CN113030312B - Method for simultaneously determining concentration of anticoagulant drug and active metabolite in blood plasma - Google Patents

Abstract

The invention discloses a method for simultaneously determining the concentrations of anticoagulant drugs and active metabolites in blood plasma, wherein the anticoagulant drugs are dabigatran etexilate, rivaroxaban, edoxaban and apixaban respectively, and the active metabolites are dabigatran. The specificity, precision, accuracy, linearity, stability and the like of the method all meet the analysis requirements of biological samples, the sensitivity is high, and the method can be used for monitoring the treatment medicines of clinical dabigatran etexilate, dabigatran, rivaroxaban, edoxaban and apixaban.

Description

A method for simultaneously determining the concentration of anticoagulant drugs and active metabolites in plasma

Technical Field

The present invention relates to the technical field of plasma drug concentration detection, and in particular to a method for simultaneously determining the concentrations of anticoagulant drugs and active metabolites in plasma.

Background Art

Dabigatran (as shown in Figure 1.1) is a highly polar amphipathic compound that is insoluble in organic solvents. Dabigatran etexilate (as shown in Figure 1.2) is a prodrug that is converted into dabigatran in the body to exert an anticoagulant effect. Dabigatran etexilate is a new type of artificially synthesized oral anticoagulant inhibitor that can reversibly and competitively inhibit thrombin activity and is a non-peptide thrombin inhibitor. After oral administration, it is rapidly absorbed through the digestive tract and can produce active metabolites dabigatran through esterase hydrolysis in plasma and liver, which exerts anticoagulant activity by inhibiting thrombin. Rivaroxaban (as shown in Figure 1.3) and apixaban (as shown in Figure 1.4) are both selective factor Xa inhibitors that do not require the participation of antithrombin III and can directly antagonize free and bound factor Xa. They are clinically used in adult patients undergoing elective hip or knee replacement surgery to prevent venous thrombosis. Edoxaban (as shown in Figure 1.5) is the fourth new oral anticoagulant approved by the US FDA after dabigatran, rivaroxaban and apixaban. It is a factor Xa inhibitor with high selectivity that can directly inhibit FXa, which can prolong the prothrombin time (PT) and activated partial thromboplastin time (APTT), ultimately inhibiting thrombosis.

Patent CN111812219A provides a method for detecting anticoagulant drug concentration in plasma, wherein provides the detection method for detecting dabigatran, rivaroxaban and apixaban, but it does not disclose the method for detecting dabigatran etcxilate, rivaroxaban, edoxaban, apixaban and dabigatran simultaneously. In addition, in this patent, the quantitative lower limit is 1ng/mL, and sensitivity is low.

Therefore, providing a method with high sensitivity that can simultaneously detect dabigatran etexilate, rivaroxaban, edoxaban, apixaban, and dabigatran in plasma is an important research direction, which will help to monitor these drugs in clinical practice.

Summary of the invention

To achieve the above object, the present invention provides a method for simultaneously determining the concentrations of four anticoagulant drugs and an active metabolite in plasma by ultra-high performance liquid chromatography-tandem mass spectrometry.

A method for simultaneously determining the concentration of an anticoagulant drug and an active metabolite in plasma, wherein the anticoagulant drugs are dabigatran, rivaroxaban, edoxaban and apixaban, and the active metabolite is dabigatran; ultra-high performance liquid chromatography tandem mass spectrometry is used to detect the anticoagulant drugs and the active metabolites in pre-treated plasma, the analyte is separated from the plasma matrix by ultra-high performance liquid chromatography, and then the tandem mass spectrometry is used for isotope internal standard quantification, a standard curve is drawn with the ratio of the standard peak area to the corresponding internal standard peak area as the Y axis and the standard concentration as the X axis, and the content of the anticoagulant drugs and the active metabolites in the plasma is calculated;

The chromatographic conditions are as follows

Stationary phase: chromatographic column model Agilent ZORBAX Eclipse XDB-C18 (3.5 μm, 2.1 × 100 mm);

Mobile phase: Phase A: 0.01-0.2% formic acid aqueous solution, Phase B: 0.01-0.2% formic acid acetonitrile solution; Mobile phase A and mobile phase B were mixed in different volumes for gradient elution;

The mass spectrometry conditions were as follows:

In electrospray ionization detection mode, multiple reaction monitoring, positive ion mode was adopted; capillary voltage was 4500V; ion source temperature was 550°C; first ion source gas, 55psi; second ion source gas, 55psi; curtain gas, 35psi.

