CN111665303A - Kit for detecting antiplatelet drugs in plasma by ultra-high performance liquid chromatography tandem mass spectrometry technology - Google Patents
Kit for detecting antiplatelet drugs in plasma by ultra-high performance liquid chromatography tandem mass spectrometry technology Download PDFInfo
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Abstract
The invention relates to a kit for detecting an antiplatelet drug in plasma by an ultra-high performance liquid chromatography tandem mass spectrometry technology, which has the advantages of simple pretreatment process, low cost, high sensitivity and strong specificity, completes the separation and detection of the antiplatelet drug within 5min, basically meets the requirements on accuracy and precision, can be used for quantitative analysis of the antiplatelet drug in clinic, and provides a reliable detection method for monitoring the treatment concentration of the antiplatelet drug in clinic.
Description
Technical Field
The invention belongs to the technical field of plasma detection, and particularly relates to a kit for detecting an antiplatelet drug in plasma by using an ultra-high performance liquid chromatography tandem mass spectrometry technology, wherein the specific drugs are as follows: ticagrelor (TIC), deshydroxyethyoxyl Ticagrelor (DHTIC), aspirin (Acetylsalicylic Acid, ASA), Salicylic Acid (Salicylic Acid, SA), Clopidogrel (Clopidogrel, CLOP) and Clopidogrel Carboxylic Acid metabolite (Clopidogrel Carboxylic Acid, CLOPC).
Background
Aspirin, a commonly used non-steroidal drug, is used for antipyretic analgesia initially, and later researches show that aspirin with a small dose can inhibit platelet aggregation and reduce the adhesion rate of platelets, so that thrombosis is prevented, 75mg of oral aspirin is used as a preventive drug and is widely used for preventing ischemic cardiovascular diseases, and meanwhile, aspirin with a low dose can be used for myocardial infarction patients. The aspirin used as a preventive medicine is often required to be used for a long time, and researches show that the aspirin used for a long time increases the risks of bleeding, neutrophilic granulocyte reduction and the like. Aspirin can be rapidly metabolized into salicylic acid in vivo, so that the aspirin content in vivo is low, and the two compounds have great concentration difference, which causes great difficulty in detecting the two compounds simultaneously. Neither the conventional LC-MS method nor the LC-UV method can detect both substances at the same time. The LC-MS/MS method can detect two compounds in vivo at the same time.
Ticagrelor (TIC) is a cyclopentyl triazolopyrimidine drug, is a unique platelet aggregation inhibitor, has a strong inhibition effect on a P2Y12 receptor, can generate a reversible inhibition effect on ADP (adenosine diphosphate) -induced platelet aggregation reaction, and has certain concentration dependence. It is often used to treat platelet-resistant, cholesterol-lowering, blood sugar-controlling, blood pressure-controlling, etc. TIC and its major metabolite deoxyethyl ticagrelor (DHTIC) both exhibit antiplatelet properties, with 30-40% of the major metabolites at absorbed doses being well tolerated by patients, with adverse reactions commonly reported including dizziness, nausea, chest pain, dyspnea and hemorrhage. The combination with statins may result in an increased risk of muscle related adverse events. However, use in combination with other drugs may have some effect leading to life threatening interactions. Therefore, it is necessary to investigate the concentration of these compounds in clinical samples, and it is therefore necessary to develop a sufficiently accurate method for the simultaneous determination of TIC, and its metabolites.
Clopidogrel (CLOP) is a thienopyridine derivative, can selectively inhibit the combination of Adenosine Diphosphate (ADP) and platelet receptors and the activation of secondary ADP-mediated glycoprotein GPI-Ib/IIIa complex, and can also block the enhancement of platelet activity of other types of huhu agonists caused by releasing ADP, thereby finally achieving the purpose of inhibiting platelet aggregation. Is often used in patients with recent stroke episodes, as well as patients with myocardial infarction with positive diagnosis of peripheral artery disease, ultimately reducing the incidence of atherosclerotic events. Clopidogrel is a prodrug, the original drug has no activity, the prodrug is mainly metabolized by liver, 85% of carboxylic acid metabolite (CLOPC) in plasma is related to drug compounds, the highest concentration occurs for about 1h after the drug is taken, and the concentration difference of clopidogrel and the clopidogrel metabolite is large among different individuals, so that accurate drug concentration monitoring is required.
At present, the comprehensive analysis of domestic and foreign documents and patents shows that the detection of the antiplatelet drugs is only to detect one or two compounds at the same time, and no report is available for detecting the six compounds at the same time.
Disclosure of Invention
The invention aims to provide a kit for detecting antiplatelet drugs in plasma by using an ultra-high performance liquid chromatography tandem mass spectrometry technology on the basis of the prior art.
The invention also aims to provide application of the kit in detecting antiplatelet drugs in plasma by using an ultra performance liquid chromatography tandem mass spectrometry technology.
The technical scheme of the invention is as follows:
a kit for detecting antiplatelet drugs in plasma by an ultra-high performance liquid chromatography tandem mass spectrometry technology,
the antiplatelet drugs are respectively: ticagrelor (TIC), Deshydroxyethoxyticagrelor (DHTIC), aspirin (ASA), Salicylic Acid (SA), Clopidogrel (CLOP), and clopidogrel carboxylic acid metabolite (CLOPC);
the isotope internal standard substances corresponding to the antiplatelet drugs are respectively as follows: ticagrelor-d 7(TIC-d7), deshydroxyethoxyticagrelor-d 7(DHTIC-d7), aspirin-d 4(ASA-d4), salicylic acid-d 4(SA-d4), and clopidogrel-d 4(CLOP-d 4).
The kit comprises the following reagents:
(1) eluent:
eluent A: 0.001 to 0.01 percent of formic acid aqueous solution; eluent B: acetonitrile;
(2) calibration solution:
preparing a mixed standard solution containing 50000ng/mL ticagrelor, 50000ng/mL deshydroxyethoxy ticagrelor, 50000ng/mL aspirin, 50000ng/mL salicylic acid, 1000ng/mL clopidogrel and 1000ng/mL clopidogrel carboxylic acid metabolites into seven calibrator solutions with different concentration points by using a blank plasma matrix, wherein the seven concentration points of the calibrator solution are as follows:
the concentrations of ticagrelor, deshydroxyethoxy ticagrelor, aspirin and salicylic acid are the same, and seven concentrations are as follows: 2500ng/mL, 1250ng/mL, 250ng/mL, 125ng/mL, 25ng/mL, 12.5ng/mL, and 5 ng/mL;
the concentrations of clopidogrel and clopidogrel carboxylic acid metabolites are the same, and seven concentrations are as follows in sequence: 50ng/mL, 25ng/mL, 5ng/mL, 2.5ng/mL, 0.5ng/mL, 0.25ng/mL, and 0.1 ng/mL.
