CN117805279A - Method for simultaneously and quantitatively analyzing rivaroxaban, ai Duosha class and dabigatran in blood sample based on liquid chromatography-tandem mass spectrometry - Google Patents
Method for simultaneously and quantitatively analyzing rivaroxaban, ai Duosha class and dabigatran in blood sample based on liquid chromatography-tandem mass spectrometry Download PDFInfo
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Abstract
The invention discloses a method for simultaneously and quantitatively analyzing rivaroxaban, ai Duosha class and dabigatran in a blood sample based on liquid chromatography-tandem mass spectrometry. The method comprises the steps of firstly establishing a standard curve, determining analysis and detection conditions of liquid chromatography and mass spectrometry, adopting an internal calibration method, taking the chromatographic area ratio of each detection substance to the corresponding internal standard as an ordinate Y-axis, taking the concentration ratio of each detection substance to the internal standard in human serum as an abscissa X-axis, respectively establishing standard curves of rivaroxaban, ai Duosha class and dabigatran, adopting liquid chromatography-tandem mass spectrometry to detect to obtain the peak area of a sample to be detected, and then calculating the concentration of rivaroxaban, ai Duosha class and dabigatran in the sample to be detected according to the respective standard curves. The method has the advantages of high sensitivity, good specificity, simple pretreatment process, analysis time of less than 5min and high efficiency, and can be used for processing a large amount of samples.
Description
Technical Field
The invention relates to a method for simultaneously and quantitatively analyzing rivaroxaban, ai Duosha class and dabigatran in a blood sample based on liquid chromatography-tandem mass spectrometry, belonging to the technical fields of analytical chemistry and drug detection.
Background
Rivaroxaban, ai Duosha class, dabigatran etexilate, all of which are novel oral anticoagulants, which inhibit only a specific coagulation factor, unlike traditional oral anticoagulants acting on multiple coagulation factors such as warfarin. The novel oral anticoagulant drugs can be particularly developed and marketed as oral Xa factor and IIa direct inhibitors, the former comprises Ai Duosha class, rivaroxaban and the like, and the latter comprises dabigatran. Both the two medicines are aimed at single active coagulation factors, compared with the traditional anticoagulants such as warfarin and the like, the anticoagulation effect is independent of antithrombin, the oral administration effect is quick, the half-life period is short, the good dose-effect relationship is achieved, the bleeding risk can be reduced to a certain extent, and the medicine is a development trend of the clinical anticoagulation medicines. However, due to the influence of individual factors of patients (age, weight, basic diseases, liver and kidney functions, etc.), the difference of blood concentration of the novel oral anticoagulant among patients is large, the anticoagulation failure may be caused by the too low concentration, the bleeding risk may be caused by the too high concentration, and the clinical medicine dosage optimization is limited only by clinical observation. Therefore, in order to better optimize the use of the novel oral anticoagulant, improve the clinical curative effect, reduce adverse reactions, and recommend the treatment drug monitoring.
Most of the currently reported methods for measuring rivaroxaban, ai Duosha class or dabigatran in human blood samples are liquid chromatography spectrophotometry, and have low sensitivity and long analysis time compared with the liquid chromatography-tandem chromatography technology. There are reports that rivaroxaban or Ai Duosha class or dabigatran in human plasma is measured by adopting a liquid chromatography-tandem mass spectrometry technology, but only a single object to be measured can be detected, and the method steps are different, if a plurality of clinical samples containing the three substances are to be detected, the detection needs multiple times, the operation is complicated, the sample turnover time (TAT) is increased, and the actual requirement of clinical examination cannot be met; the method can detect rivaroxaban, ai Duosha class and dabigatran, greatly improves the convenience of operation, effectively shortens TAT and meets the actual requirement of clinic on drug concentration monitoring.
Disclosure of Invention
The purpose of the invention is that: in order to solve the technical problems that the existing methods for measuring rivaroxaban, ai Duosha class and dabigatran in blood samples have low sensitivity and long analysis time and are different in quantitative analysis of three substances simultaneously, the invention provides a quantitative analysis method for simultaneously measuring rivaroxaban, ai Duosha class and dabigatran in blood samples, which is accurate, quick, high in sensitivity and low in cost, based on a liquid chromatography-tandem chromatography technology.
