Liquid chromatography tandem mass spectrometry detection method for clopidogrel and active metabolites thereof based on magnetic solid phase extraction

Abstract

The invention discloses a liquid chromatography-tandem mass spectrometry detection method for clopidogrel and active metabolites thereof based on magnetic solid-phase extraction, which comprises the following steps: converting the active metabolites of clopidogrel into derivatives through derivatization treatment, and then detecting the derivatives. The detection targets of the method are clopidogrel and active metabolites thereof, the metabolic condition of clopidogrel in a human body can be effectively and truly reflected, and the method has important significance for monitoring the drug concentration of clopidogrel. According to the method, detection samples of clopidogrel and active metabolites thereof are pretreated through a magnetic solid-phase extraction technology, separation and transfer of to-be-detected substances can be rapidly achieved by applying an external magnetic field, automatic pretreatment can be achieved conveniently, and meanwhile the advantages of high specificity, high extraction rate and the like of solid-phase extraction are reserved; according to the sample treatment step, interference of substances such as endogenous phospholipids, proteins and salts can be removed, and the matrix effect and the pollution risk of a chromatographic column and a mass spectrometer are reduced.

Description

Technical Field

[0001] The present invention relates to a field, and in particular to a liquid chromatography tandem mass spectrometry detection method for clopidogrel and its active metabolites based on magnetic solid phase extraction. Background Art

[0002] The content of clopidogrel and its active metabolites in plasma samples is usually very low, reaching the ng level. Among the quantitative detection methods, the most commonly used is high-performance liquid chromatography, but the instrument detection sensitivity is low and cannot meet the needs of clinical detection. In addition, the amount of plasma sample required for high-performance liquid chromatography is large, and the sample pretreatment time is long. In order to achieve the required detection sensitivity of the target analyte, it often takes a long time for chromatographic separation, and the co-eluted interfering components have a great impact on the accuracy of the results. Since many analytical methods cannot meet the requirements for quantitative analysis of clopidogrel and its active metabolites in plasma in terms of sensitivity, selectivity and analysis speed, their further promotion and development in clinical detection is limited.

[0003] Chromatography-mass spectrometry, which uses chromatography as a separation method and triple quadrupole mass spectrometry as a detection method, has become a powerful means for quantitatively detecting trace target analytes in biological samples. Therefore, it is used for the quantitative detection of clopidogrel and its active metabolites in plasma. Patent 111812223B reports a method for detecting antiplatelet drugs in plasma using ultra-high performance liquid chromatography tandem mass spectrometry, which includes clopidogrel and its carboxylic acid metabolites. The carboxylic acid metabolites are inactive metabolites of clopidogrel hydrolyzed by esterase and cannot reflect the function of clopidogrel in inhibiting platelet aggregation. At the same time, this method uses protein precipitation for pretreatment. The pretreatment process has poor specificity, low recovery rate of the test substance, and is difficult to automate. Patent 118130678A reports a liquid chromatography-mass spectrometry detection method for detecting 14 vertigo and syncope drugs in blood. The method only detects clopidogrel and does not mention the detection of its metabolites. In addition, the pretreatment process also uses a protein precipitation method. Compared with the magnetic solid phase extraction method, the detection process is cumbersome and time-consuming.

[0004] Therefore, it is necessary to improve the existing technology to provide a more reliable solution. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a liquid chromatography tandem mass spectrometry detection method for clopidogrel and its active metabolites based on magnetic solid phase extraction in view of the deficiencies in the above-mentioned prior art.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is: a liquid chromatography tandem mass spectrometry detection method for clopidogrel and its active metabolites based on magnetic solid phase extraction, wherein the active metabolite of clopidogrel is converted into a derivative by derivatization treatment, and then the derivative is detected, wherein the active metabolite has a chemical structure shown in the following formula I, and the derivative has a chemical structure shown in the following formula II:

[0007]

