CN113030312B

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

Info

Publication number: CN113030312B
Application number: CN202110240970.9A
Authority: CN
Inventors: 马春来; 张雨霏; 钟明康
Original assignee: Huashan Hospital of Fudan University
Current assignee: Huashan Hospital of Fudan University
Priority date: 2021-03-04
Filing date: 2021-03-04
Publication date: 2023-04-07
Anticipated expiration: 2041-03-04
Also published as: CN113030312A

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

Method for simultaneously determining concentration of anticoagulant drug and active metabolite in blood plasma

Technical Field

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

Background

Dabigatran (as in figure 1.1) is a strongly polar amphoteric compound, insoluble in organic solvents, dabigatran etexilate (as in figure 1.2) is a prodrug, which is converted to dabigatran in vivo to exert anticoagulant effect. Dabigatran etexilate is a novel artificial oral anticoagulant inhibitor, can reversibly competitively inhibit thrombin activity, and belongs to a non-peptide thrombin inhibitor. It can be rapidly absorbed by digestive tract after oral administration, and can generate active metabolite dabigatran by esterase hydrolysis in blood plasma and liver, and exert anticoagulant activity by inhibiting thrombin. Rivaroxaban (as in figure 1.3) and apixaban (as in figure 1.4) are both selective factor xa inhibitors that directly antagonize free and bound factor xa without the involvement of antithrombin iii and are clinically useful in adult patients with phase selective hip or knee replacements to prevent venous thrombosis. After dabigatran, rivaroxaban and apixaban (as in figure 1.5), the 4 th new oral anticoagulant approved by the FDA in the united states is a factor xa inhibitor, highly selective, directly inhibits FXa, and as a result prolongs Prothrombin Time (PT) and Activated Partial Thromboplastin Time (APTT), ultimately inhibiting thrombosis.

Patent CN111812219A provides a method for detecting the concentration of anticoagulant drugs in blood plasma, wherein detection methods for detecting dabigatran, rivaroxaban and apixaban are provided, but it does not disclose methods for simultaneously detecting dabigatran etexilate, rivaroxaban, edoxaban, apixaban and dabigatran. In addition, the lower limit of the quantification in this patent is 1ng/mL, and the sensitivity is low.

Therefore, the method which has high sensitivity and can simultaneously detect the dabigatran etexilate, rivaroxaban, edoxaban, apixaban and dabigatran is an important research direction and is beneficial to clinically monitoring the medicaments.

Disclosure of Invention

The invention provides a method for simultaneously determining the concentrations of four anticoagulant drugs and one active metabolite in blood plasma by using an ultra-high performance liquid chromatography-tandem mass spectrometry method to achieve the aim.

A method for simultaneously determining the concentrations of an anticoagulant drug and an active metabolite in blood plasma, wherein the anticoagulant drug is dabigatran etexilate, rivaroxaban, edoxaban and apixaban respectively, and the active metabolite is dabigatran; detecting an anticoagulant drug and an active metabolite in the pretreated plasma by adopting an ultra-high performance liquid chromatography tandem mass spectrometry technology, separating a substance to be detected from a plasma matrix by adopting ultra-high performance liquid chromatography, then carrying out isotope internal standard quantification by adopting a tandem mass spectrometry, drawing a standard curve by taking the ratio of the peak area of a standard substance to the peak area of a corresponding internal standard substance as a Y axis and the concentration of the standard substance as an X axis, and calculating the content of the anticoagulant drug and the active metabolite in the plasma;

the chromatographic conditions were as follows

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

mobile phase: phase A: 0.01-0.2% aqueous formic acid, phase B: 0.01-0.2% formic acid acetonitrile solution; mixing the mobile phase A and the mobile phase B in different volumes, and performing gradient elution;

the mass spectrometry conditions were as follows:

under an electrospray ionization detection mode, adopting a multi-reaction monitoring and positive ion mode; capillary voltage 4500V; the ion source temperature is 550 ℃; a first ion source gas, 55psi; a second ion source gas, 55psi; air curtain air, 35psi.

Preferably, the mobile phase: phase A: 0.1% aqueous formic acid, phase B: 0.1% formic acid acetonitrile solution; column temperature: 40 ℃; flow rate: 0.3mL/min; sample introduction amount: 2 μ L.