Preferably, the mobile phases are: phase A: 0.1% formic acid aqueous solution, phase B: 0.1% formic acid acetonitrile solution; column temperature: 40°C; flow rate: 0.3 mL/min; injection volume: 2 μL.

Preferably, the gradient elution process is as follows: in 0-1 minute, the volume ratio of mobile phase A and mobile phase B remains unchanged at 95:5; in 1-4 minutes, the volume ratio of mobile phase A and mobile phase B changes from 95:5 to 20:80 at a constant speed; in 4-4.5 minutes, the volume ratio of mobile phase A and mobile phase B changes from 20:80 to 5:95 at a constant speed; in 4.5-6 minutes, the volume ratio of mobile phase A and mobile phase B remains unchanged at 5:95; in 6-6.1 minutes, the volume ratio of mobile phase A and mobile phase B changes from 5:95 to 95:5 at a constant speed; in 6.1-8 minutes, the volume ratio of mobile phase A and mobile phase B remains unchanged at 95:5. The collection time for each sample is 8 minutes.

Preferably, the pre-treated plasma is prepared according to the following method:

Take a plasma sample, add methanol and a mixed internal standard working solution thereto, vortex and centrifuge to obtain the supernatant, blow nitrogen to dryness, dissolve the dry sample in 0.001-1% formic acid methanol solution, centrifuge, and take the supernatant and mix it with pure water.

Preferably, the pre-treated plasma is prepared according to the following method: take 100 μL of plasma sample in a 1.5 mL centrifuge tube, add 890 μL of ice-cold methanol and 10 μL of the mixed internal standard working solution of 1 μg·mL ^-1 thereto, vortex and centrifuge to obtain the supernatant, blow nitrogen to dryness, add 100 μL of 0.001-1% formic acid methanol solution to the dry sample and ultrasonically re-dissolve it, centrifuge to obtain 50 μL of supernatant, add 450 μL of pure water to mix, and wait for sampling and analysis.

Preferably, the mixed internal standard working solution contains isotope internal standards rivaroxaban-deuterium 4 and dabigatran-deuterium 3, wherein the rivaroxaban-deuterium 4 is the internal standard corresponding to rivaroxaban, edoxaban and apixaban; and the dabigatran-deuterium 3 is the internal standard corresponding to dabigatran and dabigatran etexilate.

Preferably, the mixed internal standard working solution is prepared according to the following method:

Accurately weigh about 1 mg of each of the internal standards dabigatran-deuterium 3 and rivaroxaban-deuterium 4, dissolve them in DMSO to obtain internal standard stock solutions with a concentration of 1 mg·mL ^-1 , dilute and mix a certain volume of the two internal standard stock solutions with an appropriate amount of methanol to obtain a mixed internal standard solution with a concentration of 10 μg·mL ^-1 , take 100 μL of the 10 μg·mL ^-1 mixed internal standard solution, add 900 μL of methanol solution, mix well to obtain 1 μg·mL ^-1 mixed internal standard working solution, and store it in a -20°C refrigerator.

Preferably, the standard is prepared according to the following method: take a certain amount of dabigatran etexilate, rivaroxaban, edoxaban, apixaban and dabigatran stock solution, dilute it with methanol to form a working solution of appropriate concentration, take 100 μL of this working solution, add 900 μL of blank plasma, and make the standard containing dabigatran etexilate, rivaroxaban, edoxaban, apixaban and dabigatran with concentrations of 0.1, 0.5, 1, 2, 5, 10, 50, 100, 200, 500 ng·mL ^-1 respectively.

Preferably, the blank plasma is blank plasma that does not contain anticoagulant drugs and active metabolites.

Compared with the prior art, the present invention has the following technical effects:

The detection method provided by the present invention can simultaneously detect dabigatran etexilate, rivaroxaban, edoxaban, apixaban and dabigatran in plasma. In addition, the quantitative lower limit of the present invention can reach 0.1-0.5 ng/mL, and the sensitivity is high.