[0008] The reaction mechanism of the conversion of the active metabolite of clopidogrel into its derivatives is as follows:

[0009]

[0010] The chemical structure of clopidogrel is shown in the following formula III:

[0011]

[0012] The detection method comprises the following steps:

[0013] S1. Preparation of test samples:

[0014] S1-1, take a centrifuge tube, add the human whole blood sample to be tested and the derivatization reagent thereto, mix well, and centrifuge to obtain a plasma sample;

[0015] S1-2, taking the plasma sample obtained in step S1-1, adding the internal standard solution, and mixing;

[0016] S1-3, sequentially activating and balancing the magnetic solid phase extraction filler;

[0017] S1-4, adding the magnetic solid phase extraction filler obtained in step S1-3 to the product obtained in step S1-2, and mixing;

[0018] S1-5, transferring the magnetic solid phase extraction filler in step S1-4 into the first solvent for mixing;

[0019] S1-6, transferring the magnetic solid phase extraction filler in step S1-5 to the second solvent for mixing;

[0020] S1-7, transferring the magnetic solid phase extraction filler in step S1-6 to a third solvent for mixing;

[0021] S1-8, discarding the magnetic solid phase extraction filler in the product obtained in step S1-7, and using the remaining solution as a test sample;

[0022] S2. Construct standard curve:

[0023] preparing clopidogrel standard solutions and derivative standard solutions of different concentrations for liquid chromatography tandem mass spectrometry detection, and constructing standard curves of clopidogrel and derivatives;

[0024] S3. Liquid chromatography tandem mass spectrometry is used to detect the test sample, and the clopidogrel content and derivative content in the test sample are obtained in combination with the constructed standard curve.

[0025] The detection principle of the present invention:

[0026] Pharmacokinetic studies have shown that clopidogrel itself is a prodrug, and it only plays an inhibitory role in platelet aggregation after being metabolized into active metabolites (thiol derivatives). Therefore, it is necessary to measure the active metabolites in the blood to reflect the efficacy of clopidogrel such as absorption, distribution, and metabolism. However, due to the low content of the drug's in vivo metabolic active substances and poor stability, it is difficult for ordinary in vivo drug analysis methods to meet the requirements. The present invention adopts a derivatization method to convert the active metabolites into derivatives, and then pre-treats them by protein precipitation, vortex centrifuges to take the supernatant for sampling, and detects them by liquid chromatography-tandem mass spectrometry.

[0027] Among them, the ratio of active metabolites to derivatives is 1:1, so the content of the detected derivatives represents the content of the active metabolites.

[0028] Preferably, the derivatization agent in step S1-1 is 2-bromo-3'-methoxyacetophenone.

[0029] Step S1-1 is specifically as follows:

[0030] Take 0.5-2mL of human whole blood sample to be tested and place it in a sodium heparin anticoagulation centrifuge tube, add 5-20μL of 400-600mmol / L 2-bromo-3'-methoxyacetophenone acetonitrile solution, vortex mix for 0.5-2min, centrifuge at 7000-28000rpm for 2-10min to obtain a plasma sample.

[0031] Preferably, step S1-1 is specifically:

[0032] Take 1 mL of human whole blood sample to be tested and place it in a sodium heparin anticoagulation centrifuge tube, add 10 μL of 500 mmol / L 2-bromo-3'-methoxyacetophenone in acetonitrile, vortex mix for 1 min, and centrifuge at 14000 rpm for 5 min to obtain a plasma sample.

[0033] Preferably, the first solvent is deionized water.

[0034] Preferably, the second solvent is methanol.

[0035] Preferably, the third solvent is acetonitrile.

