Detection method of platelet activation marker

Abstract

The invention relates to the technical field of medical examination and analysis, in particular to a platelet activation marker detection method which at least comprises the following steps: mixing a sample solution, a TXB2 isotope internal standard solution and a precipitator, and carrying out vortex treatment and centrifugal treatment to obtain a serum sample; a serum sample is subjected to LC-MS / MS testing, the content of thromboxane B2 in the sample is determined on the basis of a standard curve, quantitative detection of the content of TXB2 in serum is achieved by optimizing pretreatment and LC-MS / MS (liquid chromatography-tandem mass spectrometry) parameter conditions, and compared with a traditional ELISA method, the sensitivity is higher, the accuracy is higher, the result reproducibility is good, the method is more stable and reliable, and the method is suitable for large-scale popularization and application. Popularization and implementation are easy.

Description

Technical Field

[0001] This invention relates to the field of medical testing and analysis technology, specifically to a method for detecting platelet activation markers. Background Technology

[0002] Thromboboxane B2 (TXB2) is a type of prostaglandin produced by platelets. It has platelet aggregation and vasoconstriction effects, which are opposite to those of prostaglandins. The two are in dynamic balance to maintain vasoconstriction and platelet aggregation. It is the end metabolite of thromboxane A2 (TXA2) and is mainly metabolized by the kidneys. Currently, there are few detection technologies for thromboxane B2 in the field, and most focus on the detection of TXB2 metabolite 11dhTXB2 (11-dehydrothromboxane B2). Methods include radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), and liquid chromatography-tandem mass spectrometry (LC-MS / MS). For example, the ELISA kit for detecting 11-dehydrothromboxane B2 disclosed in Chinese patent application (publication number CN103592430A) uses a double antibody sandwich method. The capture antibody is a monoclonal antibody against 11-dehydrothromboxane B2, and the detection antibody is a polyclonal antibody from different species to enhance specificity. However, the ELISA method has low accuracy and poor reproducibility, and cannot meet the actual detection needs of TXB2. Summary of the Invention

[0003] To address the aforementioned issues, this invention provides a method for detecting platelet activation markers. By optimizing pretreatment and LC-MS / MS (liquid chromatography-tandem mass spectrometry) parameters, the method achieves quantitative detection of TXB2 content in serum. Compared to the traditional ELISA method, this method offers higher sensitivity, higher accuracy, better reproducibility, greater stability and reliability, and is easier to implement and promote.

[0004] This invention provides a method for detecting platelet activation markers, comprising at least the following steps: mixing a sample solution, a TXB2 isotope internal standard solution, and a precipitant, followed by vortexing and centrifugation to obtain a serum sample; performing LC-MS / MS testing on the serum sample, and determining the content of thromboxane B2 in the sample based on a standard curve.

[0005] In one embodiment, the TXB2 isotope internal standard solution is a TXB2-d9 internal standard solution or a TxB2-d4 internal standard solution.

[0006] In one embodiment, the concentration of the TXB2-d9 internal standard solution or the TxB2-d4 internal standard solution is 200-400 ng / mL.

[0007] In one embodiment, the TXB2 isotope internal standard solution is a TXB2-d9 internal standard solution with a concentration of 300 ng / mL.

[0008] This invention employs a TXB2 isotope internal standard solution to ensure the system applicability of the detection method. In particular, the use of a TXB2-d9 internal standard solution with a concentration of 300 ng / mL ensures good quality control repeatability of the detection method and guarantees the reliability of the method for analyzing the TXB2 content in serum.

[0009] In one embodiment, the precipitant is selected from at least one of acetonitrile, methanol, acetone, or aqueous perchloric acid.

[0010] In one embodiment, the volume ratio of the sample solution, the TXB2 isotope internal standard solution, and the precipitant is 1:1:(3-5).

[0011] In one embodiment, the vortex treatment is performed at a speed of 1000-3000 rpm for a duration of 1-5 min.

[0012] In one embodiment, the centrifugation process is carried out at a speed of 3000-5000 rpm for 5-15 minutes at a temperature of 3-5°C.

[0013] In one embodiment, the high-performance liquid chromatography (HPLC) conditions for the LC-MS / MS test include: column: C18 reversed-phase column; phase A: ammonium acetate-formic acid aqueous solution; phase B: lower alcohol; flow rate: 0.3-0.4 mL / min; injection time: 3-3.5 min; injection volume: 0.8-1 μL.

