20 mM dithiothreitol;

s3, detecting the sample injection sample, which comprises the following steps:

detecting the sample by using a high performance liquid chromatography-mass spectrometer, and substituting the detection result of the sample into the standard curve equation to obtain the contents of 11 antihypertensive drugs and 3 metabolites in the sample to be detected;

the conditions of the high performance liquid phase when the high performance liquid chromatography mass spectrometer is used for detection are as follows: mobile phase: the phase A is an aqueous solution containing 0.05-0.2% formic acid and 1-10 mmol/L ammonium acetate by volume percent, and the phase B is acetonitrile;

the gradient elution conditions were:

0-0.20 min, A:90%, B:10%;

0.20-0.30 min, A:90% -60%, B:10% -40%;

0.30-0.80 min, A:60%, B:40%;

0.80 to 0.90 minutes, A:60% -40%, B:40% -60%;

0.90-1.50 minutes, A:40%, B:60 percent;

1.51-2.50 minutes, A:20%, B:80%;

2.51-4.50 minutes, A:2%, B:98 percent;

4.51-4.60 minutes, A:2% -90%, B:98% -10%;

4.60 to 6.00 minutes, A:90%, B:10%.

2. The method of claim 1, wherein S1 comprises:

s11, respectively preparing an internal standard working solution and a standard working solution with gradient concentration;

S12, respectively taking the internal standard working solution and the standard working solution, adding water, uniformly mixing to obtain a standard solution with gradient concentration, adding the protein precipitant, uniformly mixing to obtain a mixed solution, adding the mixed solution into the diluent, and uniformly mixing to obtain a sample for standard curve;

and S13, detecting the standard curve sample solution by using a high performance liquid chromatography mass spectrometer to obtain a standard curve equation.

3. The method according to claim 2, wherein, in S12,

the volume ratio of the sum of the volumes of the standard working solution and the water to the volume ratio of the internal standard working solution is 5:1, a step of;

the volume ratio of the standard working solution to the water is 1:9, a step of performing the process;

the volume ratio of the sum of the volumes of the standard working solution and the water to the volume ratio of the protein precipitant is 1: 10-12 parts;

the volume ratio of the mixed solution to the diluent is 1: 1-3: 2.

4. the method according to claim 1, wherein in S2:

the volume ratio of the plasma to be tested, formic acid and dithiothreitol solution is 1000:0.5 to 1.5:0.5 to 1.5;

the volume ratio of the sample to be tested to the internal standard working solution is 5:1, a step of;

the volume ratio of the sample to be tested to the protein precipitant is 1: 10-12 parts;

The volume ratio of the supernatant to the diluent is 1: 1-3: 2.

5. the method according to claim 1 or 2, wherein the sample to be measured is a plasma sample or a serum sample, and the volume is 50 μl to 100 μl;

in S12, the sum of the volumes of the standard working fluid and the water is equal to the volume of the sample to be tested.

6. A method according to claim 1 or 2, characterized in that,

s2: the mixing condition of the internal standard working solution and the sample to be tested is 1500-2500 r/min, and vortex mixing is carried out for 30 s-1 min; s12: the conditions for uniformly mixing the internal standard working solution, the standard working solution and the water are 1500-2500 r/min for 30 s-1 min,

s2, S12: the conditions for mixing after adding the protein precipitant are as follows: vortex mixing for 5-10 min at 1500-2500 r/min; the centrifugation conditions are 12000-15000 r/min for 5-12 min; mixing conditions after adding the diluent are 1500-2500 r/min, and vortex mixing for 30 s-1 min.

