Method for synchronously determining multiple medicine components in biological sample based on LC-MS / MS (Liquid Chromatography-Mass Spectrometry / Mass Spectrometry) technology

Abstract

The invention discloses a method for synchronously determining multiple drug components in a biological sample based on an LC-MS / MS. The multiple drugs are phenytoin sodium, paliperidone, voriconazole, theophylline, fluvoxamine and lidocaine, and the method comprises the following steps: obtaining the biological sample, and pre-treating the biological sample; according to the method, phenytoin sodium, paliperidone, voriconazole, theophylline, fluvoxamine and lidocaine in a sample are synchronously detected by adopting a liquid chromatography-mass spectrometry technology. The method provided by the invention can realize rapid and synchronous determination of phenytoin sodium, paliperidone, voriconazole, theophylline, fluvoxamine and lidocaine, and has high sensitivity and accuracy.

Description

Technical Field

[0001] The invention belongs to the field of drug detection, and specifically relates to a method for synchronously determining multiple drug components in a biological sample based on LC-MS / MS technology. Background Art

[0002] Therapeutic drug monitoring (TDM) is a method of developing an individualized dosing regimen by measuring the drug concentration in the patient's body, aiming to improve the efficacy and safety of drugs. Therapeutic drug monitoring can improve the effectiveness of drug therapy, reduce adverse reactions, and provide a basis for the diagnosis and treatment of drug overdose poisoning. It is an important means to achieve individualized treatment. In the process of drug treatment for patients, it is necessary to regularly monitor the drug concentration in their bodies to adjust the dosage in time and improve the treatment effect.

[0003] Phenytoin is a widely used anti-epileptic drug and a class IB antiarrhythmic drug. It has membrane stability and can inhibit the rapid influx of sodium ions. + Channels and rapidly inactivated Ca 2+ channels, thereby inhibiting Ca 2+ Inflow and K + Outflow, effectively inhibiting abnormal high-frequency discharge. Phenytoin sodium is clinically used for anti-epileptic, treatment of trigeminal neuralgia and sciatica, anti-arrhythmia and antihypertensive effects.

[0004] Paliperidone is an atypical antipsychotic drug mainly used to treat schizophrenia and schizoaffective disorder. It exerts its antipsychotic effect by blocking central dopamine D2 receptors and serotonin 2A receptors.

[0005] Voriconazole is a broad-spectrum antifungal drug used primarily to treat invasive aspergillosis, severe infections caused by Candida albicans resistant to fluconazole, and severe infections caused by Actinomyces and Fusarium.

[0006] Theophylline is a drug commonly used to treat respiratory diseases such as bronchial asthma and asthmatic bronchitis. Theophylline mainly relaxes bronchial smooth muscles and relieves wheezing symptoms by inhibiting phosphodiesterase (PDE) and increasing the concentration of intracellular cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP). Theophylline is mainly used to treat respiratory diseases such as bronchial asthma, asthmatic bronchitis, and obstructive pulmonary emphysema to relieve wheezing symptoms. In addition, theophylline also has anti-inflammatory and immunomodulatory effects, can enhance diaphragmatic contractility, and improve respiratory function. However, the therapeutic window of theophylline is narrow, and even slight changes in blood concentrations may lead to differences between efficacy and safety. Therefore, monitoring the blood concentration of theophylline is crucial to ensure patient safety and efficacy.

[0007] Fluvoxamine is a selective serotonin reuptake inhibitor (SSRI), mainly used to treat depression and obsessive-compulsive disorder, and can also be used to treat anxiety, panic disorder, social anxiety, etc. Fluvoxamine inhibits the reuptake of 5-HT in the presynaptic membrane, increases the concentration of 5-HT in the synaptic cleft, and improves the conduction of 5-HT nerves, thereby exerting antidepressant and anti-anxiety effects.

[0008] Lidocaine is a local anesthetic and antiarrhythmic drug, commonly used for surface anesthesia, infiltration anesthesia, nerve conduction blockade and treatment of ventricular arrhythmias. Lidocaine achieves local anesthesia by blocking sodium channels and inhibiting nerve signal transmission. As a Class IB antiarrhythmic drug, lidocaine promotes potassium ion outflow in myocardial cells at low doses, reduces myocardial automaticity, and has an anti-ventricular arrhythmia effect.