[0014] In one embodiment, the C18 reversed-phase chromatographic column is a Waters 2.1*100mm HSS T3.

[0015] In one embodiment, the concentration of ammonium formate in the ammonium acetate-formic acid aqueous solution is 3-5 mM.

[0016] In one embodiment, the amount of formic acid added to the ammonium acetate-formic acid aqueous solution is 0.01-0.03 wt%.

[0017] In one embodiment, the lower alcohol is selected from at least one of methanol, ethanol, or isopropanol.

[0018] In one embodiment, the mass spectrometry (MS) conditions in the LC-MS / MS test include: an electrospray ion source; a multiple reaction monitoring (MRM) scanning mode; ion pairs Q1-Q3: 369.2-169.2; collision voltage 10-15 eV; declustering voltage: 60-66 eV; and spray gas temperature: 390-400 °C.

[0019] This invention achieves higher peak shape, improves detection sensitivity and accuracy, and better meets practical detection needs by comprehensively controlling high-performance liquid chromatography conditions and balancing separation effect and analysis time.

[0020] In one implementation, the standard curve is obtained in the following manner:

[0021] (1) Prepare standard curve samples with concentrations ranging from 2 ng / mL to 1000 ng / L;

[0022] (2) The standard curve sample was mixed with TXB2 isotope internal standard solution and precipitant, and serum sample was obtained by vortexing and centrifugation; the serum sample was tested by LC-MS / MS to obtain the response values ​​of TXB2 and internal standard in each standard curve sample.

[0023] (3) Calculate the ratio of the TXB2 response value to the internal standard response value in each standard curve sample;

[0024] (4) Plot a scatter plot with TXB2 concentration on the x-axis and the ratio of TXB2 to the internal standard response on the y-axis. Use linear regression to fit the plot and obtain the standard curve and its correlation coefficient (R²). 2 ).

[0025] In one embodiment, the matrix of the standard curve sample is a methanol-phosphate buffer solution.

[0026] In one embodiment, the amount of methanol added to the methanol-phosphate buffer solution is 40-60% (based on the volume of the phosphate buffer solution).

[0027] This invention optimizes the standard curve sample matrix to a methanol-phosphate buffer solution, making the internal standard more stable, resulting in better reproducibility and more accurate quantitative results.

[0028] The testing method provided by this invention is simple to operate, does not require additional complex pretreatment processes, greatly improves detection efficiency, reduces detection costs, and can be used for accurate quantitative detection of thromboxane B2 in human serum.

[0029] Beneficial effects

[0030] 1. This invention provides a method for detecting platelet activation markers. By optimizing the pretreatment and LC-MS / MS (liquid chromatography-tandem mass spectrometry) parameters, the method achieves quantitative detection of TXB2 content in serum. Compared with the traditional ELISA method, it has higher sensitivity, higher accuracy, better reproducibility, and is more stable, reliable, and easy to promote and implement.

[0031] 2. This invention uses TXB2 isotope internal standard solution to ensure the system applicability of the detection method. In particular, the use of TXB2-d9 internal standard solution with a concentration of 300 ng / mL ensures good quality control repeatability of the detection method and guarantees the reliability of the method for analyzing the content of TXB2 in serum.

[0032] 3. This invention achieves higher peak shape, improves detection sensitivity and accuracy, and better meets actual detection needs by comprehensively controlling high-performance liquid chromatography conditions and balancing separation effect and analysis time.

[0033] 4. This invention optimizes the standard curve sample matrix to a methanol-phosphate buffer solution, making the internal standard more stable, the results more reproducible, and the quantitative results more accurate.

[0034] 5. The testing method provided by this invention is simple to operate, does not require additional complex pretreatment processes, greatly improves detection efficiency, reduces detection costs, and can be used for accurate quantitative detection of thromboxane B2 in human serum. Attached Figure Description

[0035] Figure 1 This is the standard curve graph from Example 1.

[0036] Figure 2 This is the chromatogram from Example 1.

[0037] Figure 3 This is the chromatogram from Comparative Example 1. Detailed Implementation

[0038] Example 1

[0039] Example 1 of the present invention provides a method for detecting platelet activation markers, comprising the following steps: mixing a sample solution, a TXB2 isotope internal standard solution and a precipitant, and obtaining a serum sample by vortexing and centrifugation; performing LC-MS / MS testing on the serum sample and determining the content of thromboxane B2 in the sample based on a standard curve.