In order to achieve the aim of solving the problems, the technical scheme adopted by the invention is to provide a method for simultaneously and quantitatively analyzing rivaroxaban, ai Duosha class and dabigatran in a blood sample based on liquid chromatography-tandem mass spectrometry, which comprises the following steps:
obtaining a sample to be detected, and detecting the contents of rivaroxaban, ai Duosha class and dabigatran in the sample to be detected by utilizing liquid chromatography-tandem mass spectrometry;
wherein, the liquid chromatography conditions are as follows:
the liquid chromatography conditions were as follows:
chromatographic column: kineex F5 column;
mobile phase a: 5. Mu.M ammonium acetate in water containing 0.5% formic acid;
mobile phase B: a methanol solution;
needle washing liquid: 50% methanol;
gradient elution is adopted, the flow rate is 0.6mL/min, the column temperature is 40 ℃, and the sample injection is 20 mu L;
the gradient elution specifically comprises: 0-0.5min, the volume fraction of the mobile phase A is kept 70%, and the volume fraction of the mobile phase B is kept 30%;0.5-2.2min, the volume fraction of the mobile phase A is reduced from 70% to 10%, and the volume fraction of the mobile phase B is increased from 30% to 90%;2.2-3.2min, the volume fraction of the mobile phase A is kept 10%, and the volume fraction of the mobile phase B is kept 90%;3.21min, the volume fraction of the mobile phase A is increased from 10% to 70%, and the volume fraction of the mobile phase B is decreased from 90% to 30%; the volume fractions of mobile phase a and mobile phase B were kept to 4.2min to stop.
Preferably, the mass spectrometry conditions are as follows:
ion source: electrospray ion source, positive ion mode; capillary voltage: 5500V; ion source Temperature (TEM): 550 ℃; ion source atomizing gas (GS 1): 60psi; ion source heating assist gas (GS 2): 60psi; curtain gas (CUR): 35psi; collision gas (CAD): 8psi; scanning mode: MRM.
Preferably, the sample to be tested is obtained by: measuring an internal standard solution of a serum sample corresponding to each detection substance and precipitator acetonitrile according to the volume ratio of 1:1:2, uniformly mixing the volumes, centrifuging, sucking the supernatant, and diluting with water to obtain the sample to be detected.
Preferably, the sampling requirements of the human serum sample are as follows: collecting venous blood of a subject taking at least one of rivaroxaban, ai Duosha class and dabigatran with a blood collection tube containing separation gel to obtain a sample; and separating serum by centrifugal precipitation within 2 hr, packaging, and storing in dark place.
Preferably, the quantitative analysis adopts an internal calibration method, the concentration ratio of the standard substance of each detection substance to the corresponding internal standard substance is taken as an X axis, the peak area ratio is taken as a Y axis, linear regression analysis is carried out, standard curves of rivaroxaban, ai Duosha class and dabigatran are respectively established, the peak area obtained by detection of the sample to be detected is substituted into the standard curve corresponding to each detection substance, and the concentration of rivaroxaban, ai Duosha class and dabigatran in the sample to be detected is calculated.
Preferably, the rivaroxaban has a representative standard curve of y=0.15707x+0.01274, a linear coefficient r= 0.99849, and a detected concentration in the range of 2-500; a representative standard curve for the Ai Duosha shift is y=0.08313 x+0.00404, linear coefficient 0.99834, detected concentration range is 2-500ng/mL; the representative standard curve of dabigatran is y=0.084638 x+0.00014, linear coefficient 0.99681, detected concentration range is 2-500ng/mL.
Preferably, the standard curve sample used for establishing the standard curve is prepared by the following method:
step 1: preparing the varroa-D4, ai Duosha class-D6 containing solution of 50% v/v methanol-water as solvent,Dabigatran etexilate 13 C6, mixing the labeling working solution of 10ng/mL each;
step 2: respectively preparing stock solutions containing 2450mg/L rivaroxaban, 1898.6mg/L Ai Duosha class and 805mg/L dabigatran with 50% v/v methanol-water solution as a solvent, and diluting the stock solutions into standard working solutions containing corresponding standard substances with the concentration of 100mg/L, wherein the standard working solutions are designated as RVXB-sub1, EDO-sub1 and DB-sub1; RVXB-sub1, EDO-sub1 and DB-sub1 are diluted to obtain working solution STD1-STD7 with concentration which is 20 times of 7 concentrations, wherein the concentrations are respectively 0.04 mug/mL, 0.08 mug/mL, 0.4 mug/mL, 1.6 mug/mL, 4 mug/mL, 8 mug/mL and 10 mug/mL;
step 3: respectively taking 10 mu L of the working solution STD1-STD7 with the concentration of 20 times obtained in the step 2, and respectively adding 190 mu L of blank matrix, so as to prepare standard curve samples S1-S7, wherein the blank matrix is blank human serum; wherein, the S1-S7 concentrations are respectively as follows: 2,4, 20, 80, 200, 400, 500ng/mL.