[0009] During the treatment process, a convenient, fast and accurate separation and measurement method is needed to achieve the simultaneous measurement of the concentrations of the above multiple drugs, which will help therapeutic drug monitoring, drug interaction research and pharmacological analysis. Summary of the invention

[0010] The purpose of the present invention is to provide a method for conveniently, quickly, accurately and synchronously determining the drug components phenytoin sodium, paliperidone, voriconazole, theophylline, fluvoxamine and lidocaine in biological samples in view of the above technical problems to be solved.

[0011] In order to achieve the above invention object, the present invention provides a method for synchronously determining multiple drug components in a biological sample based on LC-MS / MS technology, wherein the multiple drug components are phenytoin sodium, paliperidone, voriconazole, theophylline, fluvoxamine, and lidocaine, and the method comprises the following steps: S1. Obtain biological samples and pre-treat them; S2. Liquid chromatography-mass spectrometry was used to simultaneously detect phenytoin sodium, paliperidone, voriconazole, theophylline, fluvoxamine, and lidocaine in the samples.

[0012] As a preferred embodiment, the biological sample is serum.

[0013] As a preferred embodiment, the pretreatment step in step S1 is as follows: add 100 μL of the mixed internal standard solution to 100 μL of the biological sample, balance for 5 minutes, add 1 mL of acetonitrile for protein precipitation, mix well, and then centrifuge at 12000 rpm for 10 minutes, then take 1 mL of the supernatant and add 500 μL of 50% methanol aqueous solution, centrifuge at 12000 rpm for 2 minutes, and take 200 μL of the supernatant in 1 mL of 50% methanol aqueous solution.

[0014] As a preferred embodiment, the preparation steps of the mixed internal standard solution are as follows: dilute the internal standard stock solution with a concentration of 100 μg / mL with methanol with a volume concentration of 50% to prepare a mixed internal standard solution of phenytoin sodium, paliperidone, voriconazole, theophylline, fluvoxamine, and lidocaine isotopes.

[0015] As a preferred embodiment, in the mixed internal standard solution, the internal standard concentration of phenytoin sodium is 0.8 µg / mL, the internal standard concentration of paliperidone is 50 ng / mL, the internal standard concentration of voriconazole is 2.0 µg / mL, the internal standard concentration of theophylline is 5 µg / mL, the internal standard concentration of fluvoxamine is 200 ng / mL, and the internal standard concentration of lidocaine is 2.0 µg / mL.

[0016] As a preferred embodiment, in step S2, the liquid chromatography conditions are as follows: the chromatographic column is Phenomenon Kinetex 100*2.10 mm, 2.6 μm; the column temperature is 40 o C; flow rate was 300 μL / min; injection volume was 5 μL; organic phase was acetonitrile; aqueous phase was 0.1% formic acid + 0.2 mM ammonium formate.

[0017] The gradient elution conditions were as follows: .

[0018] As a preferred embodiment, the mass spectrometry detection ion parameters are as follows: .

[0019] As a preferred embodiment, the gas parameters for mass spectrometry detection are as follows: curtain gas is 35 au, collision gas is 7 au, ion spray voltage is 5500 volts, temperature is 500° C., ion source gas 1 is 50 au, and ion source gas 2 is 50 au.

[0020] The method of the invention can realize the rapid and synchronous determination of phenytoin sodium, paliperidone, voriconazole, theophylline, fluvoxamine and lidocaine, and has high sensitivity and accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a chromatogram of the simultaneous detection of 6 therapeutic monitoring drugs based on LC-MS / MS technology. DETAILED DESCRIPTION

[0022] The present invention will be further described below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention, rather than to limit the scope of the present invention.

[0023] 1. Reagents 1.1 Standards Phenytoin sodium (Supelco; PHR1139, purity 96.40%) Paliperidone (CATO; 144598-75-4, purity 98.60%) Theophylline (LGC; 58-55-9, purity 98.08%) Fluvoxamine (LGC; 61718-82-9, purity 99%) Voriconazole (LGC; MM194.00, purity 99.60%) Lidocaine (J&K; 137-58-6, purity 99%) 1.2 Internal Standard Phenytoin sodium internal standard (Phe-D10) (Cerilliant; P-067-1ML, 100 μg / mL) Paliperidone internal standard (Pal-D4) (Supelco; 1020719-55-4, 100 μg / mL) Theophylline internal standard (The-IS) (CIL; 100 MILLIGRAMS, 2- 13 C,99%;1,3- 15 N 2 , 98%+) Fluvoxamine internal standard (Flu-D3) (Cerilliant; 1185245-56-0; Fluvoxamine-D3, 100 µg / mL) Voriconazole internal standard (Vor-D3) (CATO; CCAD301239, 99.7%) Lidocaine internal standard (Lid-D10) (MCE; 851528-09-1, 98.9%) 1.3 Organic solvents: Acetonitrile, methanol, chromatographic grade or better.