[0040] The TXB2 isotope internal standard solution is a TXB2-d9 internal standard solution with a concentration of 300 ng / mL. Internal standard name: [2H9]-Thromboxane B2; manufacturer: ISOREAG; number: IR-56886; serial number: 24Z642-Y1.

[0041] The precipitant is acetonitrile.

[0042] The volume ratio of the sample solution, TXB2 isotope internal standard solution, and precipitant is 1:1:4.

[0043] The vortex treatment was performed at a speed of 1500 rpm for 3 minutes.

[0044] The centrifugation process was carried out at a speed of 3470 rpm for 10 minutes at a temperature of 4°C.

[0045] The high-performance liquid chromatography (HPLC) conditions for the LC-MS / MS test included: column: C18 reversed-phase column; phase A: ammonium acetate-formic acid aqueous solution; phase B: lower alcohol; flow rate: 0.4 mL / min; injection time: 3.5 min; injection volume: 1 μL.

[0046] The C18 reversed-phase column is a Waters 2.1*100mm HSS T3.

[0047] The concentration of ammonium formate in the ammonium acetate-formic acid aqueous solution is 5 mM.

[0048] The amount of formic acid added in the ammonium acetate-formic acid aqueous solution is 0.01 wt%.

[0049] The lower alcohol is methanol.

[0050] The mass spectrometry (MS) conditions for the LC-MS / MS test include: an electrospray ion source; multiple reaction monitoring (MRM) mode; ion pairs Q1-Q3: 369.2-169.2; collision voltage: 15 eV; declustering voltage: 66 eV; and spray gas temperature: 400 °C.

[0051] The standard curve was obtained in the following way:

[0052] (1) Prepare standard curve samples with concentrations ranging from 2 ng / mL to 1000 ng / L;

[0053] (2) The standard curve sample was mixed with TXB2 isotope internal standard solution and precipitant, and serum sample was obtained by vortexing and centrifugation; the serum sample was tested by LC-MS / MS to obtain the response values ​​of TXB2 and internal standard in each standard curve sample.

[0054] (3) Calculate the ratio of the TXB2 response value to the internal standard response value in each standard curve sample;

[0055] (4) Plot a scatter plot with TXB2 concentration on the x-axis and the ratio of TXB2 to the internal standard response on the y-axis. Use linear regression to fit the plot and obtain the standard curve and its correlation coefficient (R²). 2 ).

[0056] The matrix of the standard curve sample is a methanol-phosphate buffer solution.

[0057] The amount of methanol added to the methanol-phosphate buffer solution is 50 wt% (based on the mass of the phosphate buffer solution).

[0058] The pH of the phosphate buffer solution in the methanol-phosphate buffer solution is 7.4.

[0059] Comparative Example 1

[0060] Comparative Example 1 of the present invention provides a method for detecting platelet activation markers, the specific implementation of which is the same as that of Example 1, except that the matrix of the standard curve sample is PBS solution.

[0061] Comparative Example 2

[0062] Comparative Example 2 of the present invention provides a method for detecting platelet activation markers. The specific implementation method is the same as that in Example 1, except that the TXB2 isotope internal standard solution is replaced with a 300 ng / mL cinidipine internal standard solution (cinidipine manufacturer: TLC Standard Reagent Co., Ltd.; No.: L-022).

[0063] Performance testing

[0064] 1. The methodologies provided in Example 1 and Comparative Example 2 were validated as follows.

[0065] (1) Prepare four quality control sample solutions (HQC, M2QC, M1QC and LQC) according to Table 1 and Table 2.

[0066] Table 1

[0067]

[0068]

[0069] Table 2

[0070]

[0071] (2) Four quality control sample solutions of different concentrations were tested according to the detection method provided in Example 1. Each concentration sample was measured three times in parallel to analyze its accuracy. The results are shown in Table 3.