Preferably, the gradient elution process is as follows: the volume ratio of mobile phase a to mobile phase B was kept constant at 95; in 1-4 minutes, the volume ratio of the mobile phase A to the mobile phase B is uniformly and gradually changed from 95; the volume ratio of the mobile phase A to the mobile phase B is uniformly graded from 20; the volume ratio of mobile phase a to mobile phase B was kept constant at 5; the volume ratio of the mobile phase A to the mobile phase B is uniformly graded from 5; the volume ratio of mobile phase a to mobile phase B was kept constant at 95. Each sample was collected for 8 minutes.

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

taking a plasma sample, adding methanol and mixed internal standard working solution into the plasma sample, centrifuging the plasma sample after vortex, taking supernate, blowing nitrogen to dry the supernate, dissolving the dried sample in 0.001-1% formic acid methanol solution, centrifuging the supernate, and mixing the supernate with pure water.

Preferably, the pre-treated plasma is prepared according to the following method: a plasma sample (100. Mu.L) was taken in a 1.5mL centrifuge tube, and 890. Mu.L of ice-cold methanol and 1. Mu.g.mL were added thereto ^-1 10 mu L of the mixed internal standard working solution is vortexed and centrifuged to take supernatant, nitrogen is blown to dry the supernatant, 100 mu L of methanol solution containing 0.001-1% of formic acid is added into a dry sample and ultrasonically redissolved, 50 mu L of supernatant is centrifuged and taken, 450 mu L of pure water is added and mixed evenly, and the sample injection analysis is carried out.

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

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

accurately weighing about 1mg of internal standard dabigatran-deuterium 3 and rivaroxaban-deuterium 4 respectively, and dissolving with DMSO to obtain concentrations of 1 mg. ML respectively ^-1 Diluting and mixing two internal standard stock solutions with a certain volume with a proper amount of methanol to obtain the internal standard stock solution with the concentration of 10 mu g/mL ^-1 The mixed internal standard solution of (4) was prepared in an amount of 10. Mu.g/mL, wherein 100. Mu.L of the mixed internal standard solution was prepared ^-1 Mixing the internal standard solution, adding 900 mu L of methanol solution, and uniformly mixing to obtain 1 mu g/mL ^-1 Mixing the internal standard working solution, and storing in a refrigerator at the temperature of 20 ℃ below zero.

Preferably, the standard is prepared according to the following method: taking a certain amount of dabigatran etexilate, rivaroxaban, edoxaban, apixaban and dabigatran stock solution, diluting the solution with methanol to obtain a working solution with a proper concentration, taking 100 mu L of the working solution, adding 900 mu L of blank plasma into the working solution to prepare the solution containing dabigatran etexilate, rivaroxaban, edoxaban, apixaban and dabigatran with the concentrations of 0.1,0.5,1,2,5, 10, 50, 100, 200, 500 ng.mL respectively ^-1 The standard of (1).

Preferably, the blank plasma is blank plasma without anticoagulant drugs and active metabolites.

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

the detection method provided by the invention can be used for simultaneously detecting dabigatran etexilate, rivaroxaban, edoxaban, apixaban and dabigatran in blood plasma, and the lower limit of the quantification of the invention can reach 0.1-0.5ng/mL, so that the sensitivity is high.

The linear range of four anticoagulation drugs and one active metabolite is 0.1-500 ng/mL ^-1 The correlation coefficient (r) is more than 0.997, the recovery rates under three quality control concentrations are respectively 97.0-103%,99.4-112%,93.9-106%, the RSD% is less than 14.9%, the accuracy deviation between batches is less than 15%, and the precision RSD is less than 15%. The sample is placed at 4 ℃ for 24 hours, at-80 ℃ for 90 days, and is frozen and thawed repeatedly for 3 times to keep stable. Therefore, the specificity, precision, accuracy, linearity, stability and the like of the method all meet the analysis requirements of the biological sampleThe sensitivity is high, and the method can be used for monitoring the treatment medicines of clinical dabigatran etexilate, dabigatran, rivaroxaban, edoxaban and apixaban.