Preferably, the standard curve is verified by a quality control product, and the preparation method of the quality control product is as follows:
step 1: preparing a working solution with 20 times concentration of a quality control product, wherein the working solution with 20 times concentration of the quality control product is diluted by using 50% v/v methanol-water solution of RVXB-sub1, EDO-sub1 and DB-sub1 to obtain Hs, ms, ls and LLs of corresponding standard products, and the concentrations of the working solution are 7.5 mug/mL, 1 mug/mL, 0.12 mug/mL and 0.04 mug/mL respectively; wherein the diluent is a 50% v/v methanol-water solution;
step 2: preparing a quality control product, wherein the quality control product is prepared from a working solution with 20 times concentration of the quality control product and a blank matrix (blank human serum); and respectively taking 10 mu L of Hs, ms, ls and LLs which are fully and uniformly mixed, respectively adding 190 mu L of blank matrix, and preparing high-concentration quality control (HQC), medium-concentration quality control (MQC), low-concentration quality control (LQC) and minimum quantitative limit quality control (LLOQ) samples, wherein LLOQ, LQC, MQC, HQC concentrations are respectively as follows: 2,6, 50, 375ng/mL.
Compared with the prior art, the invention has the beneficial effects that:
the method provided by the invention can be used for simultaneously detecting the concentration of rivaroxaban, ai Duosha and dabigatran, has high sensitivity, high specificity and simple pretreatment process, can be used for completing the separation and quantitative detection of rivaroxaban, ai Duosha and dabigatran in serum in a short time, basically meets the requirements on accuracy and precision, can be used for quantitative analysis of rivaroxaban, ai Duosha and dabigatran in serum, and provides a simple and quick detection method for monitoring the concentration of rivaroxaban, ai Duosha and dabigatran; the method provided by the invention has the advantages that the analysis time is less than 5min, the efficiency is high, and the method can be used for processing a large amount of samples.
Drawings
FIG. 1 is an MRM spectrum of rivaroxaban, ai Duosha class, dabigatran and corresponding internal standard;
fig. 2 is a standard graph of rivaroxaban;
FIG. 3 is a standard graph of Ai Duosha class;
fig. 4 is a standard graph of dabigatran.
Detailed Description
In order to make the invention more comprehensible, preferred embodiments accompanied with figures are described in detail below.
The test methods used in the embodiment of the invention are all conventional methods unless specified otherwise; the materials, reagents and the like used, unless otherwise specified, are those commercially available.
The instrument and the equipment adopted in the embodiment of the invention comprise:
AB SCIEX Triple Quad 4500MD high performance liquid chromatography tandem triple quadrupole mass spectrometer (AB SCIEX Co., USA);
KS-060AL ultrasonic cleaner (Shenzhen Utility ultrasonic technology Co., ltd., china);
eppendorf Centrifuge 5430R desk top high speed refrigerated centrifuge (Eppendorf, germany);
EVortex-Genie2 vortex mixer (company Scientific Industries, usa);
a Synergy UV ultrapure water preparation system (Merck Millipore, germany);
MB100-4A micro-plate constant temperature oscillator (Shanghai Luodanlun scientific instruments Co., ltd., china)
XSR204 electronic balance (METTLER toldeo company, switzerland).
The reagents and consumables used in the embodiments of the invention include: methanol: purity is not less than 99.97%, LCMS grade (product number 1.06007.4008, merk, germany); acetonitrile: purity is not less than 99.97%, LCMS grade (product number 1.00030.4008, merk, germany); 300 μl 96 well plate and gasket (Simer fly in the United states); 1.5mL centrifuge tube (Axygen, U.S.); pipettes (Eppendorf, germany).
The standard substances and internal standards used in the embodiment of the invention are as follows: ai Duosha, ai Duosha-D6 (Shanghai Seiyaka Biotechnology Co., ltd.); rivaroxaban, rivaroxaban-D4, dabigatran-o-f 13 C6 (Canada, toronto Research Chemicals). The content is changed along with different batches, and the conversion is carried out.
