CN110954630B - Method for detecting aspirin content in blood plasma and pretreatment kit - Google Patents

Abstract

The invention discloses a method for detecting aspirin content in blood plasma and a pretreatment kit, wherein the method comprises the pretreatment process of a blood plasma sample, a calibration sample and/or a quality control sample, and LC-MS/MS detection is carried out on the pretreated sample; the pretreatment process comprises the following steps: adding the sample into an extraction plate, standing for adsorption, and eluting by using an eluent, wherein the eluent is an ethyl acetate solution containing formic acid and oxalic acid. The method can accurately and quantitatively detect the aspirin in the blood plasma.

Description

Method for detecting aspirin content in blood plasma and pretreatment kit

Technical Field

The invention belongs to the field of aspirin content detection, and relates to a method for detecting aspirin content in blood plasma and a pretreatment kit.

Background

The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

Aspirin, also known as acetylsalicylic acid, belongs to a clinically common antipyretic analgesic, and has been clinically verified for many years to relieve pains such as headache, toothache, muscular soreness, rheumatalgia and the like with good effect. In recent years, the traditional Chinese medicine composition is also used for treating and preventing cardiovascular and cerebrovascular diseases such as cerebral infarction, myocardial infarction, thrombosis prevention and the like clinically. According to statistics, the number of people died due to cardiovascular diseases in China is 300 million, accounts for more than 50% of the total death causes in China each year, and becomes a main disease threatening the health of the people in China. With the increasingly serious aging of China, the incidence of cardiovascular diseases is continuously improved, so that the prevention and treatment of cardiovascular and cerebrovascular diseases become more important and become the key focus of the medical field. According to research, aspirin can prevent cardiovascular and cerebrovascular diseases by resisting platelet aggregation. A research of the American Harvard university shows that the aspirin which is insisted on taking 1 piece of aspirin every day can respectively reduce the incidence rates of myocardial infarction and fatal myocardial infarction by 44 percent and 66 percent after 5 years, and the aspirin is low in price, so that the aspirin is more commonly used and becomes a commonly used medicine which is clinically known by the general public and is used for preventing the attack of cardiovascular and cerebrovascular diseases. However, with the wide application of aspirin, adverse reactions are more and more, mainly the phenomenon of abuse of aspirin drugs. For example, after long-term administration of aspirin, renal function is impaired, mainly oxidative phosphorylation is affected, potassium ions exude from renal tubular cells, potassium deficiency is caused, uric acid content in urine is high, and the like, and vomiting, nausea, abdominal pain and the like are caused by direct stimulation of gastric mucosa, and gastric hemorrhage can occur in severe cases. If the dosage is excessive, the circulation function is inhibited by direct action and central nervous system action, thereby causing certain toxicity to heart and causing a series of nausea and vomiting. According to the research, the children influenza treatment can cause Swiss syndrome if aspirin is used, the Swiss syndrome generally occurs after virus infectious diseases and is acute encephalopathy and liver fat infiltration syndrome, so the dosage is cautiously used when the aspirin is used. In addition, improper use of aspirin also causes allergic reactions, liver damage, iron deficiency anemia, and the like. Therefore, although aspirin drugs are wide in application and economical and practical, the current situation of adverse reactions is more severe at present, and the main reason is that the drugs are unreasonably used. With the proposal of accurate medical concepts in recent years, national policies are also continuously assisting the development and layout of the accurate medical industry, so that the medical treatment of China is continuously developed towards the directions of accurate prevention and accurate treatment, and safe and effective diagnosis and treatment guidance is carried out according to individual differences. Among them, the monitoring of therapeutic drug concentration (TDM) is very significant under the current situation of drug administration, and the clinical administration scheme is individualized under the guidance of pharmacokinetics by measuring the drug concentration in blood or other body fluids, so as to improve the therapeutic effect and avoid or reduce the occurrence of toxic and side effects. Aspirin belongs to a medicine with nonlinear kinetic characteristics and large individual difference of pharmacokinetics, so that in order to change the current situation of incorrect use of aspirin medicines, the metabolism and absorption conditions of patients to the medicine are obtained by detecting the concentration in blood, and the individual administration scheme is scientifically guided and clinically formulated to realize the reasonable, safe and effective administration of aspirin, and the aspirin injection has important guiding significance.