Drawings

FIG. 1.1 shows the molecular structure of Dabigatran (Dabigatran) (C) ₂₅ H ₂₅ N ₇ O ₃ )；

FIG. 1.2 shows the molecular structure of Dabigatran etexilate (Dabigatran etexilate) (C) ₃₄ H ₄₁ N ₇ O ₅ )；

FIG. 1.3 molecular structural formula (C) of Rivaroxaban (Rivaroxaban) ₁₉ H ₁₈ N ₃ O ₅ SCl)；

FIG. 1.4 shows the molecular formula (C) of Apixaban (Apixaban) ₂₅ H ₂₅ N ₅ O ₄ )；

FIG. 1.5 shows the molecular structure of Edoxaban (Edoxaban) (C) ₂₄ H ₃₀ ClN ₇ O ₄ S)；

FIG. 2.1 is a standard curve of dabigatran etexilate in human plasma;

FIG. 2.2 is a standard curve of dabigatran in human plasma;

FIG. 2.3 is a standard curve of edoxaban in human plasma;

FIG. 2.4 is a standard curve of rivaroxaban in human plasma;

FIG. 2.5 is a standard curve of apixaban in human plasma;

figure 3.1 is a chromatogram of rivaroxaban in the plasma of a subject;

FIG. 3.2 is a chromatogram of dabigatran etexilate in a subject's plasma;

FIG. 3.3 is a chromatogram of dabigatran in the plasma of a subject;

FIG. 4.1 is a chromatogram of blank plasma and a control with dabigatran etexilate (100 ng/mL);

FIG. 4.2 is a chromatogram of blank plasma and a control with dabigatran (100 ng/mL) added;

FIG. 4.3 is a chromatogram of blank plasma and a control with edoxaban (100 ng/mL) added;

FIG. 4.4 is a chromatogram of blank plasma and a control with rivaroxaban (100 ng/mL) added;

FIG. 4.5 is a chromatogram of blank plasma and an apixaban (100 ng/mL) added control.

Detailed Description

The present invention will be described in detail and specifically with reference to the following examples to facilitate better understanding of the present invention, but the following examples do not limit the scope of the present invention.

The following instruments and chemicals were used in the specific embodiment of the invention:

instrument

An Agilent UPLC system, SCIEX Q-trap 6500 Mass Spectrometry System, analyst chromatography workstation (Version 1.7.1), agilent ZORBAX Eclipse XDB-C18 (3.5 μm, 2.1X 100 mm) chromatography column;

a low temperature high speed refrigerated centrifuge;

a nitrogen blowing instrument;

a vortex mixer;

an analytical balance;

refrigerator, thermo Fisher;

millipore ultrapure Water device Direct Q, millipore Ltd, molsheim, france

Chemical product

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

[ example 1 ] A method for producing a polycarbonate

The present embodiment provides a method for preparing a control solution, a mixed internal standard working solution, a standard curve and a quality control plasma sample:

1. preparing a reference substance solution:

accurately weighing appropriate amount of dabigatran etexilate, dabigatran, rivaroxaban, edoxaban and apixaban reference substances respectively, dissolving with methanol and fixing volume to obtain concentrations of 1 mg.mL ^-1 The dabigatran etexilate, dabigatran, edoxaban and apixaban stock solutions and the concentration of 0.75 mg/mL ^-1 The rivaroxaban stock solutions of (a) as in table 1 were stored in a refrigerator at-20 ℃. Mixing dabigatran etexilate, dabigatran, rivaroxaban,the apixaban and edoxaban stock solutions are prepared into control solutions containing 5000ng/mL of dabigatran etexilate, dabigatran, rivaroxaban, apixaban and edoxaban by using a methanol water solution.

Table 1: preparation of stock solution

2. Preparing a rivaroxaban-deuterium 4 and dabigatran-deuterium 3 mixed internal standard working solution:

accurately weighing internal standards rivaroxaban-deuterium 4 and dabigatran-deuterium 3 to be about 1mg respectively, and dissolving the internal standards with DMSO to obtain concentrations of 1 mg/mL respectively ^-1 Diluting and mixing two internal standard stock solutions with a certain volume with a proper amount of methanol to obtain the internal standard stock solution with the concentration of 10 mu g/mL ^-1 Mixed internal standard solution of (4). 100. Mu.L of 10. Mu.g/mL ^-1 Mixing the internal standard solution, adding 900 mu L of methanol solution, and uniformly mixing to obtain 1 mu g/mL ^-1 Mixing the internal standard working solution, and storing in a refrigerator at the temperature of 20 ℃ below zero.