The linear range for four anticoagulant drugs and one active metabolite was 0.1-500 ng·mL ^-1 , and the correlation coefficient (r) was greater than 0.997. The recoveries at three quality control concentrations were 97.0-103%, 99.4-112%, and 93.9-106%, respectively, and the RSD% was less than 14.9%. The intra- and inter-batch accuracy deviations were less than 15%, and the precision RSD was less than 15%. The samples remained stable after being placed at 4°C for 24 hours, -80°C for 90 days, and repeated freezing and thawing for 3 times. Therefore, the specificity, precision, accuracy, linearity, and stability of this method all meet the analytical requirements of biological samples, and it has high sensitivity and can be used for clinical therapeutic drug monitoring of dabigatran, dabigatran, rivaroxaban, edoxaban, and apixaban.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1.1 is the molecular structure of Dabigatran (C ₂₅ H ₂₅ N ₇ O ₃ );

Figure 1.2 is the molecular structure formula of Dabigatran etexilate (C ₃₄ H ₄₁ N ₇ O ₅ );

Figure 1.3 Molecular structure of rivaroxaban (C ₁₉ H ₁₈ N ₃ O ₅ SCl);

Figure 1.4 is the molecular structure formula of Apixaban (C ₂₅ H ₂₅ N ₅ O ₄ );

Figure 1.5 is the molecular structure of Edoxaban (C ₂₄ H ₃₀ ClN ₇ O ₄ S);

Figure 2.1 is the standard curve of dabigatran etexilate in human plasma;

Figure 2.2 shows the standard curve of dabigatran in human plasma;

Figure 2.3 shows the standard curve of edoxaban in human plasma;

Figure 2.4 shows the standard curve of rivaroxaban in human plasma;

Figure 2.5 is the standard curve of apixaban in human plasma;

Figure 3.1 is a chromatogram of rivaroxaban in the plasma of the subjects;

Figure 3.2 is a chromatogram of dabigatran etexilate in the plasma of the test subject;

Figure 3.3 is a chromatogram of dabigatran in the plasma of the subjects;

Fig. 4.1 is the chromatogram of blank plasma and the addition of dabigatran etexilate (100ng/mL) reference substance;

Figure 4.2 is a chromatogram of blank plasma and a reference substance added with dabigatran (100 ng/mL);

Figure 4.3 is the chromatogram of blank plasma and edoxaban (100 ng/mL) added as a reference substance;

Figure 4.4 is the chromatogram of blank plasma and reference substance added with rivaroxaban (100 ng/mL);

Figure 4.5 is the chromatogram of blank plasma and apixaban (100 ng/mL) reference substance.

DETAILED DESCRIPTION

The present invention is described in detail and specifically by specific examples below to provide a better understanding of the present invention, but the following examples do not limit the scope of the present invention.

The following instruments and chemicals are used in the specific implementation of the present invention:

instrument

Agilent UPLC system, SCIEX Q-trap 6500 mass spectrometer system, Analyst chromatography workstation (Version 1.7.1), Agilent ZORBAX Eclipse XDB-C18 (3.5 μm, 2.1 × 100 mm) chromatographic column;

Low temperature high speed refrigerated centrifuge;

Nitrogen blowing instrument;

Vortex mixer;

Analytical balance;

Freezer, Thermo Fisher;

Millipore Ultrapure Water Device Direct Q, Millipore Ltd, Molsheim, France

Chemicals

Methanol, acetonitrile, dabigatran etexilate, dabigatran, rivaroxaban, edoxaban, apixaban, dabigatran-deuterium-3, rivaroxaban-deuterium-4, formic acid.

[Example 1]

This example provides methods for preparing reference solution, mixed internal standard working solution, standard curve and quality control plasma sample:

1. Preparation of reference solution:

Accurately weigh dabigatran etcexilate, dabigatran, rivaroxaban, edoxaban and apixaban reference substance appropriate amount respectively, dissolve and constant volume with methanol, obtain concentration respectively 1mg·mL ^-1 dabigatran etcexilate, dabigatran, edoxaban and apixaban stock solution, and concentration is 0.75mg·mL ^-1 rivaroxaban stock solution, as in Table 1, be placed in -20 ℃ of refrigerators and store.Dabigatran etcexilate, dabigatran, rivaroxaban, apixaban and edoxaban stock solution are prepared into the reference substance solution containing 5000ng/mL dabigatran etcexilate, dabigatran, rivaroxaban, apixaban and edoxaban with methanol aqueous solution.