(3) Mixing internal standard solutions:
an aqueous acetonitrile solution comprising 1500ng/mL ticagrelor-d 7, 1500ng/mL deshydroxyethoxy ticagrelor-d 7, 2000ng/mL aspirin-d 4, 2000ng/mL salicylic acid-d 4, and 20ng/mL clopidogrel-d 4;
(4) protein precipitant:
a mixed solution of methanol and acetonitrile;
(5) quality control product:
blank plasma matrix containing antiplatelet drugs comprises low, medium and high concentrations of QC (L), QC (M) and QC (H), wherein,
QC (L) is the mixed standard solution diluted to 5000 times with blank plasma matrix;
QC (M) is the above mixed standard solution diluted to 500 times with blank plasma matrix;
QC (H) is the above mixed standard solution diluted 50-fold with blank plasma matrix.
In a preferred embodiment, the eluent a is 0.001% to 0.005% aqueous formic acid, preferably 0.004% aqueous formic acid.
In one scheme, the volume ratio of methanol to acetonitrile in the protein precipitator is 1: 1-5; preferably, the volume ratio of methanol to acetonitrile in the protein precipitant is 1: 4.
The mixed standard solution mentioned in the present invention is prepared as follows: preparing a mixed standard solution containing 50000ng/mL ticagrelor, 50000ng/mL deshydroxyethoxy ticagrelor, 50000ng/mL aspirin, 50000ng/mL salicylic acid, 1000ng/mL clopidogrel and 1000ng/mL clopidogrel carboxylic acid metabolite by using acetonitrile aqueous solution to prepare 2000 μ g/mL ticagrelor standard mother solution, 1000 μ g/mL deshydroxyethoxy ticagrelor standard mother solution, 2000 μ g/mL aspirin standard mother solution, 100 μ g/mL clopidogrel standard mother solution and 100 μ g/mL clopidogrel carboxylic acid metabolite mother solution.
The mixed internal standard solution mentioned in the invention is prepared according to the following method: and preparing isotope mixed internal standard solutions containing 1500ng/mL ticagrelor-d 7, 1500ng/mL desethoxylation ticagrelor-d 7, 2000ng/mL aspirin-d 4, 2000ng/mL salicylic acid-d 4 and 20ng/mL clopidogrel-d 4 from 150 ug/mL ticagrelor-d 7 isotope internal standard mother solution, 150 ug/mL desethoxylation ticagrelor-d 7 isotope internal standard mother solution, 200 ug/mL aspirin-d 4 isotope internal standard mother solution, 200 ug/mL salicylic acid-d 4 isotope internal standard mother solution and 2 ug/mL clopidogrel-d 4 isotope internal standard mother solution by using acetonitrile aqueous solutions.
When preparing a mixed standard solution and a mixed internal standard working solution, the adopted acetonitrile aqueous solution is 50-95% acetonitrile aqueous solution; preferably 70 to 90 percent of acetonitrile water solution; more preferably 80% acetonitrile in water.
In the preparation of the mixed standard solution, the blank plasma matrix is blank plasma without antiplatelet drugs.
The concentration of acetonitrile in water as referred to herein generally refers to the volume concentration.
The blood plasma mentioned in the invention is the blood plasma of human or animal.
In a preferred embodiment, the kit for detecting antiplatelet drugs in plasma by ultra performance liquid chromatography tandem mass spectrometry comprises the following reagents:
(1) eluent:
eluent A: 0.004% aqueous formic acid; eluent B: acetonitrile;
(2) calibration solution:
preparing a mixed standard solution containing 50000ng/mL ticagrelor, 50000ng/mL deshydroxyethoxy ticagrelor, 50000ng/mL aspirin, 50000ng/mL deshydroxyethoxy ticagrelor, 50000ng/mL aspirin, 50000ng/mL salicylic acid, 1000ng/mL clopidogrel and 1000ng/mL clopidogrel carboxylic acid metabolites from 2000 μ g/mL ticagrelor standard mother solution, 1000 μ g/mL deshydroxyethoxy ticagrelor standard solution, 2000 μ g/mL aspirin standard solution and 100 μ g/mL clopidogrel carboxylic acid metabolite mother solution with 80% acetonitrile water; preparing the mixed standard solution into seven calibrator solutions with different concentration points by using blank plasma without antiplatelet drugs;
(3) mixing internal standard solutions:
preparing isotope mixed internal standard solutions containing 1500ng/mL ticagrelor-d 7, 1500ng/mL desethoxylation ticagrelor-d 7, 2000ng/mL aspirin-d 4, 2000ng/mL salicylic acid-d 4 and 20ng/mL clopidogrel-d 4 from 150 ug/mL ticagrelor-d 7 isotope internal standard mother solution, 150 ug/mL desethoxylation ticagrelor-d 7 isotope internal standard mother solution, 200 ug/mL aspirin-d 4 isotope internal standard mother solution, 200 ug/mL salicylic acid-d 4 isotope internal standard mother solution and 2 ug/mL clopidogrel-d 4 isotope internal standard mother solution by using 80% acetonitrile aqueous solution;
(4) protein precipitant:
the volume ratio of methanol to acetonitrile is 1: 4;
(5) quality control product:
preparing the mixed standard solution into three different concentrations of QC (L), QC (M) and QC (H) by using blank plasma without the antiplatelet drugs, wherein the corresponding concentrations of the antiplatelet drug quality control products in the QC (L), the QC (M) and the QC (H) are shown in a table 1;
TABLE 1 corresponding concentration of quality control product for antiplatelet drugs (unit: ng/mL)
| Numbering | Components | QC(L) | QC(M) | QC(H) |
| 1 | TIC | 10 | 100 | 1000 |
| 2 | DHTIC | 10 | 100 | 1000 |
| 3 | ASA | 10 | 100 | 1000 |
| 4 | SA | 10 | 100 | 1000 |
| 5 | CLOP | 0.2 | 2 | 20 |
| 6 | CLOPC | 0.2 | 2 | 20 |
QC (L) includes: 10ng/mL ticagrelor, 10ng/mL deshydroxyethoxy ticagrelor, 10ng/mL aspirin, 10ng/mL salicylic acid, 0.2ng/mL clopidogrel, and 0.2ng/mL clopidogrel carboxylic acid metabolite.