7. The method according to claim 1 or 2, wherein the internal standard working fluid comprises an isotopic internal standard of the 10 antihypertensive drugs and 3 active metabolites:

the isotope internal standard of captopril is captopril-d ₃ ；

The isotopic internal standard of enalapril is enalapril-d ₅ ；

The isotopic internal standard of enalapril is enalapril-d ₅ ；

Isotopic internal standard of cilazapril and benazepril is benazepril-d ₅ ；

The isotopic internal standard of benazeprilat is benazeprilat-d ₅ ；

The isotope internal standard of fosinopril is fosinopril-d ₅ ；

The isotope internal standard of fosinopril is fosinopril-d ₇ ；

The isotope internal standard of ramipril is ramipril-d ₅ ；

The isotope internal standard of quinapril is quinapril-d ₅ ；

The isotope internal standard of zofenopril is zofenopril-d ₅ ；

Isotopic internal standard of perindoprilPerindopril-d ₄ ；

The isotope internal standard of the imidapril is imidapril-d ₅ ；

The isotopic internal standard of lisinopril is lisinopril-d ₅ 。

8. The method according to claim 1 or 2, wherein the conditions of the high performance liquid phase when detected using a high performance liquid chromatography mass spectrometer are:

the chromatographic column adopts a T3 chromatographic column;

flow rate: 0.4-0.5 mL/min;

column temperature: 35-45 ℃;

sample injection amount: 5-20 mu L;

analysis time: and 6 min.

9. The method according to claim 1 or 2, wherein the mass spectrometry conditions when detected using a high performance liquid chromatography mass spectrometer are:

adopting an electrospray ion source, a positive ion mode and multi-reaction monitoring;

Atomizing gas flow rate: 3.0 L/min;

DL temperature: 250 ℃;

drying gas temperature: 400 ℃;

drive gas flow rate: 10 L/min;

heating air flow rate: 10 L/min;

interface temperature: 300 ℃;

ion pairs expressed as parent/daughter ions are:

quantitative ion pair of quinapril: 439.30/117.15 qualitative ion pair of quinapril: 439.30/234.20, ion pair of quinapril internal standard: 444.30/239.20;

quantitative ion pair of perindopril: 369.30/172.35; qualitative ion pair of perindopril: 369.30/98.20; ion pair of perindopril internal standard: 373.30/176.35;

quantitative ion pair of fosinopril: 436.00/390.05 qualitative ion pair of fosinopril: 564.30/436.20 ion pair of fosinopril internal standard: 441.00/395.05;

quantitative ion pair of captopril: 218.10/116.10, qualitative ion pair of captopril: 218.10/70.15, ion pair of captopril internal standard: 221.10/73.15;

quantitative ion pair of imidapril: 406.20/234.20 qualitative ion pair of imidapril: 406.20/117.10, ion pair of the imidapril internal standard: 411.20/239.20;

quantitative ion pair of zofenopril: 430.10/105.10, qualitative ion pair of zofenopril: 430.10/280.15, ion pair of zofenopril internal standard: 435.10/110.10;

Quantitative ion pair of fosinoprilat: 436.20/109.15 qualitative ion pair of fosinoprilat: 436.20/418.20, ion pair of fosinoprilat internal standard: 443.15/153.15;

quantitative ion pair of cilazapril: 418.35/211.15, qualitative ion pair of cilazapril: 418.35/70.15;

quantitative ion pair of enalapril: 377.30/234.25, qualitative ion pair of enalapril: 377.30/117.20, ion pair of enalapril internal standard: 382.30/239.25;

quantitative ion pair of enalaprilat: 349.10/206.40, qualitative ion pair of enalaprilat: 349.10/117.15, ion pair of enalapril Li Lana standard: 354.20/211.15;

quantitative ion pair of benazepril: 425.30/351.15, qualitative ion pair of benazepril: 425.30/190.10, ion pair of benazepril internal standard: 430.30/356.15;

quantitative ion pair of benazeprilat: 397.30/351.15, qualitative ion pair of benazeprilat: 397.30/118.15, benazepril Li Lana target ion pair: 402.30/356.15;

quantitative ion pair of ramipril: 417.30/234.20, qualitative ion pair of ramipril: 417.30/117.15 ion pairs of ramipril internal standard: 422.30/239.20;

quantitative ion pair of lisinopril: 406.30/84.15 qualitative ion pair of lisinopril: 406.30/246.20, ion pair of lisinopril internal standard: 411.30/84.30.