3. Standard curve plasma sample preparation:

taking a certain amount of dabigatran etexilate, dabigatran, rivaroxaban, edoxaban and apixaban stock solutions, diluting the dabigatran etexilate, dabigatran, rivaroxaban, edoxaban and apixaban stock solutions into a series of working solutions with proper concentrations by using methanol, taking 100 mu L of the working solutions, adding 900 mu L of blank plasma into the working solutions, and preparing the dabigatran etexilate, dabigatran, rivaroxaban, edoxaban and apixaban stock solutions with the concentrations of 0.1,0.5,1,2,5, 10, 50, 100, 200, 500 ng.mL respectively ^-1 Serial standard blood samples of (a).

A specific preparation method of a standard curve plasma sample comprises the following steps:

five stock solutions were diluted with methanol to give a mixed stock solution SW of 200. Mu.g/mL. Diluting SW100 μ L and methanol 900 μ L to 20 μ g/mL to obtain working solution SW0, storing in refrigerator at-20 deg.C, and diluting SW0 four times to obtain 5 μ g/mLFollowed by gradient dilution with methanol to obtain working liquids S2 to S9. As shown in Table 2, 100. Mu.L of the corresponding working solution was added with 900. Mu.L of blank plasma to prepare a mixture containing dabigatran etexilate, dabigatran, rivaroxaban, edoxaban and apixaban at concentrations of 0.1,0.5,1,2,5, 10, 50, 100, 200, 500 ng.mL, respectively ^-1 Serial standard blood samples of (a).

Table 2: preparation method of standard curve plasma sample

4. Preparing a quality control plasma sample:

taking a certain amount of dabigatran etexilate, dabigatran, rivaroxaban, edoxaban and apixaban stock solutions, diluting the dabigatran etexilate, dabigatran, rivaroxaban, edoxaban and apixaban stock solutions into a series of working solutions with proper concentration by using methanol, taking 100 mu L of the working solutions, adding 900 mu L of blank blood plasma into the working solutions, and respectively preparing the dabigatran etexilate, rivaroxaban, edoxaban and apixaban stock solutions with the concentrations of 4, 40 and 400 ng.mL ^-1 Low, medium, high quality control plasma samples as in table 3. (same standard curve plasma sample preparation)

Table 3: preparation method of quality control plasma sample

[ example 2 ]

The embodiment provides a method for detecting anticoagulant drug dabigatran etexilate, rivaroxaban, edoxaban, apixaban and active metabolite dabigatran in pretreated blood plasma by adopting an ultra performance liquid chromatography tandem mass spectrometry technology, which specifically comprises the following steps:

step 1, plasma sample and standard curve plasma sample pretreatment

Plasma sample pretreatment:

100. Mu.L of the plasma sample was taken in a 1.5mL centrifuge tube, and 890. Mu.L of ice-cold methanol and 1. Mu.g.mL of the mixture were added thereto ^-1 Mixing 10 mu L of the internal standard solution,centrifuging after vortexing (13000g, 4 ℃,10 min), taking the supernatant, blowing nitrogen to dry, adding 100 mu L of methanol solution containing 1% formic acid into the dry sample, ultrasonically redissolving, centrifuging (13000g, 4 ℃,10 min), taking 50 mu L of supernatant, adding 450 mu L of pure water, mixing uniformly, and analyzing by sample injection.

Standard curve plasma sample pretreatment:

100. Mu.L of each concentration standard curve plasma sample was taken in a 1.5mL centrifuge tube, and 890. Mu.L of ice-cold methanol and 1. Mu.g.mL were added thereto ^-1 Mixing 10 μ L of internal standard solution, centrifuging after vortexing (13000g, 4 ℃,10 min), taking supernatant, blowing nitrogen to dry, adding 100 μ L of 1% formic acid-containing methanol solution into a dry sample, carrying out ultrasonic redissolving, centrifuging (13000g, 4 ℃,10 min), taking 50 μ L of supernatant, adding 450 μ L of pure water, mixing uniformly, and carrying out sample injection analysis.