QC (M) comprises: 100ng/mL ticagrelor, 100ng/mL deshydroxyethoxyticagrelor, 100ng/mL aspirin, 100ng/mL salicylic acid, 2ng/mL clopidogrel, and 2ng/mL clopidogrel carboxylic acid metabolites.
QC (H) includes: 1000ng/mL ticagrelor, 1000ng/mL deshydroxyethoxyticagrelor, 1000ng/mL aspirin, 1000ng/mL salicylic acid, 20ng/mL clopidogrel, and 20ng/mL clopidogrel carboxylic acid metabolites.
In a more preferred embodiment, the mixed internal standard solution is prepared as follows:
accurately transferring a certain volume of the anti-platelet drug isotope internal standard mother liquor, adding 950 microliter of 80% acetonitrile aqueous solution, and uniformly mixing to obtain 1mL of mixed internal standard solution, wherein the concentration is shown in the following table 2.
TABLE 2 preparation of mixed internal standard solutions
| Components | Mother liquor concentration (μ g/mL) | Volume removal (mu L) | Total volume (μ L) | Concentration of mixed internal standard solution (ng/mL) |
| TIC-d7 | 150 | 10 | 1000 | 1500 |
| DHTIC-d7 | 150 | 10 | 1000 | 1500 |
| ASA-d4 | 200 | 10 | 1000 | 2000 |
| SA-d4 | 200 | 10 | 1000 | 2000 |
| CLOP- |
2 | 10 | 1000 | 20 |
In a preferred scheme, the mixed internal standard solution and the protein precipitant are mixed in a volume ratio of 1:99 to prepare the protein precipitant containing the internal standard for ultra performance liquid chromatography tandem mass spectrometry detection.
In a more preferred embodiment, the calibrator solution is prepared as follows:
accurately transferring a certain volume of the anti-platelet drug standard mother liquor, adding 870 mu L of 80% acetonitrile aqueous solution, and fully and uniformly mixing to obtain 1mL of mixed standard solution, wherein the concentration is shown in the following table 3.
TABLE 3 preparation of Mixed Standard solutions
Preparing standard yeast by gradient dilution method, taking out mixed standard solution from refrigerator at-20 deg.C, vortex for 10s, preparing the highest concentration point of standard yeast with the mixed standard solution within 2min, and storing at-80 deg.C. The preparation process is as follows:
adding 10 μ L of the mixed standard solution to 190 μ L of the blank plasma matrix as a first high-value concentration point (S7); taking the first high-value concentration point (S7) and diluting the first high-value concentration point with an equal volume of blank plasma matrix to obtain a second high-value concentration point (S6); diluting the first high-value concentration point (S7) with 9 times volume of blank plasma matrix to obtain a third high-value concentration point (S5); diluting the second high concentration (S6) point with 9 times volume of blank plasma matrix to obtain a fourth high concentration point (S4); diluting the third high concentration point (S5) with 9 times volume of blank plasma matrix to obtain a fifth high concentration point (S3); diluting the fourth high concentration point (S4) with 9 times volume of blank plasma matrix to obtain a sixth high concentration point (S2); the fifth high concentration point (S3) was diluted with 4 volumes of blank plasma matrix to obtain a seventh high concentration point (S1), which was determined as shown in Table 4 below (unit: ng/mL):
TABLE 4 Standard koji preparation
The application of the kit in detecting antiplatelet drugs in plasma by using the ultra-performance liquid chromatography tandem mass spectrometry technology is also within the protection scope of the invention.
The specific detection method comprises the following steps:
a method for detecting antiplatelet drugs in plasma by ultra-high performance liquid chromatography tandem mass spectrometry technology,
the antiplatelet drugs are respectively: ticagrelor (TIC), Deshydroxyethoxyticagrelor (DHTIC), aspirin (ASA), Salicylic Acid (SA), Clopidogrel (CLOP), and clopidogrel carboxylic acid metabolite (CLOPC);
the isotope internal standard substances corresponding to the antiplatelet drugs are respectively as follows: ticagrelor-d 7(TIC-d7), deshydroxyethoxyticagrelor-d 7(DHTIC-d7), aspirin-d 4(ASA-d4), salicylic acid-d 4(SA-d4), and clopidogrel-d 4(CLOP-d 4);
detecting the antiplatelet drugs in the pretreated plasma by adopting an ultra-high performance liquid chromatography tandem mass spectrometry technology, firstly separating a target object to be detected from interfering components in a plasma matrix by utilizing the ultra-high performance liquid chromatography, then establishing a calibration curve by utilizing a mass spectrum isotope internal standard quantitative method and taking the concentration ratio of a standard substance to an internal standard substance as an X axis and the peak area ratio of the standard substance to the internal standard substance as a Y axis, and calculating the content of the antiplatelet drugs in the plasma, wherein the specific chromatographic conditions are as follows:
(1) ultra-high performance liquid chromatography conditions:
mobile phase A: 0.001 to 0.01 percent of formic acid aqueous solution; mobile phase B: acetonitrile;
the type of the chromatographic column: waters BEH C18 (2.1X 100mm, 1.7 μm);
and (3) performing gradient elution by adopting the mobile phase A and the mobile phase B as a mixed mobile phase, wherein the gradient elution process is as follows: the volume ratio of the mobile phase A to the mobile phase B is gradually changed from 95:5 to 40:60 at a constant speed within 0.0-1.0 min; the volume ratio of the mobile phase A to the mobile phase B is gradually changed from 40:60 to 2:98 at a constant speed within 1.0-2.0 minutes; the volume ratio of the mobile phase A to the mobile phase B is 2:98 within 2.0-3.0 minutes; the volume ratio of the mobile phase A to the mobile phase B is gradually changed from 2:98 to 95:5 at a constant speed within 3.0-5.0 minutes;
(2) mass spectrum conditions:
in an electrospray ionization (ESI) mode, adopting multi-reaction monitoring (MRM) to perform positive and negative switching scanning; the spray voltage was 3.0kV (ESI +)/2.5kV (ESI-); the desolvation temperature is 120 ℃; the temperature of atomizing gas is 500 ℃, the airflow speed of atomizing is 800L/h, and the airflow speed of taper hole is 150L/h; each target and its corresponding isotope internal standard were monitored simultaneously.
