CN112394132A

CN112394132A - Method for detecting aspirin active ingredient in blood plasma

Info

Publication number: CN112394132A
Application number: CN202011465562.5A
Authority: CN
Inventors: 吴�琳; 胡汉高; 李盈
Original assignee: Wuhan Haipu Biomedical Technology Co ltd
Current assignee: Wuhan Haipu Biomedical Technology Co ltd
Priority date: 2020-12-14
Filing date: 2020-12-14
Publication date: 2021-02-23

Abstract

The invention discloses a method for detecting aspirin active ingredients in blood plasma, which comprises four steps of solution preparation, pretreatment, concentration determination and quantitative analysis, wherein the whole pretreatment process is operated under a wet ice condition, and meanwhile, during the concentration determination, a specific mobile phase A is selected and matched with an isocratic gradient mode, and the content of acetylsalicylic acid and salicylic acid in the blood plasma is detected. The pretreatment is simple and easy to operate, and the acetylsalicylic acid and the salicylic acid in the blood plasma can be kept stable and cannot be converted with each other by the pretreatment setting and the use of a specific solution; and the prepared specific correction solution can also reduce the interference of human endogenous salicylic acid on the experimental result to the maximum extent.

Description

Method for detecting aspirin active ingredient in blood plasma

Technical Field

The invention belongs to the field of biological medicine detection, and particularly relates to a method for detecting an aspirin active ingredient in blood plasma.

Background

Aspirin, also called acetylsalicylic acid (ASA), is the earliest, most widely used and most common antipyretic analgesic antirheumatic. Has antipyretic, analgesic, antiinflammatory, wind-resistant, and platelet aggregation inhibiting effects, and can be used for treating common cold, fever, headache, neuralgia, arthralgia, myalgia, rheumatic fever, acute rheumatic arthritis, rheumatoid arthritis, and toothache. After oral administration, the composition is quickly and completely absorbed, the protein binding rate is low, and the half-life period is only 15-20 minutes; the active metabolite Salicylic Acid (SA) protein binding rate after hydrolysis is 65-90%, the half-life of the salicylate is determined by the dose size and urine pH, so that the clearance half-life can be 2-3 hours of low dose to 15 minutes of high dose. Salicylic acid and its metabolites are excreted mainly from the kidney.

Since 2010, EMA requires that aspirin must be simultaneously detected in bioequivalence research of raw acetylsalicylic acid and active metabolite salicylic acid, and acetylsalicylic acid is used as an equivalence evaluation index. The NMPA guiding principle of the national drug administration also clearly shows that the technical product acetylsalicylic acid and the active metabolite salicylic acid must be detected simultaneously, and the acetylsalicylic acid is used as an equivalence evaluation index. Therefore, a stable and reliable liquid chromatography-mass spectrometry (HPLC-MS/MS) method for simultaneously detecting the content of acetylsalicylic acid and salicylic acid in human plasma is urgently needed.

The prior art has the problems that acetylsalicylic acid is easily converted into an active metabolite, namely salicylic acid in blood plasma, so that an effective inhibitor needs to be searched for to prevent the conversion of acetylsalicylic acid into salicylic acid in the detection process; after the medicine is taken, the concentrations of the acetylsalicylic acid and the salicylic acid in the blood plasma are greatly different (the difference of Cmax is about 7-8 times), a set of analysis method needs to be developed to simultaneously detect two objects to be detected, and the method specifically comprises the setting of a standard curve and a quality control sample in operation and the practical operability of corresponding working solution preparation design; meanwhile, the influence of endogenous salicylic acid on the detection method cannot be eliminated, and the influence of plasma endogenous salicylic acid on the detection result cannot be eliminated.