[0036] Preferably, step S1 specifically comprises:

[0037] S1-1. Take 0.5-2 mL of human whole blood sample to be tested, place it in a sodium heparin anticoagulation centrifuge tube, add 5-20 μL of 400-600 mmol / L 2-bromo-3'-methoxyacetophenone in acetonitrile, vortex mix for 0.5-2 min, and centrifuge at 7000-28000 rpm for 2-10 min to obtain a plasma sample;

[0038] S1-2, take 50-100 μL of the plasma sample obtained in step S1-1 and add it to a 1-4 mL centrifuge tube, add 5-20 μL of the internal standard solution and 150-600 μL of the aqueous solution, and vortex mix at 500-2000 rpm for 1-4 min; the internal standard solution includes clopidogrel internal standard solution and derivative internal standard solution;

[0039] S1-3, adding 1.5-5 mg of magnetic solid phase extraction filler into 150-600 μL of acetonitrile to activate the filler, and then transferring the magnetic solid phase extraction filler into water to balance the filler;

[0040] S1-4, adding the magnetic solid phase extraction filler obtained in step S1-3 to the product obtained in step S1-2, and mixing for 75-300 seconds;

[0041] S1-5, transferring the magnetic solid phase extraction filler in step S1-4 into 200-800 μL of deionized water solution and mixing for 60-240 seconds;

[0042] S1-6, transfer the magnetic solid phase extraction filler in step S1-5 into 200-800 μL methanol and mix for 50-200 seconds;

[0043] S1-7, transfer the magnetic solid phase extraction filler in step S1-6 to 75-300 μL of acetonitrile and mix for 50-200 seconds;

[0044] S1-8. Discard the magnetic solid phase extraction filler in step S1-7, and use the remaining solution as a test sample.

[0045] Preferably, step S1 specifically comprises:

[0046] S1-1. Take 1 mL of human whole blood sample to be tested and place it in a sodium heparin anticoagulation centrifuge tube. Add 10 μL of 500 mmol / L 2-bromo-3'-methoxyacetophenone in acetonitrile solution. Vortex mix for 1 min. Centrifuge at 14000 rpm for 5 min to obtain a plasma sample.

[0047] S1-2, take 100 μL of the plasma sample obtained in step S1-1 and add it to a 2 mL centrifuge tube, add 10 μL of the internal standard solution and 300 μL of the aqueous solution, and vortex mix at 1000 rpm for 2 min; the concentration of clopidogrel in the internal standard solution is 10 ng / mL, and the concentration of the derivative of the active metabolite of clopidogrel is 50 ng / mL;

[0048] S1-3, adding 2.5 mg of magnetic solid phase extraction filler into 300 μL of acetonitrile to activate the filler, and then transferring the magnetic solid phase extraction filler into water for filler balance;

[0049] S1-4, adding the magnetic solid phase extraction filler obtained in step S1-3 to the product obtained in step S1-2, and mixing for 150 seconds;

[0050] S1-5, transfer the magnetic solid phase extraction filler in step S1-4 into 400 μL of deionized water solution and mix for 120 seconds;

[0051] S1-6, transfer the magnetic solid phase extraction filler in step S1-5 into 400 μL methanol and mix for 100 s;

[0052] S1-7, transfer the magnetic solid phase extraction filler in step S1-6 into 150 μL of acetonitrile and mix for 100 s;

[0053] S1-8. Discard the magnetic solid phase extraction filler in step S1-7, and use the remaining solution as a test sample.

[0054] Preferably, in step S3, the liquid chromatography detection conditions are:

[0055] Column: Kinetex 2.6μm C18 LC Column 100x3mm;

[0056] Mobile phase: Phase A is an aqueous solution of formic acid with a volume fraction of 0.1%, and phase B is acetonitrile;

[0057] Flow rate: 0.4 mL / min;

[0058] Injection volume: 2 μL;

[0059] Isocratic elution program: volume ratio of mobile phase A: mobile phase B = 5%:95%, elution time is 3.5 min.