In order to improve the chromatographic separation selectivity, it may be considered to adjust the polarity of the mobile phase. The formic acid is added into the mobile phase A, so that the ionization efficiency of certain target compounds can be effectively improved, and under the coordination of other conditions, compared with the prior art that an LC-MS/MS method is adopted to detect the antiplatelet drugs in the plasma, the method has the advantages of higher sensitivity, simple pretreatment process, low cost, high sensitivity and strong specificity, and the separation and detection of the antiplatelet drugs can be completed within 5 min. In a preferable embodiment, the mobile phase a is 0.001% to 0.005% aqueous formic acid solution without affecting the effect of the present invention. In a more preferred embodiment, mobile phase a is 0.004% aqueous formic acid.
In chromatography, the choice of the chromatographic column is important and the requirements for the chromatographic column: high column efficiency, good selectivity, high analysis speed and the like. The invention adopts 0.001-0.01% formic acid water solution and acetonitrile as mobile phase, the type of chromatographic column is as follows: WatersBEH C18(2.1 × 100mm, 1.7 μm), under the coordination of other conditions, the endogenous substance does not interfere the determination of the sample, the sensitivity is high, the specificity is strong, the cost is low, the pretreatment process is simple, the separation and the detection can be completed within 5.0min, and the precision and the accuracy meet the requirements.
When the internal standard method is adopted, the selection of the internal standard substance is very important work. The ideal internal standard should be capable of being added to the sample in an accurate, known amount, and have substantially the same or as consistent as possible physicochemical properties, chromatographic behavior, and response characteristics as the sample being analyzed; under chromatographic conditions, the internal standard must be sufficiently separated from the components of the sample. According to the invention, ticagrelor-d 7(TIC-d7), deshydroxyethoxy ticagrelor-d 7(DHTIC-d7), aspirin-d 4(ASA-d4), salicylic acid-d 4(SA-d4) and clopidogrel-d 4(CLOP-d4) are respectively adopted as internal standards, the deuterated internal standards and the substance to be detected have the same retention time, chemical properties and matrix effect, and the reproducibility and accuracy in the antiplatelet drug determination in blood plasma are better.
In one embodiment, the flow rate is 0.2-0.5 mL/min, preferably 0.3 mL/min.
Further, the column temperature is 35-45 ℃, and preferably 40 ℃.
Furthermore, the injection volume is 1-5 μ L, preferably 1 μ L.
In a preferred scheme, when the ultra-high performance liquid chromatography tandem mass spectrometry technology is adopted to detect the antiplatelet drugs in the plasma, the specific chromatographic conditions are as follows:
(1) high performance liquid chromatography conditions:
mobile phase A: 0.004% formic acid-water solution;
mobile phase B: acetonitrile;
the type of the chromatographic column: waters BEH C18 (2.1X 100mm, 1.7 μm);
the gradient elution mode is adopted, see table 5; the flow rate is 0.3mL/min, the column temperature is 40 ℃, and the sample injection volume is 1 mu L;
TABLE 5 mobile phase gradient elution parameters
| Time (min) | Flow rate (mL/min) | ％A | ％B | Curve |
| 0.0 | 0.3 | 95 | 5 | - |
| 1.0 | 0.3 | 40 | 60 | 6 |
| 2.0 | 0.3 | 2 | 98 | 6 |
| 3.0 | 0.3 | 2 | 98 | 6 |
| 5.0 | 0.3 | 95 | 5 | 1 |
(2) Mass spectrum conditions:
in an electrospray ionization (ESI) mode, adopting multi-reaction monitoring (MRM) to perform positive and negative switching scanning; the spray voltage was 3.0kV (ESI +)/2.5kV (ESI-); the desolvation temperature is 120 ℃; the temperature of atomizing gas is 500 ℃, the airflow speed of atomizing is 800L/h, and the airflow speed of taper hole is 150L/h; meanwhile, the antiplatelet drugs and the corresponding isotope internal standards thereof are monitored, and the mass spectrum acquisition parameters of each target substance to be detected are shown in table 6.
TABLE 6 Mass Spectrometry parameters for antiplatelet drugs
| Compound (I) | Parent ion | Daughter ions | Declustering voltage (V) | Collision voltage (V) | ESI(+/-) |
| CLOPC | 308.0 | 198.0 | 4 | 14 | ESI+ |
| CLOP | 322.1 | 212.0 | 4 | 9 | ESI+ |
| CLOP-d4 | 326.1 | 187.9 | 6 | 20 | ESI+ |
| DHTIC | 479.2 | 126.9 | 12 | 24 | ESI+ |
| DHTIC-d7 | 486.2 | 126.9 | 56 | 56 | ESI+ |
| TIC | 523.2 | 127.0 | 20 | 54 | ESI+ |
| TIC-d7 | 530.2 | 126.9 | 56 | 54 | ESI+ |
| ASA | 136.7 | 93.0 | 34 | 14 | ESI- |
| ASA-d4 | 140.6 | 97.0 | 24 | 14 | ESI- |
| SA | 136.9 | 93.0 | 38 | 14 | ESI- |
| SA-d4 | 141.0 | 97.0 | 44 | 16 | ESI- |
The blood plasma mentioned in the invention is human or animal blood plasma.
In one protocol, the pretreated plasma is prepared as follows: adding a protein precipitator containing an internal standard into the plasma, and then oscillating and centrifuging to obtain a supernatant; wherein the protein precipitator is a mixed solution of methanol and acetonitrile.
Preferably, the volume ratio of methanol to acetonitrile in the protein precipitant is 1: 1-5, without affecting the effect of the present invention, for example, the volume ratio of methanol to acetonitrile in the protein precipitant is 1: 4.
In a preferred embodiment, the pretreated plasma is prepared as follows: 50 mu L of plasma is taken and put into a 1.5mL centrifuge tube, 200 mu L of protein precipitator (the volume ratio of methanol to acetonitrile is 1: 1-5) containing internal standard is added into the centrifuge tube, and 60 mu L of supernatant is taken after centrifugation is carried out for 4-10 min at 12000-15000 r/min and 1-5 ℃. The protein precipitator containing the internal standard is prepared by mixing a mixed internal standard solution and a protein precipitator, wherein the ratio of the mixed internal standard solution to the protein precipitator is 0.1-0.3: 19.9-19.7.