The content of general medicines in a human body is low, the conventional detection method cannot meet the requirement of medicine monitoring sensitivity, and the detection methods such as an immunochemical method, a spectroscopic method, a chromatographic method, a liquid chromatography-mass spectrometry combined method and the like with high sensitivity are commonly used for determination at present. The immunochemical method mainly comprises the steps of preparing a specific antibody corresponding to a medicament, detecting the specific antibody by utilizing antigen-antibody reaction, having high sensitivity and simple and convenient operation, and being capable of being used with an automatic analyzer, but the method has poor specificity, and because the aspirin medicament (acetylsalicylic acid) and the metabolite salicylic acid have similar structures, the antibody is difficult to distinguish the prototype medicament acetylsalicylic acid and the metabolite salicylic acid, and cross interference is easy to occur, so that the detection result is high in false positive. Although low cost, spectroscopy is susceptible to interference from endogenous substances or metabolites with similar spectroscopic properties, and is less specific and less sensitive. The liquid chromatography is widely applied to the chromatography, is a widely applied chromatographic analysis technology at present, and has the characteristics of good specificity, good repeatability and good separation degree, but due to the fact that aspirin drugs have aromatic acid basic structures and contain structures such as benzene rings, carboxyl groups and ester groups, ideal separation effects are difficult to achieve in liquid with complex matrixes only by means of the liquid chromatography separation technology. In recent years, mass spectrometry technology gradually becomes an important means for monitoring therapeutic drugs, wherein liquid chromatography-mass spectrometry technology is applied more, and mainly comprises liquid chromatography-mass spectrometry (LC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS), the LC-MS is formed by connecting a liquid phase system and a single quadrupole mass spectrometry, the LC-MS/MS is formed by connecting a liquid phase system and two quadrupole mass spectrometry, the detection specificity of the latter is stronger, the determination of an analyte is carried out by double screening of parent ions and daughter ions, and the method is particularly suitable for detecting samples with complicated matrix interference clinically. LC-MS/MS is called as the 'gold standard' of clinical detection, has high sensitivity, strong specificity and high accuracy, can realize the simultaneous detection of a plurality of drugs, has less sample dosage, short analysis time and simple and convenient pretreatment process, and has more obvious advantages especially for drugs with insufficient detection by other methods. However, the research of the inventor of the invention finds that the existing liquid chromatography-mass spectrometry (LC-MS) is used for detecting the concentration of aspirin in plasma, a single quadrupole mass spectrometry is adopted, the sample pretreatment process is very complicated, the recovery rate is low, and the indexes such as the quantitative limit and the precision and the like do not reach the expected effect.

Disclosure of Invention

In order to solve the defects of the prior art, the invention aims to provide a method for detecting the content of aspirin in blood plasma and a pretreatment kit.

In order to achieve the purpose, the technical scheme of the invention is as follows:

in one aspect, the method for detecting the aspirin content in the plasma comprises a pretreatment process of a plasma sample, a calibration sample and/or a quality control sample, wherein the pretreated sample is subjected to LC-MS/MS detection;

the pretreatment process comprises the following steps: adding the sample valence into an extraction plate, standing for adsorption, and eluting by using an eluent, wherein the eluent is an ethyl acetate solution containing formic acid and oxalic acid.

The inventor of the invention finds out through research that: firstly, human plasma samples contain a large amount of phospholipid substances, phospholipid is an amphoteric compound, one end of the phospholipid is hydrophilic, and the other end of the phospholipid is lipophilic, so that traditional liquid-liquid extraction can cause layering, an emulsifying layer is generated between two phases, namely the emulsification phenomenon, and supernatant liquid is not easy to take or is not accurate after liquid-liquid extraction, so that the recovery rate or the precision of the method is influenced. The filter layer of the extraction plate adopted by the invention comprises three layers, wherein the upper part, the middle part and the lower part are respectively provided with the sieve plate, the filler and the sieve plate, the sieve plate is a filter membrane in essence, and can filter out impurities such as protein with larger particles; secondly, aspirin contains an acidic functional group, carboxyl-COOH, the stronger the electron-withdrawing ability, the stronger the acidity, and the dissociation constant pKa of aspirin is 3.41, which indicates that at a pH of 3.41, aspirin exists in two forms, each of which accounts for 50%, of-COOH and-COO, respectively^-. When the pH value of the system is slightly changed, the distribution ratio of the two conditions is influenced, and only when the pH value is out of the pKa +/-2 range, the pH value is changedOne of them exists in a form that is mainly in the form of-COOH molecules at a pH value of less than 1.41 and mainly in the form of-COO molecules at a pH value of more than 5.41^-The ionic form exists. Only in one form, aspirin is more stable, and further has higher elution efficiency, namely recovery rate, therefore, formic acid and oxalic acid are added into ethyl acetate serving as eluent, and aspirin exists in a-COOH molecular form by changing the pH value of the system, so that the recovery rate of aspirin is improved, and the molecular form is more beneficial to subsequent chromatographic separation, so that the molecular form is selected. In addition, the filler can also adsorb the interference of water-soluble matrixes in some plasma samples, so that the sample purification effect is improved.