Table 1: Stock solution preparation

2. Preparation of rivaroxaban-deuterium 4 and dabigatran-deuterium 3 mixed internal standard working solution:

Accurately weigh about 1 mg of each of the internal standards rivaroxaban-deuterium 4 and dabigatran-deuterium 3, dissolve them in DMSO to obtain internal standard stock solutions with a concentration of 1 mg·mL ^-1 , dilute a certain volume of the two internal standard stock solutions with an appropriate amount of methanol, and obtain a mixed internal standard solution with a concentration of 10 μg·mL ^-1 . Take 100 μL of the 10 μg·mL ^-1 mixed internal standard solution, add 900 μL of methanol solution, mix well to obtain a 1 μg·mL ^-1 mixed internal standard working solution, and store it in a -20°C refrigerator.

3. Preparation of plasma samples for standard curve:

A certain amount of dabigatran etexilate, dabigatran, rivaroxaban, edoxaban and apixaban stock solutions were diluted with methanol to form a series of working solutions of appropriate concentrations. 100 μL of the working solution was added to 900 μL of blank plasma to prepare a series of standard blood samples containing dabigatran etexilate, dabigatran, rivaroxaban, edoxaban and apixaban at concentrations of 0.1, 0.5, 1, 2, 5, 10, 50, 100, 200 and 500 ng·mL ^-1 , respectively.

A specific method for preparing standard curve plasma samples:

Get five kinds of a certain amount of stock solutions and dilute with methanol to obtain the mixed stock solution SW of 200 μ g/mL.Get SW100 μL, methanol 900 μL and be diluted to 20 μ g/mL to obtain working solution SW0, be placed in-20 ℃ of refrigerators and store, SW0 is diluted four times, obtains the working solution S1 of 5 μ g/mL, then with methanol gradient dilution, obtains working solution S2-S9.As in Table 2, get 100 μL corresponding working solution, add 900 μL blank plasma, make the series standard blood sample that contains dabigatran etcixic acid, dabigatran, rivaroxaban, edoxaban and apixaban concentration and be respectively 0.1,0.5,1,2,5,10,50,100,200,500ng·mL ^-1 .

Table 2: Preparation method of plasma samples for standard curve

4. Preparation of quality control plasma samples:

Take a certain amount of dabigatran, dabigatran, rivaroxaban, edoxaban and apixaban stock solutions, dilute with methanol to form a series of working solutions of appropriate concentrations, take 100 μL of this working solution, add 900 μL of blank plasma, and prepare low, medium and high quality control plasma samples containing dabigatran, dabigatran, rivaroxaban, edoxaban and apixaban with concentrations of 4, 40 and 400 ng·mL ^-1 , respectively, as shown in Table 3. (Preparation of the same standard curve plasma sample)

Table 3: Preparation method of quality control plasma samples

[Example 2]

This embodiment provides a method for detecting anticoagulant drugs dabigatran etexilate, rivaroxaban, edoxaban, apixaban and active metabolite dabigatran in pre-treated plasma using ultra-performance liquid chromatography tandem mass spectrometry, which specifically comprises the following steps:

Step 1. Plasma samples and standard curves Plasma sample pretreatment

Plasma sample preparation:

Take 100 μL of plasma sample in a 1.5 mL centrifuge tube, add 890 μL of ice-cold methanol and 10 μL of 1 μg·mL ^-1 mixed internal standard solution, vortex and centrifuge (13000g, 4°C, 10 min), take the supernatant, blow nitrogen to dryness, add 100 μL of 1% formic acid methanol solution to the dry sample and ultrasonically reconstitute, centrifuge (13000g, 4°C, 10 min), take 50 μL of supernatant, add 450 μL of pure water and mix well, and then prepare for sampling and analysis.

Standard curve plasma sample pretreatment:

Take 100 μL of plasma samples of each concentration standard curve in a 1.5 mL centrifuge tube, add 890 μL of ice-cold methanol and 10 μL of 1 μg·mL ^-1 mixed internal standard solution, vortex and centrifuge (13000g, 4°C, 10 min), take the supernatant, blow nitrogen to dryness, add 100 μL of 1% formic acid methanol solution to the dry sample and re-dissolve by ultrasonication, centrifuge (13000g, 4°C, 10 min), take 50 μL of supernatant, add 450 μL of pure water and mix well, and then prepare for sampling and analysis.