Step 2, separating the substance to be detected from the plasma matrix by ultra-high performance liquid chromatography, and then carrying out isotope internal standard quantification by tandem mass spectrometry

The chromatographic conditions were as follows:

stationary phase: (column model) Agilent ZORBAX Eclipse XDB-C18 (3.5 μm, 2.1X 100 mm);

column temperature: 40 ℃;

mobile phase: phase A: 0.1% aqueous formic acid; phase B: 0.1% formic acid in acetonitrile.

Flow rate: 0.3mL/min

Sample introduction amount: 2 μ L

The gradient elution was performed using different volume mixtures of mobile phase a and mobile phase B, under the gradient conditions shown in table 4 below. Each sample was collected for 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 an electrospray ionization detection mode, monitoring MRM by adopting multiple reactions and adopting a positive ion mode; capillary voltage 4500V; the ion source temperature is 550 ℃; a first ion source gas, 55psi; a second ion source gas, 55psi; air curtain air, 35psi. The mass transition, collision Energy (CE) and declustering voltage (DP) of the analytes are shown in table 5.

TABLE 5 MRM transition and Mass Spectrometry parameters for five anticoagulants and two internal standards

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

[ example 3 ] A method for producing a polycarbonate

The plasma samples from subjects taking anticoagulant were analyzed for anticoagulant concentration by the method of example 2, and the results are shown in FIGS. 3.1-3.3, where rivaroxaban was detected in one plasma sample and dabigatran etexilate or its metabolite dabigatran was detected in another plasma sample, and the concentrations of rivaroxaban, dabigatran etexilate and its metabolite dabigatran were further determined to be 263.7 ng.L. ^-1 、4.4ng·L ^-1 And 224.0 ng.L ^-1 。

[ test verification ]

1. Linearity

Weighted linear regression was performed on the peak area ratio (Y) of drug to internal standard versus concentration (X, ng/mL). Wherein, the weight coefficient is selected to be 1/X, the correlation coefficients r of the regression equation are all larger than 0.997, as shown in the table 6, the linearity is good, and the quantitative requirement is met.

TABLE 6 Linear Range, linear coefficient, linear equation and detection limits for five anticoagulants

Analyte	Linear range	Coefficient of linearity r	Linear equation of equations
				Dabigatran etexilate	1-500	0.99946	y＝0.00360x-0.00355
Dabigatran etexilate	0.1-500	0.99704	y＝0.008361x+0.000758087
				Edison sand class	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. Specificity

Blank plasma (without any standard substance and internal standard substance) 100 μ L is taken and subjected to pretreatment and determination according to the plasma sample pretreatment and determination method in the example 2, and the chromatogram of the blank plasma is compared with the blank plasma respectively containing 100ng/mL of dabigatran etexilate, dabigatran, edoxaban, rivaroxaban and apixaban, see figures 4.1-4.5, so that the blank plasma is confirmed to have no interference of endogenous impurities.

3. Accuracy and precision.

Preparing 5 batches of quality control plasma samples according to a quality control plasma sample preparation method, respectively taking 100 mu L of low, medium and high quality control plasma samples into a 1.5mL centrifuge tube, performing pretreatment on the quality control plasma samples according to the plasma sample pretreatment and determination method in the embodiment 2, and then determining, and calculating the average value, the standard deviation and the relative standard deviation according to the result.

The results show that: the accuracy deviation of quality control samples containing dabigatran etexilate, dabigatran, rivaroxaban, edoxaban and apixaban at three concentrations is less than 15%, and the precision RSD between batches is less than 15%, see Table 7. The results show that: the method has good precision and accuracy, and meets the analysis requirements.

TABLE 7 Intra-and Inter-batch accuracy and precision of five anticoagulants

4. Extraction recovery rate

The Low (LQC), medium (MQC) and High (HQC) quality control plasma samples are respectively taken, pretreated and then measured according to the plasma sample pretreatment and measurement method in the embodiment 2, and mixed standard working solution with corresponding concentration is taken for sample injection and measurement. And (4) dividing the peak area of the drug subjected to blood sample treatment by the peak area of the drug injected by the corresponding concentration reference substance solution to calculate the extraction recovery rate. Referring to the following table 8, the average extraction recovery rate of quality control plasma samples of various concentrations of dabigatran etexilate, dabigatran, rivaroxaban, edoxaban and apixaban is 93.9-112%, and RSD% < 14.9%.