Examples
The detection method of rivaroxaban, ai Duosha class and dabigatran based on LC-MS comprises the following steps:
(1) Preparation of internal standard liquid
rivaroxaban-D4, ai Duosha class-D6, dabigatran etexilate 13 The C6 stock solution is dissolved by 1:1 (v: v) methanol-water solution and is fixed to 10mL to obtain rivaroxaban-D4 stock solution with the concentration of 970mg/L, ai Duosha class-D6 stock solution with the concentration of 1000mg/L, dabigatran- 13 The concentration of the C6 stock solution was 500mg/L. And precisely sucking a proper amount of the stock solution, diluting with a methanol-water solution containing 1:1 (v: v) to obtain an internal standard working solution with corresponding concentration, wherein the specific reference working solution is shown in a table 1, and the actual preparation volume can be adjusted according to the requirement.
Table 1 preparation of internal standard solutions
(2) Preparation of working solution with 20 times concentration of standard curve
Rivaroxaban, ai Duosha class, dabigatran etexilate stock solution is dissolved by 1:1 (v: v) methanol-water solution and is fixed to 10mL, and the concentration of rivaroxaban stock solution is 2450mg/L, the concentration of Ai Duosha class stock solution is 1898.6mg/L, and the concentration of dabigatran etexilate stock solution is 805mg/L. The stock solution is precisely sucked up and diluted by a solution containing 1:1 (v: v) methanol-water to obtain working solution with corresponding concentration, the preparation method and the concentration are shown in Table 2, the working solution is stored in a refrigerator with the temperature of minus 80 ℃ in a sealing way, and the actual preparation volume can be adjusted according to the requirement, and the unit is mg/L.
TABLE 2 preparation of working solution at 20 times of standard curve
The standard curve sample is prepared from a working solution with 20 times of concentration of the standard curve and a blank matrix; 10. Mu.L of each of the well-mixed STD1-STD7 was added with 190. Mu.L of each of the blank substrates to prepare S1-S7. The S1-S7 concentrations are respectively as follows: 2,4, 20, 80, 200, 400, 500ng/mL.
(3) Preparation of 20-time concentration working solution for quality control product
The working solution with 20 times concentration of the quality control product is obtained by diluting rivaroxaban, ai Duosha class, dabigatran etexilate stock solution and 1:1 (v: v) methanol-water solution, the preparation method and the concentration are shown in table 3, the working solution is stored in a refrigerator at the temperature of minus 80 ℃ in a sealing way, and the actual preparation volume can be adjusted according to the requirement (the unit is mg/L).
TABLE 3 preparation of 20-fold concentration working solution for quality control product
The quality control product is prepared from a working solution with the concentration of 20 times of the quality control product and a blank matrix; 10. Mu.L of Hs, ms, ls and LLs were mixed well, and 190. Mu.L of blank matrix was added to each to prepare minimum quantitative limit quality control (LLOQ), low concentration quality control (LQC), medium concentration quality control (MQC) and high concentration quality control (HQC) samples. LLOQ, LQC, MQC, HQC concentrations are respectively: 2,6, 50, 375ng/mL.
(4) Sample collection and processing
2.5-3.0mL of venous blood of rivaroxaban, ai Duosha class or dabigatran etexilate (the user needs to take blood before and 2 hours after taking the medicine for the fourth time more than 3 days continuously) is collected by a light blue cap blood collection tube (sodium citrate anticoagulation tube), so as to obtain a serum sample; and separating serum by centrifugal precipitation (4000 rpm/min,5 min) within 2 hr, packaging, and keeping away from light.
(5) Preparation of sample to be tested
Respectively placing 50 mu L of a precise serum sample into a clean centrifuge tube; respectively adding 50 mu L of internal standard solution and 100 mu L of precipitant (acetonitrile), fully mixing for 5min, and centrifuging at 14000rpm for 10min at 4 ℃; and respectively sucking 40 mu L of supernatant, diluting with 160 mu L of water, and placing in a 96-well plate to obtain a sample to be detected, and taking 20 mu L of sample for analysis.
The standard and the quality control product are subjected to the same treatment.
(6) Instrument detection
And using a triple quadrupole mass spectrometer to analyze and detect the sample to be detected.