In a more preferred embodiment, the pretreated plasma is prepared as follows: putting 50 μ L of plasma into a 1.5mL centrifuge tube, adding 200 μ L of protein precipitant containing internal standard (volume ratio of methanol to acetonitrile is 1:4), and oscillating at high speed (maximum oscillation speed) for 5 min; centrifuging at 14000r/min at 4 ℃ for 5 min; transfer 60. mu.L of supernatant from the EP tube to a plastic lined tube in a 1. mu.L sample volume. The protein precipitant containing the internal standard is prepared by mixing a mixed internal standard solution and the protein precipitant, wherein the ratio of the mixed internal standard solution to the protein precipitant is 0.2: 19.8.
In one embodiment, the protein precipitant containing the internal standard is prepared as follows:
preparing a mixed internal standard solution containing 1500ng/mL ticagrelor-d 7, 1500ng/mL deshydroxyethoxy ticagrelor-d 7, 2000ng/mL aspirin-d 4, 2000ng/mL salicylic acid-d 4 and 20ng/mL clopidogrel-d 4 from an 80% acetonitrile aqueous solution by using 150 μ g/mL ticagrelor-d 7 isotope internal standard mother solution, 150 μ g/mL deshydroxyethoxy ticagrelor-d 7 isotope internal standard mother solution, 200 μ g/mL aspirin-d 4 isotope internal standard mother solution, 200 μ g/mL salicylic acid-d 4 isotope internal standard mother solution and 2 μ g/mL clopidogrel-d 4 isotope internal standard mother solution;
and adding 200 mu L of the mixed internal standard solution into 19.8mL of protein precipitator (the volume ratio of methanol to acetonitrile is 1:4) to obtain the protein precipitator containing the internal standard.
In one embodiment, the standard solution is prepared as follows:
preparing a mixed standard solution containing 50000ng/mL ticagrelor, 50000ng/mL deshydroxyethoxy ticagrelor, 50000ng/mL aspirin, 50000ng/mL deshydroxyethoxy ticagrelor, 50000ng/mL aspirin, 50000ng/mL salicylic acid, 1000ng/mL clopidogrel and 1000ng/mL clopidogrel carboxylic acid metabolites from 2000 μ g/mL ticagrelor standard mother solution, 1000 μ g/mL deshydroxyethoxy ticagrelor standard solution, 2000 μ g/mL aspirin standard solution and 100 μ g/mL clopidogrel carboxylic acid metabolite mother solution with 80% acetonitrile water;
preparing the mixed standard solution into seven calibrator solutions with different concentration points by using blank plasma without antiplatelet drugs, wherein the seven concentration points of the calibrator solution are as follows:
the concentrations of ticagrelor, deshydroxyethoxy ticagrelor, aspirin and salicylic acid are the same, and seven concentrations are as follows: 2500ng/mL, 1250ng/mL, 250ng/mL, 125ng/mL, 25ng/mL, 12.5ng/mL, and 5 ng/mL;
the concentrations of clopidogrel and clopidogrel carboxylic acid metabolites are the same, and seven concentrations are as follows in sequence: 50ng/mL, 25ng/mL, 5ng/mL, 2.5ng/mL, 0.5ng/mL, 0.25ng/mL, and 0.1 ng/mL;
taking 50 mu L of each concentration point sample, putting the sample into a 1.5mL centrifuge tube, adding 200 mu L of protein precipitant containing an internal standard (the volume ratio of methanol to acetonitrile is 1:4), and oscillating at high speed (maximum oscillation speed) for 5 min; centrifuging at 14000r/min at 4 ℃ for 5 min; transfer 60. mu.L of supernatant from the EP tube to a plastic lined tube in a 1. mu.L sample volume.
In one scheme, the quality control product is prepared according to the following method: taking the mixed standard solution and preparing blank plasma without antiplatelet drugs into three different concentrations of QC (L), QC (M) and QC (H):
QC (L) includes: 10ng/mL ticagrelor, 10ng/mL deshydroxyethoxy ticagrelor, 10ng/mL aspirin, 10ng/mL salicylic acid, 0.2ng/mL clopidogrel, and 0.2ng/mL clopidogrel carboxylic acid metabolite.
QC (M) comprises: 100ng/mL ticagrelor, 100ng/mL deshydroxyethoxyticagrelor, 100ng/mL aspirin, 100ng/mL salicylic acid, 2ng/mL clopidogrel, and 2ng/mL clopidogrel carboxylic acid metabolites.
QC (H) includes: 1000ng/mL ticagrelor, 1000ng/mL deshydroxyethoxyticagrelor, 1000ng/mL aspirin, 1000ng/mL salicylic acid, 20ng/mL clopidogrel, and 20ng/mL clopidogrel carboxylic acid metabolites.
By adopting the technical scheme of the invention, the advantages are as follows:
when the kit is used for detecting the antiplatelet drugs in the plasma, the pretreatment process is simple, the cost is low, the sensitivity is high, the specificity is strong, the separation and the detection of the antiplatelet drugs are completed within 5min, the accuracy and the precision basically meet the requirements, and the kit can be used for the quantitative analysis of the antiplatelet drugs in clinic and provides a reliable detection method for the monitoring of the treatment concentration of the antiplatelet drugs in clinic.
Drawings
FIG. 1 is an extracted ion current chromatogram of an antiplatelet drug standard;
fig. 2 is an extracted ion flow chromatogram of an antiplatelet drug in a plasma sample.
Detailed Description
The invention will be better understood from the following examples. However, those skilled in the art will readily appreciate that the description of the embodiments is only for illustrating the present invention and should not be taken as limiting the invention as detailed in the claims.
Example 1:
first, experimental material and instrument
1. Material
The samples were from plasma samples collected from the 12 month clinic in 2019 of the Nanjing drugstore Hospital.
(1) The instrument comprises the following steps: xevo TQ-S triple quadrupole mass spectrometer (Waters Corporation); UPLC I-Class ultra high performance liquid chromatography system (with autosampler, Waters Corporation); SCILOGEX D2012 high speed bench top centrifuge (usa); ultra pure water meter (ELGA LabWater, uk); multi-tube Vortex mixer (Vortex genie2, usa); an adjustable pipettor (Eppendorf 0.5-10 muL, 10-100 muL, 100-1000 muL); glassware, graduated cylinders, and the like. .