[0072] Quality control sample solution name Theoretical concentration (ng / mL) Actual concentration (ng / mL) Accuracy (%) LQC1 3.233 3.289 101.7 LQC2 3.233 3.383 104.6 LQC3 3.233 2.996 92.7 M1QC1 73.23 65.50 89.4 M1QC2 73.23 64.99 88.7 M1QC3 73.23 66.33 90.6 M2QC1 403.2 351.4 87.2 M2QC2 403.2 348.1 86.3 M2QC3 403.2 348.0 86.3 HQC1 703.2 611.9 87.0 HQC2 703.2 602.8 85.7 HQC3 703.2 608.8 86.6

[0073] Analysis of the data in Table 3 shows that the test method in Example 1 has high accuracy and good repeatability, and is suitable for analyzing the content of TXB2 in serum.

[0074] (3) Four quality control sample solutions of different concentrations were tested according to the detection method provided in Comparative Example 2. Each concentration sample was measured three times in parallel to analyze its accuracy. The results are shown in Table 4.

[0075] Table 4

[0076] Sample Name Theoretical concentration (ng / mL) Actual concentration (ng / mL) Accuracy (%) LQC1 9.08 9.044 99.6 LQC2 9.08 9.071 99.9 LQC3 9.08 9.570 105.4 M1QC1 79.08 66.12 83.6 M1QC2 79.08 67.89 85.9 M1QC3 79.08 71.25 90.1 M2QC1 409.1 349.5 85.4 M2QC2 409.1 349.4 85.4 M2QC3 409.1 362.5 88.6 HQC1 709.1 661.6 93.3 HQC2 709.1 607.1 85.6 HQC3 709.1 601.0 84.8

[0077] Analysis of the data in Table 4 shows that, according to the relevant ICH M10 regulations, the accuracy deviation is within ±15%, and the accuracy of the test results of the test method in Comparative Example 2 is unqualified.

[0078] 2. The chromatograms obtained in Example 1 and Comparative Example 1 are shown below. Figure 2 , Figure 3 ,contrast Figure 2 , Figure 3 It can be seen that the analyte and internal standard peak shapes in the chromatograms obtained in Example 1 are better than those in Comparative Example 1, and the quantitative results are more accurate.

Claims

1. A method for detecting platelet activation markers, characterized in that, At least the following steps are included: The sample solution, TXB2 isotope internal standard solution, and precipitant were mixed and subjected to vortexing and centrifugation to obtain serum samples. The serum samples were then subjected to LC-MS / MS testing, and the content of thromboxane B2 in the samples was determined based on the standard curve.

2. The method for detecting platelet activation markers according to claim 1, characterized in that, The TXB2 isotope internal standard solution is either TXB2-d9 or TxB2-d4.

3. The method for detecting platelet activation markers according to claim 2, characterized in that, The concentration of the TXB2-d9 internal standard solution or the TxB2-d4 internal standard solution is 200-400 ng / mL.

4. The method for detecting platelet activation markers according to claim 1, characterized in that, The precipitant is selected from at least one of acetonitrile, methanol, acetone or aqueous perchloric acid.

5. The method for detecting platelet activation markers according to claim 1, characterized in that, The volume ratio of the sample solution, TXB2 isotope internal standard solution, and precipitant is 1:1:(3-5).

6. The method for detecting platelet activation markers according to claim 1, characterized in that, The vortex treatment is performed at a speed of 1000-3000 rpm for a duration of 1-5 minutes.

7. The method for detecting platelet activation markers according to claim 1, characterized in that, The centrifugation process is carried out at a speed of 3000-5000 rpm for 5-15 minutes at a temperature of 3-5℃.

8. The method for detecting platelet activation markers according to claim 1, characterized in that, The high-performance liquid chromatography (HPLC) conditions for the LC-MS / MS test include: column: C18 reversed-phase column; phase A: ammonium acetate-formic acid aqueous solution; phase B: lower alcohol; flow rate: 0.3-0.4 mL / min; injection time: 3-3.5 min; injection volume: 0.8-1 μL.

9. The method for detecting platelet activation markers according to claim 8, characterized in that, The lower alcohol is selected from at least one of methanol, ethanol, or isopropanol.

10. The method for detecting platelet activation markers according to claim 1, characterized in that, The mass spectrometry conditions for the LC-MS / MS test include: an electrospray ion source; multiple reaction monitoring (MRM) mode; ion pairs Q1-Q3: 369.2-169.2; collision voltage: 10-15 eV; declustering voltage: 60-66 eV; and spray gas temperature: 390-400℃.

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