Method for detecting vancomycin in meat product
The proposed method addresses the challenge of detecting vancomycin in meat products by employing a preprocessing and LC-MS/MS analysis technique, offering a reliable and efficient means to quantify vancomycin residues, enhancing food safety by ensuring accurate detection and operational ease.
Patent Information
- Application Number
- CN202411506934.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-25
- Publication Date
- 2025-03-14
AI Technical Summary
The improper use of antibiotics like vancomycin in animals can lead to residues in meat products, posing health risks due to contamination, which existing technologies have not effectively addressed for detection in meat products.
A method involving sample preprocessing and analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to detect vancomycin in meat products, including steps like solvation, centrifugation, addition of PSA absorbent, ion exchange chromatography, evaporation, and mixing with methanol water solution, followed by LC-MS/MS detection with specific column and solvent conditions.
This method provides a reliable, repeatable, and efficient means to quantify vancomycin in meat products, ensuring operational simplicity and widespread applicability with high precision and speed.
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Figure CN119619328A_ABST
Abstract
Description
Technical Field
[0001] The present application relates to the technical field of analytical chemistry, and in particular to a method for detecting vancomycin in meat products. Background Art
[0002] If antibiotics are improperly used during the treatment of animals, residues of antibiotics such as vancomycin are likely to remain in their bodies, causing contamination of the raw materials of meat products, making the use of meat products risky and harmful to human health. Summary of the invention
[0003] In view of the above problems, the present application is proposed to provide a method for detecting vancomycin in meat products that overcomes the above problems or at least partially solves the above problems, comprising:
[0004] A method for detecting vancomycin in meat products comprises the steps of:
[0005] Pre-treating the test object; specifically, dissolving the test object to obtain a test object solution; centrifuging the test object solution to obtain a test object supernatant; adding a PSA adsorbent to the test object supernatant, centrifuging and taking the supernatant to a strong cation exchange column; eluting and eluting the strong cation exchange column to obtain an eluent; blowing the eluent liquid nitrogen to volatilize to obtain a re-dissolved sample, and mixing the re-dissolved sample with a methanol aqueous solution to obtain a sample test solution;
[0006] Sampling the sample test solution to obtain an on-machine test solution;
[0007] The test solution is detected by liquid chromatography-tandem mass spectrometry.
[0008] Furthermore, the step of detecting the test solution on the machine by liquid chromatography-tandem mass spectrometry uses a chromatographic column model C 18 Column, 150 mm x 2.1 mm (inner diameter), particle size 3.5 μm.
[0009] Furthermore, the step of dissolving the test object to obtain a test object solution comprises:
[0010] The analyte is mixed with a first volume of acetonitrile solution containing 0.1% methanol, and the mixture of the analyte and the acetonitrile solution containing 0.1% methanol is homogenized by a homogenizer at a rate of 10000 r / min for 2 min; and centrifuged at a rate of 4000 r / min for 5 min to obtain a first supernatant and a residue of the analyte;
[0011] The residue of the test object is mixed with a second volume of acetonitrile solution containing 0.1% methanol, and the mixture of the test object and the acetonitrile solution containing 0.1% methanol is homogenized by a homogenizer at a speed of 10000 r / min for 2 min; and centrifuged at a speed of 4000 r / min for 5 min to obtain a second supernatant and the residue of the test object;
[0012] The first supernatant and the second supernatant are mixed to obtain a test substance solution.
[0013] Furthermore, the first volume is 4 ml / g; and the volume ratio of the second volume to the first volume is 1:2.
[0014] Furthermore, the step of adding a PSA adsorbent to the supernatant of the test object, and subjecting the supernatant to a strong cation exchange column after centrifugation comprises:
[0015] Adding a PSA adsorbent to the supernatant of the analyte, wherein the mass ratio of the adsorbent to the analyte is 1:10;
[0016] Vortex mix for 2 minutes, centrifuge at 15000r / min for 10 minutes after mixing, and take 6-8ml of the supernatant and apply it to a strong cation exchange column.