In the prior art, CN110954630B discloses a method for detecting aspirin content in blood plasma and a pretreatment kit, the method can accurately determine the content of Chinese aspirin in blood plasma, the pretreatment process is simple, but the method cannot simultaneously determine the concentration of ASA and SA in the blood plasma, and cannot eliminate the influence of endogenous salicylic acid on experimental results.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a method for detecting aspirin active ingredients in blood plasma, which can simultaneously determine the concentration of ASA and SA in the blood plasma and simultaneously reduce the influence of endogenous salicylic acid in a human body on a detection result to the greatest extent, and is realized by the following technology.

A method for detecting aspirin active ingredients in blood plasma comprises the following four steps:

s1, solution preparation: the method comprises the steps of preparing a correction sample, a quality control sample and an internal standard solution (I03);

s2, preprocessing: respectively preprocessing the correction sample and the quality control sample in the S1, and preparing a blank matrix sample;

s3, concentration measurement: the calibration sample after pretreatment in S2 was determined using a liquid mass spectrometry (HPLC-MS/MS) method;

s4, quantitative analysis: respectively taking the content of ASA or SA as a horizontal coordinate (the unit is ng/mL) and the peak area as a vertical coordinate, establishing an ASA standard curve and an SA standard curve, determining a linear regression equation according to the standard curves, and verifying the linear regression equation by using a quality control sample;

in step S1, the calibration sample is prepared by: under the condition of wet ice, respectively preparing an ASA solution and an SA solution by using an acetonitrile solution (R1) containing 1% formic acid, and mixing the prepared ASA solution and the SA solution in a centrifugal tube to prepare a calibration sample; then adding blank plasma containing 5mg/mL NaF under the condition of wet ice, and mixing by vortex;

preparing the quality control sample: respectively preparing an ASA solution and an SA solution by using R1 under the condition of wet ice, mixing the prepared ASA solution and SA solution in a centrifugal tube to prepare a quality control sample, then adding blank plasma containing 5mg/mL NaF, and preserving at-90 to-60 ℃;

the pretreatment process of the calibration sample in step S2 is as follows:

transferring the calibration sample and the matrix into a centrifuge tube under the condition of P1 wet ice, and adding human blank plasma containing 5mg/mL NaF into the centrifuge tube;

p2, adding I03 and acetonitrile solution (R2) containing 0.5% formic acid to P1 under wet ice, respectively, and vortex mixing;

centrifuging the solution by using a centrifugal machine at the temperature of 0-4 ℃ under the condition of P3;

p4, transferring supernatant solution in the centrifuged solution to a 96-well plate under the wet ice condition;

p5, before sample injection analysis, storing the processed sample in an automatic sample injector or a refrigerator at 0-10 ℃;

the pretreatment process of the quality control sample is as follows:

q1, transferring the quality control sample and the matrix into a centrifuge tube under the wet ice condition, and adding blank plasma containing NaF of 5mg/mL into the centrifuge tube;

q2, adding I03 and R2 into P1 respectively under the condition of wet ice, and mixing by vortex;

q3, and centrifuging the solution in a centrifuge at the temperature of 0-4 ℃;

q4, transferring the supernatant solution in the centrifuged solution to a 96-well plate under the wet ice condition;

q5, before sample injection analysis, storing the treated sample in an automatic sample injector or a refrigerator at 0-10 ℃.

The correction sample contains ASA and SA simultaneously, and the concentration of the ASA is less than that of the SA, so that the correction sample is used for simulating the conditions of SA and SA in plasma, and the standard curves of ASA concentration-peak area and SA concentration-peak area can be more accurately determined when the plasma is used as a substrate, thereby establishing a linear regression equation; the use of R1 to formulate calibration samples minimizes the effect of endogenous salicylic acid on aspirin bioequivalence.

The quality control sample is used for testing whether the obtained linear equation can simultaneously and accurately detect the content of ASA and SA in blood plasma, and the quality control sample also simultaneously contains ASA and SA with different concentrations so as to verify the linear equation.

The I03 contains ASA and SA at the same time, and the concentration of ASA and SA is the same, so that the matrix effect and interference can be well corrected.