As the invention discovers that the pretreatment process of the sample influences the LC-MS/MS detection of the aspirin content in the human plasma, the method can more accurately detect the aspirin content in the human plasma, and in order to facilitate the pretreatment of the sample, the invention provides a pretreatment kit for detecting the aspirin content in the plasma, which comprises an extraction plate, formic acid, oxalic acid and ethyl acetate.

The invention has the beneficial effects that:

the invention has simple and fast operation, strong specificity and high accuracy, can simultaneously process a plurality of samples in batches, has high flux, can be combined with an automatic pretreatment device, is easy to popularize and popularize clinically, and is particularly suitable for medical institutions with large sample quantity. The concentration of the aspirin in the plasma of a patient is effectively monitored, scientific and reasonable medication guidance is carried out, an individual medication scheme is formulated, the optimal curative effect of the aspirin is effectively exerted, the curative effect is prevented from being reduced due to insufficient dosage, or adverse reaction and even poisoning are caused due to overlarge dosage, safe and accurate medication is achieved, and the aspirin administration monitoring system has important guiding significance.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.

FIG. 1 is a standard curve of aspirin for an embodiment of the present invention;

FIG. 2 is an aspirin chromatogram of an embodiment of the present invention;

FIG. 3 is a chromatogram of aspirin-D4 from an embodiment of the present invention.

Detailed Description

It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

In view of the difficulty in accurately quantifying aspirin in human plasma in the conventional method, the invention provides a method for detecting the content of aspirin in plasma and a pretreatment kit.

The invention provides a method for detecting aspirin content in blood plasma, which comprises a pretreatment process of a blood plasma sample, a calibration sample and/or a quality control sample, wherein the pretreated sample is subjected to LC-MS/MS detection;

the pretreatment process comprises the following steps: adding the sample into an extraction plate, standing for adsorption, and eluting by using an eluent, wherein the eluent is an ethyl acetate solution containing formic acid and oxalic acid.

Firstly, the filter layer of the extraction plate adopted by the invention comprises three layers, wherein the upper part, the middle part and the lower part are respectively provided with a sieve plate, a filler and a sieve plate, the sieve plate is a filter membrane in essence, and can filter out impurities such as protein with larger particles, the filler is solid particles and can disperse polar substances and non-polar substances in blood plasma, so that an elution solvent can be fully contacted with mutually soluble substances, the exchange efficiency is improved, and the absorption is improved, thereby the extraction effect is improved. Secondly, formic acid and oxalic acid are added into ethyl acetate serving as eluent, and aspirin exists in a-COOH molecular form by changing the pH value of the system, so that the recovery rate of aspirin is improved, and the molecular form is more beneficial to subsequent chromatographic separation, so that the molecular form is selected. In addition, the filler can also adsorb the interference of water-soluble matrixes in some plasma samples, so that the sample purification effect is improved.

In one or more embodiments of the present disclosure, the volume fraction of formic acid in the ethyl acetate solution is 0.49-0.51% o, and the volume fraction of oxalic acid in the ethyl acetate solution is 0.49-0.51% o. The pH value can be made to be less than 1.41, so that the aspirin is completely ensured to exist in a-COOH molecular form, and the aspirin recovery rate is further improved.

In one or more embodiments of this embodiment, the extraction plate is a 96-well solid phase extraction plate. 96 samples can be processed simultaneously in batches.

In one or more embodiments of the present invention, the internal standard sample is added to an extraction plate, and the mixture is allowed to stand for adsorption and then eluted with an eluent, which is an ethyl acetate solution containing formic acid and oxalic acid.