[0060] Preferably, in step S3, the mass spectrometry conditions are:

[0061] Ion source: ESI ion source, positive ion mode;

[0062] The ion source and gas parameters are as follows:

[0063]

[0064] The MRM parameters are as follows:

[0065]

[0066] Wherein, Clp represents clopidogrel, Clp-d3 represents clopidogrel internal standard, MP-H4 represents derivative, and MP-H4-13C·d3 represents derivative internal standard.

[0067] The beneficial effects of the present invention are:

[0068] (1) The detection targets of the present invention are clopidogrel and its active metabolites, which can effectively and truly reflect the metabolic status of clopidogrel in the human body and are of great significance for the drug concentration monitoring of clopidogrel.

[0069] (2) The liquid chromatography-tandem mass spectrometry detection method of clopidogrel and its active metabolites based on magnetic solid phase extraction provided by the present invention pre-treats the detection samples of clopidogrel and its active metabolites by magnetic solid phase extraction technology, and can quickly separate and transfer the analytes by applying an external magnetic field, which is convenient for realizing automated pre-treatment, while retaining the advantages of high specificity and high extraction rate of solid phase extraction;

[0070] (3) The present invention increases the contact area through dispersed adsorption of magnetic solid phase extraction fillers, improves the adsorption rate and enrichment effect, and shortens the pre-treatment time; it can avoid errors such as SPE flow rate differences and filler swelling, and reduce mass spectrometry detection variability;

[0071] (4) The sample processing step of the present invention can remove the interference of endogenous phospholipids, proteins, salts and other substances, and reduce the matrix effect, the risk of contamination of the chromatographic column and the mass spectrometer. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Figure 1 is the blank sample chromatogram;

[0073] Figure 2 is the chromatogram of the test sample;

[0074] Figure 3 is the standard curve of clopidogrel;

[0075] Figure 4 The standard curve is that of the active metabolite derivative MP-H4. DETAILED DESCRIPTION

[0076] The present invention is further described in detail below in conjunction with embodiments so that those skilled in the art can implement the invention with reference to the description.

[0077] It should be understood that the terms such as “having”, “including” and “comprising” used herein do not exclude the existence or addition of one or more other elements or combinations thereof.

[0078] The test methods used in the following examples are conventional methods unless otherwise specified. The materials and reagents used in the following examples are all commercially available unless otherwise specified. In the following examples, if no specific conditions are specified, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used, if the manufacturer is not specified, are all conventional products that can be purchased commercially.

[0079] The main instruments and reagents involved in the following examples are described as follows:

[0080] 1) SCIEX QTRAP 5500 liquid chromatography-tandem mass spectrometry system ( Figure 2 ):

[0081] The SHIMADZU liquid phase configuration is: 2 LC-20AD XR pumps, SIL-20A automatic sample injector, CTO-20A column oven, CBM-20A controller.

[0082] Other equipment: vortex machine, high-speed centrifuge (2.0mL centrifuge tubes can be used), pipette;

[0083] 3) Reagents: acetonitrile (Concord Technology Co., Ltd.), formic acid (Thermo Fisher), clopidogrel (Clp) standard and internal standard (Shanghai Zhenzhun Biotechnology Co., Ltd.), active metabolite (MP-H4) standard and internal standard (Shanghai Zhenzhun Biotechnology Co., Ltd.), 2-bromo-3'-methoxyacetophenone (MPB) (Shanghai Zhenzhun Biotechnology Co., Ltd.).

[0084] The following example LC-MS / MS data acquisition method is as follows:

[0085] 1) Liquid chromatography parameters

[0086] Instrument configuration takes Shimadzu Prominence liquid chromatography system as an example

[0087] Column: Kinetex 2.6μm C18 LC Column 100x3mm;

[0088] Mobile phase: Phase A is an aqueous solution of formic acid with a volume fraction of 0.1%, and phase B is acetonitrile;

[0089] Flow rate: 0.4 mL / min;

[0090] Injection volume: 2 μL;

[0091] Isocratic elution program: volume ratio of mobile phase A: mobile phase B = 5%:95%, elution time is 3.5 min.