The whole pretreatment process of the correction sample and the quality control sample is carried out under the wet ice condition, so that the conversion between ASA and SA can be effectively prevented, the stability of ASA and SA in blood plasma is kept, meanwhile, the pretreatment process under the wet ice condition is few, the operation is simple and easy, and the steps of concentration and the like are not needed; r1 and 5mg/mL NaF in solution were also effective in inhibiting the conversion of ASA to SA in plasma.

The ASA and SA concentrations in the calibration samples are shown in the table below:

TABLE 1 concentration of calibration samples

The ASA and SA concentrations in the quality control samples are shown in the following table, wherein HQC is a high-concentration quality control sample, MQC is a medium-concentration quality control sample, and GMQC is: a quality control sample of a geometric mean value of the low-concentration quality control sample and the high-concentration quality control sample; and LQC is a low-concentration quality control sample.

TABLE 2 concentration of quality control samples

Preferably, in the liquid chromatography-mass spectrometry method in step S3, the chromatographic conditions are:

a chromatographic column: CAPCELL PAK C18, 2.0 × 100mm, 5.0 μm, seniority;

mobile phase A: an aqueous solution containing 0.1% formic acid;

mobile phase B: acetonitrile;

autosampler wash solution: methanol;

temperature of the column oven: 35 ℃;

flow rate: 0.600 mL/min;

approximate column pressure at column equilibrium: 14.1 Mpa;

autosampler temperature: 6 ℃;

sample introduction volume: 2.00 mu L;

pressure foot lifting amount: 52 mm;

autosampler cleaning mode: cleaning before sample injection and cleaning after sample injection;

volume of needle washing with cleaning solution: 200 mu L;

soaking time when cleaning the sample injection needle of the automatic sample injector: 2 s;

chromatographic gradient:

TABLE 3 elution procedure for chromatographic gradients

Time(min)	Module	Event	Value(％)
				0.01	Pumps	Pump B Conc.	30.0
0.50	Pumps	Pump B Conc.	30.0
				1.00	Pumps	Pump B Conc.	30.0
1.10	Pumps	Pump B Conc.	30.0
				3.00	Controller	Stop

A0.1% formic acid aqueous solution is selected as a mobile phase A, and is matched with a specific chromatographic gradient, namely isocratic elution for 3min, so that the signals of ASA and SA can be obviously distinguished, the concentrations of ASA and SA in blood plasma can be more accurately and simultaneously measured, and mutual transformation between ASA and SA can be avoided in the detection process.

Preferably, in the pretreatment step P1 of the calibration sample, the matrix is EDTA-K added as anticoagulant₂The plasma of (a); in the pretreatment step Q1 of the quality control sample, the matrix is added with anticoagulant EDTA-K₂The blood plasma of (1). Anticoagulant EDTA-K₂Can effectively prevent blood plasma coagulation.

Preferably, the internal standard solution in the step S1 is an R1 solution mixed by acetylsalicylic acid-d 4 and salicylic acid-d 4; more preferably, the concentrations of acetylsalicylic acid-d 4 and salicylic acid-d 4 in the internal standard solution are both 2 mug/mL.

Preferably, in step P3 and step Q3, the rotation speed of the centrifuge is 1000g, and the centrifugation time is 5 min.

Preferably, the blank matrix in step S2 is formulated as follows:

k1, transferring blank plasma containing 5mg/mL NaF to a centrifuge tube under the wet ice condition, and adding water into a reagent blank sample;

k2, adding R1 and R2 into a centrifuge tube respectively under the condition of wet ice, and mixing by vortex;

k3, centrifuging the solution by a centrifuge at 0-4 ℃;

k4, transferring the supernatant solution in the centrifuged solution to a 96-well plate under the wet ice condition;

k5, before sample injection analysis, storing the processed sample in an automatic sample injector or a refrigerator at 0-10 ℃.