The results show that: the extraction recovery rate of the method meets the requirement of a common analysis sample.

Table 8 recovery of five anticoagulants (n = 5)

5. Stability of

(1) Short term stability test

The quality control plasma sample is taken and placed at 4 ℃, the quality control plasma sample is pretreated according to the plasma sample pretreatment and measurement method in the embodiment 2 and then is measured, and the deviation of the measurement of the plasma standard sample with each concentration after being placed in the automatic sample injector for 24 hours is less than 9.2 percent compared with the result of the instant measurement, which is shown in the following table 9.

The results show that: plasma samples containing dabigatran etexilate, dabigatran, rivaroxaban, edoxaban and apixaban are subjected to pretreatment and then placed at 4 ℃ for 24 hours to keep stable.

(2) Long term stability test

The quality control plasma samples were taken, pretreated according to the plasma sample pretreatment and measurement method in example 2, and then measured, and plasma standard samples with various concentrations are placed at-80 ℃ for 90 days, and then compared with the results of the instant measurement, the deviation is less than 8.12%, and the results are shown in Table 9.

The results show that: plasma samples containing dabigatran etexilate, dabigatran, rivaroxaban, edoxaban and apixaban remained stable when stored for 90 days at-80 ℃.

(3) Freezing and thawing experiment

Taking a quality control plasma sample, immediately measuring, standing at-80 ℃, melting at normal temperature after 24 hours, repeating the process for 3 times, and comparing with the result of the instant measurement. The results show a deviation of <9.3%, see table 9.

The results show that: plasma samples containing dabigatran etexilate, dabigatran, rivaroxaban, edoxaban and apixaban were repeatedly freeze-thawed 3 times to maintain stability.

(4) Matrix effect

And (4) taking a quality control plasma sample and a standard substance with corresponding concentration, and simultaneously carrying out pretreatment and then measuring. The results showed a matrix effect <12%. See table 9.

The results show that: plasma sample matrix effects containing dabigatran etexilate, dabigatran, rivaroxaban, edoxaban and apixaban are small.

TABLE 9 stability and matrix Effect of five novel oral anticoagulants

In conclusion, the invention provides a method for detecting anticoagulant drug dabigatran etexilate, rivaroxaban, edoxaban, apixaban and active metabolite dabigatran in pretreated blood plasma by adopting an ultra performance liquid chromatography tandem mass spectrometry technology, the specificity, precision, accuracy, linearity, stability and the like of the method accord with the analysis requirements of biological samples, the sensitivity is high, and the method can be used for monitoring treatment drugs of clinical dabigatran etexilate, dabigatran, rivaroxaban, edoxaban and apixaban.

The embodiments of the present invention have been described in detail, but the embodiments are only examples, and the present invention is not limited to the embodiments described above. Any equivalent modifications and substitutions to those skilled in the art are also within the scope of the present invention. Accordingly, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.

Claims

1. A method for simultaneously determining the concentrations of an anticoagulant drug and an active metabolite in blood plasma is characterized in that the anticoagulant drug is dabigatran etexilate, rivaroxaban, edoxaban and apixaban respectively, and the active metabolite is dabigatran; detecting an anticoagulant drug and an active metabolite in the pretreated plasma by adopting an ultra-high performance liquid chromatography tandem mass spectrometry technology, separating a substance to be detected from a plasma matrix by adopting ultra-high performance liquid chromatography, then carrying out isotope internal standard quantification by adopting a tandem mass spectrometry, drawing a standard curve by taking the ratio of the peak area of a standard substance to the peak area of a corresponding internal standard substance as a Y axis and the concentration of the standard substance as an X axis, and calculating the content of the anticoagulant drug and the active metabolite in the plasma;

the chromatographic conditions were as follows:

stationary phase: chromatography column model Agilent ZORBAX Eclipse XDB-C18,3.5 μm, 2.1X 100mm;