[0092] 2) Mass spectrometry conditions are:

[0093] Ion source: ESI ion source, positive ion mode;

[0094] The ion source and gas parameters are as follows:

[0095]

[0096] The MRM parameters are as follows:

[0097]

[0098]

[0099] Wherein, Clp represents clopidogrel, Clp-d3 represents clopidogrel internal standard, MP-H4 represents derivative, and MP-H4-13C·d3 represents derivative internal standard.

[0100] Example 1

[0101] A liquid chromatography tandem mass spectrometry detection method for clopidogrel and its active metabolites based on magnetic solid phase extraction, characterized in that the method converts the active metabolite of clopidogrel into a derivative (hereinafter referred to as the derivative of the active metabolite MP-H4) through derivatization treatment, and then detects the derivative, wherein the active metabolite has a chemical structure shown in the following formula I, and the derivative has a chemical structure shown in the following formula II:

[0102]

[0103] The detection method comprises the following steps:

[0104] S1. Preparation of test samples:

[0105] Step S1 is specifically as follows:

[0106] S1-1. Take 1 mL of human whole blood sample to be tested and place it in a sodium heparin anticoagulation centrifuge tube. Add 10 μL of 500 mmol / L 2-bromo-3'-methoxyacetophenone in acetonitrile solution. Vortex mix for 1 min. Centrifuge at 14000 rpm for 5 min to obtain a plasma sample.

[0107] S1-2, take 100 μL of the plasma sample obtained in step S1-1 and add it to a 2 mL centrifuge tube, add 10 μL of the internal standard solution and 300 μL of the aqueous solution, and vortex mix at 1000 rpm for 2 min; the concentration of clopidogrel in the internal standard solution is 10 ng / mL, and the concentration of the derivative of the active metabolite of clopidogrel is 50 ng / mL;

[0108] S1-3, adding 2.5 mg of magnetic solid phase extraction filler into 300 μL of acetonitrile to activate the filler, and then transferring the magnetic solid phase extraction filler into water for filler balance;

[0109] S1-4, adding the magnetic solid phase extraction filler obtained in step S1-3 to the product obtained in step S1-2, and mixing for 150 seconds;

[0110] S1-5, transfer the magnetic solid phase extraction filler in step S1-4 into 400 μL of deionized water solution and mix for 120 seconds;

[0111] S1-6, transfer the magnetic solid phase extraction filler in step S1-5 into 400 μL methanol and mix for 100 s;

[0112] S1-7, transfer the magnetic solid phase extraction filler in step S1-6 into 150 μL of acetonitrile and mix for 100 s;

[0113] S1-8. Discard the magnetic solid phase extraction filler in step S1-7, and use the remaining solution as a test sample.

[0114] S2. Construct standard curve:

[0115] preparing clopidogrel standard solutions and derivative standard solutions of different concentrations for liquid chromatography tandem mass spectrometry detection, and constructing standard curves of clopidogrel and derivatives;

[0116] S3. Liquid chromatography tandem mass spectrometry is used to detect the test sample, and the clopidogrel content and derivative content in the test sample are obtained in combination with the constructed standard curve.

[0117] Example 2 Methodology Verification Results

[0118] 1. Specificity / Selectivity / Interference

[0119] Experimental method: Compare the chromatograms of blank samples and blank spiked samples. There are no interfering peaks near the retention time of the compound in the chromatograms, and the method has excellent selectivity (specificity).

[0120] 1-1、Blank sample chromatogram is as follows Figure 1 As shown, the chromatogram of the test sample is as follows Figure 2 shown.