Step 2: Ultra-high performance liquid chromatography separates the analyte from the plasma matrix, and then tandem mass spectrometry is used for isotope internal standard quantification

The chromatographic conditions were as follows:

Stationary phase: (Chromatographic column model) Agilent ZORBAX Eclipse XDB-C18 (3.5 μm, 2.1 × 100 mm);

Column temperature: 40°C;

Mobile phase: Phase A: 0.1% formic acid in water; Phase B: 0.1% formic acid in acetonitrile.

Flow rate: 0.3mL/min

Injection volume: 2 μL

Different volumes of mobile phase A and mobile phase B were mixed for gradient elution, and the gradient conditions were as shown in Table 4. The collection time for each sample was 8 minutes.

Table 4 Gradient conditions

Time (min) B.conc.(%) A.conc.(%) 0 5 95 1 5 95 4 80 20 4.5 95 5 6 95 5 6.1 5 95 8 5 95

The mass spectrometry conditions were as follows:

In electrospray ionization detection mode, multiple reaction monitoring (MRM) was used, positive ion mode; capillary voltage was 4500 V; ion source temperature was 550°C; first ion source gas, 55 psi; second ion source gas, 55 psi; curtain gas, 35 psi. The mass transition, collision energy (CE) and declustering potential (DP) of the analytes are shown in Table 5.

Table 5 MRM transitions and mass spectrometry parameters of five anticoagulants and two internal standards

Step 3, with the ratio of the peak area of the standard to the corresponding internal standard peak area as the Y-axis and the concentration of the standard as the X-axis, draw a standard curve, as shown in Figures 2.1-2.5, and calculate the content of anticoagulant drugs in plasma (the standard is the standard curve plasma sample of each concentration), wherein rivaroxaban-deuterium 4 is the internal standard corresponding to rivaroxaban, edoxaban and apixaban; the dabigatran-deuterium 3 is the internal standard corresponding to dabigatran and dabigatran etexilate.

[Example 3]

Plasma samples provided by subjects taking anticoagulants were used to analyze the anticoagulant drug concentration using the method in Example 2. The results are shown in Figures 3.1-3.3. It was determined that rivaroxaban was present in one plasma sample and dabigatran and its metabolite dabigatran were present in another plasma sample. It was further determined that the concentrations of rivaroxaban, dabigatran and its metabolite dabigatran were 263.7 ng·L ^-1 , 4.4 ng·L ^-1 and 224.0 ng·L ^-1 , respectively.

【Test verification】

1. Linear

The peak area ratio (Y) of the drug to the internal standard was used for weighted linear regression of the concentration (X, ng/mL). The weight coefficient was 1/X, and the correlation coefficient r of the regression equation was greater than 0.997, as shown in Table 6. The linearity was good and met the quantitative requirements.

Table 6 Linear range, linear coefficient, linear equation and detection limit of five anticoagulants

Analytes Linear range Linear coefficient r Linear equations Dabigatran 1-500 0.99946 y＝0.00360x-0.00355 Dabigatran 0.1-500 0.99704 y＝0.008361x+0.000758087 Edoxaban 0.5-500 0.99758 y＝0.00314x-0.00315 Rivaroxaban 0.5-500 0.99975 y＝0.00991x-0.01114 Apixaban 0.5-500 0.99951 y＝0.002518x+0.08227

2. Exclusivity

Take 100 μL of blank plasma (not containing any standard and internal standard) and perform pretreatment and determination according to the plasma sample pretreatment and determination method in Example 2. Compare the chromatogram of blank plasma with blank plasma containing 100 ng/mL dabigatran, dabigatran, edoxaban, rivaroxaban and apixaban, respectively, as shown in Figures 4.1-4.5, to confirm that there is no interference from endogenous impurities in blank plasma.

3. Accuracy and precision.

Five batches of quality control plasma samples were prepared according to the quality control plasma sample preparation method. 100 μL of low, medium and high quality control plasma samples were respectively taken into 1.5 mL centrifuge tubes. The quality control plasma samples were pre-treated and then measured according to the plasma sample pretreatment and measurement method in Example 2. The mean value, standard deviation and relative standard deviation of the results were calculated.

The results showed that the accuracy deviation of the quality control samples containing dabigatran, dabigatran, rivaroxaban, edoxaban and apixaban at three concentrations was less than 15%, and the intra-batch and inter-batch precision RSD was less than 15%, as shown in Table 7. The results showed that the precision and accuracy of the method were good and met the analytical requirements.