In this series of examples, the internal standard was aspirin-D4. The physical and chemical properties of aspirin-D4 are basically consistent with those of aspirin, and the aspirin-D4 participates in the whole extraction process and the instrument detection process so as to correct various errors caused by factors such as operation, a sample matrix, an instrument and the like.

In one or more embodiments of this embodiment, the elution process is pressurized at the inlet, or depressurized at the outlet. The elution rate can be accelerated.

In one or more embodiments of this embodiment, the internal standard working solution is prepared by adding the internal standard to methanol to a constant volume.

In one or more embodiments of this embodiment, the method includes preparing a calibration sample working solution, preparing the aspirin solution and the blank plasma into different concentrations of the calibration working solution, and freeze-drying the calibration working solution. When in use, the mixture is taken out of the refrigerator and is kept at room temperature for balance, and then pure water is added for mixing for later use.

In one or more embodiments of this embodiment, the quality control sample is prepared from a blank plasma plus aspirin standard.

In this embodiment, when the quality control sample and the calibration sample are used, the lyophilized powder is equilibrated at room temperature, and then mixed with pure water for use.

In one or more embodiments of this embodiment, the mobile phase a is an aqueous ammonium formate solution comprising formic acid and the mobile phase B is a methanolic acetonitrile solution of ammonium formate comprising formic acid in a liquid chromatograph.

In this series of examples, the gradient elution procedure in liquid chromatography was: the 35% mobile phase B rose to 95% mobile phase B in 0 to 2.0 min; 95% mobile phase B in 2.0 to 3.5 min; reducing 95% of mobile phase B to 35% of mobile phase B within 3.5-4.0 min; 35% mobile phase B in 4.0 to 5.0 min.

In this series of examples, the flow rate is 0.39-0.41 mL/min, and the sample injection amount is 9.9-10.1. mu.L.

In one or more embodiments of this embodiment, the mass spectrometry conditions are: multiple ion reaction monitoring of positive ion electrospray ionization.

In the series of embodiments, the ion source parameters comprise a spraying voltage of 3400-3600V, a temperature of an ion transmission tube of 340-360 ℃, an evaporation temperature of 340-360 ℃, a sheath gas of 35-45 arb and an auxiliary gas of 4.5-5.5 arb.

In this series of examples, the MRM mass spectral parameters are as follows

In the invention, the separation is carried out by liquid chromatography, and then tandem mass spectrometry detection is carried out. Two pairs of ion pairs are respectively selected from aspirin and aspirin-D4 for mass spectrum detection, one is a quantitative ion pair (aspirin 181.1 → 101.1, aspirin-D4185.2 → 105.3), the other is a qualitative ion pair (aspirin 181.1 → 103.2, aspirin-D4185.2 → 107.5), the retention time of the qualitative ion pair and a chromatographic peak is taken as a qualitative basis, the quantitative ion pair is subjected to quantitative analysis, the internal standard method is used for quantification, a standard curve is drawn by calculating the ratio of the peak area of a calibrator to the peak area of the internal standard as an ordinate, and the concentration of the target value of the calibrator is taken as an abscissa, and a linear equation is fitted. Substituting the peak area ratio in the sample (quality control product, blood plasma sample) into the standard curve equation to calculate the aspirin concentration in the sample. The introduction of the internal standard can correct the deviation in the processes of operation, extraction, chromatographic separation, instrument detection and the like, and particularly, the internal standard of the aspirin medicament is selected, so that the correction range is wider, the effect is better, and the detection result is closer to the true value. Two ion pairs can better ensure the specificity of the detection result, particularly blood samples, the matrix is very complex, and a plurality of unknown compounds are not clearly researched. If substances with the molecular weight similar to that of aspirin in structure exist in the matrix, the substances cannot be distinguished from interferents in the matrix only by means of a single quadrupole mass analyzer, and the influence of the interferents can be reduced by adopting the tandem mass spectrometry and performing secondary detection on parent ions and daughter ions in a one-to-one correspondence manner, so that the specificity and the accuracy of a detection result are improved.

In another embodiment of the invention, a pretreatment kit for detecting aspirin content in blood plasma is provided, which comprises an extraction plate, formic acid, oxalic acid and ethyl acetate. The pretreatment of the sample can be facilitated.

In one or more embodiments of this embodiment, the volume ratio of formic acid, oxalic acid, and ethyl acetate is 0.49 to 0.51 ‰: 0.49-0.51 ‰: 1.

in order to make the technical solutions of the present invention more clearly understood by those skilled in the art, the technical solutions of the present invention will be described in detail below with reference to specific embodiments.