mobile phase: phase A: 0.01-0.2% formic acid aqueous solution, phase B: 0.01 to 0.2 percent of formic acid acetonitrile solution; mixing the mobile phase A and the mobile phase B in different volumes, and performing gradient elution;

the mass spectrometry conditions were as follows:

in an electrospray ionization detection mode, adopting a multi-reaction monitoring and positive ion mode; capillary voltage 4500V; the ion source temperature is 550 ℃; a first ion source gas, 55psi; a second ion source gas, 55psi; air curtain air, 35psi;

the pretreated plasma is prepared according to the following method: taking a plasma sample, adding methanol and mixed internal standard working solution into the plasma sample, centrifuging the plasma sample after vortex, taking supernate, blowing nitrogen to dry the supernate, dissolving the dried sample in 0.001-1% formic acid methanol solution, centrifuging the supernate, and mixing the supernate with pure water;

the lower limit of the quantification of the method is 0.1ng/mL-0.5ng/mL;

the gradient elution procedure was as follows:

the volume ratio of mobile phase a to mobile phase B was kept constant at 95; in 1-4 minutes, the volume ratio of the mobile phase A to the mobile phase B is uniformly and gradually changed from 95; the volume ratio of the mobile phase A to the mobile phase B is uniformly graded from 20; the volume ratio of mobile phase a to mobile phase B was kept constant at 5; the volume ratio of the mobile phase A to the mobile phase B is uniformly graded from 5; the volume ratio of mobile phase a to mobile phase B was kept constant at 95; each sample was collected for 8 minutes.

2. The method of claim 1, wherein the chromatographic conditions further comprise: column temperature: 40 ℃; flow rate: 0.3mL/min; sample introduction amount: 2 μ L.

3. The method of claim 1, wherein the pre-treated plasma is prepared by: a plasma sample (100. Mu.L) was taken in a 1.5mL centrifuge tube, and 890. Mu.L of ice-cold methanol and 1. Mu.g.mL were added thereto ^-1 10 mu L of the mixed internal standard working solution is vortexed and centrifuged to take supernatant, nitrogen is blown to dry, 100 mu L of methanol solution containing 0.001-1% formic acid is added into a dry sample and ultrasonically redissolved, 50 mu L of supernatant is centrifuged and taken, 450 mu L of pure water is added and mixed, and the mixture is ready for sample injection analysis.

4. The method as claimed in claim 1, wherein the mixed internal standard working solution comprises isotope internal standards of rivaroxaban-deuterium 4 and dabigatran-deuterium 3.

5. The method of claim 1, wherein the rivaroxaban-deuterium 4 is an internal standard corresponding to rivaroxaban, edoxaban, and apixaban; the dabigatran-deuterium 3 is an internal standard corresponding to the dabigatran and the dabigatran etexilate.

6. The method according to claim 4, wherein the mixed internal standard working solution is prepared according to the following method:

precisely weighing about 1mg of internal standard dabigatran-deuterium 3 and rivaroxaban-deuterium 4 respectively, and dissolving with DMSO to obtain concentrations of 1 mg. ML ^-1 Diluting and mixing two internal standard stock solutions with a certain volume with a proper amount of methanol to obtain the internal standard stock solution with the concentration of 10 mu g/mL ^-1 The mixed internal standard solution of (4) was prepared by collecting 100. Mu.L of 10. Mu.g/mL ^-1 Mixing the internal standard solution, adding 900 mu L of methanol solution, and uniformly mixing to obtain 1 mu g/mL ^-1 Mixing the internal standard working solution, and storing in a refrigerator at the temperature of 20 ℃ below zero.

7. The method of claim 1, wherein the standard is prepared according to the following method: taking a certain amount of dabigatran etexilate, rivaroxaban, edoxaban, apixaban and dabigatran stock solution, and diluting the dabigatran etexilate, rivaroxaban, edoxaban, apixaban and dabigatran stock solution into working solution with proper concentration by using methanolAdding 900 μ L blank plasma into 100 μ L working solution to obtain the final product 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 The standard of (1).

8. The method according to claim 7, wherein the blank plasma is blank plasma without anticoagulant drugs and active metabolites.