The liquid chromatography conditions were as follows:
chromatographic column: a kineex F5 column, 100 x 3.0mm, particle size 2.6 μm;
mobile phase a: 5. Mu.M ammonium acetate in water containing 0.5% formic acid;
mobile phase B: a methanol solution;
needle washing liquid: 50% methanol;
gradient elution was performed at a flow rate of 0.6mL/min, column temperature of 40℃and 20. Mu.L of sample were introduced.
The elution gradient of the gradient elution is as follows: 0-0.5min, the volume fraction of the mobile phase A is kept 70%, and the volume fraction of the mobile phase B is kept 30%;0.5-2.2min, the volume fraction of the mobile phase A is reduced from 70% to 10%, and the volume fraction of the mobile phase B is increased from 30% to 90%;2.2-3.2min, the volume fraction of the mobile phase A is kept 10%, and the volume fraction of the mobile phase B is kept 90%;3.21min, the volume fraction of the mobile phase A is increased from 10% to 70%, and the volume fraction of the mobile phase B is decreased from 90% to 30%; the volume fractions of mobile phase a and mobile phase B were kept to 4.2min to stop.
The conditions for mass spectrometry were as follows: ion source: electrospray ion source, positive ion mode; capillary voltage: 5500V; ion source Temperature (TEM): 550 ℃; ion source atomizing gas (GS 1): 60psi; ion source heating assist gas (GS 2): 60psi; curtain gas (CUR): 35psi; collision gas (CAD): 8psi; scanning mode: MRM. Mass spectrometry parameter optimization was performed by injecting a standard dilution standard solution under the conditions shown in table 4 below:
table 4 quantitative ion pair information
As shown in fig. 1, under the detection condition, the peak time of rivaroxaban and the corresponding internal standard is 2.83min; ai Duosha class and peak time of the corresponding internal standard are 2.60min; the peak time of the dabigatran and the corresponding internal standard is 1.44min.
(7) Quantitative calculation
And drawing a standard curve equation by using the detected standard substance, and carrying out linear regression analysis by taking the concentration ratio of the standard substance to the internal standard substance as an X axis and the peak area ratio as a Y axis to obtain a standard curve and a standard curve equation. The standard curve equation is shown in table 6 below; the standard curves are shown in fig. 2-4.
TABLE 5 standard curve equation
Performing cause searching on the overrun data, and performing repeated measurement analysis processing; diluting the sample exceeding the maximum point concentration of the standard curve, diluting with a blank matrix, estimating according to the test result, and selecting proper dilution factors (2 times, 5 times and 10 times); the sample with the concentration lower than the lowest point of the standard curve is reported according to the actual value; samples below the detection limit are reported as undetected.
6 samples of quality control samples of LLOQ, LQC, MQC and HQC, rivaroxaban, ai Duosha class and dabigatran were taken, and the quality control samples were processed according to the above-mentioned sample preparation method, 1 analytical batch was processed every day for three consecutive days, and analysis and detection were performed according to the above-mentioned analysis and detection conditions on the day of the processing, and the accuracy and precision were calculated, and the results are shown in Table 6. The results show that: except for the lowest limit of quantification (LLOQ), the precision RSD of both intra-and inter-batch quality control samples for rivaroxaban, ai Duosha shift, dabigatran, was less than 7% and the quality control samples at each concentration level did not deviate from their theoretical values by more than ±7%. The precision RSD of the quality control samples in the batch and the batch of the minimum quantitative rivaroxaban, ai Duosha class and dabigatran is less than 8%, and the deviation of the quality control samples from the theoretical value is not more than +/-8%. In summary, the precision and accuracy meet the requirements.
Table 6 results of measuring precision and accuracy of the measuring method of the present invention
The above-described embodiments are only preferred embodiments of the present invention, and are not intended to be limiting in any way and in nature, and it should be noted that several modifications and additions may be made to those skilled in the art without departing from the invention, which modifications and additions are also intended to be construed as within the scope of the invention.