[0017] Furthermore, the step of eluting and washing the strong cation exchange column to obtain an eluent comprises:
[0018] The strong cation exchange column is eluted with 3 ml of water and 3 ml of methanol at a rate of less than 1 ml / min respectively;
[0019] The strong cation exchange column was eluted with 3 ml of a methanol solution containing 10% ammonia water at a flow rate of less than 1 mL / min to obtain an eluate.
[0020] Furthermore, the step of volatilizing the eluted liquid nitrogen to obtain a reconstituted sample, and mixing the reconstituted sample with a methanol aqueous solution to obtain a sample test solution comprises:
[0021] The eluate was concentrated by nitrogen blowing at 40° C. until the solution was blown dry to obtain a reconstituted sample.
[0022] Dissolve the reconstituted sample in 20% methanol aqueous solution and adjust the volume to 1 ml to obtain a primary sample solution;
[0023] The primary sample solution is filtered through a 0.22 mm filter membrane to obtain a sample solution.
[0024] Furthermore, in the step of detecting the on-machine test solution by liquid chromatography-tandem mass spectrometry, the mobile phase is 0.1% formic acid aqueous solution and acetonitrile.
[0025] Furthermore, in the step of detecting the test solution on the machine by liquid chromatography-tandem mass spectrometry, the elution gradient is:
[0026] 0-5 minutes, formic acid aqueous solution 95%-80%, acetonitrile 5%-20%; 5-7 minutes, formic acid aqueous solution 80%-5%, formic acid acetonitrile solution 20%-95%; 7-9 minutes, formic acid aqueous solution 5%, formic acid acetonitrile solution 95%; 9-9.01 minutes, formic acid aqueous solution 5%-95%, formic acid acetonitrile solution 95%-5%; 9.01-15 minutes, formic acid aqueous solution 95%, formic acid acetonitrile solution 5%.
[0027] Furthermore, in the step of detecting the test solution on the machine by liquid chromatography-tandem mass spectrometry, the mass spectrometry parameters are: parent ion 725.0m / z; daughter ion: 1305.9m / z, quantitative ion 143.9m / z; cone voltage 60V; collision energy: 17eV, 20eV.
[0028] This application has the following advantages:
[0029] In the embodiments of the present application, in view of the fact that there is no detection scheme for vancomycin in meat products in the prior art, the present application fills the gap in the existing detection technology by pre-treating and setting detection conditions for the detection of the analyte. The present application provides a method for detecting vancomycin in meat products, comprising the steps of: pre-treating the analyte; specifically, dissolving the analyte to obtain a analyte solution; centrifuging the analyte solution to obtain a analyte supernatant; adding a PSA adsorbent to the analyte supernatant, and taking the supernatant to a strong cation exchange column after centrifugation; rinsing and eluting the strong cation exchange column to obtain an eluent; volatilizing the eluted liquid nitrogen to obtain a re-dissolved sample, and mixing the re-dissolved sample with a methanol aqueous solution to obtain a sample test solution; sampling the sample test solution to obtain an on-machine test solution; and detecting the on-machine test solution by a liquid chromatography-tandem mass spectrometer. The content of vancomycin in meat products can be determined by pretreating the analyte and detecting it through liquid chromatography-tandem mass spectrometry. The liquid chromatography-mass spectrometry / mass spectrometry method has excellent repeatability and reproducibility, simple operation, fast speed, and the equipment used is easy to obtain, so it is highly scalable. BRIEF DESCRIPTION OF THE DRAWINGS
[0030] In order to more clearly illustrate the technical solution of the present application, the drawings required for use in the description of the present application will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.