More preferably, in step K3, the rotation speed of the centrifuge is 1000g, and the centrifugation time is 5 min.

Preferably, the mass spectrometric conditions of the combined liquid and mass method in step S3 and the combined liquid and mass method in step S3 are as follows:

an ion source: ESI;

ionization mode: negative;

and (3) monitoring mode: MRM;

electrospray voltage: -4500V;

ion spray temperature: 500 ℃;

air curtain type: nitrogen gas;

types of atomizing gas: nitrogen gas;

the type of the auxiliary gas: nitrogen gas;

data acquisition time: 3.00 min.

Compared with the prior art, the invention has the advantages that:

1. the environment of acetylsalicylic acid and salicylic acid in a human body can be simulated, and the concentrations of the acetylsalicylic acid and the salicylic acid in blood plasma are detected at the same time, so that the active component of the aspirin is determined;

2. by performing under the condition of wet ice, acetylsalicylic acid in the plasma can be effectively prevented from being converted into salicylic acid, and the active ingredient of aspirin in the plasma can be accurately measured; meanwhile, the acetonitrile solution of 0.5 percent formic acid in the solution and 5mg/mL NaF act together to effectively prevent acetylsalicylic acid from being converted into salicylic acid;

3. the interference of endogenous salicylic acid on the detection result can be reduced to the maximum extent.

Drawings

FIG. 1 is a standard curve of ASA concentration-peak area measured by HPLC-MS/MS in example 1;

FIG. 2 is a standard curve of SA concentration-peak area measured by the HPLC-MS/MS method in example 1;

FIG. 3 is a schematic diagram of the peak pattern of example 1;

FIG. 4 is a graph showing a quantitative lower limit sample liquid mass signal in example 1;

FIG. 5 is a graph showing a signal from a sample blank liquid in example 1;

FIG. 6 is a schematic diagram of the peak pattern of a blank matrix sample in example 2;

FIG. 7 is a schematic diagram showing the peak pattern of the ASA signal detected in comparative example 1;

fig. 8 is a schematic diagram of the peak pattern of the detected SA signal of comparative example 1.

Detailed Description

The technical solutions of the present invention will be described clearly and completely below, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

(I) laboratory instruments and reagents

An experimental instrument: analytical balance: AS 60/220.R2 one-hundred-thousandth electronic balance, RAWAG, Inc.; vortex mixer: MX-S, Darongxing laboratory instruments (Beijing) Inc.; a centrifuge: TGL-16.5M high speed refrigerated centrifuge, shanghai luxiang instrument centrifuge instruments ltd; a liquid transfer device: 0.5 to 10 μ L, 2 to 20 μ L, 20 to 200 μ L, 100-; a chromatographic column: CAPCELL PAK C18, 2.0 × 100mm, 5.0 μm, seniority; high performance liquid chromatography pump: LC-20AD, Shimadzu Corp; automatic sample injector: SIL-20AC, Shimadzu Corp; column oven: CTO-20A, Shimadzu Corp; mass spectrometry: API 4000, AB Sciex Corp.

Experimental reagent: methanol and acetonitrile are chromatographically pure, Fisher Scientific company; methanol is analytically pure, Tianjin Kemi Euro Chemicals Co., Ltd; distilled water, drochen group; matrix: EDTA-K containing anticoagulant₂Human plasma of (4).

(II) HPLC-MS/MS detection of aspirin active ingredient in plasma

A method for detecting aspirin active ingredients in blood plasma comprises the following steps:

s1, solution preparation: the method comprises the steps of preparing a correction sample, a quality control sample and I03;

s4, quantitative analysis: respectively taking the content of ASA or SA as a horizontal coordinate (the unit is ng/mL) and the peak area as a vertical coordinate, establishing an ASA standard curve and an SA standard curve, determining a linear regression equation according to the standard curves, and verifying the linear regression equation by using a quality control sample.