(2) Reagent consumables: MS grade methanol (Fisher, usa); MS grade acetonitrile (Fisher, usa); HPLC grade acetonitrile (Honeywell, usa); MS grade formic acid (Fisher, usa); HPLC grade methanol (Honeywell, usa); a chromatographic column: waters BEH C18 (2.1X 100mm, 1.7 μm).
(3) And (3) standard substance: the standards and their corresponding internal standards are shown in table 7 below.
(4) Quality control product: the blank plasma matrix containing the antiplatelet drugs is divided into low, medium and high concentrations, namely QC (L), QC (M) and QC (H), which are shown in table 1.
Adding membranes on the upper and lower peripheries of the kit, performing shockproof insulation, placing the eluents A and B at the upper left, and respectively placing 11 ampoule bottles at the lower left, wherein the standard solution, the mixed internal standard solution and the quality control product are respectively contained in the ampoule bottles; to the right, 25mL of protein precipitant was placed.
Second, liquid condition
(1) Chromatographic conditions are as follows: mobile phase A: 0.004% formic acid-water solution; mobile phase B: and (3) acetonitrile. The type of the chromatographic column: WatersBEH C18 (2.1X 100mm, 1.7 μm), using gradient elution, see Table 5 for details. The flow rate was 0.3mL/min, the column temperature was 40 ℃ and the injection volume was 1. mu.L.
(2) Mass spectrum conditions: in an electrospray ionization detection mode, adopting a mass spectrum scanning mode of multi-reaction monitoring; the spraying voltage is 3.0kV (ESI +) and 2.5kV (ESI-); the desolvation temperature is 120 ℃; the temperature of atomizing gas is 500 ℃, the airflow speed of atomizing is 800L/h, and the airflow speed of taper hole is 150L/h; each target was monitored simultaneously with the isotope internal standard. The mass spectrometric acquisition parameters for each target analyte are shown in table 6.
Second, the experimental procedure
(1) Preparing a standard substance:
2000. mu.g/mL ticagrelor standard stock solution, 1000. mu.g/mL deshydroxyethoxyticagrelor standard stock solution, 2000. mu.g/mL aspirin standard stock solution, 5000. mu.g/mL salicylic acid standard stock solution, 100. mu.g/mL clopidogrel standard stock solution, and 100. mu.g/mL clopidogrel carboxylic acid metabolite standard stock solution were formulated with 80% acetonitrile aqueous solution to a mixed standard solution containing 50000ng/mL ticagrelor, 50000ng/mL deshydroxyethoxyticagrelor, 50000ng/mL aspirin, 50000ng/mL salicylic acid, 1000ng/mL clopidogrel, and 1000ng/mL clopidogrel carboxylic acid metabolite (see Table 3 for details).
The mixed standard solution was prepared into a calibrator solution (see table 4) with a blank plasma matrix (blank plasma without anti-platelet drug) at seven different concentration points, and the concentration of each calibration point is listed in table 4. The seven concentration points of the calibrator solution were:
the concentrations of ticagrelor, deshydroxyethoxy ticagrelor, aspirin and salicylic acid are the same, and seven concentrations are as follows: 2500ng/mL, 1250ng/mL, 250ng/mL, 125ng/mL, 25ng/mL, 12.5ng/mL, and 5 ng/mL;
the concentrations of clopidogrel and clopidogrel carboxylic acid metabolites are the same, and seven concentrations are as follows in sequence: 50ng/mL, 25ng/mL, 5ng/mL, 2.5ng/mL, 0.5ng/mL, 0.25ng/mL, and 0.1 ng/mL.
(2) Preparation of protein precipitant containing internal standard
150 mug/mL ticagrelor-d 7 isotope internal standard mother liquor, 150 mug/mL deshydroxyethoxy ticagrelor-d 7 isotope internal standard mother liquor, 200 mug/mL aspirin-d 4 isotope internal standard mother liquor, 200 mug/mL salicylic acid-d 4 isotope internal standard mother liquor and 2 mug/mL clopidogrel-d 4 isotope internal standard mother liquor are prepared into mixed internal standard solutions containing 1500ng/mL ticagrelor-d 7, 1500ng/mL deshydroxyethoxy ticagrelor-d 7, 2000ng/mL aspirin-d 4, 2000ng/mL salicylic acid-d 4 and 20ng/mL clopidogrel-d 4 by using 80% acetonitrile aqueous solution (see Table 2 in detail). And adding 200 mu L of the mixed internal standard solution into 19.8mL of protein precipitator (the volume ratio of methanol to acetonitrile is 1:4) to obtain the protein precipitator containing the internal standard.
(3) Preparing a quality control product:
the mixed standard solution is prepared into QC (L), QC (M) and QC (H) with three different concentrations by using blank plasma without antiplatelet drugs, and the details are shown in table 1.
QC (L) includes: 10ng/mL ticagrelor, 10ng/mL deshydroxyethoxy ticagrelor, 10ng/mL aspirin, 10ng/mL salicylic acid, 0.2ng/mL clopidogrel, and 0.2ng/mL clopidogrel carboxylic acid metabolite.
QC (M) comprises: 100ng/mL ticagrelor, 100ng/mL deshydroxyethoxyticagrelor, 100ng/mL aspirin, 100ng/mL salicylic acid, 2ng/mL clopidogrel, and 2ng/mL clopidogrel carboxylic acid metabolites.
QC (H) includes: 1000ng/mL ticagrelor, 1000ng/mL deshydroxyethoxyticagrelor, 1000ng/mL aspirin, 1000ng/mL salicylic acid, 20ng/mL clopidogrel, and 20ng/mL clopidogrel carboxylic acid metabolites.
(4) Sample processing
1) Pretreatment of a standard product: taking 50 mu L of each concentration point sample, putting the sample into a 1.5mL centrifuge tube, adding 200 mu L of protein precipitant containing an internal standard (the volume ratio of methanol to acetonitrile is 1:4), and oscillating at high speed (maximum oscillation speed) for 5 min; centrifuging at 14000r/min at 4 ℃ for 5 min; transfer 60. mu.L of supernatant from the EP tube to a plastic lined tube in a 1. mu.L sample volume.
2) Plasma sample pretreatment: putting 50 μ L of plasma into a 1.5mL centrifuge tube, adding 200 μ L of protein precipitant containing internal standard (volume ratio of methanol to acetonitrile is 1:4), and oscillating at high speed (maximum oscillation speed) for 5 min; centrifuging at 14000r/min at 4 ℃ for 5 min; transfer 60. mu.L of supernatant from the EP tube to a plastic lined tube in a 1. mu.L sample volume.