[0121] 1.2. Lower limit of quantification

[0122] According to the clinical detection requirements of clopidogrel and the sensitivity of the instrument, the quantitative lower limit of the method is 0.2 ng / mL for clopidogrel (Clp) and 0.5 ng / mL for the derivative of the active metabolite MP-H4 (i.e. the derivative mentioned above, the same below). Take three samples with concentrations close to the lower limit of quantification, test each 5 times, and calculate the deviation between the test value and the theoretical value and the coefficient of variation CV value of the test results. The test results are shown in Tables 1 and 2. It can be seen from the results that the deviation between the test value and the theoretical value is within 15%, and the coefficient of variation of the parallel samples tested is within 15%. It can be seen that the quantitative lower limit of the method is 0.2 ng / mL for clopidogrel (Clp) and 0.5 ng / mL for the derivative of the active metabolite MP-H4, which meets the requirements of clinical detection methodology.

[0123] Table 1 Results of the lower limit of quantification of clopidogrel

[0124]

[0125] Table 2 Quantitative limit results of active metabolite derivative MP-H4

[0126]

[0127] 1.3. Standard curve and quantitative range

[0128] 1) Experimental method: The calibration samples at 8 concentration levels, with concentrations shown in Table 3, were processed according to the pre-treatment method and sampled and analyzed on the instrument. The original data were automatically exported and saved from the instrument analysis software. The detection data were statistically analyzed using the data processing software, and a linear fit was automatically performed. The theoretical concentration was used as the abscissa, and the ratio of the area of ​​the analyte to the peak area of ​​the internal standard was used as the ordinate. A linear regression curve was established, and its correlation coefficient was determined.

[0129] Table 3 Standard curve concentration levels of clopidogrel and its active metabolite derivative MP-H4

[0130]

[0131] The standard curve regression equation is shown in Table 4. Figure 3 and Figure 4 shown.

[0132] Table 4 Results of the standard curve study of clopidogrel and its active metabolite derivative MP-H4

[0133]

[0134] Conclusion: The tolerance of all calibration points above LOQ is within 15% and r>0.99, and any medically relevant decision point must be within the analytical quantitative range.

[0135] 1.4. Correctness Verification

[0136] The accuracy of the method was evaluated by using a spike recovery experiment: the spike recovery rate was calculated by detecting the plasma concentration before and after spike addition, and the accuracy of the method was examined based on this. The results are shown in Table 5.

[0137] Table 5 Spiked recovery results

[0138]

[0139]

[0140]

[0141] Conclusion: The recoveries of low, medium and high concentrations were all in the range of 85.0% to 115.0%, which met the requirements.

[0142] 1.5. Precision

[0143] 1) Experimental method: Self-prepared high and low concentration samples were used as experimental objects. Five samples of each level were tested every day, and each sample was tested three times. The test was continued for three working days.

[0144] 2) The results are shown in Table 6

[0145] Table 6 Results of precision study of clopidogrel and its active metabolite derivatives MP-H4

[0146]

[0147]

[0148]

[0149] Conclusion: The precision test results all met the intra-batch CV≤15% and inter-batch CV≤15%, which fully met the requirements of the analytical method.

[0150] 1.6. Carryover / carryover effect

[0151] 1) Experimental methods:

[0152] Select self-prepared high and low concentration samples as experimental objects, first repeat the low concentration sample, then alternately inject the high concentration sample and the low concentration sample. Compare the difference in the test results of the low concentration sample and the low concentration sample after the high concentration injection, which is lower than the predetermined standard ±20%, indicating that there is no obvious carryover contamination.

[0153] Table 7 Carryover test results

[0154]

[0155]

[0156] Conclusion: The carryover contamination rate of clopidogrel and its active metabolite derivative MP-H4 was lower than the predetermined standard ±20%, which met the requirements.

[0157] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to specific details.