In step S1, the calibration sample and the quality control sample are prepared as follows:

preparation of calibration samples: respectively preparing ASA and salicylic acid solutions by using R1 under the condition of wet ice, and mixing the prepared acetylsalicylic acid and salicylic acid solutions in a centrifugal tube in proportion to prepare a correction sample; then, human blank plasma containing 5mg/mL NaF was added under wet ice conditions and vortexed.

The calibration samples were prepared as ready and the ASA and SA concentrations in the calibration samples are shown in the following table:

TABLE 4 concentration of calibration samples in example 1

Preparing a quality control sample: respectively preparing an ASA solution and an SA solution by using R1 under the condition of wet ice, proportionally mixing the prepared ASA solution and the SA solution in a polypropylene centrifugal tube to prepare a quality control sample, then adding human blank plasma containing 5mg/mL NaF, and storing at-90 to-60 ℃, wherein the ASA and SA concentrations in the quality control sample are shown in the following table:

TABLE 5 concentration of quality control sample in example 1

In step S1, the formulation procedure of I03 is as follows:

the formulation of I03 was: adding R1 into a polypropylene centrifuge tube, adding 0.100mg/mL ASA-d4 solution and 0.100mg/mL SA-d4 solution into the polypropylene centrifuge tube by using a pipette, and mixing by vortex to prepare an internal standard solution for later use, wherein the storage conditions are as follows: 0 to 10 ℃. The concentration of ASA-d4 and SA-d4 after formulation were both 2. mu.g/mL.

In step S2, the pretreatment process of the calibration sample and the quality control sample is as follows:

the pretreatment process of the calibration sample comprises the following steps:

p1, precisely transferring 40.0 μ L of calibration sample and 40 μ L of EDTA-K added anticoagulant under wet ice condition₂The human plasma is put into a centrifuge tube;

p2, adding 20.0 mu L of I02 and 400 mu L of R2 into the step P1 respectively under wet ice conditions, and mixing by vortex;

centrifuging for 5min at the speed of 10000g by a centrifuge at the temperature of 0-4 ℃ under the condition of P3;

p4, transferring 100 mu L of supernatant solution obtained in the step P3 under the condition of wet ice, adding the supernatant solution into a 96-well plate containing 400 mu L of R1 solution, and mixing in a vortex mode;

and P5, before sample injection analysis, storing the processed sample in an automatic sample injector or a refrigerator at 0-10 ℃.

The pretreatment process of the quality control sample comprises the following steps:

q1, precisely transferring 40.0 mu L of quality control sample and 40 mu L of EDTA-K added anticoagulant under the condition of wet ice₂The human plasma is put into a centrifuge tube;

q2, adding 20.0 mu L of I02 and 400 mu L of R2 into the step Q1 respectively under the condition of wet ice, and mixing by vortex;

q3, centrifuging for 5min at the speed of 10000g by a centrifuge at the temperature of 0-4 ℃;

q4, transferring 100 mu L of the supernatant solution in the step P3 under the wet ice condition, adding the supernatant solution into a 96-well plate containing 400 mu L of R1 solution, and mixing in a vortex mode;

In step S2, the process of preparing the blank matrix is as follows:

k1, precisely transferring 40 mu L of EDTA-K added anticoagulant under the condition of wet ice₂The human plasma and 40mL of distilled water are put in a centrifuge tube;

adding 20.0 mu L of R1 and 400 mu L of R2 into a centrifuge tube of K1 under the condition of K2 and wet ice respectively, and mixing by vortex;

k3, centrifuging for 5min at the speed of 10000g in a centrifuge at the temperature of 0-4 ℃;

transferring 100 mu L of the supernatant solution into a 96-well plate containing 400 mu L of R1 solution under the condition of K4 and wet ice, and mixing by vortex;