3) Pretreatment of quality control products: taking 50 μ L of quality control solution QC (L), QC (M), QC (H) in each 1.5mL centrifuge tube, and then consistent with the pretreatment of plasma samples, which is not repeated herein.
The components of the assay kit are shown in Table 8.
TABLE 8 preparation of components of kit for analysis of antiplatelet drugs (100 persons)
Remarking: the protein precipitant containing the internal standard is prepared according to the following method: and adding 200 mu L of mixed internal standard solution into 19.8mL of protein precipitant, and uniformly mixing to obtain the protein precipitant containing the internal standard.
Fourth, method verification
1. Extracting an ion current chromatogram: the peak shapes of the standard anti-platelet drug and the plasma sample are symmetrical without interfering with other peaks, which indicates that the good detection can be obtained under the condition, and fig. 1 is an extracted ion flow chromatogram of the standard anti-platelet drug; fig. 2 is an extracted ion flow chromatogram of an antiplatelet drug in a plasma sample.
2. Calibration curve: and (3) establishing a calibration curve by adopting an isotope internal standard quantitative method and utilizing TargetLynx software to calculate the concentration of the substance to be detected in the plasma by taking the concentration ratio of the standard substance to the internal standard substance as an X axis and the peak area ratio of the standard substance to the internal standard substance as a Y axis. The linear fitting equation of the antiplatelet drugs in the respective concentration ranges has good linearity, the correlation coefficient is more than 0.99, and the quantitative requirements are met, which is shown in table 9.
TABLE 9 Linear regression equation and Linear correlation coefficient for antiplatelet drugs
| Serial number | Compound (I) | Retention time (min) | Linear range (ng/mL) | Linear equation of equations | Coefficient of correlation (r) |
| 1 | CLOPC | 1.71 | 0.1-50 | Y＝0.0889533*X+0.00661122 | 0.999 |
| 2 | CLOP | 3.04 | 0.1-50 | Y＝0.492127*X+0.00365337 | 0.999 |
| 3 | DHTIC | 2.53 | 5-2500 | Y＝0.0.00688601*X+0.00134171 | 0.999 |
| 4 | TIC | 2.55 | 5-2500 | Y＝0.0189636*X+0.0180986 | 0.998 |
| 5 | ASA | 1.88 | 5-2500 | Y＝0.0115011*X-0.0183838 | 0.997 |
| 6 | SA | 2.11 | 5-2500 | Y＝0.0234423*X+0.473475 | 1.000 |
3. Accuracy survey: and evaluating the accuracy of the method by adopting a standard recovery rate test. A mixed blank serum sample is prepared, low, medium and high 3 concentrations of standard substances are respectively added, the same steps are repeated for processing and determination for 5 times, the result shows that the standard addition recovery rate of the antiplatelet drugs is between 87.64% and 112.50%, the RSD of 5 repeated tests is in the range of 1.81% to 8.01, and the statistical result is shown in Table 10.
TABLE 10 recovery of anti-platelet drug spiking results
4. And (3) precision test: taking an interference-free blank plasma sample, adding antiplatelet drug standard products with different concentrations to obtain plasma samples with low, medium and high concentrations, repeatedly processing 6 batches in one day for three days continuously, quantitatively determining the concentration of the antiplatelet drug by an isotope internal standard method, wherein the in-batch precision is 2.50-9.32%, processing 3 batches in three days, and calculating the inter-batch precision to be 2.43-14.32%, and the result is shown in Table 11.
TABLE 11 results of inter-batch precision measurements (concentration units: ng/mL)
Fifth, discuss
The invention establishes a method for simultaneously measuring antiplatelet drugs in human plasma by UPLC-MS/MS. The dosage of the blood plasma is small (only 50 mu L), the pretreatment is simple, and the analysis of various substances by one injection only needs 5min, and the method is simple and quick.
The isotope internal standard method is adopted for quantification, so that the matrix interference can be greatly eliminated, the result is not influenced by conditions such as a pretreatment process, instrument response fluctuation and the like, and accurate quantification can be achieved. The result of the accuracy of the method for evaluating the standard recovery rate by using the standard recovery rate test shows that the standard recovery rate of the antiplatelet drug is 87.64-112.50%, the RSD of 5 repeated tests is 1.81-8.01, and the accuracy is good.
The reproducibility result of the method shows that the intra-batch precision of the antiplatelet drug is 2.50-9.32%, the inter-batch precision is 2.43-14.32%, and the reproducibility of the method is good.
In a word, the method has the advantages of high sensitivity, strong specificity, accuracy and simple pretreatment process, completes the separation and detection of the compound within 5min, meets the requirements on accuracy and precision, can be used for quantitative analysis of plasma antiplatelet drugs in clinic, and provides a reliable detection method for related drug concentration monitoring.
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: modifications of the technical solutions described in the foregoing embodiments are still possible, or some technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. A kit for detecting antiplatelet drugs in plasma by an ultra-high performance liquid chromatography tandem mass spectrometry technology,
the antiplatelet drugs are respectively: ticagrelor, deshydroxyethoxy ticagrelor, aspirin, salicylic acid, clopidogrel, and clopidogrel carboxylic acid metabolites;
the kit comprises the following reagents:
(1) eluent:
eluent A: 0.001 to 0.01 percent of formic acid aqueous solution; eluent B: acetonitrile;
(2) calibration solution:
preparing a mixed standard solution containing 50000ng/mL ticagrelor, 50000ng/mL deshydroxyethoxy ticagrelor, 50000ng/mL aspirin, 50000ng/mL salicylic acid, 1000ng/mL clopidogrel and 1000ng/mL clopidogrel carboxylic acid metabolites into seven calibrator solutions with different concentration points by using a blank plasma matrix, wherein the seven concentration points of the calibrator solution are as follows:
the concentrations of ticagrelor, deshydroxyethoxy ticagrelor, aspirin and salicylic acid are the same, and seven concentrations are as follows: 2500ng/mL, 1250ng/mL, 250ng/mL, 125ng/mL, 25ng/mL, 12.5ng/mL, and 5 ng/mL;
the concentrations of clopidogrel and clopidogrel carboxylic acid metabolites are the same, and seven concentrations are as follows in sequence: 50ng/mL, 25ng/mL, 5ng/mL, 2.5ng/mL, 0.5ng/mL, 0.25ng/mL, and 0.1 ng/mL;
(3) mixing internal standard solutions:
an aqueous acetonitrile solution comprising 1500ng/mL ticagrelor-d 7, 1500ng/mL deshydroxyethoxy ticagrelor-d 7, 2000ng/mL aspirin-d 4, 2000ng/mL salicylic acid-d 4, and 20ng/mL clopidogrel-d 4;
(4) protein precipitant:
a mixed solution of methanol and acetonitrile;
(5) quality control product:
blank plasma matrix containing antiplatelet drugs comprises low, medium and high concentrations of QC (L), QC (M) and QC (H), wherein,
QC (L) is the mixed standard solution diluted to 5000 times with blank plasma matrix;
QC (M) is the above mixed standard solution diluted to 500 times with blank plasma matrix;
QC (H) is the above mixed standard solution diluted 50-fold with blank plasma matrix.