Claims

1. A method for detecting clopidogrel and its active metabolites by liquid chromatography tandem mass spectrometry based on magnetic solid phase extraction, characterized in that: The method converts the active metabolite of clopidogrel into a derivative by derivatization treatment, and then detects the derivative. The active metabolite has a chemical structure shown in the following formula I, and the derivative has a chemical structure shown in the following formula II: The detection method comprises the following steps: S1. Preparation of test samples: S1-1, take a centrifuge tube, add the human whole blood sample to be tested and the derivatization reagent thereto, mix well, and centrifuge to obtain a plasma sample; S1-2, taking the plasma sample obtained in step S1-1, adding the internal standard solution, and mixing; S1-3, sequentially activating and balancing the magnetic solid phase extraction filler; S1-4, adding the magnetic solid phase extraction filler obtained in step S1-3 to the product obtained in step S1-2, and mixing; S1-5, transferring the magnetic solid phase extraction filler in step S1-4 into the first solvent for mixing; S1-6, transferring the magnetic solid phase extraction filler in step S1-5 to a second solvent for mixing; S1-7, transferring the magnetic solid phase extraction filler in step S1-6 to a third solvent for mixing; S1-8, discarding the magnetic solid phase extraction filler in the product obtained in step S1-7, and using the remaining solution as a test sample; S2. Construct standard curve: preparing clopidogrel standard solutions and derivative standard solutions of different concentrations for liquid chromatography tandem mass spectrometry detection, and constructing standard curves of clopidogrel and derivatives; S3. Liquid chromatography tandem mass spectrometry is used to detect the test sample, and the clopidogrel content and derivative content in the test sample are obtained in combination with the constructed standard curve.

2. The method for detecting clopidogrel and its active metabolites by liquid chromatography tandem mass spectrometry based on magnetic solid phase extraction according to claim 1, characterized in that: The derivatization reagent in step S1-1 is 2-bromo-3'-methoxyacetophenone. Step S1-1 is specifically as follows: Take 0.5-2mL of human whole blood sample to be tested and place it in a sodium heparin anticoagulation centrifuge tube, add 5-20μL of 400-600mmol / L 2-bromo-3'-methoxyacetophenone acetonitrile solution, vortex mix for 0.5-2min, centrifuge at 7000-28000rpm for 2-10min to obtain a plasma sample.

3. The method for detecting clopidogrel and its active metabolites by liquid chromatography tandem mass spectrometry based on magnetic solid phase extraction according to claim 2, characterized in that: Step S1-1 is specifically as follows: Take 1 mL of human whole blood sample to be tested and place it in a sodium heparin anticoagulation centrifuge tube, add 10 μL of 500 mmol / L 2-bromo-3'-methoxyacetophenone in acetonitrile, vortex mix for 1 min, and centrifuge at 14000 rpm for 5 min to obtain a plasma sample.

4. The method for detecting clopidogrel and its active metabolites by liquid chromatography tandem mass spectrometry based on magnetic solid phase extraction according to claim 1, characterized in that: The first solvent is deionized water.

5. The method for detecting clopidogrel and its active metabolites by liquid chromatography tandem mass spectrometry based on magnetic solid phase extraction according to claim 1, characterized in that: The second solvent is methanol.

6. The method for detecting clopidogrel and its active metabolites by liquid chromatography tandem mass spectrometry based on magnetic solid phase extraction according to claim 1, characterized in that: The third solvent is acetonitrile.