[0024] 50% methanol refers to a methanol aqueous solution with a volume concentration of 50%.

[0025] 2 Preparation Operation 2.1 Preparation requirements The substances used to prepare solutions all refer to 100% content. If it is less than 100%, it should be converted according to the purity.

[0026] Conversion formula:

[0027] g 实际 : The actual mass of the raw material, in g (gram).

[0028] g 理论 : The theoretical mass of the raw material, in g (gram).

[0029] z: percentage of raw material purity, % The standard for preparing water is: resistance>10 at 25℃ 6 cm of deionized or double distilled water.

[0030] 2.2 Mobile phase preparation 2.2.1 Aqueous phase: 0.1% formic acid + 0.2 mM ammonium formate (add 1 mL formic acid and 1 mL 200 mM ammonium formate stock solution to 998 mL pure water) 2.2.2 Organic phase: pure acetonitrile 2.3 Preparation of working standards The stock solutions of drug standards were diluted with 50% methanol to prepare working standards with the drug concentrations shown in Table 1 below: Table 1: Working standard drug concentrations

[0031] 2.4 Preparation of internal standard solution Prepare the internal standard target working solution (ISWS): The drug internal standard stock solution (100µg / mL) was diluted with 50% (v / w) methanol to prepare phenytoin sodium, paliperidone, voriconazole, theophylline, fluvoxamine, and lidocaine isotope mixed internal standards with concentrations of 0.8µg / mL, 50 ng / mL, 2.0µg / mL, 5µg / mL, 200ng / mL, and 2.0µg / mL, respectively.

[0032] 3. Instruments and Equipment AB / Sciex API 5500 in series with Shimadzu LC-20AD XR liquid chromatography-mass spectrometer, centrifuge, ultrasonic cleaner, Sartorius CP225D analytical balance, French Millipore pure water system, Eppendorf pipette and magnetic stirrer, etc.

[0033] 4 Sampling and samples 4.1 Sampling: Collect blood from the elbow vein using a vacuum blood collection tube.

[0034] 4.2 Sample: Serum 5. Testing procedures 5.1 Instrument preparation 5.1.1 Liquid chromatography conditions The six drugs to be tested have different chemical and physicochemical properties. To achieve good separation effect under the same detection conditions, it is necessary to select a suitable chromatographic column and optimize the mobile phase composition and gradient elution conditions. The final optimization results are shown in Table 2 below.

[0035] Chromatographic column: Pheromon Kinetex 100*2.10 mm, 2.6 μm Mobile phase: aqueous phase: 0.1% formic acid + 0.2 mM ammonium formate; Organic phase: acetonitrile Table 2: Liquid chromatography gradient elution conditions

[0036] The detection limits and quantification limits of the six drugs to be tested are not the same. In particular, the concentrations of clinical samples of paliperidone and fluvoxamine are at the ng / mL level, while the concentrations of other drugs are at the µg / mL level. At the same time, the blood concentration ranges of these drugs are also different. To achieve simultaneous detection, it is necessary to select the best parent ion-daughter ion pair combination and optimize the detection parameters of the mass spectrometer (spray voltage, collision energy, curtain gas, etc.) to ensure that all target detection objects have sufficient sensitivity and maintain good linearity within the linear range. The final optimization results of the mass spectrometry detection ion parameters are shown in Table 3 below.

[0037] Ion source screening: ESI, positive ion mode Ion pair selection: parent ion, daughter ion.

[0038] Table 3: Ion parameters

[0039] Phe: phenytoin sodium; Phe-IS: phenytoin sodium internal standard; Pal: paliperidone; Pal-IS: paliperidone internal standard; Vor: voriconazole; Vor-IS: voriconazole internal standard; The: theophylline; The-IS: theophylline internal standard; Flu: fluvoxamine; Flu-IS: fluvoxamine internal standard; Lid: lidocaine; Lid-IS: lidocaine internal standard.