Table 7 Intra-batch and inter-batch accuracy and precision of five anticoagulants

4. Extraction recovery rate

Get low (LQC), middle (MQC), high (HQC) quality control plasma sample respectively, carry out pre-treatment after mensuration according to the method for plasma sample pre-treatment and mensuration in embodiment 2, separately get the mixed standard working solution sample introduction of corresponding concentration and mensuration.With the drug peak area after blood sample processing divided by the drug peak area of corresponding concentration reference substance solution sample introduction, calculate extraction recovery.See shown in table 8 below, the average extraction recovery of the quality control plasma sample of each concentration of dabigatran etcxilate, dabigatran, rivaroxaban, edoxaban and apixaban is 93.9-112%, RSD%＜14.9%.

The results showed that the extraction recovery rate of this method met the requirements of general analysis samples.

Table 8 Recovery rates of five anticoagulants (n=5)

5. Stability

(1) Short-term stability test

The quality control plasma samples were placed at 4°C and pre-treated and then measured according to the plasma sample pre-treatment and measurement method in Example 2. The plasma standards of various concentrations were placed in the automatic sampler for 24 hours and the results were compared with the immediate measurement results. The deviation was <9.2%, as shown in Table 9 below.

The results showed that plasma samples containing dabigatran, dabigatran, rivaroxaban, edoxaban and apixaban remained stable at 4°C for 24 hours after pretreatment.

(2) Long-term stability test

Quality control plasma samples were taken and pre-treated and then measured according to the plasma sample pre-treatment and measurement method in Example 2. The plasma standards of various concentrations were placed at -80°C for 90 days and the deviation was <8.12% compared with the results of the immediate measurement, as shown in Table 9.

The results showed that plasma samples containing dabigatran, dabigatran, rivaroxaban, edoxaban and apixaban remained stable when stored at -80°C for 90 days.

(3) Freeze-thaw experiment

The quality control plasma samples were taken, tested immediately, placed at -80°C, and thawed at room temperature after 24 hours. The process was repeated 3 times and compared with the results of the immediate test. The results showed a deviation of <9.3%, see Table 9.

The results showed that plasma samples containing dabigatran, dabigatran, rivaroxaban, edoxaban and apixaban remained stable after three repeated freeze-thaw cycles.

(4) Matrix effect

The quality control plasma samples and the standards of corresponding concentrations were taken and tested simultaneously after pretreatment. The results showed that the matrix effect was <12%. See Table 9.

The results showed that the matrix effect of plasma samples containing dabigatran, dabigatran, rivaroxaban, edoxaban and apixaban was small.

Table 9 Stability and matrix effects of five new oral anticoagulants

In summary, the present invention provides a method for detecting anticoagulant drugs dabigatran etexilate, rivaroxaban, edoxaban, apixaban and active metabolite dabigatran in pretreated plasma by ultra-high performance liquid chromatography-tandem mass spectrometry, wherein the specificity, precision, accuracy, linearity and stability of the method meet the analysis requirements of biological samples, the sensitivity is high, and the method can be used for clinical therapeutic drug monitoring of dabigatran etexilate, dabigatran, rivaroxaban, edoxaban and apixaban.

The specific embodiments of the present invention are described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions made to the present invention are also within the scope of the present invention. Therefore, the equalization changes and modifications made without departing from the spirit and scope of the present invention should be included in the scope of the present invention.

Claims (8)