[0031] Figure 1 This is a flow chart of the steps of a method for detecting vancomycin in meat products provided in one embodiment of the present application;
[0032] Figure 2 This is an operation flow chart of a method for detecting vancomycin in meat products provided in one embodiment of the present application;
[0033] Figure 3 This is a standard curve diagram of a method for detecting vancomycin in meat products provided in one embodiment of the present application;
[0034] Figure 4 It is a chromatogram of a standard curve of a method for detecting vancomycin in meat products provided in one embodiment of the present application at a concentration point of 5 μg / L;
[0035] Figure 5 It is a chromatogram of a standard curve of a method for detecting vancomycin in meat products provided in one embodiment of the present application at a concentration point of 10 μg / L;
[0036] Figure 6 It is a chromatogram of a standard curve of a method for detecting vancomycin in meat products provided in an embodiment of the present application at a concentration point of 20 μg / L;
[0037] Figure 7 It is a chromatogram of a standard curve of a method for detecting vancomycin in meat products provided in one embodiment of the present application at a concentration point of 50 μg / L;
[0038] Figure 8 It is a chromatogram of a standard curve of a method for detecting vancomycin in meat products provided in one embodiment of the present application at a concentration point of 100 μg / L;
[0039] Fig. 9 It is a graph of a method for detecting vancomycin in meat products provided in one embodiment of the present application for determining the content of vancomycin in a spiked meat sample;
[0040] Fig.10 The present invention provides a method for detecting vancomycin in meat products according to an embodiment of the present invention, which is a graph showing the content of vancomycin in a meat sample. DETAILED DESCRIPTION
[0041] In order to make the objects, features and advantages of the present application more obvious and understandable, the present application is further described in detail below in conjunction with the accompanying drawings and specific implementation methods. Obviously, the described embodiments are part of the embodiments of the present application, rather than all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present application.
[0042] The inventors have found through analysis of the prior art that if antibiotics are improperly used during the treatment of animals, residues of antibiotics such as vancomycin are likely to remain in their bodies, causing contamination of raw materials for meat products, making the use of meat products risky and harmful to human health.
[0043] Reference Figure 1-2 , showing a step flow chart and an operation flow chart of a method for detecting vancomycin in meat products provided in one embodiment of the present application:
[0044] The method comprises:
[0045] S110, pre-treating the test object; specifically, dissolving the test object to obtain a test object solution; centrifuging the test object solution to obtain a test object supernatant; adding a PSA adsorbent to the test object supernatant, centrifuging and taking the supernatant to a strong cation exchange column; rinsing and eluting the strong cation exchange column to obtain an eluent; blowing the eluent liquid nitrogen to volatilize to obtain a reconstituted sample, and mixing the reconstituted sample with a methanol aqueous solution to obtain a sample test solution;
[0046] S120, sampling the sample test solution to obtain an on-machine test solution;
[0047] S130, detecting the test solution by liquid chromatography-tandem mass spectrometry.
[0048] In the embodiments of the present application, in view of the fact that there is no detection scheme for vancomycin in meat products in the prior art, the present application fills the gap in the existing detection technology by pre-treating and setting detection conditions for the detection of the analyte. The present application provides a method for detecting vancomycin in meat products, comprising the steps of: pre-treating the analyte; specifically, dissolving the analyte to obtain a analyte solution; centrifuging the analyte solution to obtain a analyte supernatant; adding a PSA adsorbent to the analyte supernatant, and taking the supernatant to a strong cation exchange column after centrifugation; rinsing and eluting the strong cation exchange column to obtain an eluent; volatilizing the eluted liquid nitrogen to obtain a re-dissolved sample, and mixing the re-dissolved sample with a methanol aqueous solution to obtain a sample test solution; sampling the sample test solution to obtain a machine test solution; and detecting the machine test solution by liquid chromatography-tandem mass spectrometry. The content of vancomycin in meat products can be determined by pretreating the analyte and detecting it through liquid chromatography-tandem mass spectrometry. The liquid chromatography-mass spectrometry / mass spectrometry method has excellent repeatability and reproducibility, simple operation, fast speed, and the equipment used is easy to obtain, so it is highly scalable.
[0049] Next, a method for detecting vancomycin in a meat product in this exemplary embodiment will be further described.
[0050] As described in step S110, the analyte is pre-treated; specifically, the analyte is dissolved to obtain a analyte solution; the analyte solution is centrifuged to obtain a analyte supernatant; a PSA adsorbent is added to the analyte supernatant, and after centrifugation, the supernatant is taken to a strong cation exchange column; the strong cation exchange column is rinsed and eluted to obtain an eluent; the elution liquid is evaporated with nitrogen to obtain a reconstituted sample, and the reconstituted sample is mixed with a methanol aqueous solution to obtain a sample test solution.