[0040] The gas parameters are shown in Table 4 below.

[0041] Table 4: Gas parameters

[0042] 5.1.2 Instrument balance: Balance the instrument according to the set chromatographic mass spectrometry conditions, with a balance time of 30-60 minutes.

[0043] 5.1.3 Set the sample quantity and injection times, and select the set detection method.

[0044] 5.2 Sample processing Before the experiment begins, all standard working solutions, internal standard working solutions, and quality control samples should be balanced to room temperature. When sampling standard working solutions, internal standard working solutions, quality control samples, or samples to be tested, they must be mixed evenly, and try to avoid bubbles during mixing.

[0045] 5.2.1 Sample pretreatment Biological samples contain a large amount of endogenous substances, which may cause nonspecific adsorption or ion suppression, thereby affecting the accuracy of quantification. Therefore, this method uses protein precipitation method in the sample pretreatment process to remove interfering substances, optimizes the operation steps, and simplifies the sample pretreatment process. The specific pretreatment steps are as follows: 1) Add 100 μL of sample and 100 μL of mixed internal standard into a 2 mL EP tube and equilibrate at room temperature for 5 minutes; 2) Add 1 mL of acetonitrile for protein precipitation and mix well; 3) Centrifuge at 12,000 rpm for 10 minutes; 4) Take 1 mL of the supernatant and add 500 μL of 50% methanol aqueous solution; 5) Centrifuge at 12,000 rpm for 2 minutes; 6) Take 200 μL of supernatant and add it to 1 mL of 50% methanol aqueous solution.

[0046] 5.2.2 Standard curve 5.2.2.1. Select two test standard solutions and their corresponding chromatographic peaks’ peak area ratios to establish a working curve, with the chromatographic peaks’ peak area ratio as the independent variable and the concentration as the dependent variable.

[0047] 5.2.2.2 Standard curve processing The sample system is shown in Table 5 below (use 5 mL EP tube to add sample).

[0048] Table 5: Standard curve processing sample system

[0049] 5.3 Detection 5 μL of the pre-treated serum sample and mixed standard solution were respectively taken and injected into the liquid chromatography-mass spectrometer to measure and calculate the concentrations of the six drugs.

[0050] Figure 1 The chromatograms of the simultaneous detection of six therapeutic monitoring drugs based on LC-MS / MS technology are shown.

[0051] 6 Data Processing 6.1 Establishment of working curve 1) Select the mass ratio of the two test standard solutions and the peak area ratio of their corresponding chromatographic peaks to establish a working curve, in which the peak area ratio of the chromatographic peaks is the independent variable and the concentration ratio is the dependent variable. Use the SLOPE (slope) and INTERCEPT (intercept) formulas to calculate the slope a and intercept b of the linear regression fitting equation of the standard curve, and establish a working curve: y = ax + b y - Concentration of drug in the sample; x - Peak area ratio of chromatographic peaks; a - The slope of the linear regression equation of the standard curve; b - The intercept of the linear regression equation of the standard curve.

[0052] 2) Because the same amount of internal standard is added to the standard and the sample to be tested, the peak area ratio of the chromatographic peak can be obtained for the sample to be tested, and the corresponding drug concentration can be calculated through the working curve.

[0053] 6.2 Standard curve evaluation 1) Calculate the correlation coefficient r between the peak area ratio and mass ratio of the standard solution The peak area ratio of the drug / IS chromatographic peak of the standard solution was used as the independent variable, and the mass ratio was used as the dependent variable. The correlation coefficient r was calculated using the CORREL formula; 2) r>0.99 means the calibration is qualified. If it does not meet the requirements, the calibration fails and the standard curve needs to be re-established.

[0054] 7 Measurement procedure reliability analysis 7.1 Accuracy The sample detection accuracy results are shown in Table 6 below. Table 6: Sample detection accuracy results

[0055] 7.2 Precision Precision was assessed according to the Clinical and Laboratory Standards Institute (CLSI) document EP15-A3.

[0056] Serum samples with low, medium and high concentrations were measured. Intra-batch precision was evaluated by 9 parallel measurements; inter-batch and total precision were evaluated by a 5×5 method, that is, one batch was tested every day, and each batch contained 5 parallel samples. Each sample was measured 3 times and the average was taken. A total of 25 values ​​were obtained over 5 consecutive days. Subsequently, one-way ANOVA was used to calculate the imprecision between and within batches, and the coefficient of variation (CV) was usually used to evaluate precision.