Example 1

(1) Preparation of internal standard working solution

80 mu L of aspirin internal standard (aspirin-D4) with the concentration of 50 mu g/mL is added into 4mL of methanol and mixed evenly, the final concentration is 1000ng/mL, and the internal standard is used as internal standard working solution for standby.

(2) Preparation of working solution for calibrator

Preparing 100 mu g/mL aspirin stock solution by using methanol, preparing 6 gradients by using blank plasma with the concentrations of 5000, 2500, 1250, 625, 312.5 and 156ng/mL respectively as a calibrator working solution, taking 300 mu L of the calibrator working solution, subpackaging the 300 mu L of the calibrator working solution in 1.5mL freeze-drying bottles, placing the freeze-drying bottles in a freeze-dryer, pre-freezing at-55 ℃ for 4 hours, performing gradient temperature rise, vacuumizing from the first temperature rise, wherein the temperature rise is favorable for the sublimation process, the drying rate can be accelerated, and the prepared dry powder is stored at-20 ℃ for later use. When in use, the mixture is taken out of the refrigerator and is placed at room temperature for balancing for 15-20min, then 300 mu L of pure water is respectively added, and the mixture is uniformly mixed for standby.

(3) Preparation of quality control solution

The quality control method comprises three levels of low, medium and high quality control, wherein the quality control is prepared by adding aspirin standard into blank plasma, the concentration is 125 ng/mL, 500 ng/mL and 2000ng/mL respectively, 300 mu L of the quality control solution is taken and distributed into 1.5mL freeze-drying bottles, the freeze-drying bottles are placed in a freeze-drying machine, pre-freezing is carried out for 4h at the temperature of minus 55 ℃, then gradient temperature rise is carried out, vacuumizing is carried out during the first temperature rise, dry powder is prepared, and the dry powder is stored for later use at the temperature. When in use, the mixture is taken out of the refrigerator and is placed at room temperature for balancing for 15-20min, then 300 mu L of pure water is respectively added, and the mixture is uniformly mixed for standby.

(4) Plasma sample pretreatment

Add 200. mu.L of plasma sample to 96-well extraction plate, add 100. mu.L of internal standard working solution, blow and mix well. Then transferred to a 96-well extraction plate, and kept stand for 5min for adsorption. Adding 800 μ L of 0.5 ‰ formic acid 0.5 ‰ oxalic acid ethyl acetate solution, eluting twice, accelerating elution rate with positive pressure device, and collecting eluate in 96-well collecting plate. The collected eluate was dried under nitrogen and examined after redissolving with 100. mu.L of the initial mobile phase. The pretreatment process of the calibrator and quality control was consistent with that of the plasma samples.

(5) Detection on machine

Chromatographic conditions are as follows: mobile phase A: 1% per mill formic acid 5mM ammonium formate in water; mobile phase B: 1% formic acid 5mM ammonium formate in 80% methanol 20% acetonitrile; gradient elution procedure was 0-2.0min 35% mobile phase B up to 95% mobile phase B, 2.0-3.5min 95% mobile phase B, 3.5-4.0min 95% mobile phase B down to 35% mobile phase B, 4.0-5.0min 35% mobile phase B to stop, as in table 1. Flow rate 0.4mL/min, sample size 10. mu.L, column temperature: at 40 ℃.

TABLE 1 gradient elution procedure

Time (min)	Phase A ratio (%)	Phase B ratio (%)
			0	65	35
2	5	95
			3.5	5	95
4	65	35
			5	stop	stop

Mass spectrum conditions: multiple ion reaction monitoring of positive ion electrospray ionization. The ion source parameters comprise a spraying voltage of 3500V, an ion transmission tube temperature of 350 ℃, an evaporation temperature of 350 ℃, a sheath gas of 40arb and an auxiliary gas of 5 arb. The MRM mass spectral parameters are as in table 2.

TABLE 2MRM Mass Spectrometry parameters

(6) Calculation of results

And calculating a detection result. Firstly, taking the concentration of a calibrator as an abscissa and the ratio of peak areas of the calibrator and an internal standard as an ordinate, drawing a standard curve to obtain a linear equation y as bx + a, substituting the peak area ratio of the sample and the internal standard into the equation, and calculating the concentration of aspirin in the sample.