[0051] In one embodiment of the present invention, the specific process of step S110 described in "pre-treating the test object; specifically, dissolving the test object to obtain a test object solution; centrifuging the test object solution to obtain a test object supernatant; adding PSA adsorbent to the test object supernatant, and taking the supernatant to a strong cation exchange column after centrifugation; rinsing and eluting the strong cation exchange column to obtain an eluent; blowing the eluent with nitrogen to volatilize to obtain a re-dissolved sample, and mixing the re-dissolved sample with a methanol aqueous solution to obtain a sample test solution" can be further explained in combination with the following description.
[0052] As described in the following steps, dissolving the test substance to obtain a test substance solution;
[0053] In one embodiment of the present application, the analyte is mixed with a first volume of an acetonitrile solution containing 0.1% methanol, and the mixture of the analyte and the acetonitrile solution containing 0.1% methanol is homogenized by a homogenizer at a rate of 10000 r / min for 2 min; and centrifuged at a rate of 4000 r / min for 5 min to obtain a first supernatant and a residue of the analyte;
[0054] The residue of the test object is mixed with a second volume of acetonitrile solution containing 0.1% methanol, and the mixture of the test object and the acetonitrile solution containing 0.1% methanol is homogenized by a homogenizer at a speed of 10000 r / min for 2 min; and centrifuged at a speed of 4000 r / min for 5 min to obtain a second supernatant and the residue of the test object;
[0055] The first supernatant and the second supernatant are mixed to obtain a test substance solution.
[0056] It should be noted that if the volume of the analyte solution is smaller than the total volume of the first volume and the second volume, the volume of the analyte solution needs to be fixed to the sum of the first volume and the second volume.
[0057] In one embodiment of the present application, the first volume is 4 ml / g; and the volume ratio of the second volume to the first volume is 1:2.
[0058] In a specific implementation, 5 g of sample was weighed into a homogenizing tube, and 20 mL of 0.1% formic acid-acetonitrile solution was added. The sample was homogenized at 10,000 r / min for 2 min using a homogenizer, and centrifuged at 4,000 r / min for 5 min. The supernatant was taken out, and the residue was repeatedly extracted once with 10 mL of 0.1% formic acid-acetonitrile solution. The supernatants were combined and the volume was fixed to 30 mL to obtain the test solution.
[0059] As described in the following steps, PSA adsorbent is added to the supernatant of the test substance, and after centrifugation, the supernatant is taken to a strong cation exchange column.
[0060] In one embodiment of the present application, a PSA adsorbent is added to the supernatant of the analyte, wherein the mass ratio of the adsorbent to the analyte is 1:10;
[0061] Vortex mix for 2 minutes, centrifuge at 15000r / min for 10 minutes after mixing, and take 6-8ml of the supernatant and apply it to a strong cation exchange column.
[0062] In a specific implementation, 5 g of sample was extracted into a test solution, 0.5 g of PSA adsorbent was added, vortexed for 2 min, and then centrifuged at 15,000 r / min for 10 min, and 6 mL of supernatant was taken out and transferred to a strong cation exchange column.
[0063] As described in the following steps, washing and eluting the strong cation exchange column to obtain an eluent;
[0064] In one embodiment of the present application, the strong cation exchange column is eluted with 3 ml of water and 3 ml of methanol at a rate of less than 1 ml / min respectively;
[0065] The strong cation exchange column was eluted with 3 ml of a methanol solution containing 10% ammonia water at a flow rate of less than 1 mL / min to obtain an eluate.
[0066] As described in the following steps, the eluted liquid nitrogen is blown to volatilize to obtain a reconstituted sample, and the reconstituted sample is mixed with a methanol aqueous solution to obtain a sample test solution;
[0067] In one embodiment of the present application, the eluent is concentrated by nitrogen blowing at a temperature of 40° C. until the solution is blown dry to obtain a reconstituted sample.
[0068] Dissolve the reconstituted sample in 20% methanol aqueous solution and adjust the volume to 1 ml to obtain a primary sample solution;
[0069] The primary sample solution is filtered through a 0.22 mm filter membrane to obtain a sample solution.
[0070] As described in step S120, the sample test solution is sampled to obtain the on-machine test solution.