[0057] The precision determination results are shown in Table 7 below.

[0058] Table 7: Sample detection precision results

[0059] 7.3 Linear range According to the CLSI-EP6 document, high-value samples (phenytoin sodium, paliperidone, voriconazole, theophylline, fluvoxamine, and lidocaine concentrations of 20 μg / mL, 200 ng / mL, 6 μg / mL, 60 μg / mL, 2000 ng / mL, and 5.0 μg / mL, respectively) and blank serum samples (healthy subjects, whose blood did not contain the following drugs) were used to perform an 8-point serial concentration dilution (Low, 0.857L+0.143H, 0.714L+0.286H, 0.571L+0.429H, 0.429L+0.571H, 0.286L+0.714H, 0.143L+0.857H, H) to establish the linear range of clinical samples.

[0060] The results are shown in Table 8 below.

[0061] Table 8: Linear range results

[0062] 7.4 Analytical sensitivity The limit of detection (LOD) refers to the lowest concentration that can be reliably detected in a sample but may not be accurately quantified.

[0063] Limit of Quantification (LOQ) refers to the lowest concentration of a sample that can be accurately measured to a certain extent.

[0064] The present invention selected a series of low-concentration samples and blank serum samples for detection, and used the signal-to-noise ratio (S / N) ≥ 3 as the detection limit standard; and used the signal-to-noise ratio (S / N) ≥ 10 and CV ≤ 20% (n=4) as the quantification limit standard.

[0065] The test results are shown in Table 9 below.

[0066] Table 9: Detection limit and quantification limit results

[0067] 7.5 Interference Experimental tests verified that hemolysis, lipemia and jaundice had no effect on the drug detection results of this method.

[0068] 7.6 Carryover "Carryover contamination" refers to the discontinuous amount of analyte carried from one test sample reaction to another test sample reaction by the measurement system, thereby erroneously affecting the performance of another test sample, specifically referring to sample carryover. "Biochemical test system carryover contamination" refers to the process in which residual substances (biological samples, reagents, mixed reaction solutions or reaction products, etc.) from the previous biochemical test process are carried to the next biochemical test reaction through instrument components (including but not limited to probes, colorimetric cups, stirring rods, pipes, etc.), participate in the reaction, affect the reaction process, directly or indirectly interfere with colorimetry or turbidimetry, etc., and cause significant deviations in the test results.

[0069] After testing, the carryover contamination rates of high-value samples (concentrations: 5μg / mL, 200ng / mL, 8μg / mL, 30μg / mL, 800ng / mL, 8.0μg / mL) and median samples (concentrations: 2.5μg / mL, 100 ng / mL, 4μg / mL, 15μg / mL, 200 ng / mL, 4μg / mL) of 6 drugs (phenytoin sodium, paliperidone, voriconazole, theophylline, fluvoxamine, lidocaine) to low-value samples (concentrations: 0.5μg / mL, 10ng / mL, 0.5μg / mL, 2.5μg / mL, 20ng / mL, 0.5μg / mL) were all between -0.07% and 0.81%. It can be considered that there is no obvious carryover contamination under the current measurement conditions.

[0070] The above results show that the method of the present invention has good accuracy, precision and sensitivity, and can simultaneously detect the concentrations of six drugs in serum, including phenytoin sodium, paliperidone, voriconazole, theophylline, fluvoxamine and lidocaine.

[0071] Comparative Example 1 This comparative example provides an LC-MS / MS method for detecting the concentrations of multiple therapeutic monitoring drugs in biological samples, which differs from the method of the present invention only in that the protein precipitant used in the sample pretreatment uses an equal amount of methanol instead of acetonitrile.

[0072] Test results: Worst detection index R 2 The value is 0.98, the accuracy is 85%, and the precision is 12%.

[0073] Comparative Example 2 This comparative example provides an LC-MS / MS method for detecting the concentrations of multiple therapeutic monitoring drugs in biological samples, which differs from the method of the present invention only in that an equal amount of methanol is used to replace acetonitrile in the mobile organic phase.

[0074] Test results: Worst detection index R 2 The value is 0.95, the accuracy is 82%, and the precision is 15%.