Claims (7)
1. The method for simultaneously and quantitatively analyzing rivaroxaban, ai Duosha class and dabigatran in blood sample based on liquid chromatography-tandem mass spectrometry is characterized by comprising the following steps of:
obtaining a sample to be detected, and detecting the contents of rivaroxaban, ai Duosha class and dabigatran in the sample to be detected by utilizing liquid chromatography-tandem mass spectrometry;
wherein, the liquid chromatography conditions are as follows:
the liquid chromatography conditions were as follows:
chromatographic column: kineex F5 column;
mobile phase a: 5. Mu.M ammonium acetate in water containing 0.5% formic acid;
mobile phase B: a methanol solution;
needle washing liquid: 50% methanol;
gradient elution is adopted, the flow rate is 0.6mL/min, the column temperature is 40 ℃, and the sample injection is 20 mu L;
the gradient elution specifically comprises: 0-0.5min, the volume fraction of the mobile phase A is kept 70%, and the volume fraction of the mobile phase B is kept 30%;0.5-2.2min, the volume fraction of the mobile phase A is reduced from 70% to 10%, and the volume fraction of the mobile phase B is increased from 30% to 90%;2.2-3.2min, the volume fraction of the mobile phase A is kept 10%, and the volume fraction of the mobile phase B is kept 90%;3.21min, the volume fraction of the mobile phase A is increased from 10% to 70%, and the volume fraction of the mobile phase B is decreased from 90% to 30%; the volume fractions of mobile phase a and mobile phase B were kept to 4.2min to stop.
2. The method of claim 1, wherein the mass spectrometry conditions are as follows:
ion source: electrospray ion source, positive ion mode; capillary voltage: 5500V; ion source Temperature (TEM): 550 ℃; ion source atomizing gas (GS 1): 60psi; ion source heating assist gas (GS 2): 60psi; curtain gas (CUR): 35psi; collision gas (CAD): 8psi; scanning mode: MRM.
3. The method according to claim 1, wherein the sample to be tested is obtained by: measuring an internal standard solution of a serum sample corresponding to each detection substance and precipitator acetonitrile according to the volume ratio of 1:1:2, uniformly mixing the volumes, centrifuging, sucking the supernatant, and diluting with water to obtain the sample to be detected.
4. A method according to claim 3, wherein the human serum sample is sampled as follows: collecting venous blood of a subject taking at least one of rivaroxaban, ai Duosha class and dabigatran with a blood collection tube containing separation gel to obtain a sample; and separating serum by centrifugal precipitation within 2 hr, packaging, and storing in dark place.
5. The method of claim 1, wherein the quantitative analysis adopts an internal calibration method, the concentration ratio of the standard substance to the corresponding internal standard substance of each detection substance is taken as an X axis, the peak area ratio is taken as a Y axis, linear regression analysis is performed, standard curves of rivaroxaban, ai Duosha class and dabigatran are respectively established, the peak areas detected by the sample to be detected are substituted into the standard curves corresponding to each detection substance, and the concentrations of rivaroxaban, ai Duosha class and dabigatran in the sample to be detected are calculated.
6. The method of claim 5, wherein the rivaroxaban has a representative standard curve of y = 0.15707x +0.01274, a linear coefficient r = 0.99849, and a detected concentration in the range of 2-500; a representative standard curve for the Ai Duosha shift is y=0.08313 x+0.00404, linear coefficient 0.99834, detected concentration range is 2-500ng/mL; the representative standard curve of dabigatran is y=0.084638 x+0.00014, linear coefficient 0.99681, detected concentration range is 2-500ng/mL.
7. The method of claim 5, wherein the standard curve sample used in the establishment of the standard curve is formulated as follows:
step 1: preparing varoxaban-D4, ai Duosha class-D6 and dabigatran with 50% v/v methanol-water solution as solvent 13 C6, mixing the labeling working solution of 10ng/mL each;
step 2: respectively preparing stock solutions containing 2450mg/L rivaroxaban, 1898.6mg/L Ai Duosha class and 805mg/L dabigatran with 50% v/v methanol-water solution as a solvent, and diluting the stock solutions into standard working solutions containing corresponding standard substances with the concentration of 100mg/L, wherein the standard working solutions are designated as RVXB-sub1, EDO-sub1 and DB-sub1; RVXB-sub1, EDO-sub1 and DB-sub1 are diluted to obtain working solution STD1-STD7 with concentration which is 20 times of 7 concentrations, wherein the concentrations are respectively 0.04 mug/mL, 0.08 mug/mL, 0.4 mug/mL, 1.6 mug/mL, 4 mug/mL, 8 mug/mL and 10 mug/mL;
step 3: respectively taking 10 mu L of the working solution STD1-STD7 with the concentration of 20 times obtained in the step 2, and respectively adding 190 mu L of blank matrix, so as to prepare standard curve samples S1-S7, wherein the blank matrix is blank human serum; wherein, the S1-S7 concentrations are respectively as follows: 2,4, 20, 80, 200, 400, 500ng/mL.
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