[0071] As described in step S130, the on-machine test solution is detected by liquid chromatography-tandem mass spectrometry.
[0072] In one embodiment of the present invention, the specific process of "detecting the on-machine test solution by liquid chromatography-tandem mass spectrometry" in step S130 can be further explained in combination with the following description.
[0073] The instrument parameters are:
[0074] Liquid chromatography reference conditions:
[0075] Instrument: LC-MS / MS;
[0076] Chromatographic column: C18 column, 150 mm × 2.1 mm (inner diameter), particle size 3.5 μm, or equivalent;
[0077] Mobile phase: A: 0.1% formic acid aqueous solution; B: acetonitrile;
[0078] Flow rate: 0.25 mL / min;
[0079] Column temperature: 35°C;
[0080] Injection volume: 20 μL;
[0081] The flow rate gradient elution program is shown in Table 1:
[0082] Time / min Mobile phase A / % Mobile phase B / % 0.00 95 5 5.00 80 20 7.00 5 5 9.00 5 95 9.01 95 5 15.00 95 5
[0083] Table 1
[0084] The mass spectrometry reference parameters are as follows:
[0085] Ion source: electrospray ionization (ESI).
[0086] Scanning mode: multiple reaction monitoring (MRM).
[0087] The mass spectrometry reference parameters are as follows:
[0088] ESI positive ion mode;
[0089] Electrospray voltage (IS): 5500 V;
[0090] Ion source temperature (TEM): 550°C;
[0091] Atomizing gas pressure (GS1): 206.85 kPa (30 Psi);
[0092] Auxiliary gas pressure (GS2): 206.85kPa (30Psi);
[0093] Curtain air pressure (CUR): 206.85 kPa (30 Psi);
[0094] Resolution: unit resolution;
[0095] The quantitative ion pair, qualitative ion pair, cone voltage (V) and collision energy (eV) are shown in Table 2:
[0096]
[0097] Table 2, Note: Those with “*” are quantitative ions.
[0098] refer to Figure 3-8 , which is the chromatogram of each concentration point of the vancomycin standard curve measured by liquid chromatography-tandem mass spectrometry and the linearity of vancomycin, the linear range of the standard curve is: 5.0-100.0μg / L.
[0099] Fig.10 The chromatographic data of vancomycin measured by liquid chromatography-tandem mass spectrometry for a meat product. Fig. 9 The chromatogram of vancomycin measured by liquid chromatography-tandem mass spectrometry after adding a spike to the meat product sample shows that the method can achieve high-efficiency separation and stable baseline. The recovery rate is within a satisfactory range of 60%-120% through a spike recovery test on the sample. It has good repeatability and reproducibility, and accurate quantification.
[0100] It can be seen from the above embodiments that the present scheme uses LC-MS / MS (liquid chromatography tandem mass spectrometry) to detect the content of vancomycin in meat samples, which has excellent repeatability and reproducibility, is easy to operate, fast, and the equipment used is easy to obtain, and is highly scalable.
[0101] Although the preferred embodiments of the present application have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the embodiments of the present application.
[0102] Finally, it should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or terminal device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or terminal device. In the absence of further restrictions, the elements defined by the sentence "comprise a ..." do not exclude the existence of other identical elements in the process, method, article or terminal device including the elements.
[0103] The above is a detailed introduction to the method for detecting vancomycin in meat products provided by the present application. Specific examples are used in this article to illustrate the principles and implementation methods of the present application. The description of the above embodiments is only used to help understand the method of the present application and its core idea. At the same time, for those skilled in the art, according to the idea of the present application, there will be changes in the specific implementation method and application scope. In summary, the content of this specification should not be understood as a limitation on the present application.
Claims
1. A method for detecting vancomycin in meat products, characterized in that: Includes steps: Pre-treating the test object; specifically, dissolving the test object to obtain a test object solution; centrifuging the test object solution to obtain a test object supernatant; adding a PSA adsorbent to the test object supernatant, centrifuging and taking the supernatant to a strong cation exchange column; eluting and eluting the strong cation exchange column to obtain an eluent; blowing the eluent liquid nitrogen to volatilize to obtain a re-dissolved sample, and mixing the re-dissolved sample with a methanol aqueous solution to obtain a sample test solution; Sampling the sample test solution to obtain an on-machine test solution; The test solution is detected by liquid chromatography-tandem mass spectrometry.