1. a method for simultaneously measuring anticoagulant drugs and active metabolite concentration in blood plasma, characterized in that, described anticoagulant drugs are respectively dabigatran, rivaroxaban, edoxaban and apixaban, and described active metabolite is dabigatran; adopt ultra high performance liquid chromatography tandem mass spectrometry to detect anticoagulant drugs and active metabolites in the blood plasma through pre-treatment, by ultra high performance liquid chromatography, sample is separated from blood plasma matrix, then tandem mass spectrometry carries out isotope internal standard quantitative, with the ratio of standard peak area to corresponding internal standard peak area as Y axis, standard concentration is X axis, draws standard curve, calculates the content of anticoagulant drugs and active metabolites in blood plasma; The chromatographic conditions were as follows: Stationary phase: Column model Agilent ZORBAX Eclipse XDB-C18, 3.5 μm, 2.1 × 100 mm; Mobile phase: Phase A: 0.01-0.2% formic acid aqueous solution, Phase B: 0.01-0.2% formic acid acetonitrile solution; Mobile phase A and mobile phase B were mixed in different volumes for gradient elution; The mass spectrometry conditions were as follows: In electrospray ionization detection mode, multiple reaction monitoring, positive ion mode was used; capillary voltage was 4500 V; ion source temperature was 550 °C; first ion source gas, 55 psi; second ion source gas, 55 psi; curtain gas, 35 psi; The pre-treated plasma is prepared according to the following method: taking a plasma sample, adding methanol and a mixed internal standard working solution thereto, vortexing and centrifuging to obtain a supernatant, blowing nitrogen to dryness, dissolving the dry sample in a 0.001-1% formic acid methanol solution, centrifuging, and taking a supernatant and mixing it with pure water; The lower limit of quantification of this method is 0.1ng/mL-0.5ng/mL; The gradient elution process is as follows: From 0 to 1 minute, the volume ratio of mobile phase A and mobile phase B remained unchanged at 95:5; from 1 to 4 minutes, the volume ratio of mobile phase A and mobile phase B gradually changed from 95:5 to 20:80 at a uniform rate; from 4 to 4.5 minutes, the volume ratio of mobile phase A and mobile phase B gradually changed from 20:80 to 5:95 at a uniform rate; from 4.5 to 6 minutes, the volume ratio of mobile phase A and mobile phase B remained unchanged at 5:95; from 6 to 6.1 minutes, the volume ratio of mobile phase A and mobile phase B gradually changed from 5:95 to 95:5 at a uniform rate; from 6.1 to 8 minutes, the volume ratio of mobile phase A and mobile phase B remained unchanged at 95:5; the collection time for each sample was 8 minutes. 2. The method according to claim 1 is characterized in that the chromatographic conditions also include: column temperature: 40°C; flow rate: 0.3mL/min; injection volume: 2μL. 3. The method according to claim 1, characterized in that the pre-treated plasma is prepared according to the following method: take 100 μL of plasma sample in a 1.5 mL centrifuge tube, add 890 μL of ice-cold methanol and 10 μL of the mixed internal standard working solution of 1 μg·mL ^-1 thereto, vortex and centrifuge to obtain the supernatant, blow nitrogen to dryness, add 100 μL of 0.001-1% formic acid methanol solution to the dry sample and ultrasonically re-dissolve it, centrifuge to obtain 50 μL of supernatant, add 450 μL of pure water to mix, and wait for sampling and analysis. 4. The method according to claim 1, characterized in that the mixed internal standard working solution contains isotope internal standards rivaroxaban-deuterium 4 and dabigatran-deuterium 3. 5. The method according to claim 1, wherein the rivaroxaban-deuterium 4 is the internal standard corresponding to rivaroxaban, edoxaban and apixaban; and the dabigatran-deuterium 3 is the internal standard corresponding to dabigatran and dabigatran etexilate. 6. method according to claim 4 is characterized in that, described mixed internal standard working solution is prepared according to the following method: Accurately weigh about 1 mg of each of the internal standards dabigatran-deuterium 3 and rivaroxaban-deuterium 4, dissolve them in DMSO to obtain internal standard stock solutions with a concentration of 1 mg·mL ^-1 , dilute and mix a certain volume of the two internal standard stock solutions with an appropriate amount of methanol to obtain a mixed internal standard solution with a concentration of 10 μg·mL ^-1 , take 100 μL of the 10 μg·mL ^-1 mixed internal standard solution, add 900 μL of methanol solution, mix well to obtain 1 μg·mL ^-1 mixed internal standard working solution, and store it in a -20°C refrigerator. 7. method according to claim 1, is characterized in that, described standard substances is prepared according to the following method: get a certain amount of dabigatran etcxilate, rivaroxaban, edoxaban, apixaban and dabigatran stock solution, dilute with methanol and become the working solution of appropriate concentration, get 100 μ L this working solution, add 900 μ L blank plasma, make and contain dabigatran etcxilate, rivaroxaban, edoxaban, apixaban and dabigatran concentration and be respectively 0.1,0.5,1,2,5,10,50,100,200,500ngmL ^- 1 described standard substances. 8. The method according to claim 7, characterized in that the blank plasma is blank plasma that does not contain anticoagulants and active metabolites.

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