阅读说明：本技术 超高效液相色谱串联质谱技术检测血浆中抗血小板药物的试剂盒 (Kit for detecting antiplatelet drugs in plasma by ultra-high performance liquid chromatography tandem mass spectrometry technology ) 是由 成晓亮 李美娟 于 2020-06-01 设计创作，主要内容包括：本发明涉及一种超高效液相色谱串联质谱技术检测血浆中抗血小板药物的试剂盒,前处理过程简单,成本低,且灵敏度高、特异性强,5min之内完成抗血小板药物的分离和检测,准确度与精密度基本满足要求,可用于临床上抗血小板药物的定量分析,为临床上抗血小板药物的治疗浓度监测提供一种可靠的检测方法。(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.)

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.

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-d4	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.