2. The detection method according to claim 1, characterized in that: The step of detecting the test solution on the machine by liquid chromatography-tandem mass spectrometry uses a chromatographic column model C 18 Column, 150 mm × 2.1 mm (inner diameter), particle size 3.5 μm.
3. The detection method according to claim 1, characterized in that: The step of dissolving the test object to obtain a test object solution comprises: The analyte is mixed with a first volume of acetonitrile solution containing 0.1% methanol, and the mixture of the analyte and the acetonitrile solution containing 0.1% methanol is homogenized by a homogenizer at a rate of 10000 r / min for 2 min; and centrifuged at a rate of 4000 r / min for 5 min to obtain a first supernatant and a residue of the analyte; The residue of the test object is mixed with a second volume of acetonitrile solution containing 0.1% methanol, and the mixture of the test object and the acetonitrile solution containing 0.1% methanol is homogenized by a homogenizer at a speed of 10000 r / min for 2 min; and centrifuged at a speed of 4000 r / min for 5 min to obtain a second supernatant and the residue of the test object; The first supernatant and the second supernatant are mixed to obtain a test substance solution.
4. The detection method according to claim 3, characterized in that: The first volume is 4 ml / g; and the volume ratio of the second volume to the first volume is 1:
2.
5. The detection method according to claim 1, characterized in that: The step of adding a PSA adsorbent to the supernatant of the test object, taking the supernatant to a strong cation exchange column after centrifugation comprises: Adding a PSA adsorbent to the supernatant of the analyte, wherein the mass ratio of the adsorbent to the analyte is 1:10; Vortex mix for 2 minutes, centrifuge at 15000r / min for 10 minutes after mixing, and take 6-8ml of the supernatant and apply it to a strong cation exchange column.
6. The detection method according to claim 1, characterized in that: The step of eluting the strong cation exchange column to obtain an eluent comprises: The strong cation exchange column is eluted with 3 ml of water and 3 ml of methanol at a rate of less than 1 ml / min respectively; The strong cation exchange column was eluted with 3 ml of a methanol solution containing 10% ammonia water at a flow rate of less than 1 mL / min to obtain an eluate.
7. The detection method according to claim 1, characterized in that: The step of volatilizing the eluted liquid nitrogen to obtain a re-dissolved sample, and mixing the re-dissolved sample with a methanol aqueous solution to obtain a sample test solution comprises: The eluate was concentrated by nitrogen blowing at 40° C. until the solution was blown dry to obtain a reconstituted sample. Dissolve the reconstituted sample in 20% methanol aqueous solution and adjust the volume to 1 ml to obtain a primary sample solution; The primary sample solution is filtered through a 0.22 mm filter membrane to obtain a sample solution.
8. The detection method according to claim 1, characterized in that: In the step of detecting the test solution on the machine by liquid chromatography-tandem mass spectrometry, the mobile phase is 0.1% formic acid aqueous solution and acetonitrile.
9. The detection method according to claim 8, characterized in that: The step of detecting the test solution on the machine by liquid chromatography-tandem mass spectrometry, the elution gradient is: 0-5 minutes, formic acid aqueous solution 95%-80%, acetonitrile 5%-20%; 5-7 minutes, formic acid aqueous solution 80%-5%, formic acid acetonitrile solution 20%-95%; 7-9 minutes, formic acid aqueous solution 5%, formic acid acetonitrile solution 95%; 9-9.01 minutes, formic acid aqueous solution 5%-95%, formic acid acetonitrile solution 95%-5%; 9.01-15 minutes, formic acid aqueous solution 95%, formic acid acetonitrile solution 5%.
10. The detection method according to claim 1, characterized in that: The step of detecting the test solution on the machine by liquid chromatography-tandem mass spectrometry, the mass spectrometry parameters are: parent ion 725.0m / z; daughter ion: 1305.9m / z, quantitative ion 143.9m / z; cone voltage 60V; collision energy: 17eV, 20eV.