In step S3, the chromatographic conditions of HPLC-MS/MS are as follows:

a chromatographic column: CAPCELL PAK C18, 2.0 × 100mm, 5.0 μm, seniority;

mobile phase A: an aqueous solution containing 0.1% formic acid;

mobile phase B: acetonitrile;

autosampler wash solution: methanol;

temperature of the column oven: 35 ℃;

flow rate: 0.600 mL/min;

approximate column pressure at column equilibrium: 14.1 Mpa;

autosampler temperature: 6 ℃;

sample introduction volume: 2.00 mu L;

pressure foot lifting amount: 52 mm;

volume of needle washing with cleaning solution: 200 mu L;

the chromatographic gradients are shown in the table below:

TABLE 6 elution procedure for chromatographic gradient of example 1

The liquid chromatography-mass spectrometry method in step S3 has mass spectrometry conditions:

an ion source: ESI;

ionization mode: negative;

and (3) monitoring mode: MRM;

electrospray voltage: -4500V;

ion spray temperature: 500 ℃;

air curtain type: nitrogen Setting: 10;

types of atomizing gas: nitrogen Setting: 35;

the type of the auxiliary gas: nitrogen Setting: 50;

data acquisition time: 3.00 min.

FIG. 1 is a standard curve of ASA concentration-peak area measured by HPLC-MS/MS method, wherein the linear equation of ASA is that y is-2.83 × 10^-8x²+0.00156x-0.000884, r ═ 0.9979(x is the concentration of ASA in ng/mL); fig. 2 is a standard curve of SA concentration-peak area measured by HPLC-MS/MS method, and the linear regression equation of SA is y 0.0016x +0.0415 and r 0.9962(x is the concentration of SA in ng/mL).

FIG. 3 is a schematic diagram of the peak patterns of the present example, the upper is the peak pattern of ASA and the lower is the peak pattern of SA, wherein the time for the ASA to peak is 0.79min and the time for the SA to peak is 1.14 min; as can be seen from the figure, in the present embodiment, ASA and SA can be completely separated without interfering with each other, and the response is good.

FIG. 4 is a plot of the lower quantitation limit sample mass-to-liquid signal, ASA at the top and SA at the bottom, and FIG. 4 illustrates that both analyte interferences are below 20% of the lower quantitation limit; FIG. 5 is a plot of mass signal for reagent blanks, ASA on top and SA below, and FIG. 5 illustrates that neither analyte is interfering.

Examination of matrix Effect in the method of example 1

FIG. 6 is a schematic diagram of the peak patterns for a blank matrix sample, with ASA on top and SA below; figure 6 illustrates that both analyte interferences were below 20% of the lower limit of quantitation.

Comparative example 1

The present comparative example differs from example 1 in the setting of the chromatographic gradient program, and the elution procedure of the color gradient of the present comparative example is shown in the following table:

table 7 elution procedure for chromatographic gradient of comparative example 1

FIG. 7 is a schematic diagram showing the peak patterns of the ASA signal detected in this comparative example, with the ASA signal on the top and the SA signal on the bottom; FIG. 8 is a graph showing the peak patterns of the SA signal detected in this comparative example, with the ASA signal on the top and the SA signal on the bottom. As can be seen from fig. 7 and 8, in the comparative example, neither the ASA signal nor the SA signal was detected, and the peaks of ASA and SA were separated.

Claims

1. A method for detecting aspirin active ingredients in blood plasma is characterized by comprising the following four steps:

s1, solution preparation: the method comprises the steps of preparing a correction sample, a quality control sample and an internal standard solution;

s3, concentration measurement: determining the concentration of the calibration sample pretreated in the S2 by using a liquid chromatography-mass spectrometry method;

s4, quantitative analysis: respectively establishing an acetylsalicylic acid standard curve and a salicylic acid standard curve by taking the content of acetylsalicylic acid or salicylic acid as a horizontal coordinate and the peak area as a vertical coordinate, determining a linear regression equation according to the standard curves, and verifying the linear regression equation by using a quality control sample;