[0075] Comparative Example 3 This comparative example provides an LC-MS / MS method for detecting the concentrations of multiple therapeutic monitoring drugs in biological samples, which differs from the method of the present invention only in that the mobile aqueous phase uses an equal amount of 0.1% formic acid to replace "0.1% formic acid + 0.2 mM ammonium formate".

[0076] Test results: Worst detection index R 2 The value is 0.97, the accuracy is 85%, and the precision is 10%.

[0077] Comparative Example 4 This comparative example provides an LC-MS / MS method for detecting the concentrations of multiple therapeutic monitoring drugs in biological samples, which differs from the method of the present invention only in that the equilibration time of the sample and the internal standard is changed from 5 min to 30 min.

[0078] Test results: Worst detection index R 2 The value is 0.98, the accuracy is 90%, and the precision is 10%.

[0079] Comparative Example 5 This comparative example provides an LC-MS / MS method for detecting the concentrations of multiple therapeutic monitoring drugs in biological samples, which differs from the method of the present invention only in that the elution condition of the liquid chromatography is changed to isocratic elution (70% B).

[0080] Test results: Worst detection index R2 The value is 0.95, the accuracy is 80%, and the precision is 15%.

[0081] Comparative Example 6 This comparative example provides an LC-MS / MS method for detecting the concentrations of multiple therapeutic monitoring drugs in biological samples, which differs from the method of the present invention only in that the collision energy of each drug mass spectrometry detection is replaced by 20V and the dwell time is replaced by 100msec.

[0082] Test results: Worst detection index R 2 The value is 0.96, the accuracy is 85%, and the precision is 12%.

Claims

1. A method for synchronously determining multiple drug components in biological samples based on LC-MS / MS technology, characterized in that: The multiple drugs are phenytoin sodium, paliperidone, voriconazole, theophylline, fluvoxamine, and lidocaine, and the method comprises the following steps: S1. Obtain biological samples and pre-treat them; S2. Liquid chromatography-mass spectrometry was used to simultaneously detect phenytoin sodium, paliperidone, voriconazole, theophylline, fluvoxamine, and lidocaine in the samples.

2. The method according to claim 1, characterized in that The biological sample is serum.

3. The method according to claim 1, characterized in that: The pretreatment step in step S1 is as follows: add 100 μL of the mixed internal standard solution to 100 μL of the biological sample, balance for 5 minutes, add 1 mL of acetonitrile for protein precipitation, mix well, and then centrifuge at 12000 rpm for 10 minutes, then take 1 mL of the supernatant and add 500 μL of 50% methanol aqueous solution, centrifuge at 12000 rpm for 2 minutes, and take 200 μL of the supernatant in 1 mL of 50% methanol aqueous solution.

4. The method according to claim 3, characterized in that The preparation steps of the mixed internal standard solution are as follows: diluting an internal standard stock solution with a concentration of 100 μg / mL with methanol with a volume concentration of 50% to prepare a mixed internal standard solution of phenytoin sodium, paliperidone, voriconazole, theophylline, fluvoxamine, and lidocaine isotopes.

5. The method according to claim 3, characterized in that: In the mixed internal standard solution, the internal standard concentration of phenytoin sodium is 0.8µg / mL, the internal standard concentration of paliperidone is 50 ng / mL, the internal standard concentration of voriconazole is 2.0µg / mL, the internal standard concentration of theophylline is 5µg / mL, the internal standard concentration of fluvoxamine is 200ng / mL, and the internal standard concentration of lidocaine is 2.0µg / mL.

6. The method according to claim 1, characterized in that In step S2, the liquid chromatography conditions are as follows: the chromatographic column is Phenomenon Kinetex 100*2.10 mm, 2.6 μm; the column temperature is 40 o C; flow rate was 300 μL / min; injection volume was 5 μL; organic phase was acetonitrile; aqueous phase was 0.1% formic acid + 0.2 mM ammonium formate.

7. The method according to claim 1, characterized in that In step S2, the gradient elution conditions of liquid chromatography are as follows: 。 8. The method according to claim 1, characterized in that In step S2, the mass spectrometry detection ion parameters are as follows: 。 9. The method according to claim 1, characterized in that: In step S2, the gas parameters for mass spectrometry detection are as follows: curtain gas is 35 au, collision gas is 7 au, ion spray voltage is 5500 volts, temperature is 500°C, ion source gas 1 is 50 a.u., and ion source gas 2 is 50 au.

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