7. The method for detecting clopidogrel and its active metabolites by liquid chromatography tandem mass spectrometry based on magnetic solid phase extraction according to claim 1, characterized in that: Step S1 is specifically as follows: S1-1. Take 0.5-2 mL of human whole blood sample to be tested, place it in a sodium heparin anticoagulation centrifuge tube, add 5-20 μL of 400-600 mmol / L 2-bromo-3'-methoxyacetophenone in acetonitrile, vortex mix for 0.5-2 min, and centrifuge at 7000-28000 rpm for 2-10 min to obtain a plasma sample; S1-2, take 50-100 μL of the plasma sample obtained in step S1-1 and add it to a 1-4 mL centrifuge tube, add 5-20 μL of the internal standard solution and 150-600 μL of the aqueous solution, and vortex mix at 500-2000 rpm for 1-4 min; the internal standard solution includes clopidogrel internal standard solution and derivative internal standard solution; S1-3, adding 1.5-5 mg of magnetic solid phase extraction filler into 150-600 μL of acetonitrile to activate the filler, and then transferring the magnetic solid phase extraction filler into water to balance the filler; S1-4, adding the magnetic solid phase extraction filler obtained in step S1-3 to the product obtained in step S1-2, and mixing for 75-300 seconds; S1-5, transferring the magnetic solid phase extraction filler in step S1-4 into 200-800 μL of deionized water solution and mixing for 60-240 seconds; S1-6, transfer the magnetic solid phase extraction filler in step S1-5 into 200-800 μL methanol and mix for 50-200 seconds; S1-7, transfer the magnetic solid phase extraction filler in step S1-6 to 75-300 μL of acetonitrile and mix for 50-200 seconds; S1-8. Discard the magnetic solid phase extraction filler in step S1-7, and use the remaining solution as a test sample.

8. The method for detecting clopidogrel and its active metabolites by liquid chromatography tandem mass spectrometry based on magnetic solid phase extraction according to claim 7, characterized in that: Step S1 is specifically as follows: S1-1. Take 1 mL of human whole blood sample to be tested and place it in a sodium heparin anticoagulation centrifuge tube. Add 10 μL of 500 mmol / L 2-bromo-3'-methoxyacetophenone in acetonitrile solution. Vortex mix for 1 min. Centrifuge at 14000 rpm for 5 min to obtain a plasma sample. S1-2, take 100 μL of the plasma sample obtained in step S1-1 and add it to a 2 mL centrifuge tube, add 10 μL of the internal standard solution and 300 μL of the aqueous solution, and vortex mix at 1000 rpm for 2 min; the concentration of clopidogrel in the internal standard solution is 10 ng / mL, and the concentration of the derivative of the active metabolite of clopidogrel is 50 ng / mL; S1-3, adding 2.5 mg of magnetic solid phase extraction filler into 300 μL of acetonitrile to activate the filler, and then transferring the magnetic solid phase extraction filler into water for filler balance; S1-4, adding the magnetic solid phase extraction filler obtained in step S1-3 to the product obtained in step S1-2, and mixing for 150 seconds; S1-5, transfer the magnetic solid phase extraction filler in step S1-4 into 400 μL of deionized water solution and mix for 120 seconds; S1-6, transfer the magnetic solid phase extraction filler in step S1-5 into 400 μL methanol and mix for 100 s; S1-7, transfer the magnetic solid phase extraction filler in step S1-6 into 150 μL of acetonitrile and mix for 100 s; S1-8. Discard the magnetic solid phase extraction filler in step S1-7, and use the remaining solution as a test sample.

9. The method for detecting clopidogrel and its active metabolites by liquid chromatography tandem mass spectrometry based on magnetic solid phase extraction according to claim 1, characterized in that: In step S3, the liquid chromatography detection conditions are: Column: Kinetex 2.6μm C18 LC Column 100x3mm; Mobile phase: Phase A is an aqueous solution of formic acid with a volume fraction of 0.1%, and phase B is acetonitrile; Flow rate: 0.4 mL / min; Injection volume: 2 μL; Isocratic elution program: volume ratio of mobile phase A: mobile phase B = 5%:95%, elution time is 3.5 min.

10. The method for detecting clopidogrel and its active metabolites by liquid chromatography tandem mass spectrometry based on magnetic solid phase extraction according to claim 1, characterized in that: In step S3, the mass spectrometry conditions are: Ion source: ESI ion source, positive ion mode; The ion source and gas parameters are as follows: The MRM parameters are as follows: Wherein, Clp represents clopidogrel, Clp-d3 represents clopidogrel internal standard, MP-H4 represents derivative, and MP-H4-13C·d3 represents derivative internal standard.

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