2. The kit according to claim 1,
the eluent A is 0.001-0.005% aqueous formic acid solution, preferably 0.004% aqueous formic acid solution.
3. The kit according to claim 1,
the volume ratio of methanol to acetonitrile in the protein precipitator is 1: 1-5; preferably, the volume ratio of methanol to acetonitrile in the protein precipitant is 1: 4.
4. The kit according to claim 1,
the blank plasma matrix is blank plasma without antiplatelet drugs.
5. The kit according to claim 1, 2, 3 or 4,
the mixed standard solution is prepared according to the following method: preparing a mixed standard solution containing 50000ng/mL ticagrelor, 50000ng/mL deshydroxyethoxy ticagrelor, 50000ng/mL aspirin, 50000ng/mL salicylic acid, 1000ng/mL clopidogrel and 1000ng/mL clopidogrel carboxylic acid metabolite by using acetonitrile aqueous solution to prepare 2000 μ g/mL ticagrelor standard mother solution, 1000 μ g/mL deshydroxyethoxy ticagrelor standard mother solution, 2000 μ g/mL aspirin standard mother solution, 100 μ g/mL clopidogrel standard mother solution and 100 μ g/mL clopidogrel carboxylic acid metabolite mother solution.
6. The kit according to claim 5,
the mixed internal standard solution is prepared according to the following method: and preparing a mixed internal standard solution containing 1500ng/mL ticagrelor-d 7, 1500ng/mL deshydroxyethoxy ticagrelor-d 7, 2000ng/mL aspirin-d 4, 2000ng/mL salicylic acid-d 4 and 20ng/mL clopidogrel-d 4 from a acetonitrile aqueous solution by using 150 μ g/mL ticagrelor-d 7 isotope internal standard mother solution, 150 μ g/mL deshydroxyethoxy ticagrelor-d 7 isotope internal standard mother solution, 200 μ g/mL aspirin-d 4 isotope internal standard mother solution, 200 μ g/mL salicylic acid-d 4 isotope internal standard mother solution and 2 μ g/mL clopidogrel-d 4 isotope internal standard mother solution.
7. The kit according to claim 1,
the plasma is human or animal plasma.
8. The kit according to claim 6,
the kit comprises the following reagents:
(1) eluent:
eluent A: 0.004% aqueous formic acid; eluent B: acetonitrile;
(2) calibration solution:
preparing a mixed standard solution containing 50000ng/mL ticagrelor, 50000ng/mL deshydroxyethoxy ticagrelor, 50000ng/mL aspirin, 50000ng/mL deshydroxyethoxy ticagrelor, 50000ng/mL aspirin, 50000ng/mL salicylic acid, 1000ng/mL clopidogrel and 1000ng/mL clopidogrel carboxylic acid metabolites from 2000 μ g/mL ticagrelor standard mother solution, 1000 μ g/mL deshydroxyethoxy ticagrelor standard solution, 2000 μ g/mL aspirin standard solution and 100 μ g/mL clopidogrel carboxylic acid metabolite mother solution with 80% acetonitrile water; preparing the mixed standard solution into seven calibrator solutions with different concentration points by using blank plasma without antiplatelet drugs;
(3) mixing internal standard solutions:
preparing isotope mixed internal standard solutions containing 1500ng/mL ticagrelor-d 7, 1500ng/mL desethoxylation ticagrelor-d 7, 2000ng/mL aspirin-d 4, 2000ng/mL salicylic acid-d 4 and 20ng/mL clopidogrel-d 4 from 150 ug/mL ticagrelor-d 7 isotope internal standard mother solution, 150 ug/mL desethoxylation ticagrelor-d 7 isotope internal standard mother solution, 200 ug/mL aspirin-d 4 isotope internal standard mother solution, 200 ug/mL salicylic acid-d 4 isotope internal standard mother solution and 2 ug/mL clopidogrel-d 4 isotope internal standard mother solution by using 80% acetonitrile aqueous solution;
(4) protein precipitant:
the volume ratio of methanol to acetonitrile is 1: 4;
(5) quality control product:
taking the mixed standard solution and preparing blank plasma without antiplatelet drugs into QC (L), QC (M) and QC (H) with three different concentrations, wherein,
QC (L) includes: 10ng/mL ticagrelor, 10ng/mL deshydroxyethoxy ticagrelor, 10ng/mL aspirin, 10ng/mL salicylic acid, 0.2ng/mL clopidogrel, and 0.2ng/mL clopidogrel carboxylic acid metabolites;
QC (M) comprises: 100ng/mL ticagrelor, 100ng/mL deshydroxyethoxyticagrelor, 100ng/mL aspirin, 100ng/mL salicylic acid, 2ng/mL clopidogrel, and 2ng/mL clopidogrel carboxylic acid metabolites;
QC (H) includes: 1000ng/mL ticagrelor, 1000ng/mL deshydroxyethoxyticagrelor, 1000ng/mL aspirin, 1000ng/mL salicylic acid, 20ng/mL clopidogrel, and 20ng/mL clopidogrel carboxylic acid metabolites.
9. The kit of claim 8, wherein: and mixing the mixed internal standard solution with a protein precipitant in a volume ratio of 1:99 to prepare the protein precipitant containing the internal standard, wherein the protein precipitant is used for the detection of the ultra-high performance liquid chromatography tandem mass spectrometry.
10. Use of the kit according to any one of claims 1 to 9 for detecting an antiplatelet drug in plasma by means of ultra-high performance liquid chromatography tandem mass spectrometry.
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