in step S1, the calibration sample is prepared by the following method: under the condition of wet ice, respectively preparing acetylsalicylic acid solution and salicylic acid solution by using acetonitrile solution containing 1% formic acid, and mixing the prepared acetylsalicylic acid solution and salicylic acid solution in a centrifugal tube to prepare a correction sample; then adding blank plasma containing 5mg/mL NaF under the condition of wet ice, and mixing by vortex;

the preparation method of the quality control sample comprises the following steps: under the condition of wet ice, respectively preparing acetylsalicylic acid solution and salicylic acid solution by using acetonitrile solution containing 1% formic acid, mixing the prepared acetylsalicylic acid solution and salicylic acid solution in a centrifugal tube to prepare a quality control sample, and then adding blank plasma containing 5mg/mL of NaF to store at-90 to-60 ℃;

transferring the calibration sample and the matrix into a centrifuge tube under the condition of P1 wet ice, and adding blank plasma containing NaF of 5mg/mL into the centrifuge tube;

p2, under the condition of wet ice, respectively adding an internal standard solution and an acetonitrile solution containing 0.5% formic acid into P1, and carrying out vortex mixing;

the pretreatment process of the quality control sample is as follows:

q2, respectively adding an internal standard solution and an acetonitrile solution containing 0.5% formic acid into P1 under the condition of wet ice, and carrying out vortex mixing;

2. The detection method according to claim 1, wherein the chromatography conditions in the LC-MS method in step S3 are as follows:

a chromatographic column: CAPCELL PAK C18, 2.0 × 100mm, 5.0 μm, seniority;

mobile phase A: an aqueous solution containing 0.1% formic acid;

mobile phase B: acetonitrile;

autosampler wash solution: methanol;

temperature of the column oven: 35 ℃;

flow rate: 0.600 mL/min;

approximate column pressure at column equilibrium: 14.1 Mpa;

autosampler temperature: 6 ℃;

sample introduction volume: 2.00 mu L;

pressure foot lifting amount: 52 mm;

volume of needle washing with cleaning solution: 200 mu L;

the chromatographic gradient was: isocratic elution for 3min, mobile phase B30%.

3. The detection method according to claim 1, wherein the matrix is EDTA-K added as an anticoagulant in the pretreatment step P1 and the pretreatment step Q1₂The blood plasma of (1).

4. The detection method according to claim 1, wherein the internal standard solution in the step S1 is an acetonitrile solution containing 1% formic acid mixed by acetylsalicylic acid-d 4 and salicylic acid-d 4.

5. The detection method according to claim 4, wherein the concentrations of acetylsalicylic acid-d 4 and salicylic acid-d 4 in the internal standard solution are both 2 μ g/mL.

6. The detection method according to claim 1, wherein in steps P3 and Q3, the rotation speed of the centrifuge is 1000g, and the centrifugation time is 5 min.

7. The detection method according to claim 1, wherein the blank matrix in step S2 is prepared as follows:

k2, under the condition of wet ice, respectively adding acetonitrile solution containing 1% formic acid and acetonitrile solution containing 0.5% formic acid into a centrifuge tube, and carrying out vortex mixing;

k3, centrifuging the solution in a centrifuge at 0-4 ℃;

8. The detection method according to claim 7, wherein in step K3, the rotation speed of the centrifuge is 1000g, and the centrifugation time is 5 min.

9. The detection method according to claim 1, wherein the mass spectrometry conditions of the LC-MS method in step S3 are as follows:

an ion source: ESI;

ionization mode: negative;

and (3) monitoring mode: MRM;

electrospray voltage: -4500V;

ion spray temperature: 500 ℃;

air curtain type: nitrogen gas;

types of atomizing gas: nitrogen gas;

the type of the auxiliary gas: nitrogen gas;

data acquisition time: 3.00 min.