As shown in figure 1, the method of the invention is used for measuring 156-5000 ng/mL calibration substances to establish a standard curve, the linear relation in the range is good, the correlation coefficient R2 is 0.9996, and the aspirin concentration in the sample is 525ng/mL according to the equation.

As shown in fig. 2, the aspirin chromatographic peak in the sample determined by the method of the present invention.

As shown in FIG. 3, the peak of aspirin-D4 chromatogram determined by the method of the present invention.

Example 2

(1) Lower limit of quantification

According to the judgment standard that the signal-to-noise ratio is larger than or equal to 10, the repeatability Cv is smaller than or equal to 20%, and the accuracy deviation is 15% as the lower limit index of quantification, when 0.5ng/mL of aspirin is added into the blank plasma, the test is repeated for 10 times, and the analyzed data can meet the judgment standard after sample pretreatment and on-machine detection.

(2) Precision degree

The three level quality control products and the patient samples taking the aspirin medicament are repeatedly detected for 10 times for 3 days continuously, 30 detection data are obtained respectively, and the repeated Cv is calculated to obtain a result of 2.2-5.4%.

(3) Recovery rate

Adding aspirin standard solutions with the same volume and different concentrations into blank plasma to enable the final concentrations to be 200 ng/mL, 500 ng/mL and 1000ng/mL respectively, repeating the test for 3 times for each concentration, comparing the average value of 3 times with a theoretical value, and calculating the recovery rate to be 96 +/-3.1%.

(4) Stability test

The calibration material and the quality control material dry powder are stored for 30 days at the temperature of 2-8 ℃ and 60 days at the temperature of-20 ℃, the performance is still good, and the coefficient of variation Cv of observation data is calculated by regular sampling observation, wherein the coefficient of variation Cv is less than 8% under the condition. Therefore, the stability of the calibrator and the quality control product can be improved in a dry powder state, and the calibrator and the quality control product are favorable for storage and transportation.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (5)

1. A method for detecting aspirin content in plasma is characterized by comprising a pretreatment process of a plasma sample, a calibration sample and/or a quality control sample, and performing LC-MS/MS detection on the pretreated sample;

the pretreatment process comprises the following steps: adding a sample into an extraction plate, standing for adsorption, and eluting by using an eluent, wherein the eluent is an ethyl acetate solution containing formic acid and oxalic acid;

the volume fraction of formic acid in the ethyl acetate solution is 0.49-0.51 per mill, and the volume fraction of oxalic acid in the ethyl acetate solution is 0.49-0.51 per mill;

preparing aspirin solution and blank plasma into calibration product working solutions with different concentrations, and freeze-drying the calibration product working solutions;

the quality control sample is prepared by adding aspirin standard substance into blank plasma;

the conditions for LC-MS/MS detection were:

in the liquid chromatogram, a mobile phase A is an ammonium formate aqueous solution containing formic acid, and a mobile phase B is an ammonium formate methanol acetonitrile solution containing formic acid; the gradient elution procedure in liquid chromatography was: the 35% mobile phase B rose to 95% mobile phase B in 0 to 2.0 min; 95% mobile phase B in 2.0 to 3.5 min; reducing 95% of mobile phase B to 35% of mobile phase B within 3.5-4.0 min; 35% mobile phase B in 4.0 to 5.0 min; the flow rate is 0.39-0.41 mL/min, and the sample injection amount is 9.9-10.1 mu L;

the mass spectrum conditions are as follows: monitoring of multi-ion reactions of positive ion electrospray ionization; the ion source parameters comprise a spraying voltage of 3400-3600V, an ion transmission tube temperature of 340-360 ℃, an evaporation temperature of 340-360 ℃, a sheath gas of 35-45 arb and an auxiliary gas of 4.5-5.5 arb.

2. The method according to claim 1, wherein the extraction plate is a 96-well solid-phase extraction plate.

3. The method for detecting the aspirin content in the plasma according to claim 1, wherein the sample added with the internal standard is added into an extraction plate, is kept still for adsorption, and is eluted by adopting an eluent, wherein the eluent is an ethyl acetate solution containing formic acid and oxalic acid.

4. The method for detecting aspirin content in plasma according to claim 3, wherein said internal standard is aspirin-D4.

5. The method for detecting aspirin content in plasma according to claim 1, wherein during elution, the pressure is increased at the inlet or reduced at the outlet.

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